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National  Institute  of  Neurological 
Disorders  and  Stroke 


Annual  Report 
Fiscal  Year  1990 


U.S.  DEPARTMENT 
OF  HEALTH 

AND  HUMAN  SERVICES 
Public  Health  Service 
National  Institutes  of  Health 


Sational  Institute  of  Neurological 
isorders  and  Stroke  (u  sj 


Intramural 
Research 


Annual  Report 
Fiscal  Year  1990 


US.  DEPARTMENT 
OF  HEALTH 

AND  HUMAN  SERVICES 
Public  Health  Service 
National  Institutes  of  Health 


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ANNUAL  REPORT 
October  1, 1989  through  September  30, 1990 

DIVISION  OF  INTRAMURAL  RESEARCH 
National  Institute  of  Neurological  Disorders  and  Stroke 

Table  of  Contents 

TAB 

DIRECTORATE  OF  THE  DIVISION  OF  INTRAMURAL  RESEARCH 

OFFICE  OF  THE  DIRECTOR  OF  INTRAMURAL  RESEARCH  (ODIR)  1 

DIRECTOR:  Dr.  Irwin  J.  Kopin 

DIRECTOR,  BASIC  NEUROSCIENCES  PROGRAM:  Dr.  Harold  Gainer 

DEPUTY  DIRECTOR,  BASIC  NEUROSCIENCES  PROGRAM:  Dr.  Franklin  Hempel 

DIRECTOR,  CLINICAL  NEUROSCIENCES  PROGRAM:  Dr.  Mark  Hallett 

CLINICAL  DIRECTOR:  Dr.  Mark  Hallett 

BASIC  NEUROSCIENCES  PROGRAM:  DIRECTOR,  DR.  HAROLD  GAINER 

INSTRUMENTATION  AND  COMPUTER  SECTION  (BNP/ICS)  1B 

Chief:  Dr.  Bruce  M.  Smith 

ANIMAL  HEALTH  AND  CARE  SECTION  (ODIR/AHCS) 

Acting  Chief:  Dr.  E.Christopher  Staley  1C 

LABORATORY  OF  BIOPHYSICS  (LB)  2 

Acting  Chief:  Dr.  Gerald  M.  Ehrenstein 

LABORATORY  OF  CENTRAL  NERVOUS  SYSTEM  STUDIES  (CNSS)  3 

Chief:  Dr.  Carleton  Gajdusek 
Deputy  Chief:  Dr.  Clarence  J.  Gibbs,  Jr. 

LABORATORY  OF  EXPERIMENTAL  NEUROPATHOLOGY  (LENP)  4 

Chief:  Dr.  Henry  deF.  Webster 

LABORATORY  OF  MOLECULAR  BIOLOGY  (LMB)  5 

Acting  Chief:  Dr.  Franklin  Hempel 

LABORATORY  OF  VIRAL  AND  MOLECULAR  PATHOGENESIS  (LVMP)  6 

Acting  Chief:  Dr.  Monique  Dubois-Dalcq 

LABORATORY  OF  NEURAL  CONTROL  (LNLC)  7 

Chief:  Dr.  Robert  E.  Burke 

LABORATORY  OF  NEUROBIOLOGY  (LN)  8 

Chief:  Dr.  Thomas  S.  Reese 
Deputy  Chief:  Dr.  Milton  Brightman 

LABORATORY  OF  NEUROCHEMISTRY  (LNC)  9 

Chief:  Dr.  Harold  Gainer 

LABORATORY  OF  NEUROPATHOLOGY  AND  NEUROANATOMICAL  SCIENCES  (LNNS)  10 

Chief:  Dr.  Igor  Klatzo 


i-DIR/NINDS 


Table  of  Contents  (Continued) 


TAB 


LABORATORY  OF  NEUROPHYSIOLOGY  (LNP) 
Chief:  Dr.  Jeffery  L.  Barker 


11 


LABORATORY  OF  MOLECULAR  AND  CELLULAR  NEUROBIOLOGY  (LMCN) 
Rotating  Chief :  Dr.  Peter  Fishman 

CLINICAL  NEUROSCIENCES  PROGRAM:  DIRECTOR,  DR.  MARK  HALLETT 

OFFICE  OF  THE  CLINICAL  DIRECTOR  (ODIR/OCD) 

Chief:  Dr.  Mark  Hallett 


12 


1A 


BIOMETRY  AND  FIELD  STUDIES  BRANCH  (BFSB) 
Chief:  Dr.  Jonas  H.  Ellenberg 


13 


DEVELOPMENTAL  AND  METABOLIC  NEUROLOGY  BRANCH  (DMNB) 
Chief:  Dr.  Roscoe  O.  Brady 


14 


EXPERIMENTAL  THERAPEUTICS  BRANCH  (ETB) 
Chief:  Dr.  Thomas  N.  Chase 


15 


MEDICAL  NEUROLOGY  BRANCH  (MNB) 
Chief:  Dr.  Mark  Hallett 


16 


NEUROEPIDEMIOLOGY  BRANCH  (NEB) 

Chief  Designate:  Dr.  Gustavo  Roman 


17 


NEUROIMMUNOLOGY  BRANCH  (NIB) 
Chief:  Dr.  Dale  E.  McFarlin 
Deputy  Chief:  Dr.  Henry  F.  McFarland 


18 


SURGICAL  NEUROLOGY  BRANCH  (SNB) 
Chief:  Dr.  Edward  H.  Oldfield 


19 


CLINICAL  NEUROSCIENCE  BRANCH  (CNB) 
Chief:  Dr.  Irwin  Kopin 


20 


IRP,  NINDS,  Principal  Investigators 
NINDS,  Research  Projects 


Page  iii-iv 
Page  v-vii 


ii-DIR/NINDS 


ANNUAL  REPORT 

October  1 , 1 989  through  September  30, 1 990 

National  Institute  of  Neurological  Disorders  and  Stroke 

Listing  of  NINDS  Intramural  Research  Principal  Investigators 


NAME 

TAB 

PAGE 

NAME 

TAB 

PAGE 

Albers,  R.W. 

9 

6 

Ishiwara,  S. 

8 

12 

Albert,  P.S. 

13 

14 

Iwara,  R.H. 

2 

9,11 

Ali,  I.U. 

19 

41,42 

Jacobson,  S. 

18 

10 

Alkon,  D.L. 

12 

17 

Johnson,  R. 

16 

53 

Andrews,  S.B. 

8 

13 

Karlsson,  S. 

14 

16,17,22 

Arnheiter,  H. 

6 

7,8,10 

Kawai,  K. 

10 

20 

AsherD.M. 

3 

27,28 

Klatzo, I 

10 

28,31 

Astrom  K.E. 

4 

16 

Komoly,  S. 

4 

15 

Bacic,  F. 

10 

23 

Kopin,  I.J. 

20 

15,16 

Bak,  M.J. 

7 

8 

Kuikarni,  A. 

14 

20 

Barker,  J. L. 

11 

1.2 

Kusano,  K. 

9 

12 

Barton,  N. 

14 

14,15 

Lasansky,  A. 

11 

3 

Battey,  J. 

9 

10,11 

Lavine,  L. 

17 

21,22 

Biddison,  W.E. 

18 

10 

Major,  E.O. 

6 

11 

Bobo,  R.H. 

19 

28,29 

Mariani,  A. P. 

11 

5 

Brady,  R.O. 

14 

10,13,18,19 

Marks,  W.B. 

7 

10 

Brann,  M. 

5 

6 

Martin, J. R. 

4 

6 

Brown,  P. 

3 

22 

McCarron,  R. 

10 

10,22,24,29,32 

Burke,  R.E. 

7 

7,11 

McFarland,  H.F. 

18 

7,8 

Chan,  K  -F.J. 

4 

9 

McFarlin,  D.E. 

18 

9 

Chang,  C.J. 

10 

13 

Merrill,  M.J. 

19 

38-40 

Chase,  T.N. 

15 

20 

Mickel,  H. 

4 

11 

Chen,T.C. 

13 

15 

Mies,  G. 

10 

30 

Clay,  J. R. 

2 

8 

Miller,  S. P. 

14 

11,12,23 

Dalakas,  M.C. 

16 

54,55 

Mitchell,  W.J. 

4 

12 

Dambrosia,  J  M 

13 

9,10 

Mouradian,  M.M. 

15 

17 

Di  Chiro,  G. 

16 

56-58 

Nelson,  K.B. 

17 

14,23-27 

Doe,  P. 

8 

15 

Nelson,  R. 

11 

4 

Dubois-Dalcq,  M. 

6 

5,9 

Nowak,  T.S. 

10 

16,19,21 

Ehrenstein,  G 

2 

4,10 

Odenwald,  W. 

5 

8,9 

Eldridge,  R. 

17 

10,11,12 

O'Donovan,  M.J. 

7 

13 

EllenbergJ.H. 

17 

13,15-20 

Oldfield,  E.H. 

19 

27,30,46 

Emoto,  S.E. 

13 

11 

Olson,  J.J. 

19 

31 

Fedio,  P. 

16 

44-47 

O'Neill,  R.R. 

14 

21 

Fishman,  P.H. 

12 

15,16 

Pant,  H.C. 

9 

9 

Foulkes,  M.A. 

13 

12,13,16 

Plunkett,  R. 

19 

37,38 

Freese,  E. 

5 

5 

Polinsky,  R.J. 

20 

13,14 

Gainer,  H. 

9 

7,8 

Porrino,  L. 

19 

43-45 

Gajdusek,  D.C. 

3 

14,19 

Quarles,  R.H. 

12 

18-20 

Garruto,  R.M. 

3 

16,21 

Rebois,  R.V. 

12 

14 

Gibbs,  C.J.  Jr. 

3 

22-25 

Reese,  T.S. 

8 

10-12,14 

Gilbert,  D.L. 

2 

5 

Ressetar,  H.G. 

4 

14 

Grafman,  J. 

16 

51,52 

Rogawski,  M. 

16 

48-50 

Gravell,  M. 

3 

24 

Roman,  G. 

17 

13,15-20 

Hallett,  M. 

1A 

6 

Saito,  J.N. 

10 

26 

16 

39-43 

Saris,  S. 

19 

32,33 

Henken,D.B. 

4 

13 

Schmidt,  EM. 

7 

9 

Henneberry,  R.C. 

5 

7 

Hudson,  L.D. 

6 

6 

iii-DIR/NINDS 


NAME  TAB  PAGE 


Schubert,  M. 
Schwartz,  J. P. 
Sibley,  D.R. 
Simpson,  D.L. 
Smith,  B. 
Smith,  C.L. 
Smith,  T.G. 
Spatz,  M. 
Staley,  E.C. 
Stanley,  E.F. 
Stoner,  G.L 
Theodore,  W.H. 
Venter,  J.C. 
Wagner,  H.G. 
Walters,  J. R. 
Webster,  H.deF. 
Yanagihara  R 
Youle,  R.J. 
Zalewski,  A.A. 


6 

12,14 

20 

17 

15 

18 

8 

16 

1B 

1 

7 

14 

11 

6 

10 

11,14,18,25,27 

1C 

1 

2 

6,7 

4 

8 

16 

37,38 

12 

21-23 

10 

12,15,17 

15 

19 

4 

7,10 

3 

16 

18 

34,36 

7 

12 

iv-DIR/NINDS 


ANNUAL  REPORT 

October  1,  1989  through  September  30,  1990 

National  Institute  of  Neurological  and  Communicative  Disorders  and  Stroke 

INTRAMURAL  RESEARCH  PROJECTS 
Numerical  Inventory 


PROJECT  NUMBER 


TAB 


PAGE 


PROJECT  NUMBER 


TAB 


PACE 


ZOl  NS 

00200-36 

MNB 

16 

47 

ZOl 

NS 

02151-16 

LMCN 

12 

17 

Z01  NS 

00813-29 

LNC 

9 

6 

ZOl 

NS 

02160-16 

LNLC 

7 

11 

ZOl  NS 

00815-30 

DMN 

14 

10 

ZOl 

NS 

02162-16 

DMN 

14 

11 

ZOl  NS 

00969-26 

CNSS 

3 

19 

ZOl 

NS 

02163-16 

DMN 

14 

12 

ZOl  NS 

01195-26 

MNB 

16 

56 

ZOl 

NS 

02167-16 

NEB 

17 

12 

ZOl  NS 

01245-25 

MNB 

16 

46 

ZOl 

NS 

02202-15 

NIB 

18 

7 

ZOl  NS 

01282-26 

CNSS 

3 

14 

ZOl 

NS 

02203-15 

NIB 

18 

8 

ZOl  NS 

01309-25 

LMCN 

12 

16 

ZOl 

NS 

02204-15 

NIB 

18 

9 

ZOl  NS 

01424-24 

MNB 

16 

45 

ZOl 

NS 

02205-16 

NIB 

18 

10 

ZOl  NS 

01442-22 

LN 

8 

10 

ZOl 

NS 

02218-15 

LB 

2 

5 

ZOl  NS 

01658-23 

MNB 

16 

44 

ZOl 

NS 

02236-15 

MNB 

16 

38 

ZOl  NS 

01659-22 

LNP 

11 

3 

ZOl 

NS 

02240-14 

NEB 

17 

13 

ZOl  NS 

01686-22 

LNLC 

7 

7 

ZOl 

NS 

02243-14 

NEB 

17 

14 

ZOl  NS 

01687-22 

LNLC 

7 

8 

ZOl 

NS 

02254-14 

LNLC 

7 

12 

ZOl  NS 

01688-22 

LNLC 

7 

9 

ZOl 

NS 

02263-14 

ETB 

15 

18 

ZOl  NS 

01805-22 

LN 

8 

15 

ZOl 

NS 

02265-14 

ETB 

15 

20 

ZOl  NS 

01808-21 

LMCN 

12 

19 

ZOl 

NS 

02297-14 

NEB 

17 

15 

ZOl  NS 

01881-21 

LN 

8 

11 

ZOl 

NS 

02299-14 

NEB 

17 

16 

ZOl  NS 

01924-20 

NEB 

17 

10 

ZOl 

NS 

02300-14 

NEB 

17 

17 

ZOl  NS 

01927-20 

NEB 

17 

11 

ZOl 

NS 

02301-14 

NEB 

17 

18 

ZOl  NS 

01983-19 

LVMP 

6 

11 

ZOl 

NS 

02307-14 

NEB 

17 

19 

ZOl  NS 

01995-18 

LENP 

4 

7 

ZOl 

NS 

02315-13 

ODIR 

16 

58 

ZOl  NS 

02019-18 

LNP 

11 

1 

ZOl 

NS 

02318-13 

MNB 

16 

37 

ZOl  NS 

02034-18 

LVMP 

6 

5 

ZOl 

NS 

02324-14 

LNNS 

10 

10 

ZOl  NS 

02038-18 

MNB 

16 

54 

ZOl 

NS 

02330-13 

LNP 

11 

2 

ZOl  NS 

02073-17 

MNB 

16 

57 

ZOl 

NS 

02357-13 

LNNS 

10 

11 

ZOl  NS 

02079-17 

LNLC 

7 

10 

ZOl 

NS 

02365-12 

LMB 

5 

7 

ZOl  NS 

02086-17 

LN 

8 

16 

ZOl 

NS 

02366-12 

LMCN 

12 

15 

ZOl  NS 

02088-17 

LB 

2 

4 

ZOl 

NS 

02370-12 

NEB 

17 

20 

ZOl  NS 

02115-17 

CNB 

20 

13 

ZOl 

NS 

02423-11 

NEB 

17 

21 

ZOl  NS 

02139-16 

ETB 

15 

19 

ZOl  NS 

02144-16 

LN 

8 

17 

v  -  DIR/NINDS 


Intramural  Research  Projects  -  Numerical  Inventory  (Continued) 


PROJECT  NUMBER 


Z01 

NS 

02453-10 

DMN 

Z01 

NS 

02454-10 

SN 

Z01 

NS 

02483-10 

BFSB 

Z01 

NS 

02490-10 

BFSB 

ZOl 

NS 

02505-10 

BFSB 

ZOl 

NS 

02516-09 

BFSB 

ZOl 

NS 

02526-09 

LB 

ZOl 

NS 

02528-09 

LVMP 

ZOl 

NS 

02531-09 

MNB 

ZOl 

NS 

02548-09 

LNNS 

ZOl 

NS 

02549-09 

LENP 

ZOl 

NS 

02550-09 

LENP 

ZOl 

NS 

02570-08 

NEB 

ZOl 

NS 

02578-08 

ETB 

ZOl 

NS 

02594-08 

BFSB 

ZOl 

NS 

02598-08 

BFSB 

ZOl 

NS 

02603-07 

NI 

ZOl 

NS 

02606-07 

LB 

ZOl 

NS 

02608-07 

LB 

ZOl 

NS 

02609-07 

LB 

ZOl 

NS 

02610-07 

LN 

ZOl 

NS 

02623-07 

LNNS 

ZOl 

NS 

02630-06 

CNB 

ZOl 

NS 

02631-07 

LNP 

ZOl 

NS 

02652-06 

BFSB 

ZOl 

NS 

02657-06 

DMN 

ZOl 

NS 

02664-06 

DMN 

ZOl 

NS 

02667-06 

MNB 

ZOl 

NS 

02668-06 

MNB 

ZOl 

NS 

02669-06 

MNB 

ZOl 

NS 

02673-06 

SN 

ZOl 

NS 

02674-06 

SN 

ZOl 

NS 

02675-06 

ODIR 

ZOl 

NS 

02677-06 

LMB 

ZOl 

NS 

02689-06 

LNNS 

ZOl 

NS 

02697-06 

SN 

ZOl 

NS 

02698-05 

LMB 

ZOl 

NS 

02699-05 

LENP 

ZOl 

NS 

02700-05 

LN 

ZOl 

NS 

02705-05 

LNP 

ZOl 

NS 

02707-05 

SN 

ZOl 

NS 

02708-05 

SN 

ZOl 

NS 

02709-05 

LB 

ZOl 

NS 

02710-05 

LMCN 

TAB 

PACE 

14 

13 

19 

27 

13 

11 

13 

10 

13 

15 

13 

13 

2 

6 

6 

6 

16 

55 

10 

12 

4 

6 

4 

8 

17 

22 

15 

17 

13 

14 

13 

12 

18 

11 

2 

7 

2 

8 

2 

9 

8 

13 

10 

13 

20 

14 

11 

4 

13 

9 

14 

14 

14 

15 

16 

39 

16 

40 

16 

41 

19 

34 

19 

36 

1A 

6 

5 

5 

10 

14 

19 

31 

5 

8 

4 

9 

8 

14 

11 

5 

19 

40 

19 

39 

2 

10 

12 

21 

PROJECT  NUMBER 


ZOl 

NS 

02711-05 

MNB 

ZOl 

NS 

02712-06 

MNB 

ZOl 

NS 

02715-05 

NEB 

ZOl 

NS 

02716-05 

CNB 

ZOl 

NS 

02717-05 

CNB 

ZOl 

NS 

02718-05 

LNNS 

ZOl 

NS 

02720-04 

LNNS 

ZOl 

NS 

02723-04 

LNC 

ZOl 

NS 

02724-04 

LNC 

ZOl 

NS 

02725-04 

LNC 

ZOl 

NS 

02728-04 

SN 

ZOl 

NS 

02729-04 

SNB 

ZOl 

NS 

02730-04 

DMN 

ZOl 

NS 

02731-04 

DMN 

ZOl 

NS 

02732-04 

MNB 

ZOl 

NS 

02739-04 

SN 

ZOl 

NS 

02742-04 

LVMP 

ZOl 

NS 

02746-04 

NEB 

ZOl 

NS 

02747-04 

NEB 

ZOl 

NS 

02748-04 

NEB 

ZOl 

NS 

02749-04 

LNNS 

ZOl 

NS 

02750-04 

LNNS 

ZOl 

NS 

02752-04 

CNB 

ZOl 

NS 

02753-02 

LNC 

ZOl 

NS 

02754-03 

LMCN 

ZOl 

NS 

02756-03 

LVMP 

ZOl 

NS 

02757-03 

LNC 

ZOl 

NS 

02758-03 

LENP 

ZOl 

NS 

02759-03 

LENP 

ZOl 

NS 

02760-03 

SN 

ZOl 

NS 

02761-03 

SN 

ZOl 

NS 

02762-03 

SN 

ZOl 

NS 

02767-03 

LNP 

ZOl 

NS 

02768-03 

LNNS 

ZOl 

NS 

02769-03 

DMN 

ZOl 

NS 

02770-03 

DMN 

ZOl 

NS 

02771-03 

DMN 

ZOl 

NS 

02772-03 

MNB 

ZOl 

NS 

02773-03 

LNNS 

ZOl 

NS 

02774-02 

LNC 

ZOl 

NS 

02775-02 

LNNS 

ZOl 

NS 

02776-02 

LNNS 

ZOl 

NS 

02777-02 

LNNS 

ZOl 

NS 

02778-03 

SN 

ZOl 

NS 

02780-03 

LNNS 

TAB   PAGE 


16 

42 

16 

43 

17 

23 

20 

15 

20 

16 

10 

15 

10 

16 

9 

7 

9 

8 

9 

9 

19 

32 

19 

38 

14 

16 

14 

17 

16 

48 

19 

46 

6 

8 

17 

24 

17 

25 

17 

26 

10 

17 

10 

18 

20 

17 

9 

10 

12 

22 

6 

13 

9 

12 

4 

10 

4 

11 

19 

43 

19 

45 

19 

44 

11 

6 

10 

19 

14 

18 

14 

19 

14 

20 

16 

50 

10 

20 

9 

11 

10 

21 

10 

22 

10 

23 

19 

33 

10 

24 

vi  -  DIR/NINDS 


Intramural  Research  Projects  -  Numerical  Inventory  (Continued) 


'ROJECT  NUMBER 


TAB 


PAGE 


Z01  NS 

02781-03  SN 

19 

37 

Z01  NS 

02782-02  DMN 

14 

21 

Z01  NS 

0278A-02  LMCN 

12 

14 

Z01  NS 

02785-02  DMN 

14 

22 

Z01  NS 

02786-02  LMCN 

12 

18 

Z01  NS 

02787-02  LNCL 

7 

13 

Z01  NS 

02788-02  LNLC 

7 

14 

Z01  NS 

02789-02  LVMP 

6 

9 

Z01  NS 

02790-02  LVMP 

6 

10 

ZOl  NS 

02791-02  LVMP 

6 

12 

Z01  NS 

02792-02  MNB 

16 

51 

ZOl  NS 

02793-02  MNB 

16 

52 

ZOl  NS 

02794-02  MNB 

16 

53 

ZOl  NS 

02795-02  LNNS 

10 

25 

ZOl  NS 

02796-02  LNNS 

10 

26 

ZOl  NS 

02797-02  LNNS 

10 

27 

ZOl  NS 

02798-02  LNNS 

10 

28 

ZOl  NS 

02799-02  LB 

2 

11 

ZOl  NS 

02800-02  LMB 

5 

6 

ZOl  NS 

02801-02  LNNS 

10 

29 

ZOl  NS 

02802-03  LNNS 

10 

32 

NEW  INITIATIVES  FOR  1990 

4 

ZOl  NS 

02803-01  LENP 

12 

ZOl  NS 

02804-01  LENP 

4 

13 

ZOl  NS 

02805-01  LMCN 

12 

20 

ZOl  NS 

02806-01  LMCN 

12 

23 

ZOl  NS 

02807-01  LENP 

4 

14 

ZOl  NS 

02808-01  LENP 

4 

15 

ZOl  NS 

02809-01  LENP 

4 

16 

ZOl  NS 

02810-01  BFSB 

13 

16 

ZOl  NS 

02811-01  SN 

19 

30 

ZOl  NS 

02812-01  SN 

19 

29 

ZOl  NS 

02813-01  SN 

19 

28 

ZOl  NS 

02814-01  SN 

19 

41 

ZOl  NS 

02815-01  SN 

19 

42 

ZOl  NS 

02816-01  DMNB 

14 

23 

ZOl  NS 

02817-01  NIB 

18 

12 

ZOl  NS 

02818-01  LVMP 

6 

14 

ZOl  NS 

02819-01  NEB 

17 

27 

ZOl  NS 

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5 

9 

ZOl  NS 

02821-01  LNNS 

10 

30 

ZOl  NS 

02822-01  LNNS 

10 

31 

ZOl  NS 

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19 

35 

PROJECT  NUMBER 


TAB   PAGE 


vii  -  DIR/NINDS 


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ANNUAL  REPORT 

October  1, 1989  through  September  30,  1990 
Office  of  the  Director,  Division  of  Intramural  Research 
National  Institute  of  Neurological  Disorders  and  Stroke 

Table  of  Contents 

TAB 
Office  of  the  Director,  DIR  1 

Office  of  the  Clinical  Director  (OCD)  1A 

Instrumentation  and  Computer  Section  (ICS)  1 B 

Animal  Health  and  Care  Section  (AHCS)  IC 


i    -   ODIR/DIR 


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PATHOGENESIS 

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ANNUAL  REPORT  OF  THE  SCIENTIFIC  DIRECTOR 

DIVISION  OF  INTRAMURAL  RESEARCH 

National  Institute  of  Neurological  Disorders  and  Stroke 
October  1,  1989  through  September  30,  1990 

Irwin  J.  Kopin,  M.D.,  Scientific  Director 

The  Division  of  Intramural  Research  (DIR),  National  Institute  of  Neurological 
Disorders  and  Stroke,  conducts  investigations  in  a  wide  array  of  disciplines  related  to 
the  neurosciences.  The  DIR  comprises  the  Office  of  the  Director,  the  Clinical 
Neurosciences  Program  (CNP)  and  the  Basic  Neurosciences  Program  (BNP). 

The  Office  of  the  Director  includes  the  Animal  Health  and  Care  Section  (AHCS)  and 
the  Coordinator  for  AIDS  research.  At  present,  the  CNP  consists  of  eight  Branches 
and  a  Neuroimaging  Section  while  the  BNP  contains  eleven  Laboratories  and  the 
Instrumentation  and  Com-puter  Section.  The  CNP  is  located  mainly  in  the  Clinical 
Center  where  there  are  both  inpatient  facilities  and  outpatient  clinics  together  with 
supporting  laboratories.  The  BNP  is  accommodated  largely  in  Building  36  with 
important  components  in  the  Federal  Building  in  Bethesda,  the  Park  Building  in 
Rockville,  and  at  Fort  Detrick  in  Frederick,  Maryland.  Research  requiring  marine 
animals  is  conducted  during  the  summer  in  facilities  rented  from  the  Marine 
Biological  Laboratory  in  Woods  Hole,  Massachusetts. 

Federal  Government  scientists,  support  staff,  guest  researchers  and  volunteer 
workers  continue  to  contribute  new  discoveries  which  have  significant  impact  on  the 
explosive  growth  of  knowledge  about  the  nervous  system  and  its  disorders. 
Research  is  initiated  by  scientists  with  interests  ranging  from  the  fundamental  basis 
for  molecular  interactions  regulating  growth,  development,  function,  and  pharma- 
cology of  the  nervous  system  to  clinically  relevant  development  of  new  diagnostic 
and  therapeutic  procedures  targeted  on  the  early  diagnosis,  prevention,  retarda- 
tion, cure,  or  symptomatic  control  of  neurologic  disorders.  The  results  of  these 
investigations  advance  biomedical  knowledge  and  directly  or  indirectly  contribute 
to  the  prevention  or  treatment  of  human  suffering  from  disease  or  injury,  the  main 
mission  of  the  Institute  and  the  NIH.  The  important  accomplishments  and  status  of 
potentially  major  advances  toward  understanding  neuronal  function  and  dys- 
function are  summarized  in  the  Laboratory/Branch  reports  and  in  the  investigator- 
initiated  research  summaries  included  in  the  FY  I990  Annual  Report.  This  portion 
will  address  those  issues  having  a  major  impact  on  the  administration  and  resources 
(i.e.,  personnel,  available  space,  and  financing)  of  the  Institute. 

During  most  of  FY90,  management  of  the  DIR  was  a  team  effort  which  involved  the 
active  participation  of  Dr.  Mark  Hallett,  Director  of  the  CNP  (who  is  also  Clinical 
Director,  NINDS),  Dr.  Ernst  Freese,  Director  of  the  BNP,  and  the  Scientific  Director. 
The  administrative  skills,  scientific  expertise,  wise  counsel  of  the  Program  Directors, 
along  with  able  administrative  officers,  have  contributed  immensely  to  the  smooth 
operation  of  the  DIR  by  the  Director.  The  untimely  death  of  Dr.  Ernst  Freese  on 
March  30,  1990,  was  a  great  loss  for  the  Institute  as  well  as  a  personal  loss  to  his 
many  friends  and  colleagues.  Dr.  Harold  Gainer  has  succeeded  Dr.  Freese  as  Director, 
BNP,  and  is  proving  to  be  a  valuable  addition  to  the  directorship  of  the  DIR. 


1  -ODIR/DIR 


Recent  discoveries,  advances  in  technology,  and  new  initiatives  in  the  neurosciences 
require  organizational  changes  and  reallocation  of  resources.  Responses  to  new 
needs,  changes  in  staffing,  and  limited  availability  of  new  space  create  needs  for 
continual  reassessment  of  program  goals  and  progress  and  for  appropriate  evolu- 
tion of  allocations  for  optimal  utilization  of  available  resources.  These  issues  provide 
a  major  challenge  to  the  direction  of  the  DIR  so  that  both  clinical  and  fundamental 
research  can  continue  to  flourish.  Present  plans  include  the  development  of  two 
new  Branches  which  have  already  been  approved  for  development  during  the  next 
year.  The  Neuroimaging  Branch  will  exploit  more  fully  the  expertise  in  the  rapidly 
expanding  area  of  brain  imaging  by  magnetic  resonance,  positron  emission 
tomography,  and  possibly  magnetic  encephalography.  This  effort  will  be  supported 
by  some  newly  available  space  as  well  as  the  resources  of  the  Neuroimaging  Section 
formerly  in  the  Office  of  the  Director,  DIR,  and  will  continue  under  the  leadership  of 
Dr.  Giovanni  Di  Chiro.  There  are  plans  for  a  new  Stroke  Branch  and  NINDS  is  currently 
conducting  a  search  for  appropriate  leadership.  Its  resources  will  include  those 
currently  in  the  Laboratory  of  Neuropathology  and  Neuroanatomical  Science  in 
addition  to  some  additional  space  which  will  become  available  when  current  plans 
for  use  of  off-campus  space  and  space  in  Building  49  are  clarified.  Although  most  of 
the  Branches  in  the  CNP  have  been  located  in  the  Clinical  Center,  because  of  a 
shortage  of  space,  portions  have  been  accommodated  in  Building  9  as  well  as  in  the 
Park  Building  in  Rockville.  In  the  future,  some  Branch  laboratories  will  be  situated  in 
the  new  Building  49  as  well  as  the  Clinical  Center,  but  we  do  not  anticipate  that  any 
off-campus  space  will  be  required. 

Personnel 


As  in  previous  years,  the  DIR  has  utilized  fully  its  employment  ceiling.  Young 
investigators  provide  depth  of  expertise  for  future  roles  and  expansion  of  research 
leadership  in  new  high-priority  research  efforts.  They  are  particularly  important 
because  the  disparity  in  salaries  between  government  and  academic  institutions  or 
industry  makes  it  extremely  difficult,  if  not  impossible,  to  attract  senior  established 
investigators  to  the  NIH.  It  is  also  difficult  to  retain  promising  young  investigators. 
There  have  been  several  major  losses  to  the  Surgical  Neurology  Branch.  Dr.  Robert 
Plunkett,  who  was  recently  awarded  tenure  and  who  was  one  the  important  assets 
in  our  research  program  on  tissue  implants  into  brain,  has  accepted  a  position  at  the 
University  of  Buffalo  at  a  salary  in  excess  of  three-fold  greater  than  he  could  have 
earned  at  NIH.  Another  young  neurosurgeon,  Dr.  Jeffery  Olson,  also  has  declined  a 
tenure  appointment  on  completion  of  his  Senior  Staff  Fellowship  to  accept  a  much 
more  lucrative  position  at  Emory  Clinic  in  Atlanta,  Georgia.  Dr  Linda  Porrino,  who 
was  head  of  a  Neuroimaging  Section,  has  accepted  a  position  at  Bowman-Gray 
University  and  will  be  leaving  an  important  vacancy  in  the  newly-formed  Neuro- 
imaging Branch.  However,  we  have  been  successful  in  attracting  a  few  senior 
neuroscientists.  After  an  extended  search,  Dr.  Gustavo  Roman  was  recruited  to  fill 
the  position  of  Chief,  Neuroepidemiology  Branch,  and  he  is  now  begun  to  actively 
develop  a  new  program.  Dr.  David  Goldstein,  a  long-time  collaborator  with  NINDS 
scientists  interested  in  sympathetic-adrenal  function  and  regulation,  has  been  trans- 
ferred from  the  NHLBI  to  the  CNB  where  he  will  head  a  new  Section  on  Sympathetic 
Function. 

At  present,  there  are  over  570  employees  accounting  for  the  481  full-time  equivalent 
(FTE)  positions.  Requirements  for  expanded  efforts  on  research  of  AIDS  have  neces- 
sitated some  shifts  in  FTE  positions.  The  personnel  of  the  DIR  includes  171 
professional  non-tenured  FTE  employees:  28  medical  staff  fellows,  19  staff  fellows, 
48  senior  staff  fellows,  17  special  experts,  and  59  visiting  associates/visiting  scientists. 

2-ODIR/DIR 


There  are  also  86  FTE-ceilinq  exempt  scientists  (Visiting  Fellow  and  IRTA  positions; 
National  Research  Council  fellows)  who  are  subject  to  an  NIH-imposed  ceiling,  as 
well  as  guest  workers,  volunteers,  and  IPA  appointments. 

Scientists  continue  to  be  attracted  away  from  NIH,  largely  because  of  more 
advantageous  financial  arrangements  (higher  salary,  college  tuition  for  children,  less 
restriction  on  consultation  to  industry,  etc.).  Dr.  Donald  Gehlert,  a  tenure-track 
Senior  Staff  Fellow  in  the  Experimental  Therapeutics  Branch  has  accepted  an  offer  in 
private  industry.  The  relatively  low  salary  scales  at  NIH  have  frustrated  attempts  to 
appoint  a  Chief  for  the  Laboratory  of  Neuronal  Growth  and  Regeneration  (LNGR). 
The  Laboratory  has  been  disbanded  and  Dr.  Zalewski,  who  had  Deen  Acting  Chief, 
has  been  reassigned  to  the  Laboratory  of  Neural  Control  (LNC)  as  Chief  of  the  Neural 
Regeneration  Section.  Other  components  which  would  have  been  part  of  LNGR 
have  been  included  in  the  Surgical  Neurology  Branch.  Dr.  Robert  Burke,  Chief,  LNLC, 
has  established  a  new  Section  of  Developmental  Neurobiology  headed  by  Dr. 
Michael  O'Donovan. 

We  are  continuing  efforts  to  recruit  an  established  junior  investigator  to  form  a  core 
neurogenetics  group.  The  individual  selected  is  expected  to  have  a  strong  develop- 
mental neurobiology  background.  To  date,  potential  candidates  have  accepted 
other  positions. 

Dr.  Alison  Wichman  has  elected  to  join  the  Clinical  Center,  Bioethics  Office;  Dr. 
Barbara  Karp  has  been  appointed  to  replace  her  as  Chief  of  the  Neurology 
Consultation  Service. 

During  the  last  year,  three  NINDS  scientists  have  been  approved  for  tenure  as 
independent  investigators  (Drs.  Marinos  Dalakas,  MNB;  Michael  Rogawski,  MNB; 
and  Steve  Jacobson,  NIB)  and  one  as  collaborate  investigator  (Dr.  Paul  Gallant,  LN). 

Space 

Inadequacy  of  space  on  the  NIH  campus  requires  that  some  of  the  NINDS  scientists 
work  in  rented  off-campus  facilities.  Even  after  completion  of  Building  49,  this 
situation  will  persist,  and  off-campus  laboratory  space  has  been  planned  in  the 
newly  rented  former  Gillette  Laboratory  Building  on  Route  272  in  Rockville  as  well  as 
continued  occupancy  of  some  space  in  the  Park  Building.  Maintaining  a  critical  mass 
to  ensure  scientific  interactions  and  avoiding  isolation  are  important  considerations 
in  any  off-campus  facility.  At  present,  most  of  an  entire  large  laboratory  (Laboratory 
of  Molecular  and  Cellular  Neurobiology,  LMCN)  is  housed  in  the  Park  Building;  a 
portion  of  Dr.  Daniel  Alkon's  Section  on  Neural  Systems  is  accommodated  in  Building 
9.  Two  of  the  investigators  in  LMCN  will  be  given  space  in  Building  49  when 
available,  but  Dr  Alkon  will  be  relocated  to  the  Gillette  Building.  The  Section  for 
Receptor  Biochemistry  and  Molecular  Biology  under  Dr.  Craig  Venter,  as  well  as  the 
Central  DNA  Sequencing  Facility  also  under  h is  supervision,  will  remain  in  the  Park 
Building.  This  latter  assists  NINDS  laboratories  in  preparing  and  sequencing  oligo- 
nucleotides relevant  to  neuroscience  research. 

To  meet  AALAC  accreditation  standards,  all  animal  care  facilities  are  being  cen- 
tralized and  will  be  under  the  aegis  of  the  AHCS  of  the  NIH.  All  NINDS  animals  in 
Building  36  (except  primates  with  immunodeficient  virus  infections,  primates  in 
LNLC,  and  a  small  colony  of  HSV-infected  mice)  have  been  moved  into  the  combined 
animal  facility  on  the  mezzanine  level  of  Building  36.  This  new  facility  provides 
superb  holding,  procedural  space,  and  excellent  environmental  controls. 

3-ODIR/DIR 


Shortly,  Building  10A  will  be  available  to  house  all  animals  in  Building  10;  com- 
pensatory space  for  loss  of  space  in  Building  10A  by  NINDS  will  become  available. 
NINDS  will  receive  about  20  modules,  six  of  which  are  currently  occupied  by  NINDS 
staff;  five  are  to  be  renovated  during  the  next  fiscal  year  and  the  remainder  await 
transfer  after  completion  of  the  Central  Animal  Facility  in  10A.  Scientists  of  the 
Surgical  Neurology  Branch  who  are  now  working  Building  9  will  remain  there  until 
completion  of  Building  49. 

Problems  of  "swing  space"  for  ward  renovations  have  been  resolved  satisfactorily, 
and  the  two  Nursing  Units  will  be  renovated  during  FY-91. 

Fiscal  Issues 

If  current  level  of  support  is  maintained,  it  will  be  possible  to  continue  the  important 
research  operations  of  the  Institute.  Additional  funds  may  be  required  if  requests 
for  additional  personnel  are  allocated  to  expand  high-priority  research  work  on 
AIDS,  tissue  implants  and  gene  therapy,  and  to  support  additional  off-campus  space 
rental. 


4-ODIR/DIR 


Cooperation  With  Industry 

Scientists  in  NINDS  will  be  initiating,  or  already  have  initiated,  Cooperative  Research 
and  Development  Agreements  (CRADAs)  with  industrial  organizations  with  the  goal 
of  commercializing  products  developed  within  their  laboratories.  CRADAs,  nego- 
tiated in  1990  include:  (a)  Dr.  James  Battey;  Triton  Biosciences;  Dr.  Richard  Feldman; 
Isolation  of  cDNA  that  Encode  the  Murine  Gastrin  Releasing  Peptide  Receptor 
(mGRP-R);  (b)  Dr.  Richard  Youle;  Haflund  Nycomed;  Dr.  Tore  Tsjaberg;  Immuno- 
toxins  for  Central  Nervous  System  Disease;  and  (c)  Dr.  Richard  J.  Youle;  Biogen,  Inc.; 
Dr.  Roy  Lobb;  Angiogenen  Immunotoxins.  These  are  in  addition  to  eight  other 
active  CRADAs  and  two  which  are  currently  being  renewed. 


5-ODIR/DIR 


i;  : 


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■  "  .       ■  .  '.  Q  •  ■ 

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'■■■..  ■         ■  '  ■'•'■'■■ 

rbiriwowt  brse  ■•:■■   lAfi 


ANNUAL  REPORT 
October  1,  1989  through  September  30,  1990 

Office  of  the  Clinical  Director,  OD,  DIR 
Office  of  Director,  Clinical  Neuroscience  Program,  DIR 
National  Institute  of  Neurological  Disorders  and  Stroke 

Table  of  Contents 

RESEARCH  SUMMARY  1-5 

PROJECT  REPORTS  6 

Evaluation  of  Neuromuscular  Diseases 
ZOl  NS  02675-06  ODIR 


Annual  Report:  October  1,  1989  to  September  30,  1990 

Office  of  the  Director 

Office  of  the  Clinical  Director 

Clinical  Neuroscience  Program,  DIR 

Mark  Hallett,  M.D.,  Clinical  Director 

The  Office  of  the  Clinical  Director  handles  administrative  matters,  mainly  relating  to 
patient  care,  coordination  of  educational  activities,  and  delivery  of  neurologic  services. 
Service  functions  can  be  divided  into  the  EEG  Laboratory,  the  EMG  Laboratory,  the 
Consultation  Service  for  Neurology,  Neuromuscular  Services,  Neuropathology,  and 
Paraprofessional  Support  Services.  The  Otolaryngology  Consultation  Service  and  the 
Audiology  Laboratory  moved  during  the  year  to  the  National  Institute  of  Deafness  and 
Other  Communicative  Disorders  (NIDCD). 

The  two  major  educational  conferences  are  the  Clinical  Conference  (held  on  Tuesday 
afternoon),  which  is  aimed  at  the  Medical  Staff  Fellows  and  typically  reviews  a  patient 
in  detail,  and  the  NINDS  Grand  Rounds  (held  on  Friday  afternoon).  The  Clinical 
Conference  includes  some  attention  to  matters  of  patient  care  and  quality  assurance. 
The  Grand  Rounds  continues  to  offer  CME  credit. 

EEG  Laboratory,     Susumu  Sato,  M.D.,  Chief 

Diagnostic  Services: 

During  this  reporting  period,  a  significant  change  occurred  in  the  Section  staff:  one 
technician  retired  after  more  than  25  years  service  and,  therefore,  the  Section  was 
operated  by  one  technologist  alone  for  nearly  six  months.  Subsequently,  thanks  to  Title 
38,  the  special  pay  scale  program,  two  new,  well-qualified  technologists  were  recruited: 
one  in  late  1989  and  another  in  May  1990.  Thus,  the  Section  has  for  the  first  time  since 
1979  a  full  force  with  well-qualified  technologists.  This  personnel  situation  was 
reflected  in  a  slight  decrease  in  the  overall  number  of  EEG  examinations  compared  with 
that  of  the  prior  year.  It  is  anticipated  that  the  number  of  examinations  will  increase  as 
we  go  on.  The  Section  will  expand  its  diagnostic  activity  to  sleep  monitoring  and 
outpatient  video-EEG  monitoring  for  epileptic  patients. 

EEG  Evoked  Potentials 

109 
45 

37 


NINDS 

232 

NINDS  OPD 

242 

NIADDK 

1 

NICHD 

69 

NIAAA 

25 

NIMH 

113 

NCI 

73 

NHLB 

16 

NIAID 

23 

NIA 

33 

CCM,  SICU  &  CSR 

7 

OTHER  OPD 

57 

8 
3 

19 
TOTAL  891  221 

1     ODIR/DIR(OCD) 


Participation  in  Research  Activity; 

The  EEG  Section  collaborates  closely  with  the  Clinical  Epilepsy  Section  of  the  Medical 
Neurology  Branch  and  plays  an  important  role  in  evaluating  epileptic  patients.  The 
Section  staff  monitor  and  interpret  EEG  recording  during  pentylentetrazol  intravenous 
injection,  sodium  amytal  intracarotid  injection,  surgery  for  treatment  of  epilepsy 
(electrocorticography)  and  during  chronic  subdural  recording  in  epileptic  patients.  The 
Section  staff  assist  in  applying  electrodes  for  video-EEG  telemetry  recording, 
magnetoencephalographic  study  and  neuropsychological  investigation. 

The  collaboration  has  been  made  to  monitor  EEG  of  patients  with  cystinosis  and  to 
monitor  the  EEG  and  visual  evoked  responses  in  patients  who  undergo  anticancer 
chemotherapy.  The  Section  will  undertake  some  experimental  activity  in  power 
spectrum  and  brain  mapping,  intensive  EEG  monitoring  of  epileptic  patients,  and 
computer  analysis  of  epileptiform  discharges. 

The  EEG  Laboratory  provides  a  training  environment  for  a  Medical  Staff  Fellow  toward 
certification  by  the  American  EEG  Board.  The  Laboratory  Chief  continues  to  serve  as 
Associate  Examiner  on  the  EEG  Board. 

Publications 

Bromfield  EB,  Sato  S,  Charnas  L,  and  Balish  M:  Electroencephalographic  findings  in  the 
oculocerebrorenal  syndrome  of  Lowe,  (submitted). 

Fink  JK,  Brouwers  P,  Barton  N,  Malekzadeh  MH,  Sato  S,  Hill  S,  Cohen  WE,  Fivush  B  Gahl 
W:  Neurologic  complications  in  long-standing  nephropathic  cystinosis.  Arch  Neurol 
1989;46:543-49. 

EMG  Laboratory,  Roger  W.  GUliatt,  M.D.,  Chief 

EMG  ACTIVITIES     (July  1,  1989-June  30,  1990) 

Number  of  Examinations  NINDS  209  (Normal  Volunteers  39) 

OTHER  INSTITUTES  172 

Total  379 


Approximately  half  of  the  patient  referrals  to  the  EMG  Laboratory  during  the  year 
originated  within  NINDS,  and  the  other  half  were  requested  from  other  Institutes.  Part 
of  the  work  of  the  Laboratory  consists  of  routine  diagnostic  and  prognostic  studies  of 
patients  under  the  care  of  the  Clinical  Center;  the  other  part  consists  of  special  studies 
on  agreed  projects.  These  may  be  in  collaboration  with  other  Institutes  or  may  be 
initiated  from  within  the  laboratory. 

1)        Studies  on  AIDS  and  other  HIV-positive  patients  (Collaborative  studies  with  the 
National  Cancer  Institute) 

As  in  the  past,  the  section  has  continued  to  monitor  HIV-positive  patients,  including 
AIDS  and  ARC  patients,  for  signs  of  neuropathy  during  treatment  with  experimental  drug 
regimes  (AZT/DDC/DDI  combinations.) 


2    ODIR/DIR(OCD) 


2)  Patients  with  polymyositis  (Collaborative  program  with  the  National  Institute  of 
Arthritis  and  Musculoskeletal  and  Skin  Diseases.) 

These  patients  are  referred  for  the  special  treatment  of  inflammatory  muscle  disease 
with  steroids,  immunosuppression  or  plasmapheresis.  We  have  modified  the  technique  of 
quantitative  EMG  originally  described  by  Willison  (1964),  which  involves  analysis  of  the 
interference  pattern  of  the  EMG  during  sustained  muscle  contractions  against  a  standard 
load.  There  have  been  problems  with  this  technique  in  the  past  which  have  seemed  to 
limit  its  usefulness,  but  we  believe  it  has  important  advantages  over  other  quantitative 
methods,  such  as  the  measurement  of  motor  unit  duration. 

3)  Studies  on  patients  with  nephropathic  cystinosis  (Collaborative  studies  with  the 
National  Institute  of  Child  Health  and  Human  Development) 

Since  modern  treatment  has  prolonged  life  in  this  condition,  late  neuromuscular 
complications  of  the  metabolic  abnormality  have  appeared,  in  particular  an  unusual 
pattern  of  muscle  wasting  and  weakness  affecting  mainly  the  extremities,  due  to  primary 
muscle  damage  rather  than  denervation.  The  main  evidence  for  this  comes  from 
quantitative  EMG  studies. 

4)  Evaluation  of  neuromuscular  disease  (NINDS  Study  #84-N-203;  Principal 
Investigator,  Dr.  Mark  Hallett)   Studies  under  this  protocol  include: 

a.  The  effect  of  varying  the  detection  threshold  on  interference  pattern  analysis  in 
healthy  subjects  and  patients  with  primary  muscle  disease. 

b.  The  use  of  mixed  nerve  action  potentials  in  patients  with  chronic  inflammatory 
demyelinating  neuropathy,  including  the  use  of  monopolar  recording  to  detect 
temporal  dispersion. 

c.  The  use  of  medial  plantar  nerve  action  potentials  in  patients  with  axonal 
neuropathies. 

Publications: 

Meer  J,  Gilliatt  R.  Proximal  and  distal  conduction  velocity  in  the  motor  nerves  of 
patients  with  hereditary  motor  and  sensory  neuropathy  1.    Muscle  Nerve  1989;12:974-5. 

Gilliatt  R,  Meer  J.   The  refractory  period  of  transmission  in  patients  with  carpal  tunnel 
syndrome.    Muscle  Nerve  1990;13:445-50. 

La  Rocca  R,    Meer  J,  Gilliatt  R,  Stein  W,  Cassidy  CA,  Myers,  CE,  Dalakas  M.   Suramin- 
induced  polyneuropathy.   Neurology  1990;40:954-60. 

Bielawski  M,  Hallett  M.    Position  of  the  elbow  in  determination  of  abnormal  motor  c 
of  the  ulnar  nerve  across  the  elbow.    Muscle  Nerve  1989;12:803-9. 

Ravits  J,  Hallett  M,  Baker  M,  Nilsson  J,  Dalakas,  MC.  Clinical  and  electromyographic 
studies  of  postpoliomyelitis  muscular  atrophy.    Muscle  Nerve  199013:667-74. 


3    ODIR/DIR(OCD) 


Neurology  Consultation  Service,  Barbara  Illowsky  Karp,  M.D.,  Chief 

The  Neurology  Consultation  Service  consists  of  three  neurologists:  Drs.  Barbara  Karp 
(Chief),  Eric  Wassermann,  and  Alison  Wichman.  The  service  provides  emergency  and 
routine  neurologic  consultation  to  both  in:and  outpatients  at  the  Clinical  Center.  The 
consultation  services  include  initial  evaluation,  facilitation  of  procedures  and  testing  in 
other  departments  (such  as  neuroradiology  and  electrodiagnostic  studies),  and  follow-up 
neurologic  care. 

Patients  referred  to  the  consultation  service  over  the  last  year  include: 


NCI 

164 

NIAID 

78 

NHLBI 

80 

NIDDK 

67 

NIMH 

25 

NICHD 

17 

Other  (OPD,  NINDS,  NIA,  NIDR,  NEI): 

53 

TOTAL  484 

Ten  percent  of  patients  seen  by  the  neurology  consultation  were  HIV-positive. 

Participation  in  Research  Activity: 

The  consultation  service  staff  participates  in  research  activities  within  NINDS  and  in 
collaboration  with  NIMH.  Studies  being  pursued  include  those  under  protocols  84N-196 
and  87N-110  on  human  motor  control  and  on  neurologic  abnormalities  in  schizophrenia. 
Under  protocol  90M-41,  studies  on  hyponatremia  and  polydipsia  in  schizophrenics  are 
being  completed. 

Publications: 

Casanova  MF,  Prasad  CM,  Waldman  I,  Karp  BI,  Stein  B,  Weinberger  DR,  Kleinman  JE: 
Basal  ganglia  mineralization  in  schizophrenic  patients:   a  quantitative  computerized 
tomographic  study.   Biol  Psychiatry  1990;27:138-42. 

Neuropathology,  David  A.  Katz,  M.D. 

As  in  previous  years,  diagnostic  Neuropathology  Service  for  NINDS,  and  for  all  other 
Institutes,  have  been  provided  by  Dr.  Katz.  The  Neuropathology  Service  is  integrated 
with  the  Autopsy,  Surgical  Pathology,  and  Ultrastructural  Pathology  Sections  and 
residency  training  program  of  the  Laboratory  of  Pathology,  NCI;  a  high  priority  is  given 
to  resident  teaching.  The  brain  was  examined  in  a  high  percentage  of  autopsies 
performed  at  NIH  in  the  last  year.  Many  manifested  significant  primary  or  secondary 
neurologic  disease,  including  malignant  gliomas,  dementias,  neurologic  complications  of 
systemic  malignancy,  and  AIDS,  particularly  in  the  pediatric  age  group.  Braincutting, 
held  weekly,  is  scheduled  so  as  to  encourage  participation  by  interested  physicians  and 
nurses.      Relevant   neuropathologic   findings   are   presented   formally   at   gross   autopsy 


4    ODIR/DIR(OCD) 


conference  and  mortality  conferences.  Selected  cases  are  further  utilized  for  neurologic 
clinical  conferences.  Neurosurgical  specimens  include  both  in-house  and  submitted 
materials,  for  an  annual  total  of  approximately  250  cases;  intra-operative  frozen-section 
consultations  are  required  in  approximately  50  in-house  cases  per  year  (Surgical  material 
includes  primary  and  metastatic  brain  tumors,  pituitary  tumors,  spinal  tumors,  vascular 
lesions,  and  brain  biopsies  particularly  for  AIDS  cases). 

The  Neuropathology  Service  functions  in  a  collaborative  manner  to  provide  subspecialty 
expertise  in  a  range  of  clinicopathologic  investigations.  Active  consent  collaborations 
include  the  following  areas:  (1)  dementia  and  degenerative  disease:  NIMH  (Trey 
Sunderland,  M.D.),  and  NIA  (Stanley  Rapoport,  M.D.);  (2)  malignant  gliomas:  NINDS  and 
NCI;  (3)  pituitary  adenomas:    NICHD,  NIDDK,  and  NINDS;  (4)  multiple  sclerosis:  NINDS. 

Paraprofessional  Support  Services 

Publications 

Houff  SA,  Katz  D,  Kufta  CV,  Major  EO:   A  rapid  method  in  situ  hybridization  for  viral 
DNA  in  brain  biopsies  from  patients  with  AIDS.    AIDS  1990;3:843. 

McCutcheon  I,  Baranco  RA,  Katz  D,  Saris  S:   Adoptive  immunotherapy  of  intracerebral 
metastasis  in  mice.   J.  Neurosurg  1990;72:102. 

Theodore  WH,  Katz  D,  Kufta  CV,  Sato  S,  Patronas  N,  Bromfield  E:    Pathology  of 
temporal  lobe  foci.    Correlation  with  CT,  MRI,  and  PET.    Neurology  1990;40:797. 


5    ODIR/DIR(OCD) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02675-06 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Evaluation  of  Neuromuscular  Diseases 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  MarkHallett,  M.D.  Clinical  Director  OCD  ODIR  DIR  NINDS 

Others: 

Roger  Gil liatt,  M.D.  Chief,  EMG  Lab  OCD  ODIR  DIR  NINDS 

Robin  Conwit,  M.D.  Medical  Staff  Fellow  OCD  ODIR  DIR  NINDS 

Nguyet  Dang  Biomedical  Engineer 

Louis  Johnson  EMG  Technician  OCD  ODIR  DIR  NINDS 

Carlos  Luciano,  M.D.  Medical  Staff  Fellow  OCD  ODIR  DIR  NINDS 


COOPERATING  UNITS  (If  any) 

National  Institute  of  Arthritis  and  Musculoskeletal  and  Skin  Diseases/National  Cancer  Institute  and 
National  Institute  of  Child  Health  and  Human  Development 


LAB/BRANCH 

Office  of  the  Clinical  Director,  Office  of  the  Director,  CNP,  Division  of  Intramural  Research 


SECTION 

EMG  Laboratory 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  Q  g 


OTHER: 


0.1 


CHECK  APPROPRIATE  BOX(ES) 

QTj   (a)  Human  subjects  j     ]  (b)  Human  tissues  I      I  (c)  Neither 

]  (a1)  Minors 

I      |  (a2)  Interviews 
SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Understanding  of  neuromuscular  diseases  is  founded  on  careful  clinical  observation, 
electrodiaqnostic  studies  and  pathology.  This  protocol  has  been  developed  to  learn 
more  about  established  diseases,  to  characterize  new  diseases,  to  assess  current 
methodologies  and  technologies,  and  to  refine  old  methods  and  develop  new  ones. 

Studies  initiated  underthis  protocol  include: 

a.  The  effect  of  varying  the  detection  threshold  on  interference  pattern  analysis  in 
healthy  subjects  patients  with  primary  muscle  disease. 

b.  The  use  of  mixed  nerve  action  potentials  (MNAPs)  in  patients  with  chronic 
inflammatory  demyelinatinq  neuropathy  (CIDP),  including  the  use  of  monopolar 
recording  to  detect  temporal  dispersion. 

c.  The  use  of  medial  plantar  nerve  action  potentials  (NAPs)  in  patients  with  axonal 
neuropathies. 


PHS  6040  (Rev.  184) 


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ANNUAL  REPORT 

October  1,  1989  -  September  30,  1990 

Instrumentation  and  Computer  Section 
Office  of  Director,  Basic  Neurosciences  Program,  DIR 
National  Institute  of  Neurological  Disorders  and  Stroke 


Table  of  Contents  Page 

ORGANIZATIONAL  STRUCTURE  AND  SERVICES  1 

INSTR.  VENTATION  2-5 

COMPUTER  SUPPORT  5-10 


i  BNP/DIR(ICS) 


INSTRUMENTATION  AND  COMPUTER  SECTION 
Office  of  Director,  Basic  Neurosciences  Program,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 

October  1, 1989  -  September  30, 1990 

Bruce  M.  Smith,  Ph.D.,  Chief 


The  Instrumentation  and  Computer  Section  (ICS)  provides  technical  support  for  investigators 
of  NINDS  and  NIMH,  Divisions  of  Intramural  Research  (DIR's)  by  (1)  assessing  the 
instrumentation  and  computer  needs  of  the  investigator;  (2)  designing,  developing  and  constructing 
special-purpose  electronic  and  mechanical  instrumentation  systems;  (3)  designing,  specifying  and 
managing  laboratory  computer  systems  for  data  acquisition  and  processing;  (4)  designing  and 
developing  custom  software  for  scientific  and  administrative  applications;  and  (5)  managing  a  central 
computer  facility  consisting  of  a  multiuser  MicroVAX  3600,  an  image  processing  system,  and  a 
network  of  Macintosh  personal  computers  and  LaserWriter  printers.  An  additional  important  service 
provided  by  Section  personnel  is  consultation  on  a  wide  range  of  topics  in  the  areas  of 
instrumentation,  computer  science,  mathematics  and  statistics. 

When  the  services  of  the  Section  are  requested,  the  investigator  first  meets  with  the  Section 
Chief  and  other  appropriate  personnel  to  discuss  the  requirements.  On  the  basis  of  this  meeting,  a 
decision  is  made  as  to  whether  ICS  will  take  on  the  project.  If  a  commercial  product  will  satisfy  the 
requirements,  the  investigator  is  advised  to  purchase  it.  If  a  custom  design  is  required,  ICS  will 
accept  the  project  unless  we  lack  the  appropriate  expertise,  or  our  current  work  backlog  is  excessive. 
In  these  cases  the  project  may  be  contracted  to  a  private  firm,  or  the  investigator  may  be  directed  to 
the  Biomedical  Engineering  and  Instrumentation  Branch  (BEIB). 

When  the  Section  Chief  or  the  Assistant  to  the  Chief  agrees  to  accept  a  project,  a  standard 
ICS  work  request  form  is  initiated.  The  Section  member  leading  the  project  then  confers  with  the 
investigator  to  formulate  a  set  of  specifications  and  a  cost  estimate  for  the  project.  This  information 
is  recorded  on  the  work  form  which  the  investigator  and  his/her  Lab  Chief  sign  to  authorize  the 
project.  ICS  does  not  charge  for  services;  however,  upon  completion  of  the  project,  the 
investigator's  laboratory  or  branch  is  billed  for  the  cost  of  the  components  used.  Reimbursement  of 
funds  takes  place  at  the  beginning  of  the  next  fiscal  year. 


1  BNP/DIR(ICS) 


INSTRUMENTATION 

The  Section  has  a  staff  of  four  engineers,  five  computer  specialists  and  five  technicians  to 
design  and  produce  special-purpose  instrumentation.  The  availability  of  powerful,  low-cost  personal 
computers  and  single-chip  microprocessors  has  broadened  the  Section's  approach  to  instrumentation 
development.  It  is  often  appropriate  for  an  engineer,  a  technician,  and  a  computer  specialist  to  work 
together  to  combine  electronic  or  mechanical  hardware,  a  personal  computer  or  microprocessor,  and 
custom  software,  to  produce  cost-effective  solutions  to  complex  instrumentation  problems.  The 
following  are  brief  descriptions  of  the  Section's  major  projects,  taken  from  a  total  of  344  projects 
undertaken  this  year. 

Ambulatory  Patient  Activity  Monitoring  System.  The  Section  has  continued  to  develop  the 
Patient  Activity  Monitor  (PAM)  and  the  hardware  and  software  which  forms  the  system.  Intramural 
investigators  and  their  outside  collaborators  are  using  the  system  in  their  studies  and  treatment  of 
depression,  hyperactivity,  schizophrenia,  alcoholism,  sleep  and  eating  disorders,  and  animal  models 
of  Parkinson's  disease.  Monitor:  The  current  version  of  the  PAM  has  a  lK-byte  memory  and  can 
store  over  10  days  of  activity  values  accumulated  in  15-minute  recording  intervals.  Approximately 
130  monitors  are  in  use,  with  the  Section  providing  battery  changes  and  repairs  as  needed.  The 
development  of  a  new  improved  monitor  with  a  32K-byte  memory  and  one-minute  activity  intervals 
has  undergone  several  revisions  and  is  now  nearing  its  final  form.  Computer  Support:  The  Section 
supports  a  PAM  readout  system  based  on  the  Macintosh  personal  computer  coupled  to  a 
microprocessor-controlled  serial  interface.  A  comprehensive  PAM  program  has  been  written  for  the 
Macintosh  to  handle  data  readout  and  disk  filing,  graphical  data  editing,  construction  of  continuous 
data  files  and  raster  plots,  and  formation  of  tabular  data  sets  for  transfer  into  spreadsheet  and 
statistical  applications.  The  readout  program  and  the  serial  interface  have  recently  been  modified  to 
accommodate  the  new  PAM's  increased  memory  capacity  and  to  take  advantage  of  its  much  higher 
readout  speed.  IMS  CRAP  A:  In  May,  1989  the  NIMH  and  Individual  Monitoring  Systems,  Inc. 
(IMS)  entered  into  a  Cooperative  Research  and  Development  Agreement  in  order  to  work  together  to 
further  the  development  of  the  PAM  technology  for  the  benefit  of  the  NIMH  and  the  general  public. 
Thus  far  through  this  cooperative  effort,  IMS  has  produced  20  units  of  the  current  NIMH  monitor 
for  commercial  sales  to  research  and  medical  markets.  IMS  and  the  Section  have  also  utilized  their 
joint  expertise  to  finalize  the  new  PAM  design  and  are  pursuing  the  extension  of  the  PAM  technology 
to  monitor  additional  parameters. 

Ambulatory  Eye  Blink  Monitoring  Systems.  The  same  technology  employed  in  the  32K-byte 
PAM  design  has  been  used  to  develop  three  different  prototype  units  for  monitoring  eye  blinks  in 
ambulatory  subjects.  Each  device  uses  a  thin  film  piezoelectric  sensor  attached  to  the  skin  near  the 
corner  of  the  eye  to  detect  eye  blinks.  The  first  device  records  the  interval,  in  seconds,  between  eye 
blinks.  The  second  device  sounds  an  alarm  whenever  a  preselected  number  of  seconds  passes 
without  an  eye  blink,  and  records  the  number  of  alarm  occurrences  during  each  one-minute  interval. 
These  two  units  were  developed  for  use  in  sleep  deprivation  studies  for  the  treatment  of  depression 
and  manic-depressive  illness.  The  third  device,  developed  for  schizophrenia  studies,  records  the 
number  of  eye  blinks  that  occur  during  each  one-minute  interval.  The  Macintosh  PAM  program  and 

2  BNP/DIR(ICS) 


the  serial  interface  have  been  jointly  modified  to  recognize  these  three  new  types  of  monitors  and  to 
read,  store  and  display  each  data  type  correctly.  These  three  prototype  units  are  currently  being 
tested  in  the  intramural  clinics. 

PCR  Controller.  The  feasibility  of  a  highly  efficient  system  for  implementing  the  polymerase 
chain  reaction  (PCR)  method  of  DNA  amplification  is  being  investigated.  The  goal  of  this  project  is 
to  significantly  increase,  compared  to  commercial  PCR  cycler  devices,  the  number  of  samples  that 
can  be  simultaneously  processed.  A  prototype  system  has  been  developed  that  uses  temperature- 
regulated,  circulating  water  baths  to  provide  the  three  sources  of  stable  temperature  to  a  chamber  that 
holds  four  microtiter  plates;  each  plate  holds  96  samples.  A  microprocessor  system  controls  six 
solenoid  valves  to  regulate  the  cyclic  flow  of  water  from  the  three  baths  to  the  chamber.  Nine 
different  PCR  cycling  routines  can  be  individually  selected  by  the  user,  modified  as  needed,  and 
saved  for  reuse.  Continued  development  of  this  project  to  a  successful  completion  depends  on  the 
availability  of  microtiter  plates  that  have  both  a  sufficiently  high  thermal  conductivity  to  allow  rapid 
temperature  cycling  and  the  ability  to  withstand  the  94  °C  denaturing  hot  water. 

Drosophila  Activity  Monitors.  In  support  of  genetic  studies  of  the  Drosophila  response  to 
certain  anesthesias,  the  Section  has  developed  a  number  of  devices  for  assessing  activity  levels  as  a 
measure  of  anesthetic  resistance.  Last  year,  an  automated  quantification  of  the  activity  of 
Drosophila  in  a  test  tube  chamber  was  developed.  Three  additional  assays  methods  have  recently 
been  completed.  One  unit  exposes  the  flies  to  a  heated  plate  whose  temperature  can  be  adjusted  to  a 
maximum  of  40  °C  above  ambient.  A  second  stimulation  unit  energizes  a  dual-spiral  conductor  plate 
with  up  to  200  DC  volts.  When  the  spiral  circuit  is  contacted  by  a  fly,  a  shock  is  delivered  and  the 
operator  is  notified  by  a  piezo  alarm.  A  third  device  consists  of  an  airtight  chamber  equipped  with 
custom-designed  traps  to  collect  the  resistant  flies  following  anesthesia.  The  cylindrical  traps  can  be 
opened  remotely  by  turning  off  a  solenoid  which  surrounds  the  trap  opening  and  holds  a  hinged 
cover  in  place.  The  flies  are  attracted  into  the  traps  by  either  food  or  pheromones  and  by  internal 
green  light  provided  by  two  LED  lamps  which  are  activated  when  the  traps  are  opened. 

Standard  Instruments  for  Neurophysiology.  The  Section  continues  to  develop  a  variety  of 
instruments  which  find  wide  use  in  the  intramural  neurophysiology  laboratories.  These  instruments 
are  designed  with  enough  flexibility  that  only  small,  if  any,  modifications  are  required  to  satisfy 
specific  research  needs.  This  year  three  types  of  instruments  were  often  requested:  4-channel  pulse 
generators  (3  units),  4-channel  amplifiers  (5  units),  and  multichannel  valve  controllers  (6  units).  The 
pulse  generators  allow  numerous  lab  devices  to  be  sequentially  controlled  and  their  operations 
synchronized  with  data  acquisition  systems.  The  gain,  bandwidth,  offset,  and  polarity  functions  of 
the  amplifier  systems  provide  preprocessing  of  analog  signals  before  they  are  recorded  or  digitized. 
The  valve  controllers  provide  both  manual  and  remote  (gated)  control  of  up  to  nine  miniature 
solenoid  flow  valves  used  to  change  the  ionic  composition  of  the  extracellular  fluid  surrounding 
isolated  cells. 

Controllers  for  Perfusion  Studies.  The  Section  is  assisting  two  intramural  laboratories  to 
each  implement  a  different  perfusion  system  to  study  the  effects  of  extracellular  drug  concentrations 
on  cell  properties.  One  system  uses  a  linear-actuator  stepper  motor  to  rapidly  switch  the  position  of  a 

3  BNP/DIR(ICS) 


linear  array  of  nine  micropipettes.  The  solution  in  each  barrel  of  the  array  is  driven  at  the  same  flow 
rate  by  a  multichannel  pump  and  is  gated  on  and  off  by  a  nine-channel  valve  controller.  A 
microprocessor  system  is  being  developed  to  provide  the  precise  timing  between  the  stepper  motor 
movements  and  the  gating  of  the  valve  controller  so  that  complex  perfusion  sequences  can  be 
generated.  The  second  perfusion  system  is  used  to  study  slower  cellular  changes  and  requires  no 
pipette  movement.  Instead,  a  special  micropipette  holds  nine  individual  solution  tubes  whose  ends 
all  converge  very  close  to  the  tiny  common  exit  port  at  the  end  of  the  micropipette.  The  flow  of  each 
solution  is  again  controlled  by  small  valves  which  are  gated  through  a  valve  controller.  A 
programmable  sequence  generator  with  non-volatile  memory  is  being  designed  to  provide  automatic 
timing  signals  for  the  valve  controller. 

EEG  Amplifier  System.  An  improved  32-channel  EEG  amplifier  system  and  calibrator  were 
completed  this  year  for  use  in  ongoing  research  projects,  including  topographic  brain  mapping.  Both 
the  preamplifier  and  the  main  amplifier  were  redesigned  for  increased  flexibility  and  improved  60-Hz 
noise  rejection.  Considerable  interest  has  been  shown  by  other  labs  around  the  country  in 
duplicating  our  original  system.  These  improvements  should  facilitate  others  to  construct  their  own 
EEG  amplifiers  based  on  our  design.  Each  amplifier  channel  consists  of  a  preamplifier,  amplifier, 
and  a  selectable  anti-aliasing  filter.  The  system  provides  control  over  signal  bandwidth,  and 
provides  outputs  for  recording  by  a  tape  recorder  and  16-channel  polygraph,  and  for  digitizing  and 
analysis  of  the  EEG  signals  by  a  computer.  The  calibrator  for  the  system  generates  an  8-Hz,  100 
|ivolt  sine  wave  to  the  preamplifiers  of  all  32  channels  to  verify  that  everything  is  working  and  to 
adjust,  with  software,  any  small  differences  in  the  gains  of  each  channel. 

Visual  Stimulus  Generator.  A  device  has  been  designed  to  present  visual  stimuli  to 
genetically-deaf  mice  during  brain  plasticity  studies.  A  cylindrical  drum  twelve  inches  in  diameter 
with  stimulus  patterns  on  its  inside  wall  is  rotated  around  a  fixed  central  platform  which  holds  the 
animal  enclosure.  Custom  Delrin  and  Teflon  bearings  produced  a  light-weight  construction  and  low- 
noise  movement.  A  40  oz.-in.  DC  motor  coupled  to  the  drum  with  a  rubber  drive  belt  provides  an 
adjustable  rotation  speed  of  0-60  RPM. 

Personal  Computer  Interfaces.  An  interface  has  been  developed  which  allows  IBM- 
compatible  PC's  to  provide  reinforcement  during  visual  recognition  tasks  via  control  of  liquid  or 
pellet  dispensers.  Recent  modifications  to  this  design  provide  for  control  of  120  volt  AC  loads,  such 
as  infusion  pumps,  and  for  the  digital  triggering  of  stimulus  generators.  In  conjunction  with  the 
efforts  to  develop  a  versatile  Macintosh  II  data  acquisition  and  control  system,  the  Section  is 
developing  interface  units  that  provide  easy  access  to  the  A/D  and  D/A  converters  and  to  the  digital 
inputs  and  outputs.  Three  simple  units  have  been  built;  a  more  complex  design  is  being  specified  to 
provide  computer-controlled  preprocessing  of  the  analog  input  signals. 

Peripheral  Sharing  Units.  Buffered  peripheral  sharing  units  were  selected,  configured  and 
installed,  along  with  all  necessary  cabling,  in  the  NIMH,  IRP  Area  A  and  Area  B  Administrative 
Offices.  These  devices  have  increased  the  effective  use  of  office  personal  computers  by  providing  a 
means  for  sharing  common  peripherals  such  as  laser  and  dot  matrix  printers  and  modems. 


4  BNP/DIR  (ICS) 


MACHINE  SHOP  FACILITY 

The  Section  maintains  a  well-equipped  machine  shop  which  is  specialized  for  working  with 
plastics  and  other  synthetic  materials,  and  metals.  This  facility  is  critical  to  the  development  and 
fabrication  of  electronic  and  electromechanical  instrumentation  projects.  Additionally,  three  of  the 
Section's  five  technicians  utilize  this  facility  to  independently  specify,  design,  and  fabricate  a  wide 
range  of  mechanical  devices  as  part  of  the  Section's  efforts  to  provide  a  spectrum  of  services  in 
support  of  basic  and  clinical  research.  These  staff  members  are  also  available  to  advise  investigators 
on  mechanical  principles  and  techniques  and  on  the  properties  and  uses  of  materials.  Many 
investigators  and  other  intramural  staff  frequently  come  to  the  shop  for  immediate  help  with  a  small 
mechanical  problem  whose  timely  solution  is  crucial  to  their  ongoing  research.  Trained  technicians 
from  other  labs  use  our  facility  to  augment  the  limited  capabilities  in  their  own  areas. 

The  following  list  illustrates  the  range  of  mechanical  design  and  fabrication  projects  typically 
provided  by  our  machine  shop  staff:  a  wide  variety  of  chambers  for  biological  preparations, 
including  tissue  cultures,  electrophoresis  gels,  and  static  and  dynamic  temperature-controlled 
perfusion  systems;  modifications  to  micromanipulators  and  to  microscopes  and  other  optical 
devices;  modifications  to  animal  chairs,  restraining  devices  and  enclosures;  pipette  holders  and 
storage  racks,  including  radiation  shields  and  collectors;  a  variety  of  Faraday  cages  and  enclosures; 
human  and  primate  holders  and  adapters  for  brain  scanners;  and  numerous  other  adapters  for 
commercial  instrumentation. 


COMPUTER  SUPPORT 

In  addition  to  the  development  of  special  instrumentation  systems,  ICS  provides  support  for 
laboratory  and  office  computer  systems  and  maintains  central  computer  facilities  in  Bldg.  36  for 
high-capacity  data  storage,  complex  off-line  data  analysis,  image  processing,  scientific  word 
processing,  and  high-quality  printing  and  plotting.  These  support  services  are  detailed  under  the 
following  categories. 


LABORATORY  COMPUTERS 

Small  minicomputers  and  personal  computers  are  widely  used  in  the  intramural  laboratories 
for  real-time  data  acquisition  and  control,  mathematical  and  statistical  data  analysis,  graphics,  and 
word  processing.  ICS  provides  consultation  on  the  specification  and  selection  of  these  systems  and 
helps  the  scientists  in  the  procurement,  installation  and  maintenance  of  the  equipment.  Training  in 
operating  systems,  programming  languages  and  maintenance  issues  is  available  for  scientists  or 
laboratory  support  personnel.  Manpower  limitations  make  it  difficult  for  ICS  to  provide  complete 
programming  for  specific  individual  applications.  Section  computer  specialists  are  always  available 
for  consultation  and  will  aid  the  investigator  in  writing  the  difficult  time  and  data  dependent  sections 
of  real-time  programs.     Section  programming  efforts  in  support  of  laboratory  applications  are 

5  BNP/DIR(ICS) 


concentrated  on  developing  and  maintaining  a  library  of  routines  which  are  specifically  designed  to 
be  incorporated  into  investigators'  programs.  ICS  personnel  also  evaluate  commercial  software  or 
application  programs  from  other  research  facilities  to  determine  their  utility  for  intramural  laboratory 
systems. 

ICS  has  selected  the  Apple  Macintosh  family  of  computer  systems  as  our  standard  for 
support  of  scientific  applications.  The  Section  has  developed  considerable  experience  using  the 
Macintosh  Plus  to  provide  innovative  solutions  for  low- speed  laboratory  data  acquisition  projects. 
For  the  acquisition  of  real-time  data  and  control  of  laboratory  devices  at  high  speeds,  the  Section  is 
leading  an  intramural  effort  to  develop  a  Macintosh  II-based  system  which  will  provide  equivalent 
features  to  those  on  older  PDP- 1 1  systems,  and  will  offer  extended  capabilities  by  using  the 
advanced  graphical  features  of  the  Macintosh  II.  This  year,  ICS  completed  development  of  the 
backbone,  or  high-level,  portion  of  the  software  system.  A  comprehensive  Request  for  Proposal 
was  then  written  for  outside  development  of  carefully  specified  modules  tailored  to  be  compatible 
with  the  backbone  and  with  the  functionality  of  the  existing  system.  A  contract  has  been  awarded 
and  ICS  is  managing  its  execution.  Using  the  Section's  backbone  routine  as  a  framework,  the 
contractor  has  produced  a  B-test  version  of  the  final  program,  called  the  Neurophysiological  Data 
Acquisition  Program  (NDAP).  This  version  is  currently  being  evaluated  under  laboratory  conditions 
by  a  committee  of  intramural  investigators. 

NDAP  should  be  useful  in  all  disciplines  acquiring  data,  either  analog  or  discrete,  in  a 
continuous  or  event-triggered  mode.  It  contains  modules  for  event-centered  raster  displays,  signal 
averaging,  baseline  reference  monitoring,  voltage  clamping,  pre-programming  experimental 
paradigms,  maintenance  of  the  experimental  logbook,  interactive  experimental  control,  apparatus 
control,  and  high-speed,  continuous  data  acquisition. 


VAX  FACILITY 

During  this  fiscal  year,  the  Section  completed  the  replacement  of  its  DEC  VAX- 1 1/750  by 
transferring  all  accounts,  data,  software  and  user  connections  to  a  new  DEC  Micro  VAX  3600.  The 
3600  offers  2.4  times  the  processing  speed  of  the  1 1/750,  and  the  increase  in  RAM  memory  from  8 
to  32  megabytes  (MB)  greatly  reduces  "page  swapping"  of  virtual  memory  on  disk.  The  system  also 
features  a  622  MB  RA82  disk  drive,  a  TK70  296  MB  cartridge  tape  drive,  and  a  TSV05  1600  BPI 
tape  drive  to  maintain  media  compatibility  with  older  systems.  VAX/VMS  5.3  is  currently  installed 
as  the  operating  system,  and  the  Section's  Pascal  and  Fortran  compilers  have  been  installed  and 
updated.  Approximately  50  RS-232-C  hard-wired  cable  connections  have  been  moved  to  two  new 
Emulex  P4000  terminal  servers,  to  provide  serial  communication  to  the  3600  over  Ethernet.  Users 
can  also  gain  access  at  1200  or  2400  baud  on  four  dial-up  lines.  VAX  user  accounts  have  now 
increased  to  more  than  140. 

Several  additional  pieces  of  hardware  were  purchased  for  the  3600  this  year.  An  8mm  helical 
scan  tape  drive  has  been  installed  which  can  store  2.3  gigabytes  of  data  on  a  single  8mm  video 
cartridge.   A  procedure  was  written  to  allow  automated  backups  of  user  disks  every  night;  this 

6  BNP/DIR  (ICS) 


enhances  data  security  without  compromising  performance  for  users  during  the  daylight  hours. 
Three  new  664  MB  hard  drives  were  purchased  to  replace  the  old,  unreliable  drives  inherited  from 
the  VAX  1 1/750.  The  newer  drives  provide  a  total  of  125%  more  storage  space,  faster  seek  times 
and  data  transfer  rates,  and  the  industry-standard  SCSI  interface.  The  drives  can  also  be  quickly 
removed  and  replaced  with  a  spare  disk,  simplifying  maintenance  and  reducing  system  down  time. 

VAX  system  software  includes  AlisaTalk,  a  package  that  provides  central  network  file  and 
printing  services  to  personal  computers  on  the  Section's  network.  The  TCP/IP  (Transmission 
Control  Protocol/Internet  Protocol)  networking  software  was  enhanced  with  the  addition  of  Process 
Software's  Telnet  and  SMTP  programs  to  the  FTP  program  already  in  use.  FTP  (File  Transfer 
Protocol)  allows  PDP-11  laboratory  computers,  personal  computers  and  UNIX  workstations  to  do 
high-speed  file  transfers  from  and  to  the  3600  via  Ethernet.  Telnet  allows  terminal  sessions  to  the 
3600  from  personal  computers  via  Ethernet,  and  allows  3600  users  to  establish  terminal  sessions 
with  non-DEC  host  machines  on  the  network.  SMTP  (Simple  Mail  Transfer  Protocol)  uses  the 
familiar  VMS  MAIL  interface  to  allow  VAX  users  to  exchange  mail  with  other  NIH  systems  and 
world-wide  via  the  Internet  and  Bitnet. 

Currently,  the  most  popular  package  on  the  VAX  is  the  sequence  analysis  software  from  the 
University  of  Wisconsin  Genetics  Computing  Group.  This  package  includes  over  100  programs, 
extensive  documentation,  and  complete  on-line  help.  The  Section  also  provides  the  complete 
GenBank  nucleic  acid  database,  the  NBRF  Nucleic  Acid  and  PIR  Protein  databases  with  quarterly 
updates.  Other  popular  programs  on  the  VAX  are  DataPlot,  an  interactive  program  for  curve  fitting 
and  graphics,  and  UNITY,  a  System  V  UNIX  shell  with  Berkeley  extensions  that  runs  on  top  of  the 
native  VMS  operating  system. 


COMPUTER  NETWORKS 

ICS  has  continued  to  expand  its  network  linking  Macintosh,  Digital,  and  IBM-compatible 
computers  via  the  AppleTalk,  DECnet,  and  TCP/IP  protocols.  There  are  now  nine  LocalTalk 
laboratory  networks,  including  two  in  Bldg.  10,  connected  together  with  network  bridges.  The 
network's  Ethernet  portion  has  grown  to  seven  segments  connecting  laboratories  throughout  Bldg. 
36;  linked  machines  include  the  MicroVAX  3600,  15  Macintoshes,  four  IBM-compatible  PC's, 
three  PDP-ll's,  a  VAXStation  3200  and  a  Silicon  Graphics  UNIX  workstation.  All  of  the  machines 
can  share  data  via  FTP,  and  the  VAX's  and  the  Silicon  Graphics  workstation  can  support  multiple 
terminal  sessions  via  Telnet.  The  network  includes  a  Macintosh  II  AppleShare  file  server  and  an 
AlisaShare  file  server  on  the  MicroVAX  3600.  Both  provide  high-capacity  file  storage  and  high- 
speed file  access  through  the  Ethernet,  and  230  Kbits/sec.  access  for  the  LocalTalk  networks  via  a 
Kinetics  FastPath  gateway. 

In  May,  1990,  our  network  was  linked  to  DCRT's  network  via  a  dedicated  Tl  phone  line  and 
a  Cisco  router.  The  Tl  line  offers  high  speed  (1.5  megabits/sec.)  links  to  other  NIH  campus 
computers,  such  as  the  Convex  supercomputer,  the  DEC  10  and  the  IBM  3090  mainframe.  Our 
router  supports  both  TCP/IP  and  DECnet  protocols,  providing  network  terminal  sessions,  file 

7  BNP/DIR(ICS) 


transfer  services  and  electronic  mail  exchanges  with  a  wide  variety  of  machines.  TCP/IP  Telnet, 
FTP  and  SMTP  protocols  can  be  used  to  communicate  via  the  Internet,  a  vast  network  of  over 
120,000  computers  running  a  variety  of  operating  systems.  In  addition,  DCRT's  Internet/Bitnet 
gateway  provides  electronic  mail  connectivity  to  the  more  than  3,000  machines  on  the  Bitnet 
network. 


IMAGE  PROCESSING  FACILITY 

Included  in  the  Section's  central  computer  facilities  in  Bldg.  36  is  an  image  processing 
system  consisting  of  a  Macintosh  II  with  a  19-inch  color  monitor,  a  video  camera  and  lightbox,  and  a 
digital  film  recorder  for  the  production  of  presentation  quality  35mm  slides.  Several  image 
processing  and  analysis  programs  are  available  for  use  on  this  system.  The  most  popular  program, 
called  Image,  was  developed  by  Section  personnel.  The  latest  version  of  Image,  1.29,  features  a 
macro  language  useful  for  automating  complex,  or  repetitive,  image  analysis  procedures.  The 
facility  is  useful  for  numerous  applications,  including  analysis  of  CT,  MRI,  PET,  or  SPECT  images, 
receptor  binding  studies,  analysis  of  electrophoretic  gels,  and  quantitative  evaluation  of  cerebral 
blood  flow,  glucose  metabolism,  or  protein  synthesis.  Because  it  is  based  on  the  relatively 
inexpensive  Macintosh  II,  and  is  simple  to  install  and  maintain,  investigators  with  extensive  image 
analysis  requirements  can  easily  duplicate  the  Section's  facility  for  use  in  their  own  laboratories.  In 
fact,  eleven  NIMH  and  five  NINDS  intramural  laboratories  have  installed  versions  of  this  system. 


PERSONAL  COMPUTER  FACILITY 

Also  included  in  the  Section's  central  computing  facility  are  two  Macintosh  Plus  computers, 
three  Macintosh  II  computers,  a  Shiva  NetModem,  four  LaserWriter  printers,  an  Apple  flatbed 
scanner  with  optical  character  recognition  software,  and  a  Montage  slide  maker.  A  variety  of 
software  is  available  for  intramural  scientists  to  use  for  statistical  analysis,  for  communicating  with 
DCRT's  mainframes  and  MEDLINE,  and  for  word  processing,  including  creation  of  posters,  slides, 
and  publication-quality  charts  and  graphs.  All  the  Macintoshes  are  also  connected  to  the  VAX  and 
can  be  used  to  emulate  VT-100  and  Tektronix  4014  or  4105  terminals. 


COLLABORATIVE  SUPPORT 

Section  specialists  provide  collaborative  support  for  selected  research  projects  within  the 
intramural  programs.  They  provide  expertise  in  computer  applications,  software  development,  and 
statistical  analysis  and  experimental  design.  These  efforts  and  the  resulting  software  developments 
are  described  below. 

Morphological  Classification  of  Cells.  Fractal  Geometry:  A  collaborative  effort  is  in 
progress  with  LNP  and  LNC,  NINDS  and  LDN,  NICHD  to  use  fractal  geometry  as  a  mathematical 
basis  for  the  quantitative  classification  of  neural  cells  grown  in  culture.    The  group  has  now 

8  BNP/DIR(ICS) 


published  methodological  papers  on  edge  detection,  calculation  of  fractal  dimension,  and  the 
application  of  these  to  the  form  of  neuronal  cells.  Software  has  been  produced  for  PDP-1 1  and 
Macintosh  computers.  A  paper  using  fractal  analysis  to  study  the  development  of  glial  cells  has  been 
submitted  to  Neuroscience.  Fourier  Analysis:  In  collaboration  with  LNP  and  LNC,  a  method  of 
analyzing  cell  shape  has  been  developed  using  a  Fourier  transform  of  the  oudine  of  cells  produced  by 
one  of  the  edge  detecting  techniques.  This  method  is  now  being  applied  to  both  neural  and  glial  cell 
images. 

Analysis  of  Postural  Dynamics.  The  same  techniques  described  above  are  being  applied  to 
the  analysis  of  the  trajectories  of  center  of  gravity  in  normal  young,  normal  old,  and  Alzheimer 
individuals.  This  work  is  in  collaboration  with  a  team  from  Marquette  University  and  the  Veterans 
Hospital  in  Milwaukee,  Wisconsin.  Preliminary  results  will  be  reported  at  the  Society  for 
Neuroscience  meetings  in  1990.  They  show  that  the  fractal  dimension  of  the  trajectory  of  the  center 
of  pressure  on  a  plate  on  which  a  subject  stands  is  quite  different  in  the  three  classes  of  subjects.  It 
is  hoped  that  this  work  will  lead  to  a  diagnostic  tool  in  the  early  detection  of  conditions  which 
influence  the  dynamics  of  posture. 

Extensions  to  Image.  The  fractal  geometry  and  Fourier  analysis  techniques  used  in  the 
morphological  classification  of  cells  are  also  being  incorporated  into  the  Section's  Image  program. 
This  effort  will  enable  an  object  outlined  in  Image  to  be  analyzed  by  computing  its  fractal  index  or 
by  the  Fourier  technique.  In  addition,  the  fractal  index  of  any  image  displayed  on  the  screen  may  be 
computed.  A  second,  unrelated  extension  to  Image  involves  a  collaborative  effort  with  LCB, 
NIMH  to  control  the  motorized  stage  of  a  microscope.  The  objective  is  to  enable  Image  to 
automatically  scan  a  brain  section  slide  and,  either  automatically  or  through  manual  input,  identify 
and  label  cells  in  the  section.  The  location  of  the  selected  cells  will  be  stored  for  future  analysis  and  a 
plot  of  the  image  of  the  section  with  the  cells  identified  will  be  generated. 

Flow  Cytometry  Studies.  A  comprehensive  collaborative  effort  is  in  progress  with  LNP, 
NINDS  which  involves  the  use  of  voltage-sensitive  dyes  and  flow  cytometry  techniques  to  study  the 
electrical  and  pharmacological  properties  of  embryonic  rat  and  chick  CNS  neurons,  and  various 
clonal  lines.  Section  effort  is  focused  on  experimental  design,  statistical  and  graphical  analysis  of 
data,  and  the  development  of  custom  software.  Experimental  Design  and  Data  Analysis:  ICS  has 
been  intimately  involved  in  day-to-day  design  of  experiments  and  analysis  of  data  on  specific 
projects  on  development  in  rat  spinal  cord,  in  hippocampus  and  striatum,  and  in  chick  spinal  cord. 
New  methods  have  been  developed  allowing  for  quantification  of  previously  qualitative  results  on 
the  population  responses  to  neural  transmitters  and  ion  channel  agents.  Software  Development: 
Programs  have  been  developed  for  the  smoothing,  integrating,  overlaying  and  statistical  analysis  of 
distributions  of  optical  data  from  the  flow  cytometer.  These  programs  were  developed  to  run  on 
PDP-11  computers;  most  of  them  have  now  been  converted  for  use  on  LPN's  new  MicroVAX. 
Publication:  ICS  is  involved  in  writing  and  editing  papers  on  this  work  especially  as  it  applies  to 
methodology.  In  fiscal  year  1990,  papers  have  been  published,  or  are  in  press,  on  rat  spinal  cord, 
hippocampus  and  striatum,  and  a  paper  is  in  preparation  on  chick  spinal  cord.  These  will  all  appear 
in  Brain  Research  or  Developmental  Brain  Research.  They  have  all  been  reported  at  the  1989 
Society  for  Neuroscience  meetings  and  follow-up  reports  will  be  given  at  the  1990  meetings. 

9  BNP/DIR  (ICS) 


Nonlinear  Dynamics  in  Electrophysiology.  The  Section  has  begun  to  collaborate  with 
members  of  LNP,  NINDS  on  the  application  of  phase  space  analysis  of  the  nonlinear  dynamics  of 
cells  (commonly  referred  to  as  chaos  theory)  as  evidenced  by  transmembrane  voltage  measurements. 
Programs  originally  developed  at  Bryn  Mawr  College  for  the  IBM  PC  have  been  rewritten  and  are 
now  in  use  on  the  Section's  MicroVAX.  The  application  so  far  has  been  on  research  of  voltage 
recordings  in  chick  cells  exposed  to  agents  which  modify  lipid  metabolism.  Preliminary  results  will 
be  reported  this  fall  at  the  Society  for  Neuroscience  meetings  and  at  a  symposium  on  mathematical 
methods  in  medicine  in  Prague. 


10  BNP/DIR  (ICS) 


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ANNUAL  REPORT 

October  1,  1989  through  September  30,  1990 

ANIMAL  HEALTH  AND  CARE  SECTION,  OP,  DIR 

National  Institute  of  Neurological  and  Disorders  and  Stroke 

Table  of  Contents 

Summary  Report  1 

Major  Tasks  and  Accomplishments  4 

Major  Goals  for  FY  91  6 


ANNUAL  REPORT 

ANIMAL  HEALTH  AND  CARE  SECTION,  OP,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 

October  1, 1989  through  September  30, 1990 

E.  Christopher  Staley,  D.V.M.,  Chief 


The  Animal  Health  and  Care  (AHC)  Section  is  responsible  for  providing  a  full-range 
of  laboratory  animal  support  to  investigators  in  the  NINDS,  Division  of  Intramural 
Research    Included  in  this  responsibility  are:    (1)  animal  procurement/supply,  (2) 
subject  conditioning,  (3)  the  provision  of  routine  and  emergency  veterinary  care,  (4) 
provision  of  technical  support  (e.g.,  monitoring  anesthesia,  sample  collection,  etc.), 
(5)  quarantine  of  new  or  infectious  animals,  and  (6)  administration  of  an  animal 
quality  assurance/preventative  medicine  proqram 

The  AHCS  professional  staff  also  serves  in  an  advisory  capacity  in  planning  studies 
involving  the  use  of  animals.  All  animal  use  must  be  in  compliance  with  the 
regulations  promulgated  by  the  USDA  to  enforce  the  Animal  Welfare  Act,  the  NIH 
Guide  for  the  Care  and  Use  of  Laboratory  Animals,  PHS  Policy  on  Humane  Care  and 
Use  of  Laboratory  Animals,  and  the  applicable  chapters  of  the  NIH  Manual. 
Guidance  and  training  are  provided  to  investigators  and  technicians  regarding 
model  and  subject  selection,  and  animal  handling,  as  well  as  inoculation,  sampling, 
and  surgical  techniques,  etc.  In  additon,  the  Section's  professional  staff  performs 
collaborative  research  involving  animal  model  development,  refinement  of 
techniques,  and  improvement  of  animal  husbandry. 


The  Chief  serves  as  the  Institute  Veterinarian  and  as  Executive  Secretary  of  the  NINDS 
Animal  Care  and  Use  Committee  (ACUC).  In  this  capacity,  he  utilizes  his  expertise  to 
design  and  develop  the  NINDS  Animal  Care  and  Use  Program  in  accordance  with  all 
applicable  regulations,  and  to  advise  and  guide  the  Committee  in  the 
implementation  of  the  Institute's  goals  for  animal  care  and  use  practices  in 
conjunction  with  the  functions  of  the  Committee.  The  ACUC  is  responsible  for:  (1) 
review  and  approval  of  all  Animal  Study  Proposals;  (2)  maintenance  of  protocol  files; 
(3)  investigation  of  allegations  regarding  regulation  violations  involving  the  care, 
use  and  handling  of  animals,  (4)  performance  of  a  semi-annual  review  of  the  NINDS 
Animal  Care  and  Use  Program,  and  (5)  publication  of  Guidelines  and  Policies 
regarding  the  use  of  animals  in  the  DIR. 

Within  the  AHC  Section,  the  veterinarians,  laboratory  animal  technicians,  and  animal 
caretakers  are  organized  as  three  service  units:  Primate  Unit,  Small  Animal  Unit,  and 
Building  376  Unit.  The  Primate  Unit  is  responsible  for  the  support  of  DIR 
investigators  utilizing  primates  on  approved  research  protocols  in  NINDS  facilities  in 
Buildings  36,  9,  and  14on  the  Bethesda  Campus,  and  at  the  NIH  Animal  Center  in 
Poolesville,  MD.  The  Small  Animal  Unit  provides  support  to  NINDS  investigators 
utilizing  rabbits,  rodents,  amphibians  and  othersmall  animal  species  within 
Buildings  9,10,  and  36  on  the  Bethesda  Campus,  and  the  Park  5  Building  in  Rockville, 
MD.  The  Small  Animal  Unit  Veterinarian  also  serves  as  the  Facility  Veterinarian  of 
the  Building  36  Combined  Animal  Facility,  which  provides  housing  and  care  to 
investigators  from  NINDS,  NIMH,  NHLBI,  NICHD,  and  NIDCD  under  an  intraagency 
agreement.  The  Building  376  Unit  is  located  within  the  NINDS  facility  in  Building 
376,  Ft.  Detrick,  Frederick,  MD.  Its  personnel  provide  primate  care  and  research 
support  to  NINDS  investigators  within  the  facility,  and  the  Facility 


Veterinarian  serves  as  the  contract  monitor  for  the  Program  Resources,  Inc.  small 
animal  care/technical  support  contract. 

In  order  to  facilitate  the  supply  of  animals  and  animal  tissues  to  investigators  within 
the  DIR,  the  AHC  Section  maintains  breeding  colonies  of  primates  and  rodents. 
These  colonies  are  located  within  NINDS  facilities  in  Bethesda,  Frederick,  and 
Poolesville,  as  well  as  at  off-campus  contract  sites.  The  Chief,  AHCS  serves  as  the 
Project  Officer  for  the  breeding  contracts  as  well  as  a  co-Project  Officer  for  all 
intramural  contracts  involving  the  care,  housing,  and  use  of  intramurally-owned 
animals. 

The  Institute  Veterinarian  and  the  AHCS  also  provide  veterinary  support  to  the 
National  Institute  of  Deafness  and  Other  Communication  Disorders  (NIDCD)  under 
an  intraagency  agreementsigned  in  October,  1989,  following  the  formation  of  the 
NIDCD,  primarily  from  elements  of  the  NINDS. 


Major  tasks  undertaken  by  the  Animal  Health  and  Care  Section  this  Fiscal  Year: 

I.  Assumption  of  responsibility  for  animal  care/research  support  in  Building  376. 

Prior  to  this  fiscal  year,  the  caretakers  and  attending  veterinarian  in  Building  376 
were  assigned  to  the  LCNSS.  Effective  October  1,  1989,  all  animal  care  personnel 
were  reassigned  to  the  AHCS. 

II.  Renovation  of  NINDS  animal  area  in  Building  10  (ACRF  Tower). 

The  NINDS  animal  facility  in  the  ACRF  Tower  was  vacated  for  three  months,  and 
extensive  repairs/renovations  were  made  to  the  floors,  air  handling  system,  and 
lights.  The  renovations  were  successfully  completed  in  August  1990,  and  animals 
have  been  returned  to  the  facility. 

III.  Redesign  and  renovation  of  Building  36/5C  containment  area. 

The  Building  36/5C  infectious  disease  containment  area  was  redesigned  to 
eliminate  the  use  of  Horsfall  isolation  units.  The  new  design  involves  the  use  of 
state-of-the-art  Lexan  cubicles  and  incorporates  many  environmental  enrichment 
features.  This  renovation  will  continue  into  FY91. 

IV.  Establishment  of  a  clinical  diagnostic  program  with  quality  control. 

A  program  to  support  investigators  with  clinical  pathology/diagnostic  laboratory 
services  was  developed  and  initiated.  Included  in  the  capabilities  of  the  Section's 
laboratory  are  performance  of  routine  and  emergency  complete  blood  counts 
(CBC)  with  or  without  differential  cell  counts,  a  complete  panel  of  serum 
chemistry  tests,  blood  gas  determinations,  etc. 

V.  Establishment  of  a  recycling  program  for  non-human  primates. 

In  an  effort  to  conserve  NINDS  primates,  and  ensure  their  availability  to  NINDS 
investigators,  a  central  service  has  been  established  to  recycle  primates  from  non- 
terminal studies  to  other  investigators  within  the  NINDS  and  other  Institutes. 

VI.  Poolesville  AIDS  Facility  Task  Force 

The  Chief,  AHCS  serves  as  the  Chairman  of  the  Poolesville  AIDS  Facility  Task  Force, 
responsible  for  establishing  programmatic  requirements,  evaluating  design 
alternatives,  etc.,  for  a  facility  that  will  house  NINDS  and  N I  AID  non-human 
primate  on  AIDS  or  AIDS  -  related  studies. 

VII.  Collaborations  Undertaken 

A.  BMAA  Neurotoxin  evaluation 

Dr.  Barton  G.  Weick  (AHCS)  collaboration 

with  Mark  Duncan  (CNB)  February  1990  to  July  1990 

B.  Refinement  of  Audiogenic  seizure  model 

Dr.  Lowrey  Rhodes  Jr.,  and  Dr.  Lisa  T.  Pegues  (AHCS) 
collaboration  with  Dr.  Yamaguchi  (MNB).  This 
collaboration  is  expected  to  extend  into  FY91 . 


C.  Dietary  Control  of  Cholesterol  in  Lowland  Gorillas 

Dr.  E.  C.  Staley  and  Dr.  Lowrey  Rhodes  (AHCS) 
collaboration  with  Dr.  C.  J.  Gibbs  (LCNSS)  and 
Dr.  Scott  Citano  (Miami  Metrozoo) 
This  collaboration  is  expected  to  continue  into  FY91 

D.  Evaluation  of  immune  response  in  Mycobacterium  Tuberculosis  sensitized 
monkeys  (Macaca  mulatta)  following  exposure  to  each  three  measles  vaccines. 

Dr.  E.  C.  Staley  (AHCS)  collaboration  with  Dr.  Janice 
Southers  (VRP,  NCRR)  and  Dr  C.  O.  Thoen  (Iowa  State  Univ.). 
This  collaboration  is  expected  to  continue  into  FY91 


Major  Goals  for  FY91 

I.  Attain  AAALAC  accreditation  for  NINDS  Animal  Care  and  Use  Program 

Included  in  this  goal  are  the  completion  of  all  facility  renovations/repairs, 
program  evaluation  and  updating,  etc. 

II.  Continue  collaborative  efforts  and  emphasize  Animal  Health  and  Care  Section 
involvement  in  animal  model  development. 

III.  Initiation  of  NINDS  wide  program  for  the  provision  of  environmental 
enrichment/exercise  for  non  human  primates  utilized  by  NINDS  investigators. 


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ANNUAL  REPORT 

October  1,  1989  through  September  30.  1990 

Laboratory  of  Biophysics 

Basic  Neurosciences  Program.  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 

Gerald  Ehrenstein.  Ph.D.,  Acting  Chief 


Table  of  Contents 

RESEARCH  SUMMARY  1 

PROJECT  REPORTS 

Function  and  Structure  of  Membrane  Ionic  Channels  4 

Z01  NS  02088-16  LB 

The  Physiological  Role  of  Microglia  in  the  Brain  5 

Z01  NS  02218-15  LB 

Gated  Ionic  Channels  in  Membranes  6 

Z01  NS  02526-09  LB 

Calcium  Channels  in  Vertebrate  Nerve  Terminals  7 

Z01  NS  02606-07  LB 

Comparative  Aspects  of  Ionic  Conductances  in  8 

in  Nerve  and  Heart  Cell  Membranes 
Z01  NS  02608-07  LB 

Mechanism  of  Egg  Activation  Following  Fertilization  9 

Z01  NS  02609-07  LB 

Secretion  of  Neurotransmitters  and  Hormones  10 

Z01  NS  02709-05  LB 

Electro-Mechanical  Transduction  Mechanism  in  1 1 

Outer  Hair  Cells 
Z01  NS  02799-02  LB 


i  -  LB/DIR 


Annual  Report 

October  1 ,  1 989  through  September  30,  1 990 

Laboratory  of  Biophysics 

National  Institute  of  Neurological  Disorders  and  Stroke 

Gerald  Ehrenstein,  Ph.D.,  Acting  Chief 

The  Laboratory  of  Biophysics  pioneered  the  study  of  ionic  channels,  their  voltage 
dependence,  and  their  relation  to  experimentally  observed  ionic  currents.    Our  present 
research  emphasizes  the  way  in  which  channel  properties  relate  to  important  physio- 
logical processes.    Of  particular  interest  in  this  effort  are  calcium  channels,  calcium- 
activated  channels,  and  pressure-sensitive  channels.  Among  the  physiological  processes 
we  are  studying  are  transmitter  release  by  exocytosis,  control  of  the  firing  pattern  in 
heart  cells,  the  increase  in  intracellular  calcium  concentration  in  eggs  following 
fertilization,  and  the  fine-tuning  of  audio  reception. 

The  small  size  and  inaccessibility  of  vertebrate  presynaptic  nerve  terminals  has 
posed  a  major  problem  for  the  study  of  transmitter  release.  In  order  to  examine  with 
direct  recording  techniques  the  physiological  events  that  underlie  this  process  in  a 
vertebrate  system,  we  have  developed  a  calyx-type  preparation  from  the  chick  ciliary 
ganglion.    In  this  ganglion,  the  presynaptic  terminal  is  apposed  to  the  postsynaptic  cell 
over  a  relatively  large  area.    If  this  terminal  were  dissociated  from  the  surrounding 
tissue,  a  large  area  of  the  presynaptic  cell  would  be  exposed.   We  have  found  an  enzyme 
treatment  that  allows  us  to  dissociate  the  ganglia  and  to  obtain  single  ciliary  neurons 
with  attached  calyces.   With  this  treatment,  standard  patch-clamp  techniques  can  be  used 
to  study  the  channels  present  in  the  calyces. 

Using  this  approach,  we  have  characterized  the  presynaptic  calcium  channels.   The 
channels  activate  and  deactivate  very  rapidly;  they  have  little  inactivation,  even  after 
prolonged  depolarization;  and  they  are  blocked  by  cadmium  and  by  w-conotoxin,  but  not 
by  nifedipine.   The  pharmacology  of  the  channels  is  consistent  with  the  pharmacology  of 
synaptic  transmission  through  the  calyx-synapse,  as  evaluated  by  extracellular 
recording  from  the  intact  ganglion.    Thus,  the  presynaptic  calcium  channels  that  we  have 
observed  in  this  fast-transmitting  vertebrate  synapse  have  both  physiological  and 
pharmacological  properties  consistent  with  their  putative  role  as  triggers  of  acetyl- 
choline release.    Interestingly,  they  do  not  fit  neatly  into  any  of  the  previously  described 
categories. 

Fertilization  is  another  process  where  an  increase  in  the  cellular  calcium 
concentration  causes  a  number  of  important  reactions.   A  lot  is  known  about  the 
biochemistry  of  these  reactions,  but  the  primary  messenger  that  triggers  activation 
(the  increase  in  the  internal  calcium  concentration  of  the  fertilized  egg)  is  unknown. 

1  -  LB/DIR 


We  have  used  sea  urchin  gametes,  standard  preparations  for  studying  fertilization,  to 
determine  the  identity  of  the  primary  messenger  in  sperm  that  triggers  activation  of  the 
egg.    We  now  have  strong  evidence  that  the  primary  messenger  is  inositol  trisphosphate 
and  that  it  acts  on  the  internal  surface  of  the  egg.    It  had  previously  been  known  that  the 
injection  of  inositol  trisphosphate  into  the  egg  would  activate  it,  but  it  was  generally 
thought  that  the  inositol  trisphosphate  acted  as  a  second  messenger.   Our  measurement  of 
inositol  trisphosphate  in  sea  urchin  sperm,  however,  indicates  an  exceptionally  high 
concentration  (about  a  hundred-fold  higher  than  in  eggs).    Furthermore,  there  is 
evidence  that  during  the  acrosome  reaction,  the  amount  of  inositol  trisphosphate  in 
sperm  increases  several-fold.    Taken  together,  these  results  indicate  that  the  quantity  of 
inositol  trisphosphate  that  is  brought  into  the  egg  by  a  single  spermatozoon  is  compar- 
able to  the  quantity  that  has  been  shown  to  trigger  activation  by  injection.   The  use  of 
inositol  trisphosphate  as  a  primary  messenger  in  fertilization  may  be  very  general,  as 
evidenced  by  our  observation  that  the  concentration  of  inositol  trisphosphate  in  goat 
sperm  is  comparable  to  that  in  sea  urchin  sperm. 

We  also  have  independent  evidence  that  activation  occurs  at  the  internal  surface  of 
the  egg.   Eggs  were  placed  in  a  solution  containing  an  extract  of  sperm,  and  no  activation 
was  observed.   When  the  eggs  were  electrically  permeabilized  so  that  the  extract  could 
enter  the  egg,  activation  was  observed. 

A  key  issue  involving  internal  calcium  is  determination  of  the  mechanism  for 
calcium-dependent  exocytosis.    We  have  previously  addressed  this  issue  both  experi- 
mentally and  theoretically,  and  have  now  obtained  additional  support  for  a  model  that 
assigns  a  central  role  to  calcium-activated  ion  channels.    In  our  model,  a  calcium- 
activated  cation  or  anion  channel  and  a  calcium-independent  channel  that  is  permeable  to 
ions  of  the  opposite  sign  are  located  in  the  membrane  of  a  secretory  vesicle  in  the  region 
where  the  vesicle  is  apposed  to  the  plasma  membrane.   An  increase  in  intracellular 
calcium  causes  the  calcium-activated  channel  to  open,  resulting  in  a  flow  of  both  cations 
and  anions  into  the  vesicle.   This  ionic  flow  reduces  the  salt  concentration  in  the  space 
between  the  vesicle  membrane  and  the  plasma  membrane,  leading  to  a  decrease  in  the 
osmolarity  of  the  space.  The  decreased  osmolarity  causes  a  flow  of  water  out  of  the 
space,  allowing  the  two  apposed  membranes  to  move  closer  together.   It  is  this  moving 
together  of  the  two  apposed  membranes  that  triggers  membrane  fusion  and  exocytosis. 

Previous  experiments  in  several  types  of  cells  have  shown  a  very  good  correlation 
between  the  calcium  dose-response  curve  for  opening  of  calcium-activated  ion  channels 
and  the  calcium  dose-response  curve  for  secretion,  consistent  with  our  model  for 
exocytosis.    In  addition,  the  model  requires  that  secretory  vesicles  containing  calcium- 
activated  cation  channels  also  contain  calcium-independent  anion  channels  and  that 
secretory  vesicles  containing  calcium-activated  anion  channels  also  contain  calcium- 
independent  cation  channels.   We  are  measuring  the  properties  of  channels  in  secretory 
vesicles  of  the  bovine  neurohypophysis  by  reconstituting  the  channels  into  lipid 
bilayers;  our  preliminary  measurements  are  consistent  with  this  requirement  of  the 
model. 

2-LB/DIR 


We  are  studying  the  secretory  process  from  a  different  point  of  view  in  microglia, 
where  we  are  particularly  interested  in  comparing  microglia  secretion  in  trisomy  16 
mice  with  microglia  secretion  in  normal  littermates.    (Trisomy  16  mice  serve  as  an 
animal  model  for  Down's  syndrome,  which  is  caused  by  trisomy  21  in  humans.)    We 
previously  found  that  secretion  of  the  superoxide  radical  from  microglia  of  trisomy  16 
mice  is  very  much  larger  than  that  from  microglia  of  normal  littermates  subjected  to 
the  same  chemical  stimuli.   Since  neurons  and  synapses  can  be  functionally  damaged  by 
oxyradicals,  it  is  possible  that  overproduction  of  these  radicals  in  trisomy  16  mice  is 
responsible  for  the  observed  impairment  of  their  central  nervous  system.    We  are  now 
investigating  possible  causes  of  the  enhanced  secretion  of  superoxide  in  the  trisomy  16 
mice.    We  found  that  alpha  interferon  increases  the  production  of  interleukin-1  in  both 
normal  and  trisomy  16  mice,  but  that  the  effect  is  significantly  larger  for  trisomy  16 
mice.   This  raises  the  possibility  that  enhanced  secretion  of  superoxide  might  be  caused, 
at  least  in  part,  by  enhanced  production  of  interleukin-1.    Alternatively,  enhanced 
production  of  interleukin-1  might  stimulate  other  reactions,  such  as  gliosis,  that  could 
lead  to  impairment  of  the  central  nervous  system. 

Both  L-type  and  T-type  calcium  channels  have  been  found  in  heart  cells.   The  L-type 
calcium  channel,  which  is  activated  in  the  -40  to  -30  mV  range,  underlies  the  plateau 
phase  of  the  cardiac  action  potential  in  the  myocardium.   The  T-type  calcium  channel, 
which  is  activated  in  the  -70  to  -60  mV  range,  is  involved  in  diastolic  depolarization  of 
the  s-a  node,  the  primary  pacemaker  of  the  heart.   We  have  found  both  components  in 
embryonic  chick  heart  cells  during  the  first  week  of  embryonic  development.    During 
the  second  week  of  development,  however,  the  T-type  channels  are  no  longer  found  in 
atrial  or  ventricular  heart  cells.    This  suggests  that  both  types  of  channels  are  present 
in  precursor  cells,  but  that  T-type  channels  are  needed  only  in  pacemaker  cells.    These 
channels  may  be  unnecessary  or  even  undesirable  in  other  heart  cells,  and  hence  may  be 
lost  during  development. 

In  our  study  of  electromechanical  transduction  in  outer  hair  cells  from  the  organ  of 
Corti,  we  have  previously  shown  that  the  membrane  potential  of  these  cells  regulates 
cellular  movement.    Also,  it  is  known  that  hyperpolarization  of  these  cells  results  in 
elongation.  We  have  now  used  the  patch-clamp  method  to  search  for  ion  channels  in 
outer  hair  cells,  and  have  found  stretch-activated  potassium  channels  in  the  lateral  wall. 
Opening  of  these  channels  would  hyperpolarize  the  cells,  causing  elongation,  and  elonga- 
tion, in  turn,  would  stretch  the  membranes,  opening  more  stretch-activated  channels. 
Thus,  the  stretch-activated  potassium  channels  may  be  important  elements  in  a  positive 
feedback  system  providing  efficient  electromechanical  transduction. 


3-LB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02088- 17  LB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (flO  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Function  and  Structure  of  Membrane  Ionic  Channels 


PRINCIPAL  INVESTIGATOR  iUst  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:    G.  Ehrenstein,  Ph.D.  Research  Physicist  LB,  NINDS 

Others.         K  Iwasa,  Ph.D.  Special  Expert  LB,  NINDS 

N.  Moran,  Ph.D.  Visiting  Associate  LB,  NINDS 


COOPERATING  UNITS  (if  any) 

Weed  Science  Laboratory- AEQI,  Dept.  of  Agriculture,  Beltsville,  MD(C.  Baireand  C.  Mischke) 
University  of  Connecticut  (R.  Satter) 


LAB/BRANCH 

Laboratory  of  Biophysics,  BNP,  DlR 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [x~l  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

*This  project  has  been  terminated. 


4-LB/DIR 


PHSbMO(Hev   I/V4) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


201  NS 02218-15  LB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (fiO  characters  or  less    title  must  fit  on  one  line  between  the  borders.) 

The  Physiological  Role  of  Microglia  in  the  Brain* 


PRINCIPAL  INVESTIGATOR  (IJSf  other  professional  personnel  below  the  Principal  Investigator  )  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:      Daniel  L.Gilbert,  Ph.D.  Research  Physiologist  NINDS,  LB 


Others:       Liana  Harvath,  PhD 


Microbiologist 


CBER,  DBBP 


COOPERATING  UNITS  Visny) 

Georgetown  University,  Washington,  DC  (C.  Colton,  J.  Yao,  J.  Keri) 
John  Hopkins  University,  Baltimore,  MD  (ML.  Oster-Granite) 


LAB/BRANCH 

Laboratory  of  Biophysics,  BNP,  DIR 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NlH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.2 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

]   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  [IT]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Experiments  have  been  performed  on  microglia,  the  resident  macrophage  in  the  cen- 
tral nervous  system  cultured  from  cerebral  cortices  of  normal  and  trisomy  16  (Ts16) 
mice.  The  Ts16  mouse  serves  as  an  animal  model  of  humans  afflicted  with  Down's  syn- 
drome (trisomy  21).  We  found  that  interferon  (alfa  IFN),  a  cytokine,  increases  the  sensi- 
tivity of  phorbol  myristate  acetate  (PMA)  on  microglial  production  of  the  superoxide 
radical  anion  and  that  IFN  also  increases  the  production  of  interleukm-1  (IL-1),  another 
cytokine.  The  increase  in  production  of  IL-1  is  larger  for  the  Ts1 6  mouse  than  for  nor- 
mal controls. 

*Formerly:  "Effect  of  Drugs  on  Voltage-Dependent  Ionic  Conductance  in  Membranes." 


5-LB/DIR 


PHS6O40(R*v.  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02526-09  LB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (fitKharacrtrs  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Gated  Ionic  Channels  in  Membranes 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:     E.F.Stanley,  Ph.D.  Staff  Physiologist  LB,  NINDS 

Other:    A.  Atrakchi,  Ph.D.  Visiting  Fellow  LB,  NINDS 


COOPERATING  UNITS  Of  an,) 

LNN,  NICHD 


LAB/BRANCH 

Laboratory  of  Biophysics,  BNP,  DIR 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I      I   (a)  Human  subjects  ]  (b)  Human  tissues  QT]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

*This  project  has  been  terminated. 


6-LB/DIR 


PHi  6040  (Rev.  l  1)1) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02606-07  LB 


PERIODCOVEREO 

Octoberl,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (SO  characters  or  less.    Iitle  must  fit  on  one  line  between  the  border!  ) 

Calcium  Channels  in  Vertebrate  Nerve  Terminals* 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator  )  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:    E.F.Stanley,  PhD  Staff  Physiologist  LB,  NINDS 

Other:    G  Goping  Technician  LCBG,  NIDDK 


COOPERATING  UNITS  (H any) 


LAB/BRANCH 

Laboratory  of  Biophysics,  BNP,  DIR 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS.NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  1   1 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

Q   (a)  Human  subjects  □  (b)  Human  tissues  [T]  (c)  Neither 

]  (a1)  Minors 

]  (a2)  Interviews 
SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Calcium-dependent  transmitter  release  from  presynaptic  nerve  terminals  is  involved  in 
virtually  all  functions  of  neurons.  We  have  used  the  cholinergic  calyx-type  synapse  of 
the  chick  ciliary  ganglion  to  develop  a  unique  experimental  preparation  in  which  it  is 
possible  to  apply  voltage  clamp  techniques  to  the  presynaptic  nerve  terminal.  We  have 
used  this  technique  to  characterize  presynaptic  calcium  channels  by  recording  the 
voltage-dependent  calcium  current   The  presynaptic  calcium  channels  are 
dihydropyridine  blocker-insensitive,  u-conotoxin-sensitive  but  voltage-dependent- 
inactivation  resistant.  These  channels  may  represent  a  type  that  is  unique  to  fast 
transmitting  nerve  terminals. 

*Formerly:    "Chemical  Transmission  at  the  Squid  Giant  Synapse" 


7  -  LB/DIR 


PHSt040(Kev.  I  M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02608-07  LB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  ritle  must  fit  on  one  line  between  the  borders.) 

Comparative  Aspects  of  Ionic  Conductances  in  Nerve  and  Heart  Cell  Membranes 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:     J.R.Clay,  Ph.D.  Research  Physicist  LB,  NINDS 

Others:      V.  Kowtha,  Ph.D.  NRC  Fellow  LB,  NINDS 


COOPERATING  UNITS  (if an,) 

McGill  University  (A.  Shrier) 


LAB/BRANCH 

Laboratory  of  Biophysics,  BNP,  DIR 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  y  Q 


OTHER:  q  1 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)Human  subjects  ]  (b)  Human  tissues  [x~|  (c)  Neither 

J  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  is  concerned  with  a  comparative  analysis  of  ionic  current  channels  in  nerve 
and  heart  cell  membranes  and  the  relationship  of  these  channelsto  electrical  excitabil- 
ity in  both  preparations.  During  the  past  year  the  primary  experimental  preparations 
have  been  squid  giant  axons  and  embryonic  chick  heart  cells.  One  major  finding  has 
been  the  discovery  of  Wenkebach  firing  patterns  in  the  squid  axon  in  response  to  peri- 
odic current  pulse  stimulation.  This  phenomenon,  which  has  not  previously  been  char- 
acterized in  this  preparation,  may  be  relevant  to  cardiac  arrhythmias.  In  the  chick  heart 
cell  preparation,  we  have  discovered  a  developmental  loss  of  a  particular  type  of  cal- 
cium ion  channel,  the  It  component,  during  the  first  week  of  embryonic  development 
from  atrial  and  ventricular  heart  cells.  This  project  also  has  a  major  emphasis  on  com- 
puter simulations  of  electrical  activity  in  nerve  and  heart  cells.  A  major  advance  in  this 
part  of  the  project  is  the  development  of  a  model  of  the  action  potential  in  guinea-pig 
ventricular  myocytes  based  on  experimental  measurements  of  underlying  ion  current 
components  in  the  literature 


8-LB/DIR 


PHi  6040  (Rev   1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02609-07  LB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  character*  or  /ess.   Title  must  fit  on  one  line  between  the  borders.) 

Mechanism  of  Egg  Activation  Following  Fertilization 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator  )  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  K  H.  Iwasa,  Ph.D.  Special  Expert  LB,  NINDS 


Others:    G.  Ehrenstein,  PhD 
J.T.Russell,  Ph.D. 


Research  Physicist 
Research  Chemist 


LB,  NINDS 
LNN,  NICHD 


COOPERATING  UNITS  litany) 

Emory  University,  Atlanta,  GA  (  L.  J.  DeFelice) 


LAB/BRANCH 

Laboratory  of  Biophysics,  BNP,  DIR 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  10892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER 


CHECK  APPROPRIATE  BOX(ES) 

]   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  [x]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

We  have  previously  demonstrated  that  a  fertilization  membrane  forms  around  a  sea 
urchin  egg  when  it  is  injected  with  a  soluble  spermatozoa  fraction  isosmotic  with 
seawater.  We  found  that  a  spermatozoon  contains  inositol  trisphosphate  at  such  a 
high  level  as  to  trigger  calcium  elevation  leading  to  to  the  exocytosis  of  the  cortical 
granules  and  the  elevation  of  the  fertilization  membrane.  We  observed  a  similar 
amount  of  inositol  triphosphate  in  goat  sperm.  Thus,  inositol  triphosphate  plays  the 
role  not  only  of  a  second  messenger  within  eggs  as  in  other  cells  but  of  the  primary 
messenger  from  spermatozoa  to  eggs  at  fertilization. 


9-LB/DIR 


PHS  6040  (Rev.  184) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02709-05  LB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (BO  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Secretion  of  Neurotransmitters  and  Hormones 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:       G.  Ehrenstein,  Ph.D.  Research  Physicist  LB,  NINDS 


Others:       K.  Krebs,  Ph.D. 

S.  Pocotte,  Ph.D. 
E.  F.  Stanley,  Ph.  D. 


Staff  Fellow 
Staff  Fellow 
Research  Physiologist 


LB,  NINDS 
LB,  NINDS 
LB,  NINDS 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 
Laboratory  of  Biophysics,  BNP,  DIR 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


3.2 


PROFESSIONAL: 


2.9 


OTHER: 


0.3 


CHECK  APPROPRIATE  BOX(ES) 

i      ]   (a)  Human  subjects 
;     ]  (a1)  Minors 

J  (a2)  Interviews 


J  (b)  Human  tissues  j     J  (c)  Neither 


Summary  OF  WORK  (use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

In  our  model  for  calcium-dependent  secretion  of  hormones  and  neu rotransm itters,  an  important 
function  of  calcium  is  to  open  calcium-activated  ion  channels  present  in  secretory  vesicles.  The  opening 
of  these  channels  would  allow  the  transport  of  ions  from  the  intermembrane  space  between  the  plasma 
membrane  and  the  secretory  vesicle  membrane  into  the  secretory  vesicle.  The  resultant  osmotic  imbal- 
ance would  remove  water  from  the  intermembrane  space,  allowing  the  two  membranes  to  move  closer 
together  and  fuse. 

According  to  the  model,  every  secretory  granule  for  calcium-dependent  secretion  should  contain 
some  type  of  calcium-activated  channel.  In  order  to  test  this,  we  are  currently  reconstituting  channels 
from  vesicles  of  the  bovine  neurohypophysis  into  lipid  bilayers.  Preliminary  results  indicate  that  the  vesi- 
cles contain  calcium-activated  anion  channels. 

Another  approach  involves  measurement  of  the  secretion  properties  of  bovine  parathyroid  cells. 
These  cells  have  a  unique  calcium  dose-response  curve  for  secretion:  secretion  decreases  when  calcium 
concentration  increases.  We  have  previously  examined  the  vesicle  channel  properties  and  the  secretion 
properties  of  these  cells,  and  found  a  good  correlation,  as  predicted  by  our  hypothesis.  As  a  further  test, 
we  are  now  trying  to  block  ionic  flow  through  these  channels  and  to  determine  whether  this  will  block 
secretion. 

10-LB/DIR 


PHS6M0(Rev.  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02799-02  LB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (so  characters  or  less    Title  must  fit  on  one  line  between  the  borders.) 

Electro-Mechanical  Transduction  Mechanism  in  Outer  Hair  Cells 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator  )  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  K.  H.  Iwasa,  Ph.D.  Special  Expert  LB,  NINDS 


Others:    M.-XLI,  MD 
M.Jia,MD 
B.  Kachar,  MD 


Visiting  Scientist 
Visiting  Scientist 
Visiting  Scientist 


LB,  NINDS 
LB,  NINDS 
LMO,  NIDCD 


COOPERATING  UNITS  Of  an,) 


LAB/BRANCH 

Laboratory  of  Biophysics,  BNP,  DlR 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS.Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

J   (a)  Human  subjects 

]  (a1)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  QT]  (c)  Neither 


Summary  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  fast  mechanical  response  is  believed  to  be  the  cellular  basis  of  the  positive 
feedback  mechanism  required  for  the  fine  tuning  process  of  the  hearing  organ.  Using 
an  externally  applied  electric  field  with  and  without  membrane  permeabilizing  agents, 
we  demonstrated  that  the  fast  mechanical  response  of  the  outer  hair  cell  is  membrane- 
potential  dependent.  To  clarify  further  how  a  change  in  the  membrane  potential 
causes  mechanical  displacement  of  the  cell,  we  examined  the  role  of  the  calcium  ion  in 
the  fast  response.  The  result  indicated  that  the  calcium  ion  is  a  modifier  and  not  a 
messenger.  We  found  stretch-activated  potassium  channels  in  the  lateral  wall, 
indicating  that  the  lateral  wall,  as  well  as  thestereocilia,  can  act  as  a  mechanoreceptor. 


11-LB/DIR 


PHSM40  (Rev.  1/84) 


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ANNUAL  REPORT 

October  1, 1989  through  September  30, 1990 

Laboratory  of  Central  Nervous  System  Studies 

National  Institute  of  Neurological  Disorders  and  Stroke 

Table  of  Contents 

RESEARCH  SUMMARY  1  - 13 

PROJECT  REPORTS 

Neurobiology  of  Population  Isolates:  Study  of  Child  Growth,  14- 18 

Development,  Behavior  and  Learning,  and  Disease  Patterns  in 

Isolated  and  Primitive  Groups 

Z01  NS  01282-26  CNSS  (5  Subprojects) 

Chronic  Central  Nervous  System  Disease  Studies:  Slow,  19  -  28 

Latent  and  Temperate  Virus  Infections 
Z01  NS  00969-26  CNSS  (16  Subprojects) 

PUBLICATIONS  29  -  33 

CONTRACTS  34 


Z01     NS  01282-26    and  Z01  NS  00969-26 


ANNUAL  REPORT 

Laboratory  of  Central  Nervous  System  Studies 

October  1, 1989  -  September  30, 1990 

D.  Carleton  Gajdusek,  M.D. 

The  elucidation  of  the  pathogenesis  of  dementing  brain  amyloidoses,  both  the  transmissible 
type  (such  as  Kuru-Creutzfeldt-Jakob  Disease  (CJD)-Gerstmann-Straussler  syndrome  (GSS)-Scrapie, 
and  bovine  spongiform  encephalopathy  (BSE),  caused  by  slow,  unconventional  viruses)  and  the 
nontransmissible  type  (such  as  normal  aging,  Alzheimer's  disease,  and  Down's  syndrome)  continues  to 
be  our  primary  area  of  inquiry.  Our  program  now  revolves  around  the  heretic  pardigm  of  genetic 
controls  of  de  novo  generation  of  infectious  amyloid  proteins  from  host  precursors  in  kuru,  CJD,  GSS, 
scrapie  and  BSE. 

Our  repeated  assertions  that  the  unconventional  viruses  contain  no  nonhost  proteins  and  were 
replicating  polypeptides  now  appear  to  be  vindicated  by  much  more  work  from  our  own  and  many 
other  laboratories.   It  also  appears  that  the  scrapie  amyloid  precursor  protein  is  converted  to  an 
infectious  form  by  configurational  changes  in  the  secondary  and  tertiary  structure  of  the  normal  scrapie 
precursor  protein  (SPP).  On  inoculation  of  susceptible  hosts,  the  scrapie  monomer  autonucleates  and 
autopatterns  this  conversion  of  the  normal,  noninfectious  host  SPP  to  the  infectious  form.  We  also 
believe  that  most  sporadic  cases  of  CJD  arise  by  de  novo  spontaneous  conversion  of  the  normal  SPP  to 
the  infectious  form,  a  rare  event  occurring  at  the  frequency  of  one  per  million  persons  per  year  (the 
annual  incidence  of  CJD  throughout  the  world).  In  the  familial  forms  of  CJD  and  GSS  where  the 
occurrence  is  an  autosomal  dominant  trait,  we  have  found  that  each  family  has  one  of  four  different 
mutations,  three  causing  a  single  amino  acid  change  and  the  other  an  insertion  of  an  octapeptide 
repeat  (6  x  8  =  48  amino  acids).  Each  mutation  causes  a  million-fold  increased  probability  of  the 
spontaneous  configurational  change  to  an  infectious  polypeptide,  and  appears  as  an  autosomal 
dominant  trait.   Thus,  the  behavior  of  the  transmissible  cerebral  amyloidosis  parallels  completely 
that  of  the  transthretin  amyloidoses  causing  familial  amyloidotic  polyneuropathy,  in  which  there 
are  15  different  point  mutations,  each  one  of  which  increases  enormously  the  likelihood  of 
configurational  change  of  pre-albumin  to  an  amyloid  in  several  dozen  different  families. 

The  remarkable  swift  discoveries,  all  in  our  laboratory,  that  a  point  mutation  in  codon  200 
causing  a  glutamic  acid  to  lysine  replacement  is  associated  with  all  cases  of  CJD  in  the  two  high 
incidence  foci  of  CJD  in  the  Orava  and  Lucenec  regions  of  Slovakia  has  been  followed  by  our 
demonstration  that  this  same  point  mutation  is  linked  to  CJD  cases  in  Eastern  Europe  from  Poland 
through  Greece,  and  even  in  cases  in  the  United  States  and  in  South  Americans  of  Eastern  European 
Slavic  origin.  This  has  led  to  our  quickly  discovering  that  this  same  point  mutation  underlies  the 
high  incidence  of  CJD  in  Sephardic  Jews  of  Libyan  origin,  both  in  immigrants  to  Israel  and  Israeli- 
born.  Furthermore,  we  have  found  this  same  mutation  in  Sephardic  Jews  with  CJD  from  Tunis  and 
Greece  and  thus,  it  is  a  circum-Mediterranean  Jewish  trait  as  well  as  in  Eastern  European  Slavs.  Since 
the  Sephardic  Jews  moved  across  Northern  Africa  to  the  Iberian  Peninsula,  and,  in  the  15th  century, 
from  Spain  to  Greece,  this  codon  200  mutation  may  prove  to  be  a  marker  of  the  "Wandering  Jew  of  the 
Diaspora".  We  are  investigating  CJD  cases  from  Spain. 

After  six  years  of  incubation,  we  have  finally  had  transmission  of  one  of  these  Sephardic 
Jewish  CJD  cases  to  a  chimpanzee.  Obviously  the  chimpanzee  scrapie  precursor  gene  and  protein  do 
not  carry  this  point  mutation  into  their  infectious  isoform. 

The  possibility  that  synthetic  polypeptide  of  the  sequence  of  the  SPP  with  the  codon  200 
glutamic  acid  replacement  by  lysine  might  itself  be  infectious  and  serve  to  nucleate  the  autopatterned 

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Z01    NS  01282-26    and  Z01  NS  00969-26 


configurational  change  in  the  host  precursor  protein  to  its  infectious  form  is  being  investigated  by 
inoculation  of  many  species  with  such  synthetic  polypeptide.  The  codon  102  mutation  replacing  a 
proline  with  a  leucine  in  synthetic  polypeptides  is  also  being  tested  for  infectivity  as  is  also  the  48 
amino  acid  insert  mutation  which  augments  the  5  copies  of  the  octapeptide  repeat  in  the  normal 
scrapie  procurser  protein  to  11  copies.  This  perhaps  naive  oversimplification  is  nevertheless  so 
important  if  it  were  true  that  it  is  worth  the  years  of  waiting  for  long  incubation  periods  in  the 
animals  and  certainly  a  justified  experiment. 

We  are  also  investigating  the  possible  infectiousness  of  cloned  copies  of  the  human  scrapie 
amyloid  containing  these  mutation  in  Baculovirus. 

Our  concept  that  the  formation  of  brain  amyloid  from  a  normal  host  precursor  protein  underlies 
the  pathogenesis  of  Alzheimer's  disease  (AD)  has  provoked  considerable  molecular  study  of  brain 
amyloid  during  the  past  three  years.  We  were  the  first  to  characterize  the  gene  for  this  aging  brain 
amyloid  precursor  protein  (fj-protein;  A4  protein),  to  locate  it  on  chromosome  21  of  man  and  16  of  mouse, 
and  to  show  its  high  evolutionary  conservation.  This  gene  is  identical  to  that  for  an  excreted  rapidly- 
turned-over  protein  which  specifically  binds  to  the  gamma-subunit  of  nerve  growth  factor  and  many 
other  serine  proteases  and  cell  modulators  which  contain  such  serine  protease  as  binding  regions.  This 
important  negative  feedback  control  loop  may  explain  the  rapid  synthesis,  short  half-life  and  wide 
distribution  in  neurons  of  this  brain  amyloid  precursor.  Our  work  showing  that  the  gene  was  expressed 
in  several  alternatively  spliced  forms,  with  and  without  a  57  bp  or  a  76  bp  insert  which  specify  a 
serine  protease  inhibitor,  now  fits  well  with  the  identity  of  protease  nexin  II  and  the  alternatively 
spliced  forms  of  our  amyloid  precursor  protein  containing  the  serine  protease  inserts. 

Our  studies  of  mRNA  expression  in  different  brain  cells  of  normal  juveniles,  aging  brain  and 
AD  brains  have  revealed  that  all  neurons  which  develop  neurofibrillary  tangles  (NFT)  and  are  most 
vulnerable  to  loss  in  aging  and  in  AD  carry  a  very  high  level  of  turned-on  message.  Not  all  cells  with 
high  levels  of  amyloid  fi-protein  mRNA  develop  NFT,  and  thus  its  high  expression  appears  to  be  a 
necessary,  but  not  a  sufficient,  condition  for  NFT  formation.  Interestingly,  in  Guamanian  amyotrophic 
lateral  sclerosis  and  Parkinsionsim-dementia  (ALS/PD)  typical  NFT  appear  in  large  motor  neurons 
which  contain  a  high,  up-regulated  message.  We  have  also  studied  the  regulation  of  mRNA  and  the 
precursor  protein  expression  in  hippocampal  neurons  and  in  endothelial  cells  in  vitro.  Thus,  continued 
molecular  and  cell  biological  studies  of  brain  amyloid  p-protein  biosynthesis,  processing  and 
regulation  will  surely  continue  to  dominate  research  on  AD  and  aging  brain  for  some  time. 

The  discovery  that  the  subunit  of  aging  brain  amyloid  in  AD  and  Down's  syndrome  had  no 
amino  acid  homology  with  the  much  larger  subunit  of  scrapie-kuru-CJD  amyloid  in  scrapie-associated 
fibrils  (SAFs)  or  kuru-plaques  led  to  the  clear  differentiation  of  transmissible  from  non-transmissible 
cerebral  amyloidoses.  Scrapie  and  the  transmissible  dementias  (kuru,  CJD  and  its  GSS  variant)  form 
the  amyloid  of  SAF  and  scrapie  or  kuru  plaques  from  a  proteolytically  cleaved  portion  of  the  larger 
infectious  form  of  the  scrapie  amyloid  precursor  protein.  The  genes  for  aging  brain  amyloid  precursor 
and  for  the  scrapie  amyloid  precursor  show  no  sequence  homology  and  the  amyloidogenic  subunits 
cleaved  from  the  full-length  precursors  are  extremely  different.  The  subunit  protein  for  the 
nontransmissible  brain  amyloidoses  of  aging  and  AD  is  a  polypeptide  of  4.1  kDa  (42  amino  acids)  in 
size  and  is  nonglycosylated;  that  from  scrapie-kuru-CJD  is  27  kDa  in  size  and  has  two  glycosylated 
sites.  The  genes  for  aging  brain  amyloid  precursor  is  on  chromosome  21  in  man  and  for  the  scrapie 
amyloid  precursor  on  chromosome  20.  (In  mice  they  are  on  chromosome  16  and  2,  respectively). 

We  have  established  a  multifaceted  research  program  to  investigate  the  pathogenetic 
mechanisms  underlying  the  brain  amyloidoses.  These  studies  include  in  vitro  study  of  amyloid  fibrils 
formation  from  synthetic  polypeptides.  Using  the  unique  resource  of  our  tissue  bank  of  specimens  from 

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Z01     NS  01282-26    and  Z01  NS  00969-26 


experimental  animals  we  have  initiated  studies  on  the  structure  of  the  amyloid  protein  in  different 
strains  of  virus  from  the  same  host.  Virus  properties  of  pathogenesis,  incubation  time  and  host  range 
must  depend  on  secondary  and  tertiary  corifigurational  changes  in  a  host  precursor  if  no  pleomorphism 
in  amino  acid  sequence  can  be  shown  between  virus  strains  from  a  single  breed  of  host. 

Recent  experimental  data  from  our  laboratory  confirms  our  hypothesis  that  the  ALS/PD  in 
high-incidence  foci  in  the  Western  Pacific  is  caused  by  a  pathogenetic  elemental  deposition  in  the 
central  nervous  system  (CNS)which  results  from  severe  calcium  and  magnesium  deficiency.  ALS-like 
neurofibrillary  tangles  have  been  produced  in  cynomolgus  monkeys  and  rabbits  and  in  vitro  in  motor 
neurons  by  chronic  aluminum  toxicity.  This  confirmation  of  the  mineral  deposit  etiology  in  Guamanian 
ALS/PD  has  stimulated  a  renewed  worldwide  interest  in  these  high  incidence  foci  in  the  Western 
Pacific.  Interestingly,  we  have  never  been  able  to  give  serious  credence  to  extensive  claims  that  cycad 
toxicity  was  involved  in  the  high  incidence  ALS/PD  foci.  This  skepticism  is  now  vindicated. 

Research  work  on  acquired  immunodeficiency  syndrome  (AIDS)  and  related  topics  continues  to 
occupy  a  significant  portion  of  our  resources.  This  includes  studies  on  pediatric  and  adult  encephalitis 
caused  by  human  immunodeficiency  virus  (HIV),  a  major  cause  of  encephalitic  death  in  the  United 
States  today.  We  also  are  conducting  studies  in  nonhuman  primates  to  develop  an  experimental 
animal  model  for  AIDS,  and  to  evaluate  antibody  response  produced  by  potential  vaccines  against 
HIV  infection. 

Our  research  on  other  human  retroviruses,  particularly  human  T-cell  leukemia /lymphoma 
virus  type  I  (HTLV-I),  increases  in  depth  and  scope.  This  includes  intensive  investigation  of  an 
encephalomyelopathic  syndrome  called,  variously,  tropical  spastic  paraparesis,  Jamaican 
neuropathy,  Pacific  spastic  paraparesis,  and  HTLV-I-associated  myelopathy.    Our  virologic, 
immunologic,  clinical,  and  epidemiologic  research  points  to  HTLV-I  as  the  primary  cause  of  this 
syndrome,  which  occurs  in  high-incidence  foci  of  HTLV-I  infection  worldwide. 

Our  work  on  the  hantaviruses,  causative  agents  of  hemorrhagic  fever  with  renal  syndrome, 
continues  as  a  worldwide  collaborative  effort,  involving  groups  in  Europe  and  Asia.  This  year, 
however,  we  have  concentrated  on  finding  domestic  manifestations  of  hantavirus  infection,  and  on 
developing  experimental  models  of  Prospect  Hill  and  Puumala  virus  infection  in  primates. 

We  continue  our  studies  on  the  mechanism  of  language  acquisition,  including  the  naturalistic 
observation  of  extreme  polylinguality.  Our  comparative  inquiries  on  widely  divergent  styles  of 
psychosexual  development  in  children  from  diverse  cultural  milieus  continue  to  yield  new  data  on 
neurological  programming  which  departs  from  "normal"  behavior  much  further  than  previously 
imagined  by  most  psychiatrists,  psychologists,  sociologists,  and  anthropologists. 

We  have  always  studied  not  only  the  clinical  and  laboratory  aspects  of  neurologic  syndromes, 
but  also  the  social  and  public  health  implications  of  these  syndromes.  Our  long-term  data  gathering 
and  analysis  activities  concerning  kuru  in  Papua  New  Guinea,  ALS/PD  in  Guam,  and  Viliuisk 
encephalomyelitis  in  the  Soviet  Union,  as  well  as  others,  continue  to  provide  valuable  insights  on 
cultural  reaction  to  ongoing  epidemic  and  endemic  diseases,  and  to  suggest  available  social 
alternatives.  Much  that  we  learn  is  applicable,  by  extrapolation,  to  contemporary  problems  we  face 
in  the  United  States,  such  as  Alzheimer's  disease  and  senile  dementia,  the  AIDS  epidemic,  and  the 
seemingly  uncontrollable  illicit  use  of  drugs. 


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Z01    NS  01282-26    and  Z01  NS  00969-26 


Slow  Unconventional  Viruses  Causing  Transmissible  Brain  Amyloidoses 

Our  laboratory  has  concentrated  its  main  effort  on  elucidating  the  relationship  between  the 
viruses  of  kuru,  Creutzfeldt-Jakob  disease  (CJD),  and  scrapie  and  their  host-specified  precursor  proteins. 
We  now  know  that  scrapie  virus  is  the  monomeric  form  of  the  configurationally  changed  35-37  kDa  SPP 
present  in  normal  brain  and  in  infected  brain  tissues,  but  modified  by  infection  to  a  less  soluble  protease- 
resistant  form  which  is  infectious.  The  27-30  kDa  scrapie  amyloid  protein  (prion  protein;  PrP27-30)/ 
which  assembles  in  vitro  into  congophilic,  birefrigent  rods  resembling  the  scrapie-associated  fibrils 
(SAF)  of  Merz  and  into  kuru-CJD-scrapie  plaques  is  also  infectious,  even  as  a  monomer.  We  have  now 
demonstrated  that  this  normal  host  protein  modified  by  scrapie  virus  infection  is  itself  the  infectious 
agent  (an  amyloid  molecule,  autoinducing  the  modification  of  host  protein  precursor  into  its  infectious 
form).  No  nonhost  nucleic  acid  has  been  demonstrated,  even  in  highly  infectious  preparations.  The 
improbable  conjecture  that  the  entire  infectious  process  is  that  of  an  autonucleated  and  autopatterned 
conformational  change  of  a  protein  precursor  which  leads  to  a  crystallization  and  polymerization 
forming  amyloid  fibers  of  SAF  and  kuru  plaques  is  now  verified.  The  host  gene  specifying  the  35-37  kDa 
precursor  protein  has  been  fully  sequenced.  It  is  on  chromosome  20  in  man,  and  2  in  mice. 

Polyclonal  and  monoclonal  antibodies  prepared  against  synthetic  polypeptides  of  the  N- 
terminus  of  the  amyloid  of  scrapie  reveal  varying  distribution  and  patterns  of  the  epitopes  in  normal 
and  infected  tissues.  Such  antibodies  have  shown  reactivity  to  the  scrapie-associated  proteins  and,  to 
our  surprise,  to  many  purified  proteins,  including  purified  natural  and  synthetic  human  growth 
hormone.   However,  these  antibodies  to  SAFs  or  to  the  synthetic  polypeptide  specifically  label 
purified  SAFs  from  kuru-,  CJD-  and  scrapie-infected  brains.  Such  SAFs  are  not  obtainable  from  brains 
of  other  human  neurodegenerative  diseases. 

These  immunocytochemical  and  molecular  biologic  studies  on  the  scrapie/kuru/CJD- 
associated  proteins  and  their  normal  precursors  are  largely  aimed  at  preparing  them  in  high  purity 
and  sufficient  amounts  for  crystallographic  study,  and  investigation  at  the  organic  chemical  level,  of 
the  fine  structural  modification  involved  in  the  conversion  of  normal  host-protein  into  amyloid  fibers 
which  appears  to  be  the  major  pathogenic  reaction  of  these  diseases. 

Genetic  Control  of  De  Novo  Generation 
of  Infectious  Amyloid  Protein  from  Host  Precursors 

For  over  two  decades  we  have  carefully  saved  at  <-70oC  frozen  tissue  from  chimpanzees  and 
other  nonhuman  primates  affected  with  the  human  Creuztfeldt-Jakob  disease  and  Gerstmann- 
Straussler  syndrome  viruses,  kuru  virus,  and  scrapie,  and  the  frozen  tissues  collected  from  cats, 
hamsters,  guinea  pigs  and  other  animals  susceptible  to  these  viruses.  These  were  collected  and  saved 
for  eventual  biochemical  study  when  this  would  be  possible.    Newer  polymerase  chain  reaction  (PCR) 
techniques  on  long-stored  tissues  from  these  collections  from  crucial  cases  of  familial  CJD  and  GSS 
have  yielded  critical  data  on  the  several  point  mutations  underlying  the  spontaneous  generation  of 
infectious  amyloid  proteins  from  host  precursors  in  CJD  and  GSS.  This  has  led  to  the  association  of 
this  mutation  with  CJD  in  Eastern-European  Slavs  and  in  Circum-Mediteranean  Sephardic  Jews  from 
Tunis,  Greece  and  Isreal.    It  is  now  possible  to  process  these  tissues  for  PrP  27-30  protein,  SAFs,  and  the 
33-35  kDa  scrapie-specific  protein  and  its  precursor.  As  more  sophisticated  study  of  the  structure  of 
these  proteins  is  possible,  we  hope  to  determine  from  this  material  the  contribution  of  the  host  to 
these  subacute  spongiform  encephalopathy  viruses  or  slow  unconventional  viruses.  This  we  are  in  a 
unique  position  to  do,  since  it  would  take  from  two  years  to  over  a  decade  for  other  laboratories  to 
obtain  infected  brain  material  from  a  number  of  different  species  each  inoculated  with  the  same  strain 
of  virus. 

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Z01     NS  01282-26    and  Z01  NS  00969-26 


We  have  indications  that  there  are  many  strains  of  CJD  viruses.  Using  these  tissues,  it  is 
possible  to  answer  the  critical  question  of  the  relative  contributions  of  the  host  and  the  virus  strain  to 
the  pathogenesis  of  the  disorders  and  the  molecular  structure  of  the  virus  strains.  Since  we  expect  all 
strains  or  passage  lines  of  kuru-CJD-scrapie  viruses  to  replicate  by  an  infectious  transformation  of  the 
normal  host  precursor  protein  to  an  infectious  configuration,  it  follows  that  all  virus  strains  should 
produce  progeny  in  a  given  host  which  have  the  identical  host  amino  acid  sequence  in  the  infectious 
monomer.  Strain  differences  determined  by  the  host  precusor  gene  or  its  mutations  would  not  "breed 
true,"  i.e.  they  would  not  be  carried  into  the  progeny.  Thus,  since  we  do  find  strain  differences  in 
viruses  from  the  same  host,  this  would  require  a  different  explanation  than  conservation  of  genotypic 
identity.  Rather,  we  should  expect  a  conservation  of  secondary  and  tertiary  configurational  change  by 
autonucleation  and  autopatterning  epitaxial  crystal  replication  and  growth.  The  stored  frozen  brain 
passage  material,  particularly  of  different  viruses  (scrapie-kuru-CJD-GSS)  passed  into  the  same 
breed  of  host,  is  being  used  for  resolution  of  this  critical  matter. 

Non-transmissible  Brain  Amyloidoses  of 
Aging.  Alzheimer's  Disease  and  Other  Dementia 

Amino  acid  sequencing  of  the  4  kDa  polypeptide  subunit  of  the  paired  helical  filaments  of 
neurofibrillary  tangles  (NFTs),  amyloid  plaque  cores,  and  amorphous  amyloid  in  congophilic 
angiopathy  indicates  that  all  three  pathognomonic  structures  of  the  aging  brain,  Alzheimer's  disease, 
Pick's  disease,  progressive  supranuclear  palsy,  late  Down's  syndrome,  Guamanian  ALS/PD  and  von 
Economo's  encephalitis  are  composed  of  identical  4  kDa  (42  amino  acids)  subunits.  In  the  preparations 
of  purified  paired  helical  filaments  (PHF)  from  NFTs  of  Guamanian  ALS  and  PD,  no  extracellular 
amyloid  in  the  form  of  amyloid  plaques  or  vascular  amyloid  deposits  were  present  to  produce  possible 
contamination.  This  4  kDa  polypeptide  subunit  which  easily  associates  into  dimers,  tetramers, 
octamers,  and  hexadecamers,  shows  no  amino  acid  sequence  homology  to  the  infectious  scrapie 
amyloid  subunit  of  the  transmissible  cerebral  amyloidoses. 

Genetically  determined  familial  AD  is  caused  by  a  mutation  in  a  different  gene,  not  that  for 
the  B-amyloid  protein  precursor,  which  must  determine  a  molecule  involved  in  the  rapid  turn-over  of 
this  protein.   In  the  Dutch  families  with  autosomal  dominant  hereditary  cerebral  hemorrhage,  with 
amyloidotic  angiopathy,  however,  a  point  mutation  in  base  1852  produces  a  replacement  at  amino 
acid  618  of  glutamic  acid  by  glutamine,  and  this  is  amino  acid  22  in  the  amyloid  subunit. 

Hippocampal  Neuronal  and  Endothelial  Cell  Cultures 
for  In  Vitro  Study  of  ft- Amyloid  Precursor  Gene  Regulators 

Both  vascular  endothelial  cell  and  hippocampal  neurons  in  cell  culture  have  been  used  for 
studies  of  in  vitro  expression  of  the  amyloid  ft-protein  precursor  and  also  for  the  modulation  of  mRNA 
expression  of  this  precursor  by  growth  factors,  interleukin-I  and  other  regulatory  molecules.  This  work 
obviously  has  interesting  implications  for  preventative  and  therapeutic  intervention  in  the  over- 
expression  of  the  precursor. 

The  Carbohydrate  of  Glycosylated  Amyloid  Subunits 

Enzymatic  removal  of  O-  and  N-linked  carbohydrates  from  the  infectious  scrapie  amyloid  or 
the  infectious  form  of  SPP  does  not  diminish  the  infectivity  titer.  Thus,  glycosylation  is  not  critical 
for  virus  replication. 

5  -  LCNSS 


ZQ1    NS  01282-26    and  Z01  NS  00969-26 


In  Vitro  Production  of  Amyloid  Fibrils 

We  have  been  studying  the  recoiling  of  polypeptide  chains  into  different  fine  structural 
configurations  which  permit  beta-pleated  sheet  stacking  into  fibrils  resembling  the  amyloid  fibrils  of 
human  pathology.  Thus,  using  P-2-microglobulin  we  have  succeeded  in  producing  fibers  that  have  the 
congophilic  and  green  birefringent  properties  of  amyloid  fibrils,  and  also  the  electron  microscopic 
appearance  of  such  fibers  and  even  paired  twisted  fiber  structures.  More  specifically,  we  have 
produced  amyloid-like  fibers  from  potential  precursors  of  brain  amyloids,  such  as  the  200  kDa  protein 
of  neurofilament,  and  are  trying  with  MAP-tau.  The  200  kDa  neurofilament  protein  assembles  in  vitro 
into  amyloid-like  filaments  which  are  both  congophilic  and  green  birefringent,  and  in  morphology 
resemble  amyloid  fibrils;  the  amyloid-like  properties  increase  on  partial  cross-linking  with 
paraformaldehyde  fixation. 

The  possibility  that  the  fine  structural  changes  of  the  aging  or  diseased  brain  may  be 
reproduced  in  vitro  is  obviously  intriguing. 

Neurofilament  Pathology  in  Human  Neurodegenerative  Disease 

The  gene  encoding  the  amyloid  P-protein  has  been  shown  to  be  highly  conserved  in  evolution 
and  is  expressed  in  various  human  and  animal  tissues.  As  a  complex  transcriptional  unit,  it  utilizes 
alternative  splicing;  alternative  spliced  forms  of  the  amyloid  P-protein  precursor  cDNAs  contain  50% 
homology  to  the  Kunitz  family  of  serine  protease  inhibitors.  It  may  be  the  absence,  inhibition  or 
overexpression  of  these  alternative  forms  that  modify  the  host  precursor  proteins  leading  to  the 
production  of  amyloid  p-protein. 

These  modified  forms  of  the  amyloid  P-protein  in  its  microfibril  or  oligomeric  forms,  like  the 
fibril  amyloid  enhancing  factor  (FAEF)  in  AA  amyloidoses,  could  act  as  amyloid  enhancing  factors,  or 
as  nucleants  or  niduses  that  accelerate  its  own  formation  by  self  polymerization  and  copolymerization 
with  other  molecules  like  glycosaminoglycans  which  leads  to  amyloid  deposition. 

In  Alzheimer's  disease  and  Guamanian  parkinsonism-dementia,  the  42-amino  acid  subunits  of 
the  amyloid  of  the  neurofibrillary  tangles,  amyloid  plaque  core,  and  congophilic  angiopathy,  could 
themselves  serve,  in  the  form  of  oligomers  or  fibril  microfragments,  as  nuclei  that  enhance  their 
deposition  as  amyloid. 

Creutzfeldt-Takob  Disease  and  Human  Growth  Hormone 

A  further  area  of  intense  involvement  of  our  laboratory  has  been  in  the  problem  of  CJD  in 
recipients  of  human  growth  hormone  (HGH)  prepared  from  pooled  autopsy  pituitary  glands  by  the 
NIH  and  other  programs.  Thirteen  patients  are  now  known  who  developed  CJD  from  infected  or 
contaminated  hormone.   At  least  three  different  batches  of  pituitary  glands  have  been  contaminated, 
since  two  patients  occurred  in  England  and  one  in  New  Zealand  (where  none  of  the  American  products 
were  used)  and  at  least  one  batch  was  used  by  all  seven  American  patients.  Incubation  periods  have 
ranged  from  4  to  20  years.    Primates  have  been  inoculated  with  more  than  50  separate  batches  of 
HGH,  but  incubation  periods  may  be  as  long  as  five  years.  Intense  surveillance  is  under  way  of  some 
8000  other  young  people  who  received  hormone  injections,  and  one  further  probable  case  has  been 
identified.  An  Australian  case  has  followed  use  of  CJD-contaminated  pituitary  gonadotropin. 


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ZQ1    NS  01282-26    and  Z01  NS  00969-26 


Toward  a  Biochemistry  of  Silicon  and  Aluminum 

The  metabolic  adjustment  to  severe  environmental  deficiency  of  calcium  and  magnesium  which 
is  responsible  for  the  deposition  of  calcium,  aluminum,  silicon,  phosphorous  and  other  minerals  in 
brain  cells  in  early  life  in  the  high-incidence  foci  of  ALS  and  PD  and  the  early  appearance  of 
Alzheimer's  NFTs  in  isolated  populations  in  the  Western  Pacific  (Guam,  Japan,  West  New  Guinea) 
was  first  suggested  by  epidemiologic  and  ecologic  studies.  Mineral  analyses  of  environmental 
specimens  of  soil  and  water  confirmed  this  hypotheses.  Finally,  electron  probe  X-ray  microanalyses, 
using  both  energy-dispersive  and  wavelength-dispersive  spectrometry,  has  demonstrated  these  long- 
term  deposits  in  NFT-bearing  hippocampal  neurons  of  Guamanian  ALS  and  PD  patients  and  of  normal 
individual  exposed  to  the  same  environmental  deficiencies.  When  these  calcium  and  magnesium 
deficiencies  are  removed  by  increased  access  to  outside  foodstuffs,  changed  water  supply,  and 
improved  transportation  and  economy,  all  three  diseases  (Alzheimer's  ,  ALS  and  PD)  have  declined 
markedly  in  incidence  or  disappeared  within  a  period  of  two  or  three  decades.  This  discovery  of  the 
primary  cause  of  all  three  pathologic  processes  in  the  Western  Pacific  isolates  has  led  to  animal 
experiments  which  further  substantiate  the  hypotheses  (see  below)  and  stimulated  a  renewed  interest 
in  the  role  of  mineral  deposition  in  interfering  with  axonal  transport.  Even  therapeutic  and 
prophylactic  clinical  regimens  are  now  suggested  and  some  are  under  study. 

Furthermore,  the  role  of  silicon  and  its  polymers  in  altering  the  secondary  structure  of  proteins 
through  long  series  of  hydrogen  bonds  is  now  under  investigation.  Silicon  and  aluminum  compounds  can 
interact  strongly  with  phospholipids,  lipids,  carbohydrates  and  oligonucleotides  as  well  as  with 
polypeptides.   Thus,  mineral  deposits  of  montmorillonite  clays-calcium-aluminum-silicates-and 
hydroxyapatites  can  denature  and  alter  protein  fine  structure  and  conceivably  play  an  active  role  in 
degradation  of  host  precursor  proteins  to  amyloids. 

The  recent  confirmation  of  older  observations  of  silicon-containing  deposits  in  the  center  of 
purified  insoluble  amyloid  plaque  cores  from  Alzheimer's  disease  patients  and  in  Alzheimer's  NFTs 
has  greatly  stimulated  interest  in  the  possible  role  of  these  silicon  and  aluminum-containing  mineral 
deposits  as  nucleating  agents  or  even  as  autocatalytic  agents  in  the  deposition  or  crystallization  of 
such  amyloid  deposits.  The  work  and  thinking  of  this  laboratory  in  these  directions  has  had  a  major 
impact  on  determining  the  course  of  modern  inquiry  into  aging  and  the  degenerative  amyloidoses  of 
brain,  including  Alzheimer's  disease. 

Role  of  Low  Dietary  Calcium  and  Magnesium 
and  a  Neurotoxin  in  the  Evolution  of  Motor  Neuron  Disease 

Oral  administration  of  a  low  calcium  and  magnesium  diet  to  young  cynomolgus  monkeys  (Macaca 
fascicularis)  for  nearly  4  years  has  induced  degenerative  changes  and  variable  degrees  of  intracellular 
calcium  accumulation  in  the  motor  neurons  of  the  spinal  cord  and  brainstem,  and  in  the  giant  Betz  cells  of 
the  cerebral  cortex.  Supplementation  with  low-dose  aluminum  and  manganese  chloride  has  resulted  in  a 
cellular  accumulation  of  argentophilic  material  of  neurofilament  origin  in  different  areas  of  the  CNS. 
None  of  the  animals,  however,  showed  overt  clinical  signs  despite  these  neuropathologic  changes. 

Immunocytochemical  staining,  using  monoclonal  antibodies  against  neurofilaments,  has  revealed 
an  aberrant  accumulation  of  the  phosphorylated  form  of  the  200  kDa  subunit  protein  within  the 
perikarya  of  motor  neurons  in  the  spinal  cord,  mesencephalic  component  of  trigeminal  nucleus,  zona 
compacta  of  substantia  nigra,  and  of  large  pyramidal  neurons  in  the  cerebral  cortex.  This  abnormal 
accumulation  was  noted  maximally  in  animals  fed  the  low-calcium  and  magnesium  diet  supplemented 
with  aluminum. 

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In  addition  to  the  neuronal  pathology,  axonal  spheroids  were  seen  both  in  the  neuropil  of  the 
nuclear  area  and  white  matter.  As  in  ALS,  the  lateral  and  anterior  columns  of  the  spinal  cord,  the 
spinocerebellar  tracts  and  corticospinal  tracts  in  the  brain  stem  revealed  axonal  swellings,  spheroid 
formation  and  focal  axonal  loss.  Gliosis  was  conspicuously  absent. 

These  observations  support  the  hypothesis  that  low  calcium  and  magnesium  levels  interfere 
with  axonal  transport  of  the  neurofilament  subunits.  This  is  further  accentuated  by  the  addition  of 
aluminum.   It  is  believed  that  the  compact,  relatively  rigid  molecules  of  phosphorylated  200  kDa 
neurofilament  proteins  accumulate  in  the  neuronal  soma  leading  to  functional  derangement  and 
eventually  to  cytolysis.  This  nonhuman  primate  model  provides  a  means  to  understand  the 
pathogenetic  mechanism  involved  in  the  evolution  of  lesions  in  motor  neuron  disease. 

Fetal  Motor  Neuron  and  Hippocampal  Neuron  Cell  Cultures  for  In  Vitro  Studies  of  Neurofilament 
Synthesis.  Catabolism  and  Toxic  Reaction  to  Aluminum 

Within  the  past  year  a  program  of  in  vitro  neurobiologic  studies  employing  monolayer  cultures 
of  dissociated  fetal  neuronal  cells  has  been  reintroduced  to  the  laboratory.  The  impetus  underlying 
these  studies  is  the  observation  of  the  differential  morphologic  responses  among  varying  neuronal 
populations  to  heavy  metal  neurotoxins.  Exemplifying  this  is  the  virtual  absence  of  neurofilamentous 
pathology  in  rabbit  hippocampal  neurons  exposed  to  intracisternally  administered  aluminum  salts, 
contrasted  to  the  accompanying  extensive  destruction  of  anterior  horn  cells— the  hallmark  of  which  is 
aberrant  neurofilamentous  accumulations.  The  in  vitro  extension  of  these  observations  is  providing  a 
new  understanding  of  the  mechanisms  regulating  neurofilament  expression.  This  has  necessitated  the 
development  of  a  novel  technique  employing  isopyknic  centrifugation  in  Percoll  step  gradients  for  the 
purification  of  motor  neurons  from  fetal  mouse  and  rabbit  spinal  cord  homogenates.  These  neurons  co- 
cultured  with  muscle  fibers,  have  been  successfully  maintained  for  prolonged  periods  in  serum-free 
medium.  Parallel  monolayer  cultures,  of  dissociated  fetal  rabbit  hippocampal  neurons,  co-cultured  in 
serum-free  medium  over  astrocytes,  have  recently  been  successfully  introduced. 

Utilizing  these  cultures,  ongoing  studies  are  mapping  the  coexpression  of  neurofilaments  and 
neuronal  enzymes  during  the  course  of  normal  neuronal  maturation  in  vitro.  Employing 
immunohistochemical  techniques,  these  neuronal  components  are  identified  in  situ  and  with  sensitive 
neurobiologic  assays,  their  synthesis  quantified.  Subsequent  studies  will  explore  the  expression  of 
these  elements  under  aberrant  environmental  conditions. 


Retrovirus  Encephalomyelopathic  Human  Lentivirus  (AIDS) 
in  Children  and  Adults 

Our  laboratory  is  also  working  on  the  problem  of  the  primary  encephalitis  which 
characterizes  almost  all  cases  of  childhood  AIDS  acquired  congenitally  from  a  human 
immunodeficiency  virus  (HIV)-  infected  mother.  HIV-infected  mothers  are  giving  birth  to  infected 
babies  in  80%  of  their  offspring,  and  some  80%  of  these  infected  offspring  develop  clincial  AIDS.  Most 
develop  disease  within  one  to  two  years  after  birth;  a  few  are  as  delayed  as  four  to  five  years  of  age. 
All,  however,  develop  a  primary  encephalitis  characterized  by  dysarthria,  speech  impairment,  with 
eventual  aphasia,  and  severe  midline  truncal  ataxia  and  loss  of  developmental  milestones. 

We  have  demonstrated  the  virus  in  the  brains  of  these  infants  by  in  situ 
hybridization,fluorescent  antibody  localization,  and  electron  microscopy.  In  histologic  studies,  we 

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Z01     NS  01282-26    and  Z01  NS  00969-26 


have  demonstrated  that  there  is  a  specific  neuropathology  with  large,  multinucleated  macrophages 
in  the  brain,  loaded  with  virus  particles  that  are  visible  by  electron  microscopy.   Similarly,  there  are 
brain  cells  which  appear  to  be  astrocytes,  bulging  with  the  human  lentivirus  particles.  In  neurons, 
these  virus  particles  are  rarely  seen,  and  when  seen  are  few  in  number.  It  was  from  the  awareness  of 
the  primary  human  lentivirus  encephalopathy  of  infants  and  children  that  a  search  was  made  in  the 
brains  of  adults,  and  similar  pathology  was  found  in  more  than  half  the  fatal  cases  of  AIDS. 

It  is  only  in  the  last  five  years,  therefore,  that  clinicians  have  become  increasingly  aware 
that  many  adult  AIDS  patients  show  varying  degrees  of  dementia  which  is  not  due  to  opportunistic 
infection  with  mycoplasma,  mycobacterium,  yeast,  toxoplasma,  cytomegalovirus,  or  herpes  simplex 
virus. 

The  human  lentivirus  has  thus  become  the  major  cause  of  encephalitic  death  amoung  children 
and  adults  in  the  United  States.  In  adults  and  more  frequently  in  children,  primary  encephalitis  from 
human  lentivirus  infection  may  occur  without  an  immune  deficiency  syndrome.  Thus,  we  are  dealing 
with  another  example  of  transmissible  virus  infection  resulting  in  a  chronic  dementia. 

Use  of  HIV  &  HTLV-I  Infected  Chimpanzees 
to  Study  HIV  and  HTLV-I  Virus  Evolution 

Important  insights  have  been  gained  into  the  pathogenesis  and  virology  of  HIV  and  HTLV-I  infection 
from  our  experimental  infection  of  nonhuman  primates,  particularly  chimpanzees.  Using  the  earliest 
HIV-infected  chimpanzees  we  demonstrated  the  hypervariable  loop  of  the  HIV  major  envelope 
glycoprotein  to  be  a  dominant  neutralizing  epitope,  clearly  indicating  the  problem  this  would  cause  to 
conventional  schemes  of  immunization.  These  chronically  HIV-infected  chimpanzees  have  proven 
valuable  in  the  further  delineation  of  antigenic  drift  of  HIV.   This  hpervariability  to  vertex  of  the 
loop  and  the  antigenic  drift  has  been  later  confirmed  in  human  HIV  infections.  Much  has  also  been 
learned  about  HTLV-I  infection  in  humans  by  studying  chimpanzees  persistently  infected  with  HTLV- 
I,  as  well  as  HTLV-I-related  viruses  found  in  chimpanzees.  For  example,  nucleotide  sequence  analysis 
of  the  HTLV-I  pol  gene  in  chimpanzees  inoculated  six  years  earlier  with  HTLV-I  indicate  no 
hypermutability  of  the  pol  gene  for  this  oncovirus.  Simultaneous  HTLV-I  and  HIV  chronically 
infected  chimpanzees  continued  to  be  obtained. 


Search  for  an  Animal  Model  of  AIDS 

Our  laboratory  first  demonstrated  active  infection  of  chimpanzees  with  human 
immunodeficiency  virus  (HIV)  (formerly  LAV  and  HTLV-III)  and  with  primary  human  blood  and 
tissues  obtained  from  AIDS  patients.  The  animals  become  seropositive  but  do  not  develop  clinical 
disease,  and  if  there  is  any  alteration  in  immune  function,  it  is  a  transient  lymphocytosis  with 
moderate  impairment  of  lymphocyte  function,  but  not  a  helper-suppressor  ratio  change  equivalent  to 
that  in  human  AIDS.  The  animals  show  no  clinical  disease  five  years  after  inoculation  but  they 
remain  seropostive  and  viremic.  Such  chimpanzees  developing  primary  infection  on  inoculation  with 
human  brain  tissue  from  AIDS  patients  provided  the  first  demonstration  of  the  live  virus  in  the  brain 
of  AIDS  patients.  Since  such  infection  has  occurred  even  at  high  dilutions  of  suspensions  of  brain  tissue 
from  AIDS,  the  presumption  is  that  the  virus  is  in  brain  and  in  considerable  quantity. 

Many  other  species  of  nonhuman  primates  have  been  inoculated  without  producing  disease, 
primary  infection,  or  antibody  conversion;  however,  an  occasional  rhesus  monkey  inoculated  with 
these  human  viruses  has  developed  an  antibody  response.  The  human  lentiviruses  do  not  produce 

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Z01    NS  01282-26    and  Z01  NS  00969-26 


disease  in  nonhuman  primates,  even  though  they  are  very  closely  related  to  simian  immunodeficiency 
virus.  Thus,  we  are  without  a  good  experimental  model  for  vaccine  evaluation  in  small  animals  or  in 
nonhuman  primates,  and  all  that  can  be  done  at  present  is  to  test  for  the  ability  of  vaccines  to  protect 
against  primary  infection. 

Our  laboratory  first  introduced  the  studies  of  the  Icelandic  visna  and  maedi  sheep  diseases  in 
the  United  States  at  the  NINDB  Symposium  on  Slow  Viruses  in  1962,  to  which  the  Icelandic  workers 
were  invited  as  participants.  We  made  the  first  isolations  in  the  United  States  of  the  visna  virus, 
later  defined  as  the  prototype  lentivirus,  from  the  brain  of  a  sheep  with  Montana  sheep  disease.  The 
maedi  virus  previously  was  thought  to  cause  only  pulmonary  involvement  in  Montana  sheep  disease. 
It  is  now  known  to  be  the  same  virus  as  visna,  which  may  cause  maedi,  or  zoegersiekte,  in  Iceland  and 
the  Netherlands,  respectively,  the  pulmonary  forms  of  visna  virus  infection. 

AIDS  virus  (or  HIV)  belongs  to  the  subfamily  Lentivirinae  in  the  Retroviridae  family,  which 
includes  visna  virus,  equine  infectious  anemia  virus,  and  caprine  arthritis  encephalitis  virus.  We 
have  found  that  horses  inoculated  with  the  human  AIDS  lentivirus  develop  a  transient  antibody 
response,  but  no  disease. 

Human  T-cell  Lymphotopic  Viruses  (HTLV-I) 
Causing  Transverse  Myelytis  (TSP,  lamaican  Neuropathy,  HAM) 

We  have  continued  our  studies  of  tropical  spastic  paraparesis  (TSP)  in  Jamaica,  in  the  Tumaco 
area  of  southwestern  Colombia  on  the  Pacific  coast,  in  the  US.  Virgin  islands,  Barbados,  Chile  and 
the  southern  United  States.  We  have  demonstrated  an  IgG  antibody  response  in  spinal  fluid  and  serum 
to  human  T-cell  lymphotropic  virus  type  I  (HTLV-I).  By  ELISA,  antibodies  against  HTLV-I  can  be 
detected  in  cerebrospinal  fluids  (CSF)  from  most  patients  with  spastic  paraparesis  and  confirmed  the 
ELISA  results  by  Western  blot  and  radioimmunoassay.  In  the  coastal  area  of  Colombia,  the 
seroprevalence  rate  of  HTLV-I  infection  among  normal  adults  is  very  low,  less  than  one-tenth  that 
found  in  TSP  patients.  Similar  cases  have  been  found  in  Japan  but  are  only  diagnosed  as  TSP  if  they 
are  HTLV-I  positive,  so  they  are  called  HTLV-I  associated  myelopathy  (HAM).    We  now  call  the 
diseases  TSP/HAM.  In  early  studies  patients  were  identified  in  the  Tumaco  focus  and  had  a  very 
uniform  disease,  subsequently  seroepidemiologicstudies  were  done,  in  several  other  towns  on  the 
Pacific  coast  of  Colombia,  and  new  patients  with  TSP  have  been  found.  Most  recently  we  have 
identified  a  few  patients  in  the  city  of  Cali  and  also  in  some  of  the  Indian  tribes  of  Colombia. 
However,  the  prevalence  of  TSP  as  a  late  complication  of  HTLV-I  infection  is  much  higher  in  Tumaco 
than  anywhere  else  in  the  world. 

Only  a  rare  HTLV-I-infected  individual  develops  TSP  or  ATL.  We  therefore  are  looking  for 
cofactors.  Early  in  our  study  of  TSP  in  Tumaco  and  Jamaica  we  found  that  patients  had  a  much  higher 
percentage  of  treponema-positive  CSF  than  control  patients.  Chronic  treponema  infection  could  cause 
immunosuppression.  There  has  been  a  striking  decline  in  the  rate  of  treponemal  positivity  among 
patients.  The  former  higher  prevalence  of  yaws,  now  eradicated,  probably  accounted  for  the  earlier 
seropositivity.  Lyme  disease  is  also  being  investigated  and  preliminary  studies  show  the  presence  of 
antibody  to  Borrelia  burgdorferi   in  35%  of  TSP/HAM  patients. 

Family  and  household  studies  in  Tumaco  have  shown  that  relatives  of  TSP  patients  living  in  the 
same  household  have  a  much  higher  prevalence  of  antibody  to  HTLV-I  than  in  adjacent  control 
households.  Whereas  sexual  and  mother-to-child  transmission  of  the  virus  are  recognized,  sexual 
transmission  is  probably  overstated,  since  after  even  decades  of  marriage  an  infected  husband  only 

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transmits  the  infection  to  his  wife  about  half  the  time,  and  much  more  rarely  from  infected  wife  to 
uninfected  husband. 

In  Jamaica,  the  disease  occurs  islandwide,  and  new  patients  are  being  documented  each  week. 
Household  studies  have  been  done  and  the  results  are  the  same  as  found  in  Tumaco.  A  detailed 
epidemiologic  study  is  now  in  progress  at  the  University  Hospital,  Jamaica,  and  this  includes  the 
study  of  patients  with  other  neurological  diseases  and  controls.  HLA  typing  is  also  being  done  on  TSP 
patients  and  these  results  will  be  compared  with  the  findings  seen  in  adult  T-cell  leukemia  patients  in 
Jamaica.   A  protocol  for  treatment  with  steroids  has  been  established  and  these  trials  have  been 
completed  in  Jamaica  and  are  in  progress  in  Tumaco.  Several  isolations  of  HTLV-I-like  virus  have 
been  cultured  from  the  blood  and  CSF  of  Jamaican  and  Colombian  patients  with  TSP  and 
characterizations  of  these  HTLV-I  isolates  from  blood  and  CSF  samples  are  underway.   Initial  results 
suggest  that  there  are  differences  in  the  isolate  from  TSP  and  ATL  Jamaican  patients.   HTLV-I-like 
viral  particles  have  been  identified  in  the  fixed  spinal  cord  of  a  Jamaican  TSP  patient. 

In  the  study  of  spastic  paraparesis  in  the  Seychelles,  we  have  found  similar  seropositive  rates 
of  IgG  antibodies  to  HTLV-I  in  serum.  At  the  time  of  writing  last  year  year,  the  initial  report  from 
Martinique  and  our  discoveries  in  Jamaica  and  Colombia  were  supported  by  those  from  Japan,  the 
Seychelles  and  Trinidad.  We  now  have  confirmation  from  twenty-six  other  countries  and  the 
occurrence  of  TSP  in  Caucasians  who  have  not  visited  other  endemic  regions  has  also  been  reported 
from  the  temperate  zones  of  Chile,  France  and  Italy.  These  factors  serve  to  emphasize  the  need  for 
intensive  study  of  neurologic  involvement  with  HTLV-I  infection. 

In  addition,  we  discovered  that  almost  90%  of  Jamaican  patients  with  polymyositis  have  IgG 
antibodies  to  HTLV-I,  and  these  findings  were  confirmed  by  Western  blot.  We  have  now  isolated 
virus  from  three  patients  with  polymyositis  and  have  detected  HTLV-I  viral  genomic  DNA  in  the 
muscle  of  one  patient.  We  have  also  found  HTLV-I  seropositive  polymyositisin  Barbados  and  recently 
in  Tumaco. 

We  have  investigated  a  Caucasian  patient  with  mycosis  fungoides  whom  we  diagnosed  as 
having  TSP.  The  patient  died  6  months  after  onset  of  TSP,  and  we  had  autopsy  material.   Using  the 
PCR  to  amplify  the  pol  region  of  HTLV-I,  we  demonstrated  viral  genomic  DNA  in  cerebral  cortex, 
cerebellum,  thoracic  spinal  cord,  lymph  node,  kidney  and  spleen  of  this  patient.  We  were  unable  to 
detect  HTLV-I  in  fixed  spinal  cord  of  3  Jamaican  TSP  patients,  but  will  repeat  this  with  more 
material.   In  situ  hybridization  and  alkaline  phosphatase  antiphosphatase  studies  are  now  in 
progress. 

Our  previous  serologic  studies  on  CSF  and  serum  from  many  U.S.  and  European  multiple 
sclerosis  (MS)  patients  have  failed  to  show  any  HTLV-I  antibody.  Frozen  MS  brain  tissues  are  bein 
used  to  search  for  the  presence  of  genomic  sequences,  using  in  situ  hybridization  and  using  PCR.    If  we 
find  any  positive  reactions  in  the  absence  of  CSF  or  serum  antibody,  the  significance  of  such  results 
will  be  hard  to  establish. 


High  Prevalence  of  HTLV-I  Infection  in  the  Asia-Pacific  Basin 

In  our  search  for  high-prevalence  foci  of  HTLV-I  infection  in  the  Western  Pacific,  we  have 
tested  by  ELISA  and  Western  immunoblot,  more  than  4000  sera  collected  between  1956  and  1988  from  37 
population  groups.  High  prevalences  of  antibodies  against  HTLV-I,  ranging  from  14%  among  the  Touri 
to  51%  among  the  Hagahai,  were  found  among  the  coastal  and  lowland  populations  of  New  Guinea, 
the  Solomon  Islands  and  Vanuatu.  Populations  in  the  more  isolated  interior  of  Papua  New  Guinea 

11  -LCNSS 


Z01    NS  01282-26    and  Z01  NS  00969-26 


tended  to  have  no  seropositivity,  except  for  the  Genatei,  a  remote  highland  group  which  had  an  18% 
seroprevalence  before  contact  with  the  outside  world.  Similarly,  the  more  isolated  Polynesian 
outliers  of  Anuta  and  Tikopia  had  low  rates  and  the  less  isolated  outliers  of  Rennell  and  Bellona  had 
considerably  higher  prevalences.  As  confirmed  by  Western  immunoblot,  HTLV-I  seroprevalences  in 
several  Melanesian  populations  were  as  high  as  those  found  in  HTLV-I-endemic  regions  such  as 
southwestern  Japan  and  the  Caribbean  basin. 

The  high  seroprevalences  in  Melanesia  have  been  contested  because  of  the  inability  of  many 
ELISA-positive  sera  to  be  confirmed  by  Western  analysis  and  the  failure  of  such  sera  to  neutralize  a 
prototype  strain  of  HTLV-I.  However,  with  our  recent  identification  and  serologic  verification  of  a 
case  of  TSP  caused  by  HTLV-I  in  a  life-long  indigene  of  East  Guadalcanal  in  the  Solomon  Islands,  and 
our  isolation  of  HTLV-I-like  retroviruses  from  indigenous  New  Guinean  carriers  from  remote  villages, 
and  our  demonstration  of  4  strains  of  HTLV-I-like  viruses  from  Solomon  Islands  carriers  by  PCR 
amplification  and  isolation  of  fully  infectious  virus,   the  endemicity  of  HTLV-I  infection  in  Melanesia 
is  now  irrefutable.  We  have  maintained  that  the  high  frequency  of  indeterminate  Western 
immunoblots  in  Melanesia  is  indicative  of  a  closely  related  but  distinct  retrovirus.  Further 
characterization  of  our  isolates  should  settle  this  issue. 

Another  significant  finding  has  been  the  identification  of  HTLV-I  encephalomyeloneuropathy 
among  Mestizos  and  Caucausians  in  Chile,  a  temperate  zone.  HTLV-I,  serologically  indistinguishable  from 
prototype  strains  of  HTLV-I  from  the  Caribbean  basin  and  Japan,  has  been  isolated  from  several  of  these 
patients.  These  data  augment  our  concepts  of  the  geographic  and  ethnic  distribution  HTLV-I-caused  spastic 
paraperesis. 

Viliusk  Encephalomyelitis  in  Yakut  People  in  Siberia,  USSR 

We  have  just  published  a  definitive  bibliography  of  references  on  this  disease  since  most 
reprints  are  unfamiliar  to  English-speaking  neurologists.  A  complete  review  of  this  disease  in  English 
has  been  submitted  to  Brain.   A  detailed  and  comprehensive  report  of  Viliusk  encephalomyelitis  (VE) 
is  being  prepared  reporting  the  unique  CNS  pathology. 

We  have  reported  a  large  pedigree  of  Marie-type  neuropathy  in  this  Iakutsk  ASSR  of  Siberia 
in  the  Indugirka  River  villey  of  far  northern  Iakutia. 

Analysis  of  clinical  descriptions  of  248  VE  cases  and  a  comprehensive  clinical 
characterization  of  the  disease  has  been  made  in  comparison  with  the  results  of  neuropathologic  study 
of  64  cases.  The  geographic  distribution  and  epidemiologic  features  of  the  disease,  based  on  verified 
clinical  material,  were  also  studied.  VE  is  hypothesized  to  be  an  infectious  disease  with  a  strong 
inflammatory  component,  probably  slow  virus  infection.  Further  studies  on  neuropathologic  and 
etiologic  aspects  of  VE  have  been  initiated. 

Hantaviruses  and  Hemorrhagic  Fever  with  Renal  Syndrome 

We  were  first  to  isolate  an  indigenous  American  hantavirus  (Prospect  Hill  virus)  and  to 
demonstrate  its  presence  in  meadow  voles,  mice,  shrews  and  weasels,  and  its  silent  infection  of  man. 
We  also  demonstrated  that  the  massive  Chinese  epidemics  of  HFRS  occurring  during  the  past  two 
decades  were  caused  by  viruses  closely  related  to  viruses  isolated  in  Korea  and  Siberia,  We  have  also 
shown  that  nephropathia  epidemica  of  Scandanavia  was  actually  caused  by  a  hantaan-virus  related 

12  -  LCNSS 


Z01     NS  01282-26    and  Z01  NS  00969-26 


virus,  and  have  proven  that  two  serotypes  of  hantaviruses  were  responsible  for  severe  and  mild  forms 
of  HFRS  in  Yugoslavia. 

A  severe,  fatal  hemorrhagic  illnesses  with  renal  insufficiency,  of  suspected  hantavirus 
etiology  in  an  illegal  immigrant  in  Texas  prompted  us  to  investigate  the  prevalence  of  hantavirus 
infection  in  wild  rodents  in  designated  regions  in  Texas.  A  virus  anrigenically  distinct  from  other 
known  hantaviruses  has  been  isolated  from  Mus  musculus  captured  in  Leakey,  Texas.  Studies  are  in 
progress  to  further  characterize  this  isolate,  to  determine  its  role  in  human  infection  and  disease,  and 
to  clarify  if  the  seroepidemiology  of  Leakey  virus  infection  in  feral  mice  resembles  that  of  Seoul  virus 
infection  in  urban  rats. 

We  are  also  continuing  to  study  the  mild  nephropathy  produced  by  Prospect  Hill  and  Puumala 
viruses  in  cynomolgus  monkeys.  Tests  for  glomerular  filtration  (endogenous  creatinine  and 
phenosulfonophthalein  clearances)  have  been  normal  but  proteinuria  suggests  an  impaired  of 
glomerular  vascular  integrity.  Serial  renal  biopsies  are  being  planned  in  infected  monkeys.  In 
addition,  blood  and  urine  specimens  are  being  tested  for  viral  sequencies  by  the  PCR  method. 

Finally,  we  are  continuing  to  elucidate  the  epizootiology  of  hantavirus  infections  in  the 
United  States,  concentrating  on  determining  what  animals  other  than  rodents  are  important  in  the 
maintenance  of  the  enzootic  cycle.  Specifically,  predatory  small  mammals  (such  as  weasels  and 
shrews)  and  birds  (such  as  hawks  and  owls)  are  being  tested  for  evidence  of  hantavirus  infection,  and 
virus  isolation  attempts  are  being  made  from  seropositive  animals. 


13  -  LCNSS 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  01282-26  CNSS 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Neurobiology  of  Population  Isolates:  Study  of  Child  Growth,  Development,  Behavior  and  Learning,  and  Disease  Patterns  in  Isolated  and  Primitive  Groups 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  D.C.  Gajdusek,  M.D.  Chief  LCNSS 


Others:        Clarence  J.  Gibbs,  Jr.,  Ph.D. 
David  M.  Asher,  M.D. 
Paul  Brown,  M.D. 
Ralph  M.Garruto,  Ph.D. 
Richard  Yanagihara,  M.D. 


Deputy  Chief  LCNSS 

Research  Medical  Officer  LCNSS 

Medical  Director  LCNSS 

Senior  Research  Biologist  LCNSS 

Medical  Director  LCNSS 


COOPERATING  UNITS  (if  any) 

See  Sub-Project  Summaries 


LAB/BRANCH 

Laboratory  of  Central  Nervous  System  Studies,  Intramural  Reseach 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


12 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I  x  |   (a)  Human  subjects 
|  x  |  (a1)  Minors 
|~x  |  (a2)  Interviews 


[x~|  (b)  Human  tissues  ]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Studies  of  human  biology  of  vanishing  primitive  societies  focus  on  neurological  development  and 
learning  patterns  in  diverse  cultural  experiments  in  the  human  condition  found  in  such  isolated  groups. 
Opportunistic  investigation  of  problems  phrased  by  man  in  isolation  is  the  basis  of  approach  from  which 
most  of  our  studies  evolved:  kuru-CJD,  HIV  (AIDS),  HTLV-I  slow  virus  infections  of  the  CNS,  aging  and 
Alzheimer's  disease,  dementia,  ALS/PD.  Techniques  of  molecular  genetics,  biochemistry,  immunology, 
virology,  and  field  epidemiological,  clinical,  linguistic  and  behavioral  studies  in  cultural  isolates  and 
genetic  and/or  geographically  isolated  primitive  bands  yield  more  easily  interpretable  data  than  in 
cosmopolitan  societies.  Data  and  specimens  from  expeditions  to  Micronesia,  Melanesia,  Polynesia,  South 
America,  Asia  and  Africa  proved  valuable  in  recent  HIV  (AIDS),  HTLV-I,  Hantavirus,  JC  virus  of  PML  and 
herpesvirus,  CMV  and  EBV  studies.  Studies  on  nutrition,  reproduction,  fertility,  age  of  puberty  and 
aging,  genetic  distance  and  pleomorphisms,  unusual  and  odd  use  of  the  higher  cortical  functions  in 
language  learning,  cognitive  styles,  computation  (calculation  without  words  or  numbers)  and  culturally 
modified  sexual  behavior  elucidate  alternative  forms  of  neurologic  functioning  for  man  which  we  would 
be  unable  to  investigate  once  the  natural  cultural  experiments  in  primitive  human  isolates  are 
amalgamated  into  the  cosmopolitan  community  of  man.  Foci  of  high  incidence  of  kuru,  ALS/PD,  HTLV-I 
myelopathy,  epilepsy,  familial  parkinsonism,  Viliuisk  encephalopathy,  other  CNS  degenerations, 
hysterical  disorders,  schizophrenia,  neoplasms,  goiter,  cretinism,  rheumatoid  diseases,  diabetes,  asthma, 
chronic  lung  disease,  malaria,  filariasis,  leprosy,  cysticercosis,  and  other  infections  in  these  isolated 
groups  have  yielded  widely  significant  discoveries.  HFRS  caused  by  Hantaviruses  in  Asia,  USSR,  Europe 
and  newly  recognized  Hantaviruses  in  the  U.S.  are  studied.  Human  evolution  and  adaptability  to  high 
altitude,  wet  or  arid  climes,  variable  food  supply,  mineral  deficiencies,  toxic  exposures  and  responses  to 
severe  diseases  or  social/psychological  stress  are  studied  in  appropriate  population   Thus,  HTLV-1  and 
HIV  retroviruses  as  causes  of  CNS  diseasess  in  man  were  first  found  in  isolated  or  socially  segregated 
groups,  high  incidence  TSP  focus  in  Tumaco,  Colombia;  drug  using  mothers  in  Newark,  New  Jersey  and 
are  often  best  studied  in  these  isolated  or  socially  segregated  groups. 

14- LCNSS 


PHS  6040  (Rev.  1  84) 


Z01  NS  01282-26  CNSS 


I.  Mechanisms  of  dissemination  and  transmission  of  HTLV-I  in  the  Caribbean  basin,  South  America  and 
Melanesia 

PI:         Ralph  M.  Garruto,  Ph.D.  Senior  Research  Biologist 

Pamela  Rodgers-Johnson,  M.D.  Visiting  Scientist 
Carlos  A.  Mora,  M.D.  Visiting  Associate 

Richard  Yanagihara,  M.D.  Medical  Director 
Marta  Leon-Monzon,  Ph.D.  Visiting  Scientist 
Xueyun  Wu,  M.P.H.  Special  Volunteer 

Mark  A.  Miller,  M.D.  Howard  Hughes  Fellow 

Cooperating  Units: 

Owen  Morgan,  University  of  West  Indies,  Kingston,  Jamaica;  Vladimir  Zaninovic,  Universidad 
del  Valle,  Cali,  Colombia;  Luis  Cartier-Rovirosa,  Universidad  de  Chile,  Santiago,  Chile;  Carol  L. 
Jenkins,  Institute  of  Medical  Research,  Goroka,  Papua  New  Guinea;  Andrew  Ajdukiewicz,  Central 
Hospital,  Honiara,  Solomon  Islands;  Steve  Alexander,  Biotech  Research  Laboratories,  Rockville, 
Maryland 

Our  seroepidemiologic  studies  of  more  than  3000  sera,  collected  between  1956  and  1988  from  34 
Melanesian  populations,  for  antibodies  against  HTLV-I  indicate  high  prevalences  of  infection,  as 
verified  by  strict  Western  immunoblot  criteria,  in  several  remote  population  groups  having  no  contact 
with  Japanese  or  Africans  and  minimal  contact  with  Europeans  prior  to  our  bleedings.  By  contrast,  some 
Micronesian  populations  having  intense  contact  with  Japanese  for  more  than  two  decades  have  no 
evidence  of  infection,  arguing  against  the  dissemination  of  HTLV-I  in  the  Pacific  basin  by  the  Japanese 
and  calling  into  question  the  venereal  spread  of  HTLV-I.  Our  recent  identification  of  a  case  of  HTLV-I 
myeloneuropathy  in  a  life-long  resident  of  Guadacanal  in  the  Solomon  Islands  and  the  successful 
isolation  of  HTLV-I  from  unrelated  individuals  from  widely  separated  provinces  in  the  Solomon 
Islands  and  from  members  of  a  remote,  recently  contacted  hunter-horticulturist  group  in  Papua  New 
Guinea  adds  further  credibility  to  our  serologic  data.  We  are  continuing  to  investigate  the  mechanisms 
of  transmission  of  HTLV-I  in  these  remote  populations  and  to  determine  if  the  high  frequency  of 
indeterminate  HTLV-I  Western  immunoblots  in  Melanesia  results  from  the  circulation  of  novel 
retroviruses,  which  are  antigenically  related  to  but  distinct  from  HTLV-I. 

n.  Epidemiology  of  Creutzfeldt-Jakob  disease  in  recipients  of  pituitary  gland-derived  human  growth 
hormone 

PI:         Paul  Brown,  M.D.  Medical  Director 

Lev  Goldfarb,  M.D.  Visiting  Scientist 

Clarence  J.  Gibbs,  Jr.,  Ph.D.         Research  Microbiologist  (Deputy  Chief) 

Cooperating  Units: 

Judith  Fradkin,  NIDDKD,  DDEM,  Bethesda 

Continuing  surveillance  of  the  outbreak  of  Creutzfeldt-Jakob  disease  (CJD)  in  young  people  treated 
with  pituitary  gland-derived  human  growth  hormone  (hGH)  has  to  date  identified  11  subjects  (of 
whom  seven  were  treated  in  this  country)  dying  of  CJD  between  4  and  20  years  after  their  last  dose  of 
hormone.  It  now  appears  that,  worldwide,  the  risk  of  developing  CJD  in  hGH-treated  patients  is  at 
least  1  per  thousand,  and  possibly  as  high  as  1  per  hundred,  compared  to  the  risk  of  1  per  million  in  the 
general  population.  Extensive  analysis  of  processing  records  implicates  multiple  batches  of  pituitary 
glands  as  the  source  of  contamination,  and  all  patients  so  far  identified  have  been  treated  before  1976. 
Molecular  genetic  analysis  of  DNA  from  two  patients  has  so  far  not  identified  any  mutation  in  the 

15  -  LCNSS 


Z01  NS  01282-26  CNSS 


scrapie  amyloid  gene  as  a  susceptibility  factor  in  determining  which  patients  among  the  treated 
population  develop  disease. 

Additional  cases  of  iatrogenic  CJD  due  to  treatment  with  pituitary  gland-derived  gonadotropin,  and  to 
implantation  of  dura  matter  grafts,  have  also  been  identified,  and  consultations  with  the  FDA  have 
assisted  in  revised  policy  decisions. 

III.  Elucidation  of  the  cause  and  pathogenesis  of  high-incidence  motor  neuron  disease  in  different 
climatic  regions  and  among  diverse  ethnic  groups 

Co-PI:   Ralph  M.  Garruto,  Ph.D.  Senior  Research  Biologist 

Richard  Yanagihara,  M.D.        Medical  Director 

Michael  J.  Strong,  M.D.  Guest  Researcher 

Don  C.  Guiroy,  M.D.  Visiting  Associate 

Ikuro  Wakayama,  M.D.  Visiting  Fellow 

Pamela  Rodgers-Johnson,  M.D.  Visiting  Scientist 
Pedro  Piccardo,  M.D.  Visiting  Associate 

Cooperating  Units: 

Kwang-Ming  Chen,  Olivia  Cruz,  Guam  Memorial  Hospital,  Agana,  Guam;  Chris  C.  Plato,  NIA, 
Gerontology  Research  Center,  Baltimore,  Maryland 

Our  multidisciplinary  approach  to  the  study  of  high-incidence  motor  neuron  disease,  conducted  during 
the  past  three  decades  among  geographically  and  gentically  diverse  groups  in  the  Western  Pacific, 
indicates  unequivocally  that  there  is  no  genetic  cause,  but  rather  a  defect  in  mineral  metabolism, 
provoked  by  chronic  nutritional  deficiencies  of  calcium,  leads  to  increased  intestinal  absorption  of  toxic 
metals  and  the  intraneuronal  co-deposition  of  calcium,  aluminum  and  silicon,  as  aluminosilicates  and 
calcium  hydroxyapatites,  in  affected  neurons.    This  elemental  deposition  interferes  with  slow  axonal 
transport  by  altering  neurofilament  biosynthesis  and/or  catabolism,  resulting  in  excessive 
neurofilament  accumulation  in  motor  neurons,  the  ultrastructural  hallmark  of  ALS.  Epidemiologic 
studies  are  underway  to  assess  the  development  of  motor  neuron  disease  among  migrants  from  these 
high-incidence  foci  who  left  during  infancy  or  childhood,  and  to  determine  if  other  ethnic  groups  in 
different  grographic  regions  in  Asia  and  the  Pacific  are  similarly  affected.    At  present  the  greatly 
increased  life  expectancy  of  Guamanians  has  produced  the  problem  of  differentiating  the  expected 
cases  of  Alzheimer's  disease  in  aged  Guamanians  from  the  previously  more  clear-cut  cases  of 
parkinsonism-dementia  in  younger  subjects  now  no  longer  seen. 

IV.  Worldwide  epizootiology  and  epidemiology  of  hantavirus  infection:  search  for  human  disease  in 
the  face  of  a  widespread  enzootic  in  the  United  States 

PI:         Richard  Yanagihara,  M.D.        Medical  Director 

David  M.  Asher,  M.D.  Research  Medical  Officer 

Bruce  K.  Johnson,  Ph.D.  Special  Expert 

Shuyuan  Xiao,  M.D.  Visiting  Fellow 

Zayd  A.  Eldadah  Biological  Lab  Aid 

Clarence  J.  Gibbs,  Jr.,  Ph.D.  Research  Microbiologist  (Deputy  Chief) 

Mark  Godec,  M.D.  NRC  Research  Fellow 


16  -  LCNSS 


Z01  NS  01282-26  CNSS 


Cooperating  Units: 

Theodore  F.  Tsai,  CDC,  Ft.  Collins,  Colorado;  Patrick  Redig,  Raptor  Center,  St.  Paul,  Minnesota; 
Duane  Schlitter,  Carnegie  Museum  of  Natural  History,  Pittsburg,  Pennsylvania;  Robert  Traub,  Smithsonian 
Institution,  Washington,  D.C.;  Ana  Gligic,  Institute  of  Virology,  Belgrade,  Yugoslavia;  Yong  Kang, 
University  of  Ottawa,  Ottawa,  Canada;  Chin-Ming  Hsiang,  Hubei  Medical  College,  Hubei,  People's 
Republic  of  China 

We  were  first  to  isolate  hantaviruses  from  meadow  voles  (Microtus  pennsylvanicus)  and  mice  (Mus 
musculus)  captured  in  the  United  States,  to  demonstrate  that  the  massive  epidemics  of  hemorrhagic  fever 
with  renal  syndrome  (HFRS)  in  the  People's  Republic  of  China  were  caused  by  viruses  closely  related  to 
hantaviruses  isolated  in  Korea  and  Siberia,  and  to  prove  that  two  serotypes  of  hantaviruses  were 
responsible  for  severe  and  mild  forms  of  HFRS  in  Yugoslavia.  Despite  the  widespread  distribution  of 
hantaviruses  in  commensal  rats  and  indigenous  wild  rodents  in  the  United  States,  confirmed  cases  of  HFRS 
have  not  been  recognized  and  the  overall  risk  of  hantavirus  infection  in  Americans  is  low,  even  among 
individuals  who  have  frequent  exposure  to  wild  rodents.  In  an  effort  to  further  clarify  the  epizootiology, 
ecology  and  epidemiology  of  hantavirus  infection  in  the  United  States,  we  are  currently  examining  sera 
from  predatory  small  mammals  (particularly,  shrews  and  weasels)  and  birds  (such  as  owls  and  hawks),  as 
well  as  sera  from  patients  with  non-A,  non-B  hepatitis  and  from  muskrat  trappers  for  evidence  of 
hantavirus  infection.  We  are  also  investigating  the  distribution  of  these  viruses  among  indigenous 
arvicolid  and  cricetid  rodents  in  South  America  and  Canada,  and  the  epizootiology  of  Leakey  virus 
infection  in  Mus  populations. 

V.  Studies  of  high-incidence  non-neurologic  disorders  in  specific  racial  and  ethnic  groups 

PI:         Ralph  M.  Garruto,  Ph.D.  Senior  Research  Biologist 

Richard  Yanagihara,  M.D.        Medical  Director 
Mark  A.  Miller,  M.D.  Howard  Hughes  Fellow 

Cooperating  Units: 

Julianne  Imperato-McGinley  and  Ralph  Peterson,  Cornell  University  Medical  College,  New  York, 
New  York;  Charles  Weitz,  Temple  University,  Philadelphia,  Pennsylvania;  Chen-ting  Chin,  Beijing 
University  Medical  School,  Beijing,  People's  Republic  of  China 

We  have  successfully  established  the  first  high-altitude  research  station  in  southeastern  Qinghai 
province  of  the  People's  Republic  of  China  to  study  the  basic  biology  and  neurobiology  of  minority  groups 
(ethnic  isolates)  living  above  3000  meters.   Studies  initiated  among  these  sheep  and  yak-herding  pastoral 
populations  exposed  to  hypoxic  stress  include  pulmonary  and  blood  physiology,  genetic  epidemiology  and 
the  neurobiology  of  sleep,  chronic  mountain  sickness  and  aging.  Preliminary  data  indicate  that  Han 
populations  have  greater  difficulty  than  native-born  ethnic  minorities  adapting  to  high  altitude,  even 
after  emigrating  to  such  regions  nearly  two  decades  ago.  During  the  ensuing  years,  epidemiologic,  genetic, 
virologic  and  human  biologic  studies  are  expected  to  yield  important  new  insights  into  these  unique 
population  groups. 

During  the  past  25  years,  we  have  studied  a  focus  of  male  pseudohermaphroditism  among  small,  remote, 
inbred,  forest-dwelling,  hoe  and  digging-stick  horticulturalists  of  the  Simbari  Anga  linguistic  group  of  the 
Eastern  Highlands  of  Papua  New  Guinea.  Clinically,  these  male  pseudohermaphrodites  represent  a 
spectrum  of  congenital  anatomic  abnormalities  including  a  foreskin  forming  a  small  fold  above  a 
rudimentary  clitoris-like  penis  and  bilateral  scrotal  flaps  resembling  labia  enclosing  small  testes.   There  is 
a  urogenital  sinus  containing  a  urethra  and  a  blind  vaginal  pouch.  Patients  show  no  gynecomastia  or 
menses.  At  puberty,  the  clitoris-like  penis  and  testes  enlarge  with  concurrent  extensive  facial,  pubic  and 
axillary  hair  growth  and  musculature  development  greater  than  in  their  normal  male  peers.  Sera  collected 

17  -  LCNSS 


701  NS  01282-26  CNSS 


from  two  of  these  young  adult  patients  revealed  elevated  testosterone/  dihydrotestosterone  ratios    Both 
had  Weh  urinary  etiocholaolone/  androsterone,  C19  and  C21  5|3-5a  metabolite  ratios.  The  data  mdicate  a 
5  a-reductase  deficiency  similar  to  patients  studied  in  the  Dominican  Republic. 


18  -  LCNSS 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  00969-26  CNSS 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80 characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Chronic  CNS  Disease  Studies:  Slow,  Latent  and  Temperate  Virus  Infection 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:  D.C.  Gajdusek,  M.D. 

Others:        Clarence  J.  Gibbs,  Jr.,  Ph.D. 

David  M.  Asher,  M.D. 

Paul  Brown,  M.D. 

Ralph  M.  Garruto,  Ph.D. 

Richard  Yanagihara,  M.D. 

(continued  see  next  page) 


Chief  LCNSS 

Deputy  Chief  LCNSS 

Research  Medical  Officer  LCNSS 

Medical  Director  LCNSS 

Senior  Research  Biologist  LCNSS 

Medical  Director  LCNSS 


COOPERATING  UNITS  (if any) 

See  Sub-Project  Summaries 


LAB/BRANCH 

Laboratory  of  Central  Nervous  System  Studies,  Basic  Neurosciences  Program,  Intramural  Research 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


12 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I  x  |   (a)  Human  subjects 
x|  (a1)  Minors 

x|  (a2)  Interviews 


l"x~l  (b)  Human  tissues  ]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Studies  focus  on  causes  and  pathogenesis  of  chronic  degenerative  CNS  disorders  with  emphasis  on  MS; 
Parkinson's ,  Pick's,  Huntington's  and  Alzheimer's  diseases;  ALS/PD  of  Western  Pacific;  supranuclear 
palsy;  other  presenile  dementias;  spinocerebellar  ataxias;  epilepsy;  chronic  encephalitis  with  focal 
epilepsy;  Viliuisk  encephalopathy;  muscular  dystrophies;  chronic  schizophrenia;  autism;  SSPE;  PML; 
dialysis  encephalopathy;  goiterous  cretinism;  cysticercosis;  and  intracranial  neoplasm. 
We  have  defined  the  transmissible  and  nontransmissible  dementias  as  cerebral  amyloidoses  caused  by 
post-translational  modification  of  a  specific  host  precursor  protein  to  amyloid  fibril  deposits.  We  now 
recognize  the  slow  unconventional  viruses  causing  kuru-CJD-scrapie  as  replicating  polypeptides  formed 
de  novo  from  a  normal  host  precursor  protein,  specified  on  chromosome  20  in  man  and  2  in  mice.  The 
molecular  elucidation  of  the  spontaneous  configurational  change  to  infectivity,  basically  a 
crystallographic  problem,  is  now  becoming  our  major  target.  Molecular  genetic  analysis  of  familial  CJD 
already  indicates  several  point  mutations  which  enormously  increase  (x106  )  the  probability  of  this 
spontaneous  de  novo  conversion  to  an  infectious  polypeptide.  Microbiology  must  now  contend  with  a 
totally  new  paradigm  for  replicating,  infectious,  pathogenic  agents  in  the  nontransmissible  brain 
amylodoses.  Our  studies  focus  on  the  elucidation  of  the  molecular  configurational  events  conferring  the 
property  of  infectivity  on  a  previously  normal  host  precursor. 

In  normal  aging,  Alzheimer's  disease  (AD),  and  Down's  syndrome  a  different  host  precursor  protein 
(specified  on  chromosome  21  in  man,  16  in  mice)  is  a  cell  excreted  inhibitor  of  growth  factors.  Post- 
translational  degradation  of  this  normal  precursor  forms  the  42  amino  acid  amyloid  polypeptide  which 
polymerizes  to  form  the  deposits  of  amyloid  angiopathy,  amyloid  plaques  and  neurofibrillary  tangles  in 
aging,  AD  and  Down's.  This  occurs  in  all  individuals  who  reach  their  90s.  Genetic,  toxic,  and  infectious 
factors  may  accelerate  this  aging  brain  amyloid  deposition. 

Conventional  viruses  causing  slow,  infectious,  degenerative  disease  are  intensely  studied  to  elucidate  the 
neurotropism  and  mechanism  of  pathogenesis:  retrovirus  encephalomylopathies  of  HTLV-I  and  HIV  (of 
AIDS);  herpesviruses  (HSV,  CMV,  EB  and  virus  varicella-zoster);  papovavi ruses  (JC);  RSSE;  measles;  SSPE; 
and  many  chronic  virus  infections  of  amyloid. 

19- LCNSS 


PHS  6040  (Rev   1  84) 


Z01  NS  00969-26  CNSS 


I.  Molecular  pathogenesis  of  the  transmissible  cerebral  amyloidoses 

Co-PI:   Paul  Brown,  M.D.  Medical  Director 

Clarence  J.  Gibbs,  Jr.,  Ph.D.         Research  Microbiologist  (Deputy  Chief) 

Jiri  Safar,  M.D.  Visiting  Associate 

Mauro  Ceroni,  M.D.  Visiting  Associate 

Pedro  Piccardo,  M.D.  Visiting  Associate 

Don  C.  Guiroy,  M.D.  Visiting  Associate 

Pawel  P.  Liberski,  M.D.  Visiting  Fellow 

Our  work  indicates  that  the  scrapie  amyloid  precursor  protein  is  converted  into  an  infectious  form  by 
configurational  changes  of  the  normal  precursor.  In  susceptible  hosts,  the  scrapie  monomer  may  act 
much  like  the  fibril  amyloid-enhancing  factors  found  in  AA  amyloidosis  to  autonucleate  and 
autopattern  this  conversion,  resulting  in  its  own  polymerization  or  crystallization  and  precipitation  as 
insoluble  arrays  of  amyloid  fibrils.  Using  tissues  stored  from  experimentally  infected  animals,  we 
have  begun  to  determine  the  structure  and  sequence  of  the  amyloid  protein  of  different  strains  of  virus 
recovered  from  the  same  genetic  host.  In  addition,  we  have  determined  the  intracellular  localization 
of  the  amyloid  precursor  protein,  and  are  now  trying  to  define  the  pathway  of  scrapie  amyloid 
formation  and  to  interrupt  its  formation  in  vivo. 

II.  Molecular  generics  of  the  PRIP  gene  in  the  subacute  spongiform  virus  encephalophies 

Co-PI:   Paul  Brown,  M.D.  Medical  Director 

Lev  Goldfarb,  M.D.  Visiting  Scientist 

David  M.  Asher,  M.D.  Research  Medical  Officer 

Pedro  Piccardo,  M.D.  Visiting  Associate 

Cooperating  Units: 

Dmitry  Goldgaber,  State  University  of  New  York,  Stonybrook,  New  York 

We  have  demonstrated  several  mutations  in  the  open-reading  frame  of  the  gene  encoding  the  scrapie 
precursor  protein  which  seem  to  be  linked  to  the  human  transmissible  encephalopathies.  An  amino 
acid-altering  mutation  in  codon  102  was  identified  in  three  patients  with  GSS,  adding  a  large  and  well 
documented  family  of  German  origin  to  two  previously  reported  unrelated  American  and  English 
families.  Brain  tissue  from  one  of  these  patients  has  transmitted  the  disease  to  experimental  animals. 
This  mutation  was  not  found  in  8  unaffected  family  members,  25  healthy  control  individuals,  3 
transmitted  cases  of  kuru,  17  transmitted  cases  of  CJD  (4  familial  and  13  sporadic),  and  11  patients  with 
other  neurologic  disorders. 

A  different  double-allele  mutation  in  codon  129  was  found  in  2  of  3  kuru  patients,  3  of  3  patients  with 
familial  CJD  belonging  to  three  related  families,  and  2  of  2  patients  with  iatrogenic  CJD  caused  by 
treatment  with  contaminated  pituitary-derived  human  growth  hormone.  The  nucleotide  change  in  this 
codon  (ATG  to  GTG)  resulted  in  an  amino-acid  subsitution  of  valine  for  methionine;  it  also  abolished  an 
Nspl  restriction  site  and  created  a  new  Maell  site,  which  permited  the  use  of  a  restriction-endonuclease 
technique  as  a  screening  procedure  for  this  mutation.  Using  direct  sequencing  and  restriction-endonuclease 
analysis  in  additional  cases,  we  failed  to  find  this  double-allele  mutation  in  patients  with  sporadic  CJD 
or  in  healthy  controls,  but  an  identical  single-allele  codon  129  mutation  (heterozygosity)  was  found  in  3  of 
15  patients  with  sporadic  CJD  and  in  3  of  24  healthy  control  individuals.  Detection  of  a  consistent  double- 
allele  mutation  in  7  studied  patients  with  related  disorders  suggests  that  homozygosity  for  a  PRIP-129 
point  mutation  may  serve  as  the  genetic  background  for  some  cases  of  kuru  and  CJD.  It  is  likely  that  other 
amino  acid-altering  mutations  in  the  PRIP  gene  will  be  found  in  patients  with  sporadic  or  familial  CJD. 

20  -  LCNSS 


Z01  NS  00969-26  CNSS 

III.  In  vivo   and  In  vitro   models  of  ALS  pathogenesis 

PI:         Ralph  M.  Garruto,  Ph.D.  Senior  Research  Biologist 

Richard  Yanagihara,  M.D.  Medical  Director 

Michael  J.  Strong,  M.D.  Guest  Researcher 

Ikuro  Wakayama,  M.D.  Visiting  Fellow 

Vivek   R.  Nerurkar,  Ph.D.  Visiting  Fellow 

Masayuki  Yasui,  M.D.  Guest  Researcher 

Axel  V.  Wolff,  D.V.M.  Facility  Veterinarian 

Cooperating  Units: 

Charles  E.  Fiori,  BEIB,  NIH,  Bethesda;  Andres  Salazar,  Walter  Reed  Army  Medical  Center, 
Washington,  D.C.;  S.M.  Chou,  Case  Western  Reserve  University,  Cleveland,  Ohio 

As  a  direct  outgrowth  of  our  research  of  high-incidence  foci  of  motor  neuron  disease  in  the  Western  Pacific, 
we  have  implemented  a  long-term  program  to  identify  the  cellular  and  molecular  mechanisms  of 
aluminum-induced  neurofibrillary  pathology  and  disease.  We  have  established  a  chronic  model  of 
aluminum  intoxication  in  New  Zealand  white  rabbits,  the  neuropathology  of  which  we  have  recapitulated 
in  dissociated  motor  neuron  and  hippocampal  neuron  cultures  derived  from  fetal  rabbits.  In  addition,  our 
recent  discovery  of  a  novel  neurotoxin,  N-butylbenzenesulfonamide  (a  plasticizing  agent  used  in  the 
polymerization  of  polyamide  compounds),  which  induces  a  spastic  myelopathy  characterized  by 
neurofilamentous  deposits  in  motor  neurons  adds  a  further  dimension  to  models  of  ALS  pathogenesis. 
Studies  are  underway  to  systematically  investigate  neurofilament  metabolism  and  catabolism  in  these 
systems. 

IV.  Cell  biology  and  molecular  pathogenesis  of  deposition  of  aging  brain  amyloid 

PI:         Ralph  M.  Garruto,  Ph.D.  Senior  Research  Biologist 

Michael  J.  Strong,  M.D.  Guest  Researcher 

Don  C.  Guiroy,  M.D.  Visiting  Associate 

Richard  Yanagihara,  M.D.  Medical  Director 

Arne  Svedmyr,  M.D.  Visiting  Scientist 

Lev  Goldfarb,  M.D.  Visiting  Scientist 

Cooperating  Units: 

Dmitry  Goldgaber,  State  University  of  New  York,  Stonybrook,  New  York;  Ryo  Fukatsu, 
Sapporo  Medical  College,  Sapporo,  Japan 

Our  original  cloning  and  chromosomal  localization  of  the  gene  encoding  the  aging  brain  amyloid 
precursor  protein  (P-protein;  A4  protein)  was  followed  rapidly  by  the  demonstration  that  P-amyloid 
mRNA  expression  is  elevated  in  neurons  which  develop  neurofibrillary  tangles,  and  has  led  to  the 
realization  that  this  gene  is  identical  to  that  encoding  an  excreted,  rapidly  tumed-over  protein  which 
specifically  binds  to  the  y-subunit  of  nerve  growth  factor  and  many  other  serine  proteases.  We  also 
know  that  the  level  of  expression  of  amyloid  precursor  protein  (APP)  mRNA  from  which  the  amyloid 
P-protein  is  derived  varies  between  specific  neuronal  populations.  To  determine  the  conditions  under 
which  neuronal  synthesis  of  amyloid  p-protein  might  contribute  to  the  formation  of  neurofibrillary 
tangles,  we  studied  APP  mRNA  expression  in  developing  fetal  rabbit  hippocampal  neurons  in  vitro. 
Using  in  situ  hybridization  with  a  biotinylated  riboprobe  transcribed  from  a  cDNA  which  includes  the 
region  encoding  the  amyloid  P-protein,  we  observed  that  elevated  levels  of  APP  mRNA  expression 
occur  in  early  neuronal  development  in  vitro.  As  the  neuron  matures,  the  levels  and  distribution  of  APP 
mRNA  regress,  suggesting  developmental  regulation  of  APP  mRNA  expression. 


21  -  LCNSS 


Z01  NS  00969-26  CNSS 


V.  Inactivation  studies  of  the  transmissible  spongiform  encephalopathy  agents 

PI:         Paul  Brown,  M.D.  Medical  Director 

Pawel  P.  Liberski,  M.D.  Visiting  Fellow 

David  M.  Asher,  M.D.  Research  Medical  Officer 

Axel  V.  Wolff,  D.V.M.  Facility  Veterinarian 

Jiri  Safar,  M.D.  Visiting  Associate 

Ongoing  studies  of  the  resistance  of  scrapie  and  CJD  viruses  have  had  both  practical  and  theoretical 
goals.  At  the  practical  level,  methods  have  been  devised  for  general  decontamination  of  the 
workplace  and  materials  used  by  hospital  and  laboratory  personnel,  and  a  technique  has  been 
discovered  that  sterilizes  tissues  for  histopathologic  processing,  while  maintaining  histologic 
integrity.  At  the  theoretical  level,  experiments  using  combination  treatments  with  formaldehyde  and 
autoclaving,  dry  heat  up  to  temperatures  of  360°C,  sequential  enzyme  digestions,  and  polyacrylamide 
gel  electrophoresis  have  established  the  protective  effect  of  formaldehyde  upon  heat  inactivation, 
the  survival  of  small  amounts  of  infectivity  even  at  360°C,  the  primacy  of  fibrillary  amyloid  protein 
for  virus  replication,  even  stripped  of  its  post-translationally  modified  non-peptide  components. 

Further,  a  large  portion  of  infectivity  in  scrapie-infected  brain  homgenates,  has  suvived  burial  in  the 
ground  under  natural  climatic  conditions  for  a  3-year  period,  with  profound  implications  for  the 
epidemiology  of  scrapie,  BSE  and  CJD. 

VI.  In  vivo  and  in  vitro  studies  to  detect  the  etiologic  agent  of  Creutzfeldt-Jakob  disease  in  pituitary 
gland-derived  human  growth  hormone 

PI:         Clarence  J.  Gibbs,  Jr.,  Ph.D.        Research  Microbiologist  (Deputy  Chief) 

David  M.  Asher,  M.D.  Research  Medical  Officer 

Cooperating  Units: 

Judith  Fradkin,  NIDDKD,  DDEM,  Bethesda 

More  than  80  lots  of  pituitary  gland-derived  human  growthhormone  (hGH),  some  of  which  were 
prescribed  to  young  people  who  subsequently  developed  CJD,  are  being  analyzed  in  in  vivo  and  in  vitro 
studies  designed  to  detect  the  etiologic  agent  of  CJD.  Aliquots  of  each  lot  of  hormone  have  been  injected 
into  two  squirrel  monkeys  each  on  two  separate  occasions  and  several  pooled  lots  have  been  injected  into 
chimpanzees  to  assay  for  infectivity.  Additionally,  each  lot  of  hGH  and  sera  from  more  than  300 
recipients  of  one  or  more  of  the  lots  under  study,  are  being  analyzed  by  enzyme  immunoassay,  SDS- 
PAGE  and  Western  analysis.  To  date,  29  squirrel  monkeys  have  died  of  intercurrent  infections  over  a  4- 
year  period  and  the  remaining  animals  are  asymptomatic.  Antibodies  against  CJD  amyloid-associated 
protein  (PrP27-3o)  have  not  been  detected  in  the  sera  of  hGH  recipients.  By  SDS-PAGE  and  Western 
immunoblot,  both  pituitary  gland-derived  and  synthetic  recombinant  types  of  hGH  contain  proteins  in 
the  same  molecular  weight  range  as  that  of  PrP27-30- 


22  -  LCNSS 


Z01  NS  00969-26  CNSS 

VII.  Bovine  spongiform  encephalopathy:  experimental  transmission  of  scrapie  to  three  breeds  of  cattle 

PI:         Clarence  J.  Gibbs,  Jr.,  Ph.D.         Research  Microbiolgist  (Deputy  Chief) 

Jiri  Safar,  M.D.  Visiting  Associate 

Mauro  Ceroni,  M.D.  Visiting  Associate 

Alessandro  di  Martino  Guest  Researcher 

Cooperating  Units: 

J.  Hourrigan  and  W.  Clarke,  U.S.  Department  of  Agriculture,  Mission  Field  Station,  Mission, 
Texas 

In  1976,  investigators  at  the  Scrapie  Field  Station  in  Mission,  Texas,  inoculated  five  cattle  each  with  a 
Suffolk  sheep  strain  and  an  Angora  goat  strain  of  scrapie.  One  animal,  a  mixed  breed  of  Jersey  and 
Hereford,  inoculated  with  the  Suffolk  sheep  scrapie  strain,  and  two  animals,  a  Jersey  and  a  Hereford, 
inoculated  with  the  Angora  goat  strain,  developed  a  progressive  neurologic  disease  37,  27  and  36 
months,  respectively,  following  inoculation.   Histopathologic  findings  of  brains  obtained  at  autopsy 
were  reportedly  not  confirmatory  of  scrapie.  Recently,  we  have  had  the  opportunity  of  re-examining 
the  brains  of  all  10  inoculated  cattle  for  the  scrapie  amyloid-associated  protein  (PrP27-3o)/  arid  have 
successfully  detected  this  protein  in  the  brains  of  the  three  cattle  that  developed  neurologic  disease. 
Attempts  are  underway  to  transmit  the  cattle  disease  to  mice,  hamsters  and  additional  cattle.  This 
year,  in  collaboration  with  the  AIREN  Foundation,  NINDS  convened  an  International  Roundtable  on 
BSE  under  the  chairmanship  of  the  principal  investigator  of  this  study. 

VIII.  Ultrastructural  pathology  of  the  subacute  spongiform  encephalopathies:  serial  and  comparative 
studies 

Co-P'-   David  M.  Asher,  M.D.  Research  Medical  Officer 

Richard  Yanagihara,  M.D.        Medical  Director 

Pawel  P.  Liberski,  M.D.  Visiting  Fellow 

Vivek  R.  Nerurkar,  Ph.D.  Visiting  Fellow 

Pedro  Piccardo,  M.D.  Visiting  Associate 

Shuyuan  Xiao,  M.D.  Visiting  Fellow 

Kitty  L.  Pomeroy,  B.S.  Microbiologist 

Paul  Brown,  M.D.  Medical  Director 

Cooperating  Units: 

G.A.H.  Wells,  Ministry  of  Agriculture,  Fisheries  and  Food,  Surrey,  United  Kingdom;  Harash  K. 
Narang,  General  Hospital,  Newcastle-upon-Tyne,  United  Kingdom 

We  have  recently  demonstrated  parallel  arrays  of  tubulovesicular  structures,  measuring  20  to  50  nm  in 
diameter,  in  the  brain  of  a  Fresian/Holstein  cow  with  bovine  spongiform  encephalopathy  (BSE).  To 
further  characterize  these  structures,  ultramicrocryotome-cut  sections  of  brains  from  BSE-affected  cows  are 
being  examined  by  immune  electron  microscopy.  We  demonstrated  that  neuroaxonal  dystrophy  is  a 
prominent  feature  of  the  subacute  spongiform  virus  encephlopathies,  and  that  myelin  sheath  dilation  is  a 
constant  finding.  The  ultrastructural  appearance  of  myelin  ballooning  is  indistinguishible  from  that 
induced  in  spinal  cord  cultures  by  recombinant  human  tumor  necrosis  factor  (TNFa).  Using  immunocyto 
chemical  techniques,  we  have  localized  TNFa  in  hypertropic  astrocytes  in  anatomic  regions  with  striking 
myelin  dilatation  suggesting  that  this  and/or  other  cytokines  may  be  involved  in  myelin  vacuolation  in  the 
subacte  spongiform  virus  encephalopathies.     Studies  to  quantitate  TNFa  mRNA,  as  well  as  TNFa  levels, 
in  brains  of  CJD  virus-infected  mice  are  underway. 


23  -  LCNSS 


Z01  NS  00969-26  CNSS 


Finally,  attempts  are  being  made  to  characterize  the  factor  present  in  homogenates  of  immature  rat 
cerebellar  cortex  (prepared  from  10-day  old  BD-IX  rats  when  synaptogenesis  is  at  its  peak),  which 
reportedly  produces  lesions  akin  to  the  transmissible  spongiform  encephalopathies,  such  as  the 
mutlilamellated  membranes  seen  in  spider  monkeys  experimentally  infected  with  kuru. 

IX.  Attempts  to  produce  neuro-AIDS  in  experimental  animals  with  human  immunodeficiency  viruses 

PI:         Clarence  J.  Gibbs,  Jr.,  Ph.D.         Research  Microbiologist  (Deputy  Chief) 

David  M.  Asher,  M.D.  Research  Medical  Officer 

Bruce  K.  Johnson,  Ph.D.  Special  Expert 

Hiroko  Minagawa,  M.D.  Visiting  Fellow 

Mark  A.  Beilke,  M.D.  Medical  Staff  Fellow 

Gary  Stone,  M.S.  Biologist 

Maneth  Gravell,  Ph.D.  Research  Microbiologist 

Cooperating  Units: 

Prem  Sarin,  NCI,  LTCB,  Bethesda;  Jaap  Goudsmit,  University  of  Amsterdam,  Amsterdam, 
Netherlands 

In  an  effort  to  develop  an  animal  model  of  neuro-AIDS,  we  have  inoculated  multiple  species  of  New 
and  Old  world  monkeys,  chimpanzees,  domestic  horses,  goats  and  small  laboratory  rodents  with 
autopsy  tissues,  whole  blood  or  plasma  from  patients  with  AIDS  or  pre-AIDS,  as  well  as  with 
supernatant  fluids  from  cell  cultures  infected  in  vitro  with  different  strains  of  HIV-1.  Although 
chimpanzees  were  readily  susceptible  to  infection,  and  while  some  continue  to  remain  viremic  for  more 
than  five  years,  none  has  developed  clinical  AIDS.  By  contrast,  all  other  nonhuman  primate  species 
and  other  experimental  animals  were  rather  resistant  to  infection,  and  to  date,  only  one  rhesus  monkey, 
one  cynomolgus  monkey  and  six  horses  have  shown  serologic  evidence  of  subclinical  infection. 

X.  Virological  and  molecular  genetics  studies  on  simian  immunodeficiency  viruses  as  a  model  for  AIDS 

PI:         Maneth  Gravell,  Ph.D.  Research  Microbiologist 

Clarence  J.  Gibbs,  Jr.,  Ph.D.  Research  Microbiologist  (Deputy  Chief) 

Mark  A.  Beilke,  M.D.  Medical  Staff  Fellow 

Gary  Stone,  M.S.  Biologist 

Elaine  Kay  Jordan,  D.V.M.  Senior  Staff  Fellow 

Marta  Leon-Monzon,  Ph.D.  Visiting  Scientist 

Rebecca  Hamilton  Microbiologist 

Cooperating  Units: 

P.R.  Johnson,  Georgetown  University,  Washington,  D.C. 

In  an  effort  to  develop  an  animal  model  of  neuro-AIDS,  we  have  inoculated  multiple  species  of  Old 
World  monkeys  with  simian  retroviruses  and  both  rhesus  and  pigtailed  macaques  with  strains  of 
simian  immunodeficiency  virus  (SIV)  isolated  from  African  green  and  sooty  mangabey  monkeys.  Three 
of  six  rhesus  monkeys  inoculated  with  an  SrV  isolate  from  a  sooty  mangabey  monkey  have  developed 
an  AIDS-like  disease  with  impaired  motor  function  and  orofacial  dyskinesia.   Enlarged  lateral 
ventricles  and  defects  in  the  cerebral  cortex  were  found  by  MRI  brain  scan  in  one  of  these  monkeys  one 
week  before  death.  Vascular  gliosis  and  neuronal  loss  were  evident,  particularly  in  the  brain  stem,  and 


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virus  was  recovered  from  brain,  spinal  cord,  CSF,  peripheral  nerve  and  muscle.  Further  studies  are 
underway  to  clarify  the  pathogenesis  of  AIDS  encephalopathy  and  dementia. 

XI.  Prevention  of  AIDS:  antigenic  potency  and  immunogenicity  of  inactivated  HIV  vaccine  and 
synthetic  HTV  core  polypeptide  immunogen 

PI:         Clarence  J.  Gibbs,  Jr.,  Ph.D.         Research  Microbiologist  (Deputy  Chief) 

Maneth  Gravell,  Ph.D.  Research  Microbiologist 

Bruce  K.  Johnson,  Ph.D.  Special  Expert 

Hiroko  Minagawa,  M.D.  Visiting  Fellow 

Carlos  A.  Mora,  M.D.  Visiting  Associate 

Gary  Stone,  M.S.  Biologist 

Cooperating  Units: 

Jonas  Salk,  Salk  Institute,  La  Jolla,  California;  Frederick  Jensen,  Immune  Response  Corporation, 
La  Jolla,  California;  Prem  Sarin,  NCI,  LTCB,  Bethesda;  Allen  Goldstein,  George  Washington 
University,  Washington,  D.C. 

Following  repeated  doses  of  an  inactivated  whole  virus  HIV  vaccine  devoid  of  env  gp  120/160, 
chimpanzees  persistently  infected  with  HIV  developed  non-anamnestic  antibody  responses  and  were 
cleared  of  viremia  for  one  year  following  virus  challenge,  beginning  at  10  weeks  following  primary 
vaccination,  as  evidenced  by  negative  virus  isolation  attempts  and  inability  to  detect  HIV  DNA 
sequences  by  PCR.  Additional  safety,  toxicity  and  immunogenicity  studies  are  underway  using  a 
synthetic  30-amino  acid  peptide  of  a  conserved  pi 7  region  of  HIV-1.  Studies  are  in  progress  to  assess 
the  utility  of  each  of  these  vaccines  to  prevent  AIDS  in  humans  already  infected  with  HIV. 

XII.  Antigenic,  virological  and  molecular  biological  characterization  of  HTLV-I  strains  circulating  in 
high  incidence  in  the  Caribbean  basin,  South  America  and  Melanesia 

Co-PI:   Richard  Yanagihara,  M.D.        Medical  Director 

Clarence  J.  Gibbs,  Jr.,  Ph.D.        Research  Microbiologist  (Deputy  Chief) 

Ralph  M.  Garruto,  Ph.D.  Senior  Research  Biologist 

Carlos  A.  Mora,  M.D.  Visiting  Associate 

Vivek  R.  Nerurkar,  Ph.D.  Visiting  Fellow 

David  M.  Asher,  M.D.  Research  Medical  Officer 

Marta  Leon-Monzon,  Ph.D.  Visiting  Scientist 

Pawel  P.  Liberski,  M.D.  Visiting  Fellow 

Mark  A.  Miller,  M.D.  Howard  Hughes  Fellow 

Cooperating  Units: 

Prem  Sarin,  NCI,  LTCB,  Bethesda;  Michael  P.  Alpers,  Institute  of  Medical  Research,  Goroka, 
Papua  New  Guinea;  Jaap  Goudsmit,  University  of  Amsterdam,  Amsterdam,  Netherlands;  Steve 
Alexander,  Biotech  Research  Laboratories,  Rockville,  Maryland;   Andrew  Ajdukiewicz,  Ministry  of 
Health  and  Medical  Services,  Central  Hospital,  Honiara,  Solomon  Islands. 

Multiple  HTLV-I  isolates  have  been  made  from  peripheral  blood  lymphocytes  and  CSF  cells  obtained 
from  Jamaican,  Colombian  and  Chilean  patients  with  tropical  spastic  paraparesis.   Molecular  genetic 
analyses  of  some  of  these  isolates  indicate  minor  differences  from  strains  of  HTLV-I  isolated  from 
patients  with  adult  T-cell  leukemia /lymphoma  (ATLL).  Further  comparisons  between  TSP  and  ATLL 
strains  of  HTLV-I  now  in  progress  should  establish  wheither  distinct  pathogenic  markers  exist. 

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The  high  prevalences  of  HTLV-I  infection,  based  largely  on  the  results  of  screening  tests  such  as  enzyme 
immunoassay,  in  remote  population  groups  in  Melanesia,  have  been  contested  because  of  the  inability  to 
confirm  seropositivity  by  Western  analysis  in  many  Melanesian  sera,  and  the  failure  of  such  sera  to 
neutralize  prototype  strains  of  HTLV-I  We  have  now  isolated  HTLV-I  from  T-cell  lines  derived  from 
peripheral  blood  mononuclear  cells  of  Solomon  Islanders  and  a  New  Guinean  with  confirmatory 
Western  immunoblots.  By  Western  analysis,  these  isolates  exhibit  virus-specific  bands  at  15,  19,  24,  46, 
53  and  61  kda,  and  HTLV-I  sequences  have  been  detected  by  PCR  in  DNA  extracted  from  these  cell 
lines.   Further  molecular  genetic  characterization  of  these  isolates  should  establish  whether  or  not 
they  differ  significantly  from  prototype  strains  of  HTLV-I  isolated  from  Jamaica,  Colombia  and  Japan. 
In  addition,  attempts  are  being  made  to  determine  if  the  high  frequency  of  indeterminate  HTLV-I 
Western  immunoblots  among  Melanesians  is  a  direct  result  of  the  existence  of  closely  related  by  distinct 
retroviruses. 


XIII.  Experimental  HTLV-I  infection  in  nonhuman  primates  and  rabbits:  pathogenesis  and  virus 
neurotropism 

Co-PI:   Clarence  J.  Gibbs,  Jr.,  Ph.D.         Research  Microbiologist  (Deputy  Chief) 
David  M.  Asher,  M.D.  Research  Medical  Officer 

Hiroko  Minagawa,  M.D.  Visiting  Fellow 

Bruce  K.  Johnson,  Ph.D.  Special  Expert 

Carlos  A.  Mora,  M.D.  Visiting  Associate 

Maneth  Gravell,  Ph.D.  Research  Microbiologist 

Marta  Leon-Monzon,  Ph.D.  Visiting  Scientist 

Pawel  P.  Liberski,  M.D.  Visiting  Fellow 

Gary  Stone,  M.S.  Biologist 

Cooperating  Units: 

Jaap  Goudsmit,  University  of  Amsterdam,  Amsterdam,  Netherlands 

We  have  inoculated  chimpanzees,  African  green  monkeys  and  rabbits  by  various  routes,  including 
intraspinally  with  different  strains  of  HTLV-I,  some  of  which  were  isolated  from  patients  with 
tropical  spastic  paraparesis  (TSP).  All  animals  have  seroconverted  and  some  remain  viremic 
Five  chimpanzees  have  been  studied,  one  naturally  infected  with  an  HTLV-I-related  virus  and  four 
experimentally  inoculated  with  standard  strains  of  human  origin.  Two  chimpanzees  were  inoculated 
with  HTLV-I  propagated  in  human  cells:  one  was  injected  intravenously  with  complete  HTLV-I- 
infected  human  cells  and  the  other  with  cell-free  virus.  Neither  of  those  animals  became  persistently 
infected  with  HTLV-I  (repeatedly  negative  PCR  for  HTLV-I  pol  gene  in  DNA  extracted  from  their 
peripheral  blood  mononuclear  cells).  Although  they  mounted  antibody  responses  to  the  virus,  the  fact 
that  their  IgM  responses  were  of  short  duration  (<12  weeks)  and  limited  spectrum  (only  against  gag)  in 
comparison  with  other  chimpanzees  suggests  that  they  were  abortively  infected. 

Two  chimpanzees  were  inoculated  with  HTLV-I  propagated  in  chimpanzee  cells:  one  with  HTLV-I 
grown  in  its  own  cultured  peripheral  blood  mononuclear  cells  and  the  other  with  a  transfusion  of  whole 
blood  from  the  first  chimpanzee.  Both  chimpanzees  had  long  lasting  IgG  and  IgM  antibody  responses  to 
gag  and  env  antigens,  and  both  became  persistently  infected. 

The  pol  genes  of  several  HTLV-I  isolates  from  the  two  experimentally  infected  chimpanzees  were 
compared  with  those  from  the  naturally  infected  chimpanzee.  No  definite  changes  were  found  in  the 
nucleotide  sequences  of  the  area  of  pol  gene  tested  over  a  6-month  period  (one  of  209  bases  was 
apparently  altered  in  a  single  isolate  from  one  animal,  though  that  may  have  been  a  sequencing  error) 


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The  same  area  of  the  pol  gene  of  HTLV-I  isolated  from  the  naturally  infected  chimpanzee  differed 
from  the  others  by  8  nucleotides  (4%).   None  of  the  chimpanzees-naturally  infected,  abortively 
infected  or  experimentally  persistently  infected  with  HTLV-I-had  any  signs  of  acute  chronic  illness. 
The  pol  gene  of  HTLV-I  in  persistently  infected  chimpanzees  is  clearly  not  hypermutable. 

Rabbits  were  successfully  infected  with  two  strains  of  HTLV-I,  one  isolated  from  a  Colombian  patient 
with  TSP.   Rabbits  inoculated  intravenously  or  intracerebrally  with  HTLV-I-infected  autologous 
lymphocytes  were  viremic  for  more  than  40  weeks,  while  rabbits  inoculated  intraperitoneally  were 
only  intermittently  viremic.   All  animals  developed  antibodies  against  various  structural  proteins  of 
HTLV-I.   Further  manipulations  of  this  model  may  yield  additional  insights  into  the  pathogenesis  of 
HTLV-I  infection. 

XIV.  Detection  of  cryptic  viral  genomic  sequences  in  tissues  from  patients  with  chronic  neurological 
diseases  of  unknown  etiology 

PI:         David  M.  Asher,  M.D.  Research  Medical  Officer 

Richard  Yanagihara,  M.D.        Medical  Director 

Mark  S.  Godec,  M.D.  NRC  Research  Fellow 

Bruce  K.  Johnson,  Ph.D.  Special  Expert 

Lev  G.  Goldfarb,  M.D.  Visiting  Scientist 

Zayd  A.  Eldadah,  B.S.  Biological  Lab  Aid 

Kitty  L.  Pomeroy,  B.S.  Microbiologist 

Michael  P.  Sulima  Biological  Lab  Technician 

Alfred  Bacote  Biological  Lab  Technician 

Cooperating  Units: 

Steven  Feinstone,  CEBR,  FAA,  Rockville,  Maryland;   Peggy  Swoveland,  Univ.  Maryland, 
Baltimore,  Maryland;  Frederick  Andermann,  Montreal  Neurological  Institute,  Montreal,  Canada. 

We  have  developed  a  PCR  technique  for  detecting  genes  of  several  RNA  and  DNA  viruses.  Sensitivity 
has  been  increased  markedly  using  a  second  round  of  PCR  employing  oligioneucleotide  primers 
(interned)to  the  initial  compared  to  classical  virus  isolation,  the  technique  has  to  detect  incomplete, 
defective  and  degraded  viruses  in  fixed  and  embedded  tissues,  as  well  as  in  frozen  tissues  after  decades 
of  storage.  The  technique  is  highly  specific  and  is  capable  of  rapidly  distinguishing  genes  of  related 
RNA  viruses,  such  as  enteroviruses.  The  technique  is  being  applied  to  the  amplification  of  viral  RNA 
and  DNA  in  tissues  of  patients  with  chronic  neurological  disorders  of  unknown  etiology,  such  as  chronic 
encephalitis,  ALS,  parkinsonism,  and  Viliusk  encephalitis.   Thus  far,  primer  pairs  have  been 
synthesized  for  multiple  structural  genes  of  several  RNA  viruses,  including  measles,  mumps,  rubella, 
polio,  coxsackie  B,  HTLV-I,  and  St.  Louis  encephalitis  viruses,  and  DNA  viruses,  including  Epstein- 
Barr  virus,  herpes  simplex  virus  types  1  and  2,  cytomegalovirus,  varicella-zoster  virus  and  human 
herpes  virus  type  6.  Using  this  technique  we  failed  to  confirm  reports  of  others  who  used  an  in  situ 
hybridization  technique  of  dubious  specificity  and  low  sensitivity,  claiming  to  have  identified 
genomes  of  cytomegalovirus  and  Epstein-Barr  virus  in  brains  of  patients  with  chronic  encephalitis. 
Primer  pairs  for  Treponema  pallidum  and  Toxoplasma  gondii  are  also  used. 


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XV.  Search  for  an  infectious  cause  of  Alzheimer  disease  and  multiple  sclerosis 
PI:         David  M.  Asher,  M.D.  Research  Medical  Officer 

Mark  S.  Godec,  M.D.  NRC  Research  Fellow 

Cooperating  Units: 

Steven  Feinstone,  CEBR,  FAA,  Rockville,  Maryland;Stanley  Rapoport  and  Robert  Friedland, 
NIA,  LN,  Bethesda 

Futher  attempts  are  being  made  to  recover  infectious  agents  from  patients  with  Alzheimer's  disease 
and  MS.  Buffy  coat  specimens  from  more  than  100  unaffected  family  members  of  patients  with  familial 
Alzheimer's  disease  have  been  inoculated  into  LVG  hamsters  to  verify  the  claims,  by  Manuelidis  and 
his  colleagues,  of  the  successful  transmission  of  a  spongiform  encephalopathy.  In  addition,  CNS 
tissues,  intestinal  mucosa  and  peripheral  mononuclear  cells  from  patients  with  MS  are  being  examined 
for  viral  genomic  sequences  by  the  PCR. 

XVI.  Pathogenesis  of  hantavirus  infection  in  animals  and  cell  culture 

Co-PI:   David  M.  Asher,  M.D.  Research  Medical  Officer 

Richard  Yanagihara,  M.D         Medical  Director 

Shuyuan  Xiao,  M.D.  Visiting  Fellow 

Mark  Godec,  M.D.  National  Research  Council  Fellow 

Zahd  A.  Eldadah,  B.S.  Biological  Lab  Aid 

Xueyun  Wu,  M.P.H.  Special  Volunteer 

Bruce  K.  Johnson,  Ph.D.  Special  Expert 

Kitty  L.  Pomeroy,  B.S.  Microbiologist 

Axel  V.  Wolff,  D.V.M.  Facility  Veterinarian 

Cooperating  Units: 

David  J.  Silverman,  University  of  Maryland,  Baltimore,  Maryland 

We  have  developed  and  exploited  several  rodent  and  primate  systems  for  the  study  of  hantavirus 
infection,  including  infant  mice  and  weanling  rats  infected  with  Hantaan  virus,  bank  voles 
{Clethrionomys  glareolus)  infected  with  Puumala  virus  and  cynomolgus  monkeys  infected  with 
Puumala  and  Prospect  Hill  viruses.  We  have  extended  these  studies  to  include  the  use  of  the  reverse- 
tianscriptase  directed-polymerase  chain  reaction  (RT-PCR).     Using  primers  specific  for  the 
glycoprotein  2-endcoding  region  of  hantavirus  M  segment,  we  demonstrated  Hantaan  virus  sequences  in 
tissues  of  mice  experimentally  infected  with  Hantaan  virus  strain  76-118  and  HV114,  an  isolate  from 
the  urine  of  a  Chinese  patient  with  hemorrhagic  fever  with  renal  syndrome.    We  are  now  adopting 
this  method  to  detect  hantavirus  genomic  sequences  in  clinical  specimens  and  postmortem  tissues  of 
patients  with  HFRS. 


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Publications  in  Print: 


1 .  Bolis  L,  Gibbs  CJ,  Jr.  Proceedings  of  an  international  roundtable  on  bovine  spongiform 
encephalopathy.  Summary  report  and  recommendations.  J  Am  Vet  Med  Assoc  1990,196:1673. 

2.  Brown  P.  Central  nervous  system  amyloidoses:  a  comparison  of  Alzheimer's  disease  and 
Creutzfeldt-Jakob  disease.  Neurology  1989;39:1103-5. 

3.  Brown  P.    The  phantasmagoic  immunology  of  transmissible  spongiform  encephalopathy.  In 
Waksman  BH,  ed.  Immunologic  mechanisms  in  neurologic  and  psychiatric  disease.  New  York: 
Raven,  1990^05-13. 

4.  Brown  P.    Guidelines  for  high  risk  autopsy  cases:  special  precautions  for  Creutzfeldt-Jakob 
disease.  In:  Hutchins  G,  Kircher  T,  Peters  HJ,  eds.  Autopsy  Performance  and  Reporting, 
Northfield,  IL:  College  of  American  Pathologists,  1990;67-74. 

5.  Brown  P.    Transmissible  spongiform  encephalopathies  in  humans:  kuru,  Creutzfeldt-Jakob  disease 
and  Gerstmann-Straussler-Scheinker  disease.  Can  J  Vet  Res  1990;54:38-41. 

6.  Brown  P,  Jannotta  F,  Gibbs  CJ  Jr,  Baron  H,  Guiroy  DC,  Gajdusek  DC.   Coexistence  of  Creutzfeldt- 
Jakob  disease  and  Alzheimer's  disease  in  the  same  patient.  Neurology  1990;40:226-8. 

7.  Brown  P,  Liberski  PP,  Wolff  A,  Gajdusek  DC.    Conservation  of  infectivity  in  purified  fibrillary 
extracts  of  scrapie-infected  hamster  brain  after  sequential  enzymatic  digestion  or 
polyacrylamide  gel  electrophoresis.   Proc  Natl  Acad  Sci  USA  1990;87:7240-44. 

8.  Brown  P,  Wolff  A,  Gajdusek  DC.     A  simple  and  effective  method  for  inactivating  virus 
infectivity  in  formalin-fixed  samples  from  patients  with  Creutzfeldt-Jakob  disease.   Neurology 
1990;40:887-90. 

9.  Brown  P,  Wolff  A,  Liberski  PP,  Gajdusek  DC.  Resistance  of  scrapie  infectivity  to  steam 
autoclaving  after  formaldehyde  fixation,  and  limited  survival  after  ashing  at  360°C:  practical 
and  theoretical  implications.   J  Infect  Dis  1990;161:467-72. 

10.  Ceroni  M,  Piccardo  P,  Safar  J,  Gajdusek  DC,  Gibbs  CJ  Jr.  Scrapie  infectivity  and  prion  protein  are 
distributed  in  the  same  pH  range  in  agarose  isoelectric  focusing.  Neurology  1990;40:508-13. 

11.  Fox  KM,  Garruto  RM,  Gajdusek  DC,  Plato  CC.  Dermatoglyphics  of  the  isolated 
Kapingamarangese  of  Micronesia.  In:  Durham  NM,  Plato  CC,  eds.  Trends  in  dermatoglyphic 
research.   Dordrecht,  Netherlands:  Kluwer  Academic  Publishers,1990;278-287. 

12.  Fox  KM,  Plato  CC,  Garruto  RM.  Dermatoglyphics  of  the  people  of  Micronesia:  a  review. 
Proceedings  of  the  Valsik  Memoria  Symposium,  Bratislava,  September  21-21,  1987.  1990;  325-38. 

13.  Gajdusek  DC.    Raymond  Pearl  Memorial  Lecture,  1989.  Cultural  practices  as  determinants  of 
clinical  pathology  and  epidemiology  of  veneral  infections:  implications  of  predictions  about  the 
AIDS  epidemic.  Am  J  Hum  Biol  1990;2:4347-51. 

14.  Gajdusek  DC.    Fantasy  of  a  virus  from  the  inorganic  world:  pathogenesis  of  cerebral  amyloidoses 
by  polymer  nucleating  agents  and/or  "viruses".  In  Neth,  M.J. ,  ed.  Modern  trends  in  human 
leukemia  VIII.   Heidelberg:  Springer  Verlag,  1990;  481-99. 

15.  Gajdusek  DC.  Paradoxes  of  aspiration  for  and  of  children  in  primitive  and  isolated  cultures. 
Pediatr  Res  1990;27:S59-61. 

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16.  Gajdusek  DC.  Subacute  spongiform  encephalopathies:  Transmissible  cerebral  amyloidoses  caused 
by  unconventional  viruses.  In:  Knipe  DM,  Fields  BN,  eds.  Virology,  2nd  Edition.  New  York: 
Raven,  1990;2289-324. 

17.  Gajdusek  DC.  Cycad  toxicity  not  the  cause  of  high  incidence  amyotrophic  lateral 
sclerosis/parkinsonism-dementia  on  Guam,  Kii  Peninsula  of  Japan  or  in  West  New  Guinea.  In 
Hudson  AJ,  ed.  Amyotrophic  lateral  sclerosis:  concepts  in  pathogenesis  and  etiology,  Toronto: 
University  of  Toronto,  1990;  317-25. 

18.  Gajdusek  DC,  Gibbs  CJ  Jr  Brain  amyloidoses:  precursor  proteins  and  the  amyloids  of  transmissible 
and  nontransmissible  dementias:  Scrapie-kuru-CJD  viruses  as  infectious  polypedpides  or 
amyloid-enhancing  factors.  In  Goldstein  AL,  ed.  Biomedical  advances  in  aging,  New  York: 
Plenum ,  1990;  3-24. 

19.  Garruto  RM.  Cellular  and  molecular  mechanisms  of  neuronal  degeneration:  amyotrophic  lateral 
sclerosis,  parkinsonism-dementia  and  Alzheimer  disease.  Am  J  Hum  Biol  1990;1:529-43. 

20.  Garruto  RM.  Review  of  Modern  biological  theories  of  aging.  In:  Warner  HR,  Butler  RN,  Sprott 
RL,  Schneider  EL,  eds.  Ann  Hum  Biol  1990;1 6:285-6. 

21.  Garruto,  RM.  Introduction:  biocultural  Implications  of  AIDS  and  other  retroviral  infections.  Am  J 
Hum  Biol  1990;  2:,  343-5. 

22.  Garruto  RM,  Plato  CC,  Yanagihara  R,  Fox  K,  Dutt  J,  Gajdusek  DC,  Tobin  J.  Bone  mass  in 
Guamanian  patients  with  amyotrophic  lateral  sclerosis  and  Parkinsonism-dementia.  Am  J  Phys 
Anthropol  1989;80:107-13. 

23.  Garruto  RM,  Slover  M,  Yanagihara  R,  Mora  CA,  Alexander  SS,  Asher  DM,  Rodgers-Johnson  P, 
Gajdusek  DC    High  prevalence  of  human  T-lympho tropic  virus  type  I  infection  in  isolated 
populations  of  the  western  Pacific  region  confirmed  by  Western  immunoblot.  Am  J  Hum  Biol 
1990;2:439-47. 

24.  Garruto  RM,  Yanagihara  R,  Gajdusek  DC.  Models  of  environmentally  induced  neurological 
disease:  epidemiology  and  etiology  of  amyotrophic  lateral  sclerosis  and  parkinsonism  dementia 
in  the  western  Pacific.  Environ  Geochem  Health  1990;12:137-51. 

25.  Garruto  RM,  Yanagihara  R,  Strong  MJ.  Insights  from  Pacific  ALS:  development  of  models  of 
chronic  metal  induced  motor  neuron  degeneration  in  non-human  primates.  In:  Norris  FB,  Rose  FC, 
eds.  The  etiology  of  amyotrophic  lateral  sclerosis:  epidemiological  and  neurotoxicological 
aspects.  London:  Smith-Gordon,  1990;  143-56. 

26.  Gibbs  CJ  Jr.  AIDS:  the  biological,  ethical  and  moral  dilemmas  of  this  twentiety  century  plague. 
In:  Walton  CC,  ed.  Business  ethics:  other  voices,  other  perspectives.  New  York:  Plenum,  1990; 
341-65. 

27.  Gibbs  CJ  Jr.  Subcommittee  on  the  production  and  standarization  of  reference  reagents  GM1  and 
Anti-GMl  antibodies.  Ann  Neurol  1990;27:no  ppn. 

28.  Gibbs  CJ  Jr,  Safar  J,  Ceroni  M,  DiMartino  A,  Clark  WW,  Hourrigan  JL.    Experimental  transmission 
of  scrapie  to  cattle.  Lancet  1990;  336,  551-4. 

29.  Godec  MS,  Asher  DM,  Swoveland  FT,  Eldadah  ZA,  Feinstone  S,  Goldfarb  LG,  Gibbs  CJ  Jr, 
Gajdusek  DC.  Detection  of  measles  virus  genomic  sequence  in  SSPE  brain  tissue  by  the  polymerase 
chain  reaction.  J  Med  Virol  1990;  30:237-44. 

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30.  Goldfarb  LG,  Brown,  P,  Goldgaber  D,  Asher  DM,  Rubeinstein  R,  Brown  WT,  Piccardo  P,  Bocllaard 
JW,  Gajdusek  DC.  Creutzfeldt-Jakob  disease  and  kuru  patients  lack  mutation  consistently  found  in 
Gerstmann-Straussler-Scheinker  syndrome.  Exp  Neurol  1990;108:247-50. 

31 .  Goldfarb  LG,  Brown  P,  Goldgaber  D,  Garruto  RM,  Yanagihara  R,  Asher  D,  Gajdusek  DC.     An 
identical  mutation  in  unrelated  patients  with  Creutzfeldt-Jakob  disease.  Lancet  1990;336:174-5. 

32.  Goldfarb  LG,  Chumakov  MP,  Petrov  PA,  Fedorova  NI,  Gajdusek  DC.  Olivopontocerebellar 
atrophy  in  a  large  Iakut  kinship  in  Eastern  Siberia.  Neurology  1989;39:1527-30. 

33.  Goldfarb  LG,  Korczyn,  AD,  Brown  P,  Chapman  J,  Gajdusek,  DC.  Mutation  in  codon  200  of  scrapie 
amyloid  gene  is  linked  to  the  occurrence  of  Creuztfeldt-Jakob  disease  in  Sephardic  Jews  of  Libyan 
and  non-Libyan  origin.  Lancet  1990;336:  637-8. 

34.  Goldfarb  LG,  Mitrova  E,  Brown  P,  Toh  BH,  Gajdusek  DC.  Mutation  in  codon  200  of  scrapie 
amyloid  protein  gene  in  two  clusters  of  Creutzfeldt-Jakob  disease  in  Slovakia.  Lancet 
1990;336:514-5. 

35.  Goldgaber  D,  Goldfarb  LG,  Brown  P,  Asher  DM,  Brown  WT,  Lin  S,  Teener  JW,  Feinstone  SM, 
Rubenstein  R,  Kascsak  RJ,  Boellaard  JW,  Gajdusek  DC.    Mutations  in  familial  Creutzfeldt-Jakob 
disease  and  Gerstmann-Straussler-Scheinker's  Syndrome.  Exp  Neurol  1989;106:204-6. 

36.  Guiroy  DQ.Gajdusek  DC.  Modification  of  host  precursor  proteins  to  amyloid  fibrils  in 
Alzheimer's  disease.  In:  Harrison  DE,  ed.  Genetic  effects  on  aging,  Vol.  2  Caldwell  NJ:  Telford 
Press,  Jackson  Laboratory,  1989;543-56. 

37.  Johnson  PR,  Fomsgaard  A,  Alan  J,  Gravell  M,  London  WT,  Olmsted  RA,  Hirsch  VM.  Simian 
immunodeficiency  viruses  from  African  green  monkeys  display  unusual  genetic  diversity.  J  Virol 
1990;64:1086-92. 

38.  Johnson  PR,  Ono  SG,  Asher  DM,  Gibbs  CJ  Jr.  Tropical  spastic  paraparesis  and  HTLV-I 
myelopathy:  Clinical  features  and  pathogenesis.    In  Waksman  BH,  ed.  Immunologic  mechanisms 
in  neurologic  and  psychiatric  disease.  New  York:  Raven,1990;  117-30. 

39.  Lee  JW,  Fox  EP,  Rodgers-Johnson  P,  Gibbs  CJ  Jr,  Defreitas  E,  Manns  A,  Blattner  W,  Cotelingam  J, 
Piccardo  P,  Mora  C,  Safar  J,  Liberski  P,  Sausville  E,  Trepel  J,  Kramer  BS.     T-cell  lymphoma, 
tropical  spastic  paraparesis  and  malignant  fibrous  histiocytoma  in  a  patient  with  human  T-cell 
lymphotropic  virus,  type  I.    Ann  Intern  Med  1990;  110:239-41. 

40.  Liberski  PP,  Asher  DM,  Yanagihara  R,  Gibbs  CJ  Jr,  Gajdusek  DC.  Serial  ultrastructural  studies  of 
scrapie  in  hamsters.  J  Comp  Pathol  1989;101:429-42. 

41.  Liberski  PP,  Yanagihara  R,  Asher  DM,  Gibbs  CJ  Jr,  and  Gajdusek  DC.    Reevaluation  of  the 
ultrastructural  pathology  of  experimental  Creutzfeldt-Jakob  disease.    Brain  1990;113:121-37. 

42.  Liberski  PP,  Yanagihara  R,  Gibbs  CJ  Jr,  Gajdusek  DC.  Spread  of  Creutzfeldt-Jakob  disease  virus 
along  visual  pathways  after  intraocular  inoculation.   Arch  Virol  1990;111:141-7. 

43.  Liberski  PP,  Yanagihara  R,  Gibbs  CJ  Jr,  Gajdusek  DC.  Appearance  of  tubulovesicular  structures  in 
experimental  Creutzfeldt-Jakob  disease  preceeds  the  onset  of  clinical  disease.  Acta  Neuropathol 
1990;  79:349-354. 

44.  Masullo  C,  Pocchiari  M,  Mariotti  P,  Macchi  G,  Garruto  RM,  Gibbs  CJ  Jr,  Yanagihara  R,  Gajdusek 
DC.   The  nucleus  basalis  of  Meynert  in  Parkinsonism-dementia  of  Guam:  a  morphometric  study.  J 
Neuropathol  Appl  Neurobiol  1989;15:193-206. 

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45.  Morgan  OStC,  Rodgers-Johnson  P,  Mora  C,  Char  G  .  HTLV-I  and  polymyositis  in  Jamaica.  Lancet 
1989;  2  (November  18).1184. 

46.  Piccardo  P,  Safar  J,  Ceroni  M,  Gajdusek  DC,  Gibbs  CJ  Jr.    Immunohistochemical  localization  of 
prion  protein  in  spongiform  encephalopathies  and  normal  brain  tissue.  Neurology  1990;  40:518- 
522. 

47.  Plato  CC,  Garruto  RM.  Historical  notes  on  dermatoglyphics:  from  Purkinje  to  Cummins.  In: 
Durham  NM  ,  Plato  CC,  eds.  Trends  in  dermatoglyph  research.  Dordrecht,  Netherlands:  Kluwer 
Academic  Publishers,  1990;  2-10. 

48.  Rodgers-Johnson  PEB,  Garruto  RM,  Yanagihara  R,  Gajdusek  DC.  Human  T-lymphotropic  virus 
type  I:  A  retrovirus  causing  chronic  myeloneuropathies  in  tropical  and  temperate  climates.  Am  J 
Hum  Biol  1990;2:429-38. 

49.  Rodgers-Johnson  PEB,  Ono  S,  Asher  DM,  Gibbs  CJ  Jr,  Gajdusek  DC.   Tropical  spastic  paraparesis 
and  HTLV-I  myelopathy.  Clinical  features  and  pathogenesis.  In:  Waksman  BH,  ed. 
Immunologic  mechanisms  in  neurologic  and  psychiatric  disease.  New  York:  Raven,  1990;  117-30. 

50.  Rodgers-Johnson  PEB,  Ono  S,  Gibbs  CJ  Jr,  Gajdusek  DC.    Tropical  spastic  paraparesis  and  HTLV-I- 
associated  myelopathy.  Clinical  and  laboratory  diagnosis.   In  Blattner  WA,  ed.  Human 
retrovirology:  HTLV.  Raven,  New  York:  Raven,1990;205-ll. 

51 .  Safar  J,  Ceroni  M,  Piccardo  P,  Gajdusek  DC,  Gibbs  CJ  Jr.    Scrapie-associated  precursor  proteins: 
Antigenic  relationship  between  species  and  immunocytochemical  localization  in  normal,  scrapie, 
and  Creutzfeldt-Jakob  disease  brains.  Neurology  1990;40:513-17. 

52.  Safar  J,  Ceroni  M,  Piccardo  P,  Liberski  PP,  Miyazaki  M,  Gajdusek  DC,  Gibbs  CJ  Jr.  Subcellular 
distribution  and  physicochemical  properties  of  scrapie-associated  precursor  protein  and 
relationship  with  scrapie  agent  Neurology  1990;40:503-8. 

53.  Safar  J,  Wang  W,  Pagett  MP,  Ceroni  M,  Piccardo  P,  Zopf  D,  Gajdusek  DC,  Gibbs  CJ  Jr.  Molecular  mass, 
biochemical  composition  and  physiocochemical  behavior  of  the  infectious  form  of  the  scrapie  precursor 
protein  monomer.  Proc  Natl  Acad  Sci  USA  1990;87:6373-7. 

54.  Srinivasappa  J,  Asher  DM,  Pomeroy  KL,  Murphy  LJ,  Wolff  AV,  Yoon  J-W,  Gajdusek  DC,  Notkins 
AL.  Scrapie-induced  diabetes  mellitus  in  hamsters.  Microb  Pathogen  1989;7:189-94. 

55.  Strong  MJ,  Garruto  RM.  Isolation  of  fetal  mouse  motor  neurons  on  discontinuous  percoll  density 
gradients.   In  Vitro  Cell  Develop  Biol  1989,  25:939-45. 

56.  Strong  MJ,  Garruto  RM,  Wolff  AV,  Yanagihara  R,  Chou  SM,  Fox  SD.  A  novel  neurotoxic 
plasticizing  agent  -  N-butylbenzenesulfonamide  (NBBS).  Lancet  1990;  336:640. 

57.  Strong  MJ,  Svedmyr  A,  Gajdusek  DC,  Garruto  RM.    The  temporal  expression  of  amyloid  precursor 
protein  mRNA  in  vitro  in  dissociated  hippocampal  neuron  cultures.  Exp  Neurol  1990;109:171-9. 

58.  Strong  MJ,  Yanagihara  R,  Garruto  RM.  Aluminum-induced  neurofilamentous  lesions  in  dissociated 
motor  neuron  cultures.  In:  Norris  FB,  Rose  FC,  eds.  The  etiology  of  amyotrophic  lateral  sclerosis: 
epidemiological  and  neurotoxicological  aspects.  London:  Smith-Gordon,  1990;  175-80. 

59  Strong  MJ,  Yanagihara  R,  Wolff  R,  Shankar  SK,  Garruto  RM.  Experimental  neurofilamentous 

aggregates:  acute  and  chronic  models  of  aluminum-induced  encephalomyelopathy  in  rabbits.  In: 
Norris  FB,  Rose  FC,  eds.  The  etiology  of  amyotrophic  lateral  sclerosis:  epidemiological  and 
neurotoxicological  aspects.  London:  Smith-Gordon,  1990;  157-73. 

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Z01  NS  01282-26  and  Z01  NS  00969-26 


60.  Wills  PR.    Quantitative  constraints  on  mechanisms  of  replication  for  information-carrying  prions. 
In:  Iqbal,  K.,  Wisniewski,  H.M.,  Winblad,  B.,  eds.     Alzheimer's  disease  and  related  disorders. 
New  York:  Alan  R.  Liss,  1989;669-77. 

61.  Wills  PR.  Induced  frame-shifting  mechanism  of  replication  for  an  information-carrying  scrapie 
prion.  Microb  Pathogen  1989;6:235-49. 

62.  Wills  PR,  Hughes  AJ.    Stem  loops  in  HIV  and  prion  protein  mRNAs.  J  AIDS  1990;3:95-7. 

63.  Wong  T-W,  Chan  Y-C,  Joo  YG,  Lee  HW,  Lee  P-W,  Yanagihara  R.  Hantavirus  infection  in  humans 
and  commensal  rodents  in  Singapore.  Trans  R  Soc  Trop  Med  Hyg  1989;  83:248-51. 

64.  Yanagihara  R.      Hantavirus  infection  in  the  United  States:  epizootiology  and  epidemiology.   Rev 
Infect  Dis  1990;12:449-57. 

65.  Yanagihara  R,  Jenkins  CL,  Alexander  SS,  Mora  CA,  Garruto  RM.  Human  T  lymphotropic  virus 
type  I  infection  in  Papua  New  Guinea:  High  prevalence  among  the  Hagahai  confirmed  by 
Western  analysis.  J  Infec  Dis  1990;162:649-54. 

66.  Yanagihara  R,  Silverman  DJ.     Experimental  infection  of  human  vascular  endothelial  cells  by 
pathogenic  and  nonpathogenic  hantaviruses.   Arch  Virol  1990;111:281-6. 


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CONTRACTS 

University  of  Southwestern  Louisiana 
New  Iberia  Research  Center 
New  Iberia,  Louisiana 

Contract  #N01-NS-8-00931 

$562,000.00 


Program  Resources,  Inc. 
(Administration  by  NCI) 

Contract  #N01-CO-70421111 
$747,664.00 


34  -  LCNSS 


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ANNUAL  REPORT 
October  1,  1989  through  September  30,  1990 


Laboratory  of  Experimental  Neuropathology 
Basic  Neurosciences  Program,  DIR,  NINDS 

Table  of  Contents 


RESEARCH  SUMMARY  1-5 

PROJECT  REPORTS 

Herpesvirus  Infections  and  Nervous  System  Diseases 

Z01  NS  02549-09  LENP  6 

Morphological  Studies  of  Myelin  Formation,  Breakdown 

and  Regeneration 
Z01  NS  01995-18  LENP  7 

Biochemical  and  Immunologic  Mechanisms  in  Virally- 

Induced  CNS  Demyelination 
Z01  NS  02550-09  LENP  8 

Roles  of  Gangliosides  in  Neuronal  and  Myelin  Function, 

Cell  Growth  and  Differentiation,  and  Neurotoxicity 
Z01  NS  02699-05  LENP  9 

Morphological  Studies  of  Experimental  and  Human  HIV 

Infection 
Z01  NS  02758-03  LENP  10 

Lipid  Peroxidation,  Protein  Oxidation  and  Demyelination 

Z01  NS  02759-03  LENP  11 

Mechanism  of  Latency  and  Pathogenesis  of  Herpes  Simplex 

Virus  in  the  Nervous  System 
Z01  NS  02803-01  LENP  12 

Nervous  System  Regeneration  in  a  Herpesvirus  Model 

Z01  NS  02804-01  LENP  13 

JC  Human  Polyomavirus  Infection  and  Tumor  Induction  in  the 

Neonatal  Brain 
Z01  NS  02807-01  LENP  14 

The  Role  of  Protein  Kinase  C  and  raf  Protein  Kinase  in 

Cellular  Functions 
Z01  NS  02808-01  LENP  15 

Glial  Cells  in  Development  and  Viral  Disease 

Z01  NS  02809-01  LENP  16 


i/LENP/DIR 


ANNUAL    REPORT 
October    1,     1989    through    September    30,     1990 
Laboratory   of   Experimental   Neuropathology,    DIR 
National    Institute   of   Neurological   Disorders   and   Stroke 
Henry   deF .    Webster,    Chief 


The  Laboratory  of  Experimental  Neuropathology  (LENP)  includes  the 
Cellular  Neuropathology  Section  (CN)  and  the  Neurotoxicology  Section  (NT) . 
The  main  goal  of  the  Laboratory  '  s  research  program  is  to  investigate 
cellular  mechanisms  of  neurological  diseases,  especially  those  that  are 
directly  related  to  progressive  multifocal  leukoencephalopathy  (PML) ,  the 
pathogenesis  and  latency  of  herpes  simplex  virus  (HSV)  infections, 
multiple  sclerosis  (MS) ,  and  age-related  changes  in  peripheral  nerves.  Other 
closely  coordinated  projects  use  biochemical  and  immunocytochemical 
methods  to  explore  gangliosides  and  protein  phosphorylation  .  Some 
neurotoxic   actions   also   are   being   investigated. 

The  most  important  new  finding  in  LENP1  s  AIDS  research  was:  Early 
lesions  in  PML,  an  important  complication  of  AIDS,  were  shown  to  consist 
of  glial  hypertrophy  in  the  absence  of  virus  capsid  production.  In 
established  cases  of  PML,  double- labeling  showed  that  reactive  astrocytes 
resist  productive  JC  virus  (JCV)  infection,  suggesting  a  possible  new 
approach   to   treatment   of    this   usually    fatal   disorder. 

Other  important  discoveries  were:  1  )  In  the  hamster  model  of  JCV 
infection,  areas  of  gliosis  were  found  in  association  with  but  independent  of 
tumor  tissue,  suggesting  a  second  tissue  response  to  JCV  involving 
hyperplasia  without  frank  tumor  induction.  Further,  we  now  have  indica- 
tions that  endothelial  cells  are  infected  in  the  hamster  model,  and  may  play 
a  role  in  the  pathogenesis  of  disease  .  Studies  with  hybrid  JCV/SV40 
regulatory  regions  in  transgenic  mice  have  shown  that  the  regulatory 
region,  not  the  early  protein  (T-antigen) ,  determines  the  pattern  of  tumor 
induction  in  this  model .  2 )  In  adult  rat  optic  nerves,  the  a-subspecies  of 
protein  kinase  C  existed  in  astrocytes  in  both  native  and  partially  degraded 
forms .  Some  of  the  partially  proteolyzed  enzyme  was  shown  to  contain  the 
catalytic  domain.  By  contrast,  the  8- isozyme  of  protein  kinase  C  was 
always  found  as  an  intact  macromolecule  in  the  axons.  These  findings 
suggest  that  the  cellular  mechanisms  involved  in  the  regulation  of  protein 
kinase  C  activity  in  astrocytes  and  in  axons  are  different.  3)  An  HSV-2 
transcript  which  is  expressed  in  neurons  during  latent  infection  has  been 
characterized;  this  transcript  is  similar  but  not  identical  to  the  latency- 
associated  transcript  of  HSV-1  .  4)  Acute  HSV-2  infection  targets  small 
dorsal  root  ganglion  neurons;  selectivity  is  independent  of  virus  strain  and 
only  partly  dependent  on  inoculation  route.  5)  Following  corneal  HSV 
inoculation,  the  virus  targets  sensory  sympathetic  and  parasympathetic 

1/LENP/DIR 


ganglia,  and  spreads  to  the  central  nervous  system  (CNS)  by  at  least  four 
neural  routes .  6 )  When  nerve  regeneration  after  crush  injury  was  compared 
in  sciatic  nerves  of  6-month-old  and  24-month-old  mice,  unmyelinated  axon 
regeneration  was  not  significantly  different.  However,  the  number  of  myeli- 
nated fibers  in  nerves  of  24-month-old  mice  was  only  38%  of  that  seen  in 
the  young  adult  (  6-month-old)  nerves.  The  myelinated  fiber  axons  were 
smaller   and  were   surrounded  by   thinner  myelin   sheaths. 

The  following  summary  describes  these  discoveries  and  the  most 
significant   new  evidence   obtained   in  FY   1990   LENP   projects. 


Human  Polyomavirus  JC  in  Animal  Models  and  in  Human  Neurologic  Disease 
in  AIDS   and   non-AIDS   Patients. 

The  pathogenesis  of  JCV  infection  in  the  human  brain  has  resisted 
clarification  ever  since  the  recognition  of  PML  as  a  fatal  demyelinating 
disease  in  1  958 .  In  the  absence  of  clear  evidence,  opinion  has  been  about 
equally  divided  between  those  who  assume  the  disease  is  another  oppor- 
tunistic infection  of  patients  with  AIDS  in  which  the  virus  invades  tissue 
where  it  is  not  normally  resident,  and  those  who  assume  that  the  virus  is 
reactivating  frcm  a  latent  state  in  the  brain.  Clearly,  the  latter  alternative 
holds  more  interest  in  terms  of  a  possible  role  for  JCV  in  other  human 
neurologic  disease .  The  two  areas  of  greatest  interest  in  this  regard  are 
the  implications  of  JCV  latency  for  brain  tumor  induction,  particularly 
tumors  of  childhood  such  as  medulloblastomas,  and  the  possible  implications 
of  JCV  latency  for  demyelinating  diseases  of  unknown  etiology,  such  as 
MS  .  Since  much  evidence  suggests  that  JCV  is  latent  in  the  brain  and  can 
reactivate  following  immunosuppression  as  occurs  in  AIDS,  we  have  now 
explored  further  the  implications  of  latent  JCV  in  the  brain  for  demye- 
linating diseases  such  as  MS.  For  the  first  time,  a  comprehensive 
hypothesis  has  been  developed  which  has  the  potential  to  explain  all  facets 
of  the  MS  enigma.  This  hypothesis  suggests  numerous  new  avenues  of 
experimentation,  some  of  which  are  currently  being  pursued.  Our  major 
experimental  model  for  JCV  infection  continues  to  be  infection  of  the 
neonatal  hamster  brain  following  intracerebral  inoculation.  Recent  evidence 
showing  that  JCV  continues  to  express  T-antigen  in  gliotic  lesions  of  the 
hamster  brain  independently  of  tumor  induction  is  consistent  with  the 
demonstration  of  early  glial  hypertrophy  in  incipient  PML  lesions,  and 
reemphasizes  the  relevance  of  the  hamster  model  to  several  facets  of  the 
host   response   to   JCV   in   the   human   brain. 


Herpesvirus   Pathogenesis   and  Nervous   System  Disease 

Mouse  models  of  neurotropic  herpesvirus  infection  provide  important 
opportunities  to  systematically  examine  disease  mechanisms  in  the  nervous 
system.  During  FY  90,  four  areas  of  herpes  simplex  virus  (HSV)  patho- 
genesis  were   investigated. 

2/LENP/DIR 


To  determine  which  dorsal  root  ganglion  (DRG)  neuronal  subpopulations 
are  targeted  if  inoculation  route  ( footpad  and  sciatic)  and  virus  strain  ( 1 86, 
MS ,  HG52 )  are  varied,  mice  were  inoculated  with  HSV-2  strains.  Peripheral 
inoculation  targets  chiefly  small  neurons,  while  sciatic  inoculation  infects  a 
broader  range  of  cell  sizes  .  Altered  neuronal  peptide  content  was  not 
detected  in  immunohistochemical  tests .  HSV-2  strains  differing  in  virulence 
affect  the  numbers  of  DRG  neurons  that  express  viral  antigen,  but  not  the 
specific   subpopulation. 

Three  studies  were  performed  in  the  mouse  cornea  model .  In  the 
first,  an  HSV-2  transcript  which  is  expressed  in  trigeminal  ganglion  neurons 
during  latent  infection  has  been  mapped  and  described.  In  addition  to  this 
transcript ,  RNA  transcription  which  could  be  detected  and  mapped  by  in 
situ  hybridization,  occurred  adjacent  to  and  to  the  right  of  the  map 
position  of  HSV-2  LAT.  The  transcript  is  similar  but  not  identical  to  that 
found   with   HSV-1    infection. 

In  the  second,  HSV-1  spread  from  a  peripheral  (corneal)  inoculation 
site  to  tissues  targeted  by  this  injection  was  traced  in  the  whole  mouse 
head .  Results  indicate  that  a  much  wider  range  of  tissues  are  infected  in 
this  model  than  was  previously  shown.  Sensory,  sympathetic  and  para- 
sympathetic ganglia  are  infected,  and  observations  suggest  at  least  four 
neural  routes  by  which  virus  may  enter  the  intracranial  compartment. 

In  the  third,  studies  to  define  characteristics  of  latent  HSV-1  infection 
in   non-neuronal    tissues   have   been   undertaken. 

Studies  on  nervous  system  disease  caused  by  another  neurotropic 
herpesvirus,  varicella-zoster  virus  (VZV),  were  initiated.  In  an  autopsy 
case ,  a  meningoradiculopathy  developed  in  the  absence  of  detectable  skin 
lesions .  Herpesvirus  infection  was  identified  at  autopsy  and  using  immuno- 
histochemical  and  in  situ  hybridization  methods,  was  shown  to  be  due  to 
VZV  infection .  In  a  separate  study,  the  literature  on  VZV-related  arteritis 
and   angiitis   of   the   CNS   was   reviewed. 


Roles  of  Gangliosides  and  Protein  Phosphorylation  in  CNS  function  and 
Cellular   Metabolism. 

Gangliosides  are  sialic  acid-containing  glycosphingolipids  ubiquitous  in 
eukaryotes .  In  mammals ,  these  glycol ipids  are  most  abundant  in  the  CNS . 
Studies  in  this  laboratory  have  established  that  gangliosides  can  modulate 
several  protein  phosphorylation  systems  in  vitro.  Yet,  the  mechanisms 
involved  in  the  perturbation  or  mobilization  of  cellular  gangliosides  for  the 
putative  lipid  second  messenger  function  still  remain  unknown .  To  address 
this  problem,  oligosaccharides  were  generated  from  different  gangliosides 
and  their  effects  on  protein  phosphorylation  were  investigated .  Preliminary 
results  indicated  that  the  sialic  acid-containing  oligosaccharide  portion  of 
G^  could  enhance  the  autophosphorylation  activity  of  a  brain  ganglioside- 
stimulated  protein  kinase,  albeit  the  effect  is  lower  than  those  mediated  by 
ganglioside  micelles.     By  contrast,   the  oligosaccharide  derived 

3/lenp/dir 


from  asialo-Gj^i  is  completely  ineffective.  These  findings  suggest  that  the 
metabolic  turnover  of  gangliosides,  in  analogy  to  the  turnover  of  phosphati- 
dylinositols  and  other  phospholipids  such  as  phosphatidylcholine,  may  play 
an  important  role  in  regulating  cellular  signal  transduction  pathways . 


The  cellular  biochemistry  of  ganglioside  transport  also  was  studied.  In 
skeletal  and  cardiac  muscle,  several  ganglioside-binding  proteins  were 
identified .  Some  of  these  proteins  may  be  involved  in  the  transfer  of 
gangliosides  among  different  cellular  compartments .  Further  investigation 
of  ganglioside-binding  proteins  may  provide  new  insights  into  certain 
ganglioside-related  diseases .  Gangliosides  can  bind  myelin  basic  protein 
(MBP)  under  in  vitro  conditions  to  form  tight  complexes  that  remain 
undissociated  in  8  M  urea .  Phosphorylation  of  MBP  by  protein  kinase  C 
and  by  a  ganglioside-stimulated  protein  kinase  was  altered  by  this  protein- 
lipid  interaction .  Electron  microscopic  (EM)  experiments  are  in  progress  to 
define  the  exact  location  of  the  myelin  specific  ganglioside  GM1  in  vivo  and 
to  determine  whether  gangliosides  and  MBP  could  be  colocalized  in  myelin. 

Protein  kinase  C  is  a  major  Ca^+/phospholipid-dependent  protein  kinase 
in  the  brain .  The  activity  of  this  enzyme  can  be  further  regulated  by 
gangliosides .  Using  both  immunocytochemical  and  immunological  techniques, 
we  have  shown  that  different  isozymes  of  protein  kinase  C  have  different 
subcellular  distribution  in  the  optic  nerves  of  adult  rats.  The  ma j or 
isozyme  is  the  type  II  (S)  subspecies,  and  is  found  predominantly  in  the 
axons  as  intact  macromolecules .  By  contrast,  the  type  III  (a)  isozyme  is 
localized  almost  exclusively  in  astrocytes.  Some  of  type  III  isozymes  were 
in  partially  proteolyzed  forms.  Very  little  inmunostaining  of  the  type  I  (y) 
isozyme  was  observed  in  the  optic  nerves .  Because  different  protein  kinase 
C  isozymes  have  different  responses  to  modulators  such  as  arachidonic  acid, 
our  results  may  provide  a  molecular  basis  for  future  studies  on  the 
epigenetic  and  regulatory  mechanisms  of  this  protein  phosphotransferase 
system. 


Nerve   Regeneration   During   Aging 

After  inducing  Wallerian  degeneration  by  a  crush  injury,  image  analysis 
and  quantitative  EM  methods  were  used  to  compare  nerve  regeneration  in 
sciatic  nerves  of  6-month-old  and  24-month-old  mice.  Two  weeks  after  the 
crush  injury,  there  was  no  significant  difference  in  the  regeneration  of 
unmyelinated  axons  .  In  contrast,  the  number  of  regenerated  myelinated 
fibers  in  the  aging  nerves  was  only  38%  of  the  number  found  in  nerves  of 
the  young  adults ,  suggesting  that  regeneration  of  axons  destined  to  become 
myelinated  might  be  impaired.  In  addition,  the  myelinated  axons  in  the 
aging  nerves  were  smaller  and  had  thinner  myelin  sheaths .  Also,  the  aging 
nerves  had  more  single  unmyelinated  axons  surrounded  by  one  Schwann  cell, 
suggesting  that  Schwann  cell  ensheathment  and  segregation  of  single  large 
regenerating  axons  that  precedes  myelination  also  was  slowed  during  aging. 
Our  discovery  that  in  aging  mice  myelinated  fiber  regeneration  is  retarded, 

4/LENP/DIR 


is  relevant  to  the  management  of  peripheral  nerve  disease  in  older  patients . 
It  may  explain  why  older  patients  recover  function  more  slowly  in  certain 
types   of   peripheral   neuropathy   and   after   nerve    injury. 


5/LENP/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOINS  02549- 09  LENP 


PERIOD  COVERED 

October  1, 1989  through  September  30, 1990 


TITLE  OF  PROJECT  (SO  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Herpesvirus  Infections  and  Nervous  System  Diseases 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  J. R  .  Martin,  M.D.  Medical  Officer  LENP,  NINDS 

Others:  W.J.  Mitchell,  D.V.M.,  Ph.D.  Sr.  Staff  Fellow  LENP,  NINDS 

D.B.Henken,Ph.D.  Staff  Fellow  LENP,  NINDS 

H.deF.  Webster,  M.D.  Chief  LENP,  NINDS 


COOPERATING  UNITS  (if  any) 

Department  of  Neurology,  Colorado  University,  Denver,  CO;  Department  of 
Microbiology,  USUHS,  Bethesda,  MD 


LAB/BRANCH 

Laboratory  of  Experimental  Neuropathology 


SECTION 

Cellular  Neuropathology  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  1    1 


OTHER:  0.6 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  [T]  (b)  Human  tissues  EH   (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  seeks  to  identify  nervous  system  diseases  associated  with  the  neurotropic 
viruses  herpes  simplex  types  1  and  2  (HSV-1,  HSV-2)  and  varicella  zoster  virus  (VZV),  and 
to  examine  mechanisms  underlying  production  of  neural  lesions  in  experimental 
models  of  infection.  Problems  of  particular  interest  are:  (i)  the  role  of  HSV  and  VZV 
infection  in  the  production  of  CNS  and  PNS  disease,  including  acute  encephalitis  and 
chronic  demyelination,  and  (ii)  mechanisms  of  neurotropic  herpesviruses  in  CNS 
arteritis  and  stroke- 

During  FY  1990,  initial  studies  of  human  autopsy  tissues  to  examine  nervous  system 
disease  caused  by  VZV  showed  infection  of  the  CNS  and  PNS  in  an  elderly  patient  with 
neurological  signs.  Cutaneous  manifestations  of  zoster  were  not  evident,  and  VZV 
infection  was  unsuspected.  This  case  supports  the  hypothesis  that  VZV  reactivations 
may  occur  in  the  absence  of  peripheral  cutaneous  manifestations.  In  a  separate  study, 
the  literature  on  VZV-related  arteritis  and  angiitis  of  the  CNS  was  reviewed. 

In  experimental  models,  HSV-1  spread  was  traced  from  a  peripheral  (corneal) 
inoculation  site  to  other  tissue  targets  in  the  whole  mouse  head.  Virus  spread  in  this 
model,  used  for  pathogenesis  studies  by  many  investigators,  is  more  extensive  than 
previously  recognized,  including  infection  of  an  intracranial  nerve  and  associated 
artery.  This  provides  the  first  direct  evidence  linking  neural  spread  of  a  neurotropic 
herpesvirus  to  arterial  infection.  In  an  associated  study,  replication  of  HSV-1  mutants 
with  genetic  alterations  in  the  joint  region  showed  impaired  replication  together  with 
restricted  spread  in  mouse  tissues. 

6/LENP/DIR 


PHS  6040  (Rev  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBtR 


ZOI  NS0199S  18 LENP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (SO  characters  or  less.   Title  mutt  fit  on  one  line  between  the  borders.) 

Morphological  Studies  of  Myelin  Formation,  Breakdown  and  Regeneration 


PRINCIPAL  INVESTIGATOR  (list  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI: 
Others: 


H.deF.  Webster,  M.D. 
K..Tanaka,M.D. 
M.G.  Nunzi,  M.D. 


Chief 

Visiting  Fellow 
Guest  Researcher 


LENP,  NINDS 
LENP,  NINDS 
LENP,  NINDS 


COOPERATING  UNITS  (if  any) 

None 


LAB/BRANCH 

Laboratory  of  Experimental  Neuropathology 


SECTION 

Cellular  Neuropathology  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


1.5 


PROFESSIONAL: 


0.4 


OTHER: 


1.9 


CHECK  APPROPRIATE  BOX(ES) 

I      |   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


]  (b)  Human  tissues  LLJ   (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  goal  of  this  project  is  to  use  quantitative  light  (LM)  and  electron  microscopy  (EM) 
along  with  other  morphological  and  biochemical  methods  to  study  cellular  mechanisms 
of  myelin  formation,  breakdown  and  regeneration.  This  year,  to  compare  nerve 
regeneration  in  young  adult  and  aging  mice,  we  transected  sciatic  nerves  in  6-  and  24- 
month-old  mice.  After  2  weeks,  the  nerves  were  removed,  transverse  sections  were  cut 
5  mm  distal  to  the  transection  site,  and  regeneration  of  myelinated  and  unmyelinated 
axons  was  studied  quantitatively  by  LM  and  EM.  The  results  showed  that  in  sciatic 
nerves  of  aging  mice,  regeneration  of  myelinated  axons  was  retarded.  In  these  nerves 
there  also  were  more  Schwann  cells  that  surrounded  a  single  unmyelinated  axon  more 
than  one  micron  in  diameter,  suggesting  that  ensheathment  of  axons  by  Schwann  cells, 
a  process  that  precedes  the  onset  of  myelin  regeneration,  also  occurred  more  slowly 
during  aging.  Mechanisms  responsible  for  the  delayed  myelinated  fiber  regeneration 
during  aging  are  being  explored  in  current  experiments.  Another  important  finding  in 
these  experiments  was  that  regeneration  of  unmyelinated  axons  in  nerves  from  young 
adult  and  aging  mice  did  not  differ  significantly. 


7/LENP/DIR 


PHSHMdten.  l««) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOI  NS  02550-09  LENP 


PERIOD  COVERED 

Octoberl,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  /ess.   Title  must  fit  on  one  line  between  the  borders.) 

Biolochemical  and  Immunologic  Mechanisms  in  Virally-lnduced  CNS  Demyelination 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 

PI: 
Others: 


G.L  Stoner,  Ph.D. 
C.F.  Ryschkewitsch.B.S. 
H.deF.  Webster,  M.D. 
H.G.  Ressetar,  Ph.D. 


Chief,  Neurotoxicology  Section  LENP,  NINDS 

Medical  Technologist  LENP,  NINDS 

Chief  LENP,  NINDS 

Staff  Fellow  LENP,  NINDS 


COOPERATING  UNITS  (if  any) 

Dept.  of  Medical  Microbiology,  Univ.  of  Wise.  Med.  Sch.  (D.L.  Walker);  Dept.  of 
Biochemistry,  Penn.  State  Univ.  (R.J.  Frisque) 


LAB/BRANCH 

Laboratory  of  Experimental  Neuropathology 


SECTION 

Neurotoxicology  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN-YEARS: 


2.0 


PROFESSIONAL: 


1.0 


OTHER: 


1.0 


CHECK  APPROPRIATE  BOX(ES) 

J   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


[X]  (b)  Human  tissues  Lj   (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Evidence  from  several  sources  points  toward  the  existence  of  latency  by  JC  Virus  (JCV) 
in  the  CNS  in  a  significant  portion  of  the  human  population.  This  includes:  (a)  our  own 
studies  of  progressive  multifocal  leukoencephalopathy  (PML)  pathology,  (b)  The 
appearance  of  PML  in  2-5%  of  autopsied  patients  with  acquired  immunodeficiency 
syndrome  (AIDS),  (c)  Our  evidence  from  the  hamster  model  of  JCV  CNS  infection,  in 
which  JCV  inoculated  intracerebral^  in  the  neonatal  hamster  brain  localizes  in  the 
cerebellar  granular  layers,  hippocampus  and  periventricular  areas.  This  evidence 
suggests  a  mechanism  by  which  low  levels  of  JCV  entering  the  brain  through  the 
circulation  early  in  life  might  become  focally  distributed  through  replication  as  a 
minichromosome  in  synchrony  with  host  cell  division,  (d)  Preliminary  evidence  from 
studies  of  normal  brain  and  brain  with  neurological  disease  other  than  multiple 
sclerosis  (MS)  using  the  polymerase  chain  reaction  (PCR)  for  amplification  of  JCV  DNA 
suggests  that  the  virus  might  be  present  in  a  latent  state  in  brains  without  overt 
demyelinating  disease.  The  weight  of  evidence  in  favor  of  latency  by  JCV  in  the  brain 
has  led  us  to  formulate  a  theory  of  pathogenetic  mechanisms  by  which  latent  and 
partially  reactivating  (limited  to  early  region  expression  or  other  restricted  expression 
without  independent  DNA  replication)  might  lead  to  immune-mediated  demyelinating 
disease,  such  as  seen  in  MS.  It  is  proposed  that  PML  and  MS  may  be  the  result  of 
differing  host  responses  to  the  presence  of  virus  focally  distributed  in  the  brain 
following  infection  of  glial  cells  or  their  precursors  early  in  life.  While  JCV  is  a 
candidate  for  involvement  in  both  diseases,  other  DNA  viruses  with  some  specificity  for 
infection  of  and  expression  in  glial  cells  should  also  be  considered. 


8/LENP/DIR 


.  .i 


fn',  6040  (Hi-j    1  84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02699-05  LENP 


PERIOD  COVERED 

October  1, 1989  through  September  30, 1990 


TITLE  OF  PROJ  ECT  (SO  characters  or  less.  Title  must  fit  on  one  line  between  the  borders) 

Roles  of  Gangliosides  in  Neuronal  and  Myelin  Function,  Cell  Growth  and  Differentiation,  and  Neurotoxicity 


PRINCIPAL  IN  VE  STIG  A  TOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  K.-F.J.  Chan,  Ph.D.         Sr.  Staff  Fellow  LENP,  NINDS 

Others:      Y.  Liu,  M.D.  Visiting  Fellow  LENP,  NINDS 


COOPERATING  UNITS  (itany) 

None 


LAB/BRANCH 

Laboratory  of  Experimental  Neuropathology 


SECTION 

Neurotoxicoloqy  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN-YEARS:  -- 


PROFESSIONAL:  1  g 


OTHER: 


0.2 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  [T]  (D)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  main  goal  of  this  project  is  to  establish  a  functional  role  for  gangliosides  as 
multifunctional  biomodulators.  Studies  in  this  laboratory  revealed  that  gangliosides 
have  profound  modulatory  effects  on  protein  phosphorylation  and  dephosphorylation 


in  the  central  nervous  system  and  in  several  other  tissues  such  as  skeletal  muscle.  This 
lipid  second  messenger-like  action  is  mediated  in  part  through  the  regulation  of  two 
classes  of  protein  kinases,  namely,  qanqlioside-dependent  and  qanqlioside-modulated 


protein  kinases.     Preliminary  studies  revealed  that  certain  sialic  acid-containing 


oligosaccharides  derived  from  gangliosides  could  enhance  the  autophosphorylation 
activity  of  a  ganglioside-stimulated  protein  kinase.  This  finding  suggests  that 
perturbation  of  cellular  gangliosides  may  play  a  role  in  altering  metabolic  signal 
transduction  pathways 


The  mechanisms  of  intracellular  trafficking  of  gangliosides  also  were  investigated 
We  found  that  both  skeletal  and  cardiac  muscle  contain  unique  qanqlioside-bindinq 


proteins.  Further  characterization  of  these  proteins  may  provide  new  information  on 
the  biochemistry  of  cellular  gangliosides  and  perhaps  more  insights  in  the  pathogenesis 
of  ganglioside-related  diseases. 


9/LENP/DIR 


PHSiMiVw.  1141 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOI  NS  02758-03-  LENP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less,   rule  must  fit  on  one  line  between  the  borders.) 

Morphological  Studies  of  Experimental  and  Human  HIV  Infection 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  {Name,  title,  laboratory,  and  institute  affiliation) 

PI:  H.  deF.  Webster,  M.D.  ~^ef  LENP,  NINDS 

Others: 


COOPERATING  UNITS  (,f  an,) 

None 


LAB/BRANCH 

Laboratory  of  Experimental  Neuropathology 


SECTION 

Cellular  Neuropathology  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN-YEARS:  n~ 


PROFESSIONAL:  Q  Q 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

I      I   (a)  Human  subjects  ]  (b)  Human  tissues  0   (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Due  to  the  departure  of    Drs.  Lamperth  (LENP,  NINDS),  Leonard  (LMM,  NIAID)  and 
Pezan  (LMM,  NIAID)  this  project  was  terminated. 

Publications:  None. 


10/LENP/DIR 


PHS  6040  (Rev   1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBtR 


ZOINS  02759  03  LENP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  ot  less.   Title  must  fit  on  one  line  between  the  borders) 

Lipid  Peroxidation,  Protein  Oxidation  and  Demyelination 


PRINCIPAL  INVESTIGATOR  (list  other  professional  personnel  below  the  Principal  In  vestigator  )  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:        Hubert  Mickel,  M.D.  Guest  Researcher  LENP,  NINDS 


COOPERATING  UNITS  (,tany) 

None 


LAB/BRANCH 

Laboratory  of  Experimental  Neuropathology 


SECTION 

Cellular  Neuropathology  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN  YEARS. 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)Human  subjects  ]  (b)  Human  tissues         EH   (c)  Neither 

]  (a1)  Minors 

I      I   (a2)  Interviews 

SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  has  been  terminated. 

Publications: 

Mickel  HS,  Kempski  O,  Feuerstein  G,  Parisi  JE,  HdeF  Webster.  Prominent  white  matter 
lesions  develop  in  Mongolian  gerbils  treated  with  100%  normobaric  oxygen  after 
global  brain  ischemia.  Acta  Neuropathol  1990;79:465-72. 


11/LENP/DIR 


PHSbWOIRt.    1  84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOINS  02803-01  LENP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Mechanism  of  Latency  and  Pathogenesis  of  Herpes  Simplex  Virus  in  the  Nervous  System 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  W.J.  Mitchell,  D.V.M.,  Ph.D.         Sr.  Staff  Fellow  LENP,  NINDS 

Others:     J. R.  Martin,  M.D.  Medical  Officer  LENP,  NINDS 


COOPERATING  UNITS  Of  any) 

None 


LAB/BRANCH 

Laboratory  of  Experimental  Neuropathology 


SECTION 

Cellular  Neuropathology  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


10 


OTHER:  q2 


CHECK  APPROPRIATE  BOX(ES) 

[_]   (a)  Human  subjects  J  (b)  Human  tissues  E   (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  is  aimed  at  understanding  aspects  of  the  pathogenesis  of  herpes  simplex 
virus  (HSv)  infection  in  the  nervous  system  including  the  mechanism  by  which  HSV 
infections  are  regulated  within  neurons  during  acute,  latent,  and  reactivated 
infections.  These  studies  which  utilize  a  mouse  model  system  and  cultured  cells  should 
allow  a  better  understanding  of  the  pathogenesis  of  herpesvirus-induced  disease  in 
humans. 

During  FY  1990  an  HSV-2  RNA  transcript  which  is  expressed  in  neurons  during  latent 
infection  has  been  characterized;  this  transcript  is  similar  but  not  identical  to  the 
latency  associated  transcript  of  HSV-1.  Further  studies  are  in  progress  to  define  the 
differences  between  the  latency  associated  transcripts  of  HSV-1  and  HSV-2  Despite 
numerous  studies  the  role  of  these  viral  latency  associated  transcripts  in  regulation  of 
HSV  infections  is  not  clear  Studies  are  in  progress  to  define  the  characteristics  of  latent 
HSV-1  infection  in  non-neuronal  tissue.  Experiments  have  been  initiated  to  define  a 
model  for  investigating  the  role  of  nerve  growth  factor  and  other  host  gene  products 
in  the  regulation  of  viral  gene  expression  during  latent  infections  of  neurons. 


12/LENP/DIR 


PHS  6040  (Rev  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOI  NS  02804  01  LENP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJ  E  CT  (80  characters  or  less,   title  must  fit  on  one  line  between  the  borders.) 

Nervous  System  Regeneration  in  a  Herpesvirus  Model 


PRINCIPAL  INVESTIGATOR  (Ust  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  D.B.Henken.Ph.D.  Staff  Fellow  LENP,  NINDS 

Others:     J. R.  Martin,  M.D.  Medical  Officer  LENP,  NINDS 


COOPERATING  UNITS  vfmy) 

None 


LAB/BRANCH 

Laboratory  of  Experimental  Neuropathology 


SECTION 

Cellular  Neuropathology  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN-YEARS:  ? 


PROFESSIONAL:  1  q 


OTHER:  q  2 


CHECK  APPROPRIATE  BOX(ES) 

|_J   (a)  Human  subjects  ]  (b)  Human  tissues  l~x~l  (0  Neither 

]  (a1)  Minors 

]  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

One  of  the  distinguishing  features  of  alpha  herpesviruses  is  their  propensity  to  infect 
and  then  establish  latency  in  sensory  ganglion  cells.  The  initial  portion  of  this  project 
aims  at  determining  whether  there  are  specific  subpopulations  of  dorsal  root  ganglion 
(DRG)  neurons,  as  characterized  by  their  morphology,  connections  and  neuropeptide 
content,  that  initially  become  infected  following  herpes  simplex  virus  type-2  (HSV-2) 
inoculation  and  later  harbor  latent  virus.  Major  findings  are:  1)  Route  of  inoculation 
plays  an  important  role  in  determining  which  populations  of  DRG  neurons  are  acutely 
infected.  Peripheral  inoculation  establishes  HSV-2  infection  in  the  small  subpopulation, 
while  intraneural  injection  targets  a  slightly  broader  spectrum  of  cell  sizes.  2) 
Modification  of  host  cell  peptide  production  is  not  detectable  with 
immunocytochemical  methods  during  the  acute  phase  of  infection.  In  situ 
hybridization  histochemistry  for  peptide  mRNA  would  be  a  more  sensitive  indicator 
and  will  be  examined.  3)  HSV-2  strains  differing  in  virulence  affect  the  numbers  of  DRG 
cells  that  express  viral  antigen,  but  not  the  specific  subpopulation.  The  effects  of  HSV-2 
inoculation  route  and  virus  strain  on  the  latent  phase  of  DRG  infection  will  next  be 
examined.  These  experiments  lay  the  groundwork  for  the  interpretation  of 
subseguent  central  and  peripheral  nervous  system  regeneration  studies  in  a  herpesvirus 
model. 


13/LENP/DIR 


PHSMWOIRev   1  84| 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOINS  02807-01  LENP 


PERIOD  COVERED 

October!,  1989  through  September  30, 1990 


TITLE  OF  PROJECT  (80  characters  or  leu    Title  must  fit  on  one  fine  between  the  borders.) 

JC  Human  Polyomavirus  Infection  and  Tumor  Induction  in  the  Neonatal  Brain 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI: 
Others: 


H.G.  Ressetar,  Ph.D. 
G.L  Stoner,  Ph.D. 
H.deF.  Webster,  M.D. 


Staff  Fellow 
Section  Chief 
Chief 


LENP,  NINDS 
LENP,  NINDS 
LENP,  NINDS 


COOPERATING  UNITS  (it  any) 

Dept.  Medical  Microbiology,  University  of  Wisconsin  Medical  School  (D.L.  Walker);  Lab. 
of  Molecular  Oncology,  Alton  Ochsner  Med.  Foundation,  New  Orleans,  LA  (O.  Prakash) 


LAB/BRANCH 

Laboratory  of  Experimental  Neuropathology 


SECTION 

Neurotoxicoloqy  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN-YEARS: 


1.0 


PROFESSIONAL: 


1.0 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

J   (a)  Human  subjects 
]  (a1)  Minors 

J   (a2)  Interviews 


]  (b)  Human  tissues  H   (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  goal  of  this  project  is  to  examine  the  possible  role  of  the  human  polyomavirus,  JC 
virus  (JCV)  in  perinatal  brain  infection.  Childhood  acquisition  of  JCV  is  followed  by 
viral  latency  and  reactivation  of  JCV  in  the  kidney  with  shedding  of  virus  occurring 
during  pregnancy  and  the  postpartum  period.  When  injected  intracerebral^  into 
newborn  hamsters,  JCV  establishes  a  nonproductive  brain  infection  with  subsequent 
tumor  formation.  The  implications  of  this  for  human  perinatal  infection  are  being 
explored  through  three  main  projects:  a)  Studies  utilizing  the  hamster  model  have 
revealed  that  JCV  preferentially  infects  mitotic  cells  in  the  subventricular  zone  and  sites 
of  postnatal  granule  neuron  production  in  the  developing  brain.  Further  investigation 
has  suggested  that  JCV  infection  of  blood  vessel  endothelial  cells  and  local  astrogliosis 
may  be  involved  in  the  mechanisms  of  tumor  induction  Tumors  induced  in  6  -month- 
old  hamsters  exhibit  variable  expression  of  JCV  early  protein  product,  T-antigen, 
emphasizing  that  the  virus  is  not  readily  detected  in  the  cells  it  transforms,  b) 
Preliminary  studies  examining  human  pediatric  tumor  tissue  for  the  presence  of  JCV  or 
its  T-antigen  are  being  performed  using  immunostaining  and  PCR  techniques,  c)  In 
collaboration  with  Dr.  Prakash,  4  transgenic  mice  have  been  produced  that  carry  the 
hybrid  regulatory  and  coding  early  regions  from  JCV  and  SV40.  Observed  pathology 
included  an  abdominal  lymphoma  and  adrenal  tumor  in  one  mouse,  a  choroid  plexus 
papilloma  in  a  second  mouse  and  a  brainstem  tumor  in  a  third  animal.  Splenic,  thymic 
and  epidermal  abnormalities  were  also  noted. 


14/LENP/DIR 


PHS  6040  (Rev  VS4) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOI  NS  02808-01  LENP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less,   rule  must  fit  on  one  line  between  the  borders.) 

The  Role  of  Protein  Kinase  C  and  raf  Protein  Kinase  in  Cellular  Functions 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  S.  Komoly,  M.D. 

Others:  Z.  Olah,  Ph.D. 

D.VAgoston.M.D. 

E.  Mezey,  M.D. 

K.-F.  J.  Chan,  Ph.D. 

Y.  Liu,  M.D. 

H.deF.  Webster,  M.D. 


Visiting  Scientist 
Visiting  Fellow 
Visiting  Scientist 
Visiting  Scientist 
Sr.  Staff  Fellow 
Visiting  Fellow 
Chief  " 


LENP,  NINDS 
LCO,  NCI 
LCB,  NIMH 
LCB,  NIMH 
LENP,  NINDS 
LENP,  NINDS 
LENP,  NINDS 


COOPERATING  UNITS  (,fan,) 

None 


LAB/BRANCH 

Laboratory  of  Experimental  Neuropathology 


SECTION 

Neurotoxicoloqy  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN  YEARS: 


1.2 


PROFESSIONAL: 


1.2 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

I      |   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


Q  (b)  Human  tissues  [*J   (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  goal  of  this  project  is  to  study  the  role  of  protein  kinases  in  cellular  metabolism  and 
signaling  using  immunocytochemical  methods  along  with  biochemical  techniques. 
Current  studies  and  major  findings  are:  1)  We  showed  for  the  first  time  that  protein 
kinase  C  isozymes  type  II  (beta)  and  type  III  (alpha)  but  not  type  I  (gamma)  can  be  found 
in  rat  optic  nerve.  Type  II  (beta)  is  localized  in  axons,  while  type  III  (alpha)  is  expressed 
exclusively  by  the  astrocytes.  Accordingly,  in  the  retina  type  II  (beta)  isozyme  was  found 
in  the  ganglion  cells  and  nerve  fiber  layer,  type  III  (alpha)  in  the  Muller  cells  and 
interneurons.  Intracellular^  the  protein  kinase  C-like  immunoreactivity  was  associated 
in  addition  with  cell  membranes  with  cytoskeletal  structures  such  as  glial  filaments 
(alpha-isozyme),  neurotubules  and  neurofilaments  (beta-isozyme).  2)  Comparative 
studies  revealed  that  delipidation  of  the  specimens  abolishes  the  protein  kinase  C-like, 
but  not  the  raf  protein  kinase-like  immunostaining  in  frozen  and  vibratome  sections. 
Quantitative  image  analysis  of  immunostained  fibroblast  cultures  revealed  cell  density 
dependent  translocation  of  raf  protein  kinase  from  the  cytoplasm  into  the  nucleus. 
Protein  kinase  C  did  not  show  this  distribution.  In  contrast,  phorbol  ester  treatment 
caused  nuclear  translocation  and  down-regulation  in  a  dose-  and  time-dependent 
manner  of  protein  kinase  C  while  such  an  effect  of  phorbol  ester  was  very  limited  on 
raf  protein  kinase.  3)  Translocation  of  raf  protein  kinase  from  cytoplasm  into  the 
nucleus  was  also  observed  in  hippocampal  neurons  in  experimental  cerebral  ischemia. 
4)  In  preliminary  immunocytochemical  experiments  the  localization  of  GM1 
qanqlioside  was  examined  with  the  use  of  cholera  toxin  B-subunit.  In  the  brain  the  B- 
subunit  binding  sites  were  most  dense  in  myelinated  pathways,  while  in  tissue  cultures 
(fibroblast,  neuroblastoma,  embryonic  spinal  cord  cell  lines)  the  immunostaining 
suggested  highest  GM1  concentration  in  neuronal  cells.  An  increase  in  "B-subunit 
binding  capacity"  of  the  cellular  membranes  was  also  noted  with  increasing  cell 
density. 

15/LENP/DIR 


PHS  MHO  (Rev    1  84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOI  NS  02809-01  LENP 


PERIOD  COVERED 

Octoberl,  1989  through  September  30, 1990 


TITLE  OF  PROJECT  (80 characters  or  /ess.   Title  must  fit  on  one  line  between  the  borders.) 

Glial  Cells  in  Development  and  Viral  Disease 


PRINCIPAL  INVESTIGATOR  (list  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:  K.E.  Astrom,  M.D. 

Others:  G.L  Stoner,  Ph.D. 

H.deF.  Webster,  M.D. 


Visiting  Scientist 
Section  Chief 
Chief 


LENP,  NINDS 
LENP,  NINDS 
LENP,  NINDS 


COOPERATING  UNITS  Ofany) 

None 


LAB/BRANCH 

Laboratory  of  Experimental  Neuropathology 


SECTION 

Neurotoxicoloqy  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN-YEARS: 


1.0 


PROFESSIONAL: 


1.0 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I      |   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


QT]  (b)  Human  tissues  LJ   (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  long-range  goal  of  this  project  is  to  study  the  normal  early  development  of  glial  cell 
precursors  in  the  telencephalon  and  the  affect  on  of  glial  cells  by  virus,  notably  the  JC 
virus.  Major  findings  and  current  studies  are:  (1)  Methods  for  the  study  of  cellular 
shape,  growth  patterns  and  fine  structure  during  early  stages  of  development  were 
evaluated  and  modified  to  meet  certain  criteria.  These  methods  can  be  used  for 
electron  microscopic,  immunocytochemical  and  in  situ  hybridization  studies  of  early 
glial  and  neuronal  development.  (2)  The  neopallial  wall  and  area  choroidea  in  11-12- 
day  -old  fetal  rats  form  a  continuous,  nonstratified,  cohesive  monolayer  of  elongated, 
radially  oriented  polarized  cells.  During  the  early  development  of  telencephalon,  the 
epithelial  character  is  maintained  but  modified  locally  to  serve  various  functions.  Of 
particular  interest  is  that  the  appearance  of  cells  in  area  choroidea  is  similar  to  that  of 
polarized  cells  with  high  metabolic  activity  elsewhere  in  the  body,  e.g.,  the  kidney 
epithelium.  This  observation  supports  a  notion  that  JC  virus  may  enter  CNS  through  the 
apical  surfaces  of  cells  in  the  choroid  area  and  plexus.  (3)  The  study  of  two  preclinical 
cases  of  progressive  multifocal  leukoencephalopathy  (PML)  have  given  new 
information  on  the  early  appearance  and  progress  of  PML-lesions  in  the  brain.  Cells 
with  capsid-antigen  were  never  seen  outside  areas  of  myelin  destruction  and  sections 
stained  for  GFAP  showed  some  areas  which  contained  reactive  astrocytes  but  lacked 
destruction  of  myelin.  The  latter  finding  suggests  that  reaction  of  astrocytes  as 
manifestation  of  disease  may  exist  independently  of  myelin  destruction. 


16/LENP/DIR 


PHS  6040  (Rev.  184) 


> 

00 


> 
03 
O 
30 

> 

H 
O 
30 

-< 


n 

c 

r- 
> 

30 

— 

O 

i- 

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03 


ANNUAL  REPORT 

OCTOBER  1,  1989  THROUGH  SEPTEMBER  30,  1990 

LABORATORY  OF  MOLECULAR  BIOLOGY 

TABLE  OF  CONTENTS 

Page 

RESEARCH  SUMMARY  1 

PROJECT  REPORTS 

Differentiation  and  Regulation  of  Gene 
Expression  in  Astrocytes  and  Neurons 
Z01  NS  02677-06  5 

Neurotransmitter  Gene  Studies 

Z01  NS  02800-02  6 

Mechanisms  of  Neurotransmitter- 

Receptor  Interactions  in  Mammalian 

Neurons 
Z01  NS  02365-12  7 

Biology  of  Mammalian  Homeodomain 

Proteins 
Z01  NS  02698-05  8 

Cloning  and  Functional  Analysis  of  Genes 

Active  in  Neurogenesis 
Z01  NS  02820-01  9 


Annual  Report 

October  1,  1989  Through  September  30,  1990 

Laboratory  of  Molecular  Biology 

Basic  Neurosciences  Program/DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 

Franklin  G.  Hempel,  Ph.D.,  Acting  Chief 

The  Laboratory  of  Molecular  Biology  (LMB)  is  continuing  to  investigate  the  structure 
and  function  of  genes  that  code  for  enzymatic  or  structural  proteins  important  in 
neural  cells.  Enzymes  that  regulate  neurotransmitter  metabolism,  receptors  for 
neurotransmitters,  and  filament  proteins  are  included  in  these  ongoing  projects. 
The  Laboratory  has  concluded  research  studies  on  neural  excitatory  amino  acid 
receptors  and  the  effects  of  substrate  and  ionic  conditions  on  cell  survival,  while 
beginning  new  work  on  neurogenesis  and  homeodomain  proteins  in  Drosophila  and 
murine  models. 

1 .  Differentiation  and  regulation  of  gene  expression  in  neurons  and  astrocytes. 

The  synthesis  of  glutamine  from  glutamate  is  catalyzed  by  glutamine  synthetase 
(GS),  and  glutamine  in  turn  is  hydrolyzed  to  glutamate  and  NH4+  by  glutaminase 
(GA).  This  cycle  and  the  synthesis  of  GABA  from  glutamate  play  a  pivotal  role  in 
neural  function.  The  LMB  has  investigated  the  appearance  of  GS  mRNA  in  rat  brain 
astrocytes  during  development  from  embyronicday  14  (E 14)  through  adult. 
Northern  blots,  analyzed  with  full-length  rat  cDNA  probes,  show  GS  mRNA  size 
classes  of  1.4  and  2.8  kb  as  early  as  day  E14.  Message  levels  reached  near-adult  by 
postnatal  day  1 5,  when  type  II  astrocytes  differentiate.  In  primary  cultures  of  rat 
cortical  astrocytes  and  C6  glioma  cell  lines,  GS  expression  was  elevated  by 
dexamethasone  and  forskolin,  and  induced  in  astrocytes  by  glutamate  or  by 
coculturing  with  glutamatergic  rat  cerebellar  granule  cells.    Neither  human 
neuroblastoma  cells  (SKN-SH-SY5Y)  nor  PC12  cells  increased  the  GS  mRNA  pool. 

To  study  the  genomic  regions  that  regulate  GS  expression,  a  1 5  kb  fragment  of  rat 
genomic  DNA  was  cloned  from  a  Charon  4A  genomic  library.  Southern  blots  of 
restriction  digests  of  the  genomic  clone,  hybridized  with  a  lab  cDNA  clone,  iden- 
tified regions  coding  for  GS.  Seven  exons  and  a  2  kb  intron  in  the  5'  untranslated 
region  were  found.  To  determine  the  start  site  for  GS  transcription,  a  synthetic 
complimentary  oligonucleotide  was  made  for  bases  +95  to  +  1 15  in  the  first  exon. 
The  start  site  was  identified  5  bases  downstream  from  that  of  other  species.  A  TATA 
box  was  found  28  bp  upstream  from  the  start  site;  a  GC-rich  region  was  found  at  -50; 
and  at  +  65  a  CCAAT  box  was  identified. 

A  rat  brain  cDNA  library  was  constructed  in  the  pCD2  vector  and  screened  for  GA, 
using  a  500  bp  partial  cDNA  clone  for  GA.  A  4  kb  cDNA  clone  was  isolated  and  its 
identity  con-firmed  by  sequence  homology  to  the  original  GA  sequence.  This  clone 
contains  a  sequence  extending  from  a  poly  A  tail  at  the  3'  terminus  toward  the  5' 
end  for  100  bp  beyond  the  older  500  bp  clone.  The  older  GA  cDNA  clones  all 
extended  into  introns  at  their  5'  termini  as  judged  by  a  breakdown  in  the  coding 
sequence  and  a  3'  splicing  consensus  sequence.  The  new  clone  contains  divergence 
atthe  5'  end  from  that  of  the  previous  clones,  and  maintains  the  open-reading 
frame,  suggesting  that  it  is  derived  from  a  processed  message  whose  sequence 
extends  past  this  5'  intron.  By  using  the  additional  5'  sequence  past  the  intron, 
oligonucleotides  were  designed  and  used  in  a  RACE  protocol  to  generate  a  series  of 
clone-containing  sequences  from  the  5'  end  of  the  clone. 


l  -LMB/DIR 


Astrocytes  synthesize  and  secrete  only  the  B2  chain  of  laminin,  a  potent  neurite 
growth-promoting  factor  consisting  of  three  subunits:  A  chain,  B1  chain,  and  B2 
chain.  Transfection  of  cDNA  constructs  containing  gene  promoter  elements  for  B1 
and  B2  laminin  into  astrocytes  and  HepG2  cells  (which  produce  all  three  subunits  of 
laminin)  has  shown  that  laminin  subunit  synthesis  is  controlled  at  the  level  of 
transcription.  By  use  of  deletion  mutants,  the  promoter  element  of  the  B2  laminin 
gene  was  located  within  200  bp  of  the  gene. 

Astrocytes  also  exhibit  collagen  type  IV  mRNA.  Analysis  of  protein  synthesis  by 
immunoblot  and  immunoprecipitation  techniques,  and  double-labelling  immuno- 
fluorescence of  cultured  astrocytes  with  anti-GFAP  (glial  fibrillary  acidic  protein)  all 
show  expression  of  collagen  type  IV. 

GFAP,  a  major  subunit  of  intermediate  cellular  fragments,  found  exclusively  in 
astrocytes  and  used  to  identify  them,  is  expressed  by  the  gfa  gene.  Transfection  of 
cultured  cells  with  chloramphenicol  acetyltransferase  (CAT)-reporter  gene  constructs 
is  now  being  used  to  identify  cis-acting  sequences  responsible  for  cell-specific 
expression  of  the  human  gfa  gene.  A  2.2  kb  5'  fragment  has  been  found  to  drive 
expression  of  the  CAT  gene  in  the  human  glioma  cell  line  U251,  but  not  in  the 
nonglial  cell  lines  HepG2,  HeLa,  and  BHK.  Analysis  of  deletion  mutants  has  localized 
two  regions  within  the  segment  which  appear  to  interact  synergistically  to  enhance 
expression,  either  alone  supporting  little  if  any  CAT  expression.  Footprint  analysis 
shows  that  both  regions  are  strongly  bound  by  proteins.  The  distal  region  is  located 
about  1 500  bp  upstream  of  the  RNA  start  site.  Its  displays  two  closely-spaced 
footprints,  each  covering  about  30  bp  of  DNA.  The  proximal  region  is  located  about 
100  bp  upstream  of  the  RNA  start  site,  and  displays  a  single  footprint  of  about  30  bp. 
The  promoter  region  of  the  human  gfa  gene  was  found  to  contain  two  elements 
that  can  independently  specify  the  correct  transcription  startpoint.  One  of  the 
elements  is  a  TATA  box  found  25  bp  upstream  from  the  transcription  startpoint, 
while  the  other  is  located  between  10  and  50  bp  downstream  from  the  transcription 
initiation  site.  Surprisingly,  most  of  the  downstream  element  overlaps  with  the 
protein  encoding  sequence.  Binding  of  purified  transcription  factor  IID  (TFIID)  to  the 
TATA  box  was  affected  by  a  downstream  initiation  element  when  human  or  yeast 
TFIID  is  used. 

To  understand  the  genetic  mechanisms  controlling  calcium  gene  expression,  cDNA 
and  genomic  clones  for  the  calcium  channel  gene  were  obtained.  By  screening  rat 
brain  cerebral  cortex  and  hippocampus  cDNA  libraries  with  a  set  of  oligonucleotide 
probes  based  on  nucleotide  sequences  of  rabbit  skeletal  muscle  dihydropyridine 
(DHP)  receptor  a1-subunitcDNA,  the  LMB  isolated  and  sequenced  several  cDNAs 
corresponding  to  the  mRNA  encoding  a1-subunit  of  L-type  voltage-sensitive  calcium 
channel  (VSCC).  Nucleotide  sequence  analysis  of  the  cDNAsshow  at  least  four 
different  classes  of  cDNAS,  indicating  that  rat  brain  expresses  multiple  forms  of  the 
a1-subunit  of  VSCC,  probably  from  separate  calcium  channel  genes. 

Expression  in  rat  brain  of  mRNA  corresponding  to  VSSC  a1-subunit  cDNAs  was 
examined  by  in  situ  hybridization  histochemistry.  While  the  mRNA  for  one  of  the 
cDNA  clones  (BCCII)  was  widely  distributed  both  in  neurons  and  glial  cells,  the  mRNA 
for  BCCI  cDNA  was  found  only  in  brain  neurons,  such  as  the  olfactory  bulb  and 
piriform  cortex,  hippocampal  formation,  suprachiasmatic  nucleus  and  median 
preoptic  nuclei  of  the  hypothalamus,  the  pituitary  and  pineal  glands.  Northern  blot 
analysis  revealed  that  two  poly(A) +  RNA  species  of  s8  kb  and  ^1 2  kb  which 
hybridized  to  BCCI  cDNA  markedly  increased  when  intracellular  levels  of  cAMP  were 


2-LMB/DIR 


also  increased  in  NG108-15  cells.  The  BCCI  calcium  channel  gene  is  located  on  mouse 
chromosome  14  and  human  chromosome  3. 

2.  Genes  encoding  neurotransmitter  receptors. 

The  LMB  has  made  considerable  progress  in  characterizing  the  structure  and  genetic 
expression  of  acetylcholine  and  dopamine  receptors,  and  their  localization  in  the 
CNS.  Each  of  the  5  muscarinic  cholinergic  subtypes  has  been  expressed  in  CHO  and 
A9-L  cells.  In  these  cells,  muscarinic  receptors  can  be  divided  into  two  classes:  ml, 
m3,  and  m5  versus  m2  and  m4.  The  odd-numbered  receptors  increase  cAMP  levels 
and  phosphatidyl  inositol  metabolism,  and  also  open  potassium  channels  by 
coupling  to  pertussis-insensitive  G-proteins,  while  the  even-numbered  muscarinic 
receptors  decrease  cAMP  levels  by  coupling  to  pertussis  toxin-sensitive  G-proteins. 

A  region  of  strong  homology  exists  within  an  18-amino  acid  intracellular  stretch  of 
the  receptors  that  is  predictive  of  their  coupling  selectivity  of  the  odd-  versus  even- 
numbered  muscarinic  receptors.  In  chimeric  m2-m3  receptors,  this  small  domain  is 
sufficient  to  reverse  the  coupling  selectivity  of  m2  and  m3  receptors  with  respect  to 
inositol  phosphate  metabolism  as  well  as  cAMP  metabolism  and  coupling  to  G- 
protems.  The  genetic  identity  of  the  individual  G-proteins  that  mediate  functional 
responses  of  the  muscarinic  receptors  was  shown  by  coexpression  of  individual 
receptor  cDNAs  with  individual  G-protein  cDNAs.  For  m4  receptors,  coexpression  of 
the  receptor  cDNA  with  the  G-protein  Go  inhibits  adenylate  cyclase  more  than  m4 
receptors  expressed  alone. 

From  a  previously  published  dopamine  D2  receptor  sequence,  the  LMB  has  prepared 
oligodeoxynucleotide  probes  and  applied  in  situ  hybridization  procedures  to  map 
the  distribution  of  this  mRNA.  It  was  shown  that  this  mRNA  encodes  both  pre-  and 
postsynaptic  dopamine  D2  receptors  in  three  major  ascending  dopaminergic 
pathways:  the  nigrostriatal,  mesolimbicand  mesocortical  pathways.  D2  mRNA  is 
also  located  on  various  cholinergic  cell  groups  in  the  forebrain.  Using  the  same 
probes,  the  laboratory  found  that  these  mRNAs  are  present  only  in  the  inner  retina 
and  not  in  the  photoreceptors  in  rats.  A  similar  distribution  was  seem  in  monkey 
retina. 

J.  Boulter  (Salk  Institute)  supplied  the  laboratory  with  cDNAs  that  encode  for  the 
a2,  a3,   a4,and    82  subunits  of  the  neuronal  nicotinic  receptors.  These  cDNAs  were 
subcloned  into  the  expression  vector  pCD-PS,  previously  used  to  express  the 
muscarinic  acetylcholine  receptors  and  G-proteins  in  mammalian  cells.  If  the   a3  and 
a4  cDNAs  are  coexpressed  with  the   82  cDNA/then  membranes  are  obtained  that 
bind  acetylcholine.  Conversely,  if  the  DNAs  are  expressed  alone,  there  is  no  binding. 
These  data  indicate  that  functional  receptors  of  this  type  can  be  obtained  using  our 
expression  system.  This  work  represents  the  first  expression  in  mammalian  cells  of 
neuronal  nicotinic  receptors. 

From  functional  and  pharmacologic  data,  it  can  be  predicted  that  nicotinic  acetyl- 
choline and  glutamate  receptors  may  belong  to  a  homologous  family  of  genes.  To 
clone  the  glutamate  receptors,  oligodeoxynucleotide  probes  were  assembled  for  a 
region  of  the  neuronal  nicotinic  receptors  expected  to  be  in  the  receptor  channel 
lining  of  the  neuron.  Using  this  probe,  the  same  human  retinal  library  was  screened 
as  for  the  dopamine  receptors.  Full-length  human   a2,  a  3,  a  4,    82  and  84  clones, 
and  a  human  glutamate  receptor  have  been  sequenced  and  cloned. 


3  -LMB/DIR 


3.  Neurotoxicity  of  excitatory  amino  acids. 

The  LMB  has  focused  on  the  mechanisms  of  receptor-mediated  EAA  activity  and  the 
transition  of  EAAs  from  neurotransmitters  to  neurotoxins.  Such  a  transition  has 
been  postulated  as  the  possible  cause  of  neuronal  death  in  various  neuropathology 
conditions  such  as  cerebral  ischemia,  Huntington's  disease,  and  Alzheimer's  disease. 
Results  show  that  glucose  metabolism  and  ATP  production  are  necessary  to  maintain 
the  voltage-dependent  Mg2  +  block  of  the  NMDA  receptor  channel,  and  that  relief  of 
the  block  renders  the  neurons  vulnerable  to  the  toxic  effects  of  glutamate. 
Glutamate  istoxicto  cerebellar  granule  cells  in  primary  culture  when  Mg2+  is 
removed  from  the  incubation  medium.  With  Mg2  +  present  but  glucose  removed 
from  the  medium,  glutamate  is  also  toxic  due  to  partial  depolarization  resulting 
from  energy  depletion;  the  partial  depolarization  leads  to  relief  of  the  Mg2+  block 
which  is  known  from  electrophysiologic  evidence  to  be  voltage-dependent.  When 
the  neurons  are  deprived  of  oxygen  or  when  an  inhibitor  of  oxidative  phosphory- 
lation is  added,  glutamate  is  toxic  even  in  the  presence  of  both  Mg2  +  and  glucose, 
again  due  to  energy  depletion  and  partial  depolarization.  In  every  case,  the  toxicity 
of  glutamate  is  blocked  by  CPP  ((  ±  )-3-(2-carboxypiperazine-4-yl)-propyl-1-phos- 
phonic  acid)  and  D(-)-2-amino-5-phosphonovaleric  acid  (APV),  the  most  potent  and 
selective  competitive  antagonists  of  the  NMDA  receptor.  Adrenal  corticosteroids 
can  potentiate  the  neurotoxicity  of  glutamate  and  other  agonists  at  the  NMDA 
receptor.  Pretreatment  of  neonatal  rat  granule  cells  with  low  levels  of  cortico- 
sterone  permits  low  concentrations  of  NMDA  agonists  to  become  toxic  in  the 
presence  of  Mg2+  and  glucose.  Corticosterone  appears  to  potentiate  NMDA  agonist 
toxicity  by  relieving  the  NMDA  channel  Mg2+  block  via  compromised  energy 
availability. 

4.  Cloning  and  analysis  of  genes  in  neural  development. 

The  LMB  has  generated  6  lines  of  healthy  transgenic  mice  that  harbor  in  their 
genome  between  one  and  approximately  25  copies  of  a  DNA  fragment  that  contains 
1.8  kb  of  the  Mx1  promoter,  a  Hox  1.3  cDNA,  and  a  3'  noncoding  region  derived 
from  the  mouse  beta-globin  gene.  Transgenic  mRNA  levels  are  below  detectability 
in  most  organs  (i.e.,  below  endogenous  Hox  1 .3  mRNA).  However,  when  interferon 
is  injected  as  a  single  dose  into  these  transgenic  mice,  transgenic  Hox  1 .3  mRNA  and 
protein  rapidly  accumulate  to  levels  that  far  exceed  those  of  endogenous  Hox  1 .3 
transcripts  and  protein.  The  induced  protein  accumulates  in  the  nuclei  of  cells  where 
it  can  be  labelled  with  a  specific  antibody.  Western  blot  analysis  shows  that  this 
protein  comes  in  multiple  posttranslationally  modified  forms,  much  the  same  as  the 
endogenous  Hox  1 .3  protein.  Interferon  injected  into  non-transgenic  pregnant  mice 
that  carry  in  utero  transgenic  embryos,  will  activate  the  transgene  as  early  as  day  11 
of  gestation,  again  to  levels  higherthan  their  endogenous  Hox  1.3  expression.  This 
observation  opens  up  the  possibility  of  determining  the  exact  time  window  in 
development  during  which  Hox  1 .3  expression  may  affect  embryogenesis. 


4  -LMB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02677-06  LMB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  character*  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Differentiation  and  Regulation  of  Gene  Expression  in  Astrocytes  and  Neurons 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.: 
Others: 


E.  Freese,*  Ph.D.,  Chief,  LMB,  NINDS 
M.  Brenner,  Ph.D.,  Special  Expert,  LMB 
H.  Chin,  Ph.D.,  Senior  Staff  Fellow,  LMB 
Y.  Nakatani,  Ph.D.,  Visiting  Associate,  LMB 
J.  Mill,  Ph.D.,  Senior  Staff  Fellow,  LMB 
J.  Wujek,  Ph.D.,  Senior  Staff  Fellow,  LMB 


R.King,  Ph.D.,  IRTA,  LMB 
K.  Mearow,  Ph.D.,  Visiting  Fellow,  LMB 
H.  Purohit,  Ph.D.,  Visiting  Fellow,  LMB 
F.  Besnard,  Ph.D.,  Visiting  Fellow,  LMB 
V.  Kedar,  Ph  D.,  Visiting  Associate,  LMB 


COOPERATING  UNITS  Of  any) 

Dr.  Y.  Yamada,  LDBA,  NIDR,  NIH 
Dr.  R.  Wenthold,  LMO,  NIDCD,  NIH 


Dr.  M  Nirenberg,  LBG,  NHLBI,  NIH 


LAB/BRANCH 

Laboratory  of  Molecular  Biology,  BNP 


SECTION 

Developmental  Biology  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


8.3 


PROFESSIONAL: 


76 


OTHER: 


0.7 


CHECK  APPROPRIATE  BOX(ES) 

J  (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  0  (0  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  genes  for  a  number  of  neurologically  important  proteins  have  been  isolated  and  sequenced,  and 
their  control  mechanisms  are  being  studied.  In  particular,  we  want  to  understand  why  certain  genes  are 
expressed  in  neurons  but  not  in  astrocytes  and  vice  versa.  Laminin  is  a  component  of  the  extracellular 
matrix  of  astrocytes  and  promotes  neurite  outgrowth.  In  basal  lamina  cells  laminin  normally  consists  of  3 
chains  (A,  B1,  B2).  Using  chain-specific  antibodies  and  small  cDNAs,  we  have  shown  that  astrocytes  make 
only  the  B2  mRNA  and  protein  of  laminin.  The  promoter  region  responsible  for  this  specificity  has  been 
localized  within  200  bp  of  the  B2  gene.  Glial  fibrillary  acidic  protein  (GFAP)  is  an  intermediate  filament 
protein  found  only  in  mature  astrocytes.  It  is  transcribed  by  RNA  polymerase  II  but,  in  contrast  to  other 
genes,  uses  as  promoter  not  only  a  TATA  box  25  bp  upstream  of  the  start  site  but  also  a  downstream 
element  10  to  50  bp  distant.  Glutamine  synthetase  (GS)  converts  glutamate  to  glutamine  which  then 
enters  neurons  and  is  converted  to  glutamate  or  GABA.  The  promoter  for  the  GS  gene  contains  a  TATA 
box,  28  bp  upstream  from  the  transcription  start  site.  Sequences  with  homology  to  cAMP  and 
glucocorticoid  response  elements  have  also  been  found.  Deletion  mutants  with  the  GS  promoter  and  a 
CAT  reporter  gene  have  been  constructed  and  transfected  into  several  cell  lines  in  order  to  examine  the 
functional  significance  of  the  regulatory  sequences.  Genomic  clones  and  cDNAs  for  the  L-type  calcium 
channel  gene  have  been  obtained.  At  least  4  forms  of  the  a1  subunit  of  the  channel  are  expressed. 
Hybridization  experiments  reveal  high  levels  of  neuron-specific  calcium  channel  mRNA  in  the  olfactory 
bulb,  hippocampal  CA1  cells,  dentate  gyrus,  suprachiasmatic  nucleus,  and  the  medial  preoptic  nucleus. 
Other  sites  also  expressed  activity.  Work  is  continuing  toward  isolating  a  human  calcium  gene. 

*Dr.  E.  Freese  died  on  March  30,  1990. 


5-LMB/DIR 


PHSMMOtRcv.  I'M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02800-02  LMB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (SO  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Neurotransmitter  Gene  Studies 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.:  M.R.  Brann,  Ph.D.  Senior  Staff  Fellow  LMB,  NINDS 

Others:  D.  Weiner  HHMI  Fellow  LMB,  NINDS 

J.Wess,  PhD  Visisting  Associate  LMB,  NINDS 

B.  Novotny,  M.S.  Physiologist  LMB,  NINDS 

A.  Levey,  M.D.,  Ph.D.  Special  Volunteer  LMB,  NINDS 

D.  Gdula,  B.S.  Biologist  LMB,  NINDS 

S.H.Yu,  M.D.  Visiting  Associate  LMB,  NINDS 


COOPERATING  UNITS  of  any) 

S.  Jones,  J.L  Barker,  LNP,  NINDS;  W.  Simonds,  A.  Spiegel,  MPB,  NIDDK;  H.  Arnheiter,  LMG,  NINDS;  T. 
Bonner,  LCB,  NIMH;  J.  Ellis,  Univ.  VT;  S.  Jones,  N.  Nash,  T.  Stormann,  R-Gene,  CRADA 


LAB/BRANCH 

Laboratory  of  Molecular  Biology,  BNP 


SECTION 

Developmental  Biology  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 

4.7 


PROFESSIONAL:  j  7 


OTHER:  2  0 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  [T]  (b)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

_J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Our  work  involves  the  study  of  genes  encoding  receptors  that  bind  the  neurotransmitters  acetylcholine, 
dopamine,  and  qlutamate.   Previous  work  led  to  the  molecular  cloning,  sequencing  and  expression  of  a 


family  of  muscarinic  acetylcholine  receptor  subtype  genes.  We  have  continued  to  characterize  these 
subtypes  pharmacologically  with,  regard  to  agonists  and  antagonists,  anatomic  distribution,  their 
selectivity  of  coupling  to  G-proteins,  second  messengers  and  ion  channels,  and  the  development  of 
antibodies  specific  for  each  protein  subtype.  Using  the  recently  reported  sequence  of  a  rat  cDNA  that 
encodes  a  dopamine  D2  receptor,  we  have  mapped  the  distribution  of  the  corresponding  mRNA  in  rat 
retina  and  brain,  and  have  cloned,  sequenced  and  expressed  a  cDNA  that  encodes  the  human  homolog 
of  this  receptor  and  likewise  a  human  dopamine  D1  receptor.  This  work  has  provided  evidence  that 
dopamine  receptors,  like  muscarinic  receptors,  may  be  derived  from  a  family  of  genes.  We  are 
attempting  to  clone  the  other  members  of  the  family,  and  to  study  the  selectivity  of  coupling  of  the 
cloned  dopamine  receptors  to  G-proteins,  second  messengers,  and  ion  channels.  Finally,  sequences  of 
the  rat  cDNAs  that  encode  neuronal  nicotinic  acetylcholine  receptors  have  been  reported,  and  we  have 
cloned  some  of  the  human  homologs  of  these  receptors.  Because  of  a  hypothetical  evolutionary 
similarity  between  glutamate  and  nicotinic  acetylcholine  receptors,  we  have  been  attempting  to  clone 
the  qlutamate  receptors  by  homology  cloning.  As  a  result,  we  recently  cloned,  sequenced  and  expressed 
the  human  homolog  of  the  rat  glutamate  receptor. 


6-LMB/DIR 


PHS6M0(R»w.  1/MI 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02365-12  LMB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  loss.   Title  must  fit  on  one  line  between  the  borders.) 

Mechanisms  of  Neurotransmitter-Receptor  Interactions  in  Mammalian  Neurons 


PRINCIPAL  INVESTIGATOR  (I 'it  other  professional  per  sonne/  below  the  Principal  Investigator.)  {Name,  title,  laboratory,  and  institute  affiliation) 

P. I.:  R.C.  Henneberry,  Ph.D.  Chief,  Molecular  Neurobiology  Section         LMB,  NINDS 

Others:  P.G.  Lysko,  Ph.D.  Guest  Researcher 

J  A.  Cox,  Ph.D.  Visiting  Associate 

M.  Voigt,  Ph.D.  NRC  Fellow 


COOPERATING  UNITS  of  any) 

Enzyme  Chemistry  Section,  Laboratory  of  Neurochemistry,  BNP,  DIR,  NINDS 


LAB/BRANCH 

Laboratory  of  Molecular  Biology 


SECTION 

Molecular  Neurobiology  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  .   ■, 


OTHER: 


06 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [x~l  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  mechanisms  by  which  neurotransmitters  become  neurotoxins  and  the  role  of  these  toxins  in  the 
death  of  neurons  characteristic  of  neurodegenerative  disorders  such  as  Alzheimer's  disease, 
Huntington's  disease,  Parkinson's  disease,  lathyrism,  etc.  have  been  the  focus  of  our  studies  for  the  last 
five  years.  We  have  shown  that  cerebellar  neurons  cultured  from  neonatal  rats  express  several  subtypes 
of  glutamate  receptor,  including  the  N-methyl-D-aspartate  (NMDA)  receptor.  When  this  receptor  is 
occupied  by  an  appropriate  agonist,  a  receptor-gated  channel  opens,  permitting  sodium  and  calcium 
influx.  However,  in  the  healthy  brain  this  channel  is  normally  blocked  by  magnesium  in  a  voltage- 
dependent  manner,  i.e.,  magnesium  prevents  ion  influx  through  the  channel  at  normal  membrane 
potential.  Under  physiological  conditions,  the  NMDA  channel  may  only  permit  ion  flow  in  response  to 
high-frequency  stimulation.  We  have  shown  that  the  magnesium  block  is  relieved  when  neurons 
partially  depolarize  in  response  to  reduced  energy  levels  in  the  neuron;  decreases  in  adenine  nucleotide 
levels  due  to  glucose  starvation,  oxygen  deprivation,  or  metabolic  poisons  cause  sufficient 
depolarization  to  relieve  the  magnesium  block  of  the  channel.  Thus,  when  neuronal  energy  levels  are 
compromised,  endogenous  agonists  such  as  glutamate  can  persistently  open  the  NMDA  channel 
resulting  in  excess  ion  influx;  the  increased  energy  demands  by  the  pumps  involved  in  maintaining  ion 
gradients  cannot  be  met  in  the  energy-poor  neurons,  and  neuronal  death  ensues  via  a  mechanism  not 
yet  understood. 

Our  results  provide  experimental  evidence  for  a  mechanism  which  may  trigger  the  transition  of 
endogenous  glutamate  from  neurotransmitter  to  neurotoxin;  this  mechanism  may  have  important 
implications  for  a  variety  of  neurodegenerative  disorders. 

Due  to  the  retirement  of  the  principal  investigator  from  the  USPHS  this  project  was  terminated  1 
February  1990. 

7-LMB/DIR 


PHS  6040  (R»v.  1  M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02698-05  LMB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Biology  of  Mammalian  Homeodomain  Proteins* 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.:  Ward  F.  Odenwald,  Ph.D.  Staff  Fellow  LMB,  NINDS 

Others:  Shang-Ding  Zang,  M.D.  Visiting  Fellow         LMB,  NINDS 


COOPERATING  UNITS  (if  any) 

Heinz  Arnheiter,  Visiting  Scientist,  LVMP,  NINDS 


LAB/BRANCH 

Laboratory  of  Molecular  Biology,  BNP 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  -  ?[- 


PROFESSIONAL:  q  jc 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  (b)  Human  tissues  [x~|  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Most  of  the  proteins  whose  dysfunction  disturbs  embryogenesis  of  the  fly  share  a  characteristic  stretch  of 
60  amino  acids  called  the  homeodomain.  Mammals  likewise  express  homeodomain-containing  proteins. 
Based  on  their  nuclear  localization,  their  capacity  to  bind  to  specific  DNA  sequences,  and  their  relation  to 
some  well-characterized  transcription  factors,  it  is  suggested  that  homeodomain  proteins  are  involved  in 
regulating  gene  expression  in  mammals.  In  our  effort  to  determine  the  function  of  mammalian 
homeodomain  proteins,  we  have  cloned,  sequenced,  and  studied  the  expression  of  one  of  the  murine 
Antennapedia  class  genes  known  as  Hox  1.3. 

To  test  in  vivo  biofunction,  we  have  recently  generated  transgenic  mice  that  contain  either  a  single  or 
multiple  (up  to  25)  copies  of  an  inducible  Hox  1.3  transgene.  To  control  the  levels  of  ectopic  Hox  1.3 
expression,  we  have  used  the  inducible  mouse  Mx1  regulatory  element  as  our  transgene  promoter  which 
allows  the  deliberate  ubiquitous  expression  of  Hox  1.3  in  the  presence  of  its  inducers  (interferon  or 
double-stranded  RNA).  Under  physiologic  conditions,  that  is,  in  the  absence  of  induction,  the  mice 
express  their  endogenous  Hox  1.3  gene,  and  in  those  organs  tested  by  Northern  analysis,  little  or  no 
detectable  transgene  RNA  was  found.  In  the  absence  of  transgene  induction,  these  mice  appear  healthy. 
After  transgene  induction  with  either  interferon  or  double-stranded  RNA,  the  mice  start  to  express  high 
levels  of  transgenic  RNA  and  protein  in  many  different  organs.  In  addition,  intravenous  injection  of 
interferon  into  non-transgenic  females  that  carry  in  utero  transgenic  embryos  results  in  the  fetal 
induction  of  our  transgene.  Our  primary  goal  is  to  first  identify  if  and  when  during  embryonic  or  post- 
natal development  does  ectopic  expression  of  Hox  1.3  have  an  effect,  and  then  to  use  this  in  vivo  tool  to 
identify  endogenous  genes  that  are  either  up-  or  down-regulated  by  Hox  1.3. 

*Formerly  in  LVMP  Transferred  on  October  1,  1989. 


PHS  6O40  (Rev.  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02820-01  LMB 


PERIOD  COVERED 

October  1 ,  1 989  through  September  30,  1 990 


TITLE  OF  PROJECT  (80  characters  or  lets.   Title  must  fit  on  on©  line  between  the  borders.) 

Cloning  and  Functional  Analysis  of  Genes  Active  in  Neurogenesis 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.:  WardF.  Odenwald,  Ph.D.  Staff  Fellow  LMB,  NINDS 

Others:  Dervla  Mellerick-Dressler,  Ph.D.        Special  Volunteer    LMB,  NINDS 


COOPERATING  UNITS  Of  any) 

Judith  Kassis,  CBER,  DBB 


LAB/BRANCH 

Laboratory  of  Molecular  Biology,  BNP 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  - 


PROFESSIONAL:  Q  r 


OTHER: 


0.25 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  (b)  Human  tissues  [x~|  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  mechanisms  that  underlie  the  orderly  sequential  changes  in  gene  expression 
during  neurogenesis  are  unknown.  Establishment  of  form  and  pattern  within  the 
nervous  system  is  dependent  on  cellular  interactions  that  are  initiated  early  in 
development.  These  communications  regulate  cell  proliferation,  differentiation, 
neuroblast  migration,  axonal  growth  and  guidance,  target  recognition,  and, 
ultimately  synapse  formation.  The  molecular  machinery  that  relays  this  positional 
information  is  the  foundation  upon  which  neuronal  diversity  and  function  is  based. 
How  the  nervous  system  is  "wired"  during  embryogenesis  remains  a  fundamental  issue 
in  molecular  biology  today. 

We  have  initiated  our  search  for  novel  genes  involved  in  these  processes  by  identifying 
and  characterizing  genes  involved  in  the  neurogenesis  of  the  fruit  fly,  Drosophila 
melanogaster.  The  fly  was  selected  because  of  its  relatively  simple  nervous  system,  the 
availability  of  developmental  mutants,  and  the  possibility  of  genetic  analysis  of 
function.  Thus  far  we  have  identified  several  candidate  genes  and  are  focusing  our 
efforts  on  one  that  is  expressed  in  central  nervous  system  neuroblasts  during 
development.  This  gene  and  the  other  candidates  were  identified  by  the  enhancer 
detection  method  of  transposon  tagging  in  transgenic  flies.  Our  goal  is  to  use  the 
knowledge  gained  from  the  molecular  analysis  of  these  genes,  to  build  the  appropriate 
tools  to  search  for  mammalian  genes  that  carry  out  related  functions. 


9-LMB/DIR 


PHS6O40(R«v.  1/84) 


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co 


> 

CD 

O 

30 

> 

o 

30 

■< 


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30 
> 

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ANNUAL  REPORT 
OCTOBER  1,  1989  THROUGH  SEPTEMBER  30,  1990 

LABORATORY  OF  VIRAL  AND  MOLECULAR  PATHOGENESIS 
TABLE  OF  CONTENTS 

Page 

RESEARCH  SUMMARY  1 

PROJECT  REPORTS 

Biology  of  Myelin-Forming  Cells  In  Vitro  5 

and  In  Vivo  Including  Remyelination 
Z01   NS  02034-18  LVMP 

Regulation  of  Myelin  Synthesis  6 

Z01   NS  02528-09  LVMP 

Biology  of  Mammalian  Homeodomain  Proteins  7 

Z01   NS  02698-05  LVMP 

Mechanisms  of  Viral  Pathogenesis  8 

Z01   NS  02742-04  LVMP 

Neurotropism  of  Human  Retroviruses  9 

Z01   NS  02789-02  LVMP 

Expression  of  Viral  Proteins  in  Transgenic  Mice  10 

Z01   NS  02790-02  LVMP 

Molecular  Biology  of  Human  Neurotropic  Virus  11 

Infections,  HIV-1  and  JCV 
Z01   NS  01983-19  LVMP 

Replication  and  Pathogenesis  of  Enveloped  Viruses  12 

Z01   NS  02791-02  LVMP 

Neurologic  and  Systemic  Manifestations  of  Retrovirus  13 

and  Autoimmune  Mediated  Diseases 
Z01    NS  02756-03 

Pseudotypic  Defective  Interfering  HIV  Particles  as  14 

an  Antiviral  Therapy  for  AIDS 
Z01   NS  02818-01    LVMP 


ANNUAL  REPORT 

October  1,  1989  through  September  30,  1990 

Laboratory  of  Viral  and  Molecular  Pathogenesis 

National  Institute  of  Neurological  Disorders  and  Stroke 

Monique  Dubois-Dalcq  ,  M.  D.,  Acting  Chief 

The  Laboratory  of  Viral  and  Molecular  Pathogenesis  (LVMP)  was  established  2  years  ago.    It 
investigates  the  viral  and  molecular  bases  of  nervous  system  diseases  using  the  most  advanced 
techniques  of  molecular  and  cell  biology.   The  laboratory  studies  the  cell  and  molecular  biology 
of  neural  cells  in  vitro  and  in  vivo,,  characterizes  mutations  in  myelin  genes  and  studies  the 
cells  involved  in  myelin  repair  in  mice  and  men.    An  important  goal  of  LVMP  is  to  elucidate 
mechanisms  of  nerve  cell  dysfunction  in  human  diseases  caused  by  RNA  and  DNA  viruses, 
especially  human  retroviruses  (HIV,  HTLV-1)  and  other  viruses  associated  with  them,  such  as 
JC  papova  virus  and  herpes  virus.   The  mechanisms  of  demyelination  in  acquired 
immunodeficiency  syndrome,  tropical  spastic  paraparesis,  multiple  sclerosis,  and  progressive 
multifocal  leukoencephalopathy  are  being  studied.    The  laboratory  attempts  to  elucidate  how 
human  neurotropic  viruses  get  to  the  brain  and  which  viral  and  host  factors  determine  the  final 
outcome  of  infection.    Long-term  goals  are  to  find  ways  to  stop  virus  entry  in  nerve  cells,  to 
inhibit  viral  cytopathogenesis  and  persistence,  and  enhance  nervous  system  repair.    The  new 
laboratory  gathers  scientists  with  expertise  in  the  cellular  and  molecular  biology  of  glial  cells, 
and  others  with  expertise  in  the  molecular  biology  and  pathogenesis  of  neurotropic  viruses.    It 
has  maintained  the  breadth  of  expertise  in  cellular  and  molecular  biology  which  was 
characteristic  of  the  Laboratory  of  Molecular  Genetics  and  there  is  an  ongoing  discussion  of  the 
cellular  and  molecular  aspects  of  biological  phenomena  between  the  different  members  of  the 
laboratory.    In  addition,  the  laboratory  has  an  active  transgenic  mice  facility  which  has  started 
several  collaborations  with  other  NINDS  and  NIH  laboratories. 

Advances  made  this  year  in  the  area  of  neural  and  molecular  biology  of  glial  cells  and  in  the 
transgenic  mice  strategy  to  analyze  the  role  of  homeobox  containing  proteins  in  mammalian 
development  and  disease  are  described  first.     The  Studies  on  viral  pathogenesis  [involving 
viruses  other  than  HIV]  are  then  reviewed,  followed  by  a  summary  of  the  laboratory's 
approaches  to  understanding  HIV  neurotropism  and  to  designing  strategies  to  prevent  virus 
spreading. 

MOLECULAR  PATHOGENESIS 

We  have  progressed  in  our  studies  of  the  oligodendrocyte-type  2  astrocyte(0-2A)  lineage 
during  development  and  remyelination.  (Dubois-Dalcq  et  al).  First  we  showed  that  FGF  can 
modulate  the  PDGF-driven  pathway  of  oligodendrocyte  development  in  the  rat  by  up-regulating 
the  PDGF  receptor  on  0-2A  progenitor  cells.  This  receptor  is  in  fact  highly  expressed  in  rat 
brain  in  the  postnatal  period  preceding  myelination.  Second,  we  demonstrated  in  vitro  the 
directed  migration  of  these  progenitors  to  PDGF  but  not  to  FGF.   Third,  we  showed  that  0-2A 
progenitor  cells  can  be  readily  isolated  from  mice  CNS  tissue  in  remyelination  and  that  all  cells 
of  the  0-2A  lineage  can  divide  in  response  to  the  disease  process.   Moreover,  we  found  evidence 
that  the  adult  human  nervous  system  may  harbor  a  "resting"  oligodendrocyte  precursor,    an 
observation  which  bears  on  the  regeneration  potential  of  the  human  CNS  in  demyelinating 
diseases  like  Multiple  Sclerosis. 


1-LVMP/DIR 


In  our  studies  of  transcriptional  control  of  myelination  (Hudson  et  al),  we  have  delimited  a 
region  of  310  bp  in  the  PLP  promoter  which  regulates  the  expression  of  this  major  myelin  gene 
in  both  rat  and  human  glial  cells.    Four  human  DNA-binding  proteins  that  recognize  portions  of 
this  regulatory  region  have  been  isolated  from  expression  libraries.    One  of  the  DNA-binding 
proteins  which  recognizes  a  sequence  motif  present  in  PLP  as  well  as  in  other  myelin  genes, 
could  potentially  form  a  leucine  zipper  structure.     Our  continued  analysis  of  PLP  mutations, 
which  was  extended  to  identify  a  novel  mutation  in  the  dog  (the  shaking  pup),  has  revealed  that 
this  class  of  mutations  disturbs  oligodendrocyte  development.    In  preparation  for  screening 
individuals  in  families  with  the  dysmyelinating  disease,  Pelizaeus-Merzbacher,  we  have 
devised  a  strategy  for  rapidly  automated  sequencing  of  the  human  PLP  gene.  We  have  already 
received  over  30  inquiries  and  hope  that  our  request  for  special  NINDS  support  in  this  area 
will  soon  be  approved. 

In  our  collaborative  studies  on  the  role  of  homeobox  genes  in  mammalian  development 
(Arnheiter  et  al  ),  we  have  obtained  7  lines  of  transgenic  mice  expressing  the  Hox  1 .3  gene 
under  the  interferon  inducible  Mx1  promoter.  We  demonstrated  induction  of  mRNA  and  protein 
after  a  single  injection  of  interferon  in  adult  mice,  and  recently,  induction  was  obtained  at  11 
days  of  gestation.   Thus,  we  are  now  in  a  position  to  determine  the  exact  time  during  which 
Hox  1.3  can  affect  embryogenesis  and  to  identify,  in  this  induction  system,  potential  genes 
which  are  regulated  by  Hox  1.3.    In  the  course  of  our  studies  of  transgenic  animals  we  have 
come  across  an  insertional  mutant.    Mice  homozygous  for  this  insertion  show  hemivertebrae, 
spina  bifida  occulta,  fusions  of  vertebral  bodies  and  short  kinky  tails.  These  features  are 
reminiscent  of  those  seen  in  the  natural  mutation,  undulated,  alleles  of  which  have  alterations 
in  the  coding  region  of  the  Pax1  gene.  We  are  now  beginning  the  molecular  characterization  of 
the  gene  whose  function  is  disturbed  in  the  transgenic  mice.    Once  identified,  we  will  then 
determine  whether  patients  suffering  from  hereditary  malformations  of  their  vertebral  column 
also  have  mutations  in  the  corresponding  human  gene. 

VIRAL  PATHOGENESIS 

A.  RNA  and  DNA  neurotropic  viruses  other  than  HIV 

1.    Intracellular  immunization  in  vivo  (Arnheiter  et  al).  One  of  the  ultimate  goals  of 
studying  viral  pathogenesis  is  to  find  ways  to  protect  organisms  against  viral  infections.  Here 
we  report  for  the  first  time  that  through  germ  line  transformation  we  have  converted  a  mouse 
from  virus-susceptible  to  highly  resistant  to  a  virulent  infection,  influenza.    To  achieve 
resistance,  we  generated  transgenic  mice  that  express  a  potent  intracellular  anti-influenza 
protein,  Mx1,  under  control  of  the  virus/interferon-inducible  Mx1   promoter.  We  have 
obtained  several  lines  of  transgenic  mice  that  differ  in  the  level  at  which  virus  infection  can 
stimulate  Mx1  synthesis.    In  high  responder  lines,  high  levels  of  Mx1  are  induced  upon 
replication  at  the  sites  of  initial  viral  infection,  resulting  in  limited  viral  spread  and  survival 
of  the  mouse.   Low  responders  show  a  lower  degree  of  resistance,  but  paradoxically  only  to  low 
dose  infection;  when  infected  at  high  virus  doses,  they  are  as  resistant  as  high  responders.   This 
is  because  high-dose  infection  induces  higher  amounts  of  Mx1  protein  than  low-dose  infection, 
thus  apparently  overcoming  the  generally  lower  response  in  these  animals.    This  observation 
demonstrates  that  influenza  virus  pathogenesis  in  mice  results  from  a  subtle  balance  between 
the  dose  of  the  infecting  virus  and  the  levels  of  an  intracellular  antiviral  host  factor.  The  trick 
of  the  system  is  that  the  virus  itself  induces  Mx1  and  thus  controls  the  animal's  fate.  To  test 


2-LVMP/DIR 


whether  other  Mx  proteins  act  in  a  similar  way,  we  have  prepared  transgenic  mice  that  express 
human  Mx  genes.   These  mice  are  currently  being  analyzed.     It  is  anticipated  that  the  Mx1 
promoter  will  become  very  useful  for  a  number  of  studies  in  viral  pathogenesis  but  also  for 
many  other  questions  of  gene  function  in  transgenic  mice. 

2.  Expression  of  individual  viral  proteins  in  vitro  and  in  transgenic  mice.    An 
interesting  question  in  viral  pathogenesis  is  whether  individual  viral  proteins  expressed  in  the 
absence  of  any  other  viral  component  can  cause  cytopathic  changes  or  a  modified  immune 
response.  The  model  rhabdovirus  VSV  is  useful  for  such  studies.  We  have  shown  (Schubert  et 
al)  that  the  VSV  M  protein  is  solely  responsible  for  the  cytopathic  effect  observed  after  viral 
infection.  The  VSV  G  protein  of  the  virus  was  introduced  into  the  germ  line  of  mice  (Arnheiter  et 
al)  and  the  immune  response  of  these  animals  to  that  protein  was  studied.   When  challenged  with 
purified  recombinant  G  protein,  only  IgM  and  not  IgG  antibodies  able  to  neutralize  wild  type  VSV 
were  elicited.  In  contrast,  the  animals  respond  with  neutralizing  antibodies  to  the  challenge 
with  the  wild  type  virus.  Thus,  immunotolerance  breaks  down  and  autoimmunity  is  established 
in  this  case,  suggesting  that  these  mice  could  be  used  as  models  to  study  autoimmune  processes 
following  a  viral  infection. 

3.  Studies  on  neurotropic  viruses  in  vivo  and  in  vitro:   We  have  continued  our  studies 
(Major  et  al  )  on  JC  papovavirus,  the  agent  of  progressive  multifocal  leukoencephalopathy.  We 
have  developed  a  method  to  identify  viral  genomic  sequences  in  a  few  hours  in  brain  biopsy 
specimens.  Studies  on  the  JC  large  T  protein  demonstrate  that  the  retinoblastoma  protein  binds 
to  that  viral  protein,  suggesting  a  role  for  this  interaction  in  viral-induced  tumor  formation  in 
rodents  and  monkeys.  In  our  studies  of  another  DNA  neurotropic  virus,  we  showed  that 
varicella-zoster  virus  can  replicate  in  human  fetal  Schwann  cells. 

B.  HIV  infection:  prevention  and  neurotropism 

In  our  studies  of  engineering  negative  strand  viruses  using  vaccinia  virus  vectors,  we 
expressed  VSV  genes  N,  NS  and  L  in  cells  and,  with  the  help  of  the  T7  RNA  polymerase,  replicate 
defective  VSV  genome  (Schubert  et  al).   Such  studies  pave  the  way  for  the  making  of  target 
recombinant  negative  strand  virus.  Viruses  are  often  used  as  vehicles  for  other  genes,  provided 
they  are  non-cytopathic  for  the  cells  they  invade.  Our  goal  is  to  target  HIV-infected  cells  with 
recombinant  viruses  able  to  interfere  with  HIV  expression.  Toward  this  objective  we  have 
constructed  chimeric  envelope  proteins  (Schubert  et  al).  The  extracellular  portion  of  the  CD4 
molecule  has  been  fused  to  the  VSV  G  transmembrane  and  intracellular  regions  using  PCR 
technology.  Using  the  vaccinia  virus  vectors  to  express  these  genes,  we  now  have  evidence  that 
pseudotypic  virus  can  be  immunoprecipitated  from  the  supernatant  of  infected  cells  with 
anti-CD4  antibodies.  Such  a  CD4-enveloped  defective  virus  could  specifically  infect  gp120 
expressing   HIV  infected  cells.   Optimally  such  defective  virus  should  be  able  to  use  HIV  in  the 
infected  cell  as  a  helper  virus.  To  this  goal,  we  have  now  constructed  a  prototype  pseudotypic 
defective  interfering  HIV  genome.  This  prototype  contains  both  HIV  LTRs  but  the  GAG-POL-ENV 
region  has  been  replaced  by  a  new  gene  coding  for  a  CD4-ENV  chimera  protein.  In  other 
constructs  we  have  placed  a  ribozyme  that  can  cleave  the  ENV  gene  in  the  ectodomain  region  in 
order  to  render  the  HIV  particles  noninfectious. 

In  our  studies  of  HIV  neurotropism,  we  have  infected  and  transfected  human  fetal  astrocytes 
with  HIV1  (Major  et  al).  After  a  transient  period  of  viral  production,  such  cells  appear  latently 
infected  since  virus  can  be  rescued  by  coculture  with  a  CD4+  T  lymphocyte  cell  line.  This  raises 


3-LVMP/DIR 


the  question  whether  chronic  infection  of  astrocytes  and  possibly  other  nerve  cell  types  can 
occur  in  HIV1  infection  of  the  CNS  of  fetuses  carried  by  sero-positive  mothers. 

HIV  also  frequently  causes  neurological  dysfunction  and  is  abundantly  expressed  in  the  central 
nervous  system  (CNS)  of  adult  AIDS  patients  with  HIV  encephalitis  or  myelopathy.    The  virus  is 
found  mostly  in  cells  of  the  monocyte-macrophage  lineage  within  the  CNS,  but  the  possibility  of 
infection  of  other  glial  cells  has  been  raised.   Therefore,  the  effects  of  different  HIV-1  and  HIV- 
2  strains  were  studied  in  primary  cultures  of  adult  human  brain  containing  microglial  cells^ 
the  resident  CNS  macrophages,  and  astrocytes.(Dubois-Dalcq  et  al ).  These  cultures  could  be 
productively  infected  with    macrophage-adapted  HIV-1  isolates  but  not  with  T  lymphocyte- 
adapted  HIV-1  isolates  or  two  HIV-2  isolates.  As  determined  with  a  triple  label  procedure, 
primary  astrocytes  did  not  express  HIV  gag  antigens  and  remained  normal  throughout  the  three- 
week  course  of  infection.  In  contrast,  virus  replicated  in  neighboring  microglial  cells,  often 
leads  to  their  cell  fusion  and  death.  The  death  of  microglial  cells  which  normally  serve  immune 
functions  in  the  CNS,  may  be  a  key  factor  in  the  pathogenesis  of  AIDS  encephalitis  and/or 
myelopathy.  We  are  actively  investigating  the  ways  that  microglial  cells  infection  can  influence 
the  function  and  gene  expression  of  other  cell  types  such  as  astrocytes,  oligodendrocytes  and 
neurons. 

The  next  question  we  have  addressed  this  year  is:  what  is  the  receptor  used  by  HIV  for  entry 
into  microglial  cells?  (Dubois-Dalcq  et  al  ).  CD4  molecules  function  as  the  HIV  receptor  in  T 
and  B  cells,  monocytes  and  macrophages.  Although  the  CD4  receptor  is  also  expressed  in  brain, 
recent  in  vitro  studies  suggest  a  CD4-independent  pathway  of  HIV  entry  in  glial  cells 
originating  from  tumors  or  fetal  tissues.    In  our  work,  we  have  identified    CD4  transcripts  in 
primary  microglial  cell  cultures  using  the  PCR  technique.  Moreover    anti-CD4  monoclonal 
antibodies  directed  to  the  HIV  binding  site  prevent  HIV-1  infection  of  microglial  cells  .  Thus, 
infection  of  brain-derived  microglial  cells  is  CD4-mediated,  suggesting  that  therapies  aimed  at 
blocking  the  CD4  receptor  may  be  appropriate  to  inhibit  entry  and  spreading  of  HIV1  to  the 
brain. 


4-LVMP/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOI  NS  02034-18  LVMP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.    Title  must  fit  on  one  line  between  the  borders.) 

Biology  of  Myelin-Forming  Cells  In  Vitro  and  In  Vivo  Including  Remyelination 


PRINCIPAL  INVESTIGATOR  (list  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  afiihation) 

P.  I.:  M.  Dubois-Dalcq,  M  D.  Acting  Chief  LVMP,  NINDS 


Others: 


R.  McKinnon,  Ph  D 
R.Armstrong,  PhD 
H   Dorn,  B.S. 
R.  Rusten 


Sr.  Staff  Fellow 
IRTA 
Biologist 
Biol.  Lab.  Tech. 


LVMP,  NINDS 
LVMP,  NINDS 
LVMP,  NINDS 
LVMP,  NINDS 


COORPERATING  UNITS  Of  any) 

K.  V.  Holmes,  Dept.  Pathology,  USUHS,  Dr    S  Aronson,  LMG,  NC  I,  and 
Dr.  K.  Kufta,  Neurosurgery  Branch,  NINDS 


LAB/BRANCH 

Laboratory  of  Viral  and  Molecular  Pathogenesis 


SECTION 

Section  on  Neural  and  Molecular  Biology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


3.5 


PROFESSIONAL: 


2.5 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I      |   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


[  x   |  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  myelin  sheath  is  essential  for  normal  conduction  in  major  nerve  tracks.  Therefore  neurological 
dysfunction  occurs  in  demyelinating  diseases  such  as  multiple  sclerosis  (MS)  and  viral  encephalitis.  We 
study  how  myelin-forming  cells  can  develop  and  regenerate  both  in  vitro  and  in  vivo. 

Molecular  analysis  of  oligodendrocyte  type  2  astrocyte  (0-2A)  progenitor  cells  reveals  that  FGF 
modulates  the  PDGF-driven  pathway  of  oligodendrocyte  development  by  up-regulating  the  PDGF 
receptor  on  these  cells.  In  a  chemotaxis  chamber  assay  PDGF,  but  not  FGF,  can  trigger  directed  migration 
which  can  occur  in  the  absence  of  mitosis.  0-2A  lineage  cells  were  isolated  and  cultured  from  mice  with 
a  demyelinating  disease  caused  by  a  corona  virus.  Cultures  from  demyelinated  tissue  differed  in  several 
ways  from  those  of  the  controls:  (1)  the  total  number  of  0-2A  lineage  cells  was  dramatically  increased; 
(2)  the  0-2A  cells  consisted  of  a  high  proportion  of  04  positive  astrocytes  and  of  cells  with  a  mixed 
oligodendrocyte-astrocyte  lineage;  (3)  all  0-2A  cells  showed  enhanced  proliferation  in  response  to  an 
episode  of  demyelination.  In  particular  a  large  proportion  of  progenitor  cells  were  incorporating  DNA. 
Our  in  vitro  analysis  of  adult  human  brain  cells  also  suggests  the  existence  of  a  "resting" 
oligodendrocyte  precursor  cell  with  an  antigenic  pheonotype  similar  to  that  seen  in  rodents    Thus,  our 
interest  lies  in  the  characterization  of  the  oligodendrocyte  precursor  cells  present  in  the  adult  CNS  and 
their  role  in  remyelination. 


PHS  6040  (Rev   184) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02528-09  LVMP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80 characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Regulation  of  Myelin  Synthesis 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.:  L.  D.  Hudson,  Ph.D.         Staff  Scientist  LVMP:NINDS 

Others:         J  Kim,  Ph.D.  M.S.  Fellow  LVMP:NINDS 

N.  Nadon,  Ph.D.  Staff  Fellow  LVMP:NINDS 

J.  Berndt,  B.S.  Microbiologist  LVMP:NINDS 


COORPERATING  UNITS  Of  any) 

Dr.  Heinz  Arnheiter,  Section  of  Viral  Pathogenesis,  LVMP,  NINDS 


LAB/BRANCH 

Laboratory  of  Viral  and  Molecular  Pathogenesis 


SECTION 

Neural  and  Molecular  Biology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 

3.  bo 


PROFESSIONAL:  j  SR 


OTHER:  1  q 


CHECK  APPROPRIATE  BOX(ES) 

O   (a)  Human  subjects  0  (b)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  objective  of  this  project  is  to  define  the  regulatory  signals  that  control  myelination,  the  event  where 
oligodendrocytes  and  Schwann  cells  extend  processes  that  enwrap  and  ensheath  axons.  Our  search  for 
the  molecular  basis  of  transcriptional  controls  on  myelination  involves  characterization  of  both  the  trans- 
acting regulatory  factors  and  their  cognate  cis-acting  enhancer/repressor  elements  necessary  for 
expression  of  the  prototype  myelin  gene,  proteolipid  protein  (PLP).  A  combination  of  biochemical  and 
functional  assays  have  revealed  that  the  essential  cis  elements  are  clustered  in  a  small  region 
encompassing  the  PLP  promoter.  Regulation  of  this  region  most  likely  requires  an  interplay  of  various 
tissue-specific  and  ubiquitous  binding  proteins,  some  of  which  directly  interact  with  the  cis  recognition 
sequences  and  some  which  bind  a  protein-DNA  complex.  We  have  cloned  four  putative  transactivator 
factors  of  the  former  class  and  identified  features  of  DNA-binding  proteins  in  at  least  one  partially 
sequenced  clone.  The  isolation  of  these  clones  permits  a  search  for  the  growth  factors  and  other 
molecules  that  are  critical  to  the  initiation  and  maintenance  of  myelin  gene  transcription. 

PLP  is  the  most  abundant  constituent  of  central  nervous  system  myelin  and  its  loss  has  devastating  effects 
on  myelinating  cells.  After  defining  mutations  in  the  proteolipid  protein  (PLP)  gene  of  a  number  of 
animal  and  human  dysmyelinatinq  disorders,  we  have  provided  molecular  evidence  that  these  mutations 
act,  at  least  in  part,  to  interrupt  oligodendrocyte  differentiation.  Efforts  to  identify  additional  mutations 
in  PLP  and  in  other  myelin  genes  of  patients  affected  with  dysmyelinating  diseases  (such  as  Pelizaeus- 
Merzbacher)  are  underway. 


PHS  6040  (Rev.  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02698-05  LVMP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  character*  or  less.    Title  muit  fit  on  one  line  between  the  borders.) 

Biology  of  Mammalian  Homeodomain  Proteins 


PRINCIPAL  INVESTIGATOR  (L at  other  professional  personnel  below  the  Principal  Investigator  )  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  Heinz  Arnheiter,  Ph.D.       Visiting  Scientist  LVMP,  NINDS 

Others:     Ronald  J.  DeSanto,  B.S.      Biologist  LVMP,  NINDS 


COOPERATING  UNITS  (,fan„) 

Ward.  F.Odenwald,  Ph.D.       Staff  Fellow  LMB,  NINDS 


LAB/BRANCH 

Laboratory  of  Viral  and  Molecular  Pathogenesis 


SECTION 

Viral  Pathogenesis  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland,  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


0.25 


OTHER:  q  ^ 


CHECK  APPROPRIATE  BOX(ES) 

□   (ajHuman  subjects  ]  (b)  Human  tissues  [x~l  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Homeodomain  proteins  are  nuclear  proteins  that  share  a  characteristic  stretch  of  60  amino  acids,  the 
homeodomain.  In  the  fly,  members  of  this  family  of  proteins  are  important  factors  for  implementing  the 
correct  developmental  program  of  embryoqenesis.  In  vertebrates,  they  may  have  similar  roles,  but 
actual  evidence  is  scarce.    It  is  thought  that  homeodomain  proteins  function  by  regulating  the 
expression  of  other  genes,  but  we  do  not  know  which  genes  are  being  regulated. 

To  identify  potential  target  genes  regulated  by  a  representative  murine  homeodomain  protein,  we  have 
introduced  into  the  germ  line  of  laboratory  mice  a  piece  of  DNA  that  allows  the  deliberate  ubiquitous 
expression  of  Hox  1.3.  We  used  as  regulatory  element  the  Mx1  promoter  which  is  known  to  respond  to 
interferon  and  interferon-inducers  such  as  double-stranded  RNA.    Under  physiological  conditions,  that 
is,  in  the  absence  of  interferon,  the  transgenics  express  only  their  endogenous  and  not  the  transgenic 
Hox  13  protein    However,  when  stimulated  with  interferon  or  double-stranded  RNA,  they  start  to 
express  transgenic  Hox  1 .3  RNA  and  protein  at  high  levels  in  many  different  organs.  These  mice  and  their 
descendants  will  be  tested  for  up-  or  down-regulation  of  various  endogenous  genes  following 
experimental  Hox  1.3  induction. 

To  identify  potential  genes  regulated  during  embryonic  development,  we  will  test  the  effect  of 
interferon  or  interferon-inducers  given  during  pregnancy.  This  approach  may  become  extremely  helpful 
to  identify  the  time  window  during  which  Hox  1.3  expression  may  effect  organogenesis,  but  may  be 
limited  by  the  efficiency  with  which  Hox  1 .3  can  be  induced  in  embryos,  and  by  the  side  effects  interferon 
treatment  may  have  on  embryos  during  development. 


PHS6O40(Rev   1841 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOI  NS  02742-04  LVMP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Mechanisms  of  Viral  Pathogenesis 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI: 


Others: 


Heinz  Arnheiter,  M.D. 

Ellen  Meier,  Ph.D. 
Susan  Skuntz,  B.S. 
Ronald  J.  DeSanto,  B.S. 


Visiting  Scientist 

Senior  Staff  Fellow 

Biologist 

Biologist 


LVMP,  NINDS 

LVMP,  NINDS 
LVMP,  NINDS 
LVMP,  NINDS 


COOPERATING  UNITS  {if  any) 


LAB/BRANCH 

Laboratory  of  Viral  and  Molecular  Pathogenesis 


SECTION 

Viral  Pathogenesis  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


2.3 


PROFESSIONAL: 


1.5 


OTHER: 


0.8 


CHECK  APPROPRIATE  BOX(ES) 

[      |   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


J  (b)  Human  tissues  |  x   |  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

In  addition  to  generating  an  immune  response  with  its  specific  antibodies  and  T  cells,  the  vertebrate  host 
can  control  viral  infections  by  induction  of  intracellular  host  factors  that  directly  interfere  with  viral 
replication.   Our  research  focuses  on  a  family  of  such  intracellular  proteins  known  as  Mx  proteins.  One 
member  of  this  family,  the  Mx1  protein  found  in  two  strains  of  laboratory  mice,  is  induced  by  interferon 
and  arrests  growth  of  influenza  viruses  (but  not  other  viruses)  by  interfering  with  an  early  step  in  viral 
replication.  We  have  introduced  its  corresponding  cDNA  into  the  germ  line  of  Mx-negative,  influenza- 
susceptible  mice,  a  procedure  which  results  in  protection  of  the  mice  against  fatal  influenza.  This 
procedure  can  be  called  "  intracellular  immunization" ,  because  the  mice  become  immune  to  infection, 
and  because  the  antiviral  principle  is  acting  inside  cells,  unlike  antibodies  and  T  cells  which  act  on  cells  or 
viruses  from  the  outside. 

Proteins  with  homology  to  Mx1  are  expressed  in  organisms  ranging  from  yeast  to  man,  that  is,  they  are 
also  found  in  organisms  for  which  influenza  is  no  threat.  It  is  possible  that  Mx  proteins  of  such  species 
have  activities  against  other  viruses.  However,  it  is  also  possible  that  the  main  mission  of  Mx  proteins  is 
to  fulfill  some  basic  cellular  function,  and  antiviral  action  isjust  a  byproduct  of  this  basic  function. 

To  test  the  possibility  that  certain  Mx  proteins  have  activities  against  other  viruses,  we  have  analyzed  the 
Mx  system  of  the  rat.    We  found  that  a  nuclear  rat  Mx  protein  has  in  fact  activity  not  only  against 
influenza  virus,  but  also  against  vesicular  stomatitis  virus  (VSV),  a  negative-stranded  RNA  virus  against 
which  mouse  Mx1  has  no  activity.  A  cytoplasmic  rat  Mx  protein  has  activity  only  against  VSV.   A 
suggestion  for  a  basic  cellular  function  of  Mx  proteins  comes  from  the  observation  that  all  published  Mx 
sequences  contain  in  their  more  conserved  ammo-terminal  halves  a  consensus  sequence  characteristic  of 
GTP-binding  proteins.   Thus,  Mx  proteins  may  belong  to  a  family  of  GTP-bindinq  proteins. 


PHS  6040  (Rev.  184 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUM8ER 


ZOI  NS  02789-02  LVMP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.    Title  must  fit  on  one  line  between  the  borders.) 

Neurotropism  of  Human  Retroviruses 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I  M  Dubois-Dalcq,  M  D  Acting  Chief,  LVMP  LVMP,  NINDS 

Others:         B.  Watkins,  Ph.D.  Visiting  Fellow  LVMP,  NINDS 

C.  Jordan,  Ph.D.  Sr.  Staff  Fellow  LVMP,  NINDS 

H.  Dorn,  B.S.  Biologist  LVMP,  NINDS 

W  Kelly,  B.S.  Chemist  LVMP,  NINDS 


COORPERATING  UNITS  (if  any) 

Dr.  K.  Kufta,  Neurosurgery  Branch,  NINDS;  Dr.  B  Potts,  Repligen,  Boston,  Ma;  and  F.  Michaels,  Laboratory 
Tumor  Cell  Biology,  NCI. 


LAB/BRANCH 

Laboratory  of  Viral  and  Molecular  Pathogenesis 


SECTION 

Section  on  Neural  and  Molecular  Biology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  j  3 


OTHER: 


1.2 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  [V]  (b)  Human  tissues  I      I  (c)  Neither 

]  (a1)  Minors 

J   (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

In  order  to  elucidate  the  pathogenesis  of  AIDS  encephalitis  and  myelopathy,  we  have  infected  primary 
cultures  derived  from  cortex  of  patients  with  intractable  epilepsy  or  malignant  astrocytoma  with 
different  isolates  of  HIV-1  and  HIV-2.  Viral  infection  was  measured  by  reverse  transcriptase  (RT)  activity 
and  HIV-1  core  antigen  (p24)  in  cell  free  supernatant.  The  three  isolates  which  showed  signs  of 
replication  were  derived  from  either  macrophages  (HIV-1  AD87(M)and  HIV-1  BaL)  or  brain  (HIV-1jr_fl). 
whereas  T-cell  adapted  and  HIV-2  strains  did  not  replicate.  Peak  levels  of  RT  activity  (1-2x105  cpm/ml) 
and  p24  antigen  (50-100  ng/ml  in  productivity  infected  cultures  were  generally  seen  at  9-15  days  post 
infection    The  antigenic  phenotype  of  infected  cells  was  characterized  by  a  triple  label  assay  using 
antibodies  against  HIV  p24or  pi  7  gag  proteins,  glial  fibrillary  acidic  protein  (GFAP)  antibody  to  identify 
astrocytes  and  Dil-labelled  low  density  lipoprotein  (Dil-LDL),  which  binds  to  microglia,  a  cell  of  the 
macrophage  lineage  residing  in  the  brain    In  infected  cultures,  Dil-LDL  labelled  microglia  often  stained 
positively  for  gag  but  GFAP+  astrocytes  did  not.  Viral  production  was  further  confirmed  by  electron 
microscopy  which  showed  budding  of  HIV  particles  from  the  plasma  membrane  of  microglial  cells.  After 
4-5  days  post  infection  progressive  syncitia  formation  began  among  infected  microglia  and  after  3 
weeks  up  to  25  times  more  Dil  -LDL +  cells  had  died  as  compared  to  controls.  This  progressive  clustering 
and  fusion  of  microglial  cells  in  vitro  mimics  the  two  pathological  hallmarks  of  HIV  infection  in  the 
brain:  microglial  nodules  and  multi-nucleated  giant  cells.  When  cells  were  incubated  prior  to  infection 
with  large  amounts  of  anti-CD4  antibody  which  recognizes  the  gp1 20  binding  site  (25  ug/ml  Leu  3a), 
both  viral  replication  and  cytopathic  effects  were  reduced  to  control  levels.  In  addition,  microglia 
isolated  from  control  cultures  were  found  to  express  mRNA  for  CD4  by  polymerase  chain  reaction  (PCR). 
This  demonstrates  that  HIV  can  enter  microglia  via  the  CD4  receptor  as  it  does  in  T-cells  and  monocytes  . 
We  propose  that  the  death  of  microglial  cells  which  normally  serve  immune  functions  in  the  central 
nervous  system  may  be  a  key  factor  in  the  pathogenesis  of  AIDS  encephalitis  and/or  myelopathy 


f>HS6M0(fiev    1  S4| 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOI  NS  02790-02  LVMP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Expression  of  Viral  Proteins  in  Transgenic  Mice 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  Heinz  Arnheiter,  Ph.D.       Visiting  Scientist  LVMP,  NINDS 

Others:  Susan  Skuntz,  B.S.  Biologist  LVMP,  NINDS 

Ronald  J.  De  Santo,  B.S.      Biologist  LVMP,  NINDS 


COOPERATING  UNITS  </f  any) 

Rolf  M.  Zinkernagel,  Institute  for  Pathology,  University  of  Zurich,  Switzerland 
Dr.  Elisabeth  Tournier-Lasserve,  Institut  Pasteur,  Paris,  France 


LAB/BRANCH 

Laboratory  of  Viral  and  Molecular  Pathogenesis 


SECTION 

Viral  Pathogenesis  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  Q  yc 


OTHER:  q  45 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  f~x~~l  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Individual  viral  proteins  are  expressed  in  transgenic  mice  in  order  to  answer  two  fundamental  questions 
in  viral  pathogenesis:  what  are  the  mechanisms  of  induction  of  an  immune  response  against  a  virus  and 
what  effect  does  a  particular  viral  protein  have  on  the  metabolism  of  a  cell  in  vivo? 

To  address  an  example  of  the  first  question,  induction  of  an  immune  response,  the  envelope 
glycoprotein  G  of  vesicular  stomatitis  virus,  serotype  Indiana,  was  expressed  in  transgenics.  When  these 
mice  are  immunized  with  purified  G  protein,  or  when  challenged  with  a  recombinant  vaccinia  virus 
expressing  this  protein,  only  IgM  and  not  IgG  antibodies  able  to  neutralize  wildtype  Indiana  VSV  are 
formed.  However,  the  response  with  neutralizing  antibodies  is  normal  when  the  corresponding 
wildtype  Indiana  VSV  is  used.  Thus,  tolerance  breaks  down,  and  autoimmunity  is  established,  when 
wildtype  virus  is  used  for  immunization,  and  not  when  G  protein  is  presented  in  other  ways.  These  mice 
may  become  a  model  for  those  autoimmune  phenomena  of  humans  for  which  viral  infections  may  be  the 
triggering  event. 

To  address  an  example  of  the  second  question,  we  have  expressed  the  regulatory  protein  Tax  of  the 
human  retrovirus  HTLV-1  known  to  be  associated  with  certain  forms  of  T-cell  leukemias,  and  tropical 
spastic  paraparesis.  To  date  we  have  found  that  low  level  Tax  expression  in  transgenic  mice  may  cause  a 
thymic  hypoplasia  but  no  gross  pathology.  Since  in  these  mice  the  Tax  protein  is  under  the  control  of  an 
inducible  promoter,  we  will  be  able  in  the  future  to  study  the  effect  of  higher  levels  of  expression. 
However,  in  one  transgenic  line  the  Tax  transgene  is  apparently  inserted  into  a  host  gene  whose  product 
is  neccessary  for  normal  embryonic  development  of  the  vertebra:  mice  of  this  line,  when  homozygous  for 
the  transgene,  show  a  severe  malformation  of  the  vertebra  characterized  by  hemivertebras,  spina  bifida 
occulta,  fusions  of  vertebral  bodies,  and  short  kinky  tails.  The  molecular  characterization  of  the 
corresponding  gene  that  we  have  mapped  to  mouse  chromosme  1 1  is  under  way. 


10 


PHS  6040  (Rev.  1/S4) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

ZOI   NSOWaJ  191VMH 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less,   rule  must  fit  on  one  line  between  the  borders  > 

Molecular  Biology  of  Human  Virus  Infections,  HIV-1  and  JCV 


PRINCIPAL  INVESTIGATOR  (list  other  professional  personne/ below  trie  Principal  Investigator )  (Name,  title,  laboratory  and  institute  affiluti^    I 
P   I 

E.O  Major,  PhD  Section  Chief  LVMP,  NINDS 

Others: 

K.  Amemiya,  Ph.D.  Special  Expert  LVMP,  NINDS  S.  Finch,  Ph  D     Microbiologist    LVMP, NINDS 

J.  Assouline,  Ph.D.  Staff  Fellow  LVMP,  NINDS  A.  Nath,  M  D      Medical  Staff      LVMP,  NINDS 

B  Curfman,  B.S.  Microbiologist  LVMP,  NINDS  Fellow 

L.  Durham,  M.S.  Biologist  LVMP,  NINDS  R.  Traub,  B  S      Microbiologist  LVMP,  NINDS 


COOPERATING  UNITS  (if  an,) 

Microbiological  Associates,  Inc.Rockville,  MD. 
Veterans  Administration  Hospital,  Washington,  D.C. 


LAB/BRANCH 

Laboratory  of  Viral  and  Molecular  Pathogenesis 


SECTION 

Section  on  Molecular  Virology  and  Genetics 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  ?f. 


PROFESSIONAL:  4  c 


OTHER:  2  5 


CHECK  APPROPRIATE  BOX(ES) 

LJ   (a)  Human  subjects  [x~l  (b)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Progressive  multifocal  leucoencephalopathy  (PML)  and  AIDS  encephalopathy  are  viral 
induced  neurological  diseases  which  occur  almost  exclusively  in  individuals  whose 
immune  systems  are  impaired.  We  have  investigated  the  molecular  pathogenesis  of 
these  diseases  by  developing  laboratory  assays  that  examine  the  human  polyomavirus, 
JCV,  and  the  human  immunodeficiency  virus,  HIV-1  the  causes  of  PML  and  AIDS 
encephalopathy,  respectively.  We  have  developed  a  new  methodology  that  prepares 
brain  biopsy  tissue  for  in  situ  DNA  hybridization  to  detect  viral  genomes  on  the  same 
day  of  surgery.  For  example,  in  brain  biopsy  tissue  from  an  AIDS  patient  with  PML,  JCV 
DNA  can  be  detected  using  a  non-radioactive,  nucleic  acid  probe.  Data  from  this  rapid 
molecular  diagnostic  assay  facilitates  patient  management  decisions  on  a  more  timely 
basis.  We  have  also  examined  protein  interactions  between  JCV  and  infected  glial  cells 
The  non-structural  JCVT  protein  can  bind  the  cellular  retinoblastoma  protein  which 
normally  functions  for  growth  regulation.  Results  from  this  study  suggest  that  the 
oncogenic  properties  of  JCV  may  be  related  to  viral  proteins  interfering  with  normal 
cellular  metabolism.  Other  human  viruses  have  been  studied  for  their  association  with 
glial  cells  of  the  nervous  system.  Astroglial  cells  from  human  fetal  brain  respond  very 
rapidly  to  the  introduction  of  the  HIV-1  genome.  AIDS  virus  proteins  are  synthesized 
within  hours  of  transfection.  However,  HIV-1  infection  of  astrocytes  results  in  a 
persistent  infection  without  cytopathic  effects.    Varicella-zoster  virus,  the  agent  which 
causes  shingles,  can  productively  infect  Schwann  cells,  the  myelinating  cell  of  the 
peripheral  nervous  system.  VZV  proteins  are  also  produced  very  rapidly  following 
infection  and  are  efficiently  assembled  into  progeny  virions 


11 


PHS  6040  (Rev    I  nil 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  MS  02791-02  LVMP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Replication  and  Pathogenesis  of  Enveloped  Viruses 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Prin-  ipa/  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.  M.Schubert,  Ph.D.  Section  Chief  LVMP,  NINDS 


Others:  D.  Blondel,  Ph.D. 

G.  Harmison,  B.S. 
B.  Joshi,Ph.D. 


Visiting  Fellow 

Chemist 

Senior  Staff  Fellow 


LVMP,  NINDS 
LVMP,  NINDS 
LVMP,  NINDS 


COORPERATING  UNITS  Of  any) 

Dr.  Yong  Kang,  University  of  Ottawa,  Ottawa,  Canada. 


LAB/BRANCH 

Laboratory  of  Viral  and  Molecular  Pathogenesis 


SECTION 

Viral  Replication  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH.  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.8 


PROFESSIONAL: 


1.5 


OTHER: 


0.3 


CHECK  APPROPRIATE  BOX(ES) 

]    (a)  Human  subjects 
]  (a1)  Minors 

J   (a2)  Interviews 


]  (b)  Human  tissues  [jT]  (c  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  focuses  on  the  molecular  biology  of  enveloped  viruses  which  include  a  broad  group  of 
viruses  from  negative  strand  viruses  (measles,  rabies),  to  retroviruses  ( AIDS  virus),  to  DNA  viruses  (herpes 
virus).  The  goal  of  this  research  is  to  study  the  basic  molecular  mechanisms  of  viral  pathogenesis  from 
viral  entry  and  assembly  to  viral  gene  expression  and  replication.  The  understanding  of  these 
mechanisms  will  be  necessary  in  a  long  term  to  design  novel  targeted  gene  delivery  systems  or  gene 
expression  systems  with  possible  applications  in  gene  therapy  and  vaccine  development.  Our  plan  is  to 
develop  defective  virus  particles  with  tropism  for  neuronal  or  nonneuronal  cells.  So  far  our  studies  have 
centered  around  vesicular  stomatitis  virus  (VSV)  but  the  basic  strategies  can  be  applied  to  other 
enveloped  viruses. 

Towards  these  goals  we  found  that  viral  assembly  and  cytopathogenesis  of  VSV  are  most  likely 
independent  events.  We  also  found  that  the  high  affinity  of  the  polymerase  protein  NS  to  the  defective 
interfering  particle  genome  determines  the  level  of  autointerference.  These  are  important  observations 
for  the  understanding  of  viral  pathogenesis  on  one  end  of  the  spectrum  and  the  establishment  and 
maintenance  of  persistent  infections  on  the  other  end. 

For  the  targeting  of  viruses  to  specific  cells  we  were  able  to  insert  a  chimeric  HIV  receptor  CD4-VSV 
envelope  glycoprotein  into  a  majority  of  VSV  particles.  These  particles  could  be  specifically 
immunoprecipitated  with  antibodies  to  the  CD4  protein.  We  were  also  able  to  insert  normal  human  CD4 
molecule  into  the  VSV  envelope.  Experiments  are  in  progress  to  test  whether  the  efficiency  of  the 
insertion  as  well  as  viral  assembly  depend  on  the  presence  of  the  cytoplasmic  tail  region  of  the  VSV 
envelope  protein.  We  are  in  the  process  of  developing  a  system  which  allows  studing  the  molecular 
mechanisms  of  viral  assembly  when  the  viral  envelope  protein  is  absent  and/or  replaced  by  a 
recombinant  chimeric  envelope  protein.  Using  vaccinia  virus  we  were  able  to  express  the  VSV  N,  NS  and  L 
genes  and  replicate  a  defective  virus  genome  in  the  absence  of  helper  virus.  This  finding  is  particularly 
promising  in  our  efforts  to  generate  the  first  recombinant  rhabdovirus. 

12 


PHS  6040  (Rev.  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


ZOl  NS  02756-03  LVMP 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  fine  between  the  borders  ) 

Neurologic  and  Systemic  Manifestations  of  Retrovirus  and  Autoimmune  Mediated  Diseases 


PRINCIPAL  INVESTIGATOR  ft  ist  other  professional  personnel  below  the  Principal  Investigator  J  (Name,  title,  laboratory,  and  institute  affiliation) 

P.  I.:  D  M   Klinman,  M.  D.Ph.D.        Senior  Staff  Fellow         LVMP,  NINDS 


COORPERATING  UNITS  Of  any) 

None 


LAB/BRANCH 

Laboratory  of  Viral  and  Molecular  Pathogenesis 


SECTION 

Viral  Pathogenesis  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 
This  project  has  been  terminated. 


13 


(>HS  6040  (Rev    V84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02818-01  LVMP 


October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Pseudotypic  Defective  Interfering  HIV  Particles  as  an  Antiviral  Therapy  for  AIDS 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

p.l. 

M.Schubert  Ph.D.  Section  Chief  LVMP,  NINDS 

Others: 

G.  Harmisonll,  B.S.  Chemist  LVMP,  NINDS 

B.  Joshi,  Ph.D.  Sr.  Staff  Fellow  LVMP,  NINDS 

D.  Blondel.Ph.D  Visiting  Fellow  LVMP,  NINDS 

K.  Haglund  Summer  Student  LVMP,  NINDS 


COOPERATING  UNITS  Ofany) 


None 


LAB/BRANCH 

Laboratory  of  Viral  and  Molecular  Pathogenesis 


SECTION 

Viral  Replication  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


2.8 


PROFESSIONAL: 


2.5 


OTHER: 


0.3 


CHECK  APPROPRIATE  BOX(ES) 

J   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


J  (b)  Human  tissues  I  x   I  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  newly  initiated  project  has  evolved  from  the  young  project  on  the  Replication  and  Pathogenesis  of 
Enveloped  Viruses  (Z01  NS  02791-01  LVMP).  The  main  topic  covered  by  this  project  focuses  on  the 
molecular  virology  of  HIV,  the  AIDS  virus,  and  on  antiviral  strategies  designed  to  specifically  interfere 
with  the  replication  of  HIV.  It  is  our  hope  this  interference  with  virus  replication  may  cause  a  significant 
delay  in  the  onset  of  AIDS  or  possibly  prevent  it. 

We  are  currently  developing  what  we  call  pseudotypic  defective  interfering  particles  of  HIV.  The  goal  of 
this  antivirual  approach  is  to  use  these  defective  particles  to  target  and  specifically  infect  cells  which  are 
already  infected  by  HIV.  It  is  our  anticipation  that  this  cell-targeting  will  allow  deliverance  of  genes 
and/or  gene  products  specifically  into  HIV  infected  cells.  The  gene  products  are  designed  to  interfere 
with  the  replication  of  HIV  itself.  In  addition,  we  anticipate  that  these  defective  interfering  HIV  particles 
will  not  only  down-regulate  the  number  of  HIV  progeny  but  that  they  also  will  use  HIV  in  an 
opportunistic  way  as  their  helper  virus  for  their  own  assembly.  This  will  permit  repetition  of  this  cycle  in 
other  HIV  infected  cells. 

We  have  succeeded  by  using  our  gene  fusion  method  to  construct  the  first  prototype  pseudotypic 
defective  interfering  HIV  genome.  The  genome  was  assembled,  cloned  and  sequenced  and  was  found 
suitable  for  further  evaluation  as  a  targeted  antiviral  delivery  system.  We  have  also  constructed  two 
potential  ribozymes  which  are  designed  to  specifically  cleave  HIV  genomic  RNA,  inactivate  it  and  thereby 
increase  the  inferfering  potential  of  the  defective  virus.  Functional  tests  are  in  progress. 


14 


PHS  6040  (Rev.  I'M) 


> 

00 


> 

00 

O 

33 
> 

o 

30 

•< 


c 

33 
> 


n 
O 

z 

H 
33 

O 


ANNUAL  REPORT 

October  1,  1989  through  September  30,  1990 

Laboratory  of  Neural  Control,  Basic  Neurosciences  Program,  Division  of  Intramural  Research 
National  Institute  of  Neurological  Disorders  and  Stroke 

Table  of  Contents 

RESEARCH  SUMMARY  j.g 

PROJECT  REPORTS 

Motor  Control  Systems  in  the  Spinal  Cord  7 

Z01  NS  01686-22  LNLC 

Techniques  for  Making  Connections  with  the  Nervous  and  8 

Musculoskeletal  Systems 
Z01  NS  01687-22  LNLC 

Cortical  Mechanisms  of  Voluntary  Motor  Control  9 

Z01  NS  01688-22  LNLC 

Models  of  Neurophysiological  Systems  10 

Z01  NS  02079-17  LNLC 

Intrinsic  Properties  of  Motor  Units  1 1 

Z01  NS  02160-16  LNLC 

Repair  of  Injured  Nervous  Tissue  with  Foreign  Grafts  1 2 

Z01  NS  02254-14  LNLC 

Analysis  of  Network  Function  in  the  Developing  Spinal  Cord  of  the  Chick  Embryo  13 

Z01  NS  02787-02  LNLC 

Development  of  Primary  Sensory  Neurons  14 

Z01  NS  02788-02  LNLC 


i-LNLC/DIR 


ANNUAL  REPORT 

October  1,  1989  through  September  30,  1990 

Laboratory  of  Neural  Control,  Basic  Neurosciences  Program,  Division  of  Intramural  Research 

National  Institute  of  Neurological  Disorders  and  Stroke 

Robert  E.  Burke,  M.D  ,  Chief 

Introduction 

Research  work  in  the  Laboratory  of  Neural  Control  (LNLC)  is  devoted  to  studies  of  the  central  and 
peripheral  neural  mechanisms  involved  in  the  control  of  movement  in  mammals.    Our  work  emphasizes 
analysis  of  neural  organizations  at  the  level  of  the  spinal  cord  and  those  regions  of  the  brain  stem  and 
cerebral  cortex  that  project  directly  to  the  spinal  cord  in  vertebrates,  ranging  from  birds  to  primates. 
Studies  of  spinal  cord  development  mainly  involve  in  vitro  studies,  ranging  from  tissue  culture  systems  to 
whole  chick  embryos,  and  include  extra-  and  intracellular  electrophysiological,  as  well  as  optical,  recording 
techniques    Interactions  among  motoneurons,  motor  units,  and  elemental  interneuronal  circuits  are 
studied  by  single  unit  electrophysiological  methods  in  adult  cats.  The  organization  of  cortical  motor 
control  systems  is  studied  in  primates,  primarily  using  chronic  recording  techniques  in  awake,  behaving 
animals.  Neuroanatomical,  histochemical  and  immunocytochemical,  and  mathematical  modeling 
approaches  are  used  as  adjuncts  to  these  basically  physiological  studies.  The  Laboratory  also  includes  a 
Section  devoted  to  studies  of  nerve  regeneration  after  injury,  using  light  and  electron  microscopic 
anatomical  techniques.  Finally,  members  of  the  Laboratory  continue  to  be  involved  in  studies  of  neural 
prostheses  that  utilize  state-of-the-art  techniques  to  develop  systems  that  can  benefit  neurologically- 
handicapped  individuals. 

Present  Organization 

During  FY  1990,  the  staff  of  the  Laboratory  of  Neural  Control  (LNLC)  included:  thirteen  professional 
scientists  (five  permanent  senior  scientists,  one  special  expert,  and  seven  post-doctoral  fellows).  The 
permanent  staff  also  includes  eight  full-time  permanent  support  personnel  (a  physiologist,  two  engineers, 
one  computer  programmer,  one  biological  technician,  a  histology  technician,  and  one  laboratory 
secretary). 

The  FY  1990  research  effort  in  LNLC  can  be  described  under  six  general  headings: 

1 .  Electrophysiological  and  morphological  analyses  of  the  cellular  physiology  and  neuronal  circuitry 
operating  in  the  control  of  movement  at  the  spinal  cord  level  in  anesthetized  or  unanesthetized  animals 
(primarily  cats)  following  acute  destruction  of  the  supratentorial  brain  (decerebrate  preparations). 

2.  Theoretical  and  computer  modeling  studies  of  cellular  biophysical  and  morphological  features  of 
individual  neurons  and  of  information  processing  in  defined  neural  systems. 

3.  Studies  of  the  discharge  properties  of  individual  neurons  in  the  primate  motor  cortex  and 
supplementary  motor  area  (SMA)  during  performance  of  voluntary  movements. 

4.  Studies  of  the  development  of  the  vertebrate  spinal  cord,  including  maturation  of  cellular 
electrophysiological  and  morphological  characteristics,  formation  of  specific  synaptic  connections,  and 
analysis  of  the  maturation  of  intrinsic  motor  patterns  in  the  chick  embryo. 

5.  Studies  of  the  mechanisms  of  injury  repair  in  mammalian  peripheral  nerves  following  axotomy 
and  the  role  of  the  blood-nerve  barrier  in  this  process. 

6.  Activities  concerned  directly  with  the  development  of  new  methods  for  making  contact  with  the 
central  nervous  system.  Many  of  the  activities  under  this  rubric  serve  to  support  ongoing  research  projects 
within  LNLC.  During  FY  1990,  this  project  also  included  continued  work  on  the  development  of  a 
functional  prosthesis  for  stimulation  of  the  human  visual  cortex. 


1  -LNLGDIR 


Project  Summaries 

Motor  Control  Systems  and  Intrinsic  Properties  of  Motor  Units  in  the  Cat  Spinal  Cord:  Control  of 
excitatory  last-order  intemeurons:  Thissubproject  utilizes  cats,  either  anesthetized  or  after  decerebration, 
with  destruction  of  thesupratentorial  brain.  This  animal  has  been  used  as  the  ideal  model  system  for  work 
on  the  anatomy  and  physiology  of  the  spinal  cord  for  over  a  century  and  there  is  thus  a  wealth  of  detailed 
information  about  the  cat  spinal  cord  that  serves  as  the  basis  for  the  design  and  interpretation  of  new 
experiments.  In  addition,  cats  are  relatively  accessible,  have  the  ideal  body  size  for  neurophysiological 
experiments,  and  are  well-adapted  to  the  laboratory  environment.  The  neural  mechanisms  that  control 
movement  are  inferred  from  data  obtained  in  reduced,  immobile  preparations.  When  survival  surgery  is 
required,  as  in  studies  of  the  morphology  of  motor  nuclei  using  retrograde  transport  methods,  surgery  is 
performed  under  anesthesia  and  aseptic  conditions,  and  appropriate  postoperative  care  is  supplied.  The 
main  goal  of  this  project  is  to  examine  the  organization  of  neuronal  systems  in  the  mammalian  spinal  cord 
that  are  involved  in  the  control  of  movement.  Specific  topics  range  from  studies  of  cellular  properties  of 
individual  motoneurons  and  intemeurons  in  identified  neuronal  systems  to  examination  of  the 
organization  and  function  of  specific  neuronal  circuits  that  are  involved  in  movement  control,  including 
the  problem  of  central  pattern  generation  at  spinal  segmental  levels. 

During  FY  1990,  we  enlarged  earlier  results  on  the  organization  of  excitatory  intemeurons  that 
project  directly  to  motoneurons  in  the  cat  spinal  cord,  about  which  relatively  little  detailed  information  is 
currently  available.  We  have  emphasized  studies  of  the  short-latency  excitatory  pathways  from  distal  areas 
of  hindlimb  skin  to  particular  groups  of  motoneurons,  especially  those  that  innervate  the  flexor  digitorum 
longus  (FDL)  muscle.  We  have  shown  that  large,  low-threshold  afferents  from  the  dorsal  surface  and  tips 
of  the  toes,  traveling  in  the  superficial  peroneal  nerve  (SP),  as  well  as  those  from  the  ventral  surface 
traveling  in  the  medial  plantar  (PLNT)  nerve,  both  produce  excitatory  postsynaptic  potentials  (EPSPs)  with 
minimum  central  latencies  consistent  with  disynaptic  connection  to  FDL  motoneurons.  This  means  that 
that  the  fastest  pathway  from  the  respective  afferents  to  FDL  output  cells  consists  of  a  single  layer  of 
interposed  intemeurons,  which  makes  the  analysis  of  the  organization  of  the  pathways  by  conventional 
techniques  possible. 

We  previously  demonstrated  that  the  initial  excitatory  components  in  SP  PSPs  in  FDL  motoneurons 
are  strongly  facilitated  during  the  early  flexion  phase  of  fictive  locomotion  (rhythmic  patterned 
motoneuron  discharges  in  decerebrate,  paralyzed  animals  that  mimic  those  found  in  intact,  freely  walking 
animals).  This  indicates  that  excitatory  spinal  intemeurons,  including  those  in  the  disynaptic  SP  to  FDL 
pathway,  receive  convergent  excitation  from  the  spinal  central  pattern  generator  (CPG)  for  locomotion. 
The  same  spatial  facilitation  technique  was  used  to  show  that  short-latency  EPSPs  produced  in  FDL 
motoneurons  by  stimulation  of  cutaneous  afferents  in  the  PLNT  nerve  are  also  modulated  by  the 
locomotor  CPG,  but  in  a  pattern  completely  different  from  that  observed  with  SP  EPSPs.  In  contrast  to  SP 
EPSPs,  PLNT  EPSPs  are  markedly  reduced  in  amplitude  during  the  entire  flexion  phase,  with  maximum 
reduction  during  early  flexion.  In  addition,  PLNT  EPSPs  exhibit  facilitation  during  the  extension  phase  of 
fictive  stepping  in  occasional  step  cycles  in  which  the  FDL  muscle  shows  a  burst  of  activity  during  extension. 
This  facilitation  is  not  observed  in  SP  EPSPs.  These  results  indicate  that  the  last-order  intemeurons  that 
convey  excitation  from  these  two  skin  nerves  are  completely  separate  and  subject  to  independent  central 
control,  despite  the  fact  that  the  sensory  territories  of  the  two  nerves  are  contiguous.  This  model  system 
demonstrates  the  existence  of  highly  specific  cutaneous  reflex  pathways  that  presumably  subserve 
particular  functions  in  the  intact  animal.  They  also  suggest  that  particular  sets  of  excitatory  intemeurons 
that  receive  convergence  of  input  from  central  neural  systems  as  as  from  the  periphery  may  serve  multiple 
roles  in  the  generation  of  particular  patterns  of  muscle  output.  The  finesse  exhibited  by  this  system  is  quite 
at  variance  with  the  common  notion  of  cutaneous  reflexes  as  global  in  character,  as  in  the  flexion 
withdrawal  reflex. 

Studies  of  the  morphology  of  motoneuron  dendrites:  In  FY  1989,  we  began  to  examine  whether  it  is 
possible  to  define  relatively  simple  mathematical  rules  that  embody  the  complex  architecture  of 
motoneuron  dendrites.  Initial  results  using  data  from  a  large  sample  of  dendrites  from  adult  triceps  surae 


2-LNLC/DIR 


motoneurons  indicate  that  the  probability  of  branching  and  terminating  are  primarily  functions  of  local 
dendritic  diameter,  with  a  minor  dependence  on  the  length  of  dendritic  branches.  In  addition,  the  rate  of 
dendritic  taper  was  found  also  to  depend  on  local  dendritic  diameter.  During  FY  1990,  a  full  model  system 
was  implemented  in  which  relatively  simple  mathematical  rules  are  used  to  govern  the  probabilities  of 
branching  and  termination  in  individual  branches,  and  to  constrain  the  diameters  of  daughter  branches  at 
branching  points   The  rules  are  the  direct  output  from  appropriate  analyses  of  real  dendrites  and  the 
parameters  are  not  varied  beyond  the  limits  observed  in  the  data.  The  object  has  been  to  develop  a  modei 
system  that  uses  parameters  directly  obtained  form  actual  data  to  reproduce  structures  with  the 
characteristics  of  actual  dendrites.  Thus  far,  the  model  produces  simulated  "dendrites"  that  mimic  the 
characteristics  of  real  motoneuron  dendrites  in  most,  but  not  all,  respects.  Development  of  the  model  has 
forced  us  to  focus  on  very  specific  features  of  dendritic  anatomy.  The  fact  that  all  the  rules  depend 
primarily  on  local  dendritic  diameter  suggests  that  factors  correlated  with  diameter,  including  features  of 
the  local  cytoskeleton,  play  critical  roles  in  controlling  dendritic  morphology  and  maintenance. 

We  have  continued  this  work,  using  morphological  data  sets  developed  within  LNLC  and  additional 
sets  developed  in  the  Department  of  Anatomy,  Karolinska  Institutet,  Stockholm,  Sweden.  The  Karolinska 
data  includes  measurements  from  cat  alpha-motoneurons  from  newborn  and  immature  kittens,  as  well  as 
adult  animals  We  anticipate  that  application  of  our  current  analysis  methods  and  the  implementation  of 
the  mathematical  simulation  may  reveal  the  factors  that  account  for  the  morphological  changes  that 
dendrites  undergo  during  normal  postnatal  maturation. 

We  have  continued  a  similar  analysis  of  the  dendrites  of  adult  gamma  motoneurons  in  order  to  test 
our  approach  on  a  different  species  of  neuron.  It  has  been  known  for  some  time  that  gamma 
motoneurons,  although  smaller  in  total  membrane  area  than  alpha  cells,  nevertheless  have  dendrites  that 
are  equally  long,  albeit  less  highly  branched.  Our  analysis  to  date  suggests  that  the  key  parameters  that 
control  dendrite  branching  and  termination  are  relatively  similar  in  alpha-  and  gamma-motoneurons,  but 
the  rate  of  taper  is  markedly  lower  in  gamma  motoneurons.  Studies  are  underway  to  pinpoint  the  factors 
that  can  account  for  the  large  apparent  differences  in  the  morphologies  of  these  two  kinds  of 
motoneurons. 

Models  of  Neurophvsioloqical  Systems:  Work  on  this  project  during  FY  1990  has  been  closely 
connected  to  the  above  attempt  to  simulate  dendritic  morphologies  in  computer  simulations.  In  fact,  two 
parallel  approaches  have  been  used  for  the  actual  simulations  -  Monte  Carlo  and  analytical  simulations 
Both  have  given  equivalent  results  but  each  is  used  so  as  to  check  on  the  reliability  of  the  other.  Dr.  Marks 
has  undertaken  additional  work  to  examine  descriptive  rules  that  embody  the  trajectories  of  individual 
dendritic  branches.  These  can  be  adequately  simulated  using  a  smoothed  random  walk.  Motoneuron 
branches  have  an  average  fractal  dimension  of  about  1 .2.  We  are  also  exploring  the  possibility  of 
simulating  fully  branched  dendritic  trees  in  three  dimensions,  using  constraints  of  the  angles  formed  at 
branching  points.  This  direction  may  lead  to  clues  as  to  how  the  3-D  space-filling  characteristics  of 
neuronal  dendrites  can  be  quantitated  in  a  meaningful  way.  This  extremely  difficult  problem  has  no 
current  solution. 

Network  Function  in  Developing  Spinal  Cord  of  the  Chick  Embryo:  This  project  began  in  FY  1989, 
under  the  direction  of  Dr.  Michael  J.  O'Donovan.  The  primary  aim  is  to  analyze  the  properties  of  spinal 
cord  neurons  and  networks  during  embryonic  development  in  the  chick  spinal  cord.  This  model  system  is 
very  favorable  for  experimental  attack,  since  reduced  preparations  in  vitro  exhibit  spontaneous  motor 
patterns  that  are  qualitatively  similar  to  those  emitted  by  intact  embryos 

Real-time  optical  imaging  of  neuronal  activity:    A  major  focus  of  the  effort  during  FY  1990  has  been 
the  successful  development  of  a  real-time  imaging  system  to  detect  changes  in  intracellular  calcium 
concentration  during  stimulated  or  spontaneous  motor  patterns  in  chick  embryos,  using  the  calcium 
indicator,  Fura-2AM  and  fluorescence  microscopy  with  video  image  processing.  Many  spinal  neurons, 
including  both  motoneurons  and  interneurons,  exhibit  phasic  changes  in  calcium  fluorescence  during 
motor  output  bursts,  enabling  identification  particularly  of  interneurons  that  may  be  involved  in  the 
generation  of  such  bursts   Active  interneurons  are  located  mainly  dorsal  to  the  lateral  motor  column, 
including  cells  in  the  dorsal  horn.  Some  neurons  exhibit  oscillations  in  calcium  signal  in  phase  with 

3-LNLQDIR 


repetitive  motor  bursts  while  others  show  more  tonic  signal  increases.  The  identity  of  the  cells  that  give 

•sitive  signals  is  under  study  using  a  combination  of  optical  imaging  and  conventional 
electrophysiological  recording  techniques  and  by  immunocytochemistry. 

Immunocvtochemistry:  We  have  found  that  cells  loaded  with  Fura-2AM  can  be  identified  by  photo- 
oxidation  and  thus  identified  in  subsequent  histological  preparations.  Cells  thus  labeled  can  then  be 
studied  using  other  reagents,  such  as  antibodies  to  cell  constituents  specific  for  neurons  and  glial  cells,  as 
well  as  putative  neurotransmitters,  calcium-binding  proteins,  etc.  This  approach  has  now  been  successfully 
applied  to  chick  embryo  tissues  and,  in  principle,  offers  the  possibility  of  precisely  identifying  the  cellular 
elements  that  participate  in  generating  motor  patterns. 

Lesion  and  stimulation  studies  of  pattern  generation:  The  results  of  the  optical  recordings  have 
been  used  to  guide  selective  lesions  to  determine  whether  "active  versus  "inactive"  areas  are  indeed 
critical  to  pattern  generation.  We  have  found  rostro-caudal  differences  in  the  ability  to  sustain  patterned 
output,  with  greater  capacity  in  segments  rostral  to  LS3  than  caudal  to  it.  Various  components  of  the 
normal  motor  patterns  can  be  affected  in  isolation  by  restricted  lesions  within  particular  regions  of  low 
thoracic  and  upper  lumbar  cord.  In  general,  the  greatest  disruption  of  pattern  generation  occurred  when 
lesions  were  made  in  the  ventro-lateral  portion  of  the  spinal  cord,  which  is  the  region  showing  the  largest 
concentration  of  cellular  activity  in  optical  recordings. 

The  entire  chick  embryo  nervous  system  can  be  sustained  for  several  hours  in  vitro,  enabling 
experiments  to  determine  the  location  and  nature  of  descending  influences  on  motor  pattern  formation. 
Electrical  stimulation  of  the  brainstem  in  stage  37  embryos  produces  patterned  motor  output,  as  does 
superf  usion  with  NMDA.  This  preparation  offers  great  promise  in  elucidating  the  mechanisms  that 
generate  motor  output  during  early  development. 

Patch  clamp  recording  from  embryonic  neurons:  We  have  used  the  whole  cell  patch  clamp 
technique  to  record  membrane  currents  from  very  small  neurons  in  both  slice  preparations  and  intact 
spinal  cords  in  vitro.  Successful  recordings  have  been  made  as  early  as  9  days  of  development,  making  it 
possible  to  examine  changes  in  specific  membrane  currents  during  stages  of  development  during  which 
motor  patterns  mature.  It  has  been  possible  to  develop  evidence  that  extensor  motoneurons  fire  because 
of  excitatory  synaptic  input  during  patterned  motor  output,  but  flexor  cells  receive  predominant  inhibition 
early  in  development,  while  excitatory  conductances  appear  somewhat  later.  There  are  also  indications 
that  other  conductances,  as  yet  unidentified,  may  also  be  involved  in  some  cells.  The  accessibility  of  this 
preparation  and  the  ability  to  maintain  it  in  vitro  offers  great  promise  for  analysis  of  normal  motor  pattern 
generation  in  a  vertebrate  nervous  system. 

Development  of  Primary  Sensory  Neurons:  An  additional  project  in  the  Section  on  Developmental 
Neurobiology  concerns  an  attempt  to  use  a  variety  of  cytochemical,  immunocytochemical,  optical,  and 
electrophysiological  techniques  to  study  the  mechanisms  that  specify  the  identity  of  primary  sensory 
neurons  and  to  direct  their  outgrowth  to  particular  targets,  both  centrally  and  peripherally.  One 
subproject,  done  using  bullfrog  tadpoles  in  collaboration  with  investigators  at  the  University  of  Pittsburgh, 
has  shown  that  re-routing  of  thoracic  sensory  afferents  to  innervate  the  forelimb  causes  the  formation  of 
central  connections  appropriate  for  forelimb  afferents.  This  suggests  that  the  peripheral  targets  of  sensory 
neurons  can  influence  their  choice  of  targets  within  the  spinal  cord.  However,  there  are  complex 
interactions  between  the  level  of  entry  of  fibers  into  the  spinal  cord  and  the  presence  or  absence  of 
adjacent  sensory  ganglia. 

Experiments  aimed  at  determining  whether  sensory  neurons  are  also  intrinsically  specified  have 
used  the  finding  that  a  subset  of  primary  sensory  neurons  in  chick  embryos  bind  soybean  agglutinin  (SBA). 
This  binding  can  be  demonstrated  in  some  sensory  neurons  removed  from  embryos  on  the  5th  day  of 
incubation,  when  few  have  innervated  peripheral  targets,  and  grown  in  tissue  culture  without  target 
tissues.  Birthdate  studies  are  underway  to  determine  whether  the  labeled  neurons  were  remnants  of  the 
original  tissue  or  were  generated  in  culture. 


4-LNLC/DIR 


High  resolution  video  microscopy  is  being  used  to  examine  the  factors  that  control  neurite 
outgrowth  from  chick  embryo  primary  sensory  neurons  grown  in  tissue  culture.  Certain  substrates,  such  as 
laminin,  permit  these  cells  to  become  bipolar,  with  one  rapidly  growing  process  and  another  growing 
more  slowly.  These  may  be  equivalent  to  the  peripheral  and  central  neurites,  respectively,  of  cells  in  the 
embryo.  We  have  found  that  the  presence  of  nearby  neurons  in  culture  promotes  the  formation  of 
neurites  at  the  site  of  contact,  suggesting  that  neuronal  membranes  may  contain  factors  that  promote 
axon  outgrowth.  The  predictability  of  this  effect  has  enabled  us  to  use  time-lapse  video  microscopy  to 
follow  the  development  of  neurites  and  their  relation  to  other  cell  constituents.  Immunocytochemical 
studies  have  shown  that  the  neurite  outgrowths  contain  microtubules  and  GAP43,  a  molecule  known  to  be 
enriched  in  growth  cones. 

These  studies,  which  are  still  at  an  early  stage  of  development,  have  considerable  promise  for 
elucidating  the  interplay  between  intrinsic  specification  of  primary  sensory  neurons  and  the  influence  of 
the  local  and  distant  environment  in  governing  their  ultimate  structure  and  function. 

Cortical  Mechanisms  of  Voluntary  Motor  Control:  Work  in  this  project  is  designed  to  increase  our 
understanding  of  the  organization  of  neuronal  systems  in  regions  of  the  cerebral  cortex,  primarily  the 
sensorimotor  cortex  and  supplementary  motor  area  (SMA)  in  primates,  that  are  associated  with  the  control 
of  voluntary  movement  and  which  project  directly  to  the  spinal  cord  and  brainstem.  The  major  emphasis  is 
on  control  of  voluntary  arm,  wrist,  and  finger  movements.  Most  of  this  research  is  done  using  non  human 
primates  (rhesus  monkeys),  which  are  intensively  trained  to  perform  specific  tasks  while  recording  from 
individual  cortical  neurons  mounted  on  chronically  implanted  chambers.  Primates  are  used  for  this 
research  because  they  exhibit  the  same  sort  of  fine  control  of  hand  and  individual  finger  movement 
present  in  humans  and  they  can  be  trained  to  perform  fine  hand  and  finger  movements  to  receive 
desirable  food  rewards.  Macaque  monkeys  readily  adapt  to  the  laboratory  situation  and  have  been  used 
extensively  for  earlier  work  on  the  hand-arm  control  by  the  motor  cortex.  The  new  data  obtained  can  thus 
be  readily  integrated  with  the  large  body  of  existing  data  on  macaque  neuroanatomy  and  motor  cortex 
physiology.  LNLC  has  pioneered  several  new  methods  for  ensuring  the  health  of  laboratory  primates  and 
freedom  from  stress  in  the  experimental  situation. 

During  FY  1990,  we  concluded  one  phase  of  our  studies  of  the  influence  of  the  SMA  on  movement 
performance,  which  was  reported  in  previous  Annual  Reports.  We  have  made  a  major  change  in  computer 
equipment  because  of  the  increasing  fragility  and  lack  of  vendor  support  for  the  original  system.  This 
retooling  has  required  a  major  investment  of  time  and  effort.  In  addition,  we  have  begun  a  new  set  of 
experiments  to  examine  the  activity  patterns  of  motor  cortex  neurons  during  bimanual  skilled  tasks.  This 
has  required  training  of  several  animals  to  perform  skilled  control  of  wrist  flexion  and  extension  with 
either  hand,  or  both  hands  together.  When  this  training  is  complete,  cortical  recording  session  swill  begin, 
probably  in  the  first  quarter  of  FY  1991 . 

Activity  in  this  project  has  also  been  directed  to  the  development  of  a  safe  and  reliable  visual 
prosthesis  for  blind  human  subjects.  This  effort  has  extended  over  many  years  in  LNLC  and  we  have  now 
determined  that  the  LNLC  "hatpin"  electrode  design,  developed  for  primate  neurophysiology,  is  suitable 
and  safe  for  chronic  implantation  in  human  subjects.  Chronic  implantation  of  multiple  hatpin  electrodes  in 
a  monkey  for  1 05  days,  with  systematic  stimulation  protocols  at  intensities  at  and  above  those  needed  for 
production  of  visual  percepts  ("phosphenes")  have  been  shown  to  cause  minimal  tissue  reaction  and  local 
damage.  Light  and  electron  microscopic  study  of  the  tissue  was  done  in  LNLC  (see  report  of  Project  Z01  NS 
02254-14).  The  electrode  system  also  proved  to  be  mechanically  sound.  LNLC  will  continue  to  collaborate 
with  members  of  the  Surgical  Neurology  Branch  and  the  Neuroprosthesis  Program,  NINDS,  in  this  project. 
According  to  the  current  schedule,  the  first  human  volunteer  will  be  implanted  in  FY  1991. 

Mechanisms  of  Repair  Following  Injury  to  Peripheral  Nerves:  The  major  focus  of  work  in  this  project 
is  to  elucidate  the  mechanisms  by  which  peripheral  nerves  are  repaired  following  injury.  We  have 
developed  a  model  system  in  which  peripheral  nerves  regenerate  through  a  gap  within  silicone  rubber 
tubes.  Motor  axons  successfully  regenerate  through  such  gaps  but  to  do  so  they  must  traverse  a  tissue 
system  formed  de  novo  within  the  silicone  tube.  The  tube  model  thus  represents  an  exaggeration  in  both 
time  and  space  of  the  situation  at  an  end-to-end  nerve  anastomosis.  Interactions  between  fibroblasts, 


5-LNLC/DIR 


blood  vessels,  Schwann  cells,  axons,  and  extracellular  matrix  in  the  tissue  cable  that  forms  across  the  gap 
are  being  studied  using  light  and  electron  microscopic  techniques. 

Schwann  cells,  which  appear  to  migrate  mainly  from  the  distal  nerve  stump,  are  essential  for  axonai 
regeneration  within  the  cable,  since  substitution  of  tendons  for  nerve  in  the  distal  stump  prevents 
regeneration.  The  Schwann  cells  within  the  cable  are  not  organized  in  columns,  nor  are  they  surrounded 
by  basement  membrane,  as  they  are  in  the  distal  stump  of  an  end-to-end  anastomosis.  We  are  studying 
whether  axonai  regeneration  precedes  or  follows  Schwann  cell  migration  at  the  proximal  stump,  and  wr.at 
happens  when  the  fibers  meet  the  mass  of  Schwann  cells  moving  into  the  cable  from  the  distal  stump. 

Complex  interactions  occur  within  the  regenerating  cable  with  respect  to  the  endoneurial  blood- 
nerve  barrier  (BNB)  In  an  end-to-end  anastomosis  of  a  cut  or  crushed  nerve,  the  initial  breakdown  of  BNB  is 
rapidly  restored  but  there  is  long-lasting  permeability  of  BNB  in  the  tissue  cable  within  silicone  tubes,  even 
when  regeneration  is  well  advanced.  Horseradish  peroxidase  (HRP)  is  used  as  a  tracer  substance  to  study 
the  breakdown  and  repair  of  the  BNB  in  cut  or  crushed  nerves.  Electron  microscopy  is  being  used  to 
examine  the  morphological  correlates  of  BNB  changes. 

Techniques  for  Making  Contact  with  the  Nervous  System:  As  in  the  past,  this  project  includes  all 
LNLC  activities  related  to  the  development,  design,  and  fabrication  of  instrumentation,  specialized 
mechanical  equipment,  specialized  electrodes,  and  transducer  devices  used  to  support  the  research  work 
of  LNLC,  as  well  as  the  development  of  computer  software  necessary  to  handle  multiple  simultaneous 
streams  of  data  from  on-line  experiments.  The  advent  of  optical  recording  methods  and  the  combination 
of  these  with  electrophysiological  recording  and  stimulation  has  required  development  of  sophisticated, 
special-purpose  amplifiers,  tissue  chambers,  microscope  stands,  and  other  ancillary  devices   The  software 
necessary  to  store,  retrieve,  and  analyze  thousands  of  video  images  represents  a  major  effort  under  this 
project. 

Virtually  all  staff  members  of  LNLC  participate  in  one  or  more  aspects  of  this  project,  both  as  an 
adjunct  to  their  own  research  and  as  a  way  to  share  the  fruits  of  their  efforts  with  other  staff  members  and 
projects.  Many  of  the  techniques  and  instruments  developed  in  LNLC  are  new  and  without  commercial 
counterpart.  In  such  cases,  LNLC  staff  continue  to  provide  assistance  to  other  scientists  at  NIH  and  at  other 
institutions  around  the  world  who  request  information  and  advice  about  specific  data  acquisition  and 
processing  problems.  An  example  is  the  current  collaborative  effort  to  develop  a  workable  visual 
prosthesis.  The  centerpiece  of  this  project  is  the  hatpin  electrode  array  for  intracortical  microstimulation, 
which  was  developed  and  refined  over  many  years  by  LNLC  engineers  to  fit  the  requirements  of 
fundamental  research  projects.  Without  the  hatpin  electrode,  there  would  be  no  visual  prosthesis.  In 
order  to  be  used  in  humans,  we  have  had  to  refine  the  design  of  the  cable  used  to  connect  the  electrode 
with  the  outside  world.  This  required  fabrication  of  a  special  wire,  with  multiple  strands  of  stainless  steel 
for  strength  and  a  single  strand  of  gold  wire,  to  serve  as  the  flexible  link  between  the  cable  and  the 
electrode  itself.  We  have  also  been  engaged  in  evaluating  the  properties  of  the  activated  iridium  surface 
of  the  electrode  tip  after  it  is  subjected  to  the  rigorous  sterilization  procedures  needed  for  human 
implants. 


6-LNLC/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  01686-22  LNLC 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (BO  characters  or  less    Title  must  fit  on  one  line  betvteen  the  borders.) 

Motor  Control  Systems  in  the  Spinal  Cord 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (hame.  title,  laboratory,  and  institute  affiliation) 

PI:  R.E.Burke,  M.D.  Chief  LNLC,  NINDS 

Others:    A.K.  Moschovakis,  M.D.,  Ph.D.  Staff  Fellow  LNLC,  NINDS 

G.N.  Sholomenko,  Ph.D.  Visiting  Fellow  LNLC,  NINDS 


COORPERATING  UNITS  (ifny) 


LAB/BRANCH 

Laboratory  of  Neural  Control 


SECTION 

Section  on  Neural  Mechanisms 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


2.25 


PROFESSIONAL: 


1.65 


OTHER: 


0.6 


CHECK  APPROPRIATE  BOX(ES) 

I      I   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  [T]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  project  is  designed  to  provide  information  about  the  structure  and  function  of  neuronal 
mechanisms  in  the  mammalian  spinal  cord  which  produce  and  control  movement.  These  mechanisms 
include  reflex  pathways  that  convey  sensory  information  from  primary  afferents  to  alpha  motoneurons, 
interactions  between  different  reflex  pathways,  modulation  of  information  flow  through  reflex 
pathways  by  supraspinal  descending  systems,  and  the  production  of  autonomous  rhythmic  activity  by 
central  pattern  generators  within  the  spinal  cord.  Electrophysiological,  neuroanatomical,  and  computer 
modeling  approaches  are  used.  Recent  work  has  emphasized  examination  of  the  modulation  of 
transmission  through  excitatory  cutaneous  reflex  pathways  by  the  spinal  central  pattern  generator  for 
locomotion  in  order  to  clarify  the  organization  of  spinal  interneurons  that  control  the  basic  patterning  of 
muscle  activation  during  locomotion  in  the  cat. 


7-LNLODIR 


pms  |M0  (Raw.  1.&4I 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  01687-22  LNLC 


PERIOD  COVERED 

Octoberl,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Techniques  for  Making  Connections  with  the  Nervous  and  Musculoskeletal  Systems 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:             M.J.Bak  Electronics  Engineer  LNLC,  NINDS 

Others:    RE.  Burke,  M.D.  Chief  LNLC,  NINDS 

G.M.  Dold  Engineering  Technician  LNLC,  NINDS 

M.J.O'Donovan,  M.B.Ch.B.  Visiting  Scientist  LNLC,  NINDS 

E.M.Schmidt,  Ph.D.  Biological  Engineer  LNLC,  NINDS 

W.J.Yee  Biological  Engineer  LNLC,  NINDS 


COORPERATING  UNITS  (if  any) 

Fundamental  Neurosciences  Program,  NINDS  (FT.  Hambrecht) 


LAB/BRANCH 

Laboratory  of  Neural  Control 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


1.0 


PROFESSIONAL: 


0.20 


OTHER: 


0.8 


CHECK  APPROPRIATE  BOX(ES) 

]   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  0  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  is  intended  to  develop  techniques  and  instrumentation  for  the  acquisition  and  processing 
of  neuroelectric  signals  from  the  central  and  peripheral  nervous  system  in  acute  and  chronic 
neurophysiological  preparations.  Because  of  this  laboratory's  continuing  interest  in  sensorimotor  neural 
activity  during  unrestrained  movements,  the  project  also  includes  development  and  fabrication  of 
chronically  implantable  mechanical  transducers,  catheters,  and  connectors.  Due  to  the  laboratory's  new 
interests  in  doing  research  on  isolated  preparations,  such  as  the  spinal  cord  of  chick  embryos,  much 
development  work  has  been  devoted  to  improving  techiques  associated  with  the  recording  and  visual 
observations  associated  with  fluorescence  changes  of  these  preparations.  Also  included  is  the 
development  of  computer  programs  of  general  utility  for  acquisition  and  analysis  of  neuroelectric  and 
mechanical  records,  as  well  as  of  neuroanatomical  material. 


8-LNLC/DIR 


PHS6M0(Re»    1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  IMS  01688-22  LNLC 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJ  E  CT  (80  characters  or  lesi.   Title  must  fit  on  one  line  between  the  borders.) 

Cortical  Mechanisms  of  Voluntary  Motor  Control 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (nlame.  title,  laboratory,  and  institute  affiliation) 

Biological  Engineer  LNLC,  NINDS 

Electronics  Engineer  LNLC,  NINDS 

Engineering  Technician  LNLC,  NINDS 

Director,  Neuroprosthesis  Program  FNP,  NINDS 

Neurosurgeon  SNB,  NINDS 

Physiologist  LNLC,  NINDS 

Pyschologist  LNLC,  NINDS 


PI: 

E.M.Schmidt,  Ph.D. 

Others: 

M.J.  Bak 

G.M.  Dold 

FT.  Hambrecht,  M.D 

C.  Kufta,  M.D. 

J.S  Mcintosh 

M.  Pomerantz 

COORPERATING  UNITS  (If any) 

Fundamental  Neurosciences  Program,  NINDS  (FT.  Hambrecht);  Neuroprosthesis  Research  Program, 
NINDS;  Surgical  Neurology  Branch  (Dr.  Kufta,  Dr.  Oldfield) 


LAB/BRANCH 

Laboratory  of  Neural  Control 


SECTION 

Section  on  Neural  Mechanisms 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


2.8 


PROFESSIONAL: 


08 


OTHER: 


20 


CHECK  APPROPRIATE  BOX(ES) 

I  x  |   (a)  Human  subjects 
]  (a1)  Minors 
]  (a2)  Interviews 


J  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  is  designed  to  investigate  the  spatial  distribution  and  functional  properties  of  cortical 
neuron  'colonies'  in  the  primate  motor  cortex  and  supplementary  motor  area  that  project  to  the  spinal 
cord  and  are  associated  with  individual  muscles  or  closely  related  groups  of  muscles,  as  well  as  the  activity 
of  neurons  in  such  colonies  during  defined  voluntary  motor  behaviors.  Cortical  cell  discharge  patterns 
during  normal  movements  are  evaluated  in  terms  of  EMG  patterns,  and  their  responses  to  small  loading 
and  unloading  torque  perturbations.  Spinal  cord  location  of  motoneurons  innervating  selected  forelimb 
muscles  and  termination  patterns  in  the  spinal  cord  and  brain  stem  of  sensory  receptors  within  these 
muscles  are  studied  using  anterograde  and  retrograde  tracing  methods. 


9-LNLGDIR 


PHS  6040  (Rev   1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02079-17  LNLC 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJ  ECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Models  of  Neurophysiological  Systems 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  W.B.Marks,  Ph.D.  Research  Physiologist  LNLC,  NINDS 


COORPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Neural  Control 


SECTION 

Section  on  Neural  Mechanisms 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 

1.30 


PROFESSIONAL: 

0.8 


OTHER: 

0.5 


CHECK  APPROPRIATE  BOX(ES) 

Q   (a)  Human  subjects  ^]  (b)  Human  tissues  [T]  (c)  Neither 

]  (a1)  Minors 

J   (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

As  quantitative  data  become  available  for  a  particular  form  or  function  in  the  nervous  system,  it  is 
advisable  to  attempt  to  assimilate  the  information  in  a  comprehensive  model  of  the  underlying 
mechanisms  and  their  interactions.  This  project  consists  in  the  development  of  such  models  and  the 
necessary  analytical  and  mathematical  techniques  for  their  implementation  and  testing  in  several  areas 
of  experimental  investigation  carried  out  by  LNLC  members  and  in  other  laboratories. 

Drs.  Marks,  Burke,  and  Ulfhake,  guided  by  the  discovery  described  in  last  year's  Annual  Report,  that 
for  motoneuronal  dendrites,  local  dendritic  diameter  largely  determines  the  rate  of  branching, 
terminating,  and  tapering,  showed  that  (a)  these  rules,  augmented  by  the  empirical  distributions  of 
daughter  branch  diameter,  satisfactorily  predict  the  branch  length  distributions  of  dendritic  trees  of 
alpha  and  gamma  motoneurons  to  the  gastrocnemius  and  soleus  muscle,  using  the  empirical  branching 
and  tapering  rates  of  these  four  neuron  types;  (b)  the  meanderinqs  of  the  dendrites  between  branch 
points  can  be  described  as  random  walk  constrained  to  have  a  fractal  dimension  of  about  1 .2;  (c)  the 


membrane  area  beyond  any  branch  obeys  a  simple  law  implied  by  the  rules;  and  (d)  to  fit  real  neurons, 
the  rules  must  explicitly  constrain  the  growth  beyond  any  branch  point  not  to  exceed  that  allowed  by  the 


diameters  of  the  parent  branches. 


10-LNLC/DIR 


PHS  6040  (Hew.  I'M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02160-16  LNLC 


°ERIOD  COVERED 

,  October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.    Title  must  tit  on  one  line  between  the  borders  ) 

Intrinsic  Properties  of  Motor  Units 


PRINCIPAL  INVESTIGATOR  (List  other  profession*!  personnel  below  the  Principal  Investigator.)  (Name,  trtie.  laboratory,  and  institute  affiliation) 

PI:  R.E.Burke,  M.D.  Chief,  LNLC  LNLC,  NINDS 

Others:    W.B.  Marks,  Ph.D.  Research  Physiologist  LNLC,  NINDS 

A.K.  Moschovakis.  M.D. .Ph.D.  Staff  Fellow  LNLC,  NINDS 


COORPERATING  UNITS  Of  any) 

(1)  Mathematics  Research  Branch,  NIDDK 


LAB/BRANCH 

Laboratory  of  Neural  Control 


SECTION 

Section  on  Neural  Mechanisms 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


1.25 


PROFESSIONAL: 


085 


OTHER: 


04 


CHECK  APPROPRIATE  BOX(ES) 

1      1   (a)  Human  subjects 
]  (a1)  Minors 

J   (a2)  Interviews 


I      |  (b)  Human  tissues  |jT]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  designed  to  provide  information  on  the  electrophysiological  and  morphological 
properties  of  alpha  motoneurons  and  of  the  muscle  fibers  (muscle  units)  innervated  by  them.  The 
properties  of  gamma  motoneurons  are  also  being  investigated.  Methods  include  electrophysiological 
techniques  of  intra-  and  extracellular  recording,  mechanical  recording  of  muscle  unit  properties, 
neuroanatomical  techniques  of  intracellular  injection  of  horseradish  peroxidase  to  label  individual, 
functionally  identified  motoneurons,  retrograde  transport  of  lectin-conjugated  horseradish  peroxidase 
to  label  motor  nuclei,  and  computer  modeling  studies  to  analyze  the  experimental  data  produced 
Recent  work  has  emphasized  detailed  analysis  of  the  dendritic  morphology  of  alpha-  and  gamma- 
motoneurons,  with  development  of  a  computer  model  to  describe  the  structure  of  these  dendrites  using 
a  minimum  number  of  parameters 


11  -LNLC/DIR 


PHS  bMO  IRev    l/g4) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02254-14  LNLC 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  ($0  characters  or  less.   Title  must  fix  on  one  line  between  the  borders.) 

Repair  of  Injured  Nervous  Tissue  with  Foreign  Grafts 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (htame,  title,  laboratory,  and  institute  affiliation) 

PI:  A.  A.  Zalewski.M.D.  Medical  Officer  LNLC,  NINDS 

Others:    N.  A.  Azzam,  Ph.D.  Special  Expert  LNLC,  NINDS 

R.  N.Azzam  Histopathology  Tech.  LNLC,  NINDS 

J.D.  Ziemnowicz  NIH  Special  Volunteer  LNLC,  NINDS 


COORPERATING  UNITS  (if  any) 

CNS  Disorders  Research,  The  Upjohn  Co.,  Kalamazoo,  Ml  (L.R.  Williams) 


LAB/BRANCH 

Laboratory  of  Neural  Control 


SECTION 

Section  on  Neuronal  Regeneration 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


3.8 


PROFESSIONAL: 


2.00 


OTHER: 


1.8 


CHECK  APPROPRIATE  BOX(ES) 

I      |   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  QT]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Experiments  are  being  performed  in  rats  to  determine  how  a  new  peripheral  nerve  segment 
develops  in  a  silicone  tube.  We  believe  the  nerve  formation  in  an  artificial  tube  exaggerates  the  events  at 
a  nerve  anastomosis  site  in  that  they  occur  over  a  longer  distance  and  time.  Studies  are  in  progress  to 
analyze  the  early  cellular  events  that  take  place  during  the  initial  tissue  cable  formation  and  axonal 
regeneration.  We  have  prepared  cables  which,  by  light  microscopy,  have  demonstrated  that  cells 
migrate  from  both  ends  of  nerve  stumps  inserted  into  opposite  ends  of  a  14  mm  long  tube.  Our  focus  of 
interest  was  to  find  a  time  at  which  migrating  cells  had  not  yet  met  in  the  middle  of  the  tube.  We  are 
now  performing  an  electron  microscopic  analysis  of  the  growing  tips  of  axons  in  relation  to  the  non- 
neuronal  cells  (e.g.,  Schwann  cells)  and  matrix  in  the  cable.  We  have  continued  our  studies  on  the 
development  and  regeneration  of  the  permeability  barriers  of  nerve.  Previously,  we  found  that  whereas 
the  endoneurial  blood-nerve  barrier  (BNB)  of  normal  and  in  situ  regenerated  nerve  is  impermeable  to  the 
vascular  tracer  horseradish  peroxidase  (HRP),  the  BNB  of  nerve  segments  in  tubes  is  not.  However,  during 
the  early  phases  of  nerve  degeneration  and  regeneration  in  situ,  there  is  a  marked  increase  in  BNB 
permeability.  Accordingly,  we  have  begun  to  examine  crushed  and  cut  nerves  in  rats  to  elucidate  the 
cause  and  initiating  event  of  the  increased  vascular  permeability.  We  found  by  light  microscopy  that, 
within  1-2  weeks,  injured  nerves  are  permeable  to  HRP.  It  is  of  interest  that  we  did  not  see  blood-borne 
cells  migrating  through  vascular  walls,  which  might  have  accounted  for  the  increased  HRP  permeability 
An  electron  microscopic  investigation  is  in  progress  to  determine  the  route  of  HRP  passage  through  the 
endothelium  of  the  endoneurial  blood  vessels.  In  a  collaborative  project,  we  have  studied  the  effect  of 
chronic  implantation  and  stimulation  of  microelectrodes  on  the  visual  cor\ex  of  a  monkey.  Neuronal  loss 
was  minimal.  The  pia  mater  and  astocytic  foot  processes  reacted  to  ensheath  the  microelectrodes, 
isolating  them  from  the  surrounding  nervous  tissue.  The  stimulated  but  not  unstimulated  electrodes 
elicited  a  macrophage  reaction  that  included  some  giant  cells.  We  feel  that  the  pathology  observed  is 
not  severe  enough  to  contraindicate  the  use  of  our  visual  prosthesis  in  blind  people. 


12-LNLC/DIR 


PHS  6040  (Re..  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02787-02  LNLC 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders  J 

Analysis  of  Network  Function  in  the  Developing  Spinal  Cord  of  the  Chick  Embryo. 


PRINCIPAL  IN VE  STIG ATOR  (list  other  professional  personnel  below  in*  Principal  Investigator )  (Name,  title,  laboratory,  and  insniufe  affiliation) 

PI:  M.J.O'Donovan,  M.B.Ch.B.  Visiting  Scientist  LNLC,  NINDS 

Other:      M.  Antal,  M.D.,  Ph.D.  Visiting  Associate  LNLC,  NINDS 


S.  Ho,  Ph.D. 
W.B.Marks,  Ph.D. 
E.  Sernagor,  Ph.D. 
G.  Sholomenko,  Ph.D. 


Visiting  Associate 
Visiting  Fellow 
Research  Physiologist 
Visiting  Fellow 
Visiting  Fellow 


LNLC,  NINDS 
LNLC,  NINDS 
LNLC,  NINDS 
LNLC,  NINDS 


COORPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Neural  Control 


SECTION 

Section  on  Developmental  Neurobiology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


5.6 


PROFESSIONAL: 


34 


OTHER: 


22 


CHECK  APPROPRIATE  BOX(ES) 

J   (a)  Human  subjects 
]  (al)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  QT]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  project  is  concerned  with  analyzing  the  development  and  function  of  spinal  networks  in  the 
lumbosacral  cord  of  the  chick  embryo.  One  focus  of  the  study  is  on  the  synaptic  organization  of 
motoneurons  with  particular  emphasis  on  sensorimotor  and  recurrent  pathways   A  second  interest  is  in 
analyzing  the  cellular  and  network  mechanisms  responsible  for  the  genesis  of  spontaneous  motor 
activity.  All  experiments  are  performed  on  an  isolated  preparation  of  the  spinal  cord  which  is  maintained 
in  vitro. 


13-LNLC/DIR 


PHS  6O40  (Slev.  1  tW) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02788-02  LNLC 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Development  of  Primary  Sensory  Neurons 


PRINCIPAL  INVESTIGATOR  IList  other  professional  personnel  below  the  Principal  Investigator.)  (flame,  title,  laboratory,  and  institute  affiliation) 

PI:  C.L.Smith,  Ph.D.  Senior  Staff  Fellow  LNLC,  NINDS 

Other:      E.  Munro  Biologist  LNLC,  NINDS 

E.  Frank,  Ph.D.  Univ.  PA 


COORPERATING  UNITS  (Ifany) 

University  of  Pittsburgh 


LAB/BRANCH 

Laboratory  of  Neural  Control 


SECTION 

Section  on  Developmental  Neurobiology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 

2.0 


PROFESSIONAL: 

1.00 


OTHER: 

1.0 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [x~1  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  general  aim  of  this  research  is  to  provide  information  about  the  development  mechanisms 
responsible  for  the  specificity  of  the  synaptic  connections  formed  by  primary  sensory  neurons  in  the 
spinal  cord  during  embryonic  development  and  to  gain  insight  into  factors  that  might  promote 
regeneration  of  functionally  appropriate  connections  following  injury.  Developing  sensory  neurons 
innervate  peripheral  targets  before  they  form  central  connections  and  it  has  been  suggested  that  the 
central  connections  of  sensory  neurons  actually  are  determined  by  their  peripheral  targets.  This 
hypothesis  was  tested  by  forcing  sensory  neurons  in  bullfrog  tadpoles  to  innervate  a  foreign  peripheral 
target  and  then  examining  the  central  connections  of  these  neurons  by  anatomical  and 
electrophysiological  methods.  The  results  support  the  suggestion  that  central  connections  of  sensory 
neurons  are  influenced  by  their  targets  but  also  suggest  that  they  depend  on  the  axial  level  at  which  the 
central  processes  enter  the  spinal  cord  and  on  the  presence  or  absense  of  ganglia  in  adjacent  segments. 
The  formation  of  appropriate  connections  by  sensory  neurons  requires  that  they  develop  terminal  arbors 
in  specific  areas  of  the  spinal  cord.  In  order  to  identify  the  environmental  cues  that  guide  sensory  axons 
and  that  induce  them  to  form  branches,  we  are  using  videomicroscopy  to  determine  how  the  branching 
patterns  of  neurons  growth  in  vitro  are  influenced  by  contacts  with  other  cells,  purified  substrate 


molecules  and  growth  factors.  Our  initial  studies  have  focused  on  early  stages  in  axon  outgrowth  and 
the  establishment  of  neuronal  polarity. 


14-LNLC/DIR 


PHS6M0(Rev    1*4) 


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ANNUAL  REPORT 


October  1,  1989  through  September  30,  1990 

Laboratory  of  Neurobiology 

Basic  Neurosciences  Program,  DIR 

National  Institute  of  Neurological 

Disorders  and  Stroke 

Table  of  Contents 


RESEARCH  SUMMARY  1-9 

PROJECT  REPORTS 

Permeability  of  Cellular  Layers  in  the  Vertebrate  Nervous  System 

Z01  NS  01442-24  LN  10 

Structural  Basis  of  Synaptic  Transmission  and  Development 

Z01  NS  01881-20  LN  11 

Structure  of  Neuronal  Cytoplasm 

Z01  NS  02551-09  12 

Distribution  of  Mobile  and  Structural  Components  at  Chemical  Synapses 
Z01  NS  02610-07  LN  13 

Proteins  Involved  in  Axonal  Transport 

Z01  NS  02700-05  LN  14 

Membrane  Structure  of  Astrocytes 

Z01  NS  01805-22  LN  15 

Regeneration  Specificity  in  Transplanted  Neural  Tissue 

Z01  NS  02086-17  LN  16 

The  Blood-Brain  Barrier 

Z01  NS  02144-16  LN  17 


i-LN/DIR 


ANNUAL  REPORT 
October  1, 1989  through  September  30, 1990 
Laboratory  of  Neurobiology,  DIR 
Basic  Neurosciences  Program 
National  Institute  of  Neurological  Disorders  and  Stroke 
Thomas  S.  Reese,  M.D.,  Chief 

The  Laboratory  of  Neurobiology  has  two  Sections,  the  Section  on  Structural 
Cell  Biology  and  the  Section  on  Structural  Plasticity.   The  Section  on  Structural  Cell 
Biology  uses  modern  structural  and  biochemical  techniques  to  investigate  basic  cell 
biological  problems  germane  to  an  understanding  of  the  function  of  nerve  cells;  the 
Section  on  Brain  Structural  Plasticity  applies  these  and  other  appropriate 
approaches  to  problems  of  both  fundamental  and  clinical  importance  in  the 
mammalian  central  nervous  system  (CNS),  emphasizing  problems  related  to 
regeneration  and  response  to  injury.   Current  emphasis  of  the  Section  on  Structural 
Cell  Biology  is  on  the  mechanism  of  axoplasmic  transport,  axonal  growth,  and 
synaptic  function  while  the  Section  on  Structural  Plasticity  is  investigating  factors 
which  promote  establishment  of  blood-brain  barrier  function  and  neural 
connections  in  neural  tissues    implanted  in  the  brain. 

It  is  the  Section  on  Structural  Cell  Biology  that  discovered  the  molecular 
basis  of  the  directed  organelle  movements  underlying  fast  axoplasmic  transport. 
The  translocator  protein  for  fast  anterograde  transport  belongs  to  a  new  class  of 
motility  proteins  that  we  have  named  kinesin.    Kinesin  (with  ATP)  is  able  to  mimic 
the  movements  serving  fast  anterograde  transport.    Kinesin  also  occurs  in  many 
non-neuronal  cells,  and  appears  to  be  a  general  and  fundamental  effector  of  cell 
motility. 

Movement  induced  by  kinesin,  however,  is  only  unidirectional  with  respect 
to  microtubule  polarity,  which  is  away  from  the  neuronal  cell  body;  therefore, 
kinesin  must  mediate  anterograde  but  not  retrograde  axonal  transport.   There  is  a 
separate  retrograde  translocator  which  we  have  now  characterized  as  a  species  of 
cytoplasmic  dynein.    The  anterograde  kinesin-induced  movements  and  the 
retrograde  dynein-induced  movements  can  be  independently  inhibited,  suggesting 
that  they  function  as  separate  motors.   Since  organelles  can  bind  both  kinesin  and 
dynein,  the  next  question  is  how  kinesin  or  dynein  activation  is  organized  on  the 
organelle  surface. 

1-LN/DIR 


This  question  can  be  approached  using  a  new  electron  microscope  that  is  now 
available  as  a  result  of  a  major  instrumentation  development  project.    This 
instrument  is  the  field-emission  scanning  transmission  electron  microscope 
(STEM)  for  high  resolution  quantitative  mass  mapping  and  elemental 
microanalysis  of  rapidly  frozen,  isolated  macromolecules  (as  well  as  tissue 
cryosections).   This  microscope  allows,  for  the  first  time,  kinesin  and  other  protein 
motors  to  be  visualized  directly  on  the  surfaces  of  microtubules  and  organelles.   It  is 
equipped  with  a  cryotransfer  cold  stage,  a  highly  efficient  parallel  electron  energy 
loss  spectrometer  (PEELS),  and  an  ultrathin-window  energy-dispersive  x-ray 
spectrometer  which  permits  obtaining  low-dose  (<103  electron/ nm2),  dark-field 
molecular  mass  maps  with  single  electron  sensitivity,  as  well  as  performing 
sensitive  elemental  analysis  of  individual  macromolecules.    This  STEM  was  used  to 
determine  at  2  nm  resolution  the  shapes  and  mass  distributions   of  purified  squid 
brain  kinesin  molecules— both  free  and  bound  to  microtubules.    Individual  kinesin 
molecules  appeared  as  asymmetrical  dumbbell-shaped  structures  (47-51  nm  long, 
MW  =374  ±  14  kD),  with  a  large  head,  stalk,  and  smaller  globular  foot.  This 
structure  is  quantitatively  consistent  with  models  derived  from  biochemistry  and 
molecular  biology.   Maps  of  kinesin  bound  to  purified,  taxol-stabilized  bovine 
microtubules  (MW=19.2  kD/A)  by  AMP-PNP  revealed  that  both  ends  of  kinesin 
bind  to  microtubules,  indicating  that  individual  kinesin  molecules  provide  the 
bridges  responsible  for  bundling  microtubules  in  AMP-PNP.   These  results  provide 
the  first  direct  evidence  for  cross-bridging  of  microtubules  by  single  kinesins  and 
suggest  that  kinesins  in  cells  might  also  translocate  microtubules  and  therefore 
have  some  role  in  both  slow  and  fast  axonal  transport. 

Metabolic  uncouplers  of  various  classes  (e.g.,  DNP,  CCCP,  valinomycin) 
uniformly  block  organelle  movements  along  microtubules  in  vitro,  but  not 
kinesin  or  dynein  induced  movements  of  latex  beads.   Since  this  block  is  not  a  direct 
effect  on  the  translocators,  it  appears  that  an  ionic  gradient  across  the  organelle 
membrane  is  responsible  for  programming  an  organelle  to  go  in  the  anterograde  or 
retrograde  direction.   The  nature  of  this  ionic  gradient  is  now  being  investigated. 
The  structure  and  dynamic  properties  of  other  biological  motors  are  also  being 
studied  for  comparison  with  the  axonal  transport  motors.   The  bacterial  flagellar 
motor  in  E  coli  has  also  been  shown  to  depend  on  interactions  of  the  flagellar 
structure  with  membrane  proteins  although  these  motors  are  driven,  rather  than 
controlled  by,  ionic  gradients.   This  motor  system,  like  the  axonal  motor  system  can 
also  switch  direction  of  translocation.   A  new  structural  component  of  the  flagellar 

2-LN/DIR 


motor  was  discovered  which  has  led  us  to  propose  a  novel  structural  model  for  this 
motor.   A  molecular  genetic  analysis  of  the  new  component  is  underway,  which 
may  lead  to  an  understanding  of  its  function,  possibly  including  the  directional 
switching. 

The  function  of  the  microtubule-based  transport  system  in  vivo  is  also  under 
further  investigation.    Since  microtubules  do  not  extend  the  full  length  of  the  axon, 
reconstructions  of  serial  section  electron  micrographs  of  vertebrate  axons  and 
growth  cones  showed  the  distribution  of  organelles  contacting  individual 
microtubules.    Organelles  contacting  microtubules  are,  judging  from  previous  work 
on  the  squid  axon,  in  transport.   Each  organelle  in  the  vertebrate  axon  may  contact 
several  microtubules,  so  it  is  the  continuous  microtubule  bundles  which  constitute 
the  transport  pathways  down  the  axon.   An  analysis  of  microtubule  distributions  in 
CNS  cell  bodies  is  underway.   The  number  of  organelles  in  transport  along  these 
microtubules  is  being  counted  and  it  is  apparent  that  there  are  different  populations 
of  microtubules,  some  supporting  organelles  transport  in  the  cell  body  and  some 
not. 

A  preparation  of  synapses  on  cell  bodies  in  the  mammalian  brain  that  could 
be  studied  with  rapid  freeze  techniques  was  developed.   The  rostral  anteroventral 
cochlear  nucleus  (AVCN)  of  the  chinchilla   provides  a  preparation  in  which 
neuronal  cell  bodies  and  synapses  in  the  CNS  can  be  examined  after  direct  freezing 
and  freeze-substitution  of  rapidly  excised  brain  stem  slices.  The  four  types  of 
synaptic  terminals  known  to  be  in  the  AVCN  were  distinguished  and  correlated 
with  four  types  of  terminals  previously  reported  after  chemical  fixation.   Since  the 
transmitters  for  each  of  the  four  types  of  terminals  have  been  specified,  the 
transmitter  type  could  be  correlated  with  the  detailed  structure  of  the  postsynaptic 
density  in  each  chemical  type  of  synapse.  Two  types  of  filamentous  components- 
short  vertical  projections  from  the  postsynaptic  membrane  and  long  filaments 
protruding  from  these  projections— comprised  the  basic  structure  of  the  postsynaptic 
density;  the  sizes  and  distribution  of  these  components  differed  specifically  in  each 
type  of  terminal.   Thus,  the  freeze-substitution  images  have  provided  new 
information  about  structural  differences  between  receptor  arrays  and  associated 
cytoskeletal  components  at  different  types  of  CNS  synapses. 

As  part  of  a  continuing  interest  in  synaptic  secretion  by  exocytosis,  the 
formation  of  membrane  pores  induced  by  electroporation  was  studied  with  rapid 
freeze  techniques.    This  approach  provided  detailed  information  on  a  millisecond 
time  scale  regarding  the  opening  of  membrane  pores  of  our  initial  test  preparation, 

3-LN/DIR 


human  erythrocytes.   A  surprising  finding  was  that  the  pores  continued  to  open 
over  the  first  tens  of  milliseconds  before  reaching  a  maximum  size,  suggesting  that 
membrane  interactions  with  the  cytoskeleton  were  important  in  pore  formation. 

Direct  freezing,  cryosectioning  and  quantitative  x-ray  microanalysis  had 
previously  been  used  to  characterize  the  calcium-sequestering  activity  of  the 
endoplasmic  reticulum  (ER)  of  dendritic  spines  and  shafts  of  Purkinje  cells,  thereby 
demonstrating  a  role  for  these  organelles  in  intracellular  calcium  regulation. 
Presently,  the  route  of  entry  of  extracellular  calcium  remains  uncertain.    However, 
this  question  and  others  could  be  resolved  by  pharmacologic  experiments  which  are 
beyond  the  scope  of  the  present  brain  slice  preparation.  Consequently,  an  effort  has 
been  mounted  to  develop  organotypic  brain  slice  cultures  that  can  be  used  for  the 
more  complex  manipulations  of  calcium  that  are  required.    Such  cultures  of  mouse 
hippocampus  and  cerebellum  are  now  becoming  available. 

Parallel  efforts  are  directed  at  developing  fluorescent  dye  techniques  to 
investigate  the  distributions  of  internal  calcium  in  living  cells  and  cell  fractions 
where  the  time  course  of  calcium  movements  can  also  be  studied.   The  calcium 
indicator  dye  fluo-3  has  been  loaded  into  the  ER  of  a  neurosecretion  model,  the  sea 
urchin  egg  cortex,  allowing  ER  calcium  uptake  and  release  to  be  monitored  directly. 
An  RNA  staining  dye,  thiazole  orange,  stains  ribosomes  on  the  same  cortical  ER 
and  in  cultured  cells,  thereby  localizing  the  protein-synthesizing  regions  of 
cytoplasm.   These  same  regions  are  disorganized  by  microtubule  depolymerization. 
Confocal  microscopy  has  been  useful  for  studying  the  three-dimensional 
organization  of  ER  in  living  cells,  and  offers  an  opportunity  to  observe  the  response 
of  these  organelles  to  morphogenetic  and  developmental  changes  in  cell  shape. 

Recent  results  on  actively  myelinating  oligodendrocytes  in  the  CNS  have 
demonstrated  that  a  specific  isoform  (the  large,  72  kD  form)  of  the  myelin-associated 
glycoprotein  (MAG)  is  involved  in  receptor-mediated  uptake  and  retrograde 
transport  from  the  periaxonal  region  of  the  oligodendrocyte  to  the  cell  body.  Now, 
new  light  and  electron  microscopic  immunocytochemical  studies  on  optic  nerve 
and  isolated  sciatic  nerve  have  indicated  that  the  distribution  and  polarity  of 
microtubule  bundles  in  myelin-forming  cells  are  consistent  with  organelle 
transport  on  microtubules,  and  that  depolymerization  of  microtubules  reversibly 
blocks  the  transport  of  myelin  proteins  from  the  Golgi  apparatus.   These  results 
suggest  that  in  oligodendrocytes  and  Schwann  cells,  as  in  neurons,  transport  on 
microtubules  is  the  basis  of  directed  vesicular  transport. 


4-LN/DIR 


Light  and  electron  immunocytochemical  techniques  have  also  proven  useful 
for  studying  the  biogenesis  of  the  excitation-contraction  (E-C)  coupling  system  in 
skeletal  muscle.   In  normal  muscle,  the  ai  and  the  0:2  subunits  of  the 
dihydropyridine  receptor  have  been  colocalized  in  the  junctional  membrane  of  the 
T-tubule  system,  thus  providing  evidence  for  the  close  association  of  these 
polypeptides  in  the  E-C  coupling  complex.   Furthermore,  ankyrin  (a  protein 
thought  to  link  transmembrane  proteins  to  the  cytoskeleton)  has  been 
demonstrated  in  the  triad,  suggesting  a  possible  role  for  this  protein  in  the 
organization  of  this  structure.   The  localization  of  dihydropyridine  receptors  in 
normal  muscle  contrasts  with  the  expression  and  association  of  these  proteins  in 
dysgenic  (mdg/mdg)  muscle  in  vitro,  where  the  virtual  absence  of  the  oq  subunit  is 
associated  with  disorganization  of  the  (X2  subunit.  To  study  organelle  biogenesis  of 
the  T-tubule  system,  the  fluorescent  lipid  probe  [DiIC16,(3)]  has  been  used,  in 
conjunction  with  antibodies  against  T-tubules  and  plasma  membranes,  for  the 
visualization  of  T-tubules  in  living  and  fixed  cultured  muscle  cells.   These  studies 
have  shown  that  T-tubules  and  plasma  membrane  are  formed  independently  and 
that  exchange  of  specific  components  between  the  two  connected  membrane 
compartments  is  restricted. 

Membrane  renewal  is  an  important  feature  of  homeostasis  in  nondividing 
cells  such  as  neurons,  and  is  especially  prominent  in  the  light-receptive  membranes 
of  photoreceptors  which  is,  therefore,  a  suitable  model  system  for  study  of  this 
phenomenon.    Turnover  of  the  photoreceptive  membranes  was  studied  in  Limulus 
photoreceptors  maintained  in  vitro.    These  experiments  have  shown  that  there  are 
two  classes  of  ventral  photoreceptors  which  differ  in  size,  morphology,  and 
rhabdom  renewal.    Rapid,  light-stimulated  turnover  of  the  photoreceptive 
membranes  was  observed  in  the  large  photoreceptors,  but  not  in  small 
photoreceptors.   Experiments  designed  to  examine  the  role  of  the  cytoskeleton  in 
rhabdom  turnover  showed  that  light  exposure  reduced  the  numbers  of  phalloidin- 
labeled  actin  filaments  in  the  rhabdoms  of  large,  but  not  small  photoreceptors. 

The  Section  on  Brain  Structural  Plasticity  has  three  primary  objectives.    The 
first  is  to  characterize  the  neuronal  differentiation  of  PC12  pheochromocytoma  cells 
infected  with  ras  oncogene  in  order  to  select  the  type  of  neuron,  either  adrenergic  or 
cholinergic,  that  the  cells  may  be  able  most  effectively  to  replace  in  damaged  brain. 
The  second  objective  is  to  test  the  possibility  that  muscle  isografts  to  the  brain  may 
serve  as  reservoirs  for  circulating  macrophages  which  may  then  be  induced  to  enter 


5-LN/DIR 


the  brain.   The  third  is  to  explore  the  function  of  the  pituicyte,  the  astroglial  cell  of 
the  pituitary  gland's  neural  lobe. 

PCI  2  cells,  infected  with  retroviral  vectors  encoding  for  the  K-ras  oncogene, 
undergo  neuronal  differentiation  which,  unlike  that  induced  by  nerve  growth 
factor  (NGF),  is  irreversible.   Current  work  is  to  see  whether  this  profound 
difference  may  be  attributable  to  the  pathway  of  differentiation.   Does  the  path  taken 
by  the  ras  oncogene  differ  from  the  one  used  by  NGF?    We  have  now  found  that 
there  are  a  number  of  similarities  between  the  effects  of  ras  and  NGF.   Both  the  ras 
oncogene  and  NGF  down-regulate  EGF  receptors,  but  ras  is  more  effective;  by  the 
third  day  of  treatment  an  80%  loss  of  receptors  occurs  with  ras  as  compared  with  a 
56%  loss  with  NGF.   Both  ras  and  NGF  bring  about  the  development  of  Na+ 
channels  as  measured  by  the  binding  of  saxitoxin,  a  specific  ligand  for  the  Na+ 
channel.   This  binding  in  ras  -infected  cells  was  equal  to  or  greater  than  that  in 
NGF-primed  cells.  Thus,  ras-infected  PC12  cells  should  be  as  electrically  excitable  as 
the  NGF-treated  cells.   The  binding  of  tetanus  toxin  to  neurons  is  considered  to  be  a 
developmental  marker  in  the  maturation  of  neurons  in  vivo  and  in  vitro.   Like  the 
saxitoxin  binding  sites,  the  tetanus  binding  sites  are  localized  to  the  neurites  and 
growth  cones.   Both  ras-  and  NGF-treated  cells  have  a  two-  to  three-fold  increase  in 
tetanus  binding  sites  after  6  days  in  vitro.   The  remarkable  neuritic  outgrowth 
probably  entails  considerable  reorganization  of  the  cytoskeleton  and  might  involve 
a  finely  tuned  interaction  of  proteases  and  protease  inhibitors.  It  was  thus  decided 
to  examine  possible  changes  brought  about  by  either  ras  or  NGF  in  the  level  of 
calpain,  a  calcium-activated  neutral  thioprotease,  that  occurs  in  normal  rat  brain.   It 
is  known  that  calpain  activity  decreases  in  neurite-forming  PCI  2  cells  given  NGF. 
We  now  find  that  a  similar  decrease  is  brought  about  by  the  ras  oncogene. 

Although  the  effects  on  neuronal  differentiation  are  thus  very  similar  in 
PCI 2  cells  given  either  NGF  or  ras,  there  are  two  differences.   (1)  K252a,  an  inhibitor 
of  protein  kinase,  blocks  the  formation  of  neurites  in  PC12  cells  given  NGF,  but  it 
does  not  inhibit  neuritic  extension  by  PC  12  cells  infected  with  ras.  (2)  The 
glycoprotein,  NILE,  on  the  surface  of  neurons,  is  expressed  by  NGF-treated  PC12 
cells  but  not  by  ras-treated  ones.  Such  dissimilarities  suggest  that  the  pathway 
utilized  by  the  regulatory  G-protein,  P21,  that  is  encoded  by  ras  diverges  from  the 
path  taken  by  NGF-treated  PC12  cells.  It  is  not  known  whether  the  two  differing 
responses  described  are  involved  in  the  irreversibility  of  neuronal  differentiation, 
which  is  a  paramount  requirement  for  transplanted  cells. 


6-LN/DIR 


The  next  goal  is  to  examine  the  potential  of  oncogene-differentiated  PC  12 
cells  to  form  synaptic  junctions  after  being  transplanted  to  the  brain.   Such  potential 
is  more  readily  assessed,  at  first,  in  vitro.  Only  one  report  has  described  synaptic 
interaction  between  NGF-treated  PC12  cells  and  two  clonal  cell  lines  originating 
from  muscle.   We  have  begun  to  establish,  by  differential  centrifugation,  cultures  of 
myotubes  from  L-6  and  L-8  myoblast  lines,  and  have  labeled  NGF-  and  ras-treated 
PCI  2  cells  with  "cell  linker",  a  lipid  soluble  fluorescent  dye  with  a  label  retention 
half-time  of  about  12  days.   The  marked  cells  are  then  injected  into  a  recipient.   We 
have  also  begun  to  examine  the  spatial  relation  of  stained  neurite  terminals  of 
living  PC12  cells  and  the  myotube  surface.  Neurites,  varicosities  and  growth  cones, 
as  well  as  cell  bodies,  are  stained  in  PCI 2  cells  which  subsequently  appear  to  remain 
viable  in  vitro.   Myoneural  contacts,  in  the  form  of  clusters  of  acetylcholine  (ACh) 
receptors  on  the  sarcolemma  beneath  the  site  of  neuritic  contact,  are  to  be  assessed 
with  a  series  of  different  fluorescent  conjugates  of  a-bungarotoxin  conjugates. 
Concurrent  work  in  progress  is  the  stereotaxic  injection  of  ibotenic  acid  into  the 
medial  forebrain  nucleus  of  adult  rats,  which  destroys  cholinergic  neurons.   The 
objective  is  to  see  whether  ras-differentiated  PC  12  cells,  grafted  to  the  damaged  area 
emit  neurites  and  release  ACh  in  amounts  sufficient  to  overcome  the  induced 
deficiency  in  this  neurotransmitter.   We  are  also  repeating  the  measurements  of 
radiolabeled  choline  uptake,  ACh  synthesis,  and  the  K+-stimulated  release  of  ACh 
into  the  culture  medium  by  naive  PC12  cells  and  those  treated  with  NGF  and  ras  to 
evaluate  the  feasibility  of  using  the  cells  as  neuronal  replacements  in  situ. 

Autografts  of  skeletal  muscle,  inserted  into  the  fourth  ventricle,  are  the  site 
of  entry  of  blood-borne  horseradish  peroxidase.  Can  such  grafts  also  act  as 
reservoirs  of  macrophages  which  may  subsequently  be  induced  to  enter  the 
underlying  brain?    Autografts  of  superficial  neck  muscle,  about  1x2  to  1x6  mm,  are 
placed  in  the  IV  ventricle  of  normal  rats.   After  five  weeks,  peritoneal  macrophages 
are  collected  and  labeled  with  the  lipid  soluble,  fluorescent  marker  "cell  linker". 
The  labeling  of  macrophages  was  confirmed  by  immunostaining  the  same  cells 
with  MAC-I  antibody  or  by  histochemical  methods  for  the  detection  of  nonspecific 
esterase.  The  labeled  cells  are  then  repelleted  and  infused  as  a  bolus  into  an  axillary 
artery.   From  here,  the  cells  are  swept  through  a  subclavian  artery  into  a  vertebral 
artery  and  into  the  circulation  to  the  medulla.   This  means  of  perfusing  the 
medullary  vasculature  in  the  living  animal  has  not  been  tried  until  now.    In  these 
normal  rats,  nonactivated,  labeled  macrophages  remain  within  the  vessels  and 
parenchyma  of  the  muscle  graft.  No  labeled  macrophages  are  detected  within  these 

7-LN/DIR 


intact  brains,  even  near  the  site  of  the  muscle  graft.   In  contrast,  macrophages 
activated  by  incubation  with  an  endotoxin  from  E.  coli,  or  in  a  phorbol  ester, 
accumulate  in  large  numbers  within  the  graft  and  a  few  enter  the  adjacent  medulla. 
The  next  step  is  to  perfuse  the  cerebral  ventricles  with  purified  meningococcal 
endotoxin  during  circulation  of  the  marked,  activated  macrophages.   The  purpose  is 
to  entice  the  macrophages  to  enter  the  brain  substance.  We  shall  also  try  to 
differentiate  between  the  sources  of  the  intramedullary  macrophages— the 
accumulated  macrophages  from  the  graft  or  the  labeled  ones  coming  from  the 
capillary  bed  of  the  brain.  Both  graft  and  capillary  bed  may  permit  activated 
macrophages  to  enter  the  surrounding  brain  substance  in  substantial  numbers. 
Repetition  of  the  experiments  in  rats  that  do  not  bear  grafts  should  enable  the 
distinction  between  entry  directly  from  the  intrinsic  capillaries  of  the  medulla  and 
entry  from  the  transplant. 

We  are  investigating  two  aspects  of  the  pituicyte,  the  astroglial  cell  of  the 
pituitary  gland's  neural  lobe:    the  mechanism  and  conditions  under  which  pituicyte 
cell  processes  retract  and  reexpand,  and  whether  the  pituicyte  promotes  the 
ingrowth  of  regenerating  neurosecretory  axons  (NSA).   In  normal,  hydrated  rats, 
pituicyte  cell  processes  embrace  the  terminals  of  NSA  that  lie  next  to  fenestrated 
capillaries  within  the  neural  lobe.  In  dehydrated  rats,  the  processes  retract,  so  that 
the  released  secretory  products  are  free  to  reach  the  capillary  wall  without  having  to 
confront  an  intervening  pituicyte  process.    What  is  the  mechanism  of  this 
reversible  retraction  and  how  is  it  triggered?     We  have  confirmed  that  pituicytes, 
like  astrocytes  of  the  CNS,  undergo  a  pronounced  changes  in  shape  when  placed  in 
culture  medium  devoid  of  serum.   Such  pituicytes  change  from  an  epithelioid, 
scalloped  contour  to  a  highly  stellate  one.   The  responsive  cells  then  form  many 
thin  branches.   We  have  begun  to  follow  this  change  with  video-enhanced 
differential  interference  contrast  microscopy  and  by  time  lapse  photomicroscopy. 
The  alteration  is  reversed  within  60  to  90  minutes  after  addition  of  serum  to  the 
medium.   A  similar,  though  less  pronounced  branching  is  induced   by  dibutyryl- 
cAMP  when  added  to  culture  medium  containing  serum.   We  plan  to  follow  the 
changes  in  the  distribution  of  actin  with  phalloidin  staining  and  to  see  whether 
actin  is  involved  in  the  shape  change  by  adding  cytochalasin  D  to  the  medium. 
Inasmuch  as  the  shape  of  the  pituicyte  in  vivo  is  stellate  and  the  retraction  would 
involve  the  terminals  of  the  processes,  we  decided  to  bring  the  cells  to  the  stellate 
form  by  removing  serum  and  waiting  for  1-2  hours  for  the  shape  change  to  stabilize. 
It  was  then  that  we  added  various  substances  that  might  initiate  this  change  in 

8-LN/DIR 


vivo.   Such  possible  factors  are  vasopressin  and  dynorphin,  (peptides  that  are 
coreleased  during  neurosecretory  activity)  and  oxytocin.   As  dynorphin  receptors 
have  been  found  on  the  surface  of  pituicytes  in  vitro,  this  peptide  seemed  to  be  a 
particularly  good  candidate.  Other  possible  agents  are  the  neurotransmitters  or 
their  analogs  that  are  involved  in  neural  lobe  innervation:    isoproterenol, 
norepinephrine,  dopamine  and  serotonin.    The  peptides  have  not  added  any 
appreciable  change  to  the  stellate  shape  imposed  by  serum  withdrawal.   The 
neurotransmitters  have  yet  to  be  tried.   To  compare  the  dramatic  conversion  of 
epithelioid  to  stellate  forms,  with  the  change  in  shape  and  dimensions  due  to 
shrinkage  in  hyperosmotic  medium,  we  plan  to  add  arabinose  and  monitor  any 
changes  with  time  lapse  phase  microscopy.   It  is  possible  that  the  neurosecretory 
peptides  may  activate  Ca++  channels  in  pituicytes  and  we  are  to  begin  a  collaboration 
to  explore  that  possibility.  The  second  project  is  based  on:  (1)  our  previous 
observations  that  NSA  are  attracted  to  and  regenerate  within  neural  lobe  grafts, 
presumably  due  to  the  presence  of  pituicytes;  and,  (2)  the  report  that,  in  explants  of 
the  rat's  ventral  hypothalamus  from  young  fetuses,  some  magnocellular 
neurosecretory  cells  survive  but  are  only  able  to  synthesize  vasopressin  and  not 
oxytocin.   Do  pituicytes  have  a  trophic  effect  on  neurosecretory  cells  in  enhancing 
regeneration  of  their  axons  and  in  the  synthesis  of  all  their  peptides?    In  order  to 
test  the  notion  of  a  trophic  influence  exerted  by  pituicytes,  fetal  hypothalamus  is  to 
be  grown  on  extracellular  matrix  only  and  on  a  bed  of  pituicytes  that  have  grown 
out  from  explants.   The  amount  of  neurite  formation  will  be  measured  and  the 
induction  of  oxytocin  synthesis  identified  immunohistochemically. 


9-LN/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01 -NS-01442-24LN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Permeability  of  Cellular  Layers  in  the  Vertebrate  Nervous  System 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  T.S  Reese,  M.D.  Chief  LN,  NINDS 


Others: 


S  B  Andrews,  Ph.D. 


Research  Chemist 


LN,  NINDS 


COOPERATING  UNITS  frfany) 

B.  Kachar,  LNO,  NIDCD,  NI11,  Bethesda,  MD  20892.  R.  C.  Wagner,  University  of  Delaware,  Newark, 
DE.  N.  Lane,  University  of  Cambridge,  Cambridge,  England. 


LAB/BRANCH 


Laboratory  of  Neurobiology,  BNP,  DIR,  NINDS 


SECTION 


Section  on  Structural  Cell  Cell  Biology 


INSTITUTE  AND  LOCATION 

N 1 N PS,  N 111 ,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


0.1 


PROFESSIONAL: 


0.1 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

]    (a)  Human  subjects 
]  (al)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  [>T]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

How  ti^ht  junctions  might  prevent  small  charged  solutes  from  entering  the  brain 
(across  the  blood-brain  barrier)  is  made  clear  by  our  new  model  of  tight  junction 
structure  based  on  a  lipidic  backbone.  Tight  junctions  in  invertebrates  also  appear  to 
have  lipid  backbones  though  our  most  recent  observations  suggest  that  periodic 
structures,  presumably  proteins  are  intercalated  into  these  backbones.  This  new  work 
has  been  submitted  for  publication,  and  publications  describing  completed  studies  on 
the  three-dimensional  organization  of  the  vesicular  system  in  capillaries  are  appearing 
this  year.  Otherwise  this  project  is  in  abeyance. 


10-LN/DIR 


PHS  6040  (Hev    1/841 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS-0 1881  20  1,  N 


PERIOD  COVERED 

October  1 ,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  loss    Title  must  fit  on  one  line  between  the  border s) 

Structural  Basis  of  Synaptic  Transmission  and  Development 


PRINCIPAL  INVESTIGATOR  (list  other  professional  personnel  below  the  Principal  Investigator  )  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  T  S  Reese,  M.D.  Chief  LN,  NINDS 

Others:         G.  Benshalom,  Ph.D.  Visiting  Scientist  LN,  NINDS 

J.  A.  Drazba,  PhD  IRTA  Fellow  LN,  NINDS 


COOPERATING  UNITS  (if  any) 

Marine  Biological  Lab,  Woods  Hole,  MA.  H.  Tatsuoka,  Deptof  Anatonmy,  Chiba  University,  Chiba, 
Japan    I)  Chang,  Dept  of  Molecular  Physiol  and  Biophys,  Baylor  College  Medicine,  Houston,  TX 


LAB/BRANCH 

Laboratory  of  Neurobiology,  BNP,  DIR,  NINDS 


SECTION 

Section  on  Structural  Cell  Cell  Biology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH.Bethesda  Maryland  20892 


TOTAL  MAN  YEARS: 


PROFESSIONAL:  .   o 


OTHER:  q  2 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [x~\  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  rostral  anteroventral  cochlear  nucleus  (AVCN)  of  the  chinchilla  has  provided  a 
preparation  in  which  neuronal  cell  bodies  and  synapses  in  the  CNS  can  be  examined 
after  direct  freezing  and  freeze-substitution  of  rapidly  excised  brain  stem  slices.  The 
four  types  of  synaptic  terminals  known  to  be  in  the  AVCN  were  distinguished  and 
correlated  with  four  types  of  terminals  previously  reported  after  chemical  fixation. 
Since  the  transmitters  for  each  of  the  four  types  of  terminals  have  been  specified,  the 
transmitter  type  could  be  correlated  with  the  detailed  structure  of  the  postsynaptic 
density  in  each  chemical  type  of  synapse.  Two  types  of  filamentous  components 
comprising  the  basic  structure  of  the  postsynpatic  density  differed  specifically  in  each 
type  of  terminal,  thus  providing  new  information  about  structural  differences  between 
receptors  arrays  and  associated  cytoskeletal  components  at  different  types  of  central 
nervous  system  synapses.  As  part  of  a  continuing  effort  to  understand  the  basis  of 
synaptic  secretion  by  exocytosis,  the  formation  of  membrane  pores  induced  by 
electroporation  was  studied  with  rapid  freeze  techniques.  This  approach  provided 
detailed  information  on  a  millisecond  time  scale  regarding  the  opening  of  membrane 
pores  of  our  initial  test  preparation,  human  erythrocytes.  A  surprising  finding  was  that 
the  pores  continued  to  open  over  the  first  tens  of  milliseconds  before  reaching  a 
maximum  size,  suggesting  that  membrane  interactions  with  the  cytoskeleton  were  an 
important  factor  in  pore  formation.  A  new  initiative  uses  confocal  microscopy  of 
organotypic  brain  slice  cultures  to  study  dynamic  changes  in  structure  during  normal 
function  and  during  development;  reliable  cultures  and  detailed  imaging  by  confocal 
(fluorescent)  microscopy  have  now  been  achieved. 


II  I.N/DIR 


PHS  bU4U(Hev     1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01-NS-02551-09LN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Structure  of  Neuronal  Cytoplasm 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  T  S.  Reese,  M.D.  Chief  LN,  NINDS 

Others:        P.  E.  Gallant,  Ph.D.  Research  Biologist  LN,  NINDS 

J.  E  Moreira,  Ph.D.  Visiting  Scientist  LN,  NINDS 

KG.  Herman,  Ph.D.  Senior  Staff  Fellow  LN,  NINDS 


COOPERATING  UNITS  (,f  any) 

Marine  Biological  Lab,  Woods  Hole,  MA.  H.  Tatsuoka,  Department  of  Anatomy,  Chiba  University, 
Chiba,  Japan.  T.  Cheng,  State  University  of  New  York  Health  Science  Center,  Brooklyn,  NY. 


LAB/BRANCH 

Laboratory  of  Neurobiology,  BNP,  DIR,  NINDS 


SECTION 

Section  on  Structural  Cell  Cell  Biology 


INSTITUTE  AND  LOCATION 

NINDS,  Nlll,  Bethesda  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  2  3 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

Q   (a)  Human  subjects  Q  (b)  Human  tissues  |~x~]  (c)  Neither 

]  (a1)  Minors 

J   (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  determines  the  structure  of  neuronal  and  glial  cytoplasm,  particularly  as  it  pertains  to 
axoplasmic  transport.  A  protein  translocator,  kinesin,  is  responsible  for  the  anterograde  organelle 
movements  along  microtubules  which  are  the  basis  of  anterograde  fast  axonal  transport .  A  high 
molecular  weight  protein  in  squid  axoplasm,  which  we  have  characterized  as  a  cytoplasmic  dynein, 
transports  exclusively  in  the  retrograde  direction.  Since  organelles  can  bind  both  kinesin  and  dynein,  the 
next  question  is  how  kinensin  or  dynein  activation  is  programmed  on  the  organelle  surface.  Metabolic 
uncouplers  of  various  classes  uniformly  block  organelle  movements  along  microtubules  in  vitro,  but  do 
not  block  movements  of  latex  beads  induced  by  kinesin  or  dynein  so  it  appears  that  an  ionic  gradient 
across  the  organelle  membrane  is  responsible  for  programming  an  organelle  to  go  in  the  anterograde  or 
etrograde  direction.  The  function  of  the  transport  system  in  vivo  is  also  under  investigation.  Each 
organelle  contacts  several  microtubules  in  the  axon,  so  it  is  the  continuous  microtubule  bundles  which 
constitutes  the  transport  pathways  down  the  axon.  Much  of  the  pool  of  kinesin  and  dynein  in  vivo  is  in  a 
soluble  form  and  new  immunocytochemical  methods  are  being  developed  to  determine  their 
distributions  in  cytoplasm  in  relation  to  the  transport  pathways.  An  analysis  of  microtubule  distributions 
in  central  nervous  system  cell  bodies  is  underway.  The  numbers  of  organelles  contacting,  and  therefore 
in  transport,  along  these  microtubules  is  being  counted  and  it  is  apparent  that  there  are  different 
populations  of  microtubules,  some  supporting  organelle  transport  in  the  cell  body  and  some  not. 
Turnover  of  the  photoreceptive  membranes,  or  rhabdom,  was  studied  in  Limulus  photoreceptors 
maintained  in  vitro  in  order  to  investigate  interactions  of  these  membranes  with  the  cytoskeleton.  The 
distributions  of  actin  and  tubulin  were  examined  by  conventional  and  laser  confocal  fluorescence 
microscopy.  Light-induced  reduction  of  actin  labeling  suggests  a  role  for  actin  in  turnover  of  the 
microvilli,  whereas  microtubules  may  mediate  the  transport  of  shed  microvilli. 


12  LN/DIR 


PHS  6040  (Rev-  1«M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS  02610  07  LN 


PERIOD  COVERED 

October  1 ,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (do  characters  or  lest.   Title  must  fit  on  one  line  between  the  borders) 

Distribution  of  Mobile  and  Structural  Components  at  Chemical  Synapses 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  S  B  Andrews,  Ph.D.  Research  Chemist  LN,  NINDS 

Others:         TS  Reese,  M.D.  Chief  LN,  NINDS 

A  Shainberg,  Ph.D.  Visiting  Scientist  LN,  NINDS 

B  E  Flucher,  Ph.D.  Visiting  Associate  LN,  NINDS 

M  Terasaki,Ph.D.  Senior  Staff  Fellow  LN,  NINDS 

G  Benshalom.Ph.D.  Visiting  Scientist  LN,  NINDS 

M   F  O'Connell.B.S.  Biologist  LN,  NINDS 


COOPERATING  UNITS  (if  any) 

Marine  Biol  Lab,  Woods  Hole,  MA.  K.D.  Leapman,  BEIF,  NCCR,  NIH,  Bethesda,  Ml).  1). ML).  Landis,  Case-Western  Reserve  Univ 

Cleveland,  (>l  I    H.I).  Trapp,  Johns  Hopkins  Univ  Sch  of  Med,  Baltimore,  Ml).  Ml'.  Daniels,  l.BG,  NHLBI,  NIH,  Belhesda,  Ml) 


LAB/BRANCH 

Laboratory  of  Neurobiology,  BNP,  DIR,  NINDS 


SECTION 

Section  on  Structural  Cell  Biology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda   Maryland  20892 


TOTAL  MAN-YEARS:  .    . 

4.4 


PROFESSIONAL:  ^  4 


OTHER: 


10 


CHECK  APPROPRIATE  BOX(ES) 

LJ   (a)  Human  subjects  ]  (b)  Human  tissues  0  (c)  Neither 

]  (al)  Minors 

J   (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  studies  the  organization  of  structural  and  diffusible  components  of  nerve,  muscle  and  glia, 
particularly  with  respect  to  the  function  of  specialized  membrane  regions  such  as  synapses   A  new  and 
unique  instrument  -  the  low-temperature,  high-resolution,  field-emission  scanning  transmission 
electron  microscope  (STEM)  -  has  been  developed  and  shown  to  reach  new  levels  of  sensitivity  and 
resolution  for  molecular  weight  mapping  and  chemical  analysis  of  native  proteins.  This  STEM  has  now 
been  used  to  determine  the  tertiary  structure  and  molecular  weight  distribution  of  individual,  native 
molecules  of  kinesin,  as  described  in  Project  Z01-NS-02700-05  LN.  This  instrument  will  also  be  essential 
for  extending  analytical  microscopy  studies  on  calcium  regulation  in  dendritic  spines  and  shafts,  which 
have  been  advanced  by  the  development  of  suitable  organotypic  cultures  of  hippocampal  slices    In 
parallel,  uptake  and  release  of  calcium  from  the  endoplasmic  reticulum  (ER)  of  a  model  for  calcium- 
regulated  neurosecretion,  the  sea  urchin  egg  cortex,  has  been  studied  using  calcium  indicator  dyes. 
Light  and  electron  microscopy  immunocytochemistry  has  indicated  that  transport  on  microtubules  is  also 
the  basis  of  anterograde  and  retrograde  vesicular  transport  of  myelin-specific  proteins  in 
oligodendrocytes    Thus,  the  depolymerization  of  microtubule  bundles,  the  distribution  and  polarity  of 
which  support  a  role  in  organelle  translocation,  reversibly  block  transport  of  myelin  proteins  from  the 
Golgi  apparatus    Microtubule  depolymerization  also  causes  reorganization  of  organelles  involved  in 
protein  synthesis  in  cultured  cells;  in  living  cells,  the  three-dimensional  organization  of  the  ER  in  relation 
to  microtubules  is  being  revealed  by  confocal  microscopy    Immunocychemistry  has  been  used  to 
localize  the  al  and  u2  subunits  of  the  dihydropyridine  receptor  to  the  t-tubule  system  of  skeletal  muscle, 
thereby  indicating  the  association  of  these  polypeptides  in  the  excitation-contraction  (EC)  coupling 
complex    This  contrasts  with  the  distribution  of  these  receptors  in  dysgenic  muscle  (mdg/mdg)  where  the 
absence  ot  theal  subunit  is  associated  with  disorganization  of  the  remaining  a2  protein    During 
myogenesis  in  normal  muscle,  the  development  of  the  E/C  coupling  apparatus  is  characterized  by  the 
formation  ol  t-tubule  membranes  prior  to  connection  with  the  plasma  membrane. 

13-LN/DIR 


PH4il)40|Rt</    1114) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01-NS-027U0-05LN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Proteins  Involved  in  Axonal  Transport  * 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  T  S.  Reese,  M.D.  Chief  LN,  NINDS 


Others:         S    B  Andrews,  PhD. 
S.  Khan,  Ph.D. 


Research  Chemist 
Guest  Researcher 


LN,  NINDS 
LN,  NINDS 


COOPERATING  UNITS  <.f  any) 

Marine  Biological  Lab,  Woods  Hole,  MA.  R.  I).  Leapman,  BE1P,  NCCR,  NIH,  Bethesda,  MD.    B.J. 
Schnapp,  Dept  of  Physiology,  Boston  University,  Boston,  MA. 


LAB/BRANCH 


Laboratory  of  Neurobiology,  BNP,  DIR,  NINDS 


SECTION 


Section  on  Structural  Cell  Cell  Biology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


08 


PROFESSIONAL: 


0.6 


OTHER: 


0.2 


CHECK  APPROPRIATE  BOX(ES) 

J   (a)  Human  subjects 
]  (al)  Minors 
J   (a2)  Interviews 


]  (b)  Human  tissues  [xj  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  goal  of  this  project  is  to  understand  how  the  motors  which  power  fast  axonal 
transport  promote  movement.  Metabolic  uncouplers  (eg.,  DNP,  CCCP,  valinomycin) 
block  organelle  movements  along  microtubules  in  vitro,  but  do  not  block  movements  of 
latex  beads  induced  by  kinesin  or  dynein.  It  thus  appears  that  an  ionic  gradient  across 
the  organelle  membrane  is  responsible  for  programming  an  organelle  to  go  in  the 
anterograde  or  retrograde  direction.  The  next  question  is  how  kinesin  or  dynein  is 
organized  on  the  organelle  surface  and  microtubule  substrate.  A  new  scanning 
transmission  electron  microscope  (STEM),  as  described  in  Project  #  Z01-NS-02610-07- 
LN,  has  allowed  kinesin  to  be  visualized  directly  on  the  surfaces  of  microtubules  and 
organelles.  This  STEM  was  used  to  determine  at  2nm  resolution  the  shapes  and  mass 
distributions  of  purified  squid  brain  kinesin  molecules— both  free  and  bound  to 
microtubules.  Maps  of  kinesin  bound  to  purified,  taxol-stabilized  bovine  microtubules 
provided  the  first  direct  evidence  for  cross-bridging  of  microtubules  by  single  kinesins 
which  suggests  that  kinesins  in  cells  might  also  translocate  microtubules  and  therefore 
have  some  role  in  slow  as  well  as  fast  axonal  transport.  The  dynamic  properties  of  other 
biological  motors  are  being  studied  for  comparison  with  the  axonal  transport  motors;  the 
bacterial  flagellar  motor  in  E  coli  has  also  been  shown  to  depend  on  interactions  of  the 
flagellar  structure  with  membrane  proteins  though  these  motors  are  driven  by,  rather 
than  controlled  by,  ionic  gradients.  This  motor  system,  like  the  axonal  motor  system, 
can  also  switch  direction.  A  newly  discovered  component  of  the  flagellar  motor  has  led 
us  to  propose  a  novel  structure  model.  Molecular  genetic  analysis  of  the  new  structural 
components  may  lead  to  an  understanding  of  directional  switching. 

*  Formerly:  "The  Mechanochemistry  of  Proteins  Involved  in  Axonal  Transport." 

14-LN/DIK 


PHS6O40(Rev    1  tW| 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01 -NS-01 805-22  LN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  character*  or  lea.   Title  must  fit  on  one  line  between  the  border*.) 

Membrane  Structure  of  Astrocytes 


PRINCIPAL  INVESTIGATOR  (list  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  P  Doe,  M.D.  Visiting  Fellow  LN,  NINDS 

Others:         M.  W.  Brightman,  Ph.D.  Section  Chief  LN,  NINDS 


COOPERATING  UNITS  {if  any) 


LAB/BRANCH 

Laboratory  of  Neurobiology,  BNP,  DIR,  NINDS 


SECTION 

Section  on  Brain  Structural  Plasticity 


INSTITUTE  AND  LOCATION 

NINDS,  Nlll.Bethesda   Maryland  20892 


TOTAL  MAN-YEARS 


PROFESSIONAL:  ..   ? 


OTHER:  Q4 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [x~\  (0  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  pituicyte  of  the  pituitary  gland's  neural  lobe,  resembles  the  more  common 
astrocyte  of  the  CNS  in  being  perivascular  and  in  having  intramembranous,  orthogonal 
aggregates  of  particles  or  assemblies.  However,  pituicytes  subtend  fenestrated  vessels 
permeable  to  peptides  and  have  dynorphin  receptors  and  cells  processes  that  embrace 
the  neurosecretory  terminals  (NST)  of  the  neural  lobe.  It  is  known  that  in  a  dehydrated 
rat,  these  processes  retract,  exposing  the  NST  directly  to  the  fenestrated  capillaries. 
During  rehydration,  the  processes  reensheath  the  NST.  We  have  examined  explants  to 
determine  the  mechanism  of  reversible  contraction  and  to  detect  whether  Ca+  +  is 
involved.  We  have  found  that,  like  other  astrocytes,  pituicytes  change  shape  from 
epithelioid  to  stellate  in  serum-deprived  culture  medium.  If  the  pituicytes  are  initially 
kept  in  5%  fetal  calf  serum  instead  of  the  usual  10%,  the  shape  change  is  completed 
within  one  hour.  By  the  use  of  video-enhanced  differential  interference  contrast  and 
time-lapse  microscopy,  we  have  been  able  to  follow  the  conversion  of  lamellipodia  to 
thin,  varicose  branches.  A  less  pronounced  change  is  caused  by  dibutyryl  cAMP  in  the 
presence  of  serum.  If  Ca+  +  is  involved  in  process  retraction,  we  may  then  see  whether 
there  are  Ca  f  +  channels  sensistive  to  dynorphin  and  vasopressin,  that  are  co-released 
during  neurosecretion,  and  to  oxytocin  and  catecholamines  by  the  use  of  calcium- 
activated  flurochromes  (e.g.,  Fura-2)  in  a  video-enhanced  microscope  system. 


15  LN/DIR 


PHS6040|Ke.   1  t>4| 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 
Z01-NS-02086  17  1.N 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  \8Q  characten  or  toss.  Title  mutt  fit  on  one  line  between  the  bordary) 

Regeneration  Specificity  in  Transplanted  Neural  Tissue 


PRINCIPAL  INVESTIGATOR  (Lnt  other  professional  personnel  below  the  Prmapal  inwesvgator  J  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  DL  Simpson,  Ph.D.  Special  Expert  LN,  NINDS 


Others:         S  Ishihara,  M.D. 

M  W.  Brightman,  Ph  D 


Visiting  Fellow 
Section  Chief 


LN,  NINDS 
LN,  NINDS 


COOPERATING  UNITS  (ifiny) 

G.  Guroff,  IliP.Clll),  NIH,  Bethesda,  Ml) 


LAB/BRANCH 


Laboratory  of  Neurobiology,  BNP,  DIR,  NINDS 


SECTION 


Section  on  Brain  Structural  Plasticity 


INSTITUTE  AND  LOCATION 

NINDS,  N1H,  Bethesda   Maryland  20892 


TOTAL  MAN-YEARS: 


1.5 


PROFESSIONAL: 


1.3 


OTHER: 


0.2 


CHECK  APPROPRIATE  BOX(ES) 

I      I    (a)  Human  subjects 
]  (a1)  Minors 
J   (a2)  Interviews 


]  (b)  Human  tissues  \x~]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Before  PC  12  cells,  permanently  differentiated  into  neuron-like  cells  by  ras  oncogene, 
are  grafted  into  brain  as  replacements  for  missing  neurons,  it  is  necessary  to  further 
define  their  neuronal  phenotype.  We  had  found  tnatras-differentiated  PC  12  cells  in 
vitro,  are  both  catecholaminergic  and  cholinergic,  but  that  the  intracellular  amounts  of 
dopamine  and  norepinephrine,  are  less  than  that  in  naive  PC12  cells.  The  neuronal 
phenotype  induced  by  the  ras  oncogene  appears  to  follow  much  of  the  differentiation 
path  taken  by  nerve  growth  factor  (NGF).  Both  ras  infection  and  NGF  of  PC12  cells 
result  in  a  down-regulation  of  receptors  for  epidermal  growth  factor,  augmentation  of 
Na+  channels  and  tetanus  binding  sites,  and  a  decrease  in  calpain  activity.  However, 
the  effects  of  the  two  agents  diverged  in  two  aspects.  While  NGF  induced  the  production 
of  a  surface  glycoprotein,  NILE,  ras  did  not.  The  protein  kinase  inhibitor  K252a,  which 
prevents  neurite  formation  in  NGF-treated  PC12  cells  has  no  such  effect  on  ras-treated 
cells.  The  pathway  of  differentiation  in  PC12  cells  treated  with  ras  thus  diverges  from 
that  taken  by  NGF.  The  regulatory  G-protein,  P21,  encoded  by  the  ras  oncogene  acts  at 
a  different  point  from  that  of  NGF. 

As  the  Schwann  cell  (SC)  is  a  neighbor  of  the  PC12  cell  progenitor,  the  chromaffin  cell  of 
the  adrenal  gland,  we  have  examined  the  interactions  between  the  PC12  and  SC  in 
vitro.  SC,  derived  from  sciatic  nerve,  forms  clusters  upon  which  naive  PC12  cells 
aggregate.  This  affinity  for  SC  is  expressed  even  earlier  by  ras  -PC12  cells.  There  is  no 
affinity  for  fibroblasts  in  the  same  co-cultures,  or  for  astrocytes  or  endothelial  cells. 
Neurite  extension  is  supported  by  SC  cells  or  their  matrix  and  some  SC  ensheath  PC12 
neurites.  In  co-cultures,  choline  acetyltransferase  increases  while  acetylcholinesterase 
is  largely  unchanged. 

16-LN/DIR 


CriS  b04lj  [lUv    I  ts.ll 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01-NS-02144-16LN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (tit)  characters  or  less.    Title  must  fit  on  one  line  between  the  borders  ) 

The  Blood-Brain  Barrier 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:  S  Ishihara,  M.D. 

Others  D.l    Simpson,  PhD 

M  W    Brightman,  PhD 


Visiting  Fellow 

Special  Expert 
Section  Chief 


LN,  NINDS 

LN,  NINDS 
LN,  NINDS 


COOPERATING  UNITS  Of  3ny) 

M.  Sawada,  I, DMA,  N1DR,  Nlll,  Belhesda,  Ml) 


LAB/BRANCH 


Laboratory  of  Neurobiology,  BNP,  DIR,  NINDS 


SECTION 


Section  on  Brain  Structural  Plasticity 


INSTITUTE  AND  LOCATION 

NINDS,  Nlll,  Belhesda  Maryland  2U892 


TOTAL  MAN  YEARS: 


1.8 


PROFESSIONAL: 


1.4 


OTHER: 


04 


CHECK  APPROPRIATE  BOX(ES) 

j    (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  QT]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Does  a  piece  of  skeletal  muscle,  transplanted  to  the  brain's  surface,  provide  an  entry  into 
the  brain  for  blood-borne  macrophages?  Pieces  of  neck  muscle,  2-3  mm  x  1  mm,  are 
grafted  to  the  dorsal  surface  of  the  medulla  in  mature  rats.  Two  to  12  weeks  later,  the 
rats  macrophages  are  withdrawn  from  the  peritoneal  cavity,  pelleted  and  labeled  with  a 
long-lasting  fluorochrome  "Cell  linker,"  which  is  presumably  a  carbocyanin  dye.  The 
macrophages  are  then  infused  as  a  single  bolus  into  the  rat's  axillary  artery.  This 
vessel,  which  has  not  been  used  heretofore  as  a  means  of  perfusing  the  vertebral  artery, 
leads  the  cells  into  the  brain  stem  circulation.  A  few  labeled  macrophages  accumulate 
in  the  graft  and  surrounding  choroid  plexus;  very  few  enter  the  medulla  under  the 
muscle  graft.  It  is  to  be  tested  whether  macrophages  will  enter  the  brain  primarily  from 
the  intrinsic  capillaries  of  the  CNS  or  from  the  graft,  when  endotoxin  is  perfused 
through  the  cerebral  ventricles  prior  to  introducing  the  macrophages. 


17  1,N/1)1R 


PHS6M0(Rev    184) 


Laboratory  of  Neurobiology 
Publications 


Brightman  MW,  Simpson  DL,  Tao-Cheng  JH,  Bressler  J,  Okuda  O,  Jhang  L. 
Some  neuronal  properties  of  PC  12  cells  infected  with  kirsten-ras  orcogene.  J 
Neurocytol  1990;  20,  in  press. 

Brightman  MW,  Tao-Cheng  JH.    Mutually  imposed  structural  changes  in 
plasma  membranes  of  astroglia  and  brain  endothelium.    In:  Levi  G,   ed. 
Differentiation  and  function  of  glial  cells.    New  York:  AR  Liss,  1990;107-15. 

Chang  DC,  Reese  TS.    Changes  in  membrane  structure  induced  by 
electroporation  as  revealed  by  rapid-freezing  electron  microscopy.    Biophys  J 
1990;58:1-12. 

Flucher  BE,  Morton  ME,  Froehner  SC,  Daniels  MP.    Localization  of  the  ai  and 
a2  subunits  of  the  dihydropyridine  receptor  and  ankyrin  in  skeletal  muscle 
triads.    Neuron  1990;  in  press. 

Herman  KG.  Two  classes  of  Limulus  ventral  photoreceptors.  J  Comp  Neurol 
1990;  in  press. 

Kerman  KG.    Light  stimulated  rhabdom  turnover  in  Limulus  ventral 
photoreceptors  maintained  in  vitro.  J  Comp  Neurol   1990;  in  press. 

Kadota  Y,   Pettigrew  KD,  Brightman  MW.     Regrowth  of  damaged 
neurosecretory  axons  to  fenestrated  vessels  of  implanted  peripheral  tissues. 
Synapse  1990;5:175-89. 

Landis  DMD,  Reese  TS.    Substructure  in  the  assemblies  of  intramembrane  particles  in 
astrocytic  membranes.  J  Neurocytol  1989;18(6):819-31. 

Leapman  RD,  Andrews  SB.    High-resolution  biological  microanalysis  in  the  field- 
emission  STEM.    Proc  Xllth  Int  Congress  Electron  Microsc  1990;154-55. 

Leapman  RD,  Andrews  SB.    Analysis  of  directly  frozen  macromolecules  and 
tissues  in  the  field-emission  STEM.   J  Microsc  1990;  in  press. 

Reese  T.    Molecular  basis  of  axonal  transport:  Kinesin  and  other  transport  proteins.  In: 
Fidia  Foundation  Neuroscience  Award  Lectures    1989;3:99-119. 

Schnapp  BJ,  Crise  B,  Sheetz  MP,  Reese  TS,  Kahn  S.  Interaction  between  nucleotide 
bonding  sites  during  kinesin-based  microtubule  movement.  Proc  Natl  Acad  Sci  1990; 
in  press. 

Schnapp  BJ,  Reese  TS.    Dynein  is  the  motor  for  retrograde  axonal  transport  of 
organelles.   Proc  Nad  Acad  Sci,  USA  1989;86:1548-52. 


Simpson  D,   Dickens  G,  Doll  S,   Koizumi  S,   Okuda  O,  Oshima  I,  Rudkin  B, 
Brightman  MW,  Guroff  G.     Differentiation  of  PC12  Cells  with  K-ras; 
Comparison  with  nerve  growth  factor.   J  Neurosci  Res  1990;  in  press. 

Tao-Cheng  JH,  Nagy  Z,  Brightman,  MW.     Astrocytic  orthogonal  arrays  of 
intramembranous  assemblies  are  modulated  by  brain  endothelial  cells  in  vitro.    J 
Neurocytol  1990;19:143-53. 

Tatsuoka  H,  Reese  TS.    New  structural  features  of  synapses  in  the  anteroventral 
cochlear  nucleus  prepared  by  direct  freezing  and  freeze-substitution.   J  Comp  Neurol 
1989;290:343-57. 

Terasaki  M.    Recent  progress  on  structural  interactions  of  the  endoplasmic 
reticulum.    Cell  Motility  and  the  Cytoskeleton  1990;15:71-5. 

Trapp  BD,  Andrews  SB,  Cootauco  C,  Quarles  RH.    The  myelin-associated 
glycoprotein  is  enriched  in  multivesicular  bodies  and  periaxonal  membranes  of 
actively  myelinating  oligodendrocytes.   J  Cell  Biol  1989;109:2417-26. 

Wagner  RC,  Andrews  SB.    Cryofixation  of  vascular  endothelium.    J  Electron 
Microsc  Tech  1990;  in  press. 

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ANNUAL  REPORT 

October  1 ,  1 989  through  September  30,  1 990 

Laboratory  of  Neurochemistry 
National  Institute  of  Neurological  Disorders  and  Stroke 

Table  of  Contents 

RESEARCH  SUMMARIES 

Section  on  Cellular  and  Developmental  Neurobiology  l 


3 
4 


Section  on  Enzyme  Chemistry 
Section  on  Molecular  Neuroscience 

PROJECT  REPORTS 

Enzymological  Aspects  of  Neural  Functions  6 

Z01-NS-00813-29LNC 

Peptides  in  the  Adult  and  Developing  Vertebrate  Nervous  systems  7 

Z01-NS-02723-04LNC 

Molecular  Mechanisms  in  Neuronal  Structure  and  Function  8 

Z01-NS-02724-04LNC 

Calcium  Metabolism  and  Protein  Phosphorylation  in  Neuronal  Systems  9 

Z01-NS-02725-04LNC 

Molecular  Biology  of  the  Genes  Encoding  Prohormones  for  Bombesin-Like  Peptides        10 
Z01-NS-02753-02LNC 

Molecular  Cloning  of  the  Bombesin  Receptor  H 

Z01-NS-02774-02  LNC 


Analyses  of  Peptide  Receptors 
Z01-NS-02757-03LNC 


12 


ANNUAL  REPORT 

October  1,  1989  through  September  30,  1990 

Laboratory  of  Neurochemistry,  Division  of  Intramural  Research 

National  Institute  of  Neurological  Disorders  and  Stroke 

Harold  Gainer,  Ph.D.,  Chief 


The  laboratory  is  concerned  with  the  development  and  functional  organization  of 
the  nervous  system,  with  a  special  focus  on  molecular  mechanisms.  The  approach  of 
the  laboratory  is  cellular  and  molecular  biological  in  nature  and  utilizes  a  wide 
variety  of  techniques  and  concepts  from  a  number  of  disciplines,  e.g.,  physiology, 
biochemistry,  biophysics,  morphology,  immunology,  and  molecular  biology. 
Specifically,  we  study  neuropeptides  and  other  neurotransmitters,  neuropeptide 
receptors,  plasma  membrane  and  intracellular  membrane  systems,  cytoskeletal 
proteins,  and  various  enzymes  (e.g.,  Na,K-ATPase,  protein  kinases,  and  proteases) 
which  are  found  in  the  nervous  system  and  are  essential  to  its  development  and 
function. 

The  Laboratory  of  Neurochemistry  is  composed  of  three  Sections:    Cellular  and 
Developmental  Neurobiology,  Enzyme  Chemistry,  and  Molecular  Neuroscience. 

I.  Section  on  Cellular  and  Developmental  Neurobiology 

Studies  on  the  role  of  neuropeptides  in  nervous  system  development  and 
function,  and  the  molecular  mechanisms  underlying  the  unique  morphologies  of 
neurons  constitute  the  major  activities  in  this  Section.  Our  studies  focus  on  selected 
neuronal  populations  in  the  hypothalamus  and  peripheral  nervous  system  which  we 
beiieve  are  models  for  the  analysis  of  the  regulation  of  peptideric  phenotype  and 
neuronal  morphology.  These  are  the  luteinizing  hormone-releasing  hormone 
(LHRH),  arginine  vasopressin  (AVP),  and  oxytocin  (OT)-synthesizing  neurons  in  the 
Hypothalamus,  and  the  peptidergic  sensory  neurons  in  dorsal  root  and  trigeminal 
ganglia.  Some  questions  we  are  addressing,  using  these  systems,  are:  1)  What  are 
the  ontogenetic  histories  of  these  neuronal  populations?  How  do  their  cell  lineage 
relationships  and  migratory  patterns  during  development  relate  to  their 
differentiated  phenotypes  (e.g.,  specific  peptide  expression)?  2)  What  are  the 
mechanisms  which  underlie  the  establishment  of  the  differentiated  neuronal 
phenotypes?  These  issues  include  considerations  of  the  heterogeneity  of 
differentiated  properties  of  the  populations  (subpopulations),  their  membrane, 
receptor,  and  signal  transduction  systems,  unique  morphologies  and  relevant 
intrinsic  cytoskeletal  proteins,  and  axonal  outgrowth  and  nerve  terminal 
distributions.  3)  What  are  the  regulatory  elements  in  the  genes  uniquely  expressed 
by  these  neurons  during  development  and  homeostatically  after  maturation?  and  4) 
To  what  extent  do  these  neuronal  populations  exhibit  "plasticity"? 

Most  peptidergic  neurons  in  the  central  nervous  system  (e.g.,  OT  and  AVP 
neurons)  derive  from  precursor  cells  in  the  ventricular  germinal  zone.  Recent  studies 
in  our  section,  however,  have  demonstrated  that  LHRH  neurons  normally  located  in 
the  adult  forebrain  are  in  fact  derived  from  progenitor  cells  outside  the  central 
nervous  system  proper,  i.e.,  in  the  olfactory  placode,  and  subsequently  migrate 
along  "tracks"  in  the  nasal  area  towards  the  brain.  These  cells  first  accumulate  at 
the  base  of  the  telencephalon,  after  which  they  penetrate  the  brain  and  migrate 
towards  their  final  resting  destinations  in  the  forebrain.    Our  recent  observations 


1  -LNC/DIR 


suggest  that  the  LHRH  neurons  associate  with  an  neural  cell  adhesion  molecule  (N- 
CAM)-rich  "track"  during  migration,  but  appear  to  avoid  fibronectin-rich  areas. 
Transgenic  mice  containing  a  construct  of  a  human  LHRH  promotor  connected  to  the 
SV-40  T-antigen,  appear  to  produce  tumor  cells  in  the  migratory  pathway.  When 
these  cells  are  cultured,  they  express  LHRH  in  vitro.  Although  formation  of  the  LHRH 
system  is  a  prenatal  event,  its  function  is  primarily  postnatal;  i.e.,  occurring  after 
puberty.  The  analysis  of  postnatal  development  and  function  requires  an  in  vitro 
model  system.  Recently,  organotypic  slice  explant  cultures  have  been  developed  in 
our  Section  which  allow  for  the  study  of  LHRH  neurons  (and  other  peptidergic 
neurons,  e.g.,  AVP,  OT,  corticotropin-releasing  hormone  (CRH),  and  thyrotropin- 
releasing  hormone  (TRH)-containing  neurons).  Studies  of  the  regulation  of  peptide 
gene  expression  in  these  neurons  under  rigorously  controlled  environmental 
circumstances  are  currently  in  progress,  and  we  have  found  thus  far  that  estrogen 
inhibits  LHRH  gene  expression  in  a  subset  of  cells,  whereas  inhibition  of  spontaneous 
electrical  activity  inhibits  overall  LHRH  gene  expression  in  vitro. 

In  previous  studies  we  have  found  that  various  neuroendocrine  cells  (e.g.,  CRH, 
AVP  and  OT  cells)  in  the  hypothalamus  exhibit  neuropeptide  coexistence  which  can 
have  profound  effects  when  the  coexisting  peptides  (in  contrast  to  each  peptide 
alone)  are  secreted  near  targets.  A  central  issue  of  peptide  coexistence  is  the 
elucidation  of  the  molecular  mechanisms  which  underlie  the  various  combinations 
of  peptide  coexistence  in  individual  neurons.  Sensory  ganglion  neurons  exhibit  a 
remarkable  degree  of  peptide  coexistence,  and  studies  on  the  regulation  of  specific 
peptide  gene  expression  in  these  neurons  in  dissociated  cell  culture  is  a  major  activity 
in  this  Section.  In  addition  to  containing  neurons  which  secrete  specific  peptides,  a 
functional  network  must  also  contain  peptide  receptors.  In  our  Section,  the  analysis 
of  peptide  receptors  has  focused  on  two  candidates:  bombesin-like  peptide  family 
(gastrin-releasing  peptide  and  neuromedin  B)  receptor,  and  the  vasopressin  (V1) 
receptor.  Messenger  RNAs  from  cultured  cell  lines  containing  these  receptors  have 
been  injected  into  Xenopus  oocytes,  which  then  express  these  receptors  and  are 
assayed  by  pharmacological/electrophysiological  methods.  Efforts  to  clone  these 
receptors  are  in  progress,  using  the  Xenopus  assay  for  identification  of  relevant 
clones  (see  Section  on  Molecular  Neuroscience  summary  for  more  details).  Both 
tumor  cell  lines  and  primary  tissue  cultured  neurons  which  contain  the  above 
receptors  are  studied  by  patch-clamp  electrophysiological  techniques.  The  central 
issues  here  are  the  elucidation  of  signal  transduction  pathways  interposed  between 
activation  of  the  receptors  and  the  mobilization  of  membrane  effecters. 

At  present,  the  study  of  cytoskeletal  proteins  in  relation  to  neuronal  morphology 
in  our  Section  is  restricted  to  the  study  of  intermediate  filament  proteins.  Studies  on 
the  posttranslational  regulation  of  neurofilament  proteins  have  shown  that  these 
proteins,  which  appear  to  stabilize  axonal  structures,  are  highly  phosphorylated. 
Using  monoclonal  antibodies  and  immunocytochemistry  we  have  shown  that  the 
higher  molecular  weight  neurofilament  proteins  (NF-M  and  NF-H)  are  principally 
phosphorylated  in  axons  at  later  stages  of  development.  We  have  also  found  that 
the  specific  phosphorylated  sites  (epitopes)  found  in  NF-H  are  not  universally  used  by 
all  neurons.  Analyses  of  phosphorylated  epitopes  in  the  rat  spinal  cord 
demonstrates  that  myelinated  axons  of  comparable  diameter  in  the  cuneate  and 
corticospinal  tracts  selectively  phosphorylate  different  sites  on  NF-H.  Probably  the 
most  significant  issue  with  respect  to  neurofilaments  is  what  role  these  polymers  and 
their  phosphorylation  play  in  neuronal  structure  and  function.  The  most  popular 
hypothesis  at  present  is  that  these  molecules  stabilize  axonal  structure  and  are 
involved  in  increasing  axonal  caliber.  We  have  examined  their  function  by  injecting 
antibodies  to  specific  neurofilament  subunits  and  forms  into  one  blastomere  of 


2-LNC/DIR 


Xenopus  embryos  at  the  two-cell  stage.  With  subsequent  development  these 
antibodies  partition  selectively  into  only  those  cells  which  derive  from  the  injected 
blastomere.  In  Xenopus,  this  leads  to  a  bilateral  difference  in  the  antibody 
distribution  and  also  appears  to  modify  axonal  outgrowth  and  branching  only  in 
those  neurons  which  contain  the  antibodies,  suggesting  that  neurofilaments  may 
play  a  role  in  axonal  outgrowth  and  elongation.  We  have  also  found  that 
neurofilament  phosphorylation  appears  to  protect  the  neurofilaments  from 
degradation  by  calpain  (a  calcium-dependent  protease  presumed  to  act  on  NFs  in 
nerve  terminals),  and  that  both  squid  axoplasm  and  bovine  spinal  cord  contain 
unique  protein  kinases  related  to  casein  kinase  I  that  can  phosphorylate 
neurofilament  proteins.  Experiments  employing  mammalian  sensory  ganglion 
explants  are  currently  underway  to  investigate  the  effects  of  specific  protein  kinase 
activations  on  the  phosphorylation  of  specific  serine/threonine  residues  in  NF-L,  M, 
and  H  and  peripherin  in  cell  bodies  versus  axons.  The  significance  of  site  specific 
phosphorylation  on  neurofilament  assembly  and  function  remains  an  enigma  and  a 
major  focus  of  this  Section. 

II.  Section  on  Enzyme  Chemistry 

The  major  projects  in  the  Section  on  Enzyme  Chemistry  are  concerned  with  the 
relationships  between  structure,  function  and  the  mechanism  of  the  ATP-dependent 
cation  transport  proteins.  The  ability  of  cells  to  use  metabolic  energy  to  create  and 
maintain  gradients  of  Na  +  ,K  +  ,Ca2  +  and  protons  depends  directly  on  these 
transport  proteins.  In  the  case  of  the  sodium-potassium  pump,  some  general 
principles  of  the  transport  mechanism  and  considerable  structural  information  are 
now  available,  but  relatively  little  is  known  about  the  relationships  between 
structure  and  function.  Current  projects  in  the  Section  on  Enzyme  Chemistry  are 
directed  toward  (1)  resolving  certain  mechanistic  questions,  and  (2)  gaining 
information  about  the  relationships  between  structure  and  function. 

We  have  been  developing  a  series  of  antibodies  against  synthetic  peptides  that 
correspond  to  strategically  located  segments  of  sodium  pump  proteins.  We  are 
studying  the  isoforms  of  the  sodium  pump  that  are  expressed  in  rat  brain  tissue.  All 
three  isoforms  are  expressed  in  a  single  neuronal  cell  type,  i.e.,  primary  cultures  of 
the  cerebellar  granule  cell.  Studies  of  the  selective  regulation  of  these  isoforms  in 
pituitary  and  pineal  glands  are  in  progress. 

The  sodium  pump  consists  of  equimolar  a-  and  B-subunits.  Little  is  known  of  the 
function  of  the  8-subunit,  which  is  heavily  glycosylated.  Although,  as  yet,  there  is 
good  evidence  for  only  a  single  message  for  the  principal  B-subunit,  the  expressed 
protein  displays  a  marked  degree  of  microheterogeneity.  We  have  recently  shown 
that  much  of  this,  in  Electrophorus  electric  organ,  can  be  ascribed  to  the  sialic  acid 
component.  Current  studies  are  directed  at  determining  the  sites  of  glycosylation  in 
the  Electrophorus  B-subunit.  Because  a  second  form  of  the  B-subunit  has  been 
identified  in  rat  brain,  we  are  planning  to  examine  the  physiological  inter- 
relationships among  the  isoforms  of  the  a-  and  B-subunits. 

We  are  also  extending  previous  studies  on  the  mechanism  through  which  the 
catalytic  phosphorylation  of  the  sodium  pump  is  coupled  to  cation  transport.  This 
involves  a  combination  of  steady-state  kinetic  studies  of -ATPase  and  phosphatase 
activities  and  radiometric  studies  of  cation-binding. 


3  -  LNC/DIR 


Several  aspects  of  these  studies  are  interrelated.  Our  recent  kinetic  studies  of  the 
sodium  pump  from  Electrophorus  electric  organ  indicate  that,  even  in  a  preparation 
that  appears  to  consist  of  a  single  isoform,  the  kinetics  are  complicated  by  oligomeric 
interactions.  This  raises  questions  about  cells  that  express  more  than  one  isoform. 
Do  isoforms  interact  within  the  same  cell  to  form  functional  hybrids?  Are  the  B- 
subunits  that  combine  with  different  isoforms  identical?  Are  these  variables 
functionally  significant?  Some  of  the  techniques  to  address  such  questions  are  now 
at  hand. 


III.  Section  on  Molecular  Neuroscience 

The  goal  of  this  Section  is  to  use  a  molecular  approach  to  explore  the  structure, 
function,  and  regulation  of  neuropeptide  hormone  and  neuropeptide  receptor 
genes  in  the  mammalian  nervous  system.  The  current  effort  is  focused  on  the 
mammalian  bombesin-like  peptides  (gastrin-releasing  peptide  (GRP)  and 
neuromedin  B  [NMB])  and  their  receptors.  The  bombesin  family  of  peptides  is 
expressed  in  many  brain  nuclei,  the  dorsal  root  ganglia,  and  the  posterior  spinal 
cord.  They  are  potent  neuropeptides,  eliciting  a  variety  of  central  homeostatic  and 
behavioral  responses  including  poikilothermia,  regulation  of  blood  glucose  levels, 
anorexia,  alterations  in  gastric  acidity,  and  scratching  behavior.  These  peptides  have 
also  been  detected  in  the  intrinsic  neurons  of  the  gut,  where  they  regulate  smooth 
muscle  contraction,  stimulate  the  release  of  gastrin  from  G  cells  of  the  antral  gastric 
mucosa,  and  function  as  secretogogues  for  a  variety  of  gastroenteropancreatic 
hormones.  Bombesin-like  peptides  are  mitogens  for  growth-arrested  murine 
embryonal  Swiss  3T3  fibroblasts  in  culture,  G  cells  in  neonatal  rat  antral  mucosa,  and 
autocrine  growth  factors  for  human  small  cell  lung  cancer,  indicating  that  under 
some  circumstances  these  peptides  can  regulate  cell  division  in  addition  to 
transducing  a  secretory  or  neuromodulatory  signal. 

We  have  obtained  cDNA  and  genomic  clones  for  the  prohormones  encoding  the 
two  known  mammalian  bombesin-like  peptides,  GRP  and  NM-B.  The  rat  prepro  GRP 
gene  is  expressed  in  many  brain  nuclei,  most  prominently  in  the  suprachiasmatic 
nucleus  of  the  hypothalamus.  Two  forms  of  the  mRNA  are  found  in  brain:  a  more 
abundant  1.1  kb  form  which  initiates  in  both  central  and  peripheral  neurons,  and  a 
less  abundant  1.5  kb  mRNA  form,  whose  initiation  sites  are  heterogeneous,  located 
several  hundred  bases  upstream  of  the  1.1  kb  initiation  site,  and  is  used  only  in  spinal 
cord  and  a  subset  of  brain  nuclei  expressing  prepro  GRP  mRNA.  Studies  performed 
on  cultured  cells  expressing  the  human  prepro  GRP  gene  indicate  that  gene 
regulation  occurs  primarily  at  the  level  of  transcription  initiation,  and  involves 
chromatin  structural  changes  resulting  in  DNase  hypersensitive  sites.  Transfection  of 
GRP  promotor-luciferase  reporter  gene  constructs  into  these  cells  have  identified 
two  cis  promotor  elements  of  importance  in  regulating  prepro  GRP  transcription. 
These  regions  will  be  precisely  mapped  and  characterized,  with  the  central  goal  of 
obtaining  cDNA  clones  for  any  novel  transcription  factors  which  specifically  bind 
these  cis  acting  elements.  The  molecular  mechanisms  responsible  for  cell-type 
specific  regulation  of  the  prepro  GRP  gene  constitute  an  area  of  intense  interest  that 
we  plan  to  pursue  in  detail  in  the  future. 

We  have  isolated  and  characterized  cDNA  and  genomic  clones  for  the  rat  prepro 
NM-B  gene.  The  gene  has  a  three-exon  structure  analogous  to  that  described 
previously  for  the  prepro  GRP  gene,  consistent  with  the  view  that  the  two  genes 
diverged  from  a  common  ancestral  precursor  gene.  Similarity  between  the  two 
genes  is  observed  only  over  the  carboxyl  ten  amino  acids  of  the  GRP  and  NM-B 


4-LNC/DIR 


peptides,  which  is  the  region  of  the  peptide  necessary  and  sufficient  for  high  affinity 
binding  to  bombesin  receptors.  The  sequences  of  the  two  genes  in  the  promoter 
region  show  no  regions  of  similarity,  suggesting  that  the  two  genes  are 
independently  regulated.  Expression  of  the  gene  results  in  a  1.0  kb  mRNA  species 
that  is  most  abundantly  expressed  in  brain  and  gut.  The  initiation  site  in  the  brain 
appears  to  be  heterogeneous,  and  not  TATA-directed.  In  situ  hybridization  studies 
localizing  NM-B  mRNA  in  the  brain  indicate  that  the  distribution  of  cells  and  loci 
expressing  NM-B  is  quite  distinct  from  those  expressing  GRP,  and  more  limited.  Both 
genes  contribute  independently  to  the  bombesin-like  immunoreactivity  described 
previously  in  the  brain,  indicating  that  these  two  peptides  modulate  different 
physiologic  processes.  Neuromedin  B  mRNA  is  expressed  at  high  levels  in  trigeminal 
and  dorsal  root  ganglia  neurons.  In  collaboration  with  Dr.  Gainer,  we  are  currently 
exploring  the  possibility  that  primary  cultures  derived  from  these  ganglia  may 
provide  a  population  of  neurons  expressing  the  neuromedin  B  gene.  These  cells 
would  be  appropriate  hosts  for  promoter-reporter  fusion  gene  transfection  studies 
done  in  collaboration  with  Dr.  Gainer's  lab  to  define  transcription  regulatory 
elements  in  the  neuromedin  B  promoter. 

In  collaboration  with  Dr.  Richard  Feldman  at  Triton  Biosciences,  and  Drs.  Kiyoshi 
Kusano  and  Hagit  Shapira  of  the  Laboratory  of  Neurochemistry,  we  have  isolated 
cDNA  clones  encoding  the  GRP  receptor  expressed  in  Swiss  3T3  murine  embryonal 
fibroblasts.  Amino  acid  sequences  derived  from  an  internal  tryptic  fragment  of  the 
purified  GRP  receptor  protein  were  used  to  design  oligonucleotide  probes,  which  in 
turn  were  used  to  screen  a  subtracted  cDNA  library  enriched  for  Swiss  3T3  GRP 
receptor  cDNA  clones.  Structural  analysis  of  cDNA  clones  encoding  the  entire  open- 
reading  frame  show  that  the  receptor  is  a  member  of  the  G-protein  coupled  receptor 
superfamily  with  seven  predicted  transmembrane  domains  and  numerous  sites  for 
N-linked  glycosylation.  In  vitro  transcripts  from  cloned  cDNA  templates 
enrompassing  the  predicted  protein  coding  domain  express  functional  GRP 
receptors  when  injected  into  Xenopus  oocytes. 

Low-stringency  screening  of  cDNA  libraries  derived  from  rat  esophagus  and 
olfactory  bulb  using  the  Swiss  3T3  GRP-preferring  receptor  probe  identified  a  clone 
sharing  50%  amino  acid  identity,  which  is  an  excellent  candidate  for  an  additional 
bombesin  receptor  subtype  (NMB-preferring)  known  to  exist  in  these  tissues. 
Expression  of  this  candidate  receptor  in  Xenopus  oocytes  will  confirm  this  result, 
since  there  is  an  antagonist  available  which  selectively  blocks  the  GRP  receptor  from 
Swiss  3T3  cells  but  does  not  block  the  NMB-preferring  bombesin  receptor  found  in 
the  esophagus.  These  clones  will  form  the  basis  for  site-specific  mutagenesis 
experiments  probing  the  domains  and  residues  involved  in  specific  ligand  binding 
and  coupling  to  G-proteins.  Expression  of  cDNA  clones  in  various  model  systems  will 
allow  the  production  of  large  quantitites  of  this  relatively  rare  protein,  facilitating 
the  identification  of  subtype-specific  antagonists  which  will  prove  useful  in 
dissecting  the  functions  of  the  bombesin-like  peptides  and  their  receptors  in  the 
nervous  system.  In  addition,  these  antagonists  may  prove  useful  as  therapeutic 
agents  in  the  subset  of  human  small  cell  lung  carcinomas  which  show  bombesin- 
dependent  growth. 


5  -  LNC/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01-NS-00813  29LNC 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  /ess.  Title  must  fit  on  one  line  between  the  borders.} 

Enzymological  Aspects  of  Neural  Functions 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  Robert  W.  Albers,  Ph.D.  Chief,  Enzyme  Chemistry  LNC,  NINDS 

Others:        William  T.  Link,  Ph.D.  Senior  Staff  Fellow  LNC,  NINDS 

CharleneP.  Osborn,  B.S.  Biological  Technician  LNC,  NINDS 

Paul  M.  Rowe,  Ph.D.  Senior  Staff  Fellow  LNC,  NINDS 


COORPERATING  UNITS  lit  an,) 

J. P.  Froehlich,  Ph.D.,  M.D.,  NIA,  NIH,  Baltimore,  MD; 

E.  Bamberg,  Max-Planck-lnstitut  fur  Biophysik,  Frankfurt,  FRG;  D.  Klein,  Ph.D.,  NICHD,  NIH 


LAB/BRANCH 

Neurochemistry,  DIR,  NINDS 


SECTION 

Enzyme  Chemistry 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS:  .  n 

4.0 


PROFESSIONAL:  o  q 


OTHER: 


1.0 


CHECK  APPROPRIATE  BOX(ES) 

Q   (a)  Human  subjects  ^]  (b)  Human  tissues  [x~|  (c)  Neither 

]  (a1)  Minors 
J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  is  comprised  of  research  into  the  structure  and  functioning  of  ion  transport  systems.  There 
are  currently  4  active  subprojects:  1)  Studies  of  the  transient-state  kinetics  of  phosphorylation  and 
dephosphorylation  of  the  Na,K-ATPase  catalytic  site,  utilizing  rapid  quenching  techniques.  2)  A  study  of 
the  regulation  and  expression  of  isoforms  of  the  Na,K-ATPase  utilizing  site-directed  antibodies  raised 
against  synthetic  peptides  as  identifying  probes.  3)  A  study  of  the  relation  of  the  glycosylation  state  of 
the  B-subunit  of  the  Na,K-ATPase  to  the  expression  and  function  of  the  sodium  pump.  4)  A  study  of  the 
interactions  between  the  catalytic  and  the  ionophoric  domains  of  the  sodium  pump  utilizing  steady- 
state  kinetic  and  radiometric  binding  techniques. 


6-LNGDIR 


■HS6CM0(Row    1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01NS  02723-04  LNC 


PERIODCOVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  fAO characters  or  less    Title  must  fit  on  one  line  between  the  borders.) 

Peptides  in  the  Adult  and  Developing  Vertebrate  Nervous  Systems 


PRINCIPAL  INVESTIGATOR  tList  other  profession*/  personnel  below  the  Principal  Investigator.)  {Name,  title,  laboratory.  *nd  institute  affiliation) 

PI:  Harold  Gainer,  Ph.D.  Chief  LNC,  NINDS 

Others:        Carolyn  Bondy,  M.D.  Senior  Staff  Fellow  LNC,  NINDS 

MonaCastel.Ph  D  Visiting  Scientist  LNC,  NINDS 

Sharon  Key,  B.S.  Biologist  LNC,  NINDS 

Susan  Wray,  Ph.D.  Senior  Staff  Fellow  LNC,  NINDS 


COORPERATING  UNITS  (,fanf) 

Dr.  W.J.  Schwartz,  University  of  Massachussetts,  School  of  Medicine;  Dr.  W.S.  Young,  NIMH; 
Dr.  S.  ^adovick,  NIDDK 


LAB/BRANCH 

Laboratory  of  Neurochemistry,  DIR,  NINDS 


SECTION 

Section  on  Cellular  and  Developmental  Neurobiology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN-YEARS:  .  n 

4.0 


PROFESSIONAL:  o  Q 


OTHER: 


1.0 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [~x~l  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  nor  exceed  the  space  provided.) 

Studies  of  the  extracellular  matrix  over  the  track  upon  which  the  LHRH  cells  migrate  from  the  olfactory 
placode  to  the  CNS  have  shown  that  the  appearance  of  N-CAM  directly  correlates  with  the  appearance  of 
LHRH  cells,  whereas  fibronectin  does  not.  LHRH  cells  are  present  in  tissue  cultures  deriveJ  from 
embryonic  nasal  regions  of  mice.  A  transgenic  line  was  established  using  a  human  LHRH  promoter  joined 
to  the  SV40  t-antiqen  construct.  These  animals  show  abnormal  reproductive  function  and  on 
examination  very  few  LHRH  immunopositive  cells  were  detected  in  the  forebrain.  Transgenic  mice 
containing  constructs  which  have  both  AVP  and  OT  genes  together  exhibit  cell-specific  expression, 
whereas  transgenic  mice  with  only  one  of  these  genes  exhibit  only  ectopic  expression.  Studies  of  aldose 
reductase  mRNA  in  lens  and  kidney  show  that  expression  of  this  gene  is  inhibited  by  galactosemia  and 
hyperglycemia.  Rat  brain  slice-explant  cultures  have  been  used  to  show  that  estrogen  and  inhibition  of 
spontaneous  electrical  activity  by  TTX  leads  to  inhibition  of  LHRH  gene  expression.  Exaggerated 
coexistence  of  CGRP  and  substance  P  peptides  in  postnatal  sensory  neurons  in  vitro  was  found. 


7-LNC/DIR 


■"!'.  blUOIK.v    1*4) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01 -NS-02724-04LNC 


PERIOD  COVERED 

Octoberl,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Molecular  Mechanisms  in  Neuronal  Structure  and  Function 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  Harold  Gainer,  Ph.D.  Chief  LNC,  NINDS 

Others:        Margi  Goldstein,  Ph.D.  Senior  Staff  Fellow  LNC,  NINDS 

Philip  Grant,  Ph.D.  Expert  Consultant  LNC,  NINDS 

Shirley  House,  B.S.  Biologist  LNC,  NINDS 

Ben  Szaro,  Ph.D.  Senior  Staff  Fellow  LNC,  NINDS 


COORPERATING  UNITS  of  any) 

L.  Charnas,  M.D.,  NICHD;  V.M.Y.  Lee,  Ph.D.,  University  of  Pennsylvania 


LAB/BRANCH 

Laboratory  of  Neurochemistry,  DIR,  NINDS 


SECTION 

Section  on  Cellular  and  Developmental  Neurobiology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  j  5 


OTHER: 


1.0 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [~x~~|  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Monoclonal  antibodies  directed  against  neurofilament  (NF-M)  proteins  in  Xenopus,  have  been  injected 
into  individual  blastomeres  of  Xenopus  embryos  at  the  two-cell  stage.  These  antibodies  remain 
restricted  in  the  intracellular  space  so  that  only  cells  derived  from  the  injected  blastomere  contain  the 
antibody  (i.e.,  about  half  the  cells  in  the  developing  embryo  contain  antibody).  The  results  are  that  the 
axons  derived  from  neurons  containing  these  antibodies  show  a  variety  of  dramatic  deficiencies  in 
axonal  outgrowth  and  elongation,  representing  the  first  demonstration  in  vivo  that  perturbation  of  a 
neurofilament  protein  has  structural  consequences.  Studies  on  rat  central  and  peripheral  nervous  system 
neurons  in  vivo  and  in  vitro  have  shown  that  different  post-translational  modifications  (i.e., 
phosphorylation  of  specific  sites)  of  neurofilaments  are  characteristic  of  specific  nerve  pathways  and 
neuronal  types,  and  not  necessarily  correlated  with  axonal  caliber  or  their  central  or  peripheral  origins. 
Dissociated  sensory  ganglion  cells  from  postnatal  rats  survive  in  defined  media  in  the  absence  of  NGF, 
but  are  deficient  in  general  growth,  neuropeptide  and  neurofilament  expression  and  neuritic 
outgrowth. 


8-LNGDIR 


•v-ioMOIRev.  1,84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01-NS-02725-04LNC 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80 character s  or  Ion    Title  must  tit  on  one  line  between  the  borders  ) 

Calcium  Metabolism  and  Protein  Phosphorylation  in  Neuronal  Systems 


PRINCIPAL  INVESTIGATOR  (List  other  professional  per  sonnel  6e'ow  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation} 

PI:  HarishC.  Pant,  Ph.D.  Research  Chemist  LNC,  NINDS 

Others:         Ayse  Dosemeci,  Ph.D.  Visiting  Fellow  LNC,  NINDS 

Carl  C.  Floyd,  PhD  IRTA  Fellow  LNC,  NINDS 

Ben  G.Szaro,  Ph.D.  Senior  Staff  Fellow  LNC,  NINDS 

James  Battey,  M.D.,  Ph.D.  Chief,  Molecular  Neurosciences  Section  LNC,  NINDS 

James  Way,  B.S.  Biologist  LNC,  NINDS 

Alexander  Wheaton  Biological  Laboratory  Technician  LNC,  NINDS 


COOPERATING  UNITS  (if  an,) 

Dr.  P.E.  Gallant,  LN,  NINDS;  Dr.  Jan  Metuzals,  University  of  Toronto 


LAB/BRANCH 

Neurochemistry,  DIR,  NINDS 


SECTION 

Cellular  and  Developmental  Neurobiology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS:  .  .. 

4.3 


PROFESSIONAL:  ,  j 


OTHER: 


1.1 


CHECK  APPROPRIATE  BOX(ES) 

LJ   (a)  Human  subjects  ]  (b)  Human  tissues  ["*!  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Studies  have  been  continued  to  understand  the  regulation  and  role  of  neurofilament  phosphorylation  in 
the  nervous  system.  Axonal  neurofilament-rich  preparation  from  squid  giant  axon  contains  second 
messenger-independent  protein  kinases  that  phosphorylate  high  molecular  weight  >400  and  220  kDa 


squid  neurofilament  protein  subunits,  as  well  as  exogenous  substrates.  Two  major  kinase  activities  were 
separated  and  characterized  in  this  preparation.  One  of  these  strongly  phosphorylated  kemptide  and 
was  inhibited  by  the  selective  inhibitors  of  cAMP-dependent  kinase,  wiptide.  The  second  kinase  activity 
effectively  phosphorylated  alpha-casein  and  was  not  inhibited  by  wiptide  and  heparin.  The  alpha-casein 
phosphorylating  activity  was  the  principal  activity  responsible  for  neurofilament  protein 
phosphorylation  and  was  not  inhibited  by  various  kinase  inhibitors.  A  newly  synthesized  isoquinohne 
derivative  (CKI-7)  that  specifically  inhibited  purified  casein  kinase-l  was  the  effective  inhibitor  of  the 
axonal  neurofilament  protein  kinase.  The  physical,  biochemical  and  pharmacological  studies  indicated 
that  the  major  kinase  activity  associated  with  axonal  neurofilaments  is  like  a  casein  kinase  I.  The 
mammalian  neurofilament  associated  kinases  appear  to  be  more  complex.  There  was  only  a  partial 
inhibition  of  bovine  neurofilament  kinase  activity  by  casein  kinase  I  inhibitor  and  the  tryptic  peptide 
maps  of  neurofilament  protein  subunit  (NF-M)  after  its  phosphorylation  by  bovine  neurofilament  kinase 
showed  some  similarities  when  phosphorylated  by  purified  casein  kinase  I  or  casein  kinase  II.  The  cAMP- 
dependent  kinase  phosphorylated  distinct  peptides  on  NF-M.  We  have  determined  the  sequence  of  a 
squid  neuronal  intermediate  filament  protein,  approximately  59  kDa,  using  a  cDNA  library  made  from 
squid  optic  lobe.  This  protein  is  specific  to  neural  tissue,  and  is  present  in  the  axoplasm  of  the  giant  axon. 
Structurally,  its  rod  domain  possesses  features  common  to  mammalian  Type  IV  (neurofilament)  and  Type 
III  (vimentin,  GFAP)  intermediate  filament  proteins  as  well  as  Lamms.  By  highlighting  those  sequences  of 
NF-proteins  that  are  conserved  between  these  two  animals,  which  diverged  around  570  million  years 
ago,  the  sequence  of  this  mRNA  will  help  elucidate  functionally  important  domains  in  NF-proteins. 


9-LNC/DIR 


^HS  bM  |H«v    1  Ml 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01-NS-02753-02LNC 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (SO  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Molecular  Biology  of  the  Genes  Encoding  Prohormones  for  Bombesin-like  Peptides 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  James  Battey,  M.D.,  Ph.D.  Section  Chief  LNC,  NINDS 

Others:         Zahra  Fathi,  Ph.D.  IRTA  Fellow  LNC.  NINDS 

Etsuko  Wada,  M.D.,  Ph.D.  Visiting  Fellow  LNC,  NINDS 

James  Way,  B.S.  Biologist  LNC,  NINDS 


COORPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Neurochemistry,  DIR,  NINDS 


SECTION 

Section  on  Molecular  Neuroscience 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN-YEARS: 


1.8 


PROFESSIONAL: 


1.3 


OTHER: 


0.5 


CHECK  APPROPRIATE  BOX(ES) 

I   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  0  (0  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  goal  of  this  project  is  to  understand  aspects  of  the  structure,  function,  and  regulation  of  the  genes 
encoding  the  mammalian  counterparts  to  bombesin-like  neuropeptides.  Two  mammalian  bombesin-like 
peptides  have  been  identified  to  date,  gastrin-releasinq  peptide  (GRP)  and  neuromedin  B  (NM-B).  Both 
peptides  are  found  in  a  subset  of  central  and  peripheral  neurons,  and  share  a  structural  motif  at  their 
carboxyl-terminal  domains,  which  is  necessary  and  sufficient  for  binding  to  high-affinity  cell  surface 
bombesin  receptors.  There  are  several  subprojects  being  actively  pursued:  1)  cDNA  and  genomic  clones 
for  rat  and  human  prepro-GRP  and  prepro-NMB  genes  have  been  isolated  and  characterized  to  identify 
potential  regulatory  motifs  in  the  promoter  regions.  2)  In  situ  hybridization  technigues  are  being 
utilized  to  determine  the  localization  of  GRP  and  NMB  mRNA  at  the  cellular  level  in  brain;  3)  Promoter- 
reporter  gene  constructs  will  be  introduced  into  cultured  cell  hosts  by  transfection  to  map  the  elements 
in  the  promoter  region  of  the  human  GRP  gene  responsible  for  the  cell-type  specific  regulation. 


10-LNODIR 


■'•  "■>  bWD  IR.w   1.84J 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS02774  02  LNC 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  lesi.    Title  must  fit  on  one  line  between  the  borders.) 

Molecular  Cloning  of  Bombesin  Receptors 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Hame.  title,  laboratory,  and  institute  affiliation) 

PI:  James  Battey,  M .D.,  Ph.D.  Section  Chief  LNC,  NINDS 

Others:         Martha  Corjay,  Ph.D.  Staff  Fellow  LNC,  NINDS 

Kiyoshi  Kusano,  Ph  D  Visiting  Scientist  LNC,  NINDS 

HagitShapira,  Ph.D.  Visiting  Fellow  LNC,  NINDS 

Etsuko  Wada,  M.D.,  Ph.D.  Visiting  Fellow  LNC,  NINDS 

James  Way,  B.S.  Biologist  LNC,  NINDS 


COORPERATING  UNITS  (if  any) 

Richard  Feldman,  Ph.D.,  Triton  Biosciences,  1 501  Harbor  Way  Parkway,  Alameda,  CA  94501 


LAB/BRANCH 

Neurochemistry,  DIR,  NINDS 


SECTION 

Section  on  Molecular  Neuroscience 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS:  ^  n 


PROFESSIONAL:  j  Q 


OTHER:  1  q 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [x~l  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  goal  of  this  project  is  to  use  molecular  genetic  techniques  to  study  the  structure  and  function  of 
mammalian  bombesin  receptors.  We  plan  to  accomplish  this  objective  by  first  obtaining  a  full-length 
cDNA  clone  for  the  murine  bombesin  receptor  found  on  Swiss  3T3  fibroblasts  in  relatively  high  numbers. 
The  cDNA  will  be  sequenced  to  determine  the  predicted  amino  acid  sequence  and  general  structural 
features  of  the  receptor.  Specific  antisera  will  be  generated  against  synthetic  peptides  to  allow  further 
characterization  of  the  receptor  protein.  This  cDNA  clone  can  then  be  used  in  site-specific  mutagenesis 
studies  where  the  effects  of  structural  perturbations  can  be  examined  after  expression  of  the  mutant 
receptors  in  Xenopus  oocytes  and  DNA-mediated  transfection  into  murine  fibroblasts  which  do  not 
normally  express  the  receptor  Genomic  clones  will  be  isolated  to  examine  the  gene  structure  and 
promoter  region.  The  cDNA  coding  domain  will  be  used  as  a  low-stringency  probe  to  screen  cDNA 
libraries  from  appropriate  sources  for  other  distinct  but  structurally  related  bombesin  receptor  cDNA 
clones,  including  a  distinct  subtype  found  on  esophageal  smooth  muscle  and  in  the  olfactory  bulb  of  the 
brain. 


11  -LNC/DIR 


'-'.bWD|>t>.  1  »4) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS-02757-03LNC 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Analyses  of  Peptide  Receptors 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  Kiyoshi  Kusano,  Ph.D.  Visiting  Scientist  LNC,  NINDS 

Others:        James  Battey,  M.D.,  Ph.D.  Chief  Section  on  Molecular  Neurosciences  LNC,  NINDS 

Harold  Gainer,  Ph.D.  Chief  LNC,  NINDS 

HagitShapira,  Ph.D.  Visiting  Fellow  LNC,  NINDS 


COORPERATING  UNITS  Of  any) 

Dr.  M.J.  Brownstein  and  Dr.  L.  Mahan,  LCB,  NIMH;  Dr.  I.  Tasaki,  LCB,  NIMH; 
Dr.  R.  Wenthold,  LND,  NINDS 


LAB/BRANCH 

Laboratory  of  Neurochemistry,  DIR,  NINDS 


SECTION 

Section  on  Cellular  and  Developmental  Neurobiology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS:  c 

1 .0 


PROFESSIONAL:  .,  g 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  fx~|  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 
SU  MMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  study  on  the  cellular  and  molecular  mechanisms  of  peptide  receptor  signal  transduction  processes 
has  used  two  approaches.  The  first  approach  is  to  analyze  the  electrophysiological  responses  to 
application  of  neuropeptides  in  selected  peptide  receptor-bearing  cells:  Swiss  3T3  cells  for  bombesin 
(BN)  receptors  and  AR42J  cells  for  cholecvstokinin  (CCK)  receptors.  The  second  approach  is  to  study  the 
peptide  receptor  signal  transduction  process  in  detail  using  the  Xenopus  oocyte  surrogate  system,  after 
injection  of  mRNAs  extracted  from  the  above  cells.  Whole  cell  voltage-damp  and  patch-clamp 
techniques  are  applied  to  those  cultured  cells.  The  Swiss  3T3  cells  do  not  have  profound  voltage  sensitive 
K-channels,  but  some  cells  possess  a  transient  type  of  Ca-current  (lca(t>)  channels.  Application  of 
bombesin  to  3T3  cells  induces  a  K-conductance  increase  by  activating  a  Ca-mediated  K-current  (l«(Ca)) 
flow.  AR42J  cells  possess  voltage-sensitive  Ik,  l|\ia.  'ca(t)  ancl  'ca(i).  ancl  Ca-sensitive  lci(Ca)-  Application  of 
CCK  to  AR42J  cells  induces  a  conductance  increase  to  CI  ions,  whereas  Na  and  K  ions  do  not  show 
significant  effects.  In  both  cell  lines,  ligand  binding  with  receptors  triggers  a  rise  in  intracellular  Ca  ion 
concentration  by  releasing  from  intracellular  sources.  The  two-electrode  voltage  clamp  technique  has 
been  applied  to  assay  the  functionally  transcribed  CCK  and  bombesin  receptors  in  the  Xenopus  oocytes, 
after  injection  of  their  respective  mRNAs.  Size-fractionated  mRNAs  obtained  by  the  sucrose  gradient 
technique  are  further  assayed  in  order  to  clone  and  sequence  these  receptor  cDNAs  and  genes. 
Pharmacological  analysis  of  BN  receptors  expressed  by  injecting  mRNAs  of  various  tissues  indicates  that 
there  are  subtypes  in  brain  and  esophageal  tissues. 


12-LNC/DIR 


.  ',010  (Rev   1  811 


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03 
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30 
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30 


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ANNUAL  REPORT 


October  1, 1989  through  September  30, 1990 


Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 

Basic  Neurosciences  Program,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 


Table  of  Contents 

RESEARCH  SUMMARY  1-9 

PROJECT  REPORTS 

Blood-Brain  Barrier:  In  Vitro  Model  for  the  Study  of  10 

Cerebrovascular  Endothelial  Permeability 
Z01  NS  02324-14  LNNS 

Cerebral  Ischemia  and  Monoamines  11 

Z01  NS  02357-13  LNNS 

Evaluation  of  Electrical  Impedance  in  Cerebral  Ischemia  12 

Z01  NS  02548-09  LNNS 

Cerebial  Ischemia  and  Edema:  Tryptophan  Uptake  and  13 

Metabolism 

Z01  NS  02623-05  LNNS 

Regulation  of  Carbohydrate  Metabolism  in  14 

Cerebromicrovascular  Cultures 
Z01  NS  02689-06  LNNS 

Study  of  Cerebral  Electrical  Activity  Associated  with  1 5 

Ischemia 

Z01  NS  02718-05  LNNS 

Stress  Protein  Induction  in  Gerbil  Brain  After  Ischemia  16 

Z01  NS  02720-04  LNNS 

The  Measurement  of  Cerebral  Blood  Flow  by  Laser  1 7 

Doppler  Velocimetry 
Z01  NS  02749-04  LNNS 

Cultures  of  Mouse  Capillary  Endothelium:  Establishment 

Z01  NS  02751-04  LNNS  18 


Postischemic  Accumulation  of  Calcium  in  Brain  Tissue  19 

Z01  NS  02768-03  LNNS 

Observations  on  Global  Cerebral  Ischemia  in  Rats  20 

Z01  NS  02773-02  LNNS 

Blood-Brain  Barrier  Changes  Following  Repeated  21 

Ischemic  Insults 

Z01  NS  02775-02  LNNS 

Mechanism  of  Production  of  Experimental  Allergic  22 

Encephalomyelitis 

Z01  NS  02776-02  LNNS 

Human  Cerebromicrovascular  Endothelium:  23 

Studies  in  Vitro 

Z01  NS  02777-02  LNNS 

Cerebral  Vascular  Endothelial  Cell-Specific  24 

Monoclonal  Antibodies 
Z01  NS  02780-03  LNNS 

Human  Cerebromicrovascular  Endothelial  Culture:  25 

Cholinergic  and  Histaminergic  Receptors 
Z01  NS  02795-02  LNNS 

Transneuronal  Effects  of  Cryogenic  Brain  Injury  on  26 

Calcium  Uptake  and  Blood-Brain  Barrier  Changes 
Z01  NS  02796-02  LNNS 

Cultures  of  Human  Cerebromicrovascular  Endothelium:  27 

Establishment,  Growth  and  Characterization 
Z01  NS  02797-02  LNNS 

Ultrastructural  Observations  on  Distribution  of  28 

Calcium  in  Cerebral  Ischemia 
Z01  NS  02798-02  LNNS 

Interactions  Between  Cerebrovascular  Endothelial  29 

Cells  and  Immune  Lymphocytes 
Z01  NS  02801-02  LNNS 

Dynamics  of  Postischemic  Calcium  Accumulation  and 

Protein  Synthesis  in  Brain 

Z01  NS  02821-01  LNNS  30 

Glutamate  Microdialysis  During  Repeated  Ischemia 

and  Cold  Lesions. 

Z01  NS  02822-01  LNNS  31 

Immune  Mechanisms:  Regulation  of  la  Antigen  Expression  32 

Z01  NS  02802-03  LNNS 


ANNUAL  REPORT 

October  1#  1989  through  September  30, 1990 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 

Basic  Neurosciences  Program,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 


Igor  Klatzo,  M.D.  Chief 


During  the  past  year,  the  Laboratory  of  Neuropathology  and  Neuroanatomical 
Sciences  has  continued  its  effort  on  elucidating  the  mechanisms  operative  in  the 
pathophysiology  of  ischemia  and  traumatic  brain  injury. 

In  the  Section  on  Cerebrovascular  Pathology,  the  main  interest  has  been  directed  to 
define  and  evaluate  extent  to  which  neuroexcitatory  mechanisms  were  involved  in  the 
final  development  of  postischemic  and  posttraumatic  lesions. 

In  1975,  our  laboratory  was  first  to  describe  a  selective,  delayed  destruction  of  CA1 
pyramidal  neurons  of  the  hippocampus  following  a  short-lasting  cerebral  ischemia  (Ito  et 
al.  1975).  Later  we  were  able  to  associate  the  development  of  this  injury  with  a 
protracted  period  of  neuroexcitation  of  pyramidal  CA1  neurons  by  a  direct  recording  of 
axonal  discharges  with  microelectrodes  (Suzuki  et  al.  1983).  At  the  same  time  the 
neuroexcitatory  nature  of  CA1  lesions  was  further  confirmed  by  Benveniste  et  al.  (1984), 
who  demonstrated  by  microdialysis  a  marked  extracellular  release  of  glutamate  and 
aspartate  during  and  after  an  ischemic  insult.  The  demonstration  of  neuroexcitation  as  a 
primary  mechanism  of  ischemic  injury  has  long  been  confined  to  the  hippocampus.  More 
recently,  however,  it  became  increasingly  apparent  that  excitatory  damage  may  involve 
different  transmitter  systems  and  may  affect  distant  neuronal  locations,  according  to 
connecting  circuitries.  Thus,  an  excitatory  effect  of  the  dopaminergic  system  on  ischemic 
injury  was  demonstrated  by  Globus  (1987)  who  reported  a  significant  reduction  of 
striatal  damage  by  lesioning  the  substantia  nigra. 

One  of  our  three  experimental  models  of  cerebral  ischemia  in  which  we  assume  a 
widespread  involvement  of  excitatory  mechanisms  is  repetitive  ischemia  in  gerbils.  Our 
studies  on  the  effect  of  three  2-minute  occlusions  at  one-hour  intervals  revealed  a 
pattern  of  dense  accumulations  of  45Ca  in  various  structures  at  different  time  sequences. 
The  earliest  accumulations  appeared  in  the  ventral  parts  of  the  thalamus,  particularly  in 
the  nucleus  reticularis  thalami,  then  in  the  lateral  portions  of  caudate,  and  the  cerebral 
cortex,  and  after  4  days  in  the  medial  geniculate  bodies  and  substantia  nigra.  There  was 
an  obvious  correlation  between  caudate  and  substantia  nigra,  since  the  unilateral 
preponderance  of  calcium  uptake  in  the  caudate  corresponded  to  increased  45Ca  on  the 
same  side  in  the  substantia  nigra.  The  abnormal  uptake  of  calcium  correlated  with  the 
appearance  of  histological  neuronal  changes  which,  however,  were  usually  of  a 
moderate,  chronictype,  with  exception  of  CA1  pyramidal  neurons,  which  showed  severe 
destruction  after  4  days. 

The  abnormal  calcium  uptake  correlated  also  with  changes  in  blood-brain  bar Tier 
(BBB)  permeability  to  serum  proteins,  expressed  by  the  noticeable  presence  of 
extravasated  albumin  in  the  cytoplasm  of  ischemically  affected  neurons,  which  was 
demonstrated  by  staining  with  conjugated  anti-gerbil  albumin  serum.  The  abnormal 
accumulation  of  calcium  and  intracytoplasmic  albumin  uptake  were  associated  with 


1  -LNNS/DIR 


morphological  picture  of  rather  moderate  neuronal  injury  and  were  absent  in  regions 
showing  severe,  irreversible  damage  of  the  nerve  cells. 

The  involvement  of  neuroexcitatory  mechanisms  in  repetitive  ischemia  was 
supported  also  by  our  observations  using  microdialysis  to  assess  levels  of  glutamate 
during  and  following  repetitive  ischemic  insults.  The  release  of  extracellular  glutamate 
was  evaluated  by  cortical  microdialysis  followed  by  an  enzymatic  cycling  assay  employing 
glutamate  dehydrogenase  and  glutamate  B-oxaloacetate  transaminase.  Our  assay 
revealed  in  some  animals,  during  and  after  repeated  5-minute  occlusions,  a  progressive 
increase  in  glutamate  release  after  the  second  and  third  ischemic  insult,  often  reaching 
concentrations  of  glutamate  in  the  dialysate  comparable  to  those  observed  after  a  single 
1 5-minute  occlusion.  Such  progressively  higher  levels  of  glutamate  may  constitute  the 
main  explanation  for  a  cumulative  effect  of  repeated  insults,  whereas  this  cumulative 
effect  appeared  in  our  previous  studies  to  be  totally  unrelated  to  the  reduced  cerebral 
blood  flow  (hypoperfusion)  which  develops  after  recirculation,  or  to  energy  metabolism 
disturbances  during  the  immediate  postischemic  periods  (Hossmann  et  al.  1990). 

Studies  on  the  the  formation  of  secondary  foci  of  injury  following  a  cryogenic 
cortical  lesion  in  rat  have  further  supported  to  the  assumption  of  neuroexcitatory 
mechanisms  operative  in  the  development  of  such  lesions.  The  delayed  appearance  of 
secondary  lesions  in  the  ipsilateral  thalamus  or  bilaterally  in  CA1  sectors  of  the 
hippocampus,  when  the  cortical  injury  was  extended  to  include  the  entorhinal  cortex, 
was  characterized  by  the  presence  of  4$Ca  accumulation,  neuronal  uptake  of  serum 
albumin,  and  morphological  changes  indicating  a  moderate  neuronal  injury.  To 
establish  whether  these  changes  originate  from  neuroexcitation  at  the  margin  of  the 
cryogenic  focus,  the  cold  lesion  was  produced  in  proximity  to  the  rat  visual  cortex  from 
which  the  evoked  potentials  were  measured  following  stroboscopic  light  stimulation. 
Our  observations  indicate  a  significant  reduction  in  latency  time  which  appeared 
approximately  4  hours  after  induction  of  the  cortical  lesion.  This  reduction  in  the  latency 
period  could  be  abolished  by  pretreatment  with  the  noncompetitive  glutamate 
antagonist  of  MK-801.  Further  indication  of  glutamate  involvement  was  obtained  from 
direct  application  of  I  mM  glutamate  solution  on  the  exposed  cortex,  which  reduced  the 
latency  period  similar  to  that  following  cold  injury.  The  demonstrated  reduction  in  the 
latency  time  would  be  in  line  with  facilitation  of  neuronal  excitability.  Another 
observation,  which  would  support  an  assumption  that  neuroexcitation  may  be  primarily 
responsible  for  the  development  of  secondary  lesions,  was  provided  by  comparative 
evaluation  of  volumes  of  the  areas  of  the  cerebral  cortex  and  the  thalamus  showing 
dense  4$Ca  accumulation.  Such  assessments,  done  by  Dr.  G.  Mies,  indicated  that  the 
thalamic  foci  tended  to  be  of  uniform  volume,  whereas  the  cortical  45Ca  areas  varied 
greatly  in  volume,  without  any  correlation  to  the  thalamic  foci.  This  observation 
indicates  against  a  possibility  that  the  thalamic  lesions  were  merely  a  product  of  a 
retrograde  neuronal  degeneration. 

An  assumption  of  neuroexcitatory  involvement  was  further  strengthened  in  our 
studies  on  the  pathophysiology  of  global  ischemia  produced  by  compression  of  the  major 
heart  vessels.  This  model  was  characterized,  as  in  repetitive  ischemia,  by  45Ca  marking  of 
various  anatomic  locations  affected  by  ischemia  which  appeared  after  different  time 
delays.  The  most  striking  was  sharply  outlined  very  dense  marking  of  the  nuclei 
reticularis  thalami  as  early  as  6  hours  after  10  minute  global  ischemia,  thus  appearing  as 
the  first  structure  affected  by  ischemia.  This  may  have  etiologic  significance,  since  the 
ischemic  affection  of  this  nucleus  which  consists  entirely  of  GABAergic  neurons,  may 
remove  inhibitory  function  on  the  otherthalamic  nuclei  and  other  structures, 
contributing  thus  to  development  of  ischemic  lesions  in  "uninhibited"  areas.  After 
24  hours  accumulation  of 45Ca  calcium  was  noticeable  in  other  regions  such  as  cerebral 
cortex,  caudate,  ventral  thalamic  nuclei,  medial  geniculate  and  substantia  nigra.  In  the 
hippocampus,  CA1  neurons  showed  abnormal  calcium  uptake  only  after  48  hours.  At  the 

2-LNNS/DIR 


initial  postischemic  periods  the  abnormal  calcium  uptake  was  associated  with  neuronal 
changes  assessed  with  cresyl  violet  stain.  After  one  week  the  abnormal  calcium  deposits 
persisted  in  the  CA1  sector  of  hippocampus  and  in  the  dorsolateral  portions  of  the 
caudate,  which  later  revealed  severe  neuronal  damage.  In  the  other  areas,  animals 
examined  after  2  weeks  and  later  showed  fading  or  absent  abnormal  45Ca  radioactivity. 
Otherwise  those  regions  revealed  an  improved  neuronal  appearance,  the  nerve  cells 
being  either  well-preserved  or  showing  a  minimal  injury.  These  observations  provide 
another  indication  that  following  ischemic  injury  although  neurons,  may  show  marked 
histological  changes,  such  as  hyperchromasia,  loss  of  Nissl  bodies  and  cytoplasmic 
vacuolization,  nonetheless  they  preserve  their  capacity  for  recovery,  this  constitutes  the 
most  important  research  challenge  to  find  the  means  to  facilitate  such  neuronal 
recovery. 

Concerning  the  abnormal  deposition  of 45Ca,  it  clearly  requires  participation  of 
living  cells,  since  it  does  not  occur  in  regions  where  neurons  suffer  irreversible  injury, 
with  exception  of  CA1  sector  of  the  hippocampus  which  may  show  persistently  dense 
45Ca  radioactivity,  even  a  few  months  after  ischemic  injury.  In  such  cases,  the  increased 
4$Ca  content  may  be  related  to  ultrastructurally  observed  dense  calcium  precipitates  in 
the  surving  pyramidal  cells  and  interneurons  as  seen  by  EM  with  the  oxalate- 
pyroantimonate  method. 

Simultaneous  demonstration  of  4$Ca  uptake  and  protein  synthesis  in  the  same 
sections  brought  some  insight  into  the  problems  of  ability  and  extent  of  neuronal 
recovery  from  ischemic  insults.  The  separate  demonstration  of  4$Ca  ar|d  3H-leucine 
radioactivities  was  accomplished  by  exposure  of  the  same  sections  to  emulsions 
selectively  sensitive  to  these  two  markers.  Observations  with  this  procedure  revealed 
that  the  abnormal  45Ca  uptake  was  frequently  preceded  by  reduction  of  protein 
synthesis  in  the  ischemic  regions,  followed  later  by  an  inversely  proportional  increase  in 
calcium  uptake  and  reduction  in  protein  synthesis.  With  regard  to  the  CA1  sector, 
changes  in  protein  synthesis  were  varied.  An  initial  supression  of  synthesis  during  the 
first  recirculation  days  was  superceded  by  an  improvement  evident  at  7  days,  which  was 
followed  by  a  secondary  reduction  of  protein  synthesis  several  months  after  global 
ischemia.  Interestingly,  in  rats  sacrificed  after  one  year  protein  synthesis  appeared  to 
show  recovery;  this  was  supported  by  histological  sections  showing  a  remarkable 
number  of  well-preserved  pyramidal  neurons. 

In  summary,  our  studies  on  repetitive  and  global  ischemia,  as  well  as  on  cryogenic 
cortical  injury  have  further  advanced  the  assumption  that  neuroexcitatory  mechanisms 
play  a  significant  role  in  the  pathophysiology  of  resulting  injuries  in  these  models.  Our 
current  effort  is  directed  primarily  towards  quantitative  evaluation  of  changes  in 
excitatory  transmitters  during  and  after  ischemic  or  traumatic  insults.  We  intend  to  carry 
out  these  evaluations  as  regionally  as  possible,  in  order  to  define  the  sequence  of 
excitatory  changes  with  regard  to  neuronal  structures,  such  as  cortex,  striatum, 
hippocampus  and  thalamus.  In  addition  to  glutamate,  we  intend  to  evaluate,  mostly  by 
immunohistochemical  methods,  changes  involving  other  systems,  such  as  the  GABAergic 
and  dopaminergic  ones.  In  view  of  the  striking  early  changes  involving  the  nuclei 
reticularis  thalami  in  global  ischemia,  we  intend  to  elucidate  more  thoroughly  the  nature 
of  these  changes  and  their  possible  effect  on  other  interconnected  structures.  Finally, 
with  the  information  acquired  we  intend  to  test  various  compounds  which,  by 
interferring  with  neuroexcitatory  mechanisms,  will  be  able  to  ameliorate  the 
development  of  secondary  excitatory  lesions.  The  studies  of  Dr.  Nowak  regarding 


3  -  LNNS/DIR 


changes  in  gene  expression  after  ischemia  and  other  brain  insults  are  increasingly 
relevant  to  the  framework  outlined  above,  in  that  there  is  considerable  evidence 
suggesting  that  at  least  some  aspects  of  the  changes  may  result  from  activation  of  signal 
transduction  mechanisms  coupled  to  neuronal  activity.  It  is  particularly  striking  that  the 
initial  distribution  of  hsp  70  mRNA  induction  in  gerbil  brain  after  ischemia,  identified  by 
in  situ  hybridization,  is  identical  to  that  of  the  mRNA  encoding  the  proto-oncogene  c-fos. 
It  has  been  proposed  that  c-fos  induction  may  provide  an  index  for  mapping  functional 
neuronal  activity,  and  excitatory  amino  acids  have  been  demonstrated  to  induce  c-fos  in 
a  number  of  experimental  systems.  While  c-fos  induction  appears  to  occur  in  response  to 
relatively  mild  stimuli  it  would  appearthat  additional  input  is  required  to  induce  hsp  70, 
making  it  a  potential  marker  for  pathologic  activation.  One  goal  of  future  studies  is  to 
determine  the  precise  signal  transduction  mechanisms  responsible  for  hsp  70  induction. 
The  most  striking  feature  of  hsp70  mRNA  induction  after  ischemia  is  its  prolonged 
expression  in  severely  challenged  neurons,  such  as  those  in  the  CA1  sector  of  the 
hippocampus.  While  these  cells  fail  to  express  immunoreactive  hsp70  protein  after  a 
5-min  ischemic  insult,  they  show  a  prolonged  expression  of  hsp70  mRNA.  Both  of  these 
observations  may  be  linked  to  the  persistent  reductions  in  protein  synthesis  in  these  cells, 
with  a  superinduction  of  hsp70  mRNA  in  the  absence  of  the  functional  protein.  These 
results  suggest  the  using  hsp70  mRNA  expression  as  a  marker  for  vulnerable  neurons; 
and  this  hypothesis  is  currently  being  tested  in  a  variety  of  experimental  injury  models. 

One  interesting  aspect  of  these  studies  is  the  entirely  neuronal  distribution  of  hsp70 
induction  after  transient  ishemia  and  its  predominantly  glial  and  vascular  localization 
after  mild  hyperthermic  insults,  with  an  increasing  neuronal  component  under  more 
severe  conditions.  Focal  injury  models  have  been  reported  by  others  to  induce 
expression  in  diverse  cell  types.  Future  efforts  will  continue  to  expand  the  range  of 
probes  used  in  these  studies  to  include  those  expected  to  have  complementary  neuronal 
and  glial  localizations,  in  order  to  evaluate  the  contributions  of  altered  gene  expression 
in  specific  cell  populations  to  the  evolution  of  brain  injury. 

The  continuous  goals  of  the  Section  of  Neurocytobiology  have  been  to:  (a)  develop 
and  utilize  model  systems  for  the  investigations  of  basic  mechanisms  operative  on  the 
level  of  normal  and  pathologically  altered  blood-brain  barrier  (BBB),  cerebral  blood  flow 
(CBF)  and  hypertension;  (b)  to  study  metabolic  processes  occurring  in  ischemia  and 
ischemic  edema,  their  prevention  and  therapy;  and  (c)  to  evaluate  the  influence  of 
genetic  and  immunologic  factors  on  the  generation  of  experimental  autoimmune 
diseases  and  ischemia  involving  the  central  nervous  system. 

During  the  last  year,  cultured  endothelial  cells  (EC)  derived  from  dissociated 
microvesselsof  human,  mice  and  rat  brains  have  been  very  useful.forthe  continuous 
studies  of  cerebromicrovascular  function.  Three  different  aspectsof  cerebral  EC 
properties  related  to  BBB  function,  regulation  of  CBF  and  hypertension  have  been 
further  investigated  in  the  in  vitro  models  using  EC  cultures:  A)  assess  the  susceptibility 
of  human  cerebromicrovascular  EC  to  oxygen-derived  species  (like  H2O2)  and  compare  it 
with  that  of  EC  obtained  from  animal  brains;  B)  characteriza-tion  of  dopaminergic 
receptors  and  interaction  with  a-adrenergic  and  5-HT  receptors  in  human  EC;  and  C) 
interaction  between  cerebral  EC  and  immune  cells. 

Formation  of  free  radical  species  has  been  thought  to  affect  the  BBB  permeability 
and  formation  of  brain  edema.  To  determine  whether  exogenous  H2P2  may  alter  may 
alter  permeability,  we  examined  its  effect  on  cultured  endothelium  derived  from 
cerebral  microvessels  from  human  and  animals.  Release  of  5Kr  from  labeled 


4-LNNS/DIR 


endothelium  exposed  to  these  substances  was  used  as  a  main  marker  for  the  assessing 
of  endothelial  injury.  In  general,  the  51  Cr-release  from  EC  exposed  to  glucose/glucose 
oxidase  (representing  a  continous  H2C>2-generated  system  was  time-  and  dose- 
dependent.  Both  human  and  murine  EC  exposed  to  0.001  U/ml  glucose  oxidase  in  the 
presence  of  glucose  showed  no  loss  of  the  isotopic  marker,  whereas  the  same 
concentration  of  these  substances  caused  5iCr-releasefrom  rat  EC.  atalase  blocked  the 
release  of  51  Cr-labeled  EC  exposed  to  all  concentrations  of  glucose  oxidase  (0.1-0.01 
U/ml)  irrespective  of  EC  origin.  However,  catalase  inhibition  was  less  effective  in  EC  of 
rats.  SOD  had  no  effect  on  the  51  Cr  release  from  the  EC.  Addition  of  NaN3  or 
aminotriazole  (catalase  blocker)  increased  (2-7  fold)  the  51  Cr-release  from  all  EC.  A 
higher  dose  of  diethylmaleate  (which  depletes  the  reduced  glutathione)  was  required  to 
affect  the  release  of  51  Cr  from  human  than  from  murine  EC.  The  study  represents  the 
first  demonstration  of  human  brain  EC  response  to  H2O2  insult.  The  results  support  the 
existence  of  a  balance  between  the  endogenous  anti-oxidant  properties  of  cerebral  EC 
and  exogenous  oxidants.  The  disturbance  of  these  parameters  results  in  EC  injury.  Thus, 
the  altered  EC  function  may  contribute  to  the  development  and/or  progression  of  brain 
edema. 

Dopamine  (DA^  is  a  well  known  neurotransmitter  and  vasoactive  substance.  It  may 
affect  the  metabolism  of  the  central  nervous  system  (CNS)  and  reactivity  of  both  central 
and  peripheral  vessels.  It  has  been  suggested  that  DA  may  interact  with  specific 
receptors  in  cerebrovascular  smooth  muscle  to  alter  their  diameter,  thus  modulating 
blood  flow  and  blood  pressure.  Since  a  marked  DA  induced  enhancement  of  endothelial 
AC  activity  was  detected  in  cultured  endothelium  derived  from  human  microvessels 
(Spatz  et  al.,  1989),  we  characterized  the  endothelial  DA  receptors  and  elucidated  their 
relationship  to  a-adrenergic-and  5-HT  receptors.  Cultured  endothelium  derived  from 
three  fractions  of  human  cerebral  microvessels  was  used  to  characterize  DA  receptors 
linked  to  adenylate  cyclase  activity.  DA  or  Di-agonists  [(SKF-38393,  R(  ± )  SKF- 
82958(±)HBR)J  stimulated  endothelial  cAMP  formation  in  a  dose-dependent  manner. 
The  selective  Di-antagonist  [(SCH-23390,  R(  ± )]  dose-dependently  inhibited  the  DA  or  Di- 
agonists  stimulated  production  of  cAMP.  The  selective  62-antagonist  (sulpiride,  S(-)) 
inconsistently  augmented  the  DA  stimulatory  effect  on  cAMP  formation  in  all  three 
endothelial  fractions.  The  sensitivity  of  the  endothelial  adenylate  cyclase  was  greater  in 
endothelium  derived  from  large  and  small  microvessels  than  from  capillaries.  Agonists  of 
ai-adrenergic  receptors  (phenylephrine,  6-fluoronorepinephrine)  or  5-HT  which 
stimulated  the  production  of  cAMP  by  itself  did  not  augment  the  DA-stimulated  cAMP 
formation.  On  the  contrary,  ai-adrenergic  agonists  or  5-HT  incubated  with  DA  reduced 
the  cAMP  production,  which  could  be  partially  blocked  by  the  5-HT2  or  D2  antagonists, 
respectively.  These  findings  represent  the  first  demonstration  of  Di  (stimulatory)  and  D2 
(inhibitory)  receptors  linked  to  the  adenylate  cyclase  in  microvascular  endothelium 
derived  from  human  brain.  The  data  also  indicate  that  dopaminergic  receptors  can 
interact  with  either  ai-adrenergic  or  5-HT  receptors  in  endothelium  on  the  AC  level.  The 
observed  presence  of  endothelial  D1  and  D2  receptors  positively  and  negatively  linked  to 
AC  system  strongly  supports  the  contention  of  an  existing  central  microvascular 
dopaminergic  mechanism  which  may  be  affected  by  dopaminergic  agents  and  interact 
with  adrenergic  and  serotoninergic  substances. 

A  continuous  effort  has  been  to  examine:  interactions  between  lymphocytes  and  EC 
in  order  to  establish  possible  mechanisms  leading  to  alterations  in  BBB  permeability  and 
the  migration  of  peripheral  blood  immune  cells  across  the  BBB  into  the  CNS;  and  the 
role  of  class  II  MHC  antigens  in  the  pathogenesis  of  neuroimmunologic  disorders. 

The  perivascular  infiltration  of  peripheral  blood  lymphocytes  and  macrophages  into 
the  CNS  is  a  histopathologic  feature  of  human  demyelinating  diseases  such  as 


5-LNNS/DIR 


multiple  sclerosis  and  the  animal  model  disease,  experimental  allergic  encephalomyelitis 
(EAE).  To  elucidate  the  mechanisms  which  could  be  involved  in  this  event,  we  examined 
the  capacity  of  EC  to  function  as  targets  for  lysis  by  T  cells  capable  of  transferring  EAE. 
Lysis  was  proportional  to  E/T  ratios,  dependent  upon  the  expression  of  la  antigen  and  the 
presence  of  specific  antigen.  Continuously  cultured  antigen-specific  T-cell  lines 
ultimately  lost  the  ability  to  lyse  EC  but  continued  to  proliferate  to  MBP.  These  cell  lines 
also  lost  the  ability  to  transfer  EAE  suggesting  a  relationship  between  lytic  potential  and 
encephalitogenicity.  Another  aspect  of  these  studies  was  concerned  with  the 
observations  that  cerebral  vascular  EC  inhibited  antigen-induced  proliferation  by  MBP- 
specific  lymphocytes.  Attempts  to  characterize  the  inhibition  indicated  that  IL-1  induced 
synthesis  of  prostacyclin  (PGI2)  by  EC.  The  amount  of  PGL2  synthesized  in  these 
experiments  was  sufficient  to  account  for  the  inhibitory  response  observed.  These  data 
indicate  that  EC-lymphocyte  interactions  may  result  in  effects  on  both  EC  (i.e.,  lysis)  and 
lymphocytes  (i.e.,  anti-proliferative  response).  Such  interactions  may  result  in  alterations 
of  the  BBB  permeability  leading  to  vascular  egression  into  the  CNS,  which  are  pathologic 
hallmarks  of  neuroimmunologic  disorders  such  as  EAE  and  multiple  sclerosis. 

In  EAE,  la  antigen  expression  by  cerebral  vascular  EC  (which  comprise  the  BBB)  is 
believed  to  play  an  important  role  in  migration  of  peripheral  immune  cells  into  the  CNS. 
In  addition,  la  antigen  expression  by  resident  CNS  cells  (astrocytes,  microglia,  pericytes, 
and  smooth  muscle  cells)  also  plays  important  roles  in  the  expression  of  pathogenesis. 
The  investigations  focused  on  induction  of  la  antigen  on  microvascular  human 
endothelium  which  has  been  successfully  cultured  in  our  laboratory.  Cerebral  cap;llary 
EC  isolated  from  human  brain  biopsy  tissue  demonstrated  the  absence  of  class  II  MHC 
antigen  expression  on  cultured  EC.  However,  EC  could  be  induced  to  express  HLA-DR,  - 
DP  and  -DQ  antigens  by  culture  with  the  recombinant  human  IFN.  The  degree  of  HLA-DR 
antigen  expression,  as  determined  by  ELISA,  immunofluorescence,  FACS  analysis  and 
Northern  blot  analysis,  was  proportional  to  both  time  in  culture  and  concentration  of 
IFN-y   (20-200  U/ml).  This  represents  the  first  demonstration  of  class  II  MHC  antigen 
expression  by  cultured  human  cerebral  vascular  EC.  Preliminary  evidence  indicated  that 
IL-1  treatment  down-regulates  IFN-induced  expression  of  these  antigens.  These  results 
indicate  that  human  cerebral  vascular  EC  may  participate  in  MHC  class  ll-restricted 
interactions  with  peripheral  immune  cells.  Such  interactions  may  play  a  role  in  the 
passage  of  immune  cells  across  the  BBB  into  the  CNS  which  is  a  marker  of  certain  CNS 
autoimmune  disorders. 

The  continuous  studies  of  cerebral  ischemia,  its  pathophysiology,  prevention  and 
therapy  have  been  focused  on:  (a)  the  relationship  between  ischemic  changes  in  the 
cerebral  content  of  energy-related  metabolites  and  biogenic  amines  in  adult  and  young 
gerbils;  and  (b)  in  vivo  detection  of  regional  ischemic  release  of  monamines  into 
extracellularfluid.  Changes  in  the  brain  content  of  biogenic  amines  are  one  of  the  main 
factors  implicated  in  the  pathogenesis  of  ischemic  CNS  damage.  We  have  recently 
demonstrated  a  lack  of  correlation  between  the  cortical  ischemic  changes  of  energy- 
related  metabolites  and  noradrenergic  metabolites  in  adult  orthe  young  gerbil  brains. 
This  study  was  extended  to  the  striatum  (which  is  more  vulnerable  to  ischemia  than 
cortex)  to  further  elucidate  the  relationship  between  the  ischemic  cerebral  monoamine 
changes  and  the  lesser  susceptibility  of  younger  than  adult  animals  to  ischemic  insult. 
Bilateral  ischemia  of  5  to  1 5  minutes  duration  with  and  without  1  hour  reflow  similarly 
affected  the  glucose  and  energy-related  metabolites  in  both  groups  of  animals.  A 
reduction  in  striatal  norepinephrine  (NE)  level  was  detected  only  in  adults  after  each 
period  of  ischemia.  In  adults,  1 5  minutes  but  not  5  minutes  of  ischemia  reduced  the 
striated  DA  content  which  also  decreased  during  recirculation.  In  young  animals,  the 
same  striatal  DA  changes  were  seen  after  1 5  min  ischemia  with  reflow.  The  delayed 
alteration  of  monoamines  in  the  striatum  of  the  young  animals  observed  in  reflow  but 


6-LNNS/DIR 


not  during  ischemia  as  seen  in  adults  resembles  the  described  "maturation" 
phenomenon  in  the  adult  brain.  The  late  manifestions  of  monoaminergic  changes  in 
young  as  compared  to  adult  brain  structures  most  likely  reflect  a  lesser  extent  of  ischemic 
injury  in  the  young  than  adult  animal.  These  observations  suggest  that  the  relative 
resistance  of  young  animals  to  ischemia  may  be  related  to  the  function  of 
neurotransmitters  in  the  CNS. 

To  further  elucidate  monoamines  participation  in  selective  regional  vulnerability  to 
ischemia,  we  determined  and  compared  the  cortical  release  into  extracellular  fluid  (ECF) 
of  biogenic  amines  with  those  of  striatum  exposed  to  the  same  ischemic  insult.  Ischemia 
induced  a  marked  release  of  DA  and  5-HT  from  the  cortex  and  striatum.  The  levels  of  DA 
released  into  ECF  from  the  striatum  was  4-2000  fold  higher  than  in  the  cortex.  Moreover, 
the  release  of  DA  from  both  structures  was  4,000-20,000-fold  higher  than  that  of  5-HT. 
DA  and  5-HT  release  from  the  cortex  peaked  at  15  minutes  of  ischemia.  The  levels  of  5- 
HT  remained  higher  than  that  of  DA  during  1 5  minutes  of  reperf  usion  and  normalized  at 
30  min  of  reflow.  A  similar  pattern  e*  DA  release  was  seen  in  thedialysate  of  the 
striatum.  However,  the  level  of  the  released  5-HT  during  ischemia  remained  the  same 
after  1 5  minutes  of  reflow  and  returned  to  preischemic  value  at  30  minutes  of 
recirculation.  DOPAC,  HVA  and  5-HIAA  showed  a  similar  pattern  in  the  perfusate.  These 
metabolites  decreased  during  ischemia  and  their  accumulation  above  the  preischemic 
level  was  observed  between  60-120  minutes  after  recirculation,  3-methyl  tyrosine 
accumulated  in  reflow.  The  findings  support  the  idea  that  selective  vulnerability  to 
ischemia  may  at  least  be  in  part  related  to  ischemic  disturbances  of  biogenic 
neurotransmitters.  The  results  of  this  investigation  confirm  data  previously  obtained  by 
indirect  pharmacologic  techniques  concerning  the  behavior  of  monoamines  in  ischemia. 
Thisdirect  method  allowing  in  vivo  monitoring  of  the  ischemic  release  of  monoamines 
will  be  very  useful  for  elucidating  the  pathomechanisms  and  prevention  of  ischemic 
sequelae. 

Dr.  McCarron  continues  to  study  the  mechanism  responsible  for  the  production  of 
chronic  relapsing  EAE  in  F1  animalsand  examines  the  role  of  parental  immune  response 
antigens  in  the  induction  of  relapses  in  animals  with  disease.  A  better  understanding  of 
the  regulation  of  immune  response  gene  products  expression  in  the  F1  mice  (i.e.,  .I-A,s,  I- 
Au  and  l-As/u  hybrid  molecules)  is  also  a  major  objective  of  this  project.  MBP  residues  90- 
101  are  encephalitogenic  in  SJL(H-2s),  while  residues  1-9  areencephalitogenic  in  PL  mice 
(H2u).  Utilizing  SJLxPL(F1)  mice  the  relationship  between  encephalitogenicity  and 
genetic  background  were  previously  examined.  It  was  found  that  the 
encephalitogenicity  of  antigen  was  dependent  upon  l-A  haplotype  of  the  antigen 
presenting  cell.  T-cellsfrom  F1  mice  immunized  with  MBP  fragment  1-37  (containing  1-9 
fragment)  were  positively  selected  on  PL  (l-Au)  parental  macrophages.  These  cells 
transferred  disease  to  100%  of  the  recipient  naive  F1  mice.  Examination  of  spleen  cell 
populations  during  the  course  of  disease  in  mice  revealed  the  presence  of  T-cells 
responsive  to  1-37,  as  well  as  other  MBPfractions  (i.e.  89-169).  In  addition  to  l-A" 
restricted  responses,  l-As  restricted  responses  were  also  observed.  These  findings 
demonstrated  that  T-cells  with  novel  epitope  specificities  and  class  II  MHC  restriction 
requirements  were  generated  during  the  course  of  disease.  Such  cells  may  be  involved  in 
the  pathogenesis  of  chronic  disease  (i.e.,  initiation  of  relapses). 


7-LNNS/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02324-1 3  LNNS 


PERIOD  COVERED 

October  1,  1989  to  September  30,  1990 


TITLE  OF  PROJECT  (SO  characters  or  less.  Trt/e  must  fit  on  one  line  between  the  borders.) 

Blood-Brain  Barrier:  In  Vitro  Model  for  the  Study  of  Cerebrovascular  Endothelial  Permeability 


PRINCIPAL  INVESTIGATOR  (Ust  other  professional  personnel  below  tne  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Pi:  McCarron  Ph.D.,  Senior  Staff  LNNS,  NINDS 

Other:  M.Spatz  M.D.,  Section  Chief  LNNS,  NINDS 


COOPERATING  UNITS  (if  any) 

Sumio  Uematsu  and  M.  Delong,  John  Hopkins  Hospital,  Baltimore,  MD 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neurocytobioloqy 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  q  n 


PROFESSIONAL: 


0.6 


OTHER:  Q  2 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)  Human  subjects  ]  (b)  Human  tissues  0  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Arachidonic  acid  release  from  tissue  membranes  and/or  formation  of  free  radical 
species  have  been  thought  to  affect  the  blood  brain  barrier  permeability  and  formation 
of  brain  edema.  To  determine  whether  exogenous  arachidonic  acid  or  H2O2  may  alter 
blood  brain  barrier  permeability,  we  examined  their  effect  on  cultured  endothelium 
derived  from  cerebral  microvessels  of  human  and  animals.  Release  of  5iCr  from  labeled 
endothelium  exposed  to  these  substance  was  used  as  a  main  marker  for  the  assessment 
of  endothelial  injury.  The  results  of  these  studies  indicate  that  the  endothelial  cells  are 
susceptible  to  exogenous  arachidonic  acid  or  H2O2  insult  irrespective  of  their  origin. 
However,  human  endothelial  cells  are  less  affected  than  animal  endothelial  cells  by  the 
H202-generated  system.  The  findings  suggest  that  a  disturbance  of  the  existing 
balance  between  the  endogenous  antioxidant  properties  of  endothelial  cells  and 
exogenous  oxidant  leads  to  EC  injury. 


10-LNNS/DIR 


PHS  6040  (Rev.  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  •  PUBUC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

201  NS  02357-12  LNNS 


PERIOD  COVERED 

i  October  1, 1989  to  September  30, 1990 


TITLE  OF  PROJECT  (tOdttntUn  v Im.  Tut*  man  M  an  m km Mmm  «*•  aoraMJ 

Cerebral  Ischemia  and  Monoamines 


PRINOPAL  INVESTIGATOR  (an  •ttarptffcuiaail jmommIMm  u»  ^vk*</  tmm§Hm )  (*•««.  in*  te*or«i<y>.  •/>«  mnnvM  i»jiw) 

PI:  M.Spatz,  M.D.  Section  Chief  LNNS,  NINDS 

Others:  C.J.Chang  Visiting  Fellow  LNNS,  NINDS 


COOPERATING  UNITS  (Htn,) 

Institute  of  Biochemistry,  Faculty  of  Medicine,  Belgrade,  Yugoslavia,  (Bogomir.  Mrsulja) 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neurocytobioloqy 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□  (ajHuman  subjects  □  (b)  Human  tissues  HH  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Uit  flAncUrd  unreduced  type  bo  not  txct*d  the  ipict  proved .) 

Changes  in  the  brain  content  of  biogenic  amines  constitute  one  of  the  main  factors 
implicated  in  the  pathogenesis  of  ischemic  central  nervous  system  damage.  We  have 
recently  demonstrated  a  lack  of  correlation  between  the  cortical  ischemic  changes  of 
energy  related  metabolites  and  noradrenergic  metabolites  in  the  adult  or  the  young 
gerbil  brains.  This  study  was  extended  to  the  striatum  (which  is  more  vulnerable  to 
ischemia  than  cortex)  to  further  elucidate  the  relationship  between  the  ischemic 
cerebral  monoamine  changes  and  the  lesser  susceptibility  of  young  than  adult  animals 
to  ischemic  insult. 

Bilateral  ischemia  of  5  to  15  minutes  duration  with  and  without  1  hour  reflow  similarly 
affected  the  glucose  and  energy-related  metabolites  in  both  groups  of  animals.  A 
reduction  in  striatal  norepinephrine  (NE)  level  was  only  detected  in  adults  after  each 
period  of  ischemia.  In  adults,  15  minutes  but  not  5  minutes  of  ischemia  reduced  the 
striatal  dopamine  (DA)  content  which  also  decreased  during  recirculation.  In  young 
animals,  the  same  striatal  DA  changes  were  seen  after  1 5  minutes  ischemia  with  reflow. 
The  delayed  alternation  of  monoamines  in  the  striatum  of  the  young  animals  observed 
in  reflow  but  not  during  ischemia  as  seen  in  adults  resembles  the  described 
'maturation"  phenomenon  in  the  adult  brain.  The  late  manifestations  of 
monoaminergic  changes  in  young  as  compared  to  adult  brain  structures  most  likely 
reflect  a  lesser  intensity  of  ischemic  injury  in  the  young  than  adult  animal.  These 
observations  suggest  that  the  relative  resistance  of  young  animals  to  ischemia  may  be 
related  to  the  function  of  neurotransmitters  in  the  central  nervous  system. 


ll-LNNS/DIR 


•MSiMtflU*.  I«4| 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02548-09  LNNS 


PERIOD  COVERED 

October  1,  1989  to  September  30,  1990 


TITLE  OF  PROJ  ECT  (so  dimeters  or  less.  Title  must  fir  on  one  line  between  the  borders.) 

Evaluation  of  Electrical  Impedance  in  Cerebral  Ischemia 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  H.G.Wagner,  M.D.  Scientist  Emeritus  LNNS,  NINDS 


Others: 


S.Xu,  M.D. 
I.  Klatzo,  M.D. 


Visiting  Fellow 
Chief,  LNNS 


LNNS,  NINDS 
LNNS,  NINDS 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Cerebrovascular  Pathology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


0.3 


PROFESSIONAL: 


0.3 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I      [   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


]  (b)  Human  tissues  QT]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Further  study  was  made  of  cerebral  electrical  impedance  (CEI)  during  the  phase  of 
hypoperfusion.  In  the  gerbil  no  substantial  decrease  in  the  CEI  was  found  during  this 
phase  after  a  single  5  minute  bilateral  carotid  occlusion.  This  was  in  spite  of  noticeable 
swelling  of  the  brain  and  probable  compression  of  the  capillaries. 


This  project  was  terminated  because  of  the  retirement  of  the  prinicipal  investigator. 


12-LNNS/DIR 


WIS  6M0  (Dm.  1/M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02623-07 
LNNS 


PERIOD  COVERED 

i  October  1 , 1 989  to  September  30, 1 990 


TITLE  OF  PROJ  ECT  (SO  characters  or  less.  Trite  must  fit  on  one  lint  bthntr.  fh«  borders.} 

Cerebral  Ischemia  and  Edema:  Extracellular  Biogenic  Amines 


PRINCIPAL  IN  VE  STIGATOR  (Uu  other  professional  personnel  below  the  frinapal  Investigator.)  (Name,  title.  laboratory,  end  institute  affiliation) 

Pi:  C.J.  Chang.  M.D.  Visiting  Fellow  LNNS,  NINDS 

Others:  M.  Spatz,  M.D.  Section  Chief  LNNS,  NINDS 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neurocytobioloqy 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)  Human  subjects  ]  (b)  Human  tissues  l"x~l  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  cortical  and  '".  iaiai  .lease  of  biogenic  amines  induced  by  ischemia  has  been 
evaluated  in  dialysates  of  extracellular  fluid.  Mongolian  gerbils  subjected  to  bilateral 
common  carotid  artery  occlusion  for  1 5  minutes  with  a  reflow  up  to  2  hours  served  as  a 
model  for  transient  ischemia  of  brain.  These  studies  are  still  incomplete,  but  the  results 
indicate  that  ischemia  of  short  duration  causes  a  marked  release  of  dopamine  and 
serotnin  (5-HT)  from  both  structures.  The  level  of  the  released  amines  was  higher  in 
the  striatum  than  in  the  cortex.  Both  metabolites  of  dopamine  and  5-HT  decreased 
during  ischemia  and  increased  in  reflow.  The  findings  support  the  idea  that  the 
selective  vulnerability  to  ischemia  may  at  least  be  in  part  related  to  ischemic 
disturbances  of  biogenic  neurotransmitters. 

*Formerly  Cerebral  Ischemia  and  Edema:  Tryptophan  uptake  and  metabolism. 


13-LNNS/DIR 


PHS&IMOiRev.  1/M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02689-06 

LNNS 


PERIOD  COVERED 

Octoberl,  1989  to  September  30, 1990 


TITLE  OF  PROJ  ECT  (SO  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Reg u I ation  of  Carbohydrate  Metabolism  in  Cerebromicrovascular  Cultures 


PRINCIPAL  INVESTIGATOR  (Ujt  other  professional  personnel  below  the  frindpal  Investigator.)  (Name,  title,  laboratory,  and  instrtute  affiliation) 

Pi:  M.Spatz,  M.D.  Section  Chief  LNNS,  NINDS 


COOPERATING  UNITS  (if  any) 

David  Lust,  Case  Western  Reserve  University,  Cleveland,  Ohio 

B.B.  Mrsulja,  CVD  Research  Group,  Institute  of  Biochemistry,  Belgrade,  Yugoslavia 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neurocytobioloqy 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  fxl  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Studies  in  vitro  related  to  neurogenic  regulation  of  cerebromicrovascular  function 
showed  an  involvement  of  B2-adrenergic  system  in  controlling  norepinephrine- 
inducible  glycogenosis  in  separately  cultured  cerebromicrovascular  cellular  elements. 
The  present  investigation  focused  on  the  responsiveness  of  phosphorylase  A  and  B  to 
adrenergic  agonists  and  antagonists  in  order  to  elucidate  the  possible  mechanisms 
responsible  for  norepinephrine-inducible  glycogenosis. 

The  study  is  still  in  progress  and  no  sufficient  data  were  generated  for  proper 
evaluation. 


14a  -  I.NNS/DIR 


FHS6M0(Rev.  1/M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02718-05  LNNS 


PERIOD  COVERED 

October  1,  1989  to  September  30,  1990 


TITLE  OF  PROJ  ECT  (so  dtmmcttrs  or  hss.  Tit/*  must  fit  on  on*  Ina  Worn  tn*  bordvi.) 

Study  of  Cerebral  Electrical  Activity  Associated  with  Ischemia  and  Brain  Injury 


PRINCIPAL  INVESTIGATOR  (Ust  otr»r  pmtosionil  DWTOnntt  b»low  th*  trtnaotl  InnstiomtorJ  (Htm*,  trtl*.  Itborttory,  mnd mstrt </»•  IffilHtion) 

PI:  H.G.Wagner,  M.D.  Scientist  Emeritus  LNNS,  NINDS 

Other:  S.Xu.M.D.  Visiting  Fellow  LNNS,  NINDS 


COOPERATING  UNITS  (rttny) 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neuropathology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  ft  „ 


PROFESSIONAL:  q  q 


OTHER:  0_0 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [x~]  (c)  Neither 

]  (al)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Our  initial  efforts  to  detect  and  analyze  single  neurons  in  the  cortex  were 
unsatisfactory  for  the  purposes  of  analyzing  the  effects  of  ischemia  and  cold  lesions  to 
the  cortex.  Improved  success  followed  comparison  of  power  spectra  of  occipital  and 
frontal  EEG  in  the  rat  subjected  to  a  parietal  cold  lesion.  Normally,  the  dominant 
frequency  was  about  5  Hz  with  higher  frequency  components.  Rats  subjected  to  a 
parietal  cold  lesion  showed  immediate  reduction  in  EEG  amplitude  and  loss  of  high 
frequency  components.  The  dominant  frequency  in  power  spectrum  analysis  of  the 
EEG  showed  a  shift  to  1-3Hz  and  remained  there  for  more  than  1  week  of  animal 
survival.  The  amplitude,  however,  recovered  rapidly  within  about  20  minutes.  Visually 
evoked  potentials  were  also  examined  using  flash  stimulation  and  extradurally  placed 
electrodes  over  visual  area  I.  The  cold  lesions  were  applied  as  in  the  aforementioned 
lesions. 

A  most  striking  result  was  a  reduction  of  latency,  starting  at  about  4  hours  and 
reaching  a  maximum  at  24  hours  to  3  days.  On  the  assumption  that  this  reflected 
heightened  excitability  from  excessive  presence  of  glutamate,  a  non-competitive 
antagonist  of  glutamate,  MK-801  was  given  intraperitoneal^  (2  mg/kg)  at  24  hours 
after  the  cold  lesion.  The  effect  of  this  treatment  was  a  prolongation  of  the  latency 
beginning  within  about  5  minutes  of  injection  and  lasted  about  4  hours.  This  was 
associated  with  a  marked  reduction  of  amplitude  of  the  evoked  response.  This  effect 
of  a  direct  application  of  glutamate  (IM)  resulted  in  reduction  of  latency,  similar  to  the 
cold  lesion..  This  provides  further  support  to  the  hypothesis  that  glutamatergic 
mechanisms  were  involved  in  the  generation  of  the  evoked  response. 


15-LNNS/DIR 


mi  M40  <K»v.  1/M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02720-04  LNNS 


PERIOD  COVERED 

October  1 ,  1 989  to  September  30,  1 990 


TITLE  OF  PROJ  ECT  (80  characters  or  less.  T7f/e  must  fit  on  one  tine  between  the  borders.) 

Stress  Protein  Induction  in  Gerbil  Brain  After  Ischemia 


PRINCIPAL  INVESTIGATOR  (Ust  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  T.S.  Nowakjr.  M.D.  Special  Expert  LNNS,  NINDS 

Others:  K.  Kawai,  M.D.  Visiting  Fellow  LNNS.NINDS 

N.Saito,  M.  D.  Visiting  Fellow  LNNS.NINDS 

G.Mies  Visiting  Associate  LNNS.NINDS 


COOPERATING  UNITS  (if  any) 

(j.  F.  Mill,  LMB,  NINDS.  A.M.  Marini,  CNB.  NINDS;  S.  Nadi,  LNP,  NINDS;  P.  Lindsberg.  Dept.  Neurol.,  USUHS;  M.  Blake  and  N. 
Holbrook,  LMG,  NIA;  M.  Heyes,  LCS,  NIMH;  M.  Jacewia  and  W.  Pulsinelli,  Dept.  Neurol,  Cornell  University  School  of  Medicine. 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Cerebrovascular  Pathology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  n? 


PROFESSIONAL:  q  y 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  {x~\  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

In  situ  hybridization  studies  of  mRNA  encoding  the  major  70  kDa  heat  shock/stress 
protein,  hsp70,  have  demonstrated  its  prolonged  expression  in  vulnerable 


lippocampal  CA1  neurons  after  transient  global  ischemia.  These  cells  also  experience  a 
asting  impairment  of  protein  synthesis  that  may  contribute  to  such  a  superinduction 
phenomenon.  The  proto-oncoqene,   c-fos  shows  only  transiently  increased  expression 
but  with  a  distribution  identical  to  that  of  hsp70  mRNA  at  early  recirculation  intervals. 
These  results  suggest  that  related  signal  transduction  mechanisms  may  be  responsible 
forthe  induction  of  these  RNAs,  and  support  the  utility  of  hsp  70  hybridization  asa 
possible  indicator  of  some  aspect  of  persistent  neuronal  activation.  A  threshold  of  2 
minutes  ischemia  has  been  established  for  induction  of  hsp70  in  the  gerbil. 

A  more  complex  pattern  of  induction  is  observed  after  hyperthermia,  with  a 
predominantly  glial  and  vascular  distribution  of  immunoreactive  protein  but  a 
superimposed  neuronal  localization  of  hsp70  mRNA  during  increasingly  severe 
hyperthermia. 

The  major  direction  of  future  studies  is  to  utilize  changes  in  hsp70,  c-fos  and  other 
mRNAs  and  proteins  as  tools  for  identifying  the  precise  contributions  of  individual  cell 
types  to  the  evolution  of  pathophysiology  following  diverse  brain  insults,  and  to 
elucidate  the  specific  signal  transduction  mechanisms  involved. 

16-LNNS/DIR 


PHS  6040  (Rev.  1(84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02749-04  LNNS 


PERIOD  COVERED 

October  1,  1989  to  September  30,  1990 


TITLE  OF  PROJECT  (80  chirectert  or  less,    title  must  fit  on  one  Itne  between  the  borders.) 

The  Measurement  of  Cerebral  Blood  Flow  by  Laser  Doppler  Velocimetry 


PRINCIPAL  IN ViS7\GA70R  (Ust  other  professiontl personnel  below  the  rrinapet  Investigator.)  (Njrne.  title,  tiborttory,  end  institute  tffilietion) 

PI:  H.G.Wagner,  M.D.  Scientist  Emeritus  LNNS,  NINDS 

Other:  S.  Xu,  M.D.  Visiting  Fellow  LNNS,  NINDS 


COOPERATING  UNITS  (rfeny) 

Biomedical  Engineering,  Research  Services,  NIH,  R.F.  Bonner 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neurocytobioloqy 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  -  q 


PROFESSIONAL:  q  q 


OTHER: 


0,0 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [x~l  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

A  study  has  been  made  of  cerebral  blood  flow  (CBF)  in  gerbils  by  laser  Doppler 
velocimetry  (LDV).  The  gerbils  were  subjected  to  repeated  bilateral  carotid  occlusions 
and  studied  for  up  to  3  hours.  Continuous  measurement  of  the  CBF  was  made  of  both 
parietal  cortices  simultaneously  using  2  probes  separately  located.  The  purpose  of 
these  experiments  was  to  examine  the  agreement  between  the  two  sides.  The  essential 
finding  was  that  while  the  CBF  dynamics  were  usually  similar,  they  were  not  always  the 
same  and  the  differences  coula  be  substantial  in  time  of  recovery,  magnitude  of 
recovery  and  in  pattern  of  recovery. 

This  project  is  terminated  because  prinicipal  investigator  retired. 


17-LNNS/DIR 


P«i6O40(»e.    1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02751-04 
LNNS 


PERIOD  COVERED 

October  1, 1989  to  September  30, 1990 


TITLE  OF  PROJECT  (60  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.} 

Cultures  of  Mouse  Capillary  Endothelium:  Establishment,  Growth  and 


PRINCIPAL  INVESTIGATOR  (Ust  other  professional  personnel  below  trie  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Pi:  M.Spatz,  M.D.  Section  Chief  LNNS,  NINDS 

Others:  R.  M.  McCarron,  M.D.  Senior  Staff  Fellow        LNNS,  NINDS 

L.Wang,  M.D.  Guest  Researcher  LNNS,  NINDS 


COOPERATING  UNITS  (if  any) 

Dale  E.  McFarlin,CNP,  NIB,  NINDS 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neurocytobiology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:         «  -, 


PROFESSIONAL: 


0.2 


OTHER: 


0.5 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (fc>)  Human  tissues  [T]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  study  represents  a  continuous  effort  to  develop  and  establish  the  most  reliable  and 
reproducible  method  for  culturing  cerebromicrovascular  endothelium  derived  from  SJL 
mice  susceptible  to  experimental  allergic  encephalomyelitis. 

The  focus  of  the  present  investigation  has  been  to  analyze  the  nutritional  requirements 
needed  forsustained  long-term  growth  and  propagation  of  the  endothelium.  High 
concentration  of  endothelial  cells  grow  equally  well  in  medium  containing  10-20% 
fetal  calf  serum  (FCS).  They  required  endothelial  cell  growth  factor  (ECGF)  for  maximal 
proliferation.  Inclusion  of  heparin  synergistically  increased  the  proliferative  response 
of  the  cells.  In  the  presence  of  high  concentration  of  FCS  (20%),  heparin  surpassed 
ECGF  in  the  ability  to  support  increased  proliferative  response  of  the  endothelium.  A 
more  detailed  analysis  of  various  nutrients  is  still  required  for  establishment  and 
characterization  of  long-term  endothelial  culture  for  expediting  the  investigation 
concerned  with  interaction  between  capillary  endothelium  and  immune  cells. 

Formerly:  "Interaction  Between  Cerebral  Capillary  Endothelium  and  Immune  Cells" 

This  investigation  could  not  be  continued  in  this  fiscal  year  (1989-1990)  due  to  fire 
destruction  of  the  57.6  mouse  breeding  facilities. 


18aLNNS/DIR 


f>HS  6M0  (Rev.  1*4) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02768-03  LNNS 


PERIOD  COVERED 

October  1,  1989  to  September  30,  1990 


TITLE  OF  PROiiCT  (»0  chtrtcttrs  or  less.  Title  musl  tn  on  ont  lint  bttwvtn  tht  bordtn.) 

Postischemic  Accumulation  of  Calcium  in  Brain  Tissue* 


PRINCIPAL  INVESTIGATOR  (Usl  othtt  proftsuontl penonntl  btlow  tht  Frinopal  Innitigttor.)  (Manx.  Irt It.  laboratory,  tnd inttitutt  ttfilittion) 


PI: 

T.S.  Nowak,  Jr.  M.D. 

Expert 

LNNS.NINDS 

Others: 

K.  Kawai.M.D. 

Visiting  Fellow 

LNNS,  NINDS 

N.Saito,  M.D. 

Visiting  Fellow 

LNNS.NINDS 

J.  Lohr,  M.D. 

Lab.  Technician 

LNNS.NINDS 

C.  Ruetzler,  M.D. 

Biologist 

LNNS.NINDS 

1.  Klatzo,  M.D. 

Chief 

LNNS.NINDS 

COOPERATING  UNITS  (rftny) 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  on  Cerebrovascular  Pathology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.0 


PROFESSIONAL: 


0.7 


OTHER: 


0.3 


CHECK  APPROPRIATE  BOX(ES) 

I     1   (a)  Human  subjects 
]  (a1)  Minors 

]  (a2)  Interviews 


J  (b)  Human  tissues  [xT|  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 


Observations  on  the  distribution  and  the  time  sequences  of  calcium  accumulation 
following  single  and  repetitive  ischemic  insults  using  45calcium  autoradiography 
revealed  significant  differences  in  the  pattern  of  calcium  uptake  between  single  and 
repeated  occlusions.  A  striking  feature  of  calcium  accumulation  in  repeated  ischemia 
was  intense  uptake  of  Ca  +  +  in  thalamus,  striatum  and,  later,  in  the  medial  geniculate 
and  substantia  nigra.  Also,  a  striking  finding  was  an  intense  Ca  +  +  uptake  in  CA1 
when  the  pyramidal  neurons  were  virtually  destroyed.  Our  observations  suggest  that 
abnormal  accumulation  of  calcium  may  be  due  to  other  causes  than  a  direct  ischemic 
injury  and  may  not  necessarily  indicate  irreversible  neuronal  damage. 

*Formerly:  "Post-lschemic  Accumulation  of  Calcium  in  Brain  Tissue  and  Histological 
Ischemic  Injury." 


19-  LNNS/DIR 


PHS6O40(Rev.  1*a| 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02773-02  LNNS 


PERIOD  COVERED 

October  1, 1989  to  September  30, 1990 


TITLE  OF  PROJECT  (SOchjracferjortesi  Title  must  fit  on  one  line  betvireen  the  borders.) 

Observations  on  Global  Cerebral  Ischemia  in  Rats 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Frindpal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI: 
Others: 


K.  Kawai,  M.D. 

N.Saito,  M.D. 

T.S.  Nowak,Jr.,M.D. 

G.Mies,  M.D. 

J.  Lohr,  M.D. 

C.  Ruetzler,  M.D. 

I.  Klatzo,  M.D. 


Visiting  Fellow 

Visiting  Fellow 

Expert 

Visiting  Associate 

Lab.  Technician 

Biologist 

Chief 


LNNS,  NINDS 
LNNS,  NINDS 
LNNS,  NINDS 
LNNS,  NINDS 
LNNS,  NINDS 
LNNS,  NINDS 
LNNS,  NINDS 


COOPERATING  UNITS  (rf  any) 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  on  Cerebrovascular  Pathology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.3 


PROFESSIONAL: 


1.0 


OTHER: 


0.3 


CHECK  APPROPRIATE  BOX(ES) 

]   (a)  Human  subjects 
]  (a1)  Minors 
]  (a2)  Interviews 


]  (b)  Human  tissues  0  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 


Our  studies  on  global  cerebral  ischemia  in  rats  by  compression  of  major  cardiac  vessels 
revealed  striking  early  changes  in  the  nucleus  reticularis  thalami,  which  suggests  a 
possibility  that  loss  of  inhibitory  function  of  this  GABAergic  nucleus  may  contribute  to 
aggravation  of  ischemic  injury  in  the  thalamus  and  in  other  structures.  Morphological 
observations  in  this  study  indicate  that  neurons  in  CA1  sector,  cerebral  cortex  and 
thalamus  can  survive  very  long  and  then  recover. 


20  -  LNNS/DIR 


PHS  60*0  (R*v.  1/M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02775-02  LNNS 


PERIOD  COVERED 

October  1,  1989  to  September  30,  1990 


TITLE  OF  PROJECT  (SO  charecters  or  less.   Trtte  must  fit  on  on*  lint  between  the  borders.) 

Blood  -Brain  Barrier  Changes  Following  Repeated  Ischemic  Insults 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  Mow  the  frinapal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  T.S.  Nowak,  Jr.  Expert  LNNS,  NINDS 

Others:  N.  Saito  Visiting  Fellow  LNNS,  NINDS 

C.  Ruetzler  Biologist  LNNS,  NINDS 

I.  Klatzo  Chief  LNNS,  NINDS 


COOPERATING  UNITS  (if  an,) 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Cerebrovascular  Pathology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  -  , 

U.b 


PROFESSIONAL:  n  g 


OTHER: 


0.3 


CHECK  APPROPRIATE  BOX(ES) 

LJ   (a)  Human  subjects  ]  (b)  Human  tissues  PH  (c)  Neither 

]  (a1)  Minors 


J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Rabbit  antisera  have  been  raised  against  purified  gerbil  albumin  and  used  for  the 
immunocytochemical  localization  of  the  extravasated  protein  in  brain  following 
repeated  ischemic  results.  A  striking  accumulation  of  albumin  immunoreactivity  was 
evident  in  thalamus  as  early  as  6  hours  after  a  series  of  repeated  5  minute  carotid  artery 
occlusions  while  positive  staining  was  first  evident  in  this  region  at  24  hours  following 
repeated  2  minute  occlusions.  At  longer  recirculation  intervals,  thalamic  staining  was 
intensified  and  theCAl  region  of  hippocampus  showed  strong  immunoreactivity  which 
accompanied  the  degeneration  of  neurons  in  this  vulnerable  area.  In  general,  albumin 
immunoreactivity  was  closely  correlated  with  the  distribution  of  enhanced  45calcium 
45uptake  as  well  as  with  the  appearance  of  reactive  glia,  as  evidenced  by 
immunocytochemical  visualization  of  glial  fibrillary  acidic  protein.  The  similarity  of 
intraneuronal  uptake  of  albumin  to  the  endocytotic  uptake  of  a  protein  tracer, 
observed  in  epileptic  convulsions,  suggests  that  neuroexcitatory  mechanisms  may  be 
involved  in  uptake  of  proteins  from  the  extracellular  space  in  both  conditions. 


21-LNNS/DIR 


l>HS  t(M0  (Rev  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02776-02 

LNNS 


PERIOD  COVERED 

October  1, 1989  through  September  30, 1990 


TITLE  OF  PROJECT  (SO characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Mechanism  of  Production  of  Experimental  Allergic  Encephalomyelitis 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  R.M.  McCarron,  M.D.  Senior  Staff  Fellow  LNNS,  NINDS 

Others:  L.Wang,  M.D.  Guest  Researcher  LNNS,  NINDS 


COOPERATING  UNITS  (rtan,) 

Dale  McFarlin,  Chief,  NIB,  NINDS 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neurocytobioloqy 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  n    . 


PROFESSIONAL: 


0.4 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  |      |  (b)  Human  tissues  [7]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

MBP  residues  90-101  are  encephalitogenic  in  SJL  mice  (H-2*),  while  residues  1-9  are 
encephalitogenic  in  PL  mice  (H  2u).  Utilizing  SJLx  PL(F1)  mice  the  relationship  between 
encephalitogenicity  and  gentic  background  were  previously  examined.  It  was  found 
that  the  encephalitogenicity  of  antigen  was  dependent  upon  l-A  haplotype  of  antigen 
presenting  cell.  T-cells  from  F1  mice  immunized  with  MBPfragment  1-37  (containing  1- 
9  fragment)  were  positively  selected  on  PL  (l-Au)  parental  macrophages.  These  cells 
transferred  disease  to  100%  recepient  naive  F1  mice.  Examination  of  spleen  cell 
populations  during  the  course  of  disease  in  the  mice  revealed  the  presence  of  T-cells 
responsive  to  1-37,  as  well  as  other  MBP  factions  (i.e.  89-169).  In  addition  to  l-Au- 
restricted  responses,  l-As-restricted  responses  were  also  observed.  These  findings 
demonstrated  that  T-cells  with  novel  epitope  specificities  and  class  II  MHC  restriction 
requirements  were  generated  during  the  course  of  disease.  Such  cells  may  be  involved 
in  the  pathogenesis  of  chronic  disease  (i.e.,  initiation  of  relapses). 

Significant  progress  on  this  project  was  not  possible  due  to  the  inability  to  obtain 
sufficient  Fl  mice.  These  mice  are  solely  distubuted  by  Jackson  Labs  which  suffered  a 
debilitating  fire  resulting  in  loss  of  many  breeder  pairs  including  the  F1  mouse  stock. 
Attempts  to  breed  cur  own  were  successful,  but  resulted  in  only  a  limited  number  of 
mice  available  for  research.  The  supply  at  Jackson  has  been  reestablished  and  we  are 
currently  performing  experiments  with  these  mice. 


22  -  LNNS/DIR 


PHS  6040  (Re».  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02777-02 
LNNS 


PERIOD  COVERED 

October!,  1989  to  September  30, 1990 


TITLE  OF  PROJECT  (SO character*  or  less.   Title  must  fit  on  on*  line  between  «/>•  border*.) 

Human  Cerebromicrovascular  Endothelium:  Studies  in  Vitro 


PRINCIPAL  INVESTIGATOR  (Ijji  other  profession*!  personnel  below  the  rnnapal  Investigetor.)  (Heme,  title,  laboratory,  end  institute  effilutron) 

PI:  F.  Bacic,  M.D.  Visiting  Fellow  LNNS,  NINDS 


Others: 


R.M.  McCarron,  M.D. 
M.Spatz,  M.D. 


Senior  Staff  Fellow 
Section  Chief 


LNNS,  NINDS 
LNNS,  NINDS 


COOPERATING  UNITS  (rf,n,) 

The  Johns  Hopkins  Hospital,  Baltimore,  MD  (Donlin  Long  and  Sumio  Uematsu) 


LAB7B  RANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neurocytobioloqy 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


0.8 


PROFESSIONAL: 


0.5 


OTHER: 


0.3 


CHECK  APPROPRIATE  BOX(ES) 

I      1   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


LD 


]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Cultured  endothelium  derived  from  three  fractions  of  human  cerebral  microvessels 
was  used  to  characterize  dopamine  (DA)  receptors  linked  to  adenylate  cyclase  activity. 
Dopamine  or  Di  -agonists  [SKF  -38393,  R(  ± ),  SKF  -82958(  ± )  HBR]  stimulated 
endothelial  cAMP  formation  in  dose  -  dependent  manner.  The  selective  Di  -antagonist 
[SCH  -23390,  R(  ± )]  dose  -dependency  inhibited  the  DA  or  Di  -agonists  stimulated 
production  of  cAMP.  The  selective  D2  -antagonist  [sulpiride,  S  (-)]  inconsistently 
augmented  the  DA  stimulatory  effect  on  cAMP  formation  in  all  three  endothelial 
fractions.  The  sensitivity  of  endothelial  adenylate  cyclase  was  greater  in  endothelium 
derived  from  large  and  small  microvessels  than  from  capillaries.  Agonists  of  ai  - 
adrenergic  receptors  (phenylephrine,  6  -fluoronorepinephrine)  or  serotonin  (5-HT) 
which  stimulated  the  production  of  cAMPby  itself  did  not  augment  the  DA-stimulated 
cAMP  formation.  On  the  contrary,  ai-adrenergic  agonists  or  5-HT  incubated  with  DA 
reduced  the  cAMP  production  which  could  be  partially  blocked  by  5HT2-  or  D2- 
antagonists,  respectively. 

These  findings  represent  the  first  demonstration  of  Di  -(stimulatory)  and  D2  - 
(inhibitory)  receptors  linked  to  adenylate  cyclase  in  microvascular  endothelium  derived 
from  human  brain.  The  data  also  indicate  that  dopaminergic  receptors  can  interact 
with  either  ai-adrenergic  or  5-HT  receptors  in  endothelium  on  the  adenylate  cyclase 
level. 


23-LNNS/DIR 


•HSMXXHRe..  I'M! 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02780-03 
LNNS 


PERIOD  COVERED 

October  1#  1989  through  September  30, 1990 


TITLE  OF  PROJECT  (80  character*  or  less,  mie  must  fit  on  one  line  between  the  borders.) 

Cerebral  Vascular  Endothelial  Cell-Specific  Monoclonal  Antibodies 


PRINCIPAL  IN  VESTIGATOR(UitotrierproreM«>n*/  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI: 
Others: 


R.M.  McCarron,  M.D. 

M.Spatz,  M.D. 
J.  Bembry,  M.D. 
N.  Merkel,  M.D. 


Senior  Staff  Fellow 

Section  Chief 

Biologist 

Chemist 


LNNS,  NINDS 

LNNS,  NINDS 
LNNS,  NINDS 
LNNS,  NINDS 


COOPERATING  UNITS  (rf  any) 

Dale  McFarlin,  Chief,  NIB,  NINDS 

Laura  Quigley,  Lab  Technician,  NIB,  NINDS 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neurocytobiology 


INSTITUTE  AND  LOCATION 

NINDS,  N1H,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


0.4 


PROFESSIONAL: 


0.4 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

1      |   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


□  (b)H 


uman  tissues 


["*~l  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

In  vitro  studies  on  the  role  of  the  blood-brain  barrier  (BBB)  in  neuroimmunological 
disorders  involving  migration  of  cells  across  the  BBB  have  been  hampered  by  the 
inability  to  establish  long-term  cultures  of  cerebral  vascular  endothelial  cells  (EC),  the 
main  component  of  the  BBB.  Using  hybridoma  technology,  we  have  established  a 
panel  of  hybridomas  (fusion  of  Lewis  rat  and  mouse  myeloma  NS-1  cells)  which  secrete 
antibodies  which  recognize  EC.  Characterization  of  these  antibodies  by 
immunofluorescence  microscopy,  FACS  analysis  and  ELISA  techniques  demonstrate 
antibodies  which  react  with  only  murine  cerebral  vascular  EC,  with  EC  isolated  from 
other  than  of  the  mice,  or  react  with  EC  from  other  species.  These  antibodies  were  also 
characterized  according  to  their  type  (IgG  vsIgM). 

The  results  demonstrate  the  isolation  of  pure  monoclonal  antibodies  to  CNS-EC 
which  will  be  useful  to  further  progress  on  establishment  of  long-term  EC  cultures  and 
other  studies  utilizing  cerebral  vascular  EC. 


24-LNNS/DIR 


»HSM«0(R*v.  1/M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02795-02LNNS 


PERIOD  COVERED 

October  1 ,  1 989  to  September  30,  1 990 


TITLE  OF  PROJECT  (BOcharaaers  or  less.  Title  must  fit  on  one  Sim  bettveen  the  borders.; 

Human  Cerebromicrovascular  Endothelial  Culture:  Cholinergic  and  Histaminergic  Receptors 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  M.Spatz,  M.D.  Section  Chief  LNNS,  NINDS 

Others:  F.  Bacic,  M.D.  Visiting  Fellow  LNNS,  NINDS 

R.M.  McCarron,  M.D.  Senior  Staff  Fellow  LNNS,  NINDS 


COOPERATING  UNITS  (if  an,) 

The  Johns  Hopkins  Hospital,  Baltimore,  MD,  (Donlin  Long  and  Sumio  Uematsu) 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neurocytobioloqy 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  Ac 

0.0 


PROFESSIONAL:  n  j 


OTHER:  q  3 


CHECK  APPROPRIATE  BOX(ES) 

[7]   (a)Human  subjects  [7]  (b)  Human  tissues  ]  (c)  Neither 

J  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Acetylcholine  and  histamine  are  known  as  vasoactive  substances  which  may  be  involved 
in  regulatinq  cerebral  blood  flow  (CBF)  and/or  blood-brain  barrier  (BBB)  functions  This 
supposition  has  been  supported  by  detection  of  cholinergic  and  histaminergic  fibers  in 
the  close  vicinity  of  cerebral  vessels.  Moreover,  receptors  for  both  substances  were 
described  in  microvessels  isolated  from  brain  of  animals  but  not  from  man.  The  aim  of 
this  investigation  was  to  examine  the  effect  of  acetylcholine,  carbachol  and  histamine 
on  adenylate  cyclase  (AC)  activity  in  the  endothelium  derived  from  three  fractions  of 
human  microvessels. 

The  response  of  adenylate  cyclase  to  acetylcholine,  carbachol  and  histamine  was  seen  in 
all  examined  homogenates  of  endothelium  (4th-i  1th  passage)  derived  from  large 
microvessels.  Pirenzepine  (Mi-muscarinic  inhibitor)  blocked  the  acetylcholine  and 
carbachol  stimulated  formation  of  cAMP  while  methoctramine  (M2  muscarinic 
inhibitor)  had  no  effect  on  AC  of  endothelium  derived  from  large  microvessels. 
Preliminary  investigations  demonstrated  a  dose-dependent  cholinergic  stimulation  of 
cAMP  production  in  endothelium  derived  from  capillaries  and  small  microvessels.  The 
response  of  endothelial  AC  activity  to  histamine  was  inhibited  (30-100%)  by  cimetidine 
indicating  the  AC  linkage  to  ^-receptors.  These  findings  represent  the  first 
demonstration  of  cholinergic  and  histaminergic  receptors  linked  to  the  AC  system  in 
cerebromicrovascular  endothelium  of  human  brain. 

The  loss  of  endothelial  cultures  due  to  toxic  human  serum  prevented  the  completion  of 
this  project. 

25-LNNS/DIR 


**ti  WU  (««v-  1/M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02796-02  LNNS 


PERIOD  COVERED 

October  1,  1989  to  September  30, 1990 


TITLE  OF  PROJ  ECT  (80  characters  ci  lass.  Title  must  fit  an  one  line  between  the  borders.) 

Transneuronal  Effects  of  Cryogenic  Brain  Injury  on  Calcium  Uptake  and  Blood-Brain  Barrier  Changes 


PRINCIPAL  IN  VE  STIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

LNNS,  NINDS 
LNNS,  NINDS 
LNNS,  NINDS 
LNNS,  NINDS 
LNNS,  NINDS 
LNNS,  NINDS 


PI: 

JN.  Saito 

Visiting  Fellow 

Others: 

T.S.  Nowak,  Jr. 

Expert 

G.  Mies 

Visiting  Associate 

1,  Lohr 

Lab.  Technician 

C.  Ruetzler 

Biologist 

I.  Klatzo 

Chief 

COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  on  Cerebrovascular  Pathology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


0.9 


PROFESSIONAL: 


0.6 


OTHER: 


0.3 


CHECK  APPROPRIATE  BOX(ES) 

]   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


]  (b)  Human  tissues  0  (0  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Development  of  transneuronal,  secondary,  injuries  developing  in  distant  but 
anatomically  connected  regions  following  primary,  direct  injury  to  the  cerebral  cortex 
was  shown  to  be  associated  with  an  abnormal  uptake  of  calcium,  and  blood-brain 
barrier  (BBB)  permeability  changes  in  those  areas.  The  delayed  onset  of  these  changes, 
pattern  of  calcium  uptake  into  dendritic  structures,  and  intracytoplasmic  uptake  of 
extravasated  albumin  suggest  a  neuroexcitatory  nature  of  mechanisms  involved.  If 
correct,  studies  will  be  developed  to  ascertain  whether  suppression  or  reduction  of 
neuroexcitation  by  some  agents  could  substantially  ameliorate  or  prevent  development 
of  secondary  transneuronal  injuries.  This  could  be  of  relevance  to  the  treatment  of 
brain  trauma  patients. 


26-LNNS/DIR 


•-HS6M0  (Rev.  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02797-02 

LNNS 


PERIOD  COVERED 

October  1, 1989  to  September  30, 1990 


TITLE  OF  PROJ  ECT  (80  dttntatrs  or  hss.  rah  must  fit  on  on*  lint  t»tn>Mn  th»  borders.; 

Cultures  of  Human  Cerebromicrovascular  Endothelium:  Establishment,  Growth  and  Characterization 


PRINCIPAL  IN VIST\GA70R  (LHt  other  proftssnnil p*?onr*l b*k>w  th*  rnnaiHl  lnv*itio*tor.)  (Hrm.  trtJe.  laboratory,  •nd  irutrtufe  fffitalion) 

PI: 


Others: 


M.Spatz,  M.D. 

R.M.  McCarron,  M.D. 
L  Wang,  M.D. 


Section  Chief 

Senior  Staff  Fellow 
Guest  Researcher 


LNNS,  NINDS 

LNNS,  NINDS 
LNNS,  NINDS 


COOPERATING  UNITS  (if my) 

Drs.  Doniin  Long  and  Sumio  Uematsu,  The  John  Hopkins  Hospital,  Baltimore,  MD 
Dr.  Ronald  F.  Dodson,  University  of  Texas,  Health  Center,  Tyler,  TX 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neurocytobioloqy 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


0.9 


PROFESSIONAL: 


0.5 


OTHER: 


0.4 


CHECK  APPROPRIATE  BOX(ES) 

I      |   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


I  x   [  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  ( Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  study  has  been  concerned  with  the  development  and  establishment  of  endothelial 
cell  cultures  derived  from  three  microvascular  fractions  of  human  brain.  The  purity  of 
the  endothelial  cultures  was  found  to  be  greater  than  95%  since  most  of  the  cells 
expressed  human  Factor  Vlll-related  antigen  and  only  an  occasional  cell  stained  for  glial 
fibrillary  acidic  protein.  Both  light  and  electronmicroscopy  showed  that  the  separately 
cultured  endothelium  derived  either  from  the  capillaries  (EC)  or  small  [(<100ym)  ES] 
and  large  [  >  100ym)  EL]  arterioles  and  venules  exhibited  the  same  appearance  and 

?rowth  pattern.  The  maximal  proliferative  response  of  endothelium  was  seen  on  the 
irst  day  after  exposure  to  fresh  medium.  A  direct  relationship  was  found  between 
various  components  of  growth  medium  and  individual  proliferative  response  of 
endothelium  dependent  on  its  origin  indicating  different  growth  requirements.  The 
characterization  of  the  cultured  endothelium  is  still  incomplete  and  in  progress. 

Nevertheless  the  successfully  developed  technique  for  cultivating  pure 
cerebromicrovascular  endothelium  derived  from  human  brain  (dependent  on  the 
availability  of  brain  tissue)  provides  a  model  in  vitro  for  investigation  of  endothelial 
properties  related  to  the  normal  or  altered  function  of  blood-brain  barrier  and  cerebral 
blood  flow  in  man. 

The  loss  of  endothelial  cultures  due  to  toxic  human  serum  prevented  the  completion  of 
this  project. 


27-LNNS/DIR 


•MS  tOa(MB.»    V»J| 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02798-02  LNNS 


PERIOD  COVERED 

October  1 , 1 989  to  September  30, 1 990 


TITLE  OF  PROJECT  (SO  character*  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Ultrastructural  Observations  on  Distribution  of  Calcium  in  Cerebral  Ischemia 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  I.  Klatzo,  M.D.  Chief  LNNS,  NINDS 

Others:  J.  Lohr,  M.  Lab.  Technician  LNNS,  NINDS 

C.  Ruetzler,  M.D.  Biologist  LNNS,  NINDS 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Cerebrovascular  Pathology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.0 


PROFESSIONAL: 


0.2 


OTHER: 


0.8 


CHECK  APPROPRIATE  BOX(ES) 

I      |  (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


]  (b)  Human  tissues  [x~]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Using  the  pyroantimonate  method  for  precipitating  calcium  to  electron-dense  calcium 
pyroantimonate,  the  ultrastructural  distribution  of  calcium  was  evaluated  following 
single  and  repeated  ischemic  insults,  produced  by  bilateral  occlusions  of  common 
carotid  artery  in  gerbils.  Our  observations  revealed  an  excessive  accumulation  of 
calcium  deposits  in  the  mitochondria  of  pyramidal  cell  dendrites  preceding  their 
destruction.  Following  the  disappearance  of  pyramidal  cells,  which  becomes  evident 
approximately  3  days  after  ischemic  insults,  a  striking  accumulation  of  electron-dense 
calcium  deposits  was  observed  in  the  dendrites  of  the  interneurons,  whereas 
mitochondria  of  these  dendrites  were  free  of  calcium.  Occasionally  the  reverse  was 
true  and  calcium  deposits  were  conspicuous  in  the  center  of  mitochondria  with  well- 
preserved  cristae,  whereas  there  was  no  calcium  in  the  cytosol  of  the  dendrites.  The 
pattern  of  calcium  distribution  in  the  dendrites  appears  similar  to  that  observed 
following  convulsive  seizures  and  indicates  a  possibility  of  neuroexcitatory  mechanisms 
playing  an  important  role  in  accumulation  of  calcium  in  injured  by  ischemia  neurons. 
Otherwise,  the  intraneuronal  presence  of  calcium  itself  does  not  seem  to  be  invariably 
associated  with  a  neuronal  death. 


28-LNNS/DIR 


PHS  MW0  (Rev.  1/84] 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02801-02 
LNNS 


PERIOD  COVERED 

October  1, 1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  character!  or  less.  Trtle  must  fit  on  one  line  tennn  the  borden.) 

Interactions  Between  Cerebrovascular  Endothelial  Cells  and  Immune  Lymphocytes 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator .)  (Name,  title,  laboratory.  and  institute  aft station) 

P.I.  R.  M.  McCarron,  M.D.  Senior  Staff  Fellow  LNNS,  NINDS 

Other:  M.Spatz,  M.D.  Section  Chief  LNNS,  NINDS 


COOPERATING  UNITS  (rfany) 

Dale  McFarlin,  Chief,  NIB,  NINDS 

Michael  Racke,  Senior  Staff  Fellow,  NIB,  NINDS 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neurocytobioloqy 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  q  -, 


PROFESSIONAL: 


0.7 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [T]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  studies  were  designed  to  examine  interactions  between  lymphocytes  which 
induce  the  model  autoimmune  disease  experimental  allergic  encephalomyelitis  (EAE) 
and  cerebral  vascular  endothelial  (EC)  which  comprise  the  blood-brain  barrier  (BBB). 

The  first  part  of  these  experiments  examined  the  capacity  of  EC  to  function  as 
targets  for  lysis  by  T-cells  capable  of  transferring  EAE.  Lysis  was  proportional  to  E/T 
ratios,  dependent  upon  the  expression  of  la  antigen  and  the  presence  of  specific 
antigen.  Continuously  cultured  antigen-specific  T-cell  lines  ultimately  lost  the  ability  to 
lyse  EC  but  continued  to  proliferate  to  MBP.  These  cell  lines  also  lost  the  ability  to 
transfer  EAE  suggesting  a  relationship  between  lytic  potential  and  encephalitogenicity. 

The  second  part  of  these  experiments  concerned  observations  that  cerebral  vascular 
EC  inhibited  antigen-induced  proliferation  by  MBP-specific  lymphocytes.  Attempts  to 
characterize  the  inhibition  indicated  that  IL-I  induced  synthesis  of  prostacyclin  (PGI2)  by 
EC.  The  amount  of  PGI2  synthesized  in  these  experiments  was  sufficient  to  account  for 
the  inhibitory  response  observed. 

These  data  indicate  that  EC-lymphocyte  interactions  may  result  in  effects  on  both  EC 
(i.e.,  lysis)  and  lymphocytes  (i.e.,  antiproliferative  response).  Such  interactions  may 
result  in  alterations  of  BBB  permeability  leading  to  vascular  egression  into  the  CNS, 
which  are  pathologic  hallmarks  of  neuroimmunological  disorders  such  as  EAE  and 
multiple  sclerosis. 


29-LNNS/DIR 


»HS6O60(l>*»  1*4) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02821-01 
LNNS 


PERIOD  COVERED 

October  1, 1989  through  September  30, 1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 


Dynamics  of  Postischemic  Calcium  Accumulation  and  Protein  Synthesis  in  Brain  Tissue 


PRINCIPAL  INVESTIGATOR  (list  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.  G.  Mies,  M.D.  Visiting  Associate  LNNS,  NINDS 


Other:  T.S.  Nowak,  Jr.,  M.D. 

K.  Kawai,  M.D. 
N.Saito,  M.D. 
I.KIatzo,  M.D. 


Expert 

Visiting  Fellow 
Visiting  Fellow 
Chief 


LNNS,NINDS 
LNNS.NINDS 
LNNS/NINDS 
LNNS/NINDS 


COOPERATING  UNITS  (if  an,) 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Cerebrovascular  Pathology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


0.8 


PROFESSIONAL: 


0.8 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

I      |   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


J  (b)  Human  tissues  I  x   |  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

After  10  minute  global  of  ischemia  in  rat  brain,  a  marked  increase  in  abnormal 
calcium  uptake  and  suppression  of  protein  synthesis  in  nuclei  reticularis  of  thalamus 
was  observed  as  early  as  6  hours  postischemia,  followed  by  a  significant  increase  in 
abnormal  accumulation  of  calcium  at  moderately  reduced  rates  of  protein  synthesis  in 
the  ventral  thalamic  nucleus  and  inferior  colliculus  which  lasted  up  to  7  days 
postischemia.  In  hippocampal  CA1  sector,  protein  synthesis  of  CA1  neurons  was 
severely  suppressed  until  4  days  postischemia,  but  recovered  significantly  to  subnormal 
levels  of  protein  synthesis  after  7  days  postischemia.  Metabolic  amelioration  of 
hippocampal  CA1  neurons  correlated  with  their  improvement  of  morphological 
appearance.  Our  findings  suggest  1)  that  early  postischemic  calcium  accumulation 
occurring  predominantly  in  GABAergic  brain  areas  appears  to  reflect  the  consequences 
of  inhibition  failure  of  GABAergic  interneurons  which  may  result  in  a  subsequent 
'disinhibition'  of  interconnected  projection  areas  and  the  development  of  remote 
neuron  injury;  and  2)  that  following  a  phase  of  chronic  neuronal  injury,  hippocampal 
CA1  neurons  are  able  to  recover  from  an  ischemic  insult. 


30-LNNS/DIR 


MfSWXO  (Rev.  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02822-01 LNNS 


PERIOD  COVERED 

October  1,  1989  to  September  30,  1990 


TITLE  OF  PROJ  ECT  (BO  ctortcfn  or  Itss.  7/tte  must  ftlOfiOMlint  betwn  (h«  borders.) 

Giutamate  Microdialysis  During  Repeated  Ischemia  and  Cold  Lesions 


PRINCIPAL  INVESTIGATOR  (Ust  other  professional  ptrsonnel  below  the  frinaptl  Investioetor.)  (Name,  title,  laboratory,  end  institute  affiliation) 

PI:  I.  Klatzo,  M.D.  Chief,  LNNS,  NINDS 

N.Saito,  M.D.  Visiting  Fellow  LNNS.NINDS 

K.  Kawai.M.D.  Visiting  Fellow  LNNS.NINDS 

T.S.  NowakJr.,  M.D.  Specital  Expert  LNNS.NINDS 


COOPERATING  UNITS  (if  en,) 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Cerebrovascular  Pathology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


0.6 


PROFESSIONAL: 


0.6 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

J   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  [T]  (0  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Previous  studies  have  demonstrated  a  cumulative  effect  of  repeated  ischemic  insults  in 
thegerbil.  The  present  studies  have  been  designed  to  address  the  potential  role  of  the 
amino  acid,  giutamate,  in  this  phenomenon,  since  giutamate  is  known  to  be  released 
during  ischemia  and  has  been  suggested  to  contribute  to  excitotoxic  damage  following 
ischemia  and  other  insults.  Extracellular  giutamate  was  monitored  in  several  brain 
regions  by  in  vivo  cerebral  microdialysis.  Current  results  demonstrate  that  a  burst  of 
giutamate  release  occurs  during  each  ischemic  insult,  but  that  this  appears  to  be 
cleared  more  rapidly  than  the  larger  increase  seen  after  single  longer  occlusions. 
Further  studies  will  attempt  to  determine  whether  the  timing  of  giutamate  release 
plays  a  critical  role  in  the  increased  pathogy  seen  after  repeated  occlusions. 


31  -LNNS/DIR 


PHSWMDIBtv    1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02802-03 
LNNS 


PERIOD  COVERED 

October  1, 1989  through  September  30, 1990 


TITLE  OF  PROJECT  (so  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Immune  Mechanisms:  Regulation  of  la  Antigen  Expression 


PRINCIPAL  INVESTIGATOR  (LJst  other  profession*!  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:  R.  M.  McCarron,  M.D. 

Others:  M.Spatz,  M.D. 

L  Wang,  M.D. 


Senior  Staff  Fellow 

Section  Chief 
Guest  Researcher 


LNNS,  NINDS 

LNNS,  NINDS 
LNNS,  NINDS 


COOPERATING  UNITS  (if  any) 

Masami  Tanaka,  Brain  Research  Institute,  Niagata  University,  Niagata  City,  Japan 
Elliot  Cowan,  Special  Expert,  NIB,  NINDS 


LAB/BRANCH 

Laboratory  of  Neuropathology  and  Neuroanatomical  Sciences 


SECTION 

Section  of  Neurocytobioloqy 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.4 


PROFESSIONAL: 


1.4 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I      I  (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


|x|  (b)  Human  tissues  _J  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  role  of  class  II  MHC  antigen  expression  in  neuroimmunological  disorders  is  being 
studied  using  the  model  autoimmune  disease  experimental  allergic  encephalomyelitis 
(EAE)  in  SJL  mice.  Studies  are  focused  on  the  expression  of  la  antigen  by  SJL  cerebral 
vascular  endothelial  cells  (EC)  which  comprise  the  blood-brain  barrier  (BBB).  Previous 
experiments  characterized  expression  of  la  antigen  by  EC  and  functional  significance 
(i.e. ,  antigen-presentation).  The  potential  role  of  la-positive  EC  in  the  extravasation  of 
peripheral  blood  lymphocytes  (across  the  BBB  into  the  CNS)  is  examined  by  analyzing 
regulation  of  EC  la  antigen  expression  in  vitro. 

Observations  include  inhibition  of  IFN-induced  la  antigen-expression  by  cytokines  IL- 
1  and  TNF.  Similar  experiments  utilizing  human  cerebrovascular  EC  indicate  that  IFN 
induced  the  expression  of  HLA-DR,  -DP,  and-  DQ  antigens.  Preliminary  experiments 
indicate  that  cytokines  may  regulate  expression  of  human  class  II  MHC  antigens  as  was 
observed  in  mice. 

The  mechanism  of  IFN-induction  of  la  antigen  expression  was  studied  by  examining 
effect  of  agents  which  alter  secondary  messangers  (i.e.,  cAMP  and  protein  kinases), 
which  are  involved  in  IFN-induced  up-regulation  of  la  antigen.  Results  demonstrate 
that  agonists  and  antagonists  of  adrenergic  receptors  modulate  induction  of  la 
expression.  The  mechanism  involves  interaction  between  the  two  signal  transduction 
pathway  leading  to  activation  of  protein  kinase  a  (and  cAMP  formation)  and  protein 
kinase  C. 


32  -  LNNS/DIR 


»HSSO40<R«v.  1*4) 


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> 
o 

30 

> 

H 

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30 

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V/1 


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ANNUAL  REPORT 

October  1, 1989  through  September  30, 1990 

Laboratory  of  Neurophysiology 

Basic  Neurosciences  Program,  DIR 

National  Institute  of  Neurological 

Disorders  and  Stroke 

Research  Summary II-rV 

Project  Reports 

Physiological  Properties  Developing  on  CNS  Cells  1 

Z01NS  02019-18  LNP 

Cell  Biological  Studies  of  Developing  CNS  Cells  2 

Z01NS  02330-13  LNP 

Synaptic  Contacts  of  Retinal  Neurons    3 

Z01NS  01659-22  LNP 

Structure  and  Function  in  Retinal  Neurons    4 

Z01NS  02631-07  LNP 

Anatomical  Studies  of  Neurons,  Neurotransmitters  and     5 

Neurotransmitter  Receptors 
Z01NS  02705-05  LNP 

Image  Processing  and  Analysis  of  Cellular  Structures 6 

Z01  NS  02767-03  LNP 


I-LNP,  BNP,  Dm 


Annual  Report 
October  1, 1989  through  September  30, 1990 

Laboratory  of  Neurophysiology,  BNP,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 

Jeffery  L.  Barker,  M.D.,  Chief 

The  Laboratory  of  Neurophysiology's  research  program  has  been  divided 
primarily  between  application  of  well-established  techniques  in  electrophysiology  to 
generate  new  insight  into  intercellular  communication  and  signal  transduction 
mechanisms  and  the  refinement  of  relatively  novel  strategies  in  physiological 
recording  techniques  to  expedite  the  rate  at  which  such  insights  can  be  obtained. 
Most  of  the  research  endeavors  are  related  to  the  physiology  of  intercellular 
communication  and  signal  transduction  mechanisms  expressed  by  vertebrate, 
Central  Nervous  System  (CNS)  cells  or  cells  that  are  primary  targets  of  CNS  signals. 

The  broad  and  long-term  goal  of  this  program  is  to  systematically  elucidate 
details  regarding  the  development,  differentiation  and  cellular  distribution  of 
specific  circuits  in  vertebrate  nervous  systems.  All  CNS  cells  and  their  peripheral 
targets  exhibit  receptors,  ion  channels  and  signal  transduction  mechanisms  that 
mediate  synaptic  and  extrasynaptic  types  of  intercellular  communication.  How, 
when  and  where  specific  excitable  membrane  properties  develop  in  vertebrate 
nervous  systems  is  the  focus  of  much  of  our  research  activities.  Multidisciplinary 
study  of  the  differentiation  of  transmitters,  receptors,  ion  channels  and  signal 
transduction  mechanisms  during  development  should  reveal  fundamental  insights 
into  the  complex  process  of  distributing  such  mechanisms  among  cells  and  their  roles 
both  in  differentiating  and  in  differentiated  circuits.  In  time,  we  aim  to  provide  a 
useful  reference  for  studying  specific  pathophysiologies  of  the  vertebrate  CNS  that 
involve  dysfunctions  of  specific  circuits  and  signals. 

All  the  projects  are  at  cellular  or  molecular  levels  of  study.  Experimental 
substrates  and  lines  of  investigation  include:  2)  monolayers  of  primary  neuronal  and 
glial  cells  cultured  from  embryonic  and  early  postnatal  mammalian  and  avian  CNS; 
2)  monolayers  of  cultured  fibroblasts  transfected  with  genes  encoding  specific 
transmitter  receptors;  3)  acutely  isolated  central  elements  from  specific  regions  of 
the  embryonic  and  postnatal  CNS;  4)  retinal  eye  cup  and  intact  retina;  5) 
quantitative  electrophysiological  analysis  of  cellular  excitability  expressed  in  short 
(hours)  or  long-term  (days- weeks)  cultured  embryonic  CNS  neurons,  as  well  as 
tumoral  and  primary  pituitary  cells;  6)  quantitative  electrophysiological  analysis  of 
cellular  excitability  resident  in  retinal  circuits;  7)  flow  cytometric  analysis  of 
physiological  properties  exhibited  in  populations  of  acutely  suspended  central 
elements;  8)  light-  and  electron-microscopic  resolution  of  cellular  form  and 
subcellular  structure  in  normally  developed  mammalian  CNS  tissues  including 
retina  and  spinal  cord;  9)  cytochemical  and  biochemical  characterization  of  specific 
transmitter  phenotype  expressions  in  vivo  and  in  monolayer  culture;  and 
10)  quantitative  optical  recordings  of  physiological  signals  emitted  by  embryonic 
neurons  or  associated  with  the  morphology  of  differentiating  glia  and  neurons. 
Conceptually,  these  diverse  strategies  and  lines  harmonize  to  allow  quantitative 
resolution  of  receptor  functions,  ion  channel  expression  and  signal  transduction 


E-LNP,  BNP,  DIR 


mechanisms.  The  strength  of  the  Laboratory's  research  lies  in  the  spectrum  of 
contemporary  and  innovative  strategies  at  single-cell,  circuit  and  population  levels, 
and  the  opportunity  for  multidisciplinary  and  collaborative  study  of  basic  problems 
into  the  physiology  of  intercellular  communication  emerging  in  the  embryonic  CNS. 

Several  accomplishments  this  year  have  long-lasting  implications  with  regard  to 
the  role  of  y-aminobutyric  acid  (GABA)  and  its  receptors  in  the  differentiation  of 
embryonic  neurons  and  circuits,  which  has  become  a  major  focus  of  LNP  research 
activity. 

Dr.  Marc  Walton,  in  collaboration  with  Dr.  Anne  Schaffner,  has  applied  voltage- 
sensitive  dye  to  cultured  embryonic  rat  spinal  cord  cells,  and  probed  the  emergence  of 
functional  Na  +  channels  and  amino  acid  receptors,  which  may  be  among  the  first  to 
develop  in  postmitotic  elements.  Their  results  demonstrate  that  depolarizing 
GABAa  receptors  are  widespread  and  co-expressed  with  functional  Na+  channels 
and  kainic  acid  receptors  during  the  early  postmitotic  period.  This  multicellular 
recording  strategy  provides  an  especially  insightful  perspective  for  examining 
excitable  membrane  properties  of  intact  cells  differentiating  for  hours  to  weeks  in 
monolayer  culture. 

The  early  embryonic  appearance  of  functional  GABAa  receptors  has  prompted 
parallel  study  of  the  synthesis  and  release  of  GABA  during  embryogenesis. 
Dr.  Suzan  Nadi  has  used  biochemical  methods  to  study  GABA  synthesis  and  release 
during  rat  and  chick  CNS  development.  She  has  found  that  polyamines  precede 
glutamate  as  a  source  of  GABA  during  embryogenesis.  Preliminary  data  also 
indicate  that  GABA  can  be  released  from  embryonic  cortical  neurons  cultured  by 
Ms.  Smallwood. 

Ms.  Toby  Behar  and  Dr.  Schaffner  have  used  immunological  techniques  to 
demonstrate  that  during  the  course  of  rat  spinal  cord  development  many  neurons  (up 
to  40%  )  contain  GABA  along  with  proteins  related  to  the  enzyme  that  decarboxylates 
glutamate  to  GABA  (GAD).  Immunoblot  analysis  of  spinal  cord  proteins  probed  with 
anti-GAD  antibodies  reveals  that  low  molecular  weight  GAD-related  proteins 
precede  the  developmental  appearance  both  of  high  molecular  weight  forms  and  of 
GABA.  Ms.  Behar  and  Dr.  Walton  have  found  that  depolarizing  GABAa  receptors 
precede  the  appearance  of  GABA  immunoreactivity,  and  that  virtually  all  GABA  + 
neurons  express  functional  GABAa  receptors.  Together  these  results  suggest  that 
GABAa  receptors  may  play  differentiating  roles  in  the  emergence  of  functional,  high 
molecular  weight  GAD  proteins  and  the  synthesis  of  GABA  in  spinal  cord  neurons. 

Dr.  Anita  Prasad  has  cultured  embryonic  chick  spinal  cord  neurons,  which,  like 
embryonic  rat  spinal  cord  neurons  also  express  depolarizing  GABAa  receptors,  in  the 
presence  of  the  GABAa  antagonists  bicuculline  and  picrotoxin,  and  discovered  that 
pharmacological  block  of  GABAa  receptor  function  prevents  cell  adherence  and 
neurite  outgrowth.  These  toxic  actions  are  neither  shared  by  the  Na+  channel 
antagonist  tetrodotoxin  nor  by  the  kainic  acid  receptor  antagonist  CNQX,  and  can  be 
reversed  by  co-application  of  muscimol,  a  GABAa  agonist.  Thus,  GABAa  receptors 
may  be  critical  to  morphogenesis. 

m-LNP,  BNP,  DIR 


Postnatal  GABAa  receptors  consist  of  various  permutations  and  combinations  of 
subunits.  What  is  the  molecular  composition  of  depolarizing  GABAa  receptors? 
Dr.  Michael  Poulter,  in  collaboration  with  members  of  the  Laboratory  of  Cell  Biology, 
NIMH,  has  used  in  situ  hybridization  techniques  to  begin  to  elucidate  the 
developmental  appearance  of  GABAa  receptor  mRNA.  His  preliminary  results 
indicate  that  {5-subunit  mRNA  is  more  abundant  in  the  embryonic  rat  CNS  than  a- 
subunit  mRNA. 

The  presence  of  embryonic  GABAa  receptors  has  led  to  an  electrophysiological 
analysis  of  GABAa  receptor  functions  at  membrane  and  cytoplasmic  levels. 
Drs.  Alexander  Valeyev  and  Ricardo  Cruciani  have  used  whole-cell  recording 
techniques  to  study  GABAa  receptor  functions  on  cells  cultured  from  embryonic 
hippocampal  tissue  by  Ms.  Smallwood.  Their  preliminary  results  indicate  that  the 
GABAa  receptors  couple  to  a  CI-  ion-selective  conductance  whose  kinetics  are 
complex.  Using  cell-attached  recording  techniques,  Dr.  Jan  Suszkiw  has  observed 
that  the  response  to  GABA  in  otherwise  intact  cells  is  functionally  excitatory, 
triggering  action  potentials  in  early  postnatal  septal  neurons  cultured  by 
Dr.  Schaffner.  Suszkiw's  results  are  consistent  with  the  depolarizing  effects  of 
GABAa  receptor  activation  indirectly  recorded  by  Dr.  Walton  in  cultured  spinal 
neurons  and  by  others  in  this  laboratory  (Drs.  Nadi,  Wu  Ma,  and  Keiichi  Torimitsu 
all  working  with  Ms.  Smith)  using  flow  physiological  recording  techniques  applied  to 
embryonic  and  early  postnatal  neurons  dissociated  from  spinal  and  supraspinal 
regions  of  the  rat  and  chick  central  nervous  systems.  Finally,  Dr.  Torimitsu  has 
recorded  Ca2  +  -indicator  dye  signals  in  response  to  GABAa  receptor  stimulation 
using  cells  suspended  from  the  embryonic  chick  telencephalon  in  a  fluorescence 
spectrophotometer.  GABAa  receptors  depolarize  responsive  embryonic  elements 
leading  to  Na+  influx  as  well  as  Ca2+  increases. 

From  all  these  results  it  is  clear  that  functional  GABAa  receptors  emerge  soon 
after  terminal  cell  division  in  the  early  period  of  postmitotic  differentiation 
throughout  rat  and  chick  CNS,  well  in  advance  of  functional  synaptic  circuits. 
Continued  systematic  and  multidisciplinary  analysis  of  GABA  should  reveal  what 
roles  GABA  and  its  receptors  play  in  the  differentiation  of  specific  cellular 
phenotypes,  including  GABAergic  neurons  and  their  synapses. 


D7-LNP,  BNP,  DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS  02019-18  LNP 


PERIOD  COVERED 

October  1 ,  1989  through  September  30, 1990 


TITLE  OF  PROJ  ECT  (SO  chincters  or  less    Till*  must  fit  on  one  line  between  the  borders.) 

Physiological  properties  developing  on  CNS  cells* 


PRINCIPAL  INVESTIGATOR  (List  other  profession*!  personnel  betom  the  frinaptl  Investigator  )  (Mime,  title,  laboratory,  and  institute  affiliation) 

PI:  J.L.  Barker,  Chief,  LNP,  BNP,  DIR,  NINDS.  Others  (LNP,  BNP,  DIR,  NINDS):  A.E.  Schaffner, 
Biologist;  M.K.  Walton,  Senior  StaffFellow;  A.  Y.  Valeyev,  Visiting  Scientist;  R.  Cruciani,  Visiting 
Associate;  MO  Poulter,  Visiting  Fellow;  K.S.  Madden,  NRC  Fellow;  J.  Suszkiw,  1PA;  K.  Torimitsu, 
Special  Volunteer;  R.E.  Study,  Special  Volunteer;  B.  Dufy,  Special  Volunteer;  N.  Hardegen,  Electronics 
Technician;  L.  Mahan  (LCB,  NIMH). 


COOPERATING  UNITS  litany) 

G.D.  Lange  (RSB,  NINDS);  J.M.H.  ffrench-Mullen  (1CI  Pharmaceuticals,  Wilmington,  DE) 


LAB/BRANCH 

Laboratory  of  Neurophysiology,  BNP,  DIR,  NINDS 


SECTION 

Office  of  the  Chief 


INSTITUTE  AND  LOCATION 

NINDS,  N1H,  Bethesda,  Maryland  20892 


TOTAL  MAN  YEARS:  „  . 

8.5 


PROFESSIONAL:.  -  g 


OTHER:  j  q 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [T\  (c)  Neither 

]  (a1)  Minors 

]  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Electrophysiological  and  optical  recording  techniques  are  used  primarily  to  elucidate 
the  development,  differentiation  and  cellular  distribution  of  physiologically  important 
properties  expressed  in  vitro  by  vertebrate  CNS  neurons  or  by  fibroblasts  transfected 
with  transmitter  receptor  genes.  Electrical  studies  involve  direct,  high-fidelity 
amplification  of  ion  fluxes  generated  in  single  cells  or  in  synaptically  coupled  pairs  of 
cells  maintained  in  monoloayer  culture.  Optical  recordings  include  simultaneous 
indirect  measurements  of  membrane  potential  or  of  intracellular  ion  concentration  in  5- 
50  cells  in  culture.  Principal  findings  this  year  include:  1)  depolarizing  GABAa 
receptors  are  co-expressed  with  functional  voltage-dependent  Na+  channels  and 
depolarizing  kainic  acid  receptors  in  the  earliest  period  of  postmitotic  differentiation  of 
the  rat  spinal  cord;  2)  GABAa  receptors  couple  to  CI-  ion  selective  conductances  in 
hippocampal  neurons  during  the  embryonic  period;  3)  GABAa  receptors  activated  on 
physiologically  intact  early  postnatal  septal  neurons  are  functionally  excitatory, 
triggering  action  potentials;  4)  dose-response  curves  indicate  that  submicromolar 
agonist  concentrations  are  effective  and  that  a  Hill  coefficient  close  to  1  describes  the 
dose-response;  5)  pharmacological  effects  of  anesthetic  and  naturally  occurring  steroids 
at  GABAa  receptors  can  be  detected  before  birth  on  embryonic  hippocampal  neurons;  6) 
fluctuation  analysis  of  GABAa  receptor-coupled  CI-  conductances  shows  complex,  two- 
component  kinetics  of  ion  channel  behavior  both  in  embryonic  hippocampal  neurons  and 
in  fibroblasts  expressing  specific  GABAa  receptor  subunit  combinations;  7)  septal 
neurons  express  a  full  complement  of  electrical  and  chemical  excitability  during  the 
first  week  postnatal;  8)  TRH  activates  K  +  -ion  selective  conductances  in  tumoral 
pituitary  cells  via  1,4,5-inositol  trisphosphate-  and  diacylglycerol-induced  mobilization 
of  Ca^+  from  intracellular  stores;  9)  fluctuation  analyses  of  endogenous  voltage 
trajectories  in  cultured  embryonic  chick  spinal  cord  cells  at  the  resting  membrane 
potential  reveals  complex  behavior  suggestive  of  a  deterministic  system. 

l-T.NP  R\TP,  DTR 


PHSMMOfRtv   1  M 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 
Z01NS  02330-13  LNP 


PERIOD  COVERED 

October  1, 1989  through  September  30, 1990 


TITLE  OF  PROJECT  (80  characters  orless.  Title  must  fit  on  one  lute  between  the  borders.) 

Cell  Biological  Studies  of  Developing  CNS  Cells 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  J.L.  Barker,  Chief,  LNP,  BNP,  DIR,  NINDS.  Others  (LNP,  BNP,  DIR,  NINDS):  S.  Nadi,  Special 
Expert;  A.E.  Schaffner,  Biologist;  K.  Thor,  Senior  Staff  Fellow;  W.  Ma,  Senior  Staff  Fellow;  M.  Fiszman, 
Visiting  Fellow;  A.  Prasad,  Staff  Fellow;  MO.  Poulter,  Visiting  Fellow;  K.  Torimitsu,  Special  Volunteer; 
T.N.  Behar,  Microbiologist;  S.V.  Smith,  Biologist,  N.  Hardegen,  Electronics  Technician.;  V.  Smallwood, 
Bio.  Lab.  Technician;  J.  Cuebas,  Lab.  Technician. 


COOPERATING  UNITS  (it any) 

G.D.  Lange  (RSB,  NINDS):  S.  Lolait  (LCB,  NIMH) 


LAB/BRANCH 

Laboratory  of  Neurophysiology 


SECTION 

Office  of  the  Chief 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  PH  (c)  Neither 

]  (a1)  Minors 

]  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Flow  cytometry  and  immunochemical  methods  are  applied  to  embryonic  rat  spinal 
and  supraspinal  regions  to  study  the  development,  differentiation  and  cellular 
distribution  of  transmitters  and  their  corresponding  receptor-coupled  alterations  in 
membrane  excitability.  At  present  this  multidisciplinary  investigation  is  focused  on 
elucidation  of  a  developmental  calendar  outlining  the  expression  of  specific  transmitters 
and  their  functional  receptors  with  a  primary  focus  on  the  development  of  the  GABA 
phenotype  and  a  secondary  focus  on  the  glutamate  phenotype.  Principal  observations 
this  year  include:  1)  immunological  detection  of  GAD-related  proteins  during  embryonic 
development  of  the  rat  spinal  cord  with  low  molecular  weight  proteins  apparent  in  many 
neurons  before  the  advent  of  GABA,  the  product  of  GAD  enzyme  activity;  2)  progressive 
appearance  of  GABA +  fibers,  then  GABA+  neurons  during  embryogenesis  along  rostro- 
caudal  and  ventrodorsal  axes  in  the  spinal  cord  coincident  with  the  appearance  of  high 
molecular  weight  GAD  proteins;  3)  postnatal  emergence  of  a  second  set  of  GAD-related 
proteins  with  a  subcellular  distribution  and  colchicine  sensitivity  characteristic  of 
vesicular  transport  in  neurons  also  immunoreactive  for  embryonic  GAD  proteins;  4) 
biochemical  detection  of  GABA  transiently  derived  from  ornithine  and  increasingly 
synthesized  by  GAD  in  embryonic  rat  cortex;  5)  sizable  secretion  of  GABA  in  vitro  under 
resting  conditions  from  embryonic  cortical  neurons;  6)  early  embryonic  appearance  of 
functional  voltage-gated  Na+  channels  and  depolarizing  GABAa  progesterone 
metabolite  receptors  in  rat  cortex,  cerebellum  and  thalamus;  7)  in  situ  hybridization 


evidence  of  GABAa  receptor  subunit  (5  receptor  message  preceding  a  subunit  message 
throughout  the  embryonic  rat  CNS;  8)  emergence  of  depolarizing  GABAa  receptors  along 
a  rostrocaudal  gradient  throughout  the  early  embryonic  chick  CNS;  9)  critical 
requirement  of  GABAa  receptors  for  chick  spinal  cord  neuron  adherence  in  vitro  and 
process  formation;  10)  arrival  of  spinothalamic  tract  projections  in  the  thalamus  about 
the  time  of  birth  in  the  rat. 

2-LNP,BNP,DIR 


?HS£IW<">" 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  01659-22  LNP 


PERIOD  COVERED 

October  1,  1989  through  September  30, 1990 


TITLE  OF  PROJECT  (SO  chanctari  ot  luii.   Tnl9  murt  frt  on  on*  hna  kMMM  tha  burden .) 

Synaptic  Contacts  of  Retinal  Neurons 


PRINCIPAL  \NVISTIGA70R  (Lm  olhar  enfatsmnal partonnal baltn*  tht  Principal ln¥*s1iQa<oc.)  (Him*,  tnla.  laboratory,  and  mstituta  affiliation) 

PI:  A.  Lasansky,  Unit  Chief,  LNP,  BNP,  DIR,  NINDS 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Laboratory  of  Neurophysiology,  BNP,  DIR,  NINDS 


SECTION 


Unit  on  Cell  Biology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN  YEARS: 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  fxl  (c)  Neither 

]  (a1)  Minors 

]  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  current-voltage  relationship  of  salamander  depolarizing  bipolar  cells  displays  a 
strong  outward  rectification.  As  a  result  of  this  membrane  nonlinearity,  increases  in 
the  intensity  of  bright  lights  cause  relatively  smaller  amplitude  increases  in  the  voltage 
than  in  the  current  responses  and  the  latter  have  a  proportionally  smaller  after  effect. 
The  light-evoked  responses  appear  to  consist  of  at  least  two  components:  a  chloride- 
dependent  on-off  increase  in  membrane  conductance  and  a  faster  depolarizing  input 
that  is  lost  through  diffussional  exchange  between  the  cytosol  and  the  content  of  patch 
pipettes.  Because  of  its  on-off  pattern,  the  chloride-dependent  component  is  thought  to 
reflect  input  from  amacrine  or  interplexiform  cells. 


3-LNP,  BNP,  DIR 


PHSMMOIitev   lltl 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01-NS-02631-07 

LNP 


PERIOD  COVERED 

October  1, 1989  through  September  30, 1990 

TITLE  OF  PROJECT  (80  characters  or  lass.   Title  must  fit  on  one  line  between  the  borders..) 

Structure  and  Function  in  Retinal  Neurons 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.:         Ralph  Nelson,  Unit  Chief,  LNP,  NINDS 

Others:  Michael  Freed,  StaffFellow,  LNP,  NINDS;  Renate  Pflug,  Instructor,  U.  of  Vienna;  Helga  Kolb, 
Professor,  U.  of  Utah;  Steven  Baer,  Asst.  Prof,  U. of  Arizona,  Tempe 


COOPERATING  UNITS  (if  my) 

Physiology,  University  of  Vienna,  Austria  (Renate  Pflug);  Physiology,  University  of  Utah  School  of 
Medicine,  Salt  Lake  City  (Helga  Kolb);  Mathematics,  Arizona  State  University,  Tempe  and 
Mathematical  Research  Branch,  NIDDK  (Steven  Baer) 


LAB/BRANCH 

Laboratory  of  Neurophysiology,  DIR,  NINDS 


SECTION 

Neural  Circuitry  Unit 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  2  Q 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)Human  subjects  □  (b)  Human  tissues  [71  (c)  Neither 

J  (a1)  Minors 

]  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

.  Neural  organization  and  neural  interactions  in  mammalian  retinas  are  investigated 
using  intracellular  recording  and  staining  techniques,  electron-microscopic  (EM)  and 
pharmacological  approaches.  In  the  inner  plexiform  layer  of  cat  retina  amacrine  cells 
with  depolarizing  responses  to  both  onset  ant  offset  of  photic  stimuli  (ON-OFF  cells)  are 
filled  with  horseradish  peroxidase  and  identified  as  wide-field,  monostratified  types 
(A19).  In  addition  to  a  medium-field  dendritic  arbor  surrounding  the  soma,  A19 
amacrine  cells  have  multiple  axon-like  processes  extending  from  dendritic  tips. 
Impulses  of  variable  height  accompany  depolarizations,  and  also  occur  spontaneously. 
Current  clamp  reveals  that  depolarizations  result  from  a  conductance  increase.  In  the 
EM  A19  cells  synapse  on  alpha  ganglion  cells  (GC's),  and  may  energize  their  nonlinear 
subunits. 

OFF-beta  cat  retinal  GC's  reveal  about  equal  numbers  of  amacrine  and  bipolar  cell 
inputs  whereas  OFF-alpha  GC's  receive  mainly  amacrine  input,  as  seen  in  serial  EM 
reconstructions.  OFF-alpha  cells  receive  input  from  only  one  type  of  bipolar  cell 
whereas  OFF-beta  cells  receive  input  from  two  types.  OFF-depolarizations  arise  from  a 
conductance  increase.  Investigations  of  ON-alpha  GC's  reveal  about  2000  chemical 
synapses  distributed  in  dome-like  fashion  across  the  dendritic  field.  This  dome-shaped 
synaptic  weighting,  as  convolved  with  dendritic,  and  electrotonic  factors,  contributes  to 
the  Gaussian  profile  of  alpha-GC  receptive-field  sensitivity. 

In  the  outer  plexiform  layers  of  cat  and  rabbit  retinas,  the  dopaminergic  agonist 
apomorphine  depolarizes  horizontal  cells  and  both  suppresses  and  delays  cone-flicker 
signals.  The  effect  is  mimicked  by  SKF38393  and  may  be  Dl-like.  The  potentiating 
effects  on  cone  signals  of  rod-desensitizing  backgrounds  is  modelled  by  a  feedback 
circuit  in  which  dark-adapted,  depolarized  horizontal  cells,  release  a  substance  that 
antagonizes  cone  feed-forward  synaptic  transmission. 

4-LNP,  BNP,  DIR 


WtMOIBt,   IM, 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02705-05  LNP 


PERIOD  COVERED 

October  1, 1989  through  September  30,  1990 


TITLE  OF  PROJECT  ffiO  characters  or  Ian.    Title  rnuif  fit  on  one  line  b«l»««n  rh«  borOe'i  ) 

Anatomical  Studies  of  Neurons,  Neurotransmitters  and  Neurotransmitter  Receptors 


PRINCIPAL  INVESTIGATOR  (Usx  other  prorfuskmai  personnel  below  the  Principal  tnvestiaetor.)  (Name,  title,  laboratory,  and  institute  affiliation) 

A. P.  Mariani,  Expert,  LNP,  BNP,  D1R,  NINDS 


COOPERATING  UNITS  Of  my) 

Ohio  State  University  College  of  Medicine  (N.H.  Neff  and  M.  Hadjiconstantinou) 


LAB/BRANCH 

Laboratory  of  Neurophysiology 


SECTION 

Office  of  the  Chief 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)Human  subjects  □  (b)  Human  tissues  [x~1  (c)  Neither 

J  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Amacrine  cells  of  the  retina  are  central  nervous  system  interneurons.  Knowledge  of 
their  morphology,  connections  and  neurotransmitters  could  aid  in  the  understanding  of 
the  local-circuit  neurons  in  the  brain.  Amacrine  cells  of  the  rhesus  monkey,  Macaca 
mulatto.,  were  studied  in  38  retinas  Golgi-impregnated  as  whole,  flat  preparations.  By 
using  criteria  of  dendritic  morphology,  span  of  arborization,  and  level  of  arborization  in 
the  inner  plexiform  layer,  26  types  of  amacrine  cell  ranging  in  size  of  dendritic  span 
from  30  mm  to  2  mm  were  identified.  This  study  provides  evidence  for  an  unprecedented 
number  of  amacrine  cell  types  in  the  primate  retina.  The  similar  morphologies  of 
different  types  of  amacrine  cell  types  within  a  group  suggests  other  common  features 
within  these  groups  such  as  neurotransmitter  phenotype.  Two  types  of  amacrine  cell 
(type  1CA  and  type  2CA)  immunoreactive  for  tyrosine  hydroxylase  (TH),  the  rate- 
limiting  enzyme  in  the  catecholamine  (CA)  synthetic  pathway,  are  present  in  the  retina 
of  the  rhesus  monkey,  Macaca  mulatta.  Although  type  2CA  cells  are  more  numerous 
than  type  1CA  amacrines,  type  2  cells  contain  3.5  times  less  TH  than  the  type  1  cells. 
Electron  microscopy  of  retinal  tissue  immunoreactive  for  TH  by  the  peroxidase- 
antiperoxidase  (PAP)  method  revealed  synaptic  input  from  amacrine  cells  at 
conventional  synapses,  and  bipolar  cells  at  ribbon  synapses  onto  the  type  2CA  amacrine 
cells.  Curiously,  although  the  synaptic  input  is  comparatively  easily  found,  the  output 
synapses,  or  synapses  of  the  type  2CA  amacrine  cells  onto  other  neuronal  elements,  are 
rarely  found.  Some  synapses  of  the  type  2CA  cells  onto  non-immunoreactive  amacrine 
cells  have  been  identified  however.  This  unusual  pattern  of  synaptic  organization,  with 
many  identifiable  input  synapses  but  few  morphologically  characterizable  output 
synapses,  suggests  a  paracine  role  for  nonsynaptic  release  of  dopamine  by  the  type  2CA 
amacrine  cells  in  the  primate  retina. 

5-LNP,  BNP,  DIR 


PHS  WMOIBev    1  gdl 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02767-03  LNP 


PERIOD  COVERED 

October  1,  1990  through  September  30, 1991 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Image  Processing  And  Analysis  of  Cellular  Structures 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  T.G.  Smith,  Jr.,  Unit  Chief,  LNP,  BNP,  DIR,  NINDS. 

Others  (LNP,  BNP,  DIR,  NINDS):  Kathleen  Madden,  Guest  Researcher;  Anita  Prasad,  Staff  Fellow; 

T.N.  Behar,  Technician 


COOPERATING  UNITS  lifmy) 

G.D.  Lange,  (IACS,  NINDS),  W.B.  Marks  (LNLC,  NINDS),  E.A.  Neale  (LDB,  NICHD);  W.H.  Sheriff,  Jr. 
(1ACS,  NINDS) 


LAB/BRANCH 

Laboratory  of  Neurophysiology 


SECTION 

Unit  on  Sensory  Physiology 


INSTITUTE  AND  LOCATION 

NINDS,  N1H,  Bethesda,  Maryland  20892 


rOTAL  MAN-YEARS: 


PROFESSIONAL:  2  fj 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [x~|  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

We  have  continued  to  employ  the  concepts  of  Mandelbrot's  fractal  geometry  to  the 
quantitative  studies  of  central  nervous  system  neurons,  Glia  and  other  cell  types  grown 
in  tissue  culture  or  from  whole  animals.  We  do  this  by  employing  image  processing 
techniques  to  measure  the  fractal  dimension  (FD),  which  is  a  quantative  measure  of  the 
complexity  of  the  structure  under  investigation.  In  particular,  the  FD  relates  to  the 
degree  ofpranching  (e.g.  of  dendrites),  the  ruggedness  of  borders,  and  the  degree  of 
space-lilling  of  theobject  of  interest. 

We  have  undertaken,  in  separate  studies,  how  the  fractal  dimension  changes  during 
the  differentiation  and  growth  of  glial  and  neuronal  cells  in  tissue  culture.  We  have 
found  that  optic  nerve-derived  oligodendrocytes  differentiate  faster  and  to  a  greater 
extent  than  do  nerve-derived  astrocytes.  A  subsequent  study  has  found  that  nerve- 
derived  glia  also  differentiate  taster  and  to  a  greater  extent  than  do  brain-derived  glia. 
interestingly,  the  rates  of  differentiation  as  measured  by  the  FD  can  be  described  by  a 
single  time  constant.  The  work  on  cultured  spinal  neurons  is  not  yet  complete,  but  it 
appears  that  they  differentiate  in  a  similarly  simple  lashion.  We  nave  proposed  the  FD 
is  a  useful,  quantitative  measure  of  morphological  differentiation. 

We  have  begun  separate  studies  1)  of  the  development  of  the  internal  structures  of 
cultured  chick  spinal  cord  neurons  with  fluorescence  microscopy,  2)  of  the  phase-plane 
plots  of  the  electrical  activity  of  spinal  cord  activity  by  measuring  their  FD's,  and 
6)  Courier  methods  of  analysing  cellular  structure. 


6-LNP,  BNP,  DIR 


PHS6M0(Rev   1'WI 


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ANNUAL  REPORT 

October  1,  1989  through  September  30,  1990 

Laboratory  of  Molecular  and  Cellular  Neurobiology 

Basic  Neurosciences  Program,  DIR 

Table  of  Contents 

Page  #'s 

RESEARCH  SUMMARY  2-13 

PROJECT  REPORTS 

Investigation  of  the  Structure  and  Function  of  14 

Gonadotropins  and  their  Receptors 
Z01  NS  02784-02  LMCN 

Regulation  of  Hormone-Responsive  Adenylate  Cyclase  1 5 

Z01  NS  02366-12  LMCN 

Biosynthesis  and  Function  of  Glycosphingolipids  and  16 

Othtr  Glycoconjugates 
Z01  NS  01309-25  LMCN 

Information  Processing  in  Simple  Nervous  Systems  17 

Z01  NS  02151-16  LMCN 

Antibodies  to  Glycoconjugates  in  Neurological  Diseases  18 

Z01  NS  02786-02  LMCN 

Glycoproteins  of  Myelin  in  Development  and  Disease  19 

Z01  NS  01808-21  LMCN 

Molecular  and  Immunological  Aspects  of  Myelin  Abnormalities  20 

in  Neuro-AIDS 

Z01  NS  02805-01  LMCN 

Molecular  Biology  of  Neurotransmitter  Receptors  21 

Z01  NS  02710-05  LMCN 

Megabase  DNA  Sequencing  22 

Z01  NS  02754-03  LMCN 

Human  Brain  cDNA  Project  23 

Z01  NS  02806-01  LMCN 


1     LMCN/DIR 


ANNUAL  REPORT 

October  1, 1989  through  September  30,  1990 

Laboratory  of  Molecular  and  Cellular  Neurobiology 

National  Institute  of  Neurological  Disorders  and  Stroke 

Peter  H.  Fishman,  Ph.D. ,  Chief  (Rotating) 

The  Laboratory  of  Molecular  and  Cellular  Neurobiology  consists  of  four  independent 
Sections,  each  of  which  is  engaged  in  a  comprehensive  research  program  in  the 
neurosciences.  Thus,  there  is  overlap  in  areas  of  interest,  especially  in  biochemistry, 
cell  biology  and  molecular  biology.  Our  recently  renovated  library  and  conference 
room  provides  a  focal  point  for  staff  interactions  and  the  development  of 
collaborative  endeavors.  The  future  status  of  the  Laboratory,  however,  remains 
unclear.  It  is  envisioned  that  at  least  two  of  the  Sections  will  return  to  the  main 
campus  upon  completion  of  the  Child  Health  and  Neurosciences  Building  (Building 
49)  whereas  the  other  Sections  may  remain  off  campus. 

Membrane  Biochemistry  Section 

The  Membrane  Biochemistry  Section  actively  investigates  the  structure,  biosynthesis 
and  regulation  of  cell  membrane  components  involved  in  various  recognition 
phenomena  and  incellular  signaling.  These  include  complex  glycoconjugates  such  as 
gangliosides  and  the  receptor-coupled  adenylate  cyclase  and  phospholipase  C 
systems  which  mediate  the  cellular  responses  to  various  hormones, 
neurotransmitters,  and  growth  factors  as  well  as  pathological  toxins  and  viruses. 
Most  of  our  studies  involve  using  cultured  cell  lines  which  express  these  components 
and  respond  to  physiological  and  environmental  signals. 

1.  Role  of  Ganqlioside  Lipid  Moiety  in  Action  of  Cholera  Toxin. 

In  an  extensive  series  of  experiments,  we  had  previously  established  that  ganglioside 
GM1  is  the  only  endogenous  cell  surface  receptor  for  cholera  toxin,  the  active  agent 
in  the  disease  cholera.  The  toxin  intoxicates  target  cells  by  ADP-ribosylation  of  the 
stimulatory  G  protein,  Gs,  of  adenylate  cyclase  which  thus  results  in  a  persistent 
activation  of  adenylate  cyclase.  We  recently  demonstrated  that  Givn-type 
"ganglioproteins"  were  unable  to  serve  as  functional  receptors  for  cholera  toxin 
even  though  they  were  able  to  bind  the  toxin.  In  order  to  further  explore  the  role  of 
the  ceramide  lipid  moiety  of  Gmi,  we  synthesized  a  series  of  neoglycolipids  by 
attaching  the  oligosaccharide  of  GM1  to  cholesterol,  aliphatic  amines  and 
phospholipids.  We  incubated  the  derivatives  with  rat  C6  glioma  cells  which  are 
deficient  in  GM1,  bind  only  traces  of  cholera  toxin  and  are  poorly  responsive  to  the 
toxin.  C6  cells  exposed  to  the  various  derivatives  exhibited  an  increase  in  both 
iodinated  cholera  toxin  binding  and  toxin  activation  of  adenylate  cyclase.  There 
were  differences  among  the  neoglycolipids  in  terms  of  the  amounts  of  cyclic  AMP 
formed  per  amount  of  toxin  bound  to  the  treated  cells.  Those  derived  from  long- 
chain  aliphatic  amines  and  cholesterol  were  more  efficient  as  toxin  receptors  than 


2    LMCN/DIR 


GM1  whereas  those  derived  from  phospholipids  were  less  efficient .  When  the 
distance  between  the  oligosaccharide  and  the  phospholipid  was  increased  by  adding 
spacers,  the  efficiency  decreased  even  more.  Thus,  the  lipid  moiety  of  GM 1  is 
important  for  the  action  of  cholera  toxin,  and  these  neoglycolipids  may  be  useful  as 
probes  of  GM  1  bioactivity  in  other  systems  such  as  the  neuritogenic  and 
neuronotrophic  effects  of  the  ganglioside. 

2.   Receptors  for  Neurotransmitters  and  Neuropeptides 

We  are  continuing  to  mak  progress  on  the  identification,  characterization  and 
regulation  of  neurotransmitter  and  neuropeptide.  These  include  ^-adrenergic,  Di 
dopaminergic,  and  neuropeptide  Y  receptors.  Large  advances  have  been  made  by  us 
and  others  to  understand  the  mechanisms  of  desensitization  and  down-regulation 
of  0-adrenergic  receptors.  Although  there  are  two  major  subtypes  of  P-adrenergic 
receptors,  most  of  the  progress  has  been  directed  toward  the  {^-subtype  as  it  is  the 
subtype  found  on  all  the  established  ^-adrenergic  responsive  cell  lines  to  date.  We 
have  been  able  to  demonstrate  that  human  neurotumor  SK-N-MC  cells  express  only 
01-adrenergic  receptors.  Agonist  stimulation  of  cyclic  AMP  accumulation  was 
inhibited  more  potently  by  {$r  than  by  [32-selective  antagonists  as  was  binding  of 
labeled  antagonists  to  both  intact  cells  and  cell  membranes.  Northern  blot  analysis 
of  mRNA  from  SK-N-MC  cells  using  cDNA  probes  for  {5i-  and  {32-adrenergic  receptors 
revealed  only  the  presence  of  {ii-adrenergic  receptor  mRNA.  When  SK-N-MC  cells 
were  exposed  to  isoproterenol,  there  was  no  loss  of  maximum  responsiveness  to  the 
agonist  upon  rechallenge  during  the  first  hour.  After  longer  exposure, 
desensitization  slowly  occurred  and  the  Pi-adrenergic  receptors  slowly  down- 
regulated  to  50%  of  control  levels  by  24  hours.  We  explored  this  unusual  resistance 
to  regulation  by  preparing  membranes  from  control  and  agonist-treated  cells  and 
assaying  them  for  adenylate  cyclase  activity.  There  was  no  loss  of  maximum 
stimulation  of  adenylate  cyclase  by  isoproterenol  in  membranes  from  cells  exposed 
to  the  agonist  for  1  hour.  There  was,  however,  a  3-fold  shift  to  the  right  in  the  dose 
response.  As  receptor  phosphorylation  has  been  implicated  in  desensitization,  we 
determined  which,  if  any,  protein  kinases  might  be  involved.  Cells  were  rendered 
permeable  and  loaded  with  either  an  inhibitor  of  cyclic  AMP-dependent  protein 
kinase  A  or  heparin,  an  inhibitor  of  the  p-adrenergic  receptor-specific  kinase. 
Inhibiting  protein  kinase  A  but  not  the  receptor-specific  kinase  blocked  the  shift  in 
the  agonist  dose  response.  Furthermore,  membranes  incubated  with  ATP  and  the 
purified  catalytic  subunit  of  protein  kinase  A  exhibited  a  similar  shift.  The  shift, 
however,  was  not  observed  in  cells  treated  with  dibutyryl  cyclic  AMP.  Thus,  in  intact 
cells,  this  unusual  desensitization  requires  both  agonist-occupied  receptors  and 
activation  of  protein  kinase  A.  We  are  now  generating  antipeptide  antibodies 
against  specific  sequences  in  the  B-adrenergic  receptor  and  will  use  these  to 
determine  the  state  and  site  of  phosphorylation  of  the  receptor  in  control  and 
desensitized  cells.  A  key  issue  to  explore  is  whether  the  receptor  is  being 
phosphorylated  by  the  receptor-specific  kinase  and  if  not,  why  not.  We  have 
preliminary  evidence  that  the  dopamine  Di  receptor-coupled  adenylate  cyclase 
undergoes  a  normal  desensitization  when  SK-N-MC  cells  are  exposed  to  dopamine. 
As  the  current  dogma  predicts  that  the  receptor-specific  kinase  will  phosphorylate 
and  initiate  desensitization  of  Di  receptors,  it  would  appear  that  this  kinase  is 
present  in  SK-N-MC  cells. 

Using  our  recently  developed  procedures  for  solubilizing  and  purifying  the  Di 
receptor  from  rat  striatum,  we  have  scaled  up  the  procedure  and  obtained  sufficient 
receptor  protein  for  partial  amino  acid  sequencing  and  the  raising  of  antibodies, 
both  of  which  will  be  used  to  screen  a  rat  striatal  cDNA  library  by  standard 


3    LMCN/DIR 


techniques.  With  the  availability  of  suitable  molecular  probes  for  the  Di  receptor, 
we  will  be  able  to  investigate  both  human  and  animal  models  for  defects  in  the 
receptor  gene.  One  potential  possibility  is  the  spontaneous  hypertensive  rat  model. 
Kidney  proximal  convoluted  tubules  contain  a  dopamine-sensitive  adenylate  cyclase 
and  it  is  believed  that  dopamine  is  involved  in  the  regulation  of  sodium  transport. 
We  found  that  in  tubules  from  spontaneous  hypertensive  rats,  adenylate  cyclase 
activity  was  not  stimulated  by  dopamine  Di  agonists  but  exhibited  a  normal 
response  to  guanine  nucleotides,  forskolin  and  parathyroid  hormone.  Maximum 
binding  activity  and  affinity  for  a  Di  selective  antagonist  was  the  same  in  both 
control  and  hypertensive  tissues  as  was  the  apparent  molecular  weight  of  the  Di 
receptor  as  determined  by  photoaffinity  labeling  and  separation  by  sodium  dodecyl 
sulfate-polyacrylamide  gel  electrophoresis.  Di  agonists,  however,  were  able  to 
compete  more  effectively  for  antagonist  binding  in  control  than  in  hypertensive 
specimens.  We  believe  that  these  results  are  consistent  with  a  defect  in  the  Di 
receptors  in  proximal  kidney  tubules  of  hypertensive  rats  and  this  defect  impairs 
receptor  coupling  to  G  proteins. 

Neuropeptide  Y  (NPY)  is  the  most  abundant  neuropeptide  in  the  mammalian 
nervous  system.  It  is  widespread,  found  in  both  central  and  peripheral  neurons, 
often  colocalized  with  catecholamines,  and  has  several  physiological  effects, 
including  appetite  stimulation  and  vasoconstriction.  Little  is  known  about  its  mode 
of  action  or  its  receptors.  We  have  previously  established  that  human  neurotumor 
cells  express  functional  NPY  receptors  and  thus  are  useful  to  investigate  various 
aspects  of  NPY  bioactivity.  In  this  regard,  we  found  that  an  NPY  analog  in  which 
amino  acid  residues  7-17  had  been  replaced  with  an  amino-octanoicacid  moiety  and 
further  stabilized  with  an  intramolecular  disulfide  bridge,  [D-Cys7-Aoc817- 
Cys20]pNPY,  both  competed  for  [125I]NPY  binding  to  SK-N-MC  cells  and  inhibited 
isoproterenol-stimulated  cyclic  AMP  accumulation  in  these  cells,  but  with  slightly 
lower  affinity  and  potency  than  native  pNPY.  Thus,  the  central  a-helix  of  the 
molecule  does  not  appear  to  be  required  for  either  binding  or  biological  activity  but 
may  be  important  for  stabilizing  the  N-and  C-terminal  regions  of  the  peptide. 
Recently,  NPY  receptors  have  been  classified  into  Yi  and  Y2  subtypes  which  can  be 
distinguished  both  physiologically  and  pharmacologically.  Y1  receptors  bind  only 
the  full-length  NPY  and  are  associated  with  vasoconstriction;  Y2  receptors  will  also 
bind  the  C-terminal  fragment  of  NPY  (residues  1 3-36)  and  mediate  the  inhibition  of 
norepinephrine  release  from  presynaptic  neurons.  Whereas  SK-N-MC  cells  and  its 
subclone  MC-IXC  cells  have  only  Y1  receptors,  another  human  neurotumor  cell  line, 
SMS-MSN,  contains  only  Y2  receptors.  We  have  used  these  two  cell  lines  to 
investigate  the  mechanism(s)  of  signal  transduction  mediated  by  these  two  NPY 
receptor  subtypes.  High  affinity  binding  of  NPY  to  membranes  from  both  cell  lines 
was  sensitive  to  pertussis  toxin,  indicating  that  the  receptors  are  linked  to  G  proteins. 
The  toxin  catalyzed  the  ADP-ribosylation  of  a  41,000  dalton  protein  in  both  cell  lines. 
NPY  inhibited  effector-stimulated  cyclic  AMP  production  in  MC-IXC  cells  but  not  in 
SMS-MSN  cells.  In  contrast,  fluoroaluminate,  a  general  activator  of  Gj  in  intact  cells, 
inhibited  cyclic  AMP  production  in  both  cell  lines.  Western  blot  analysis  of  G 
proteins  with  specific  antibodies  showed  that  MC-IXC  cells  contained  Gs  and  Gj 
whereas  SMS-MSN  cells  contained  Gs,  Gj  and  G0-  Whereas  Gj  is  involved  in  coupling 
receptors  to  the  inhibition  of  adenylate  cyclase,  G0  has  been  implicated  in  coupling 
receptors  to  the  modulation  of  ion  channels.  Our  results  suggest  that  Y1  and  Y2 
receptors  mediate  cellular  signaling  through  different  G  proteins.  We  are  now 
exploring  which  ion  channels  may  be  coupling  to  the  latter  receptor  subtype. 
Initially,  we  will  investigate  Ca2+  and  K+  channels  which  have  been  implicated  for 
other  inhibitory  neurotransmitters. 


4    LMCN/DIR 


3.  Differential  Activation  of  G  Proteins  in  Cells  and  Membranes 

It  is  well-established  that  many  receptors  for  hormones  and  neurotransmitters  are 
coupled  to  their  effector  systems  through  guanine  nucleotide  binding  proteins  (G 
proteins).  Thus,  stimulatory  receptors  activate  adenylate  cyclase  through  Gs  and 
inhibitory  receptors  inhibit  adenylate  cyclase  through  Gj.  These  G  proteins  are 
heterotrimers  with  common  By  and  distinct  a  subunits.  It  is  believed  that  hormone- 
bound  receptors  activate  their  respective  G  proteins  by  promoting  the  exchange  of 
GDP  with  GTP  which  results  in  dissociation  of  the  heterotrimers  and  release  of  the  a 
subunits  containing  bound  GTP.  In  time,  the  bound  GTP  is  hydrolyzed  to  GDP  by  the 
intrinsic  GTPase  activity  of  the  a  subunit  which  then  reassociates  with  the  By 
subunits  to  complete  the  cycle.  Hydrolysis-resistant  analogues  of  GTP  and 
f  luoroaluminate  also  can  persistantly  activate  G  proteins  as  the  GTPase  "turnoff "  is 
no  longer  operating.  We  have  shown  previously  that  fluoroaluminate  is  a  potent 
inhibitor  of  hormone-stimulated  adenylate  cyclase  in  intact  cells  but  by  itself 
activated  adenylate  cyclase  in  permeable  cells  and  membranes.  Our  results  indicated 
that  the  interactions  or  relationships  between  the  G  proteins  and  the  catalytic 
subunit  of  adenylate  cyclase  are  altered  when  the  cell  membrane  is  perturbed. 

We  now  have  applied  another  probe  to  explore  these  interactions.  The  diterpene 
forskolin,  is  a  potent  activator  of  the  catalytic  subunit  of  adenylate  cyclase  but  its 
effect  is  potentiated  by  the  presence  of  Gs.  We  observed  that  different  cells  varied  in 
their  responses  to  forskolin.  Some  cell  lines  accumulated  large  amounts  of  cyclic 
AMP  whereas  as  others  accumulated  very  little  when  exposed  to  it.  We  found  that 
even  in  the  "unresponsive"  cells,  forskolin  potentiated  the  stimulation  of  cyclic  AMP 
production  by  hormones  and  neurotransmitters.  Thus,  the  drug  was  not  being 
excluded  by  "unresponsive"  cells.  Forskolin,  however,  was  able  to  activate 
adenylate  cyclase  in  membranes  prepared  from  both  "responsive"  and 
"unresponsive"  cells.  Again,  it  appears  that  the  relationships  between  G  proteins 
and  the  catalyst  are  altered  upon  cell  disruption.  Although  most  studies  have  used 
preparations  of  cell  membranes  or  purified  components  (receptors,  G  proteins, 
catalyst)  reconstituted  into  lipid  vesicles,  it  is  clear  that  a  better  understanding  of  the 
biological  effects  of  hormones  and  neurotransmitters  as  well  as  drugs  such  as 
forskolin  will  require  the  use  of  methods  to  elucidate  the  interactions  of  these 
components  in  intact  cells. 

Neural  Systems  Section 

The  Neural  Systems  Section  takes  a  multidisciplinary  approach  to  the  question  of 
how  information  is  stored  during  associative  learning  and  how  it  is  made  available 
for  later  recall.  Biophysical  and  molecular  mechanisms  of  associative  learning  are 
being  analyzed  in  parallel  for  a  mollusc  (the  sea  snail  Hermissenda  crassicornis),  the 
rabbit,  and,  most  recently,  the  rat.  Parallel  analyses  offer  the  important  opportunity 
for  uncovering  general  cellular  principles  of  learning  and  memory  -  principles  which 
have  been  conserved  over  the  course  of  evolution  and  which  therefore  could  have 
relevance  for  human  cognition.  Parallel  analyses  also  permit  exploitation  of  critical 
experimental  advantages  unique  to  diverse  species.  For  Hermissenda  we  have 
demonstrated  the  first  causal  relationship  of  biophysical  and  molecular 
transformations  within  individual  neurons  to  Pavlovian  conditioning  of  a  living 
animal.  Casual  relationships  of  cellular  physiology  and  associative  learning  have  not 
yet  been  approximated  for  any  vertebrate  preparation.  Nevertheless,  we  have 
found  evidence  of  biophysical  transformations  which  are  common  to  both  mollusc 
and  mammal.  An  identified  group  of  neurons,  the  CA1  cells  (rather  than  individual 


5    LMCN/DIR 


identified  neurons)  was  shown  to  have  a  distribution  of  conditioning-specific 
modification  of  K  +  channels  within  hippocampal  slices  removed  from  rabbits  on 
days  after  they  had  been  conditioned.  Such  slices  provide  vastly  greater  amounts  of 
tissue  (than  does  the  snail)  for  biochemical  studies.  Indeed,  we  have  already 
observed  conditioning-specific  translocation  of  protein  kinase  C  (PKC)  not  only  in  the 
hippocampus  CA1  neurons,  but  also  in  a  restricted  region  of  the  cerebellar  cortex 
called  "H6".  PKC  regulation  of  identical  K+  channels  occurs  in  both  Hermissenda 
and  hippocampal  neurons.  In  Hermissenda  this  regulation  is  being  investigated  with 
isolated  membrane  patches  whose  intracellular  surfaces  are  accessible  to  precise 
ionic  and  biochemical  manipulations.  The  distribution  of  PKC-mediated  neural 
changes  with  associative  learning  in  large  neuronal  arrays  is  currently  being 
analyzed  autoradiographically.  Within  the  past  year,  image  analyses  have  revealed 
changes  within  neuronal  populations  of  conditioned  but  not  control  brain  areas. 
Also  recently,  PKC  regulation  has  been  linked  to  conditioning-specific  modification 
of  Hermissenda  mRNA  metabolism.  This  molecular  storage  step  occurs  within  a 
specific  temporal  window  during  the  retention  period  of  the  associative  memory. 
Convergence  of  CS  and  UCS  pathways  activated  during  associative  conditioning  of 
the  nudfibranch  mollusc  Hermissenda  involves  inhibition  of  type  B  photoreceptors  by 
hair  cells  caudally  located  in  statocysts.  Recently  we  demonstrated  that  this  GABA- 
mediated  inhibition  is  entirely  transformed  into  excitation  when  stimulation  of  the 
visual  pathway  is  precisely  timed  in  relation  to  stimulation  of  the  vestibular  pathway. 
The  pairing-specificity  of  this  vestibular-visual  synapse  has  important  implications 
not  only  acquisition  for  associative  memory  in  the  snail  Hermissenda  but  for 
mammalian  systems  as  well.  Furthermore,  theoretical  constructs  formulated  on  the 
basis  of  this  unique  synaptic  function  have  been  extremely  successful  in  improving 
the  design  of  artificial  learning  networks.  Conditioning-specific  changes  of 
particular  proteins  have  been  related  to  mRNA  changes  during  the  last  year.  The 
functional  roles  of  these  proteins  as  well  as  their  identitiesare  now  being  explored. 
One,  for  example,  has  been  linked  to  GTP-binding  protein  signal  transduction. 
Another  may  have  more  importance  for  cell  structure.  These  learning-induced 
changes  of  protein  availability  may  represent  an  important  step  for  consolidating, 
i.e.,  making  more  permanent  the  physiologic  memory  trace  which  could  have 
expression  in  conditioning-specific  structural  changes  of  Hermissenda  neurons. 
These  latter  changes,  as  studies  with  the  formation  of  Hermissenda  associations, 
involve  an  apparent  reorganization  of  the  cell's  terminal  branches  on  which  synaptic 
interactions  occur. 

The  experimental  psychology  program  of  the  Section  uses  associative  learning 
paradigms  to  produce  persistent  behavioral  changes  in  the  nudibranch  mollusc 
Hermissenda  crassicomis  as  well  as  vertebrate  such  as  rabbits  and  rats.  Quantitative 
assessments  are  made  of  the  animals'  responses  to  the  conditioned  and 
unconditioned  stimuli  before  and  after  classical  conditioning  paradigms.  These 
assessments  include  precise  dissection  of  generalized  behavioral  transformations 
into  modification  of  individual  muscular  components  of  the  behaviors.  A  full  range 
of  psychological  manipulations  has  been  used  to  clearly  establish  the  sensitivity  of 
the  learning  behavior  to  the  exact  temporal  relationship  of  the  stimuli  which  are 
associated  during  learning  acquisition.  Also  of  interest  to  psychologists  is  the  close 
linkage  of  the  learning  behaviortothe  specific  stimuli  associated  and  discriminative 
functions  involving  those  stimuli  not  associated.  Recently  this  aspect  of  the  program 
has  been  extended  to  include  more  cognitively  oriented  learning  tasks  involving 
spatial  mapping  of  an  organism's  environment. 

The  Section's  neurophysiology  program  is  concerned  first  with  the  definition  of 
those  neural  systems  relevant  to  the  learning  capability.  Multiple  intracellular 


6    LMCN/DIR 


recordings  from  pre-  and  postsynaptic  neurons  have  been  employed  within  the 
visual,  vestibular  and  chemosensory  pathways  of  Hermissenda  to  establish  a  working 
knowledge  of  the  critical  neural  systems  and  to  describe  how  information  flows  in  a 
stepwise  fashion  beginning  with  sensory  cells  at  the  output.  A  similar  approach  is 
being  taken  with  the  rabbit  hippocampus,  and  more  recently  the  cerebellum,  and 
critical  afferent  and  efferent  pathways  within  these  structures.  Neurophysiological 
correlates  are  then  obtained  for  conditioned  (as  well  as  a  variety  of  control)  animals. 
These  neurophysiological  correlates  are  recorded  in  intact  animals,  isolated  nervous 
systems,  and  isolated  neuronal  membranes.  Based  on  such  correlates, 
electrophysiological  sequences  are  constructed  to  trace  the  transformation  of  the 
information  in  electrical  terms  of  the  neural  system. 

The  Section's  biophysics  program  measures  persistent  modification  of  specific  ionic 
channels  during  and  following  the  learning.  In  the  past,  a  two  microelectrode 
voltage  clamp  was  employed  to  characterize  genetically  specified  membrane 
currents  within  identified  neurons  which  were  demonstrated  to  play  a  causal  role  in 
the  acquisition  and  retention  of  associative  learning.  More  recently  the  patch-clamp 
technique  has  been  used  in  both  the  cell-attached  and  "inside-out"  configurations 
to  determine  which  subcellular  biochemical  processes  (e.g.,  Ca2  +  -dependent 
phosphorylation)  are  critical  for  regulating  those  ionic  channels  which  change 
during  learning.  All  these  biophysical  approaches  have  also  been  applied  to 
demonstrate  unequivocally  that  it  is  persistent  modification  of  specific  ionic 
channels  which  encode  a  learned  association  for  later  recall. 

The  biochemistry  research  of  the  Section  seeks  to  uncover  the  molecular  basis  for  the 
persistent  ionic  channel  modifications  shown  to  underlie  associative  learning  (both 
in  Hermissenda  and  the  rabbit).  A  variety  of  biochemical  and  molecular  biological 
methods  are  being  used  for  this  purpose.  Microgel  analysis  of  phosphorylation  of 
individual  neuronal  proteins,  for  example,  has  revealed  that  Ca2  +  -dependent 
phosphorylation  of  a  specific  low-molecular  weight  protein  changes  within  certain 
neurons  of  conditioned  animals  but  not  those  exposed  to  control  paradigms. 
Exposure  of  neurons  to  prolonged  depolarization,  which  simulated  the  integrated 
visual-vestibular  network  effects  on  identified  neurons  during  conditioning,  is  also 
followed  by  long-lasting  phosphorylation  differences  for  particular  low-molecular 
weight  proteins.  More  recently,  learning-induced  differences  in  the  amount  and/or 
synthesis  of  these  and  other  proteins  have  been  detected.  Recently,  it  has  been 
possible  to  isolate  specific  proteins  and  inject  them  into  individual  neurons,  thereby 
reproducing  the  actual  learning  effects  on  membrane  channels.  Furthermore,  a 
number  of  intracellular  manipulations  have  supported  the  hypothesis  that  learning- 
induced  modification  of  ionic  channels  involves  Ca2  +  -calmodulin-dependent  and 
Ca2  +  -lipid-dependent  phosphorylation.  Such  manipulations  include  iontophoretic 
injection  of  Ca2  +  -calmodulin-dependent  protein  kinase,  inositol  trisphosphate  or 
PKC,  or  preincubation  with  PKC  activators  such  as  phorbol  esters  or  OAG.  Modern 
molecular  biological  techniques  are  enabling  the  Section  to  use  monoclonal 
antibodies  to  enzymes  implicated  in  the  learning  process.  Other  antibodies  may 
also  be  helpful  to  reconstruct  of  the  biochemical  and  associated  biophysical 
sequences  which  make  biological  records  of  memory  possible.  Recombinant 
techniques  may  be  particularly  helpful  in  identifying  specific  proteins  whose 
synthesis  is  modified  as  a  result  of  the  recently  observed  conditioning-specific 
increase  in  mRNA  turnover. 

The  cellular  anatomy  aspects  of  the  Section's  programs  contributes  in  several  ways  to 
the  various  levels  of  inquiry  into  the  learning  process  already  mentioned. 
Ultrastructural  measurements  of  the  cells  and  their  synaptic  interaction  have 


7    LMCN/DIR 


provided  further  definition  of  the  relevant  neural  systems.  Activity-dependent 
uptake  of  radioactive  labels  within  these  systems  has  been  monitored  by 
autoradiographic  methods.  Morphometric  techniques  together  with  serial 
sectioning  and  computerized  reconstruction,  have  recently  uncovered  structural 
manifestations  of  the  biophysical  and  biochemical  changes  already  shown  for 
neurons  within  conditioned  but  not  control  animals.  Differential  absorption 
spectrophotometry  allows  intracellular  localization  of  fluctuations  of  cytosolic  Ca2  +  - 
dependent  modulation  of  the  channelsduring  learning. 

Finally,  the  developmental  biologists  within  the  Section  have  established  laboratory 
strains  of  Hermissenda.  Such  strains  permit  assessment  of  how  genetic  and 
environmental  factors  may  interact  to  determine  individual  differences  in  the  ability 
of  the  animals  to  undergo  associative  learning.  Furthermore,  critical  biochemical 
steps  have  now  been  identified  which  may  serve  potent  cell-transforming  functions 
in  learning,  developmental,  and  oncogenic  contexts. 

Perhaps  most  important  in  all  these  efforts  is  the  accumulated  evidence  that  a 
remarkable  similarity  exists  between  means  of  encoding  learned  associations  in  the 
snail,  rabbit,  and  now  the  rat.  The  same  learning-induced  reduction  of  well- 
characterized  K  +  currents  has  been  found  to  provide  such  encoding  in  Hermissenda 
as  it  does  within  identified  neurons  of  rabbit  hippocampal  slices.  Similar  regulation 
of  these  channels  appears  to  occur  atthe  molecular  level  for  both  the  mollusc, 
rabbit,  and  rat.  Such  parallel  mechanisms  may  ultimately  provide  the  basis  for 
clinical  intervention  and  thereby  the  amelioration  of  pathologic  symptoms. 

Section  on  Myelin  and  Brain  Development 

Research  in  the  Section  on  Myelin  and  Brain  Development  is  divided  into  three 
related  projects.  The  first  project,  entitled  "Glycoproteins  of  Myelin  in  Development 
and  Disease,"  has  been  existed  for  many  years  and  emphasizes  the  myelin-associated 
glycoprotein  (MAG).  However,  other  myelin  proteins  and  lipids  are  also  studied  with 
the  ultimate  objective  of  understanding  molecular  mechanisms  of  myelin  formation 
and  breakdown.  The  second  project,  entitled  "Antibodies  to  Glycoconjugates  in 
Neurological  Diseases"  grew  out  of  the  first  as  a  consequence  of  the  discovery  that 
monoclonal  IgM  antibodies  to  MAG  in  patients  with  neuropathy  cross  reacted  with 
other  glycoproteins  and  glycolipids.  This  research  emphasizes  the  occurrence  and 
pathogenic  significance  of  antibodies  to  MAG  and  to  sphingoglycolipids  in  patients 
with  neurological  diseases,  especially  peripheral  neuropathies.  Very  recently,  when 
it  became  apparent  that  a  significant  aspect  of  the  neuropathology  in  acquired 
immunodeficiency  syndrome  (AIDS)  involves  white  matter,  a  third  area  of  research 
was  undertaken  to  elucidate  molecular  mechanisms  involved  in  the  neurological 
aspects  of  this  disease.  This  new  project  is  entitled  "Molecular  and  Immunological 
Aspects  of  Myelin  Abnormalities  in  Neuro-AIDS". 

1.  Structure  and  function  of  the  myelin-associated  glycoprotein  (MAG).  The 
localization  of  MAG  in  periaxonal  Schwann  cell  and  oligodendroglial  membranes  of 
myelin  sheaths,  its  membership  in  the  immunoglobulin  (Ig)  superfamily  together 
with  other  neural  adhesion  molecules  and  experiments  directly  demonstrating  its 
capacity  to  promote  cell-cell  interactions  all  suggest  that  this  glycoprotein  functions 
in  the  interaction  of  myelin-forming  cells  with  the  axolemma.  MAG  is  also  present  in 
Schmidt-Lanterman  incisures,  lateral  loops  and  the  outer  mesaxons  of  peripheral 
nervous  system  (PNS)  myelin  sheaths  where  it  appears  to  play  a  role  in  maintaining 
the  spacing  of  adjacent  Schwann  cell  membranes  in  these  structures  where 


8    LMCN/DIR 


extracellular  membrane  surfaces  are  separated  by  12-14  nm  and  there  is  always 
cytoplasm  at  the  inner  membrane  surface.  In  addition,  during  active  myelination, 
oligodendrocytes  contain  numerous  multivesicular  bodies  (M VBs)  that  are  enriched 
in  MAG.  MVBs  are  known  to  be  endocytosed  structures  in  other  cells,  and  this  raises 
that  possibility  that  these  MAG-  enriched  bodies  are  carrying  a  signal  from  the 
periaxonal  regions  back  to  the  oligodendrocyte  cell  bodies. 

The  myelin-associated  glycoprotein  has  a  single  membrane  spanning  domain 
separating  its  C-terminal  tail  from  a  heavily  glycosylated  N-terminus  that  is 
composedof  5  domains  related  in  amino  acid  sequence  to  each  other  and  to 
members  of  the  immunoglobulin  superfamily  such  as  neural  cell  adhesion  molecule 
(N-CAM).  These  extracellular  immunoglobulin-like  domains  must  mediate  the 
function  of  MAG  in  glia-axon  interactions,  possibly  by  specifically  interacting  with 
another  member  of  the  superfamily  on  the  axolemma.  Homophilic  MAG  binding 
may  be  involved  in  the  interactions  of  adjacent  Schwann  cell  membranes  in  incisures, 
lateral  loops,  and  mesaxons.  An  important  factor  that  has  become  apparent  in  the 
past  few  years  is  that  the  functional  binding  properties  possessed  by  the  native  MAG 
molecule  may  be  lost  when  the  glycoprotein  is  purified  by  conventional  methods 
involving  strong  surface  active  agents  such  as  lithium  diiodosalicylate.  Therefore, 
experiments  are  underway  to  isolate  native  MAG  for  functional  studies  by  using 
milder  detergents. 

MAG  has  8  extracellular  sites  for  N-linked  glycosylation,  and  detailed  investigation 
of  the  oligosaccharides  are  underway  utilizing  the  Dionex  BioLC  Carbohydrate 
System  with  an  amperometric  detector  that  is  capable  of  quantifying  low  picomole 
amounts  of  carbohydrate.  Adult  rat  brain  MAG  contain  a  heterogeneus  mixture  of 
complex  bi-,  tri-  and  tetraantennary  oligosaccharides,  many  of  which  are  highly 
negatively  charged  due  to  sialic  acid  moieties  and  sulfate  groups.  Differences  in 
oligosaccharide  structure  may  modulate  the  functioning  of  MAG  in  membrane- 
membrane  interactions.  MAG  is  abnormally  glycosylated  in  the  dysmyelinating 
quaking  mutant,  and  this  may  lead  to  some  of  the  abnormalities  of  myelin  structure 
in  this  mutant. 

MAG  occurs  in  two  developmental^  regulated  isoforms  differing  in  the  lengths  of 
their  C-terminal  domains  and  arising  by  alternative  splicing  of  the  primary  mRNA 
transcript.  The  larger  isoform  (L-MAG)  is  most  prominent  at  the  time  of  active 
myelination  in  rodent  brain,  whereas  nearly  all  the  MAG  in  rodent  peripheral  nerve 
is  the  shorter  isoform  (S-MAG)  at  all  ages.  Both  CNS  and  PNS  MAG  are 
phosphorylated  on  their  cytoplasmic  domains,  but  the  sites  of  phosphorylation  are 
different.  Oligodendrocytes  phosphorylate  MAG  on  serine,  threonine  and  tyrosine 
residues  in  L-MAG,  whereas  Schwann  cells  phosphorylate  MAG  primarily  on  serine 
residues  of  S-MAG.  This  may  be  relevant  to  cellular  differences  in  the  process  of 
myelination  in  the  CNS  and  PNS,  respectively.  Regulation  of  expression  of  the  two 
forms  of  MAG  as  well  as  phosphorylation  of  the  cytoplasmic  domains  may  modulate 
interactions  with  cytoskeletal  components.  Overall,  the  MAG  molecule  seems  well- 
suited  to  mediate  interactions  between  intracellular  cytoskeletal  elements  and 
adjacent  extracellular  membrane  surfaces.  In  this  manner,  MAG  could  play  a  key 
role  in  transmitting  the  chemomechanical  forces  involved  in  generating  spiraled 
myelin  sheaths. 

An  investigation  of  MAG  in  cultured  oligodendrocytes  and  Schwann  cells  is  under 
way  with  the  ultimate  objective  of  determining  factors  that  control  its  expression 
and  probing  its  function  as  an  adhesion  molecule.  Cultured  oligodendrocytes 
constitutively  express  MAG  and  other  myelin  proteins.  Recent  experiments  show 


9    LMCN/DIR 


that  cultured  oligodendrocytes  isolated  from  myelinated  bovine  brain  appear  to 
express  more  MAG  than  those  enriched  from  primary  cultures  of  embryonic  rat 
brain,  and  therefore  appear  to  provide  the  preferable  system  for  studies  on  the  cell 
biology  of  MAG.  Cultured  Schwann  cells  do  not  constitutively  express  MAG,  but  can 
be  induced  to  express  a  small  amount  by  elevating  cAMP  levels  with  forskolin. 
Recently,  an  immortalized  line  of  Schwann  cells  (S-1 6)  has  been  generated  by 
multiple  passaging  that  expresses  a  remarkably  high  level  of  MAG,  similar  to  that  in 
adult  sciatic  nerve.  These  cells  appear  to  resemble  Schwann  cells  early  in  the  process 
of  myelination,  since  they  are  also  galactocerebroside  positive,  but  express  little  or 
none  of  the  major  myelin  proteins,  PO  glycoprotein  or  myelin  basic  protein.  The 
MAG  expressed  in  the  S-1 6  cells  resembles  that  in  vivo  since  it  is  primarily  the  shorter 
isoform,  is  glycosylated  to  about  the  same  extent  and  is  both  sulfated  and 
phosphorylated  .  Most  of  the  MAG  expressed  by  the  cells  is  on  the  surface,  and  it  is 
expected  that  these  immortalized  Schwann  cells  will  be  useful  for  studying  the  cell 
biology  of  MAG  and  its  function  in  cell-cell  interactions. 

2  MAG  in  multiple  sclerosis  (MS)   Previous  studies  have  demonstrated  a  greater  loss 
of  MAG  than  other  myelin  proteins  at  the  periphery  of  MS  plaques,  suggesting  an 
important  role  for  MAG  in  the  molecular  pathology  of  this  disease.  Currently,  our 
favored  hypothesis  to  explain  the  selective  loss  of  MAG  involves  its  high 
susceptibility  to  a  myelin-associated,  calcium  -activated,  neutral  protease.  This 
enzyme  converts  the  glycoprotein  to  a  partially  soluble,  lower  Mr  weight  derivative, 
dMAG,  that  is  missing  tne  C -terminus.  Experiments  are  planned  to  further  define 
the  reason  for  the  elevated  activity  of  this  enzyme  in  tissue  from  MS  patients. 

3.  Antibodies  to  qlycolipids  in  peripheral  neuropathies  This  area  of  investigation 
began  with  the  demonstration  of  monoclonal  anti-MAG  antibodies  in  patients  with 
polyneuropathies  occuring  in  association  with  IgM  gammopathy.  It  was 
subsequently  demonstrated  that  these  anti-MAG  antibodies  cross-reacted  with  the 
newly  discovered  sphingoglycolipid,  sulfate-3-glucuronyl  paragloboside  (SGPG). 
Further  studies  showed  the  monoclonal  IgM  antibodies  in  other  neuropathy  patients 
did  not  react  with  MAG  and  SGPG,  but  that  they  did  react  with  various  ganglioside 
antigens.  Patients  with  motor  neuropathies  and  monoclonal  antibodies  to  GM 1 
ganglioside  have  been  of  particular  interest  in  recent  years.  Overall,  about  80%  of 
patients  with  neuropathy  occurring  in  association  with  IgM  paraproteinemia  have  a 
monoclonal  antibody  that  reacts  with  SGPG  or  a  ganglioside  antigen,  suggesting 
that  glycolipids  may  be  important  target  antigens  in  this  type  of  neuropathy.  An 
extension  of  the  research  on  monoclonal  gammopathies  has  been  the 
demonstration  that  some  patients  with  neuropathy  in  the  absence  of  a  monoclonal 
antibody  have  polyclonal  antiganglioside  antibodies.  For  example,  10  to  20%  of 
patients  with  Guillain-Barre  syndrome  have  high  levels  of  anti-ganglioside 
antibodies  that  may  play  a  pathogenic  role.  Also,  high  levels  of  polyclonal 
antibodies  reacting  with  GM1  ganglioside  in  some  patients  with  primarily  motor 
neuropathies  have  been  of  special  interest. 

Recent  research  has  revealed  differences  in  the  fine  specificities  of  both  the  anti- 
MAG  antibodies  in  the  paraproteinemic  neuropathies  and  the  anti-GM1  antibodies 
in  association  with  motor  neuropathies.  For  example,  experiments  with  chemically 
modified  derivatives  of  SGPG  revealed  that  some  of  the  MAG/SGPG  antibodies 
absolutely  require  the  sulfate  for  reactivity,  others  require  the  carboxyl  group  of 
glucuronic  acid,  whereas  still  others  will  react  as  long  as  one  or  the  other  of  the  two 
negatively  charged  groups  is  present.  The  fine  specificities  of  anti-GMI  antibodies  in 
patients  with  motor  neuropathy  also  vary  as  revealed  by  differing  cross-reactivity 
with  other  gangliosides.  However,  since  GM1  is  the  only  common  antigen  in  all  the 


10    LMCN/DIR 


patients  examined  by  us,  the  results  suggest  that  it  could  be  the  pathogenically  most 
significant  target  antigen  in  motor  nerves.  Nevertheless,  differences  in  fine 
specificity  and  the  spectrum  of  cross-reacting  neural  antigens  could  affect  the  clinical 
presentation  in  these  patients.  It  should  be  emphasized  that  our  research  and  that 
of  others  indicates  that  serum  antibodies  occurring  in  association  with  neuropathy 
should  be  analyzed  both  qualitatively  by  TLC-overlay  and  quantitatively  by  ELISA, 
since  their  pathogenic  significance  appears  to  depend  both  on  their  specificity  and 
titer.  Although  anti-MAG/SGPG  antibodies  have  been  demonstrated  to  be  capable 
of  causing  neurological  disease,  an  important  priority  for  the  future  is  to  evaluate 
the  capacity  of  anti-ganglioside  antibodies  to  induce  neural  damage  or  interfere 
with  normal  function  in  appropriate  animal  models  or  tissue  culture  systems. 

4.  Biochemical  neuropathology  in  acquired  immunodeficiency  syndrome  (AIDS). 
Relevant  areas  of  our  research  described  in  the  preceding  section  are  being 
extended  in  an  attempt  to  determine  the  molecular  basis  for  some  of  the 
abnormalities  of  myelin  occurring  in  neuro-AIDS  including  myelin  pallor,  vacuolar 
myelopathy  and  multifocal  demyelination  in  the  CNS  as  well  asdemyelinating 
peripheral  neuropathy.  This  research  has  just  begun  and  results  are  very  preliminary. 
Specific  aspects  of  the  research  include  the  following:  1)  Postmortem  CNS  tissue 
from  AIDS  patients  is  being  analyzed  for  quantitative  and  qualitative  alterations  of 
myelin  proteins.  2)  Since  the  myelin-associated  glycoprotein  (MAG)  and  the  CD4 
receptor  for  HIV  are  both  in  the  immunoglobulin  superfamily  and  have  a  modest, 
but  statistically  significant,  sequence  homology,  possible  interactions  between  MAG 
and  GP1 20  are  being  tested.  3)  Sera  from  AIDS  patients  with  neuropathy  are  being 
tested  for  antibodies  to  spingoglycolipids  as  have  been  observed  in  other  types  of 
neuropathy. 

Receptor  Biochemistry  and  Molecular  Biology  Section 

The  Section  of  Receptor  Biochemistry  and  Molecular  Biology  conducts  research  on 

?iene  super  families  of  neurotransmitter  receptors,  including  receptor  structure, 
unction  and  evolution.  The  Section  has  major  programs  underway  on  genomic 
structure  and  evolution  and  on  the  the  genetic  basis  of  neurological  diseases.  New 
personnel  have  been  recruited  to  the  Section  in  a  number  of  important  areas.  Dr. 
Mark  Dubnick,  a  molecular  biologist  and  computer  programmer,  joined  the 
laboratory  in  January  1990  as  a  Staff  Fellow.  He  has  already  made  significant 
contributions  along  with  Senior  Staff  Fellow  Dr.  Anthony  Kerlavage,  Chief,  Unit  on 
Molecular  Structure  and  Evolution,  and  John  Powell  of  DCRT,  in  the  area  of  data 
handling  and  analysis.  They  have  established  a  computer  network  consisting  of  six 
IBM,  fifteen  Macintosh,  two  Sun  4  and  two  Silicon  Graphics  workstations  in  the 
laboratory  and  networked  these  machines  to  the  NIH  Convex,  Cray  and  other 
computers  essential  for  the  analysis  of  large  genomic  sequences;  and  have 
developed  software  to  facilitate  data  analysis.  Dr.  Mark  Adams  joined  the 
laboratory  from  the  University  of  Michigan  where  he  recently  completed  his  Ph.D.  in 
biochemistry.  Dr.  Adams  is  the  lead  scientist  on  a  major  new  project  in  the 
laboratory  to  isolate  and  sequence  all  the  genes  expressed  in  the  human  brain  (see 
below).  Working  with  Dr.  Adams  is  another  recruit  to  the  laboratory,  Dr.  Bjorn  Olde, 
a  recent  graduate  of  University  of  Stockholm.  Dr.  Antonia  Martin  was  recruited 
from  Praxis  Biologies  to  head  up  a  new  unit  established  on  gene  therapy.  Dr.  Martin 
has  lead  a  team  at  NIH,  in  collaboration  with  Dr.  Francis  Collins  of  the  University  of 
Michigan,  to  sequence  several  exons  of  the  neurofibromatosis  gene  from 
chromosome  17,  and  is  working  on  the  genomic  structure  of  the  alpha-3  subunit  of 
the  GABA  receptor  mapped  to  Xq28  and  the  mytonic  dystrophy  region  of 
chromosome  19  (see  below). 


11    LMCN/DIR 


Dr.  W.  Richard  McCombie,  Chief,  Unit  on  Neurological  Disease  Gene  Structure  and 
Function,  is  the  lead  scientist  on  sequencing  three  cosmid  clones  (40kb  each)  isolated 
from  the  region  on  chromosome  4  where  the  Huntington's  disease  gene  has  been 
mapped.  The  cosmid  clones  were  provided  as  part  of  a  collaboration  with  the 
Huntington's  disease  collaborative  group.  Dr.  McCombie  has  also  established  a 
collaboration  with  Lev  Goldfarb  in  LCNSS  to  sequence  DNA  from  patients  with 
various  spongiform  encephalopathies,  in  search  of  point  mutations  associated  with 
these  disorders.  This  project  involves  sequencing  multiple  clones  from  each  of  43 
patients,  or  over  600,000  base  pairs  of  raw  sequence  data. 

Jeannine  Gocayne,  microbiologist,  has  integrated  the  new  robotics  added  to  the 
laboratory.  These  completely  automate  DNA  sequencing  reactions.  This  work  is  part 
of  a  CRADA  with  Applied  Biosystems,  Inc. 

The  laboratory  has  completed  over  2  million  bases  of  raw  DNA  sequence  and  over 
300  kb  of  finished  sequence  this  year,  including  the  following  projects.  Over  15  kb 
from  the  disease  gene  for  neurofibromatosis  I  (NF1)  have  been  sequenced, 
identifying  several  new  exons.  Determining  the  intron-exon  boundaries  will  allow 
us  to  develop  polymerase  chain  reaction  (PCR)  probes  to  sequence  exons  from  NF1 
patients  in  search  of  point  mutations  associated  with  disease  symptoms.  Three 
cosmids  from  the  Huntington's  region  of  chromosome  4  have  been  sequenced. 
These  cosmids  total  over  1 20  kb  of  finished  sequence  and  comprise  one  of  the  two 
largest  segments  of  human  chromosomal  DNA  sequenced  to  date.  The  sequence 
from  this  region  is  currently  being  analyzed.  The  second  largest  region  which  has 
been  sequenced  by  the  laboratory  in  collaboration  with  the  DOE  and  Applied 
Biosystems,  is  a  three  cosmid  contig  of  over  100  kbfrom  chromosome  19.  This  region 
is  where  a  number  of  DNA  repair  enzymes  and  the  gene  for  mytonic  dystrophy  have 
been  tentatively  mapped.  Lambda  and  cosmid  clones  have  been  obtained  from  the 
human  Xq28  region  which  contains  several  loci  of  neurological  significance 
including  the  alpha-3  subunit  of  the  GABA  receptor. 

Progress  on  the  project  to  characterize  the  gene  and  protein  structure  and  evolution 
of  neurotransmitter  receptors  belonging  to  two  major  multigene  families  continues. 
The  first  gene  family  contains  adrenergic,  muscarinic,  opsin,  serotonin  and 
octopamine  receptors;  the  nicotinic  cholinergic,  GABA/benzodiazepine,  NMDA,  and 
glycine  receptors.  To  data  we  have  cloned  and  sequenced  a  significant  number  of 
receptor  genes  from  both  multigene  families.  These  include  several  alpha  and  beta- 
adrenergic  receptors  from  human  and  rat  cDNA  and  genomic  libraries;  muscarinic 
cholinergic  receptors  from  human,  rat,  and  Drosoph'na  genomic  libraries; 
octopamine  receptors  from  Drosophila;  human  alpha  and  beta  subunitsof  the 
GABA/benzodiazepine  receptor;  and  nicotinic  receptors  from  locust  and  C.  elegans. 
For  example,  the  human  GABA  beta-1  subunit  has  been  cloned,  sequenced  and 
localized  to  chromosome  4.  This  gene  is  over  65  kb  and  comprises  9  exons.  The 
exons  and  splice  sites  are  highly  conserved  in  other  GABA  receptor  subunits  from 
humans  and  lower  species.  Permanent  cell  lines  expressing  the  unique 
neurotransmitter  receptor  proteins  provide  key  new  information  concerning  the 
mechanism  of  receptor  activation  by  neurotransmitters.  Extensive  site  directed 
mutagenesis  studying  on  adrenergic  and  muscarinic  cholinergic  receptors  have 
identified  a  number  of  conserved  amino  acid  residues  essential  for  ligand  binding 
and  receptor  activation. 

Another  major  new  project  within  the  laboratory  has  the  goal  of  isolating, 
sequencing  and  characterizing  all  genes  expressed  in  the  human  brain.  As  many  as 


12    LMCN/DIR 


half  of  the  over  50,000  human  genes  are  believed  to  be  expressed  in  the  brain. 
While  sequencing  the  human  genome  is  expected  to  take  over  1 5-20  years, 
sequencing  a  large  number  of  cDNA  clones  can  readily  provide  coding  sequence  data 
on  genes  expressed  in  tissues.  We  are  building  a  large  library  of  clones  and 
sequences  of  human  brain  cDNAs.  Sequence  data  will  be  obtained  on  1000  human 
brain  clones  in  the  first  3-4  months  of  this  project.  Over  half  the  genes  isolated  are 
new  ones,  not  previously  identified  or  cloned.  An  additional  percentage  are  new 
human  genes,  having  been  previously  isolated  from  other  species.  Computer 
analysis  of  the  protein  sequences  at  primary  and  secondary  structural  levels  is 
assisting  in  clone  identification.  DNAand  predicted  protein  sequences  will  be 
examined  for  the  presence  of  conserved  primary  and  secondary  structure  motifs  and 
relationships  to  previously  sequenced  genes.  Further  characterization  of  potentially 
interesting  clones  will  include  chromosome  localization,  examination  of  tissue 
distribution  of  expression,  and  evolutionary  conservation.  The  availability  of  a 
broad-based  library  of  cDNA  sequences  will  also  facilitate  identification  of  coding 
regions  in  genomic  sequences  as  well  as  provide  a  starting  point  for  individual 
cloning  projects.  A  variety  of  approaches  are  being  used  to  select  brain-specific 
clones  and  to  eliminate  highly  represented  sequences. 

The  Section  of  Receptor  Biochemistry  and  Molecular  Biology  also  runs  the  NINDS 
DNA  facility.  This  facility  synthesizes  oligonucleotides  for  institute  laboratories  and 
branches  and  performs  DNA  sequencing.  During  FY  1990  the  DNA  synthesis  facility 
under  the  direction  of  Mike  FitzGerald  will  have  synthesized  over  900 
oligonucleotides  for  eight  institute  laboratories  and  branches.  The  DNA  facility  has 
also  sequenced  over  50,000  bases  for  other  laboratories. 


13    LMCN/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01NS02784-02LMCN 


PERIOD  COVERED 

October  1, 1990  through  September  31,  1990 


TITLE  OF  PROJECT  (aOchtrtcUrtorlta.  Tnlt  mull  tn  on  sot  lint  Minan  Hit  boiOtrt ) 

investigation  of  the  Structure  and  Function  of  Gonadotropins  and  their  Receptors 


PRINCIPAL  INVESTIGATOR  (tol  olhw  ontmtueotl  pmHonntl btlew  Uw  fruiaptl  kiwiiiuttoi )  (Htm;  till*.  Itbotttary,  tnd  instituf  tttilmion) 

PI:  R.  V.  Rebois,  Ph.D.  Head,  Unit  on  Receptor  Structure  and  Function        LMCN,  NINDS 

OTHERS:      S.  Okuya,  M.D..  Ph.D.     Visiting  Associate  LMCN,  NINDS 

V.  J.  B.  Reddy,  Ph.D.       Visiting  Fellow  LMCN,  NINDS 


COOPERATING  UNITS  (itt*,) 


LAB/BRANCH 

Laboratory  of  Molecular  and  Cellular  Neurobiology,  BNP 


SECTION 

Membrane  Biochemistry  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Park  Building,  Room  408,  Bethesda,  Maryland   20892 


TOTAL  MAN-VEARS: 


2.5 


PROFESSIONAL: 


25 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

I      I    (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


GO  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  sundsrd  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  carbohydrate  moieties  of  glycopeptide  hormone  human  chorionic  gonadotropin  (hCG)  have  sialic 
acid  (NeuAc)  residues  which  are  required  for  full  biological  activity.  hCG  stimulates  adenylate  cyclase  in 
murine  Leydig  tumor  MLTC-1  cells,  and  we  used  this  system  as  a  model  to  study  the  role  of  NeuAc  in  the 
biological  activity  of  hCG.  Removing  NeuAc  produced  asialo-hCG  which  had  only  50%  of  the  biological 
activity  of  hCG.  The  NeuAct  was  replaced  with  a  modified  form  of  NeuAc  or  with  the  carbohydrate 
moiety  of  the  ganglioside  Gmi  which  also  contains  NeuAc.  Both  derivatives  had  the  same  activity  as  the 
asialo-hCG  indicating  the  importance  of  both  position  and  structure  of  NeuAc  for  biological  activity.  2. 
The  drug  forskolin  activates  adenylate  cyclase,  but  we  found  that  different  cells  varied  in  their  response 
to  the  drug  as  measured  by  cyclic  AMP  accumulation.  Cyclic  AMP  accumulation  in  cells  with  a  strong 
response  to  forskolin  was  blocked  by  activating  the  inhibitory  G  protein  (G,),  with  fluoroaluminate. 
Forskolin  potentiated  cyclic  AMP  accumulation  stimulated  by  hormones  that  activate  the  stimulatory  G 
protein  (Gj),  even  in  cells  the  responded  poorly  to  forskolin  alone.  Activating  G,  with  fluoroaluminate 
prevented  the  hormone  response,  as  well  as  its  potentiation  by  forskolin.  Forskolin  stimulated  adenylate 
cyclase  activity  in  membranes,  even  if  the  membranes  were  prepared  from  cells  that  responded  poorly  to 
the  drug.  Activating  G,  with  GTP  analogues  inhibited  forskolin-stimulated  adenylate  cyclase  activity  in 
membranes.  The  results  indicate  that  interactions  between  Gs  G„  and  the  catalytic  subunit  of  adenylate 
cyclase  affect  the  ability  of  forskolin  to  stimulate  cyclic  AMP  accumulation.  The  observation  that  some 
cells  responded  well  to  forskolin  and  other  responded  poorly  suggests  that  these  interactions  are  not  the 
same  in  all  cell  types.  Furthermore,  differences  in  the  response  of  cells  and  their  membranes  to  forskolin 
suggest  that  changes  in  these  interactions  occur  during  the  preparation  of  membranes. 


14a-LMCN/DIR 


'HStMSIfto  I'M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01NS02366-12LMCN 


PERIOD  COVERED 

October  1 ,  1 989  through  September  31,1 990 


TITLE  OF  PROJECT  fwchmctwtartau  hti»  i»«»i  tit  an  on»  iu>*  Mm**  iht  txxant.) 

Regulation  of  Hormone-Responsive  Adenylate  Cyclase 


PRINCIPAL  \NV  ISHG  AT  OR  tun  olhw  *nt*ui»*tl  ftunnml  *»lem  th»  tiituittl  IniHHigttoi.)  (Mam*,  into.  Itkoftat/.  tn4  inmtufttilHtion) 


PI: 

P.  H.  Fishman,  Ph.D. 

Chief,  Membrane  Biochemistry  Section 

LMCN,  NINDS 

OTHERS: 

A.  Sidhu.Ph.D. 

Biochemist  (IPA) 

LMCN,  NINDS 

E.A.Gordon,  Ph.D. 

IRTA  Fellow 

LMCN,  NINDS 

X.-M.  Zhou,  M.D..  Ph.D. 

Visiting  Fellow 

LMCN,  NINDS 

M.  Lee 

Special  Volunteer 

LMCN,  NINDS 

T.L.  Miller 

Biologist 

LMCN,  NINDS 

COOPERATING  UNITS  fi'myj 


LAB/BRANCH 

Laboratory  of  Molecular  and  Cellular  Neurobiology 


SECTION 

Membrane  Biochemistry  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Park  Building,  Room  411,  Bethesda,  Maryland   20892 


TOTAL  MAN-YEARS: 


4.3 


PROFESSIONAL: 


3.3 


OTHER: 


1.0 


CHECK  APPROPRIATE  BOX(ES) 

I      I   (a)  Human  subjects 
]  (a1)  Minors 

J   (a2)  Interviews 


f"x~1  (b)  Human  tissues  ]  (c)  Neither 


SUMMARY  OF  WORK  (Ut*  lUniUrd  unnduttf  typ«.  Do  not  M(Nd  !/)•  ijmc*  provldtd.) 

Neuropeptide  Y  (NPY)  binds  to  Y  ^ -subtype  receptors  on  MC-IXC  cells  and  to  Y2  receptors  on  SMS-MSN, 
two  human  neuroblastoma  cell  lines.  NPY  inhibits  adenylate  cyclase  in  MC-IXC  but  not  SMS-MSN  cells. 
NPY  binding  in  both  cell  lines  is  inhibited  by  pertussis  toxin,  which  ADP-ribosylates  41  kDalton  G  proteins 
in  both.  Western  blotting  with  specific  antibodies  to  Ga  subunits  showed  that  MC-IXC  has  Gia,  the 
inhibitory  G  protein  of  adenylate  cyclase  whereas  SMS-MSN  has  both  Gio  and  Goa,  which  has  been 
implicated  in  activation  of  ion  channels.  Thus,  Yt  receptors  appear  to  mediate  inhibition  of  adenylate 
cyclase  and  Y:,  activation  of  ion  channels.   In  contrast  to  other  0-adrenergic  responsive  cell  lines  which 
have  B?-adrenerqic  receptors  (02AR),  SK-N-MC  have  BjAR  and  undergo  an  unusual  desensitization  when 
exposed  to  agonists.  There  is  no  loss  of  SiAR  or  maximum  agonist-stimulated  adenylate  cyclase  activity 
but  a  shift  in  the  dose  response  which  can  be  mimicked  by  incubating  membranes  with  ATP  and  the 
catalytic  subunit  of  cyclic  AMP-dependent  protein  kinase  (PKA).  Desensitization  is  blocked  in  permeable 
cells  by  a  specific  inhibitor  of  PKA  but  not  an  inhibitor  of  BAR  kinase.  A  large-scale  purification  of  the 
dopamine  Pi  receptor  from  rat  striatum  has  been  carried  out  and  the  purified  receptor  is  being  used  to 
obtain  peptide  sequences,  antibodies,  and  eventually  the  receptor  gene.  The  latter  will  be  useful  for 
identifying  defects  in  the  receptor  that  occur  in  disease  states.  In  kidney,  Oy  receptors  coupled  to 
adenylate  cyclase  have  been  implicated  in  regulation  of  sodium  transport   Dopamine  fails  to  stimulate 
adenylate  cyclase  in  proximal  convoluted  ('joules  from  spontaneous  hypertensive  rats  although  the 
enzyme  responds  normally  to  other  effectors.  As  the  binding  activity  and  apparent  molecular  weight  of 
the  kidney  receptors  from  the  hypertensive  rats  are  normal,  it  appears  that  the  D  i  receptors  are  defective 
in  their  ability  to  couple  to  the  stimulatory  G  protein  of  adenylate  cyclase. 


15a-LMCN/DIR 


f  Hi  tiwu  man  I  Ml 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01NS01309-25LMCN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (aochtrtatn  01  iiu-  Till*  null  lit  on  on*  lm*  Mnui  th*  bo/0*i>.) 

Biosynthesis  and  Function  of  Glycosphingolipids  and  Other  Glycoconjugates 


PRINCIPAL  INVESTIGATOR  dm  oth*r  pronuUonsI p*nenn*l  a*low  th*  ftlnxla*! kiy*UhJ»toi.)  {Ntm*.  ml*.  Itoo/ttory.  tndlnsMul*  ttftlntlon) 


PI:  P.H.  Fishman,  Ph.D. 

OTHERS:  T.  Pacuszka,  Ph.D. 

D.R.  Critchley,  Ph.D. 

R.M.  Bradley 


Chief,  Membrane  Biochemistry  Section  LMCN,  NINDS 

Visiting  Scientist  LMCN,  NINDS 

Visiting  Scientist  LMCN,  NINDS 

Chemist  LMCN,  NINDS 


COOPERATING  UNITS  (it*n,) 


LAB/BRANCH 

Laboratory  of  Molecular  and  Cellular  Neurobiology 


SECTION 

Membrane  Biochemistry  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Park  Building,  Room  41 1,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.9 


PROFESSIONAL: 


1.3 


OTHER: 


0.6 


CHECK  APPROPRIATE  BOX(ES) 

I      I  (a)  Human  subjects 
(al)  Minors 

(a2)  Interviews 


I      I  (b)  Human  tissues  [T]  (c)  Neither 


SUMMARY  05  WORK  (Us*  sundsrd  unreduced  typ*.  Do  not  txifd  th*  ipse*  provided.) 

Ganqlioside  Gmi  is  the  cell  surface  receptor  for  cholera  toxin.  The  oligosaccharide  chain  of  Gmi  is 
recognized  by  the  B  or  binding  subunits  of  the  toxin  whereas  the  A  subunit  of  the  toxin  activates 
adenylate  cyclase  by  ADP-ribosylation  of  the  stimulatory  G  protein  of  the  cyclase  system.  Less  is  known 
about  the  role(s)  that  the  lipid  moiety  of  Gmi  plays  in  toxin  action.  We  synthesized  derivatives  of  Gmi  in 
which  the  oligosaccharide  was  coupled  to  different  lipids  such  as  phospholipids,  cholesterol  and  aliphatic 
amines.  We  tested  the  derivatives  on  rat  glioma  C6  cells  which  are  deficient  in  Gmi  .  bind  only  traces  of 
the  toxin  and  are  poorly  responsive  to  it.  C6  cells  incorporated  the  neoglycolipids  into  the  plasma 
membranes  as  measured  by  the  increase  in  cholera  toxin  binding.  However,  neoglycolipids  consisting  of 
GMi-oligosacchande  attached  to  short  chain  aliphatic  amines  were  not  taken  up  by  the  cells;  at  least  a 
dodecylamine  was  required.  The  neoglycolipids  also  conferred  enhanced  cholera  toxin  responsiveness 
ci  the  cells  as  measured  by  increased  stimulation  of  adenylate  cyclase.  There  were  differences  among 
the  neoglycolipids  in  terms  of  the  amounts  of  cyclic  AMP  formed  per  amount  of  toxin  bound  to  the 
treated  cells.  Those  derived  from  long-chain  aliphatic  amines  and  cholesterol  were  more  efficient  than 
Gm  i  as  toxi  n  receptors  whereas  those  derived  from  phosphol  i  pids  were  less  efficient  that  Gmi-  When  the 
distance  between  the  oligosaccharide  and  the  phospholipid  was  increased  by  adding  spacers,  the 
efficiency  as  receptors  decreased.  Thus,  the  lipid  moiety  of  Gmi  is  important  for  the  action  of  cholera 
toxin  and  these  neoglycolipids  may  be  useful  as  probes  of  Gmi  bioactivity  in  other  systems  such  as  the 
neuritoqenic  and  neuronotrophic  effects  of  the  ganglioside. 


16a-LMCN/DIR 


PHi  MM0  <«•«  1<M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


HKOJtU  NUMbtK 


Z01  NS02151-16 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  fMdtWMMnarJtu  T"l»  mvU  tit  on  on*  bna  b*tm—n  iht  boio*ri.) 

Memory  Storage  in  Neural  Networks 


PRINCIPAL  IN  VE  STlGATOR  (tin  olli*i  otantmontl  a*iMnn*l  »*tom  I'm  hinuotl  Inmitiotioi)  <*u<m.  Ulh.  Utbtunoif.  »no"  MKilul*  tffilislion) 

PI:  D  L  Alkon  Medical  Officer        LMCN,  NINDS 

Others:        LMCN,  NINDS:  C  Collin,  Vis.  Fellow;  P  Huddie,  Vis.  Fellow;  C  Bramham,  Vis.  Fellow;  R 

Etcheberrigaray,  Vis.  Fellow;  JV  Sanchez-Andres,  Vis.  Fellow;  E  Yavin,  Vis.  Scientist;  D  Lester, 
Vis.  Assoc;  E  Maduh,  Vis.  Fellow;  L  Matzel,  IRTA  Fellow;  B  Schreurs,  Staff  Fellow;  I 
Lederhendler,  Staff  Fellow;  T  Nelson,  Staff  Fellow;  J  Olds,  Staff  Fellow;  D  McPhie,  Biologist;  J 
Blaszcyk,  Sp.  Volunteer;  AM  Craig,  Spec.  Volunteer;  S  Moshiach;  Spec.  Volunteer;  L  Keith, 
Spec.  Volunteer;  S  Hyman,  Guest  Researcher;  E  Yamoah,  Guest  Researcher. 


COOPERATING  NITS<ir«w 

Marine  Biological  Laboratory,  Woods  Hole,  MA  02S43  (A.  Kuzirian);  California  Institute  of  Technology  (C. 
Chen);  Medical  Research  Council,  Canada  (AM.  Craig) 


LAB/BRANCH 

Laboratory  of  Molecular  and  Cellular  Neurobiology,  BNP,  DIR,  NINDS 


SECTION 

Neural  Systems  Section 


INSTITUTE  AND  LOCATION 

Park  Building,  Room  431  and  Building  9,  Room  1W125,  NINDS,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


10.0 


PROFESSIONAL: 


9.0 


OTHER: 


1.0 


CHECK  APPROPRIATE  BOX(ES) 

I      I   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


I     I  (b)  Human  tissues  [Tj  (c)  Neither 


SUMMARY  OF  WORK  {Use  standard  Unreduced  type.  Do  not  tk-ctd  iht  tpact  provided.) 

The  principal  objective  of  the  program  is  to  define  molecular  and  biophysical  mechanisms  of  learning 
and  memory.  Emphasis  is  placed  on  learning  and  memory  which  can  be  related  to  human  cognition. 
Ultimate  goals  of  such  research  are  to  arrive  at  clinically  meaningful  interventions  and  to  design  and 
construct  artificial  intelligence  which  has  advanced  learning  and  memory  capabilities.  With  human 
cognitive  function  as  the  principle  frame  of  reference,  the  research  focuses  on  associative  processes  (such 
as  Pavlovian  conditioning)  rather  than  non-associative  behavioral  modifications  (such  as  sensory 
adaptation,  habituation,  arousal,  and  sensitization).  The  biological  basis  of  learning  and  memory  is  of 
interest  at  several  levels  of  complexity:  behavioral  phenomena,  neuronal  systems,  neuronal 
membranes  and  molecular  transformations.  To  literally  reconstruct  the  physiology  involved  (and  to 
model  it  in  artificial  contexts)  it  is  necessary  to  use  both  "simple  system"  preparations  such  as  the 
nudi  branch  mollusc  Hermissenda  crasiicomis  as  well  as  "complex  system"  preparations  such  as  rabbits 
and  rats.  The  molluscan  work  thus  far  has  yielded  the  first  unequivocal  biological  record  of  an 
associative  memory.  This  record  consists  of  persistent  transformations  of  specific  ionic  channels.  Because 
these  records  have  been  found  within  the  membranes  of  identified  single  neurons  it  has  proven  possible 
to  define  biochemical  pathways  which  regulate  such  long-term  membrane  modifications  as  well  as  to 
analyze  how  this  biological  memory  record  is  expressed  by  the  integrative  functions  of  an  entire 
neuronal  system.  The  work  on  the  vertebrate  brain  offers  two  essential  opportunities.  First,  the 
generality  of  mechanisms  determined  for  much  less  evolved  species  can  be  tested.  Remarkably,  the  same 
ionic  channel  transformations  have  been  shown  in  our  program  to  record  associative  memory  in  the 
rabbit  as  were  found  in  Htrmiutnd*.  Rabbit  and  now  rat  neural  systems  have  also  provided  sufficient 
quantities  of  tissue  so  that  conditioning-specific  alterations  of  critical  enzymatic  (e.g.,  protein  kinase  C) 
pathways  which  control  membrane  excitability  have  recently  been  demonstrated. 


17a-LMCN/DIR 


WMMIM>    1  Ml 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 
Z01NS02786-02LMCN 


PERIOD  COVERED 

Octoberl,  1989  through  September  30,  1990 


TITLE  OF  PROJ  ECT  (ao  chtracfrt  or  Mm.  fill*  must  hi  on  on*  lint  Mi»no  iht  bortory) 

Antibodies  to  Glycoconjugates  in  Neurological  Diseases 


PRINCIPAL  IN VESTI6ATOR(UitofnwpranuiioiutpwsoflfM/s*k>i*l/w*rlMvfltanni9ilwJ  (Mtrn*.  Ml*.  Memory,  tndmttituftfiltttion) 

PI:  Richard  H.Quarles,  Ph.D.  Section  Chief     LMCN.NINDS 

Others:        Genevieve  Daune,  Ph.D.  Visiting  Fellow  LMCN,  NINDS 

Antonio  Noronha,  Ph.D.  Staff  Fellow      LMCN.NINDS 

Carl  Lauter  Chemist  LMCN,  NINDS 

Jeffrey  Hammer  Biologist  LMCN.NINDS 


COOPERATING  UNITS  Oltny) 

Medical  Neurology  Branch,  NINDS;  Dept.  Neurology,  Johns  Hopkins  Univ.,  Baltimore,  MD; 
The  Kennedy  Institute,  Baltimore,  Maryland 


LAB/BRANCH 

Laboratory  of  Molecular  and  Cellular  Neurobiology,  BNP 


SECTION 

Section  on  Myelin  and  Brain  Development 


INSTITUTE  AND  LOCATION 

Park  Building,  Rm.  425,  NINDS,  NIH  Bethesda,  MD  20205 


TOTAL  MAN-YEARS:  ,  c 

1 .0 


PROFESSIONAL:  1  , 


OTHER: 


0.3 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  [T]  (b)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

_J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  st»nd»rd  unreduced  type.  Do  not  exceed  the  sptce  provided.) 

This  area  of  investigation  began  with  the  demonstration  of  monoclonal  anti-MAG  antibodies  in  patients 
with  mixed  motor-sensory  polyneuropathies  occuring  in  association  with  IqM  paraproteinemia.  It  was 
subsequently  demonstrated  that  these  anti-MAG  antibodies  were  all  directed  toward  carbohydrate 
epitopes  in  MAG  and  cross-reacted  with  19  to  28  kD  glycoproteins  of  PNS  myelin  and  a  sphinqoqlycolipid. 
sulfate-3-qlucuronvl  paraqloboside  (SGPG).  Although  all  these  human  anti-MAG  antibodies  have  similar 
specificities  since  they  react  with  the  same  family  of  glycoconjugates  in  nerve,  experiments  with 
chemically  modified  derivatives  of  SGPG  have  revealed  differences  in  fine  specificities.  Some  of  the 
human  antibodies  and  the  related  HNK-1  antibody  absolutely  require  the  sulfate  for  reactivity,  others 
require  the  free  carboxyl  group  of  glucuronic  acid,  whereas  still  others  will  react  if  either  one  or  the 
other  of  the  two  negatively  charged  groups  is  present.  The  fine  specificity  of  the  anti-MAG  antibodies 
may  affect  the  clinical  presentation  in  these  patients.  Our  research  in  recent  years  has  emphasized 
antibodies  to  GM1  qanqlioside  in  patients  with  motor  nerve  disorders.  Four  patients  with  multifocal 
motor  neuropathy  were  found  to  have  high  titers  of  polyclonal  antibodies  reacting  with  GM1 
qanqlioside.  The  fine  specificities  of  these  antibodies  also  differed  in  the  individual  patients  as  revealed 
by  identification  of  different  cross  reacting  gangliosides.  Since  GM 1  was  the  only  common  antigen  in 
these  patients,  GM1  may  be  the  pathogenically  significant  target  antigen  in  motor  nerves.  Although 
these  findings  reveal  a  strong  correlation  between  the  occurrence  of  antibodies  to  GM  1  and  one  type  of 
motor  nerve  disorder,  we  did  not  find  high  levels  of  antibodies  to  GM1  in  motor  neuron  disease  or 
amyotrophic  lateral  sclerosis.  Preliminary  results  have  revealed  moderately  elevated  antibodies  to 
gangliosides  in  some  cases  of  adrenoleukodvstrophy  which  may  play  a  role  in  variable  secondary 
autoimmune  aspects  of  this  genetic  disease  caused  by  a  defect  in  the  peroxisomal  degradation  of 
saturated,  very  long  chain  fatty  acids.  Finally,  it  should  be  emphasized  that  our  work  and  that  of  others 
indicates  that  serum  antibodies  in  patients  with  neurological  diseases  should  be  analyzed  both 
qualitatively  by  TLC  overlay  and  quantitatively  by  ELISA.  since  the  pathogenic  significance  of  antibodies 
to  glycolipids  appears  to  depend  both  on  their  specificity  and  titer. 

18a-LMCN/DIR 


f>HS  6040  (»•«.  I/M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECTNUMBER 


Z01NS01808-21  LMCN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  faoctaracun  or  itti   Tula  muif  ni  on <ww  lint  tuMtn  i/m  botdtnj 

Glycoproteins  of  Myelin  in  Development  and  Disease 


PRINCIPAL  INVESTIGATOR  (List  o<nu  orotmuiontl  ptnonnil  btlow  tho  trinaptt  In— ttiottor )  (Htm*,  utk.  Itbotnory.  tnd  mmtutt  ttfilittion) 

PI:  Richard  H.Quarles,  Ph.D.   Section  Chief    LMCN,  NINDS 

Others:  Antonio  Noronha,  Sr.  Staff  Fellow,  LMCN,  NINDS;  Kenichi  Toda,  Vis.  Fellow,  LMCN, NINDS 

Johanna  Moller,  Sr.  Staff  Fellow,  LMCN,  NINDS;  Carl  Lauter,  Chemist,  LMCN,  NINDS 

Sung  Hye  Yim,  Special  Expert,  LMCN,  NINDS;  Jeffrey  Hammer,  Biologist,  LMCN,  NINDS 

Judy  Small,  Sr.  Staff  Fellow,  LMCN,  NINDS;  Ritu  Khanna,  Biologist,  LMCN,  NINDS 
Shuichiro  Goda,  Vis.  Fellow,  LMCN,  NINDS 
Zbigniew  Bartoszewicz,  Vis.  Fellow,  LMCN,  NINDS 


COOPERATING  UNITS  lifm/i 

Dept.  Neurology,  Johns  Hopkins  Univ.,  Balto.,  MD;  Dept.  Ped.,  Washington  Univ.,  St.  Louis,  MO;  Lab. 
Exp.  Neuropath.,  NINDS;  Developmental  and  Metabolic  Neurology  Br.,  NINDS;  Lab.  Clinical  Sci.,  NIMH 


LAB/BRANCH 

Laboratory  of  Molecular  and  Cellular  Neurobiology 


SECTION 

Section  on  Myelin  and  Brain  Development 


INSTITUTE  AND  LOCATION 

Park  Building,  Rm.425,  NINDS,  NIH I,  Bethesda,  MD.  20892 


TOTAL  MAN-YEARS:  ca 


PROFESSIONAL:  e  2 


OTHER: 


1.7 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  [T]  (h)  Human  tissues  □  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  sundard  unreduced  type.  Do  not  exceed  the  iptce  provided.) 

The  myelin-associated  glycoprotein  (MAG)  is  localized  in  the  periaxonal  membranes  of  PNS  and  CNS 
myelin  sheaths  where  it  appears  to  be  involved  in  glia-axon  interactions.  MAG  is  a  member  of  the 
immunoglobulin  gene  superfamily  along  with  other  neural  adhesion  proteins,  and  alternative  splicing  of 
its  mRNA  generates  two  developmental!  v  regulated  isoforms  with  differing  C-terminal  tails.  The  longer 
isoform  (L-MAG)  is  the  principal  form  synthesized  during  active  myelination  early  in  CNS  development, 
whereas  the  shorter  isoform  (S-MAG)  is  the  principal  form  synthesized  in  the  PNS  at  all  ages.  MAG  is 
phosphorylated  both  in  the  CNS  and  PNS,  but  the  sites  of  phosphorylation  by  oligodendrocytes  and 
Schwann  cells  differ.  The  extracellular  domains  of  the  two  forms  of  MAG  are  identical  with  5 
immunoglobulin-like  domains  and  8  potential  sites  for  N-linked  glycosylation.  The  carbohydrate  consists 
of  a  mixture  of  neutral  and  negatively  charged,  complex  oligosaccharides  whose  structures  are  now 
under  detailed  investigation.  MAG  appears  to  be  abnormally  glycosylated  in  dvsmyelinating  guaking 
mice.  The  expression  of  MAG  in  cultured  oligodendrocytes  and  Schwann  cells  is  being  studied  with  the 
ultimate  objectives  of  identifying  factors  that  control  its  synthesis  and  probing  its  function  in  cell-cell 
interactions.  Cultured  oligodendrocytes  from  adult  bovine  brain  constitutively  express  a  substantial 
amount  of  MAG  on  their  surface.  Although  cultured  Schwann  cells  do  not  normally  express  MAG  in  the 
absence  of  neurons,  an  immortalized  Schwann  cell  line  generated  in  our  laboratory  expresses  a 
remarkably  high  level  of  MAG  but  little  or  none  of  the  major  myelin  proteins.  Furthermore,  the  post- 
translational  glycosylation.  sulfation  and  phosphorylation  of  MAG  occuring  in  these  immortalized  cells  is 
similar  to  that /nv/vo,  and  this  continuous  cell  line  expressing  a  lot  of  MAG  on  its  surface  should  be  useful 
for  investigating  the  cell  biology  and  function  of  this  glycoprotein.  MAG  and  other  myelin  proteins  were 
studied  during  the  demyelination  and  remyelination  occurring  following  cuprizone  treatment  of  mice. 
Finally,  a  novel  autoantibody  was  identified  in  an  apparently  normal  rabbit  that  specifically  reacts  with 
the  shorter  isoform  of  2'.3'-cvclic  nucleotide  3'-phosphodiesterase  (CNP). 


19a-LMCN/DIR 


'HiUWJIHi.  114) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  •  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01NS02805-01LMCN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  chmrtavt  or 1***,  ntl*  mutt  fit  on  on*  lint  botw—n  tnt  batdmrt.) 

Molecular  and  immunological  aspects  of  myelin  abnormalities  in  neuro-AIDS 


PRINCIPAL  INVESTIGATOR  tUttothoiont*umt»lo*rtonnoloolowth*hlndp*llnnsttg*tor,)  (ktmt.  tit*.  l»borttory,  tnd Inn/tut*  ttillittton) 


PI:  Richard  H.  Quarles.  Ph.D. 

Others:       Johanna  Moller,  M.D. 

Antonio  Noronha,  Ph.D. 

Mary  McLenigan 

Jeffrey  Hammer 

Ritu  Khanna 


Section  Chief 
Sr.  Staff  Fellow 
Sr.  Staff  Fellow 
Chemist 
Biologist 
Biologist 


LMCN,  NINDS 
LMCN,  NINDS 
LMCN,  NINDS 
LMCN,  NINDS 
LMCN,  NINDS 
LMCN,  NINDS 


COOPERATING  UNITS  otm,) 

Dept.  Neurology,  Johns  Hopkins  University,  Baltimore,  Maryland;  Medical  Neurology  Branch,  NINDS 


LAB/BRANCH 

Laboratory  of  Molecular  and  Cellular  Neurobiology,  BNP 


SECTION 

Section  on  Myelin  and  Brain  Development 


INSTITUTE  AND  LOCATION 

Park  Building,  Rm.  425,  NINDS,  NIH  Bethesda,  Maryland   20892 


TOTAL  MAN-YEARS: 


1.7 


PROFESSIONAL: 


0.7 


OTHER: 


1.0 


CHECK  APPROPRIATE  BOX(ES) 

[      I   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


|~x  |  (b)  Human  tissues  ^]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  typo.  Do  not  exceed  the  space  provided.) 

This  is  a  new  project  undertaken  to  elucidate  biochemical  and  immunological  aspects  of  myelin  disorders 
associated  with  neuro-AIDS  including  myelin  pallor,  vacuolar  myelopathy  and  multifocal  demyelination 
in  the  CNS  as  well  asdemyelinatinq  peripheral  neuropathy.  Specific  areas  to  be  investigated  fall  into 
three  catagories:  1)  Postmortem  CNS  tissue  from  AIDS  patients  will  be  analyzed  for  quantitative  and 
qualitative  alterations  of  myelin  proteins  for  comparison  to  results  previously  obtained  in  other 
demyelinating  diseases.  Adjacent  sections  of  tissue  will  be  examined  histologically  and 
immunocytochemically  to  evaluate  the  pathological  changes  for  correlation  with  the  biochemical  data. 
Preliminary  biochemical  results  have  already  demonstrated  a  loss  of  myelin  proteins  in  some  areas  of 
AIDS  CNS;  2)  Since  the  myelin  associated  glycoprotein  (MAG)  and  the  CD4  receptor  for  HIV  a  re  both  in 
the  immunoglobulin  superfamily  and  have  a  modest,  but  statistically  significant,  sequence  homology, 
possible  interactions  between  MAG  and  GP120will  tested.  The  capacity  of  GP120  to  interfere  with  the 
normal  role  of  MAG  in  olia-axon  interactions  will  be  studied  as  will  the  possible  role  of  MAG  as  a  neural 
cell  receptor  for  HIV.  The  effects  of  GP120  on  cultured  oligodendrocytes  will  also  be  examined.  3)  Since 
high  levels  of  antibodies  to  qangliosides  and  other  qlvcoconjuqates  were  previously  detected  in  patients 
with  some  types  of  neuropathy  (including  a  few  that  were  HIV-positive),  a  larger  number  of  sera  from 
AIDS  patients  with  neuropathy  will  be  examined  systematically  for  antibodies  to  qlycoconiuoates. 


20a-LMCN/DIR 


'HSKMOIRk  1/W) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS02710-05LMCN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (w<Mnmnwl<ii    Mrt/nurt  hi  on  ont  lint  b»t~9*n  ihtbordtn.) 

Molecular  Biology  of  Neurotransmitter  Receptors 


PRINCIPAL  INVESTIGATOR  (l«f  olntr  enHiuontl pwwoMl  b*low  tl»  frinaptl  m ^^tV)ilot )  (htmt.  IfUft  Ijfco/jeory.  «ntfin«nut«  i«.iui.on) 

PI:  J.C.  Venter,  Ph.D.,  Chief,  RBMB,  LMCN,  NINDS.  OTHERS  (LMCN,  NINDS):  E.  Kirkness,  Ph.D.,  Fogarty 
Fellow;  T.  Onai,  M.D.,  Fogarty  Fellow;  S.  Arakawa,  M.D.,  Special  Volunteer;  J.  Flemming,  B.S.,  Special 
Volunteer;  J.  Kusiak,  Ph.D.,  Staff  Scientist;  M.  Fitzgerald,  B.A.,  Biologist;  R.  Bevan,  Ph.D.,  Special 
Volunteer;  T.  Saverese,  Ph.D.,  Special  Volunteer;  J. -P.  Giacobino,  Ph.D.,  Special  Volunteer;  M.  Adams, 
Ph.D.,  IRTA;  A.  R.  Kerlavage,  Ph.D.,  Sr.  Staff  Fellow;  W.R.  McCombie,  Ph.D.,  Sr.  Staff  Fellow;  J.  Gocayne, 
M.S.,  Microbiologist;  A.  Martin,  Ph.D.,  Sr.  Staff  Fellow 


COOPERATING  UNITS  <>'»»>; 

Section  of  Neurobiology,  LPPS,  NIAAA  (C.  M.  Fraser,  Ph.D.  &  M.  Buck,  Ph.D.,  IRTA  Fellow); 
Department  of  Biochemical  Pharmacology,  SUNY  at  Buffalo  (L.M.  Hall,  Ph.D.,  Professor) 


LAB/BRANCH 

Laboratory  of  Molecular  and  Cellular  Neurobiology,  BNP,  DIR,  NINDS 


SECTION 

Section  of  Receptor  Biochemistry  and  Molecular  Biology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  ,  . 


PROFESSIONAL:  5  g 


OTHER: 


0.2 


CHECK  APPROPRIATE  BOX(ES) 

I      I   (a)  Human  subjects  ]  (b)  Human  tissues  [T]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  is  to  characterize  the  gene  and  protein  structure  and  evolution  of  neurotransmitter 

receptors  belonging  to  two  major  multigene  families.  The  first  gene  family  comprises  adrenergic. 


muscarinic,  opsin,  serotonin  and  octopamine  receptors  and  the  second  comprises  nicotinic  cholinergic. 


GABA/benzodiazepine  NMDA.  and  glycine  receptors.  The  specific  aims  are  to  clone  and  seouence  the 


genes  for  receptors  in  these  two  multigene  families;  to  obtain  high  density  receptor  expression  and  to 
use  the  expressed  proteins  to  determine  the  complete  receptor  structure  in  part  by  computer  enhanced 
molecular  modeling;  to  determine  the  evolution  of  the  neurotransmitter  receptor  gene  families;  and  to 


search  for  and  characterize  receptor  gene  polymorphisms.  To  date,  we  have  cloned  and  sequenced  a 
significant  number  of  receptor  genes  from  both  multigene  families.  These  include  several  alpha'  and 
beta-adrenergic  receptors  from  human  and  rat  cDNA  and  genomic  libraries;  muscarinic  cholinergic 
receptor  from  human,  rat  and  Drosophila  genomic  libraries.  Octopamine  receptors  for  Drosophila, 
human  alpha  and  beta  subunits  of  the  GABA/benzodiazepine  receptor;  and  nicotinic  receptors  from 
locusts  and  C.  e/eqans.  For  example  the  human  GABA  beta  1  subunit  has  been  cloned  and  sequenced 
and  localized  to  chromosome  4.  This  gene  is  over  65kb  and  is  composed  of  nine  exons.  Theexonsand 
splice  sites  are  highly  conserved  in  other  GABA  receptor  subunits  from  humans  and  lower  species. 
Permanent  cell  lines  expressing  the  unique  neurotransmitter  receptor  proteins  are  providing  key  new 
information  concerning  the  mechanism  of  receptors  activation  by  neurotransmitters. 


21a-LMCN/DIR 


►  Hi  WKOIHt.  I'M  I 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

201  NS  02754-03  LMCN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (BO  cn*r*c1*r<  or  /•>>.  nt/»  must  fit  on  on*  lint  btftmn  <rt*  bordtn ) 

Megabase  DNA  Sequencing 


PRINCIPAL  INVESTIGATOR  (Um  otbtr  pnttiuonil  prtonnti octet*  lh» hmaptl  lnmtig»tor.)  (M*m*.  mi*,  ijooMiory.  tnd msiitui* Mttihttion) 

PI:      J.C.  Venter,  Ph.D.,  Chief,  RBMB,  LMCN,  NINDS .  OTHERS  (LMCN,  NINDS):  W.R.  McCombie, 
Ph.D.,  Sr.  Staff  Fellow;  A.  Martin,  Ph.D.,  Sr.  Staff  Fellow;  A.R.  Kerlavage,  Ph.D.,  Sr.  Staff  Fellow;  M. 
Dubnick,  Ph.D.,  Staff  Fellow;  M.  Adams,  Ph.D.,  IRTA;  J.  Gocayne,  M.S.,  Microbiologist;  J.  Kelley,  M.S., 
Microbiologist;  S.  Trapp,  B.S.,  Microbiologist;  M.  FitzGerald,  B.A.,  Biologist;  E.  Kirkness,  Ph.D.,  Fogarty 
Fellow;  J.  Kusiak,  Ph.D.,  Staff  Scientist;  J.  Xin,  Ph.D.,  Fogarty  Fellow 


COOPERATING  UNITS  (lfiV) 

DCRT  (M.  Hunkapiller);  University  of  Michigan  (F.  Collins,  M.D.);  DOE  (A.  Corrano,  DOE);  L.  Goldfarb, 
LCNSS; 


LAB/BRANCH 

Laboratory  of  Molecular  and  Cellular  Neurobiology,  BNP 


SECTION 

Section  of  Receptor  Biochemistry  and  Molecular  Biology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Park  Building,  Room  405,  Bethesda,  MP  20892 


TOTAL  MAN-YEARS:  „- 


PROFESSIONAL:  e  1 


OTHER: 


2.9 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  HH  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  has  as  its  goal  the  acquisition  and  analysis  of  the  sequence  of  genomic  DNA  that  is  associated 
with  receptor  multigene  families  and  neurological  disorders  and  to  understand  genomic  structure  at  the 
sequence  level.    To  accomplish  this,  sequencing  strategies  and  technologies  are  being  developed  that 
will  allow  the  sequencing  of  megabase  regions  of  DNA.  Our  use  of  four  Applied  Biosystems  automated 
DNAseguencers  allows  the  generation  of  up  to  48  kilobases  of  raw  sequence  per  day.  A  computer 
network  consisting  of  Macintosh,  Sun  4,  and  Silicon  Graphics  computers  in  the  laboratory  connecting  to 
the  NIH  Convex  and  Cray  computers  is  assisting  the  analysis  of  large  genomic  sequences.  The  use  of  Tag 
polymerase  and  PCR  cycle-sequencing  reactions  has  greatly  enhanced  the  quantity  and  the  quality  of  the 
sequence  data  generated.  New  automation  has  been  implemented  in  the  laboratory  by  the  addition  of 
new  robotics  which  completely  automate  DNA  sequencing  reactions.  These  techniques  have  been 
applied  to  sequencing  clones  associated  with  several  important  neurological  disease  gene  regions  of 
human  chromosomes.  The  laboratory  has  completed  over  2  million  bases  of  DNA  sequence  this  year, 
including  the  following  projects.  Over  1 5kb  from  the  disease  gene  for  neurofibromatosis  [  has  been 
sequenced,  identifying  several  new  exons.  Having  the  intron-exon  boundaries  has  allowed  us  to  develop 
PCR  probes  to  sequence  exons  from  NF1  patients  in  search  of  point  mutations  associated  with  disease 
symptoms.  A  three  cosmid  contig  from  the  Huntington's  region  of  chromosome  4  has  been  sequenced. 
This  region  of  approximately  1 0Okb  is  one  of  the  two  largest  segments  of  human  chromsomal  DNA 
sequenced  to  date  and  is  currently  being  analyzed.  The  second  largest  region  is  a  1 0Okb  region  of 
chromosome  19  which  has  been  sequenced.  This  region  is  where  a  number  of  DNA  repair  enzymes  and 
the  gene  for  mytonic  dystrophy  have  been  mapped.  Cosmid  clones  have  been  obtained  from  the  human 
Xq28  region  which  contains  several  loci  of  neurological  significance  including  the  alpha3  subunit  of  the 
GABA  receptor.  These  clones  are  currently  being  processed  for  sequencing.  We  have  begun  sequencing 
clones  obtained  from  patients  with  spongiform  encephalopathies  in  collaboration  with  Lev  Goldfarb. 
This  project  will  involve  sequencing  about  240  clones  from  40  patients.  This  will  require  obtaining  about 
600,000  bases  of  raw  sequence  data. 

22a-LMCN/DIR 


►  HS  MM0(«*v.  1*4) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS02806-01LMCN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  f*0cn«r*ct*r<o/ Mis.  Titltmult  Monona  lint  MMM  th*  oorote'l) 

Human  Brain  cDNA  Project 


PRINCIPAL  INVESTIGATOR  (Urt out*/ prvttiuon*!  p»nonn»l potow  fn*  *rinao<( Inriiitttoi.)  (Manx,  inn,  laboratory,  •no' mnituf  tttiiuuon) 

PI:  J.C.  Venter,  Ph.D.,  Chief,  RBMB,  LMCN,  NINDS;  OTHERS  (LMCN,  NINDS):        M.  Adams,  Ph.D.,  IRTA; 
B.  Olde,  Fogarty  Fellow;  W.R.  McCombie,  Ph.D.,  Sr.  Staff  Fellow;  A.R.  Kerlavage,  Ph.D.,  Sr.  Staff  Fellow;  J. 
Gocayne,  M.S.,  Microbiologist;  J.  Kelley,  M.S.,  Microbiologist;  S.  Trapp,  B.S.,  Microbiologist;  M. 
FitzGerald,  B.A.,  Biologist 


COOPERATING  UNITS  V*»n,) 

A.  Corrano,  Lawrence  Livermore  (DOE);  G.  Evans,  Salk  Institute; 


LAB/BRANCH 

Laboratory  of  Molecular  and  Cellular  Neurobiology,  BNP,  DIR,  NINDS 


SECTION 

Receptor  Biochemistry  and  Molecular  Biology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  .  5 


PROFESSIONAL: 


1.1 


OTHER: 


0.4 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  |T]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  goal  of  this  project  is  to  isolate,  sequence,  and  characterize  the  genes  expressed  in  human  brain.  As 
many  as  half  of  the  more  than  50,000  human  genes  are  believed  to  be  expressed  in  the  brain.  While 
sequencing  the  human  genome  is  expected  to  take  over  1 5-20  years,  sequencing  a  large  number  of  cDNA 
clones  can  readily  provide  coding  sequence  data  on  genes  expressed  in  tissues.  We  are  building  a  large 
library  of  clones  and  sequences  of  human  brain  cDNA  clones.  One  thousand  human  brain  sequences 
have  been  completed  in  the  first  3-4  months  of  this  project.  Over  half  of  the  genes  isolated  are  totally 
new  genes,  not  previously  identified  or  cloned.  An  additional  percentage  are  new  human  genes,  having 
been  previously  isolated  from  other  species.  Computer  analysis  of  the  protein  sequences  at  the  primary 
and  secondary  structural  level  is  assisting  clone  identification.  DNAand  predicted  protein  sequences  will 
be  examined  for  the  presence  of  conserved  primary  structure  motifs  and  relationships  to  previously 
sequenced  genes.  Further  characterization  of  potentially  interesting  clones  will  include  chromosome 
localization,  examination  of  tissue  distribution  of  expression,  and  evolutionary  conservation.  The 
availability  of  a  broad-based  library  of  cDNA  sequences  will  also  facilitate  identification  of  coding 
regions  in  genomic  sequences  as  well  as  provide  a  starting  point  for  individual  cloning  projects.  A  variety 
of  approaches  are  being  used  to  select  brain-specific  clones  and  to  eliminate  highly  represented 
sequences. 


23a-LMCN/DIR 


KMiWHOIMv   1/14) 


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ANNUAL  REPORT 

October  1,  1989  through  September  30,  1990 

Biometry  and  Field  Studies  Branch 
Division  of  Intramural  Research 
Clinical  Neurosciences  Program 
National  Institute  of  Neurological  Disorders  and  Stroke 


Table  of  Contents 


RESEARCH  SUMMARY  1  -  7 


CONTRACT  NARRATIVES: 

Statistical  and  Collaborative  Biomedical  Research  8 

Data  Management  Support 

PROJECT  REPORTS 

Statistical  Collaboration  and  Consultation  9 

Z01  NS  02652-06  BFSB 

Research  in  Statistics  10 

Z01  NS  02490-10  BFSB 

Predictive  Value  of  the  EEG  in  Febrile  11 

Seizures 
ZOl  NS  02483-10  BFSB 

Stroke  Data  Bank  12 

ZOl  NS  02598-08  BFSB 

Traumatic  Coma  Data  Bank  13 

ZOl  NS  02516-09  BFSB 

Factors  Predictive  of  Reading  and  Writing  14 

Skills  in  the  Congenitally  Deaf 
ZOl  NS  02594-08  BFSB 

Headache  in  Pregnant  Women  15 

ZOl  NS  02505-10  BFSB 

Statistical  Coordinating  Center  for  Collaborative  16 

Clinical  Studies 
ZOl  NS  02810-01  BFSB 

Publications  17  -  19 


i  -  BFSB/DIR 


ANNUAL  REPORT 
October  1,  1989  through  September  30,  1990 

Biometry  and  Field  Studies  Branch 
Division  of  Intramural  Research 
Clinical  Neurosciences  Program 
National  Institute  of  Neurological  Disorders  and  Stroke 

Jonas  H.  Ellenberg,  Ph.D.,  Chief 

The  Biometry  and  Field  Studies  Branch  (BFSB)  supports  a  program  in 
biostatistics  to  advance  the  mission  of  NINDS  in  the  areas  of  neurologic 
disorders.   The  Branch  participates  in  a  wide  range  of  intramural  and  extramural 
collaborative  projects,  including  large-  and  small-scale  observational  studies, 
clinical  trials  and  laboratory  studies.   These  collaborative  studies  have  been 
conducted  both  through  direct  staff  research  and  research  and  development 
contracts.   In  addition  to  collaborative  work,  the  Branch  has  an  important 
research  component  in  statistical  methodology. 

The  Branch  is  composed  of  an  Office  of  the  Chief  and  three  Sections.  Dr  James  .  . 
Dambrosia  is  Deputy  Chief  of  the  Branch.   In  that  capacity  he  assists  the  Branch 
Chief  in  policy  decisions  and  assumes  part  of  the  administrative  responsibilities 
of  running  the  Branch.   The  Mathematical  Statistics  Section,  headed  by  Dr.  James 
Dambrosia  is  the  main  statistical  consulting  unit  for  other  branches  and 
laboratories  in  the  Division  of  Intramural  Research,  the  Extramural  Research 
Divisions  as  well  as  neuroscience  units  outside  of  NINDS.   This  Section  is  also 
responsible  for  research  in  statistical  methodology.   The  Computer  Applications 
Section  is  currently  occupied  primarily  with  the  activities  of  the  Stroke  and 
Traumatic  Coma  Data  Banks.   Dr.  Mary  Foulkes  is  Acting  Chief  of  this  Section,  and 
Project  Director  for  both  of  the  Data  Banks.   With  the  completion  of  the  enormous 
data  management  and  data  collection  activities  and  of  the  Data  Banks ,  the  Section 
is  focusing  on  analysis  of  the  data  from  these  projects  as  well  as  the  development 
of  statistical  collaborative  initiatives  for  clinical  studies  in  other  areas  such 
as  the  clinical  trial  on  treatment  of  AIDS  Dementia  Complex.   A  change  in  the  name 
of  the  Section  to  the  Collaborative  Studies  Section  has  been  proposed  to  reflect 
the  current  focus  of  the  Section's  activities  and  a  permanent  Section  Chief  will 
be  appointed.   The  Data  Processing  Section  provides  computer  programming,  systems 
analysis,  and  data  management  support  to  the  Branch.   The  Branch  Chief  is  the 
Acting  Chief  of  this  Section. 

Over  the  past  two  years,  the  Branch  has  aggressively  recruited  six  recent  Ph.D. 
statisticians  for  positions  through  the  Staff  Fellow  Program,  and  a  Senior 
Statistician.   As  a  result  of  these  efforts,  we  have  been  able  to  attract  only  two 
candidates,  Dr.  Paul  Albert  of  Johns  Hopkins  University  and  Dr.  Lisa  McShane  of 
Cornell  University,  to  accept  our  Staff  Fellow  positions.   With  the  departure  of 
Dr.  Young  Jack  Lee  to  take  a  position  as  the  Biometry  Branch  Chief  in  NICHD  and 
the  departure  of  Dr.  Sherrie  Emoto  on  extended  maternity  leave,  the  Branch  is 


BFSB/DIR 


currently  recruiting  for  three  positions:  a  Visiting  Scientist  and  two  Staff 
Fellows.   Our  recruitment  efforts  to  fill  vacant  positions  for  entry  level 
biostatisticians  have  been  hampered  by  increased  competition  for  well -trained 
Ph.D.  level  statisticians  and  a  major  differential  in  Staff  Fellow  salaries  as 
compared  to  salaries  offered  by  academic  institutions  and  industry. 

Several  of  our  previous  projects  were  long-term  and  labor  intensive,  requiring  a 
large  amount  of  effort  for  routine  activities  such  as  data  entry,  data  editing  and 
day-to-day  monitoring  of  protocol  compliance.   These  activities,  although 
essential,  consume  an  excessive  amount  of  staff  time,  with  the  drawback  that 
little  time  is  left  for  statistical  methodological  research  and  expansion  of  our 
collaborative  efforts  into  new  areas.   BFSB  collaboration  on  new  large-scale 
projects  with  a  substantial  data  management  component  will  depend,  in  large  part, 
on  whether  routine  data  management  operations  of  such  studies  can  be  contracted 
out  under  our  supervision.   To  support  ongoing  projects  and  other  future 
collaborative  studies,  an  R&D  contract,  "Statistical  and  Collaborative  Biomedical 
Research  Data  Management  Support"  (NOl-NS-9-2325) ,  was  awarded  to  Information 
Management  Services,  Inc.  on  December  31,  1988.   This  R&D  contract,  with  an 
initial  funding  period  of  three  years,  will  provide  expertise  in  statistical 
programming,  data  entry,  data  monitoring,  and  systems  analysis,  in  partial  support 
of  collaborative  projects  and  statistical  methodological  research. 

I.   STATISTICAL  COLLABORATION  AND  CONSULTATION 

Our  current  program  of  collaborative  research  has  developed  primarily  in  response 
to  requests  for  collaboration  from  intramural  and  extramural  scientists  at  NINDS 
and  from  researchers  outside  of  NIH.   Typically,  BFSB  assumes  responsibility  for 
the  statistical  design,  data  management,  statistical  analysis,  and  interpretative 
aspects  of  the  projects,  with  the  subject  matter  specialists  providing  the  project 
initiatives,  subject  matter  expertise,  and  overall  leadership.   The  Branch  selects 
projects  on  the  basis  of  scientific  merit,  a  high  probability  of  successful 
completion,  and  potential  for  scientific  contributions  consistent  with  the  goals 
of  the  DIR. 

In  collaboration  with  the  Division  of  Convulsive,  Developmental  and  Neuromuscular 
Disorders,  (DCDND) ,  BFSB  was  the  statistical  coordinating  center  for  the  clinical 
trial  of  behavioral  and  cognitive  side  effects  of  phenobarbital  used  for  the 
prevention  of  febrile  seizure  recurrence.   This  trial  required  extensive 
monitoring  of  patient  accrual,  extensive  data  quality  control  and  several  interim 
data  analyses  for  the  trial's  Safety  and  Data  Monitoring  Committee.   Patient 
accrual  was  completed  in  December  1985  with  217  children  with  febrile  seizures 
randomized  to  treatment  and  150  seizure  free  controls  recruited  for  the  study. 
The  two  and  one-half  year  follow-up  of  the  last  patients  was  completed  in  July 
1988.   The  primary  results  of  this  clinical  trial  have  been  published  (NEJM  1990; 
332: (6)  364-9)  and  the  results  of  other  analyses  such  as  the  effect  of 
phenobarbital  on  sleep  of  children  with  febrile  seizures,  and  the  prediction  of 
recurrence  of  febrile  seizures,  have  been  submitted  for  publication. 

A  second  collaborative  effort  with  the  DCDND  and  the  Neuroepidemiology  Branch  is  a 
population-based  study  of  the  prognostic  value  of  the  EEG  for  subsequent  seizure 
activity  in  children  who  experienced  a  febrile  seizure.   The  cooperating  medical 

2  -  BFSB/DIR 


center  is  the  Pediatric  Clinic  in  Skopje,  Yugoslavia.   The  recruitment  of  new 
cases  ended  in  December  1984,  and  follow-up  (including  repeat  EEGs  and  neurologic 
and  physical  examinations)  continued  through  December  of  1989.   The  study  includes 
400  children  with  a  normal  or  non-specific  abnormal  EEG  following  a  first  febrile 
seizure,  as  well  as  about  300  children  with  a  specific  abnormal  EEG  following  a 
seizure.   The  major  outcomes  of  the  study  are  recurrent  febrile  and  afebrile 
seizures  and  their  relationship  to  the  initial  EEG,  subsequent  EEG  changes,  and 
the  influence  of  other  medical  and  demographic  factors.   Univariate  statistical 
analysis  of  the  data  for  the  baseline  visit  examined  a  large  number  of  potential 
factors  predictive  of  abnormal  specific  EEG  classification.   For  example,  number 
of  previous  febrile  seizures  was  associated  with  an  increased  rate  of  EEG 
abnormality:  18%  in  children  with  no  previous  seizures  and  63%  in  those  with  four 
or  more  prior  attacks.   When  these  factors  were  considered  jointly  in  a  logistic 
regression  model  the  significant  prognostic  factors  for  abnormal  specific  EEG 
were:  age  at  initial  EEG;  number  of  prior  febrile  seizures;  focal  febrile 
seizures;  and  motor  activity  abnormalities.   Data  editing  and  further  analyses  are 
in  progress . 

The  BFSB  continues  to  collaborate  with  many  Branches  and  Laboratories  in  the 
Division  of  Intramural  Research  (DIR) .   The  feasibility  of  a  randomized  controlled 
trial  of  treatment  with  anti-convulsant  medication  following  a  first  convulsion  in 
subjects  presenting  for  care  to  the  Beijing  Tiantan  Hospital  is  being  evaluated. 
This  collaborative  study  involving  BFSB,  the  Neuroepidemiology  Branch,  and  the 
Tiantan  Hospital  in  the  Peoples  Republic  of  China  will  address  the  issue  of 
whether  early  treatment  after  a  first  seizure  can  reduce  the  likelihood  of 
developing  chronic  epilepsy.   A  similar  protocol  is  being  considered  for 
implementation  with  a  consortium  of  hospitals  in  Israel.   BFSB  would  collaborate 
as  the  statistical  coordinating  center  for  these  projects. 

BFSB  is  collaborating  with  the  Medical  Neurology  Branch  on  two  clinical  trials  of 

felbamate:  one  trial  is  evaluating  the  effect  of  felbamate  in  controlling  seizures 

in  adults  with  intractable  partial  epilepsy;  the  other  is  assessing  efficacy  in 
children  with  Lennox -Gas taut  syndrome. 

Other  collaborative  studies  in  DIR  include:  development  of  optimal  sampling 
procedures  for  estimation  of  the  size  of  a  population  of  neuron  cells  (CN) ; 
evaluation  of  the  effect  of  the  levorotatory  forms  of  S-Hydroxytryptophan  in  the 
amelioration  of  gait  and  limb  ataxia  (MN) ;  mapping  of  the  cerebral  cortex  using 
EMG  amplitude  and  latency  responses  to  electro-magnetic  stimuli  to  the  scalp  (MN) : 
preliminary  evaluation  of  environmental  and  occupational  risk  factors  for  multiple 
system  atrophy  (CN) ;  clinical  trial  of  high  dose  prednisone  in  the  treatment  of 
post-polio  muscular  atrophy  (MN) ;  examination  of  psychopathology  of  epileptics 
(MN,  NIMH);  analysis  of  time  to  stroke  using  time  dependent  covariates  in  a 
proportional  hazards  regression  model  (NE) ;  a  case-control  study  of  the  potential 
association  of  serologically  confirmed  infection  during  pregnancy  with  morbidity 
in  the  child;  analysis  and  comparison  of  the  amplitude  of  blink  responses  evoked 
by  mechanical  or  electrical  stimuli  for  normal  controls  and  spasmodic  dysphonic 
patients  (MN) ;  examination  of  catecholamine,  neuropeptide  and  amino  acid  levels  in 
epilepsy  patients  at  baseline  and  ictal  periods  (MN) ;  survey  of  attitude  and 
potential  behavior  of  patients  with  von  Recklinghausen's  neurofibromatosis  with 
respect  to  genetic  screening  (NE) ;  clustering  of  occurrences  of  somatic  and 
affective  symptoms  in  epilepsy  patients(MN) ;  nature  of  parkinsonism-dementia 

3  -  BFSB/DIR 


complex  on  Guam  (NE) ;  prevalence  of  neurological  diseases  in  the  Navajo  tribe 
(MN) ;  hyperarousal  in  chronic  insomnia  patients  (MN) ;  study  of  epilepsy 
progression  to  generalized  tonic-clonic  seizures  (MN) ;  an  examination  of  seizure 
frequency  in  patients  with  intractable  complex  partial  seizures  (MN) ;  the  effect 
of  dexamethasone  suppression  tests  in  medicated  patients  with  poorly  controlled 
partial  seizures  (MN) ;  clinical  evaluation  of  Ceredase^  glucocerebrosidase 
infusion  for  treatment  of  Gaucher 's  disease  (DMN) ;  and  determination  of  the  effect 
of  time  from  last  seizure  and  seizure  type  on  the  dynamics  of  inter- ictal 
metabolic  change  (MN) . 

BFSB  will  act  as  the  statistical  coordinating  center  for  a  collaborative  study 
with  MNB  and  the  National  Naval  Medical  Center  on  a  clinical  trial  involving  a 
dose  comparison  of  3'-Azido-2',  3' -Dideoxy thymidine  (zidovudine)  in  the  treatment 
of  Human  Immunodeficiency  Virus  Type  1  infection  of  the  nervous  system.   This 
double-blind  clinical  trial  is  designed  to  compare  the  efficacy  of  two  different 
daily  doses  of  zidovudine  in  the  treatment  of  mild/moderate/severe  AIDS  Dementia 
Complex  (ADC)  and  will  involve  the  treatment  and  one  year  follow-up  of 
approximately  124  ADC  patients.   This  study  is  now  in  the  planning  and  approval 
stage. 

Collaborative  research  with  biomedical  research  units  not  in  NINDS  includes:  the 
comparison  of  expert  systems,  AI ,  and  statistical  classification  for 
differentiation  of  cerebral  infarction  and  intracerebral  hemorrhage;  development 
of  laboratory  quality  control  procedures  for  the  measurement  of  selenium; 
evaluation  of  the  effects  of  weather  and  ambient  light  on  mood  in  patients  with 
seasonal  affective  disorders;  area  surveys  for  epidemiologic  studies  of  neurologic 
disorders  in  Latin  America,  India  and  Italy;  study  on  the  incidence  of  primary 
intracranial  neoplasms  in  Israel;  and  publication  of  a  monograph  on  the 
epidemiology  of  neurologic  disorders. 

A  major  commitment  has  been  made  for  collaboration  with  the  Parkinson's 
Epidemiology  Research  Committee  (PERC) ,  a  group  comprised  of  individuals  from  the 
fields  of  movement  disorders  (neurology),  biometry,  epidemiology,  occupational 
health,  chemistry,  toxicology  and  neuropathology.   PERC  is  charged  with  the 
development  of  research  protocols  for  identifying  industrial,  agricultural  and 
naturally  occurring  environmental  chemicals  and  compounds  that  might  play  a  role 
in  Parkinson's  disease  (PD) ,  and  with  the  assessment  of  past  and  current 
epidemiologic  efforts.   PERC  has  evolved  beyond  its  original  "think  tank"  mission 
to  become  a  working  group,  and  the  members  took  full  advantage  of  the 
multidisciplinary  nature  of  the  committee.   PERC  has  taken  on  the  feasibility 
evaluation  or  initiation  of  several  specific  projects,  such  as  (1)  Studying  cases 
of  Parkinson's  disease  that  occur  in  both  husband  and  wife  (conjugal  Parkinson's 
disease);  (2)  Establishing  a  central  registry  to  study  Parkinson's  disease 
clusters;  (3)  Using  the  resources  of  the  Mayo  Clinic  for  studies  of  etiology  and 
progression  of  Parkinson's  disease;  (4)  Identifying  chemicals  in  natural  products 
that  may  cause  Parkinson's  disease;  (5)  Determining  if  occupational  exposure  to 
MPTP-like  chemicals  increases  the  risk  of  Parkinson's  disease;  (6)  Examining 
available  data  bases  to  determine  their  utility  in  studying  either  the  etiology  of 
Parkinson's  disease  or  the  progression  of  Parkinson's  disease;  and  (7)  Summarizing 
what  is  known  about  the  etiology  of  Parkinson's  disease  in  a  comprehensive, 
critical  overview  of  the  literature. 


BFSB/DIR 


BFSB  has  taken  the  primary  role  for  projects  (6)  and  (7).   To  date,  the  vital 
registration  system,  the  provider  based  surveys  and  the  population  based  surveys 
of  the  National  Center  for  Health  Statistics  (NCHS)  have  been  examined  in  project 
(6)  with  regard  to  ascertainment  bias  of  PD  cases,  correctness  of  diagnosis, 
completeness  of  records,  recall  bias,  etc.   From  among  the  myriad  of  NCHS  data 
bases,  the  National  Death  Record  System  and  the  National  Health  and  Nutrition 
Examination  Survey  have  been  identified  for  possible  studies  of  the  association  of 
PD  with  occupation  and  diet  respectively.   Other  ongoing  assessments  of  available 
data  bases  include  the  Twins  Registry  of  the  Medical  Follow-up  Agency  (MFA)  of  the 
Institute  of  Medicine  (16,000  white  male  twin  pairs,  of  service  age  during  World 
War  II)  and  the  MFA  roster  of  Parkinson's  disease  patients. 

We  have  now  compiled  approximately  700  papers  related  primarily  to  the  etiology  of 
PD  and  the  critical  review  and  annotation  for  project  (7)  is  in  progress.   The 
published  studies  have  not,  in  general,  been  definitive  due  to  such  problems  as: 
difficulty  of  diagnosis;  modest  prevalence;  and  retrospective  and  environmental 
histories  requiring  long-term  memory  recapture  in  PD  patients  or  their  surrogates. 
We  are  proceeding  with  the  evaluation  and  annotation  of  the  previous  research, 
based  on  stated  hypotheses,  appropriateness  of  study  design,  and  inference.   An 
annotated  bibliography  along  with  the  assessment  of  the  knowledge  base  for 
etiology  of  PD  will  be  published. 

II.   CLINICAL  DATA  BANKS 

BFSB  continues  its  responsibility  for  the  management  and  operation  of  the  Stroke 
and  Traumatic  Coma  Data  Banks.   Each  data  bank  was  a  collaborative  effort  between 
BFSB,  which  was  the  statistical  coordinating  center,  and  four  hospital  centers. 
The  data  banks  were  involved  in  the  collection  of  prospective,  observational, 
clinical  and  laboratory  data  at  the  multiple  clinical  centers  using  a  common  set 
of  data  forms.   These  data  banks  have  provided  a  resource  for  addressing  research 
questions  on  the  characteristics,  clinical  course,  and  outcome  of  hospitalized 
stroke  and  traumatic  head  injury  patients. 

For  the  duration  of  both  data  bank  projects,  resources  will  be  allocated  to  ensure 
the  continued  maintenance,  updating  and  easy  access  by  the  principal  investigators 
and  BFSB  to  complete  and  to  provide  accurate  databases.   BFSB  will  continue  in  its 
primary  scientific  leadership  role  in  the  collaborative  analysis  efforts  of  these 
projects,  providing  both  statistical  collaboration  and  oversight  of  the 
preparation  of  the  scientific  reports. 

Stroke  Data  Bank 

Data  collection  for  the  main  phase  of  the  Stroke  Data  Bank  began  in  FY  1983. 
By  the  end  of  new  patient  accrual  in  June  1986,  1,805  patients  had  been  entered. 
All  acute  care  data  were  entered  by  the  centers,  edited  and  corrected  by  BFSB  and 
the  centers,  and  a  final  acute  care  data  file  was  created  in  September  1986. 
Primary  analysis  of  acute  care  data  began  at  that  time.   Collection  of  follow-up 
data  continued  until  March  1987,  and  the  final  edited  data  base  was  completed  in 
May  1987.   A  priority  for  analysis  and  publication  has  been  established  to  focus 
on  the  areas  of  primary  interest.   A  policy  for  publication  and  a  Publication 
Committee  were  established  to  review  and  critique  all  potential  publications. 

5  -  BFSB/DIR 


Examples  of  research  studies  being  addressed  include:   the  investigation  of  racial 
and  sexual  differences  in  stroke  type,  site  and  vascular  territory;  the 
examination  of  stroke  severity  to  determine  whether  motor  weakness  and/or  sensory 
loss  can  be  predicted  by  the  location  or  size  of  the  CT  scan  abnormality  in 
infarcts;  evaluation  of  trends  in  institutionalization  of  post-stroke  survivors 
has  shown  that  race,  sex  and  marital  status  are  predictive  of  institutional- 
ization; and  a  homunculus  profile  analysis  is  in  progress  which  will  demonstrate 
the  association,  or  lack  thereof,  between  lesion  location  and  corresponding  motor 
deficits,  using  digital  mapping  of  CT  scan  data  on  lesion  location. 

Traumatic  Coma  Data  Bank 

Data  collection  for  the  main  phase  Traumatic  Coma  Data  Bank  (TCDB)  began  in 
FY  1984.   By  the  end  of  September  1987,  1,030  severely  head- injured  patients  were 
enrolled  in  this  project.   Patient  follow-up  ended  in  February  1988,  and  data 
editing  was  completed  in  July  1988. 

The  first  major  analyses  began  after  the  completion  of  data  collection  and 
editing.   Reports  submitted  for  publication  include:  outcome  following  severe  head 
injury;  the  influence  of  age  on  outcome;  verbal  learning  deficits  following  severe 
head  injury;  initial  CT  scan  findings  in  patients  with  severe  head  injury;  the 
impact  of  intracranial  pressure  instability  and  hypotension  on  outcome;  and 
several  methodologic  reports  on  TCDB  diagnostic  classifications,  intracranial 
pressure  monitoring  methods,  and  longitudinal  neurobehavioral  assessments 
following  severe  head  injury.   Additional  publications  are  also  in  progress,  and 
these  include  the  relationship  between  intracranial  pressure  and  Glasgow  Coma 
Outcome  Score;  post-injury  recovery  of  memory  and  attention;  time  related 
prognostic  factors  for  outcome  in  severe  head  injury;  recovery  from  a  vegetative 
state;  obstreperousness  and  depression;  and  the  role  of  secondary  injury  in 
determining  outcome. 

III.   METHODOLOGICAL  RESEARCH  IN  STATISTICS 

BFSB  statisticians  continue  to  develop  new  statistical  methodology  and  derive 
innovative  modifications  of  statistical  techniques  to  meet  the  needs  of  the 
Institute  for  the  design  of  experiments  and  field  studies,  analysis  of  data,  and 
statistical  modeling  of  biological  processes  and  phenomena.   Most  of  the 
statistical  problems  addressed  arise  from  collaborative  studies  with  the  Division 
of  Intramural  Research  and  neuroscience  units  outside  of  NINDS .   In  general,  there 
are  two  objectives  associated  with  these  various  statistical  activities  of  BFSB. 
The  primary  objective  is  the  development  and  improvement  of  statistical 
methodology  to  meet  the  needs  of  the  Institute.   The  secondary  objective  is  to 
make  contributions  to  the  development  of  statistical  methodology  which  may  be  more 
generally  useful  in  neurologic  and  other  medical  research. 

A  partial  listing  of  areas  in  which  BFSB  staff  is  developing  new  statistical 
applications  to  neurologic  problems  includes:  statistical  analysis  of  shapes  with 
spatial  dependencies;  growth  functions  for  responses  with  contributions  from  two 
compartments;  sampling  strategies  for  rare  neurologic  disorders;  determination  of 
the  order  of  categories  in  attribute  data;  methods  of  inference  on  frequency  of 
events  in  follow-up  data;  analysis  of  longitudinal  data  with  missing  observations; 

6  -  BFSB/DIR 


statistical  designs  for  two-state  episodic  diseases  using  follow-up  data;  and 
statistical  design  and  analysis  of  randomized  clinical  trials  in  neurology. 

Theoretical  statistical  work  has  included:   an  empirical  Bayes  approach  for 
examining  multiple  time  series;  the  effect  of  informative  and/or  prognostic 
censoring  on  survival  analysis;  comparison  of  common  parametric  and  nonparametric 
methods  for  survival  analysis  in  the  presence  of  mismodeling;  proportional  hazard 
model  estimators  for  transitions  in  discrete  state  semi-Markov  processes; 
derivation  of  consistent,  efficient  estimators  for  the  bivariate  Weibull 
distribution;  mixture  models  for  time  series  count  data;  regression  models  for 
interval  censored  time-to-event  data;  optimal  checking  methods  for  laboratory 
quality  control;  and  a  new  Weibull  survival  function  for  dependent  censoring. 

IV.   BRANCH  RECOGNITION  AND  OTHER  PROFESSIONAL  ACTIVITIES 

Several  of  our  staff  have  been  active  on  important  national  and  inter- 
national review  committees,  and  have  participated  in  major  meetings  or  received 
other  peer  recognition  this  fiscal  year.   Dr.  Ellenberg  continues  as  an  officer  of 
the  International  Biometric  Society  (immediate  past  President).   He  is  also  a 
member  of  the  Parkinsonism  Epidemiology  Research  Committee;  the  NINDS ,  Monitoring 
Committee  for  the  Deprenyl  and  Tocopherol  Antioxidative  Therapy  of  Parkinsonism 
Clinical  Trial  ( DAT ATOP ) ;  the  NINDS,  DIR  Clinical  Review  Board;  and  the  NIH,  Ad 
Hoc  Epidemiologist  and  Statistician  Review  Panel.   Dr.  Dambrosia  is  the  Biometrics 
Society  (Eastern  and  Western  North  American  Region)  representative  to  the  AAAS 
Medical  Science  Section  (N)  and  Chair  of  the  Regional  Advisory  Board  of  the 
Eastern  North  American  Region  of  the  Biometrics  Society.   Dr.  Anderson  serves  on 
the  American  Statistical  Association's  Committee  on  Committees  and  will  be  an 
invited  lecturer  in  Bressanone  at  the  First  International  Workshop  on  Statistics 
in  Epidemiologic  and  Pharmacologic  Research  speaking  on  case  finding  strategies 
for  neurologic  disorders.   Dr.  Foulkes  is  a  member  of  the  Monitoring  Committee  for 
the  VA  Cooperative  Study  of  Carbamazepine  versus  Valproic  Acid  for  Treatment  of 
Partial  Seizures,  and  organized  and  chaired  a  workshop  at  the  annual  meeting  of 
the  Society  for  Clinical  Trials  entitled  "When  a  Coordinating  Center  Assumes  an 
Ongoing  Trial."   Drs .  Ellenberg,  Anderson  and  Dambrosia  were  invited  chapter 
contributors  for  medical  monographs. 

In  summary,  BFSB  is  involved  in  a  strong  program  of  collaborative  research.   Our 
collaboration  extends  throughout  the  Institute  on  projects  with  both  Intramural 
and  Extramural  scientists,  and  also  involves  collaboration  with  scientists  outside 
of  NINDS.   The  scope  of  our  research  activity  ranges  from  small,  one-on-one 
collaboration  with  intramural  scientists,  to  the  conduct  of  large-scale, 
multicenter  clinical  studies.   BFSB  also  makes  an  important  and  continuing 
contribution  to  statistical  methodology  applicable  to  neurological  research. 


BFSB/DIR 


CONTRACT  NARRATIVE 

Biometry  and  Field  Studies  Branch,  CNP,  DIR,  NINDS 

Fiscal  Year  1990 

Information  Management  Services.  Inc..  Rockville.  Maryland 

CNOl-NS-9-2325') 


Title:   Statistical  and  Collaborative  Biomedical  Research 
Data  Management  Support 

Date  Contract  Initiated:   December  30,  1988 

Contractor's  Project  Director:   William  Lake,  Jr. 

Current  Annual  Level  FY  90:   $69,500 

Objectives :  To  provide  statistical  programming  and  data  management  support 
for  both  collaborative  research  projects  and  the  development  of  statistical 
methodology. 

Major  Findings:   This  contract  provides  statistical  programming  and  data 
management  support  for  data  entry,  editing,  quality  control  and  report 
generation  for  all  BFSB  collaborative  projects.   Software  for  new  statistical 
methods  as  well  as  all  data  management  support  is  developed,  tested  and 
implemented  by  the  Contractor  on  the  NIH  computer  system. 

Significance  to  the  NINDS  Program  and  Biomedical  Research:   The  statistical 
staff  of  BFSB  engages  in  collaborative  biomedical  research  and  conducts 
statistical  research  evolving  generally  from  problems  encountered  in  these 
collaborative  studies.   The  Contract  provides  timely  and  efficient  systems 
development,  data  management  (including  data  entry),  data  processing  and 
programming  for  both  ongoing  and  future  collaborative  studies.   Statistical 
programming,  an  essential  element  of  biostatistical  research,  is  provided 
under  the  direction  of  the  Branch.   New  statistical  methods  developed  by  BFSB 
are  coded,  evaluated  and  then  made  compatible  with  existing  interactive 
statistical  software  packages  by  the  Contractor. 

Proposed  Course  of  the  Project:   The  Contract  began  on  December  30,  1988  and 
continues  through  December  29,  1991. 

Publications :   None 


8  -  BFSB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02652-06  BFSB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  tit  on  one  line  between  the  borders  ) 

Statistical  Collaboration  and  Consultation 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:     James  M.  Dambrosia,  Ph.D. 

Co-PI  :  Jonas  H.  Ellenberg,  Ph.D. 
Others:  Paul  S.  Albert,  Ph.D. 

Dallas  Anderson,  Ph.D. 

Sherrie  E.  Emoto,  Ph.D. 

Mary  A.  Foulkes ,  Ph.D. 

Lisa  McShane,  Ph.D. 


Chief,  Mathematical 
Statistics  Section 
Chief 

Mathematical  Statistician 
Mathematical  Statistician 
Mathematical  Statistician 
Mathematical  Statistician 
Mathematical  Statistician 


BFSB, 

DIR, 

NINDS 

BFSB, 

DIR, 

NINDS 

BFSB, 

DIR, 

NINDS 

BFSB, 

DIR, 

NINDS 

BFSB, 

DIR, 

NINDS 

BFSB, 

DIR, 

NINDS 

BFSB, 

DIR, 

NINDS 

COOPERATING  UNITS  (if  any) 

Bombay  Hospital,  India  (Dr.  N.  Bharucha;  Dr.  Z.  Fu,  Dr.  Z.  Zhang,  D.  S.  Li  (PRC) 
Y.  Leibowitz  (Israel);  Dr.  J.  de  Pedro  Cuesta  (Sweden),  National  Institute  of 
Mental  Health  (Dr.  Norman  Rosenthal) 


LAB/BRANCH 

Biometry  and  Field  Studies  Branch 


SECTION 

Mathematical  Statistics  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS 

6.2 


PROFESSIONAL 


4.2 


OTHER 


2.0 


CHECK  APPROPRIATE  BOX(ES) 

B  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

This  project  encompasses  a  wide  scope  of  statistical  collaboration  and 
consultation  with  Laboratories  and  Branches  within  the  Division  of  Intramural 
Research  (DIR),  and  with  other  units  outside  of  NIH.   Particular  consideration  i^ 
given  to  statistical  planning  and  design  of  experiments,  statistical  analysis  of 
data,  and  statistical  inference.   Our  collaboration  has  involved  seven 
Laboratories/Branches,  and  the  scope  of  the  studies  has  ranged  from  the  coordin- 
ation and  statistical  management  of  clinical  trials  to  consultation  on  the 
appropriateness  of  the  statistical  analysis  used  for  small  laboratory  experi- 
ments.  Examples  of  studies  with  DIR  include:   randomized  clinical  trials  of 
felbamate  for  the  treatment  of  intractable  complex  partial  seizures  (MN) ; 
development  of  time  series  models  for  the  effect  of  weather  and  ambient  light  on 
mood  in  patients  with  seasonal  affective  disorder  (NIMH) ;  measurement  of  the 
effect  of  time  from  last  seizure  and  seizure  type  on  the  dynamics  of  inter- ictal 
metabolic  change  (MN) ;  clinical  trial  of  predisone  for  the  treatment  of  post 
polio  muscle  atrophy  (MN) ;  optimal  sampling  procedures  to  estimate  the  size  of  a 
population  of  neuron  cells  (CN) ;  identification  of  risk  factors  for  febrile 
seizures  with  a  population  based  case-control  study  of  six  cities  in  China  (NE) ; 
statistical  analysis  of  shape  and  spatial  relationships  of  maps  of  the  cerebral 
cortex  based  on  EMG  responses  to  electomagnetic  stimulation  of  the  scalp  (MN) ; 
clinical  course  and  outcome  of  patients  with  Gaucher's  disease  (DMN) ;  clinical 
evaluation  of  Ceredasera  glucocerebrosidase  in  Gaucher's  diseases  (DMN); 
prevalence  of  neurological  diseases  in  the  Navajo  tribe  (MN) ;  use  of  quasi- 
likelihood  models  to  demonstrate  that  seizure  frequencies  are  not  random  in  time 
(MN) ;  study  of  epilepsy  progression  to  general  tonic-clonic  seizures  (MN) ;  and 
the  use  of  hyperarousal  scores  for  diagnosis  of  chronic  insomnia  (MN) . 

9   -  BFSB/DIR 


PHS  6040  (Rev    1/84) 


CPO   SI  4-S16 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02490-10  BFSB 


PERIOD  COVERED 

October    1,     1989    through    Sppt-pmhpr    ^0,     1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  fit  on  one  line  between  the  borders.) 
Research    in    Stafisfir'; 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:      James  M.  Dambrosia,  Ph.D. 


Co -PI  :   Jonas  H.  Ellenberg,  Ph.D. 
Others:   Paul  S.  Albert,  Ph.D. 

Dallas  W.  Anderson,  Ph.D. 

Sherrie  E.  Emoto,  Ph.D. 

Mary  A.  Foulkes ,  Ph.D. 

Lisa  M.  McShane. ,  Ph  D 


Chief,  Mathematical 
Statistics  Section 
Chief 

Mathematical  Statistician 
Mathematical  Statistician 
Mathematical  Statistician 
Mathematical  Statistician 

Mat-hpmafiral Stati  sM  rian 


BFSB,  DIR,  NINDS 

BFSB,  DIR,  NINDS 

BFSB,  DIR,  NINDS 

BFSB,  DIR,  NINDS 

BFSB,  DIR,  NINDS 

BFSB,  DIR,  NINDS 

BFSB,  nTR,  NTNDS 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Biometry  and  Field  Studies  Branch 


SECTION 

Mathematical  Statistics  Section 


INSTITUTE  AND  LOCATION 

NINDS.    NIH,    Bethesda, 


Maryland    7089? 


TOTAL  MAN-YEARS: 

2.5 


PROFESSIONAL: 


2.0 


OTHER. 


0   5 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□  (b)  Human  tissues  H  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  addresses  statistical  problems  generated  from  collaboration  with 
scientists  in  other  program  areas  and  general  statistical  problems  of  current 
interest.   This  project  is  a  continuing  activity  of  the  Section  on  Mathematical 
Statistics.   Papers  have  been  submitted,  are  in  review  or  were  published  in  FY 
1990  on  the  following  statistical  subjects:   estimation  of  the  joint  survival 
and  censoring  distribution  in  the  presence  of  dependent  censoring;  statistical 
planning,  design  and  analysis  of  randomized  clinical  trials  in  neurology; 
development  of  a  Weibull  model  for  survival  data  with  dependent  censoring;  an 
empirical  Bayes  procedure  for  examining  the  relationships  between  multiple  time 
series;  establishing  statistical  quality  control  methods  for  biomedical 
laboratories;  design  of  panel  studies  under  alternating  Poisson  process 
assumptions.   Other  work  in  progress  includes:  selection  criteria  for  use  of 
the  Kaplan-Meier  or  parametric  MLE  for  survival  analysis;  influence  of  missing 
data  in  randomized  clinical  trials;  methods  to  improve  coverage  in  surveys; 
analysis  of  time-to-event  data  with  non-regular  censoring;  estimation  of 
time-to-event  with  interval  data  in  the  presence  of  left  and  right  censoring; 
-  site  selection  for  epidemiological  surveys;  adjustments  for  covariates  in  the 
analysis  of  categorical  data;  two-state  models  for  analyzing  time  series  count 
data;  analysis  of  response  surface  data  with  both  spatial  and  temporal 
components;  development  of  sampling  strategies  for  count  data  in  the  presence 
of  multiple  types  of  clustering;  estimation  of  hazard  functions  with  time- 
dependent  covariates  in  the  presence  of  competing  risks. 


10   -  BFSB/DIR 


PHS  6040  (Rev.  1/84) 


GPO  614-818 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02483-10  BFSB 


PERIOD  COVERED 


October  1,  19ft9  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  lit  on  one  line  between  the  borders  ) 

Pre.rii  rHve  Value  of   the   EEG   in  Febrile   Seizures 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:      Sherrie  E.  Emoto,  Ph.D 

Others:   Jonas  H.  Ellenberg,  Ph.D. 
Deborah  G.  Hirtz,  M.D. 
Karin  B.  Nelson,  M.D. 
Jack  Panossian 


Mathematical  Statistician  BFSB,  DIR,  NINDS 


Chief 

Pediatric  Neurologist 

Medical  Officer 

Programmer 


BFSB,  DIR,  NINDS 
DNB,  DCDND,  NINDS 
NEB,  DIR,  NINDS 
BFSB,  DIR,  NINDS 


COOPERATING  UNITS  (It  any) 

Cerebral  Palsy  and  Other  Motor  Disorders  Section,  DNB,  DCDND,  NINDS; 
Pediatric  Clinic,  University  of  Skopje,  Yugoslavia  (Nikola  Sofijanov) 


LAB/BRANCH 


Biometry  and  Field  Studies  Branch 


SECTION 

Mathematical Statistics    SecfcJ 


1  on 


INSTITUTE  AND  LOCATION 

NINDS.  NTH,  Bethesria, 


Maryland 7089? 


ry 

5FE 


TOTAL  MAN-YEARS 


0.35 


PROFESSIONAL 


0    IS 


OTHER 


0.20 


CHECK  APPROPRIATE  BOX(ES) 

LS  (a)  Human  subjects 
S   (a1)  Minors 
□  (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

This  population  based  study  will  evaluate  the  significance  of  the  EEG  as  a 
predictor  for  recurrence  of  seizures  in  those  children  who  have  had  a  simple 
febrile  convulsion.   Outcomes  reported  are  febrile  seizure  recurrence  and 
afebrile  seizure  occurrence.   The  evolution  of  the  EEG  pattern  will  be  described, 
and  patterns  will  be  correlated  with  the  clinical  outcome.   The  clinical  study  is 
being  carried  out  in  Skopje,  Yugoslavia,  at  the  Pediatric  Clinic  of  the 
University  of  Skopje. 

The  study  began  in  FY  1982  and  final  follow-up  visits  will  be  completed  in 
FY  1991.   Patient  accrual  was  completed  in  December,  1984,  by  which  time 
approximately  400  patients  with  a  febrile  seizure,  no  prior  complex  or  multiple 
seizures  and  with  a  normal  or  nonspecific  abnormal  EEG,  were  registered  into  the 
study  and  began  the  study  protocol  and  follow-up.   An  additional  300  patients 
with  a  specific  abnormal  EEG  were  entered  for  baseline  information  and  follow- 
up.   Data  editing  and  file  creation  are  continuing.   After  an  on-site  review 
visit,  it  was  determined  that  additional  efforts  by  the  clinical  center  were 
needed  to  collect  data  from  those  patients  lacking  a  return  visit  and  those  who 
did  not  have  return  visits  after  24  months  following  study  entry.   This  will 
facilitate  study  of  long-term  recurrence  of  febrile  seizures,  change  in  EEG,  and 
the  predictive  qualities  of  EEG  for  febrile  seizure  occurrence.   The  extended 
follow-up  effort  is  now  in  progress.   Statistical  analysis  of  baseline  EEG  and 
its  association  with  characteristics  of  the  child  and  family  and  the  clinical 
characteristics  of  the  seizure  has  been  completed  and  a  manuscript  has  been 
submitted  for  publication.   The  master  file,  including  follow-up  data  at  all 
visits  for  all  patients,  should  be  completed  in  FY  1991,  at  which  time  further 
analyses  will  be  undertaken. 

11    -   BFSB/DIR 


PHS  6040  (Rev.  1/84) 


GPO   91  4-916 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS   02598-08   BFSB 


PERIOD  COVERED 

October  1.  1989 


through  September  ^n,  iQQn 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  HI  on  one  line  between  the  borders.) 

Stroke   Data   Bank 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:     Mary  A.  Foulkes,  Ph.D. 
Others:  James  M.  Dambrosia,  Ph.D. 

Margaret  Meadows 
Rebecca  Rohde 
Jack  Panossian 
Alan  Polls 


Mathematical  Statistician 
Chief,  Mathematical 
Statistics  Section 
Statistician  Asst. 
Statistician 
Programmer 
Comp.  Sys .  Analysis 


BFSB,  DIR,  NINDS 


BFSB, 

DIR, 

NINDS 

BFSB, 

DIR, 

NINDS 

BFSB, 

DIR, 

NINDS 

BFSB, 

DIR, 

NINDS 

BFSB, 

DIR, 

NINDS 

COOPERATING  UNITS  (If  any) 

Departments  of  Neurology:   Boston  University  Medical  Center,  Michael  Reese 

Hospital,  Neurological  Institute  -  Columbia  University,  and  University 

of  Maryland . 


LAB/BRANCH 

Biometry  and  Field  Studies  Branch 


SECTION 

Computer  Applications  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Be thesda.  Maryland  20892 


TOTAL  MAN-YEARS: 

2.4 


PROFESSIONAL: 


1.6 


OTHER 


0-8 


CHECK  APPROPRIATE  BOX(ES) 

H  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  Stroke  Data  Bank  is  a  prospective  observational  study  which  collected  data 
on  hospitalized  newly  diagnosed  stroke  patients,  at  four  clinical  centers.   The 
collaborating  clinical  centers  were  responsible  for  the  collection  of  acute  care 
and  longitudinal  follow-up  information  using  common  definitions  and  procedures, 
under  contracts  NO1-NS-2-2302 ,  2398-9,  N01-NS-5-2384.   The  general  objective  for 
the  project  was  to  provide  a  comprehensive  body  of  data  for  clinical  research  on 
the  factors  influencing  survival .  morbidity  and  quality  of  life  following  onset 
of  a  stroke.   The  BFSB  served  as  the  statistical  coordinating  center  for  the 
project.   The  data  collection  phase  was  completed  in  FY  1988  including  follow- 
up,  with  a  final  cohort  of  1805  patients.   Now  the  BFSB  is  responsible  for 
statistical  collaboration  with  the  clinical  investigators  for  the  analysis  of 
the  research  questions.   An  observational  study  reporting  the  occurrence  within 
the  Stroke  Data  Bank  cohort  of  dementia  both  at  first  exam  post- stroke  onset  and 
incident  dementia  over  the  first  year  of  follow-up  summarizes  the  factors 
associated  with  dementia  within  this  cohort.   An  homunculus  profile  analysis  is 
in  progress  which  will  indicate  the  association,  or  lack  thereof,  between  lesion 
location  and  corresponding  motor  deficit.   Institutionalization  after  hospital 
discharge  is  the  focus  of  an  ongoing  investigation  which  indicates  that  race, 
sex  and  marital  status  may  be  associated  with  institutionalization  among  stroke 
survivors.   An  investigation  of  the  factors  predictive  of  recurrence  within  two 
years  post-stroke  indicated  that  prior  stroke,  diabetes  mellitus,  blood  pressure 
on  admission,  and  an  identifiable  cause  of  infarct  were  predictive  of  recurrent 
infarction.   Two  analyses  of  lacunar  infarctions  contrast  these  with  other 
infarction  types  and  with  lacunar  infarctions  from  the  Schlaganfall  -  Datenbank 
from  Austria. 

12   -  BFSB/DIR 


PHS  6040  (Rev.  1/84) 


GPO   » 14-81  6 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02516-09  BFSB 


PERIOD  COVERED 


October    1,     1 989    through    SpptPmhpr    30,     1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  tit  on  one  line  between  the  borders  ) 
Traumatic    C.nma    Data    Rank 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator  j  (Name,  title,  laboratory,  and  institute  affiliation) 


PI: 

Others: 


Mary  A.  Foulkes,  Ph.D.   Mathematical  Statistician  BFSB,  DIR,  NINDS 


Margaret  Meadows 
Rebecca  Rohde 
Jack  Panossian 
Alan  Polis 


Statistician  Asst. 

Statistician 

Programmer 

Comp.  Sys .  Analysis 


BFSB,  DIR,  NINDS 

BFSB,  DIR,  NINDS 

BFSB,  DIR,  NINDS 

BFSB,  DIR,  NINDS 


COOPERATING  UNITS  (if  any) 

Departments  of  Neurosurgery:   Medical  College  of  Virginia,  University  of 
California  -  San  Diego,  University  of  Texas  -  Galveston,  University  of  Virginia 


LAB/BRANCH 


Biometry  and  Field  Studies  Rranr.h 

SECTION 


Computer    Applications;    SprM'nn 


INSTITUTE  AND  LOCATION 

NINDS.  NIH.  Bethesda. 


TOTAL  MAN-YEARS 

2.2 


Maryland  2089? 


PROFESSIONAL 


1     U 


OTHER 


0    8 


CHECK  APPROPRIATE  BOX(ES) 

E  (a)  Human  subjects 
H   (a1)  Minors 
SI   (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  Traumatic  Coma  Data  Bank  is  a  prospective  observational  study  which 
collected  data  on  severely  head  injured  patients  at  four  clinical  centers. 
The  collaborating  centers  were  responsible  for  the  collection  of  acute  care 
and  longitudinal  follow-up  information  using  common  definitions  and  procedures, 
under  contracts  N01-NS- 3-2339-42 .   The  research  objectives  for  the  project  were 
formulated  by  a  Steering  Committee  composed  of  the  principal  investigators  from 
the  clinical  centers,  other  outside  experts,  and  BFSB  staff,  with  the  concur- 
rence of  the  BFSB  Advisory  Committee.   The  research  objectives  were  the  basis 
for  determining  the  specific  data  to  be  collected,  the  format  of  the  data 
collection  forms  and  the  data  collection  procedures.   The  general  objective  for 
the  project  was  to  provide  a  comprehensive  body  of  data  for  clinical  research  on 
the  factors  influencing  survival .  morbidity  and  quality  of  life  following  a 
severe  head  injury.   The  BFSB  was  the  statistical  coordinating  center  for  the 
project.  Accrual  of  1030  patients  was  completed  in  September  1987,  and  patient 
follow-up  was  completed  in  January  1988.   Reports  are  being  prepared  for 
publication  on  such  topics  as  the  relationship  between  intracranial  pressure  and 
Glasgow  Coma  Score,  post- injury  recovery  of  memory  and  attention,  time  related 
prognostic  factors  for  outcome,  recovery  from  a  vegetative  state, 
obstreperousness  and  depression,  and  the  role  of  secondary  injury  in  determining 
outcome . 


13    -  BFSB/DIR 


PHS  6040  (Rev   1/84) 


GPO  814-Sia 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02594-08  BFSB 


PERIOD  COVERED 


October    1,     1989    through    Sepi-emher  /*",     19QQ 


TITLE  OF  PROJECT  (80  characters  or  less   Title  must  tit  on  one  line  between  the  borders.) 

— Factors  Predictive  nf  Reading  and  Writing  Skills  in  the  Congeni tally  Deaf* 

PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:       Paul  S.  Albert,  Ph.D. 


Others:   Christy  Ludlow,  Ph.D. 
Judith  Cooper,  Ph.D. 


Staff  Fellow 


Speech  Pathologist 
Speech  Pathologist 


BFSB,  DIR,  NINDS 


DCSD,  NIDCD 
DCSD,  NIDCD 


COOPERATING  UNITS  (it  any) 


Central  Institute  for  the  Deaf,  St.  Louis,  MO  (Ann  Geers) ; 
Gallaudet  College,  Washington,  D.C.  (Donald  Moores) 


LAB/BRANCH 

Biometry  and  Field  Studies  Rranrh 


SECTION 

Mathematical  Statistics  Section 


INSTITUTE  AND  LOCATION 

NINDS.    NIHT    Bethesda, 


Maryland ?089? 


TOTAL  MAN-YEARS: 

0.05 


PROFESSIONAL 


n  ns 


OTHER: 


0.00 


CHECK  APPROPRIATE  BOX(ES) 

SI  (a)  Human  subjects 
S   (a1)  Minors 
D9   (a2)  Interviews 


D  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  consists  of  the  statistical  and  data  editing  and  file  creation 
aspects  of  a  NIDCD  project.   Collaboration  includes  design  of  data  collection 
and  monitoring  procedures,  and  statistical  analysis  of  study  data. 

The  study  examined  factors  that  may  be  associated  with  development  of  reading  and 
writing  skills  in  the  congenitally  deaf.   Study  subjects  comprised  three  groups 
of  deaf  16-  to  17 -year-olds ,  with  65  subjects  in  each  group.   Each  group  included 
only  subjects  who  received  their  preschool  language  training  through  one  of  three 
approaches:   aural-oral,  total  communication,  and  American  Sign  Language.   Data 
were  collected  on  the  audiologic,  familial,  and  educational  background  of  the 
subjects,  and  on  their  present  language  skills.   Data  will  be  examined  for  their 
association  with  present  reading  and  writing  skills  of  the  subjects.   Familial 
and  educational  data  for  the  main  phase  have  been  received  and  entered  onto  the 
NIH  computer  system.   Substantial  amounts  of  missing  data  dictated  the  need  for 
new  efforts  to  obtain  the  information  required  for  the  study. 

*[This  project  is  the  BFSB/NINDS  support  of  the  NIDCD  contract  study  NIH-NINDS- 
84-19.   The  project  officer  is  Dr.  Judith  Cooper,  NIDCD.   This  project  has  been 
subsumed  under  Statistical  Collabor?tion  and  Consultation  (Z01  NS  02652-06).] 


14   -  BFSB/DIR 


PHS  6040  (Rev.  1/84) 


GPO  914-918 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  •  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01   NS   02505-10   BFSB 


PERIOD  COVERED 


October  1.  1989  through  SeptPrnhpr  3D,  1  990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  tit  on  one  line  between  the  borders  ) 

Headache  in  Pregnant  Womp.n 


PRINCIPAL  INVESTIGATOR  (List  other  protessionai  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  attiliation) 


PI:  Ta-Chuan  Chen,  Ph.D. 

Other:  Jonas  H.  Ellenberg,  Ph.D. 


Mathematical  Statistician 
Chief 


BFSB,  DIR,  NINDS 
BFSB,  DIR,  NINDS 


COOPERATING  UNITS  (it  any) 

Boston  Children's  Hospital  (Dr.  Alan  Leviton) 


LAB/BRANCH 

Biometry  and  Field  Studies  Branch 


SECTION 

Office  of  the  Chief 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda. 


Maryland  2089? 


TOTAL  MAN-YEARS 

0.35 


PROFESSIONAL 


0-30 


OTHER 


0.05 


CHECK  APPROPRIATE  BOX(ES) 

E  (a)  Human  subjects 
H   (a1)  Minors 
□   (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

This  project  investigates  the  relationship  between  migraine  headache  and 
other  diseases  based  on  the  data  collected  from  the  large  group  of  gravidae 
in  the  Collaborative  Perinatal  Project.  The  report  of  the  investigation  of  the 
influence  of  smoking  on  associations  between  migraine  and  other  diseases  such  as 
heart  and  thrombotic  diseases  and  some  respiratory  and  allergic  diseases  was 
published  in  the  Archives  of  Neurology,  in  FY  '1988. 

We  are  currently  examining  the  possible  association  of  maternal  migraine  in 
pregnant  women  with  the  health  status  of  their  children.   Subgroups  of  women 
characterized  by  the  absence  and  presence  of  migraine  and  other  recurrent 
headaches  prior  to  or  during  pregnancy,  were  identified.   Children  of  mothers 
with  a  history  of  migraine  appear  to  have  higher  incidence  of  some  infectious  and 
allergic  diseases  than  children  born  to  mothers  in  the  non-migraine  group. 
Statistical  investigation  of  the  latter  results  has  revealed  an  association  of 
occurrence  of  bronchial  asthma  in  children  born  to  mothers  with  migraine,  even 
after  partitioning  out  the  potential  influence  of  maternal  asthma  and  allergies. 
The  risk  of  occurrence  of  bronchial  asthma  in  children  born  to  mothers  with  a 
history  of  migraine  was  estimated  to  be  1.8  times  larger  than  in  the  children 
whose  mothers  did  not  have  migraine  headaches.   A  manuscript  has  been  accepted 
for  publication  in  the  Archives  of  Neurology. 

Further  studies  examined  the  frequency  and  pattern  of  headache  attacks  during 
pregnancies  of  the  women  with  a  history  of  migraine  to  test  the  hypothesis  of 
improvement  of  headache  during  pregnancy,  and  the  possible  association  of 
pregnancy  complications  such  as  uterine  bleeding  with  migraine  and  headache 
medication. 

This  project  will  be  subsumed  under  Statistical  Collaboration  and  Consultation 
(Z01  NS  02652-06) . 

15   -   BFSB/DIR 


PHS  6040  (Rev    1/84) 


GPO    SI  4-9  1  f 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02810-01  BFSB 


PERIOD  COVERED 


Or.rnhp.r  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 
Sfafi  Sti  cal Cnnrriinat-ing    Center    for    Col  1  ahnraf.i  vs    Clinical     Studies 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:     Mary  A.  Foulkes. 

Others :  Marinos  Dalakas . 
Jordan  Graf man, 
George  Grimes 
Rebecca  Rohde 
Jack  Panossian 
Alan  Polis 


Ph.D.   Mathematical  Statistician 
M.D.    Chief 
Ph.D.    Chief 

Pharmacist 

Statistician 

Programmer 

Comp.  Sys .  Analyst 


BFSB,  DIR,  NINDS 
NDS,  MNB,  DIR,  NINDS 
CNS,  MNB,  DIR,  NINDS 
CC,  PHAR,  NIH 
BFSB,  DIR,  NINDS 
BFSB,  DIR,  NINDS 
BFSB,  DIR,  NINDS 


COOPERATING  UNITS  (if  any) 

Depts .  Of  Neurology  and  Psychiatry:   National  Naval  Medical  Center,  Bethesda, 
and  Oak  Knoll  Naval  Medical  Center,  Oakland 


LAB/BRANCH 


Biometry  and  Field  Studies  Branch 


SECTION 


Computer  Applications  Section 


INSTITUTE  AND  LOCATION 


NINDS.    NIH,     Bethesda,    Maryland 2QM1 


TOTAL  MAN-YEARS: 

UL 


PROFESSIONAL: 


1  .6 


OTHER: 


0-4 


CHECK  APPROPRIATE  BOX(ES) 

S  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  encompasses  all  statistical  coordinating  center  responsibilities  for 
collaborative  clinical  studies  undertaken  by  this  Section.   Currently  the  major 
collaborative  clinical  study  is  a  double-blind  clinical  trial,  designed  to 
compare  the  efficacy  of  two  different  daily  doses  of  zidovudine  in  the  treatment 
of  mild/moderate/severe  AIDS  Dementia  Complex  (ADC) .   It  is  a  collaborative  trial 
between  the  National  Institute  of  Neurological  Disorders  and  Stroke  (NINDS),  the 
National  Naval  Medical  Center  (NNMC) ,  and  Naval  Hospital,  Oakland.   The  major 
objective  of  the  trial  is  to  compare  the  effects  of  two  different  daily  doses  of 
zidovudine  on  the  rate  of  progression  of  HIV-1  infection  of  the  brain.   The  trial 
is  designed  to  determine  if  zidovudine  at  a  reduced  dose  offers  a  therapeutically 
effective  alternative  to  zidovudine  at  1250  mg  daily  dose  in  the  management  of 
patients  with  mild/moderate/severe  ADC.   One  hundred  twenty- four  patients  will  be 
enrolled,  62  in  each  of  two  treatment  arms.   All  patients  will  either  be  Depart- 
ment of  Defense  (DOD)  health  care  beneficiaries  who  meet  established  criteria  for 
ADC  stage  1  (mild) ,  stage  2  (moderate) ,  or  stage  3  (severe)  and  who  are  currently 
being  treated/monitored  at  Naval  hospitals  in  Bethesda  or  Oakland,  or  non-DOD 
health  care  beneficiaries  with  stage  1,2,  or  3  ADC  who  reside  in  the  Washington- 
Baltimore  area  and  are  under  the  care  of  local  private  practitioners.   All 
patients  will  be  followed  for  one  year  from  the  time  of  enrollment  with  periodic 
studies  to  monitor  their  response  to  the  dose  assigned.   These  studies  will 
include  routine  clinical  evaluations  to  detect  adverse  response  to  the  drug  as 
well  as  specialized  clinical,  neuropsychological,  imaging,  neurochemical, 
neurovirologic ,  and  immunologic  studies  to  measure  the  efficacy  of  the  drug 
regimens  employed  in  this  trial. 


16   -  BFSB/DIR 


PHS  6040  (Rev.  1/84) 


GPO  91  4-818 


BIBLIOGRAPHY 


ZOl  NS  02652-06  BFSB 


Gilboe  DD,  Kintner  DB,  Emoto  SE,  Fitzpatrick  JH.  Brain  intracellular  pH  during 
graded  hypoxia  and  subsequent  reoxygenation.  Am  J  Physiol;  (in  press). 

Gilboe  DD,  Fitzpatrick  JH,  Kintner  D,  Emoto  SE,  Bazar  NG,  Braquet  P. 
Biochemical  changes  in  normoxic  and  post  ischemic  brain  tissue  following 
treatment  with  BN52021.   In:  Braquet  P,  ed.  Ginkgolides  -  chemistry,  biology, 
pharmacology  and  clinical  perspectives,  vol  2.  Barcelona:  JS  Prous  Science; 
(in  press) . 

Eldridge  R,  Denkla  MB,  Bien  E,  Myers  S,  Kaiser  MI,  Pikus  A,  Schlesinger  SL, 
Parry  DM,  Dambrosia  JM,  Zasloff  MA,  Mulvihill  JJ .   von  Recklinghousen 
neurofibromatosis:   neurological  and  cognitive  assessment.   Am  J  Dis  Child 
1989;  143:933-7. 

Von  Lubitz  DKEJ ,  Dambrosia  JM,  Redmond  DJ .   Protective  effect  of  cyclohexyl 
adenosine  in  the  treatment  of  cerebral  ischemia  in  gerbils.   Neuroscience 
1989;  30:451-62. 

Nelson  KB,  Ellenberg  JH.   Prenatal  and  perinatal  antecedents  of  febrile 
seizures.   Ann  Neurol  1990;  27:127-31. 

Zhang  Z-X,  Anderson  DW,  Lavine  L,  Mantel  N.   Patterns  of  acquiring 
parkinsonism- dementia  complex  on  Guam:  1944  through  1985.   Arch  Neurol;  (in 
press) . 

Zang  Z-X,  Anderson  DW,  Mantel  N.   Geographic  patterns  of  parkinsonism- dementia 
complex  on  Guam:  1956  through  1985.  Arch  Neurol;  (in  press). 

Zhao  F,  Emoto  SE,  Lavine  L,  Nelson  KB,  Wang  C,  Li  S,  Cheng  X,  Bolis  CL, 
Schoenberg  BS .   Risk  factors  for  febrile  seizures  in  the  People's  Republic  of 
China.   Epilepsia;  (in  press). 

McShane  LM,  Clark  LC ,  Combs  GF,  Turnball  BW.   Reporting  the  accuracy  of 
biochemical  measurements  for  epidemiologic  and  nutrition  studies.   Am  J  Clin 
Nutr ;  (in  press) . 

Kessler  M J ,  London  WT,  Madden  DC,  Dambrosia  JM,  Hillard  JK,  Soike  KF,  Rawlings 
RG .  Serological  survey  for  viral  diseases  in  the  Cayo  Santiago  rhesus  Macaque 
population  course.   Puerto  Rico  Health  Sci  J  1989;8:95-7. 

Farwell  JR,  Lee  YJ ,  Hirtz  DG,  Sulzbacher  SI,  Ellenberg  JH,  Nelson  KB. 
Phenobarbital  for  febrile  seizures:  effects  on  intelligence  and  on  seizure 
recurrence.   NEJM  1990;  322: (6)  364-9. 


17  -  BFSB/DIR 


Z01  NS  02490-10  BFSB 


Emoto  SE,  Matthews  PC.   A  Weibull  model  for  dependent  censoring.   Ann  Stat; 
(in  press) . 

Albert  PS.   A  two-state  mixture  model  for  a  time  series  of  epileptic  seizure 
counts.  Biometrics;  (in  press). 

Lee  Y J .   Nonparametric  statistical  methods  for  Salmonella/Ames  mutagenesis 
assay.   Proceedings  of  the  First  International  Conference  on  Statistical 
Computing  1990;  (in  press). 

Dambrosia  JM.   Statistical  and  epidemiological  considerations  for  clinical 
trials  in  neurology.   In:  Porter  R J ,  Schoenberg  BS ,  eds .   Controlled  clinical 
trials  in  neurology.   Boston:  Kluwer  Academic,  1990;  31-57. 

Foulkes  MA.   Design  issues  in  chemosensory  trials.   Arch  Otolaryngol  Head  Neck 
Surg  1990;  116:65-68. 

Ellenberg  JH.  Clinical  trials.   In:  Theodore  W,  ed.  Clinical  Neuro- 
pharmacohology.  Philadelphia:  WB  Saunders;  1990;  vol.8, (1)  15-30. 

Anderson  DW,  ed.  Neuroepidemiology .  A  tribute  to  Bruce  Schoenberg.  Boca  Raton: 
CRC  Press;  (in  press). 

Gourie-Devi  M,  Anderson  DW,  Rao  VN.   Neuroepidemiologic  survey  in  a  developing 
country:   some  questions  and  answers.   In:   Anderson  DW,  ed. 
Neuroepidemiology.  A  tribute  to  Bruce  Schoenberg.   Boca  Raton:  CRC  Press, 
1990;  (in  press). 

Ellenberg  JH.   Biostatistical  collaboration  in  medical  research  (with 
discussants).   Biometrics  1990;  46,  1-32. 

Dambrosia  JM.   Comments  on  some  personal  remarks  on  clinical  trials.  Brazilian 
J  Probability  Stat  1990;  (in  press). 

Albert  PS,  Brown  CH.   The  design  of  a  panel  study  under  an  alternating  Poisson 
process.   Biometrics;  (in  press). 


Z01  NS  02598-08  BFSB 


Tatemichi  TK,  Foulkes  MA,  Mohr  JP,  Hewitt  JR,  Hier  DB,  Price  TR,  Wolf  PA. 
Dementia  in  stroke  survivors  in  the  Stroke  Data  Bank  cohort.   Stroke  1990; 
21:858-66. 

Kittner  SJ ,  Sharkness  CM,  Price  TR,  Plotnick  GD,  Dambrosia  JM,  Wolf  PA,  Mohr 
JP,  Hier  DB,  Kase  CS ,  Tuhrim  S.   Infarcts  with  a  cardiac  source  of  embolism  in 
the  NINDS  Stroke  Data  Bank:   historical  features.   Neurology  1990;  40:281-4. 


18  -  BFSB/DIR 


Project  No.  Z01  NS  02505-10  BFSB 


Significance  to  NINDS  Program  and  Biomedical  Research:   Headache  is  an  area  of 
interest  to  the  NINDS  Program.   It  has  been  reported  in  the  published  medical 
literature  that  there  are  notable  indications  of  the  association  of  migraine  with 
many  other  diseases  such  as  hypertensive  disorders,  functional  heart  diseases, 
cerebral  vascular  accident,  hayfever  etc.   However,  the  results  of  these  studies 
have  not  been  consistent  because  (1)  the  clinical  groups  of  migraine  patients 
under  study  might  often  have  been  biased  by  their  mechanism  of  selection  or  (2) 
the  reports  were  produced  based  on  insufficient  medical  observations.   Further 
studies  with  sufficient  data  and  unbiased- sampling  groups  of  subjects,  therefore, 
are  still  needed. 

Proposed  Coures:   This  project  will  be  subsumed  under  Statistical  Collaboration 
and  Consultation  (Z01  NS  02652-06). 

Publications :   Chen  TC,  Leviton  A.   Asthma  and  exzema  in  children  born  to  women 
with  migraine.   Arch  Neurol;  (in  press). 


19  -  BFSB/DIR 


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ANNUAL  REPORT 

October  1,  1989  through  September  30.  1990 

Developmental  and  Metabolic  Neurology  Branch 

Clinical  Neurosciences  Program,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 


TABLE  OF  CONTENTS 

RESEARCH  SUMMARY  1 

CONTRACTS  5 

PROJECT  REPORTS 

Metabolism  of  Complex  Lipids  of  Nervous  Tissue 

Z01  NS  0081  5-30  DMN  10 

Synthesis  of  Compounds  Analogous  to  Glycolipids 

Z01  NS02162-16DMN  11 

Development  of  Analytical  Methods  for  the  Use  of 

Reseaix  i  Sphingolipidoses 

Z01  NS02163-16DMN  12 

Gaucher's  Disease:  Biochemical  and  Clinical  Studies 

Z01  NS  02453-10  DMN  13 

Molecular  and  Genetic  Studies  of  Niemann-Pick  Disease 

Z01  NS  02657-06  DMN  14 

Clinical  Studies  of  Neurogenetic  Diseases 

Z01  NS  02664-06  DMN  15 

Retroviral  Mediated  Transfer  of  Human  Globin  Genes 

Z01  NS  02730-04  DMN  16 

Gene  Therapy  of  Inherited  Enzyme  Deficiencies 

Z01  NS  02731-04  DMN  17 

Exploration  of  Strategies  for  the  Treatment  of  AIDS 

Z01  NS  02769-03  DMN  18 

Strategies  for  the  Treatment  of  Autoimmune  Neuropathies 

Z01  NS  2770-03  DMN  19 

Modification  of  Growth  Factor  Genes  by  Genes  Targeting 

Z01  NS  02771-02  DMN  20 

Preparation  of  Transgenic  Murine  Analogs  of  Human 

Metabolic  Storage  Disorders 

Z01  NS  02782-02  DMN  21 


Generation  of  Mice  with  Sickle  Cell  Anemia 

Z01  NS  02785-02  DMN  ll 

Synthesis  of  Inhibitors  of  N-Myristoyltransferase 

Z01  NS  02816-01  DMN  zs 


ii 


ANNUAL  REPORT 


OCTOBER  1,   1989  THROUGH  SEPTEMBER  30,  1990 

DEVELOPMENTAL  AND  METABOLIC  NEUROLOGY  BRANCH,  DIR 

NATIONAL  INSTITUTE  OF  NEUROLOGICAL  DISORDERS  AND  STROKE 


ROSCOE  O.  BRADY,  M.D.,  CHIEF 


The  principal  activities  of  the  Branch  include  the  following  areas  of  investigation : 
1.  Basic  studies  of  lipid  and  mucopolysaccharide  synthesis,  catabolism,  and 
enzymatic  abnormalities  in  hereditary  human  disorders.  2.  Investigations  of  the 
molecular  basis  of  metabolic  storage  disorders.  3.  Pathogenic  and  therapeutic 
investigations  in  animal  models  of  disorders  of  metabolism.  4.  Development  of 
transgenic  analogs  of  human  disorders.  5.  Clinical  investigations  of  lysosomal 
storage  disorders  and  neurogenetic  diseases.  6.  Development  of  therapy  for 
patients  with  heritable  diseases.  7.  Development  of  gene  replacement  technology. 

I.  BASIC  INVESTIGATIONS  IN  HEREDITARY  METABOLIC  DISORDERS 

A.  Gaucher's  Disease 

Fundamental  studies  are  in  progress  to  examine  the  effects  of  lipid  activators 
and  small  molecular  weight  protein  "cohydrolases"  on  sphingolipid  degrading 
enzymes.  It  is  anticipated  that  appropriate  combinations  of  these  substances  will 
improve  the  efficiency  of  exogenous  enzymes  administered  to  patients  with  lipid 
storage  disorders.  Maximally  effective  mixtures  of  these  agents  have  been 
identified.  Their  application  to  the  treatment  of  Gaucher's  disease  is  being 
examined  in  animal  and  tissue  culture  modelsof  thisdisorder. 

B.  TypeC  Niemann-Pick  Disease 

We  have  extended  our  investigation  of  the  effect  of  dimethylsulfoxide  (DMSO) 
on  the  intracellular  accumulation  of  unesterified  cholesterol  in  cultured  skin 
fibroblasts  obtained  from  patients  withType  C  Niemann-Pick  disease.  This 
information  is  being  used  in  our  phase  I  clinical  trial  of  DMSO  in  patients  with  this 
disorder. 

C.  Fabry's  Disease 

A  comprehensive  investigation  is  underway  concerning  the  targeting  of 
exogenous  ceramidetrihexosidase  to  cells  in  which  ceramidetrihexoside  accumulates 
in  patients  with  Fabry's  disease.  This  strategy  has  been  employed  with  extraordinary 
success  in  enzyme  replacement  in  Gaucher's  disease.  When  the  pertinent  tissue 
lectins  have  been  conclusively  identified,  a  clinical  trial  with  carbohydrate-modified 
ceramidetrihexosidase  will  be  undertaken  in  Fabry's  disease. 


1   DMN/DIR 


II.  INVESTIGATIONS  OF  THE  MOLECULAR  BASIS  OF  METABOLIC  STORAGE 
DISORDERS 

A.  Type  C  Niemann-Pick  Disease 

Recent  studies  have  provided  exceptionally  promising  leads  concerning  the 
molecular  defect  in  Type  C  Niemann-Pick  disease.  A  protein  polymorphism  has  been 
discovered  in  subcellular  particles  in  an  Hispanic  pedigree  with  severely  affected 
individuals  with  this  disorder.  Investigations  are  underway  to  determine  whether 
this  molecular  variant  is  involved  in,  or  closely  linked  to  Type  C  Niemann-Pick  disease. 

III.  PATHOGENIC  ANDTHERAPEUTIC  INVESTIGATIONS  IN  ANIMAL  MODELS 

A.  Murine  Analog  of  Type  C  Niemann-Pick  Disease 

The  treatment  of  patients  with  Type  C  Niemann-Pick  disease  is  currently  based  on 
the  concept  that  cholesterol  is  the  principal  offending  metabolite  in  this  disorder. 
Investigations  with  a  spontaneously  occurring  murine  analogue  of  the  human 
disease  revealed  that  modifying  the  quantity  of  dietary  cholesterol  can  affect  the  life 
span  of  affected  mice.  Restricting  cholesterol  led  to  increased  longevity  whereas 
cholesterol  supplements  hastened  morbidity  and  mortality.  These  studies  provided 
the  basis  for  our  attempts  to  alter  the  cholesterol  burden  in  patients  with  Type  C 
Niemann-Pick  disease. 


IV.  DEVELOPMENT  OFTRANSGENIC  ANIMAL  MODELS  OF  HUMAN  DISORDERS 

A.  Gaucher's  Disease 

Progress  has  been  made  in  developing  a  transgenic  murine  model  of  Gaucher's 
disease  using  genomic  clones  of  the  mouse  glucocerebrosidase  isolated  by  DMNB 
scientists.  Retroviral  constructs  have  been  made  with  specific  mutations  in  the  gene 
in  orderto  produce  a  transgenic  mouse  analogue  of  Gaucher's  disease. 

B.  Sickle  Cell  Anemia 

Transgenic  mice  have  been  generated  that  contain  the  abnormal  Antilles  beta- 
sickle  globin  gene.  The  human  alpha-globin  gene  isalso  inserted  in  orderto 
generate  mice  that  produce  high  levels  of  beta-sickle  Antilles  and  human  alpha- 
globins.  Transgenic  mice  have  been  produced  in  which  human  globin  chains  are 
produced  at  a  level  between  1 5  and  20  percent  of  mouse  globin.  These  transgenic 
mice  will  be  mated  with  alpha-thalassemic  mice.  In  addition,  founder  mice  will  be 
mated  to  obtain  mice  homozygous  for  the  sickling  trait.  Investigations  with  these 
mice  will  greatly  accelerate  studies  of  therapeutic  agents  to  correct  sickling. 

C.  Transgenic  Mice  Containing  a  Defective  Interleukin  3   (IL-3)  Gene 

A  principal  objective  of  this  investigation  is  to  determine  the  consequences  of 
altering  the  functional  state  of  genes  that  control  cell  growth  and  maturation.  This 
goal  will  be  examined  through  studies  that  include  the  introduction  of  the  IL-3  gene 


2   DMN/D1R 


into  an  IL-3-dependent  cell  line.  Other  attempts  will  be  made  to  develop  transgenic 
mice  with  a  defective  IL-3  gene.  If  such  animals  can  be  produced,  studies  will  be 
undertaken  to  correct  the  induced  growth  and  maturation  problems  through 
homologous  recombination  with  a  normally  functioning  IL-3  gene. 

V.     CLINICAL  INVESTIGATIONS  OF  METABOLIC  DISORDERS  AND  NEUROGENETIC 
DISEASES 

A.  Familial  Diurnally  Variant  Dystonia 

Genetic  linkage  studies  and  an  examination  of  the  mode  of  inheritance  of  this 
condition  that  is  associated  with  low  cerebrospinal  fluid  biopterin  levels  have  been 
carried  out.    Beneficial  effects  of  supplemental  biopterin  have  been  documented  in 
one  of  these  patients. 

B.  Fabry's  Disease 

Symptomatic  therapies  have  been  devised  to  correct  the  autonomic  nervous 
system  dysfunction  that  occurs  in  patients  with  this  disorder.  Specific  treatment 
regimens  are  being  developed  that  relieve  the  painful  acroparesthesias  without 
exacerbating  signs  and  symptoms  of  autonomic  dysfunction.  Additional  studies  are 
directed  toward  correcting  gastric  hypomotility  in  Fabry  patients. 

C.  Lennox-Gastaut  Syndrome 

Potential  metabolic  alterations  in  patients  with  this  syndrome  are  under 
investigation.  Two  patients  with  this  disorder  that  exhibit  non-ketotic 
hyperglycinemia  have  been  identified.  Such  studies  may  lead  to  the  development  of 
specific  remedial  therapy  in  patients  with  this  clinical  presentation. 

D.  Type  C  Niemann-Pick  Disease 

The  incidence  of  seizures  and  the  occurrence  of  cataplexy  have  been  determined 
in  patients  with  this  disorder.  A  neurological  staging  system  to  evaluate  disease 
progression  is  under  development.  The  effect  of  modifying  dietary  cholesterol  and 
drugs  that  alter  cholesterol  production  and  its  intracellular  disposition  are  under 
investigation  in  Type  C  Niemann-Pick  patients. 


VI.  THERAPY  FOR  HEREDITARY  METABOLIC  DISORDERS 

A.  Gaucher's  Disease 

1 .  We  completed  a  pharmacodynamic  investigation  of  the  effect  of  varying 
quantities  of  intravenously  injected  glucocerebrosidase  targeted  to  macrophage 
storage  cells  in  patients  with  Gaucher's  disease.  Reduction  of  hepatic 
glucocerebroside  and  ultrastructural  improvement  of  the  liver  was  observed 
following  infusion  of  this  enzyme.  The  threshold  quantity  of  enzyme  at  which 
these  changes  occur  was  determined.  This  study  provided  the  guideline  for  the 
selection  of  the  therapeutic  dose  of  mannose-terminated  glucocerebrosidase  in 
the  clinical  efficacy  trial  summarized  in  the  following  paragraph. 


3  DMN/DIR 


2.  A  phase  ll/lll  enzyme  replacement  trial  was  carried  out  in  which  the  efficacy  of 
enzyme  replacement  was  evaluated  in  1 2  patients  with  Gaucher's  disease. 
Dramatically  beneficial  responses  followed  biweekly  infusions  of  60  I.U./kg  of 
mannose-terminated  human  placental  glucocerebrosidase.  In  all  patients,  there 
was  striking  improvement  of  the  anemia  and  thrombocytopenia  that  are 
characteristic  of  Gaucher's  disease.  In  addition,  there  was  a  surprisingly  large 
reduction  in  the  size  of  the  spleen  in  the  recipients.  Further,  a  significant 
decrease  of  hepatomegaly  was  also  observed  in  all  of  the  patients.  Evidence  of 
skeletal  improvement  was  seen  in  four  patients.  Major  skeletal  improvement  was 
documented  in  another  patient  with  Gaucher's  disease  treated  with 
macrophage-targeted  glucocerebrosidase  over  a  four-year  period.  These  salutary 
responses  indicate  that  enzyme  replacement  is  effective  and  that  it  is  the  therapy 
of  choice  for  Gaucher's  disease  at  this  time.  These  benefits  augur  well  for  the 
successful  enzyme  replacement  therapy  in  additional  human  metabolic  disorders. 

B.  Type  C  Niemann-Pick  Disease 

A  phase-l  clinical  protocol  to  examine  the  therapeutic  potential  of  orally 
administered  dimethylsulfoxide  (DMSO)  and  other  agents  known  to  modify  organ 
and  tissue  cholesterol  in  patients  with  Type  C  Niemann-Pick  disease  is  nearing 
completion.  The  results  obtained  in  this  investigation  will  be  used  to  develop  a 
protocol  to  examine  their  therapeutic  efficacy  in  Type  C  Niemann-Pick  disease. 


VII.  DEVELOPMENT  OF  GENE  REPLACEMENT  TECHNOLOGY 

A.  Gaucher's  Disease 

High-titer  recombinant  retroviruses  have  been  constructed  that  contain  the 
human  glucocerebrosidase  gene.  The  gene  has  been  transferred  to  human 
hematopoietic  cell  lines  and  progenitor  cells.   Glucocerebrosidase  activity  has  been 
increased  to  normal  levels  following  gene  transfer  into  hematopoietic  progenitor 
cells  obtained  from  patients  with  Gaucher's  disease.  These  results  provide  a  sound 
experimental  basis  for  further  exploration  of  gene  replacement  for  the  treatment  of 
Gaucher's  disease. 

B.  Human  qlobin  disorders 

Investigations  with  retroviral  vectors  containing  the  human  beta-globin  gene 
resulted  in  regulated  expression  of  human  globin  mRNA  and  protein  in  murine 
erythroleukemia  cells.   A  novel  high-titer  retrovirus  vector  was  produced  that 
effectively  transfers  the  human  beta-globin  gene  into  CFU-S  spleen  colonies  that 
express  the  human  beta-gene.  In  addition,  human  beta-globin  protein  has  been 
produced  in  long-term  reconstituted  mice.    Bone  marrow  from  a  primary  murine 
recipient  that  contained  the  human  beta-globin  gene  for  more  than  a  year  was 
transplanted  into  a  secondary  recipient.  The  latter  mouse  expressed  the  human 
beta-globin  gene  in  erythroid  cells  more  than  three  months  after  transplantation. 
This  study  proves  that  the  initial  gene  transfer  was  targeted  to  repopulating  bone 
marrow  stem  cells  and  provides  the  basis  for  further  exploration  of  gene  transfer 
technology  to  correct  human  globin  disorders  such  as  beta-thalassemia. 


4  DMN/DIR 


CONTRACT  NARRATIVE 

DEVELOPMENTAL  AND  METABOLIC  NEUROLOGY  BRANCH 

DIVISION  OF  INTRAMURAL  RESEARCH,  NINDS 

OCTOBER  1,  1989  THROUGH  SEPTEMBER  30,  1990 


Contractor:  GENZYME  CORPORATION,  BOSTON,  MA.  (N01-NS-9-2358) 
Title:  Preparation  of  Ceramidetrihexosidase  from  Human  Placental  Tissue 
Contractor's  Project  Director:  F.  Scott  Furbish 
Current  Annual  Level  of  Support:  $100,000 

Objectives:  To  isolate  human  placental  ceramidetrihexosidase  in  sufficient  purity 
and  quantity  for  use  in  enzyme  replacement  trials  in  patients  with  Fabry's  disease. 

Major  Findings:  A  procedure  has  been  developed  for  the  large-scale  purification  of 
human  placental  ceramidetrihexosidase  in  sufficient  purity  and  specific  catalytic 
activity  so  that  it  can  be  safely  administered  to  patients  with  Fabry's  disease.  Its 
carbohydrate  portion  has  been  analyzed.  We  are  developing  methods  to  target  the 
enzyme  to  cells  and  tissues  where  ceramidetrihexoside  is  stored. 

Significance  to  Biomedical  Research  and  to  the  Program  of  the  Institute: 
A  principal  mission  of  the  Institute  is  to  develop  effective  therapy  for  human 
diseases.  If  salutary  clinical  results  can  be  obtained,  an  extraordinary  milestone  will 
have  been  accomplished  regarding  this  type  of  human  genetic  disease. 

Proposed  Course  of  the  Contract:  We  have  made  significant  progress  in  our  effort  to 
increase  the  delivery  of  this  enzyme  to  specific  cells  in  which  ceramidetrihexoside 
accumulates.  When  this  preclinical  investigation  is  completed,  we  shall  resume 
enzyme  replacement  trials  in  patients  with  Fabry's  disease.  We  shall  examine  the 
effectiveness  of  the  enzyme  with  regard  to  clearance  of  accumulated 
ceramidetrihexoside  in  the  liver,  kidney  and  blood,  and  monitor  clinical  responses  in 
thistherapeutictrial. 


5  DMN/DIR 


CONTRACT  NARRATIVE 


DEVELOPMENTAL  AND  METABOLIC  NEUROLOGY  BRANCH 

DIVISION  OF  INTRAMURAL  RESEARCH,  NINDS 

OCTOBER  1,1989  THROUGH  SEPTEMBER  30,  1990 


Contractor:  GENZYME  CORPORATION,  BOSTON,  MA.    (N01-NS-9-2360) 
Title:  Preparation  of  Sphingomyelinase  from  Human  Placental  Tissue 
Contractor's  Project  Director:  F.  Scott  Furbish 
Current  Annual  Level  of  Support:  $100,000 

Objectives:  To  isolate  human  placental  sphingomyelinase  in  sufficient  purity  and 
quantity  for  use  in  enzyme  replacement  trials  in  patients  with  Niemann-Pick  disease. 

Major  Findings:  A  procedure  is  being  developed  for  the  large-scale  isolation  of 
human  placental  sphingomyelinase.  The  final  purification  steps  will  be  performed  at 
NIH.  The  highly  enriched  enzyme  will  be  administered  to  patients  with  Type  B 
Niemann-Pick  disease  and  its  effect  on  liver  and  blood  sphingomyelin  levels  will  be 
determined. 

Significance  to  Biomedical  Research  and  to  the  Program  of  the  Institute: 
A  principal  mission  of  the  Institute  is  to  develop  effective  therapy  for  human 
diseases.  If  salutary  biochemical  results  can  be  obtained,  a  clinical  efficacy  trial  will 
be  conducted  to  determine  the  response  of  patients  with  Type  B  Niemann-Pick 
disease  to  enzyme  replacement. 

Proposed  Course  of  the  Contract:  We  have  carried  out  experiments  to  isolate  human 
placental  sphingomyelinase  in  a  high  degree  of  purity.  Large  quantities  of  a 
partially  purified  preparation  are  required  in  order  to  obtain  sufficient  enzyme  for 
an  investigation  of  its  effect  on  stored  sphingomyelin  in  Niemann-Pick  patients. 
When  a  sufficient  quantity  of  pure  sphingomyelinase  is  available,  we  shall  determine 
if  it  causes  a  reduction  of  accumulated  lipid  in  the  liver  and  blood  of  patients  with 
this  disorder. 


6  DMN/DIR 


CONTRACT  NARRATIVE 

DEVELOPMENTAL  AND  METABOLIC  NEUROLOGY  BRANCH 

DIVISION  OF  INTRAMURAL  RESEARCH,  NINDS 

OCTOBER  1,  1989  THROUGH  SEPTEMBER  30,  1990 


Contractor:  GENZYME  CORPORATION,  BOSTON,  MA.  (N01-NS-9-2365) 
Title:  Preparation  of  Glucocerebrosidase  from  Human  Placental  Tissue 
Contractor's  Project  Director:  F.  Scott  Furbish 
Current  Annual  Level  of  Support:  $300,000 

Objectives:  To  isolate  human  placental  glucocerebrosidase  in  sufficient  purity  and 
quantity  for  use  in  enzyme  replacement  trials  in  patients  with  Gaucher's  disease. 

Major  Findings:  A  procedure  has  been  developed  for  the  large-scale  purification  of 
human  placental  glucocerebrosidase  targeted  to  cells  in  which  glucocerebroside 
accumulated  in  patients  with  Gaucher's  disease.  The  intravenous  infusion  of  this 
enzyme  causes  a  decrease  in  the  quantity  of  glucocerebroside  stored  in  the  liver  and 
in  association  with  erythrocytes  in  the  blood.  Long-term  administration  of  the 
enzyme  has  caused  significant  hematologic  improvement,  reduction  of 
hepatosplenomegaly,  and  skeletal  benefit  in  13  patients  with  Gaucher's  disease. 

Significance  to  Biomedical  Research  and  to  the  Program  of  the  Institute: 

A  principal  mission  of  the  Institute  is  to  develop  effective  therapy  to  treat  human 

diseases.  The  results  indicated  in  the  preceding  paragraph  indicates  that  we  have 

achieved  this  goal  in  Gaucher's  disease,  the  most  prevalent  human  metabolic  storage 

disorder. 

Proposed  Course  of  the  Contract:  We  shall  determine  the  minimal  amount  of 
exogenous  glucocerebrosidase  required  to  stabilize  patients  in  whom  the  beneficial 
responses  indicated  above  have  occurred.  We  shall  also  determine  the  minimal 
amount  of  enzyme  required  to  bring  about  these  salutary  changes.  In  addition,  we 
shall  examine  enzyme  replacement  therapy  in  patients  with  Type  3  Gaucher's 
disease,  the  chronic  neuropathic  form  of  this  disorder. 


7   DMN/DIR 


CONTRACT  NARRATIVE 

DEVELOPMENTAL  AND  METABOLIC  NEUROLOGY  BRANCH 

DIVISION  OF  INTRAMURAL  RESEARCH,  NINDS 

MARCH  1,  1990  THROUGH  SEPTEMBER  30,  1990 


Contractor:     LIPITEK,  INC.    (NO1-NS-0-2386) 
Title:   Production  of  Radiolabeled  Sphingolipids 
Contractor's  Project  Director:  Alexander  L.  Weis,  Ph.D. 
Current  Annual  Level  of  Support:  $85,200 

Objectives:  To  prepare  glucocerebroside,  sphingomyelin,  and  ceramidetrihexoside 
labeled  with  14C  in  critical  portions  of  the  molecule  for  diagnostic  tests  for  Gaucher's 
disease,  Niemann-Pick  disease,  and  Fabry's  disease. 

Maior  Findings:  Synthetic  chemical  procedures  have  been  developed  to  incorporate 
radioactive  carbon-14  jn  specific  portions  of  sphingolipid  molecules.  These 
compounds  are  used  to  diagnose  patients  with  the  sphingolipid  storage  disorders 
listed  above,  to  identify  heterozygous  carriers  of  these  conditions,  to  diagnose  these 
disorders  prenatally,  and  to  monitor  enzyme  isolation  procedures  for 
glucocerebrosidase,  sphingomyelinase,  and  ceramidetrihexosidase. 

Significance  to  Biomedical  Research  and  to  the  Program  of  the  Institute: 
The  ability  to  diagnose  patients,  identify  heterozygotes,  and  to  monitor  pregnancies 
at  risk  for  sphingolipid  storage  disorders  represents  major  contributions  to  the 
control  of  the  incidence  of  these  diseases.  These  procedures  are  in  wide  use  at  the 
present  time. 

Proposed  Course  of  the  Contract:  The  contract  has  recently  been  negotiated. 
Contractor's  performance  will  be  monitored  for  compliance  under  a  fixed-price 
agreement  concerning  the  reguisite  deliverables. 


8  DMN/DIR 


CONTRACT  NARRATIVE 


DEVELOPMENTAL  AND  METABOLIC  NEUROLOGY  BRANCH 
DIVISION  OF  INTRAMURAL  RESEARCH,  NINDS 
JULY  16,  1990  THROUGH  SEPTEMBER  30,  1990 

Contractor:  UNIVERSITY  OF  NEW  MEXICO  (NO1-NS-0-2394) 

Title:  Preparation  of  Homogeneous  Human  Liver  Sterol  Carrier  Protein-2 
(SCP-2)  And  Production  of  High-Titer  Antibody  TO  SCP-2 

Contractor's  Project  Director:    Terence  J.  Scallen,  M.D.,  PH.D. 

Current  Annual  Level  of  Support:  $74,577 

Objectives:  To  isolate  homogeneous  SCP-2  from  human  livertissue  and  prepare 
anti-SCP-2  monospecific  polyclonal  antibody  for  investigations  in  Type  C  Niemann- 
Pick  disease. 

Major  Findings:  An  isoform  of  SCP-2  appears  to  be  lacking  in  tissue  specimens 
obtained  from  patients  with  Type  C  Niemann-Pick  disease.  We  need  to  determine 
whether  this  finding  is  consistent  in  all  of  the  patients  we  are  monitoring,  and 
whether  exogenous  SCP-2  can  correct  the  impairment  of  cholesterol  homeostasis  in 
cultured  skin  fibroblasts  and  other  specimens  derived  from  patients  with  this 
disorder.  If  these  results  are  positive,  we  will  need  to  determine  the  amino  acid 
seguence  of  SCP-2  and  to  clone  the  gene  for  this  protein  in  order  to  evaluate  the 
molecular  investigations  in  Type  C  Niemann-Pick  disease. 

Significance  to  Biomedical  Research  and  to  the  Program  of  the  Institute: 

The  ability  to  identify  biochemical  and  molecular  abnormalities  in  patients  with 

previously  unsolved  metabolic  disorders  is  a  major  goal  of  the  Institute.  Acguisition 

of  this  information  is  likely  to  be  required  to  develop  effective  therapy  for  such 

conditions. 

Proposed  Course  of  the  Contract:  The  contract  has  just  been  awarded  and  SCP-2  and 
its  antibody  will  be  used  in  metabolic  and  molecular  investigations  in  Type  C 
Niemann-Pick  disease. 


9  DMN/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS00815-30DMN 


PERIOD  COVERED 

October  1,  1989  to  September  30,  1990 


TITLE  OF  PROJ  ECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Metabolism  of  Complex  Lipids  of  Nervous  Tissue 


PRINCIPAL  INVE  STIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:      R.O.  Brady,  M.D. 
Others:     P.G.  Pentchev,  Ph.D. 

R.R.  O'Neill,  Ph.D. 

J.M.  Quirk,  M.S. 

C.  Roff,  Ph.D. 

E.Goldin,  Ph.D. 

M.  Comly,  B.S. 
A.  Coonev.  B.S. 


Chief 

DMN 

NINDS 

Section  Chief 

DMN 

NINDS 

Senior  Staff  Fellow 

DMN 

NINDS 

Biochemist 

DMN 

NINDS 

Special  Expert 

DMN 

NINDS 

Visiting  Fellow 

DMN 

NINDS 

Biologist 

DMN 

NINDS 

Bioloaist 

DMN 

NINDS 

COORPE  RATING  UNITS  Of  any) 

Laboratory  of  Cellular  and  Developmental  Biology,  NIDDKD,  Laboratory  of  Biochemistry,  Faculty  of 
Medicine,  Lyon-Sud,  France 


LAB/BRANCH 

Developmental  and  Metabolic  Neurology  Branch 


SECTION 

Molecular  and  Cellular  Pathophysiology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 

6.0 


OTHER: 

2.0 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  QT]  (b)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Summary:  The  metabolic  defect  in  patients  with  Types  C  and  D  Niemann-Pick  disease  has  been  shown  to 


be  due  to  abnormal  intracellular  cholesterol  homeostasis.  The  molecular  lesion  in  these  disorders  results 
in:  (1)  failure  to  down-regulate  LDL  receptors  on  cell  membranes;  (2)  lack  of  down-regulation  of 
HMGCoA  reductase,  a  key  enzyme  in  cholesterol  biosynthesis;  and  (3)  inability  to  up-regulate  acyl 
cholesterol  acyl  CoA  transferase,  the  enzyme  that  catalyzes  the  esterification  of  intracellular  cholesterol. 
Tests  have  been  developed  and  introduced  into  medical  practice  for  the  diagnosis  of  Types  C  and  D 
Niemann-Pick  disease  and  the  identification  of  heterozyqotes,  and  the  prenatal  diagnosis  of  these 


conditions.  Current  emphasis  is  on  the  development  of  effective  therapy  for  patients  with  this  disorder 
and  the  elucidation  of  the  molecular  basis  of  this  novel  metabolic  disorder. 


10  DMN/DIR 


PHSMMOIRev    1  84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS02162-16DMN 


PERIOD  COVERED 

October  1,  1989  to  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  'ess.   Title  must  fit  on  one  line  between  the  border*.) 

Synthesis  of  Compounds  Analogous  to  Glycolipids 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator J  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:     S  P.  Miller,  Ph.D. 
Others:    S.  A.  French,  B.S. 


Senior  Staff  Fellow 
Chemist 


DMN  NINDS 
DMN  NINDS 


COORPERATING  UNITS  Of  an,  ) 


LAB/BRANCH 

Developmental  and  Metabolic  Neurology 


SECTION 

Neurochemical  Methology  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD      20892 


TOTAI  MAN-YEARS: 


08 


PROFESSIONAL: 


0.5 


OTHER: 


0.3 


CHECK  APPROPRIATE  BOX(ES) 

| J   (a)  Human  subjects 

]  (a1)  Minors 
J   (a2)  Interviews 


[~x  |  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Summary:  The  object  of  this  work  is  to  prepare  artificial  substrates  of  sphinqolipid  hydrolases  that  can 
detect  enzyme  activity  within  intact  cells    Chromogenic  substrates  synthesized  earlier  in  this  section  have 
proven  highly  useful  for  enzyme  measurements  in  vitro.  However,  the  requirements  of  in  vivo 
measurement  of  enzyme  activity  necessitate  the  preparation  of  fluoroqenic  glycolipids.  The  substrates 
required  for  such  an  approach  should  be  nonfluorescent  prior  to  enzymatic  cleavage  of  the  glycoside 
bond.  The  released  product  should  then  be  fluorescent  at  lysosomal  pH.  A  group  of  strongly  fluorescent 
phenols,  obtained  by  0-4  monoalkylation  of  2,3-dicyanohydroquinone  (DCH)  were  synthesized  for  this 
project.  These  were  converted  into  gjucosides  and  qalactosides  which  were  then  tested  as  enzyme 
substrates.  Most  interesting  was  the  beta-qalactoside  of  a  lysosomotropic  derivative  of  DCH.  The 
membrane-permeant  tetraacetate  of  this  substrate  was  hydrolysed  within  intact  human  fibroblasts. 
Moreover,  beta-galactosidase  deficient  fibroblasts  derived  from  patients  with  Gmi  gangliosidosis 
hydrolysed  this  compound  at  6-17%  of  the  rate  seen  with  normal  cells.  Attempts  to  apply  these 
techniques  to  the  beta-glucocerebrosidase  which  is  deficient  in  Gaucher's  disease  are  in  progress. 
Ultimately,  these  probes  will  be  used  to  measure  the  efficiency  of  gene-transfer  experiments  with 
lysosomal  hydrolase  genes.  In  this  setting,  jn  vivo  fluorogenic  substrates  have  the  potential  to  greatly 
increase  the  sensitivity  for  detection  of  gene  expression  in  transformed  cells.  In  conjunction  with 
fluorescence  activated  cell  sorting,  fluorogenic  probes  should  also  expand  the  possibilities  for  study  of 
the  developmental  biology  of  these  recombinant  cells. 


11   DMN/DIR 


PHSSMOIRe-   184) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS02163-16DMN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Development  of  Analytical  Methods  for  the  Use  of  Research  of  Sphingolipidoses 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  S. P.  Miller,  Ph.D.  Senior  Staff  Fellow  DMN      NINDS 


COORPERATING  UNITS  (if  my) 


LAB/BRANCH 

Developmental  and  Metabolic  Neurology 


SECTION 

Neurochemical  Methodology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD.  20892 


TOTAL  MAN-YEARS: 


0.2 


PROFESSIONAL: 


0.2 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I      I   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


[x~l  (b)  Human  tissues  ]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

New  analytical  techniques  were  developed  and  used  in  enzymatic  research  and  in  clinical  investigations 

of  lipidoses.  Quantitation  of  all  major  phospholipid  and  neutral  lipid  species  was  performed  on  hepatic 

biopsy  specimens  from  Type  C  Niemann-Pick  patients.  Analytical  techniques  were  applid  to  the  study  of 

glycerolphosphorylcholine  requlation  in  kidney  cells.  Structural  studies  of  polyglycosylated  saponins 

were  carried  out.  Techniques  were  developed  for  the  quantitation  and  study  of  new  fluorescent  enzyme 

substrates. 


12  DMN/DIR 


PHS  6040  (Rev.  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS02453-10DMN 


PERIOD  COVERED 

Octoberl,  1989  to  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Titlemust  fit  on  one  line  between  the  borders.) 

Gaucher's  Disease:  Biochemical  and  Clinical  Studies 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator  )  (Name,  title,  laboratory,  and  institute  affiliation) 

Chief 

Section  Chief 
Special  Volunteer 
Biologist 

Special  Volunteer 
Special  Volunteer 
Special  Volunteer 


PI: 

R  0  Brady,  M  D 

OTHERS: 

N  Barton,  M.D.,Ph  D 

G.  Murray,  PhD 

G.  Zirzow,  B.S. 

S.  Reisz,  B.S. 

K.  Howard,  B.S. 

F.S.Jin,  M.D. 

DMN 

NINDS 

DMN 

NINDS 

DMN 

NINDS 

DMN 

NINDS 

DMN 

NINDS 

DMN 

NINDS 

DMN 

NINDS 

COORPERATING  UNITS  (.f  any) 

Massachusetts  Gen.  Hospital,  Dept.  of  Orthopedic  Surgery,  Boston,  MA:  (H  Mankin,  D  Rosenthal, 
S.  Doppelt);  Children's  Hospital,  Washington,  D.  C.  (P.  Guzetta) 


LAB/BRANCH 

Developmental  and  Metabolic  Neurology 


SECTION 

Clinical  Investigations  &  Therapeutics  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


5.5 


PROFESSIONAL: 


35 


OTHER: 


2.0 


CHECK  APPROPRIATE  BOX(ES) 

I      I   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


|~x  |  (b)  Human  tissues  ]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Summary:  Successful  therapy  for  Gaucher's  disease  depends  upon  a  comprehensive  clinical  and  basic 
scientific  knowledge  of  the  disorder.  Patients  have  been  extensively  studied  and  complications 
identified    Research  on  qlucocerebrosidase  addresses  the  biochemistry,  cell  biology,  and  molecular 
genetics  of  the  enzyme.  A  pharmacodynamic  assessment  of  mannose-terminated  human  placental 
glucocerebrosidase  has  been  completed.    Extraordinarily  gratifying  results  have  been  obtained  with 
enzyme  replacement  therapy  in  patients  with  Gaucher's  disease. 


13  DMN/DIR 


PHS  6040  (Rev.  1/841 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS02657-06DMN 


PERIOD  COVERED 

October  1 ,  1 989  to  September  30,  I990 


TITLE  OF  PROJECT  [80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Molecuar  and  Genetic  Studies  of  Niemann-Pick  Disease 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:      N.  Barton,  M.D., Ph.D.  Section  Chief  DMN      NINDS 

OTHER:       K.  Oliver,  B.S.  Biologist  DMN      NINDS 


COORPERATING  UNITS  (•!  any) 


LAB/BRANCH 

Developmental  and  Metabolic  Neurology 


SECTION 

Clinical  Investigations  and  Therapeutics 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD. 


20892 


TOTAL  MAN-YEARS: 


0.9 


PROFESSIONAL: 


0.3 


OTHER: 


0.6 


CHECK  APPROPRIATE  BOX(ES) 

I  x  |   (a)  Human  subjects 
|  x  1  (a1)  Minors 

J  (a2)  Interviews 


I  x   |  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Niemann-Pick  disease  is  a  proqressly  debilitating,  neurogenetic  disorder  which  is  characterized 
biochemically  by  the  accumulation  of  sphingomyelin  in  several  tissues  and  organs  in  conjunction  with 
deficiency  of  the  lysosomal  hydrolase,  sphingomyelinase.  Detailed  description  of  various  phenotypes  in 
terms  of  cellular  pathochemistry  and  molecular  genetics  has  not  been  accomplished  to  date.  A  major 
obstacle  in  this  area  has  been  the  absence  of  reproducible  techniques  for  the  isolation  of  homogeneous 
preparations  of  sphingomyelinase.  Employing  novel  detergent  and  chromography  systems,  we  have 
purified  sphingomyelinase  to  homogeneity.  The  purified  enzyme  migrates  with  an  apparent  molecular 
weight   of  67,000  daltons  is  SDS-polyacrylamide  gels  under  both  reducing  and  nonreducing  conditions. 
Kinetic  analyses  and  determinations  of  the  primary  protein  structure  and  carbohydrate  composition  are 
in  progress.  Polyclonal  antibodies  have  been  raised  to  the  purified  enzyme  that  will  assist  in  cloning  the 
c-ene  for  sphingomyelinase.  Characterization  of  the  phenotypes  of  Niemann-Pick  disease  in  terms  of 
protein  polymorphisms  and  specific  mutations  at  the  DNA  level  will  be  undertaken. 


14  DMN/DIR 


PHS6(M0(Kev   1/M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS02664-06DMN 


PERIOD  COVERED 

October!.  1989  to  September  30.  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  fit  on  one  line  between  the  borders } 

Clinical  Studies  of  Neurogenetic  Diseases 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  In  vestigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:     N.    Barton,  M.D., Ph.D.  Section  Chief  DMN  NINDS 

OTHERS:     R.     Brady,  M.D.  Chief  DMN  NINDS 

J.     Fink,  M.D.  Sen.  Staff  Fellow  DMN  NINDS 

K.    Yu,  M.D.  Visiting  Associate  DMN  NINDS 

R.    Grewal,  M.D.  Visiting  Associate  DMN  NINDS 


COORPERATING  UNITS  OUn,) 

Armed  Forces  Institute  of  Pathology,  (  K.  Ishak) 
and  S  Doppelt) 


Massachusetts  General  Hospital,  (H.  Mankm  and 


LAB/BRANCH 

Developmental  and  Metabolic  Neurology  Branch 


SECTION 

Clinical  Investigations 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,   MD.  20892 


TOTAL  MAN-YEARS 


7.0 


PROFESSIONAL: 


65 


OTHER 


05 


CHECK  APPROPRIATE  BOX(ES) 

1  x  |   (a)  Human  subjects 
|  x  |  (a1)  Minors 
J   (a2)  Interviews 


[x~|  (b)  Human  tissues  ]  (c)  Neither 


Summary  OF  WORK  (use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

A  number  of  novel  neurogenetic  diseases  have  been  identified  by  studies  carried  out  under  this  project. 
Genetic  linkage  studies  are  underway  in  familial  diurnally  variant  dystonia  associated  with  low  CSF 
biopterin  levels  and  a  beneficial  effect  of  supplemental  biopterin  has  been  documented  in  one  of  these 
patients.  Corrective  therapy  has  been  developed  for  autonomic  nervous  system  dysfunction  associated 
with  Fabry's  disease.    Potential  metabolic  alterations  in  the  Lennox-Gastaut  syndrome  have  been 
examined.  The  incidence  of  seizures  and  cataplexy  has  been  determined  in  patients  with  Type  C 
Niemann-Pick  disease.  The  effect  of  modifying  dietary  cholesterol  and  the  responses  of  patients  to 
drugs  that  alter  cholesterol  synthesis  are  under  investigation  in  this  disorder. 


15   DMN/D1R 


miHMII(«t»   I'M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS02730-04DMN 


PERIOD  COVERED 

October  1,  1989  to  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Retroviral  Mediated  Transfer  of  Human  Globin  Genes 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:  S.  Karlsson,  M.D.,Ph.D 

OTHERS:  D.  Bodine,  Ph.D. 

L.  Perry,  B.S. 
A.  Nienhuis,  M.D. 


Acting  Chief,  M&MG 
Sen.  Staff  Fellow 
Biologist 
Chief 


DMN  NINDS 

CHB  NHLBI 

DMN  NINDS 

CHB  NHLBI 


COORPERATING  UNITS  i,h-.,i 

National  Heart,  Lung  and  Blood  Institute 


LAB/BRANCH 

Developmental  &  Metabolic  Neurology 


SECTION 

Molecular  &  Medical  Genetics  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD. 


20892 


TOTAL  MAN-YEARS: 


2.3 


PROFESSIONAL: 


1.5 


OTHER: 


0.8 


CHECK  APPROPRIATE  BOX(ES) 

J   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


]  (b)  Human  tissues  [T]  (c)  Neither 


Summary  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Retroviral  vectors  can  be  used  to  transfer  genes  effectively  into  cell  lines  and  primary  cells.  Less  is  known, 
however,  whether  these  vectors  can  be  used  to  transfer  genomic  genes  into  tissue  culture  cells  and 
animals  to  yield  tissue-  specific  expression  of  the  transferred  gene.  We  have  now  shown,  that  retroviral 
vectors  carrying  the  human  beta-globin  gene  can  lead  to  regulated  expression  of  human  globin  RNA  and 
protein  in  murine  erythroleukemia  cells.  In  addition,  we  have  recently  designed  a  new  high  titer 
ecotropic  retroviral  vector  that  has  been  used  to  transfer  the  human  beta  globin  gene  into  CFU-S  murine 
spleen  colonies.  The  human  beta-  globin  gene  is  expressed  in  a  vast  majority  of  the  CFU-S  colonies 
indicating  preferential  integration  at  transcriptionally  active  sites,  the  expression  level  of  the  transferred 
human  beta  globin  gene  was  found  to  be  1-5%  of  the  mouse  beta  globin- genes. 

Infected  bone  marrow  was  transplanted  into  genetically  anemic  WAA/v    mice  resulting  in  production  of 
h;jman  beta  globin  chains  in  circulating  red  cells  of  long  term  reconstituted  mice.   The  human  beta- 
globin  gene  is  detected  in  all  hematopoietic  lineages  of  these  mice  and  it  is  expressed  in  a  tissue-specific 
manner.  Bone  marrow  from  a  primary  recipient  that  had  contained  the  beta-  globin  gene  for  a  whole 
year  was  transplanted  into  a  secondary  recipient.  The  secondary  recipient  contains  and  expresses  the 
human  beta-globin  gene  in  erythroid  cells  more  than  three  months  after  transplantation  proving  that 
the  initial  gene  transfer  was  targeted  to  repopulating  bone  marrow  stem  cells.  Furthermore  it  has  been 
demonstrated,  that  the  growth  factors  IL-3  and  IL-6  preserve  stem  cell  function  in  culture  and  enhance 
retroviral-mediated  gene  transfer  into  murine  hematopoietic  stem  cells. 


16   DMN/DiR 


PHSbMO(Rev.  VIM) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS02731-04DMN 


PERIOD  COVERED 

October  1, 1989  to  September  30,  1990 


TITLE  OF  PROJECT  (SO  characters  or  less    Title  must  fit  on  one  fine  between  rne  borden.) 

Gene  Therapy  of  Inherited  Enzyme  Deficiencies 


PRINCIPAL  INVESTIGATOR  (i/srofne/,pror'ess/ona/pe/'sonne/6e/otvfhePnnc/pa//nvesf/gafor  J  (Name.  title,  laboratory,  and  institute  affiliation) 


PI: 

S.  Karlsson,  M.D. 

,Ph.D. 

Acting  Chief,  M&MG 

OTHERS: 

J.  Fink,  M.D. 

Senior  Staff  Fellow 

L.  Perry,  B.S. 

Biologist 

P.  Correll.B.S. 

Special  Volunteer 

Y  Kew,  B.S. 

Biologist 

R.  Brady,  M.D. 

Chief 

DMN 

NINDS 

DMN 

NINDS 

DMN 

NINDS 

DMN 

NINDS 

DMN 

NINDS 

DMN 

NINDS 

COORPERATING  UNITS  (.( *nY> 


LAB/BRANCH 

Developmental  and  Metabolic  Neurology 


SECTION 

Molecular  and  Medical  Genetics 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland 


20892 


TOTAL  MAN-YEARS: 


9.0 


PROFESSIONAL: 


4.5 


OTHER: 


4.5 


CHECK  APPROPRIATE  BOX(ES) 

I «   ]   (a)  Human  subjects 
]  (a1)  Minors 

J   (a2)  Interviews 


I      |  (b)  Human  tissues  ]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 
Gaucher's  disease  is  an  inherited  disorder  caused  by  a  mutation  of  the  gene  for  the  enzyme 
qlucocerebrosidase.  The  normal  gene  for  this  enzyme  has  been  cloned  by  several  laboratories.  We  have 
constructed  high-titer,  helper-free  recombinant  retroviruses  containing  this  gene    We  have  shown  that 
infection  of  cell  lines  from  normal  individuals  and  patients  with  Gaucher's  disease  with  this  retroviral 
vector  results  in  increased  glucocerebrosidase  activity.  The  glucocerebrosidase  gene  has  been  transferred 
efficiently  into  progenitor  cells  and  repopulating  stem  cell  of  mouse  bone  marrow  and  is  expressed  at  the 
RNA  and  protein  level  in  the  progeny  of  CFU-S  multipotential  progenitor  cells  following  gene  transfer. 
The  gene  has  also  been  transferred  efficiently  into  murine  hematopoietic  stem  cells  that  can  be  used  to 
repopulate  secondary  transplant  recipients.  The  vector  genome  can  be  detected  in  all  hematopoietic 
lineages  and  produces  human  glucocerebrosidase  RNA  in  all  hematopoietic  tissues  tested.  The  human 
glucocerebrosidase  gene  has  been  introduced  into  human  hematopoietic  progenitor  cells  with  a  high- 
degree  of  efficiency  (at  least  35%).  Vector  transduced  hematopoietic  progenitors  from  Gaucher's 
patients  produce  progeny  cells  with  glucocerebrosidase  enzyme  values  similar  to  those  of  normal 
individuals. 


17   DMN/DIR 


PHUMIKIItv    1,114) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS02769-03DMN 


PERIOD  COVERED 

October  1,  1989  to  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  trie  borders.) 

Exploration  of  Strategies  for  the  Treatment  of  AIDS 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


Pi:     R.  O.  Brady,  M.D. 
OTHERS:     R.  R.  O'Neill,  Ph.D. 


/  the  Principal , 

Chief 
Staff  Fellow 


DMN 
DMN 


NINDS 
NINDS 


COORPERATING  UNITS  (if  any) 


LAB/BRANCH 

Developmental  and  Metabolic  Neurology 


SECTION 

Enzymology  and  Genetics 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD.      20892 


TOTAL  MAN-YEARS: 


1.5 


PROFESSIONAL: 


0.5 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

]   (a)  Human  subjects 
]  (a1)  Minors 

]  (a2)  Interviews 


J  (b)  Human  tissues  [  x   |  (c)  Neither 


SUMMARY  OF  WORK  (use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 
Two  novel  approaches  for  the  treatment  of  AIDS  will  be  examined.  The  first  encompasses  the 
development  of  anti-retroviral  agents  that  traverse  the  blood-brain  barrier.  The  second  utilizes 
molecular  biological  approaches  to  inhibit  virus  replication. 


18  DMN/DIR 


PHSf>(MO(Kev   1.84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01-NS2770-03DMN 


PERIOD  COVERED 

Octoberl,  1989  to  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  lea.    title  must  fit  on  one  line  between  the  border*  I 

Strategies  for  the  Treatment  of  Autoimmune  Neuropathies 


PRINCIPAL  INVESTIGATOR  R/tt  other  professional  petvonne/ 6-  'iwrhe  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:  R    O    Brady,  M.D 

OTHERS:  R     H.  Quarles,  Ph  D 

M.C.  Dalakas,  PhD 
N.  W.  Barton,  M.  D., 
A.    L.    Weis,  PhD 


Ph  D 


Chief 
Chief 
Unit  Chief 
Section  Chief 


DMN 

NINDS 

LMCN 

NINDS 

MN 

NINDS 

DMN 

NINDS 

Eastman  Kodak  Co 


COORPERATING  UNITS  (,fany) 

Laboratory  of  Molecular  and  Cellular  Neurobiology 

Eastman  Kodak  Company  Resarch  Laboratories,  Rochester,  NY. 


LAB/BRANCH 

Developmental  and  Metabolic  Neurology  Branch 


SECTION 

Enzymology  and  Genetics 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,   Bethesda,  Maryland 


20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


1.5 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I  x   I   (a)  Human  subjects 
]  (a1)  Minors 
]  (a2)  Interviews 


~]  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  (use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

A  number  of  patients  with  peripheral  neuropathy  associated  with  benign  monoclonal  qammopathy  have 
been  shown  to  have  circulating  antibodies  to  specific  glycoconjugates    The  principle  immunoreactive 
epitopes  in  a  number  of  these  individuals  have  been  identified    It  is  proposed  that  immunoaffinity 
columns  be  prepared  with  appropriate  ligands  for  selective  apheresis  trials  to  remove  the  reactive 
immunoglobulin  from  the  patients's  serum  and  to  assess  the  effect  tof  this  procedure  on  the  clinical 
course  of  the  patients. 


19  DMN/DiR 


PHSbMOIltev    1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS02771-02DMN 


PERIOD  COVERED 

October  1 ,  1 989  to  September  30,  1 990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Modification  of  Growth  Factor  Genes  by  Gene  Targeting 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:    A.  Kulkarni.Ph.D. 
OTHERS:    S.  Karlsson,  M.D.,  Ph.D. 
L.  Perry,  B.  S. 
L.  Cairns,  Ph.D. 


Sen.  Staff  Fellow 

DMN 

NINDS 

Acting  Section  Chief 

DMN 

NINDS 

Biologist 

DMN 

NINDS 

Visiting  Fellow 

DMN 

NINDS 

COORPERATING  UNITS  (if  any) 


LAB/BRANCH 

Developmental  and  Metabolic  Neurology  Branch 


SECTION 

Molecular  and  Medical  Genetics 


INSTITUTE  AND  LOCATION 

NINDS,  NiH.Bethesda,  MD.   20892 


TOTAL  MAN-YEARS: 


2.5 


PROFESSIONAL: 


2.0 


OTHER: 


0.5 


CHECK  APPROPRIATE  BOX(ES) 

]   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  QT]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type-  Do  not  exceed  the  space  provided.) 

Gene  targeting  by  homologous  recombination  can  be  used  to  activate  or  inactivate  cellular  genes  in 
eukaryotic  cells.  The  objective  of  this  project  is  to  alter  the  functional  status  of  genes  that  control  growth 
and  maturation  of  specific  tissues  and  study  the  biological  consequences  of  these  molecularly  defined 
alterations.  The  interleukin  3  gene  has  been  chosen  for  this  study  as  it  is  a  well- characterized  gene 
whose  biological  funtions  are  well  known.  Activation  of  the  interleukin  3  (IL-3)  gene  will  be  attempted 
in  a  cell  line  (32-D)  which  is  IL-3  dependent  for  growth.  Successful  activation  will  therefore  lead  to  a 
selective  phenotype  which  will  minimize  screening  and  iidentification  of  the  cells  where  the  gene  has 
been  targeted  to  the  correct  location.  Similarly,  the  biological  importance  of  the  IL-3  gene  to  its 
will  be  studied  in  the  context  of  a  whole  organism  by  targeting  a  defective  IL-3  gene  to  its  cognate 
counterpart  in  embryonic  stem  cells  by  homologous  recombination.  These  cells  will  subsequently  be 
used  to  generate  transgenic  mice  containing  a  defective  IL-3  gene.     Using  the  same  approach,  attempts 
are  being  made  to  target  the  transforming  growth  factor  beta  gene  in  embryonic  stem  cells  in  order  to 
better  define  the  biological  role  of  this  growth  factor  gene. 


20  DMN/D1R 


PHS  6040  (Rev.  1/114) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS02782-02DMN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (SO  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Preparation  of  Transgenic  Murine  Analogs  of  Human  Metabolic  Storage  Disorders 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  R.R.  O'Neill,  Ph.D.  Senior  Staff  Fellow  DMN  NINDS 

Others:  S  Karlsson,  M.D.,  Ph.D.  Acting  Section  Chief  DMN  NINDS 

E.Carstea,  Ph.D.  Staff  Fellow  DMN  NINDS 

L.  Cairns,  PhD.  Visiting  Fellow  DMN  NINDS 

A.  Kulkarni.Ph.D.  Senior  Staff  Fellow  DMN  NINDS 

R.  Brady,  M.D.  Chief  DMN  NINDS 


COORPERATING  UNITS  f/f  any; 


LAB/BRANCH 

Developmental  and  Metabolic  Neurology 


SECTION 

Enzymology  and  Genetics 


INSTITUTE  AND  LOCATION 

NINDS,  NIH.Bethesda,  MD.  20892 


TOTAL  M",f    "EARS: 


3.0 


PROFESSIONAL: 


3.0 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I      [   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


J  (b)  Human  tissues  I  x   |  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Summary:  Molecular  genetic  principles  will  be  used  to  produce  murine  analogs  of  inherited  human 
diseases  for  which  spontaneous  mutants  are  not  available.  Development  of  a  transgenic  mouse  model  of 
Gaucher's  disease  is  a  high  priority  aspect  of  this  project.  Murine  genomic  and  cDNA  clones  were 
obtained,  characterized,  and  mapped  to  mouse  chromosome  3  and  introduced  into  murine  embryonic 
stem  cells  grown  in  cell  culture.  Methods  have  been  devised  using  the  PCR  and  Southern  blot  techniques 
to  identify  which  few  clones  of  the  108  progenitor  cells  that  were  electroporated  are  the  cells  carrying  an 
inactivated  glucocerebrosidase  gene. 


21   DMN/DIR 


PHS  6M0  (Rev   1«4| 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS02785-02DMN 


PERIOD  COVERED 

October  1,  1989  to  September  30,  1990 


TITLE  OF  PROJ  ECT  (SO  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Generation  of  Mice  with  Sickle  Cell  Anemia 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  S.  Karlsson,  M.D.Ph.D. 

OTHERS:  B.  Dropulic,  Ph.D. 

L.  Perry,  B.S. 
A.  Schecter,  M.D. 
C.  Noguchi.Ph.D. 
F.  Shafer.Ph.D 


Acting  Chief,  M&MG 

DMN 

NINDS 

Visiting  Fellow 

DMN 

NINDS 

Biologist 

DMN 

NINDS 

Chief, 

LCB 

NINDS 

Sen.  Scientist 

LCB 

NINDS 

Staff  Fellow 

LCB 

NINDS 

COORPERATING  UNITS  Ofsny) 

Laboratory  of  Chemical  Biology,  NIAID 
Dept.  of  Biology,  Univ.  of  S.  Carolina,  INSERM,  CreteM,  France 


LAB/BRANCH 

Developmental  and  Metabolic  Neurology  Branch 


SECTION 

Molecular  and  Medical  Genetics 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD.  20892 


TOTAL  MAN-YEARS: 


3.7 


PROFESSIONAL: 


2.5 


OTHER: 


1.2 


CHECK  APPROPRIATE  BOX(ES) 

]   (a)  Human  subjects 
]  (a1)  Minors 
]  (a2)  Interviews 


J  (b)  Human  tissues  [  *   |  (c)  Neither 


SUMMARY  OF  WORK  (use  standard  unreduced  type.  Do  not  exceed  the  space  provided) 

Transgenic  mouse  technology  can  be  utilized  to  produce  animals  expressing  a  foreign  gene  to  high  levels 
of  its  RNA  and  protein.  The  objective  of  this  project  is  to  generate  mice  that  contain  an  abnormal  beta- 
globin  gene  (beta-  sickle  Antilles).  This  gene  has  two  mutations  and  generates  sickle  cell  anemia  in  a 
heterozygous  individual.  High  level  expression  of  the  gene  is  obtained  by  inserting  the  dominant  control 
region  from  the  human  beta  globin  locus  in  cis  to  the  Antilles  gene.  This  dominant  control  region  allows 
high  level  globin  expression  to  occur.  The  human  alpha- globin  gene  is  also  inserted  in  cis  in  order  to 
generate  mice  that  can  produce  high  levels  of  beta  sickle  Antilles  and  human  alpha  globins.  Three 
independent  transgenic  mouse  lines  that  express  both  human  globin  genes  in  a  coordinated  fashion  have 
been  generated.  The  production  levels  of  the  human  globin  chains  in  the  founder  mice  is  15-20% 
compared  to  the  mouse  globin  levels.  The  founder  mice  exhibit  sickling  erythrocytes  in  vitro  but  not  m 
vivo.  To  generate  in  vivo  sickling,  the  levels  of  human  globins  will  be  increased  by  breeding  the  animals 
to  homozygosity  and  by  mating  them  with  thalassemic  mice. 


I'd  DMN/DIR 


PHSbMOfllev  l'Bfl) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01NS02816-01DMN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  fit  on  one  line  between  the  borders.) 

Synthesis  of  Inhibitors  of  N-Myristoyltransferase 


PRINCIPAL  \NV£S7\GATOR  (List  other  professional  personnel  belo*  the  Principal  Investigator  )  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  S.P.Miller,  Ph.D.  Senior  Staff  Fellow  DMN  NINDS 

Others:  S.A.  French,  BS  Chemist  DMN  NINDS 

J. M.  Quirk,  M  S.  Biochemist  DMN  NINDS 

R.R.  O'Neill,  Ph.D.  Senior  Staff  Fellow  DMN  NINDS 

R.O.  Brady,  M.D.  Chief  DMN  NINDS 


COORPERATING  UNITS  dtany) 


LAB/BRANCH 

Developmental  and  Metabolic  Neurology 


SECTION 

Neurochemical  Methodology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH.Bethesda,  MD     20892 


TOTAL  MAN-YEARS: 


0.8 


PROFESSIONAL: 


0.4 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

]   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


[x~1  (b)  Human  tissues  ]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Summary:  This  research  project  was  begun  this  year  with  the  object  of  preparing  new  inhibitors  of  the 
enzyme  myristoyl-CoA:  protein  N-myristoyltransferase  (NMT).  This  enzyme  catalyzes  the  covalent 
modification  of  specific  proteins  by  acylation  of  the  N-terminal  amino  group  with  myristoyl-CoA. 
Biomedically  important  proteins  which  are  myristoylated  include  oncoproteins  such  as  p60srcand  the  gag 
polyproteins  of  a  number  of  retroviruses  including  HIV.  Interference  with  myristoylation  has  been  shown 
to  greatly  reduce  the  transforming  potential  of  p60src  without  affecting  its  tyrosine  kinase  activity. 

Blocking  of  the  myristoylation  of  the  HIV  gag  protein  by  site-directed  mutagenesis  (N  terminal  glycine 
alanine)  prevents  viral  particle  release.  Thus,  potent  chemical  inhibitors  of  NMT  should  be  useful  for 
studies  of  the  biochemical  function(s)  of  protein  myristoylation,  and  could  potentially  have  therapeutic 
significance    A  group  of  target  structures  have  been  identified  as  potential  transition  state  or  combined 
product  inhibitors  of  NMT.  Both  irreversible  and  competitive  inhibitors  are  sought.  Synthesis, 
purification  and  characterization  has  been  completed  for  12  new  compounds.  These  are  being  tested  for 
inhibition  of  NMT  both  in  vitro  and  with  cells  in  tissue  culture 


23  DMN/DIR 


PHSMMOIRav   l  «4) 


> 

w 

VI 


X 

m 
30 


30 
> 

5 


H 
00 


Annual  Report 

October  1,  1989  through  September  31,  '990 

Experimental  Therapeutics  Branch 

Clinical  Neurosciences  Program,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 


Table  of  Contents 


RESEARCH  SUMMARY 


page 


Pharmacology  and  Cellular  Biology  of  17 

Peptidergic  Neurons 
Z01  NS  02578-08  ET 

Biochemical  and  Pharmacological  Studies  18 

of  Dopamine  Receptors 
Z01  NS  02263- 14  ET 

Pharmacology  and  Physiology  of  the  19 

Substantia  Nigra  and  Basal  Ganglia 
Z01  NS  02139-16  ET 

Pharmacology,  Biochemistry  and  Physiology  20 

of  Central  Neurotransmitters 
Z01  NS  02265-14  ET 


ANNUAL  REPORT 

October  1,  1989  through  September  31,  1990 

Experimental  Therapeutics  Branch 

Clinical  Neurosciences  Program,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 

Thomas  N.  Chase,  M.D.,  Chief 

The  goal  of  the  Experimental  Therapeutics  Branch  is  to  develop  improved 
pharmacotherapies  for  neurologic  disease.  To  this  end  the  Branch  operates  a 
vertically  integrated  program  of  research  extending  from  basic  neurobiology 
to  clinical  trials.  The  focus  of  these  investigative  efforts  remains  on 
neurodegenerative  disorders  which  impair  motor  and  cognitive  function. 

The  Branch  is  organized  into  four  highly  interactive  operating  components: 
1)  The  Molecular  Pharmacology  Unit,  directed  by  Dr.  David  Sibley,  conducts 
molecular  and  biochemical  investigations  to  characterize  central  transmitter 
receptors  and  information  transduction  processes;  2)  The  Genetic  Pharmacology 
Unit,  since  July  of  1990  led  by  Dr.  M  Maral  Mouradian,  seeks  at  the  molecular 
level  to  develop  pharmaceutical  approaches  to  the  selective  regulation  of 
gene  expression  within  the  mammalian  central  nervous  system  (CNS);  3)  The 
Neurophysiologic  Pharmacology  Section,  operating  under  the  leadership  of  Dr. 
Judith  Walters  at  the  neuronal  network  level,  studies  basal  ganglia  function 
especially  in  relation  to  dopamine  receptor  mechanisms  and  the  effect  of 
drugs  that  influence  motor  behavior;  and  4)  The  Clinical  Pharmacology  Section 
under  Dr.  Thomas  Chase  works  with  neurologic  patients  and  in  animal  models  to 
elucidate  pathophysiologic  mechanisms  and  evaluate  novel  pharmaceutical 
interventions. 

Genetic  Pharmacology  Unit 

Molecular  Regulation  of  Transmitter  and  Receptor  Genes 

The  goal  of  the  Genetic  Pharmacology  Unit  is  the  study  of  molecular 
regulation  of  neurotransmitter  and  transmitter  receptor  genes,  and  through 
this  understanding,  develop  novel  strategies  in  the  design  of  improved 
therapeutic  agents  for  neurologic  disorders.  The  major  thrust  of  the  Unit 
over  the  past  six  years  has  been  examination  of  the  regulation  of 
neuropeptide  gene  expression  using  the  pro-opiomelanocortin  (POMC)  gene  as  a 
model  system.  In  FY90,  the  Unit  initiated  a  program  to  study  the  genetic 
regulation  of  the  D1  and  D2  dopamine  receptor  genes  of  relevance  to  basal 
ganglia  diseases  and  their  treatment. 

In  FY88,  experiments  were  begun  concerning  the  regulatory  elements  in 
the  51  upstream  region  of  the  mouse  POMC  gene.  The  promoter  region  of  this 
gene  was  subcloned  into  pGEM  and  used  in  exonuclease  protection  assays  to 
determine  protein  binding  sequences  that  are  involved  in  modulating 
transcription.  Initial  experiments  conducted  in  FY88-89  have  identified 
several  protein  binding  sites  in  the  -1000  to  +1  bp  region  of  the  mouse  POMC 
promoter.  One  of  these  sites  located  between  -119  and  -106  base  pairs  (bp) 
upstream  from  the  transcription  start  site,  was  found  to  be  homologous  to  the 
consensus  sequence  of  AP-2  (a  DNA-binding  protein  thought  to  convey  second 


1  -  ET/IR 


messenger  signals  to  transcriptional  machinery).  In  FY89,  this  putative  AP-2 
site  was  characterized  in  detail  using  gel  retardation  experiments.  Results 
indicated  that  this  site  binds  to  nuclear  factor(s)  present  in  AtT-20  cells 
in  a  specific  manner,  completely  abolished  with  excess  DNA  of  the  authentic 
AP-2.  In  addition,  this  putative  AP-2  site  appears  physiologically  relevant, 
with  its  ability  to  bind  to  nuclear  factor(s)  dependent  on  phosphorylation. 

In  FY90,  another  strong  exonuclease  stop  site  located  between  -137  and 
-131  bp  from  the  transcription  initiation  site  of  the  POMC  gene  was 
characterized  further  because  of  its  sequence  homology  to  AP-1  binding  site 
and  the  previously  shown  inducibility  of  POMC  transcription  in  AtT-20  cells 
with  phorbol  esters.  Gel  shift  studies  using  a  double-stranded 
oligonucleotide  containing  three  copies  of  this  sequence  and  AtT-20  cell 
nuclear  extract  revealed  that  a  heat  labile  factor(s)  exhibited  specific 
binding  to  this  sequence.  Although  this  DNA  sequence  had  about  70%  homology 
to  the  consensus  sequence  for  AP-1,  its  interaction  to  the  nuclear  factor(s) 
was  unaffected  by  an  oligonucleotide  containing  authentic  AP-1  sequence. 
Furthermore,  using  specific  antibodies  against  each  peptide  component  of  AP- 
1,  c-fos  and  c-Jun,  no  significant  changes  were  noted.  Current  studies 
address  the  role  of  phorbol  ester  treatment  and  phosphorylation  in  the 
interaction  of  nuclear  factor(s)  with  this  new  protein  binding  sequence. 

Also  in  FY90,  studies  of  AtT-20  cell  signal  transduction  machinery 
involved  in  POMC  gene  expression  were  continued.  Based  on  findings  from  this 
laboratory  in  FY88-89  about  the  role  of  protein  kinase  C  (PKC)  in 
corticotroph  releasing  hormone  (CRH)-elicited  8-endorphin  secretion,  signal 
transduction  pathways  in  relation  with  other  secretagogues  were  studied.  The 
cytokine,  interleukin-1  (IL-1),  which  has  been  shown  to  stimulate  8-endorphin 
release  from  AtT-20  cells  and  to  potentiate  the  effects  of  other  agents  such 
as  CRH,  forskolin  and  TPA,  were  studied  in  detail.  Unlike  CRH,  IL-1-induced 
B-endorphin  release  was  found  to  be  independent  of  PKC.  Prolonged  treatment 
with  TPA,  which  was  shown  to  desensitize  an  87-KDa  protein  (a  substrate  for 
PKC),  had  no  effect  on  IL-1-induced  phosphorylation  of  20-,  60-  and  87-KDa 
proteins,  indicating  that  the  phosphorylation  of  these  proteins  does  not 
involve  PKC.  Treatment  of  AtT-20  cells  with  IL-1  markedly  phosphorylated  19-, 
20-  and  60-KDa  proteins  within  minutes,  presumably  by  early  activation  of 
protein  kinases.  Since  IL-1  does  not  generate  cAMP  in  AtT-20  cells,  cAMP- 
dependent  protein  kinase  is  not  involved  either.  The  presence  of  IL-1  was 
required  only  initially  for  a  short  time  to  induce  late  secretion  of  8- 
endorphin  in  AtT-20  cells  suggesting  that  once  IL-1  generates  an  early 
signal,  its  presence  is  no  longer  necessary  for  the  subsequent  secretion  of 
8-endorphin.  Based  on  recent  observations  that  c-fos  and  c-jun  have  an 
important  physiologic  role  in  IL-1-induced  8-endorphin  secretion,  the 
possible  involvement  of  these  proto-oncogenes  in  relation  to  CRH  and  other 
secretagogues  are  currently  being  investigated  using  antisense 
oligonucleotides. 

In  FY90,  studies  aimed  at  investigating  the  molecular  regulation  of 
dopamine  receptor  genes  were  begun.  For  the  D2  receptor,  the  cDNA  of  which 
has  been  recently  cloned  by  several  groups,  efforts  are  currently  underway  to 
clone  the  gene,  including  the  5'  upstream  elements.  Because  the  D2  receptor 
gene  has  long  introns,  and  the  available  cDMA  clones  are  not  full-length,  two 
strategies  are  adopted  to  obtain  genomic  clones  having  the  most  5'  sequences. 
One  strategy  uses  a  5'  stretch  cDNA  library  subjected  to  polymerase  chain 


2  -  ET/IR 


reaction  (PCR),  while  the  second  uses  a  full-length  cDNA  clone  ootained  from 
an  initial  screen  of  a  rat  striatal  cDNA  library  with  oligonucleotide  probes 
homologous  to  the  published  D2  sequence.  We  have  completely  sequenced  the  5' 
region  of  this  cDNA  clone  which  is  upstream  from  all  published  sequences  thus 
far.  In  addition,  this  cDNA  clone  was  subjected  to  PCR  analysis  to  obtain  a 
DNA  copy  of  this  51  region.  Northern  blot  analysis  using  rat  striatal 
poly(A)+  mRNA  has  indicated  that  this  product  hybridizes  with  a  message  of 
the  same  size  as  the  known  rat  D2  mRNA.  Subsequently,  this  PCR  product  was 
used  to  screen  a  rat  genomic  library,  yielding  several  candidate  clones  that 
are  currently  being  characterized.  For  the  D1  dopamine  receptor  which  appears 
to  be  intronless,  the  recently  cloned  cDNA  for  the  rat  D1  receptor  (by  the 
Molecular  Pharmacology  Unit  of  the  Experimental  Therapeutics  Branch)  was  used 
as  a  probe  to  screen  a  human  genomic  library;  this  screen  has  yielded  13 
positive  clones  that  are  now  being  characterized  using  Southern  blots, 
restriction  analysis  and  DNA  sequencing. 

Molecular  Pharmacology  Unit 

The  Molecular  Pharmacology  Unit  continued  investigating  the  biochemical, 
molecular,  and  pharmacologic  properties  of  dopamine  receptors  in  FY90.  The 
long  term  goal  of  the  Unit  is  to  characterize  neurotransmitter  receptor- 
mediated  information  transduction,  and  its  regulation,  across  neuronal 
membranes.  Dopamine  receptors  are  used  as  representative  model  systems  for 
the  large  class  of  neurotransmitter  receptors  which  are  linked  to  their 
biochemical  effectors  via  guanine  nucleotide-binding  regulatory  (G)  proteins. 
In  order  to  characterize  the  D1  and  D2  receptors  at  biochemical  and  molecular 
levels  and  study  their  regulation,  there  are  two  major  interrelated  lines  of 
research  which  are  ongoing:  1)  investigation  of  the  cell  biology,  function 
and  regulation  of  the  receptors  at  the  protein  level;  and  2)  the  molecular 
cloning  of  the  receptor  genes  and  investigation  of  gene  structure  and 
regulation  in  normal  and  pathophysiological  states. 

1.  Cell  Biology  and  Regulation  of  Dopamine  Receptors. 

Our  efforts  in  FY90  have  been  to  capitalize  on  our  discovery  of  various 
mammalian  cell  lines  that  express  either  D1  or  D2  receptors  and  conduct 
studies  of  dopamine  receptor  regulatory  mechanisms.  For  D1  receptors  we  have 
been  utilizing  the  NS20Y  murine  neuroblastoma  cell  line  which  expresses  high 
levels  of  D1  receptors  functionally  coupled  to  the  Gs  protein  and  the 
adenylate  cyclase.  Experimentation  in  FY90  has  indicated  that  acute  exposure 
of  these  cells  to  dopamine  results  in  a  profound  desensitization  of  the  D1 
receptor-stimulated  adenylate  cyclase  activity  as  well  as  a  loss  in  D1 
receptor  radioligand  binding  activity.  This  agonist-induced  desensitization 
response  appears  to  be  homologous  in  nature  as  only  the  dopamine-mediated 
response  is  attenuated.  This  process  also  appears  to  be  multimechanistic 
involving  receptor  uncoupling,  internalization  and  degradation.  We  have  also 
found  in  FY90  that  the  D1  receptors  in  these  cells  can  undergo  a  cAMP- 
mediated  heterologous  form  of  desensitization  where  the  response  to  multiple 
hormones  as  well  as  nonhormonal  stimulators  is  attenuated.  We  are  currently 
investigating  the  underlying  biochemical  and  molecular  mechanisms  associated 
with  these  desensitization  processes  and  are  also  investigating  the  effects 
of  chronic  (days)  agonist  or  antagonist  exposure  as  well  as  evaluating  the 
withdrawal  periods  to  detect  a  potential  supersensitization  response.  In 
addition,  we  are  examining  the  effects  of  treating  the  cells  with  other 


3  -  ET/IR 


agents  such  as  steroid  hormones  to  see  if  they  may  also  modulate  receptor 
activity. 

For  D2  receptors,  we  have  been  characterizing  the  Y-79  human 
retinoblastoma  cell  line  which  expresses  significant  quantities  of  functional 
D2  dopamine  receptors  after  attachment  and  differentiation  with  sodium 
butyrate.  In  FY90  we  have  found  that  pretreatment  of  the  cells  with  dopamine 
results  in  a  diminution  in  the  subsequent  ability  of  dopamine  to  inhibit 
adenylate  cyclase  activity.  This  effect  is  time-dependent,  reaching  maximal 
desensitization  after  ~24  hr.  Preincubation  of  the  cells  with  dopamine  also 
promoted  a  time-dependent  increase  (~3  fold)  in  the  Kd  for  [3H]methylspiperone 
with  no  change  in  its  Bmax-  *n  contrast,  after  dopamine  preincubation,  the 
KD  for  p25i]sulpride  is  unchanged  while  its  Bmax  is  reduced  by  -50?  upon 
maximum  desensitization.  These  results  suggest  that  agonist  preincubation 
results  in  a  functional  uncoupling  of  the  D2  dopamine  receptor  as  well  as  a 
loss  in  its  ligand  binding  activity.  We  have  thus  established  in  FY90  that 
the  Y-79  cell  line  serves  as  an  ideal  model  system  to  study  agonist 
regulation  of  D2  dopamine  receptors  and  are  currently  investigating  the 
underlying  biochemical  and  molecular  mechanisms  associated  with  this 
process(es) . 

In  FY90,  we  completed  development  of  an  irreversible  D2  receptor 
antagonist.  Previously,  we  synthesized  N-(p-isothiocyanatophenethyl) 
spiperone  (NIPS)  a  derivative  of  the  high  affinity  D2  selective  antagonist, 
spiperone.  The  isothiocyanate  moiety  in  this  ligand  renders  it  a  potential 
affinity  probe  of  the  D2  receptor.  This  property  was  evaluated  in  FY90.  In 
vitro  evidence  was  obtained  using  rat  striatal  membranes  for  an  irreversible 
inhibition  of  D2  receptors.  Experiments  in  vivo  indicated  that  NIPS  can 
almost  completely  inactivate  D2  receptors  without  affecting  D1  dopamine 
receptors  in  the  striatum  or  alphal,  alpha2,  or  muscarinic  cholinergic 
receptors  in  the  frontal  cortex  or  5-HTia  receptors  in  the  hippocampus. 
These  results  suggest  that  NIPS  is  a  highly  selective  irreversible 
inactivator  of  D2  dopaminergic  receptors  and  may  prove  useful  in  in  vitro  and 
in  vivo  functional  studies  of  this  receptor  subtype. 

In  FY90  we  continued  our  synthesis  program  for  novel  fluorescently- 
labeled  ligands  with  high  affinity  and  specificity  for  D1  and  D2  dopamine 
receptors.  The  interaction  of  these  fluorescent  ligands  with  dopamine 
receptors  was  evaluated  by  examining  their  ability  to  compete  for  the  binding 
of  the  D1  and  D2  selective  radiolabeled  antagonists.  In  addition,  we 
evaluated  their  affinities  for  5HTia,  5HTic,  and  5HT2  serotonin  receptors. 
In  order  to  further  evaluate  the  utility  of  these  fluorescent  probes  for  the 
cellular  visualization  of  dopamine  receptors,  we  have  collaborated  with  Dr. 
Majorie  Ariano  who  has  successfully  demonstrated  receptor-specific  regional 
and  cellular  fluorescent  labeling  of  D1  and  D2  receptors  in  slices  of  rat 
forebrain,  pituitary,  retina,  and  superior  cervical  ganglion.  The  regional 
localization  of  the  dopamine  receptors  reflect  previous  work  ascertained 
using  in  vitro  receptor  autoradiography.  We  are  currently  using  these 
probes  in  simultaneous  labeling  experiments  to  ascertain  whether  or  not  D1 
and  D2  receptors  can  be  shown  to  colocalize  in  the  same  neurons  within  the 
striatum  and  other  brain  regions. 

In  FY90  we  initiated  a  project  to  develop  antibodies  to  the  D2  dopamine 
receptor  by  raising  antibodies  to  synthetic  peptides  derived  from  the  cloned 


4  -  ET/IR 


cDNA  sequence  of  this  receptor  (see  below).  This  approach  of  preparing 
antibodies  against  synthetic  peptides  has  proven  most  successful  in  preparing 
antibodies  against  native  proteins  of  which  the  sequence  is  known  but  not 
enough  of  the  protein  purified  to  use  directly  as  an  immunogen. 
Immunohistochemical  and  immunocytochemical  localization  of  the  D2  receptor  in 
rat  brain  slices  was  performed  using  the  antisera  raised  against  the  NH2 
terminal  18  residues.  Within  the  striatum,  about  50%  of  the  medium-sized 
neurons  were  labeled  as  well  as  an  occasional  large  cholinergic  interneuron. 
Electron  microscopic  examination  of  the  striatal  neuropil  demonstrated  that 
numerous  reactive  synaptic  structures  were  present  of  both  symmetrical  and 
asymmetrical  classifications.  The  data  show  that  the  D2  dopamine  receptors 
can  be  localized  in  the  projection  neurons  and  also  in  the  two 
morphologically  defined  aspiny  interneuron  populations  of  the  striatum. 

2.  Molecular  Cloning  and  Expression  of  Dopamine  Receptors. 

In  FY90  we  continued  investigations  related  to  our  discovery  of  RNA 
splice  variants  of  the  D2  dopamine  receptor.  These  RNA  splice  variants 
result  in  long  (D2L)  and  short  (D2S)  receptor  isoforms.  It  should  be  noted 
that  this  finding  represents  the  first  known  example  of  alternative  mRNA 
splicing  giving  rise  to  two  different  neurotransmitter  receptor  isoforms.  To 
investigate  the  functional  properties  of  the  short  (D2S)  and  long  (D2L) 
isoforms  of  the  D2  receptor,  we  have  stably  expressed  both  rat  cDNAs  in  CHO 
cells  in  FY90.  Following  transfection,  several  clonal  cell  lines  were 
isolated  which  exhibited  D2  receptor  binding  with  Bmax  values  ranging  from 
0.5  to  5  pmol/mg  protein  as  determined  by  [3H]methylspiperone  binding.  Both 
receptor  isoforms  revealed  a  Kd  for  [3H]methylspiperone  of  -60  pM. 
Competition  assays  for  [3H]methylspiperone  binding  by  dopamine  antagonists 
to  both  D2L  and  D2S  exhibited  identical  affinities  and  rank  orders  of 
potency.  Inhibition  of  [3H]methylspiperone  binding  by  dopamine  revealed  the 
presence  of  high  and  low  affinity  agonist  binding  states  with  both  receptor 
forms  exhibiting  similar  values  for  the  affinities  (KH  and  KL)  and 
proportions  of  the  agonist  binding  states.  In  addition,  both  receptor 
isoforms  show  a  complete  loss  of  high  affinity  agonist  binding  with  the 
addition  of  guanine  nucleotides.  Both  of  the  D2  receptor  isoforms  expressed 
in  CHO  cells  exhibit  dopamine- induced  inhibition  of  forskolin-stimulated  cAMP 
accumulation  by  -60%  with  EC50  values  of  -100  nM.  In  each  case,  dopamine 
inhibition  of  cAMP  accumulation  is  prevented  by  pretreatment  of  the  cells 
with  pertussis  toxin.  Finally,  for  both  receptor  isoforms,  pretreatment  of 
the  cells  with  100  uM  dopamine  for  4  to  24  hours  results  in  desensitization 
of  the  ability  of  dopamine  to  inhibit  cAMP  accumulation.  These  data  suggest 
that  the  D2L  and  D2S  receptors  are  functionally  similar  in  their  ligand 
binding  properties  and  linkage  to  adenylate  cyclase  inhibition. 

In  FY90,  we  have  collaborated  with  Dr.  Chris  Felder  in  the  Laboratory  of 
Cell  Biology,  NIMH  to  examine  what  role  the  D2  receptor  might  play  in  the 
production  of  the  second  messenger,  arachidonic  acid.  In  transfected  CHO 
cells  expressing  the  rat  D2L  receptor,  the  calcium  ionophore,  A23 1 87 , 
stimulated  the  release  of  arachidonic  acid  (AA)  and  this  was  potentiated  by 
dopamine  in  a  dose-dependent  fashion.  Dopamine  alone,  however,  had  no  effect 
on  basal  AA  release.  Quinpirole,  a  D2-selective  agonist,  augmented  A23187- 
stimulated  AA  release,  and  sulpiride,  a  D2-selective  antagonist  blocked  this 
augmentation.  Forskolin,  prostaglandin  E2,  dibutyryl  cAMP,  8-(4- 
chlorophenylthio)  cAMP  and  pertussis  toxin  all  had  no  appreciable  effect  on 


5  -  ET/IR 


both  A23l87-stimulated  AA  release  and  on  the  dopamine  enhancement. 
Inhibition  of  PKC  using  long-term  phorbol  ester  desensitization  and 
pharmacologic  inhibitors  diminished  the  dopamine  potentiation  of  AA  release. 
Preliminary  evidence  with  CHO  cells  expressing  the  D2S  receptor  indicates 
that  this  form  of  the  receptor  also  augments  AA  release.  These  results 
suggest  that  both  forms  of  the  D2  receptor  may  be  increasing  the  release  of 
AA  by  a  mechanism  involving  PKC  but  adenylate  cyclase  independent. 

In  FY90  we  continued  our  investigation  of  a  novel  D1  receptor  subtype 
using  functional  expression  assays  in  Xenopus  oocytes  that  have  been  injected 
with  rat  striatal  poly(A)+  RNA  (mRNA).  We  have  found  that  dopamine  is  able 
to  induce  Ca2+  mobilization  as  detected  by  stimulation  of  45ca2+  efflux  in 
striatal  mRNA-inJected  oocytes.  This  response  was  mimicked  by  SKF-38393  and 
blocked  by  the  D1-selective  antagonist,  SCH-23390.  No  efflux  was  observed 
with  quinpirole  and  the  dopamine  response  was  not  blocked  with  domperidone,  a 
D2-selective  antagonist.  Assay  of  size-fractionated  striatal  mRNA  using  the 
oocyte/45Ca2+  efflux  assay  revealed  a  single  peak  of  dopamine-stimulated 
activity  corresponding  to  an  mRNA  size  of  2.5-3.0  kb.  Oocytes  which  were 
injected  with  the  peak  mRNA  fractions  then  prelabeled  with  myo[3H]inositol , 
additionally  showed  a  4-fold  increase  in  IP3  production  in  response  to 
dopamine.  In  contrast,  oocytes  injected  with  the  peak  mRNA  showed  no 
elevation  of  cAMP  levels  in  response  to  dopamine  application.  Moreover, 
exogenously  applied  dibutyryl  cAMP  did  not  elicit  the  electrophysiological  or 
the  45ca2+  efflux  response.  These  data  thus  indicate  the  existence  of  a  D1 
dopamine  receptor  subtype  which  is  coupled  to  phosphatidyl  inositol  (PI) 
turnover  and  Ca2+  mobilization  in  addition  to  a  D1  receptor  subtype  which 
activates  adenylate  cyclase. 

In  FY90  we  have  begun  to  clone  this  novel  D1  receptor  subtype  coupled  to 
PI  turnover.  We  have  constructed  a  cDNA  library,  using  the  size-fractionated 
rat  striatal  mRNA  discussed  above,  in  the  lambda  SWAJ-2  vector.  This  vector 
contains  RNA  polymerase  sites  flanking  the  polylinker  and  thus  the  cDNA 
insert  region.  It  is  thus  possible  to  transcribe  RNA  from  the  cDNA  off  the 
vector  and  inject  it  into  oocytes  for  translation  and  expression  of  the 
protein.  The  cloning  strategy  consists  of  dividing  the  cDNA  library  into  a 
number  of  pools  consisting  of  10,000-100,000  clones  each,  running  mRNA  off 
each  pool  individually  and  injecting  it  into  oocytes  and  assaying  for  a 
positive  45ca2+  efflux  response.  Once  a  positive  pool  is  identified,  it  is 
subdivided  into  daughter  pools  and  the  process  is  repeated  until  a  pure  clone 
is  obtained.  This  approach  has  been  validated  by  the  fact  that  it  has  been 
recently  used  to  clone  the  serotonin  5HTic  and  the  substance  K  receptors, 
both  of  which  are  coupled  to  PI  turnover  and  Ca2+  mobilization. 

In  FY90  we  have  used  NS20Y  cells,  which  express  D1  dopamine  receptors 
linked  to  adenylate  cyclase  activation,  to  investigate  the  molecular  biology 
of  this  receptor  subtype.  In  order  to  clone  this  D1  receptor,  the  polymerase 
chain  reaction  (PCR)  method  was  used  to  selectively  amplify  a  cDNA  sequence 
from  NS20Y  cell  mRNA.  Poly  (A)+  RNA  was  used  to  synthesize  cDNA  by  reverse 
transcription  followed  by  PCR  amplification  with  sets  of  highly  degenerate 
primers  derived  from  the  transmembrane  sequences  of  the  previously  cloned 
adrenergic,  D2  dopaminergic,  and  serotonin  receptors.  This  amplification 
produced  a  novel  cDNA  fragment  which  exhibits  considerable  sequence  homology 
to  previously  cloned  G  protein-coupled  receptors.  In  order  to  characterize 
this  cDNA  further,  a  full-length  clone  was  isolated  from  a  rat  striatal 


ET/IR 


library  using  the  cDNA  fragment  as  a  probe.  Sequence  analysis  of  this  cDNA 
clone  indicated  that  it  is  indeed  a  member  of  the  G  protein-coupled  receptor 
family  and  exhibits  greatest  homology  wLth  the  previously  cloned 
catecholamine  receptors.  Northern  blot  analysis  in  various  neural  tissues 
revealed  a  transcript  size  of  ~U  kb  which  was  predominantly  located  in  the 
striatum  with  lesser  amounts  in  the  cortex  and  retina.  In  contrast,  no  mRNA 
was  detected  in  the  cerebellum,  hippocampus,  olfactory  bulb,  mesencephalon  or 
pituitary.  In  situ  hybridization  analysis  also  revealed  a  high  abundance  of 
mRNA  in  the  striatum  as  well  as  in  the  olfactory  tubercle.  To  establish  the 
identity  of  this  cDNA,  we  performed  transient  expression  experiments  in  COS-7 
cells.  [3HJSCH-23390,  a  D1  selective  radioligand,  exhibited  specific, 
saturable  binding  only  in  cells  which  were  transfected  with  this  cDNA. 
Competition  binding  analysis  with  a  variety  of  dopaminergic  ligands 
demonstrated  a  D1  dopaminergic  pharmacology.  In  addition,  dopamine  as  well 
as  other  D1 -selective  agonists  stimulated  cAMP  accumulation  in  transfected 
COS-7  cells.  We  have  concluded  that  we  have  cloned  a  cDNA  encoding  the  D1 
dopamine  receptor  linked  to  the  activation  of  adenylate  cyclase  activity. 
Importantly,  we  have  found  that  when  RNA  is  transcribed  from  this  D1  receptor 
cDNA  clone  and  injected  into  Xenopus  oocytes,  dopamine  will  stimulate  cAMP 
accumulation  but  is  incapable  of  producing  a  Ca2+  mobilization  response. 
Consequently,  we  propose  that  the  D1  receptor  subtypes  linked  to  the 
activation  of  adenylate  cyclase  and  phospholipase  C  be  designated  D1A  and 
D1B,  respectively. 

Neurophvsiological  Pharmacology  Section 

Focus  in  the  Neurophysiological  Pharmacology  Section  is  on  the 
physiology  of  the  basal  ganglia  and  the  potential  for  correcting  basal 
ganglia  dysfunction  with  drugs.  We  are  concerned  with  the  organization  of 
the  various  components  of  the  basal  ganglia  with  one  another  and  with  the 
roles  of  the  individual  nuclei  in  modulating  basal  ganglia  function.  We  are 
especially  interested  in  the  role  of  the  nigrostriatal  dopamine  system  in 
regulating  basal  ganglia  output.  Dysfunction  of  these  neuronal  systems  has 
been  implicated  in  the  etiology  of  many  neurologic  diseases,  including 
Parkinson's  disease,  tardive  dyskinesia,  Huntington's  chorea,  and  torsion 
dystonia. 

1)   D1  and  D2  dopamine  receptors  in  the  basal  ganglia. 

In  the  past  year,  the  section  has  continued  to  study  the  roles  of  the 
different  dopamine  receptor  subtypes  in  the  substantia  nigra  and  basal 
ganglia  and  the  consequences  of  selectively  stimulating  various  combinations 
of  these  receptor  subtypes.  We  have  previously  found  that  it  is  necessary 
for  postsynaptic  Dl  and  D2  receptors  to  be  simultaneously  stimulated  for  the 
induction  of  processes  once  thought  to  be  independently  mediated  by  the  D2 
receptor.  This  observation  led  to  a  fundamentally  new  and  exciting  perception 
about  the  relative  roles  of  the  D1  and  D2  receptors  located  in  the  basal 
ganglia  and  has  had  important  implications  for  the  use  of  dopamine  agonists 
and  antagonists  in  the  treatment  of  neurologic  disease  which  are  currently 
being  explored  by  ETB  clinicians. 

A  current  area  of  focus  is  the  mechanism  underlying  the  synergistic 
interactions  of  the  D1  and  D2  receptors.  A  key  question  is  whether  the  D1  and 
D2  receptors  are   segregated  from  one  another  with  respect  to   their 


7  -  ET/IR 


Localization  on  the  different  types  of  striatal  neurons,  or  whether  they 
coexist  on  the  same  cells.  Our  electrophysiologic  data  has  indicated  that  D1 
receptors  exert  a  greater  effect  on  the  str iatonigral  pathway  than  do  D2 
receptors,  while  both  appear  to  regulate  the  str iatopallidal  neurons.  Very 
recent  mRNA  localization  studies  carried  out  by  others  in  the  ETB  support  our 
conclusions.  Evidence  for  some  degree  of  segregation  of  the  receptor  subtypes 
provides  an  exciting  challenge  for  the  neurophysiologist  because  it  implies 
that  multisynaptic  processes  involving  multiple  neurotransmitters  and  their 
receptors  mediate  the  complex  synergistic  interactions  between  the  dopamine 
receptor  subtypes. 

During  the  past  year  we  have  made  a  very  significant  addition  to  our 
technical  capabilities  by  establishing  two  intracellular  recording  setups  in 
the  Section.  This  has  allowed  us  to  begin  to  investigate  how  D1  and  D2 
receptor  stimulation  affects  the  physiology  of  striatal  neurons  and  alters 
local  synaptic  transmission  in  this  nucleus.  Striatal  neurons  in  our  slice 
preparation  exhibit  a  membrane  potential  and  resistance  at  rest  which  is 
similar  to  that  reported  by  other  investigators.  Baseline  striatal 
recordings  generally  reveal  no  fluctuations  in  membrane  potential  or 
spontaneous  action  potentials  while  accumbal  and  septal  neurons  are 
spontaneously  active.  We  have  studied  the  apparently  lower  excitability  of 
striatal  neurons  by  applying  cathodal  pulses  to  elicit  neuronal  activity. 
Two  types  of  response  have  been  observed.  One  group  of  striatal  neurons 
responds  with  repetitive  spiking  activity.  The  evoked  spikes  exhibit  a 
characteristic  waveform  beginning  with  slow  depolarization  followed  by  a  fast 
spike  and  then  repolarization  below  baseline.  A  second  group  of  striatal 
neurons  do  not  exhibit  spikes  with  steps  of  constant  depolarizing  current. 
However,  electrically-evoked  EPSPs  may  elicit  a  fast  action  potential  in 
these  cells  without  the  slow  convex  ramp  of  depolarization  which  is 
characteristic  of  cathodally-evoked  spikes  in  the  former  group  of  neurons. 
Steady-state  current-voltage  data  obtained  using  slowly  depolarizing  ramps  of 
current  has  also  provided  evidence  of  different  membrane  properties  in 
responsive  and  silent  groups  of  striatal  neurons.  These  differences  raise 
interesting  questions  as  to  whether  groups  of  striatal  neurons  possess 
different  ionic  channels  (intrinsic  membrane  properties)  and  what  the 
functional  significance  of  such  differences  may  be  with  regard  to  striatal 
organization.  Different  levels  of  membrane  excitability,  for  instance,  may 
be  required  in  projection  neurons  subserving  striatonigral  and 
striatopallidal  pathways.  The  application  of  practical  anatomical  and 
electrophysiological  techniques  to  identify  neurons  in  our  slice  preparation 
is  an  issue  currently  being  explored. 

We  have  also  used  focal  electrical  stimulation  in  the  slice  studies  to 
induce  neurotransmitter  release  from  local  nerve  terminals.  We  have  found 
this  stimulation  evokes  synaptic  potentials  in  the  recorded  neurons  as  well 
as  slower  changes  in  current  voltage  relationships.  The  EPSP  elicited  in 
striatal  neurons  by  focal  stimulation  is  composed  of  at  least  three 
potentials  with  different  latencies.  We  are  investigating  the  possibility 
that  these  potentials  are  mediated  by  separate  transmitters.  The  potentials 
may  be  mediated  directly  by  an  excitatory  amino  acid  or  indirectly  by  amino 
acid  driven  release  of  another  excitatory  transmitter  in  our  slice 
preparation.  The  fastest  potential  appears  to  involve  a  non-NMDA  receptor. 
The  slower  potentials  can  be  blocked  by  an  NMDA  receptor  antagonist. 


8  -  ET/IR 


Following  focal  electrical  stimulation  of  slices,  we  have  also  observed 
changes  in  current-voltage  relationships  suggesting  increased  membrane 
resistance  near  rest.  Current-voltage  changes  are  also  mduced  by 
superfusion  of  dopamine  agonists,  but  these  appear  to  be  different  from  those 
produced  by  electrical  stimulation  of  the  slice,  suggesting  endogenous 
dopamine  is  not  mediating  the  latter  effects.  When  dopamine  agonists  are 
superfused,  however,  the  evoked  EPSPs  are  depressed  with  a  slow  time  course 
similar  to  that  associated  with  changes  in  the  I-V  relationship.  D1  and  D2 
agonists  could  be  acting  pre-  and  postsynaptically  to  produce  this  depression 
and  may  have  mechanistically  different  actions  on  each  of  the  EPSPs  we  have 
recorded  in  striatal  neurons.  From  these  first  results,  dopamine  appears  to 
be  a  modulator  of  voltage-dependent  conductances  and  may  regulate  repetitive 
firing  in  striatal  neurons.  While  dopamine  does  not  appear  to  be  "the 
modulatory  factor"  liberated  by  low  frequency  electrical  stimulation  of  our 
slice  preparation,  we  believe  that  dopamine  may  act  together  with  other 
neuromodulator  candidates  (e.g.  enkephalin,  substance  P,  dynorphin, 
somatostatin)  as  possible  comediators  of  this  action.  Pharmacologic 
characterization  of  this  dopaminergic  action  and  the  affected  membrane 
conductances  using  selective  agonists  and  antagonists  is  needed,  however,  to 
establish  the  specificity  of  this  action  and  significance  in  neuronal 
function.  Future  studies  will  extend  these  investigations  to  determine 
whether  concurrent  D1  and  D2  stimulation  is  necessary  for  full  expression  of 
dopaminergic  effects  and  will  attempt  to  address  the  mechanism  of  this  action 
at  a  cellular  level. 

2)  D2  Autoreceptor/D2  Postsynaptic  Receptor  Studies 

It  has  long  been  thought  that  one  site  where  D2  dopamine  receptors  are 
located  in  the  substantia  nigra  and  basal  ganglia  is  on  the  dopamine  cells 
themselves.  The  existence  of  these  receptors,  termed  autoreceptors,  has  been 
supported  by  a  large  body  of  experimental  evidence  and  now  appears  confirmed 
by  localization  of  mRNA  for  the  receptor  protein.  The  autoreceptors  on  the 
dopamine  cell  body  induce  inhibition  of  dopamine  cell  firing  and  those  on  the 
dopamine  terminals  induce  a  decrease  in  dopamine  synthesis  and  release.  The 
possibility  that  these  D2  autoreceptors  might  be  functionally  or 
fundamentally  different  from  those  located  on  the  postsynaptic  neurons  has 
been  one  area  of  active  interest  in  both  preclinical  and  clinical  areas  in 
the  Branch. 

The  recent  discovery  that  the  dopamine  autoreceptor  is  genetically 
identical  to  the  D2  receptor  located  on  the  neurons  postsynaptic  to  the 
dopamine  cells  rules  out  fundamental  receptor  differences  between  the 
autoreceptor  and  postsynaptic  D2  receptors;  attention  has  now  turned  to 
understanding  the  mechanisms  which  underlie  the  apparent  functional 
differences  between  the  D2  receptors  at  the  two  sites.  Our  demonstration  of 
the  existence  of  a  greater  receptor  reserve  at  dopamine  autoreceptor  sites 
relative  to  postsynaptic  D2  dopamine  receptor  sites  has  provided  an 
explanation  for  the  properties  of  the  so  called  "autoreceptor  selective" 
agonists.  Differences  in  spare  receptor  number  at  the  different  sites  can 
allow  a  partial  agonist  to  act  like  a  fully  efficacious  agonist  at  the  D2 
autoreceptors  while  providing  a  "clamp"  at  postsynaptic  D2  receptors.  By 
occupying  but  only  weakly  stimulating  at  postsynaptic  sites  where  there  are 
fewer  spare  receptors,  a  partial  agonist  can  provide  some  low  level  chronic 
stimulation  while  blocking  detrimental  fluctuations  or  excessive  stimulation 


9  -  ET/IR 


from  endogenous  neurotransmitter.  This  could  have  advantages  over  trying  to 
maintain  therapeutically  appropriate  levels  of  receptor  stimulation  or 
blockade  by  careful  adjustment  of  blood  levels  of  more  efficacious  agonists 
or  antagonists.  We  have  recently  evaluated  N-0923  and  N-0924  mth  respect  to 
these  properties.  The  former  is  a  highly  potent  and  apparently  efficacious 
dopamine  D2  agonist,  but  the  latter  is  less  potent  and  also  less  efficacious, 
acting  as  a  partial  agonist.  The  Pharmacology  Section  is  investigating  the 
therapeutic  properties  of  these  drugs  in  Parkinson  patients. 

3)  Consequences  of  Dopamine  Cell  Degeneration  in  the  Basal  Ganglia 

A  third  focus  of  investigation  this  year  has  been  the  neurophysiological 
consequence  of  dopamine  cell  degeneration.  The  effects  of  stimulating  the 
individual  dopamine  receptor  subtypes  in  the  basal  ganglia  are  altered  by 
chronic  dopamine  denervation.  Moreover,  we  have  shown  that  lesion  of  the 
dopaminergic  nigrostriatal  pathway  differentially  affects  dopamine  receptor 
mediated  effects  on  the  striatopallidal  versus  the  striatonigral  pathways. 
Consequences  of  intermittent  administration  and  chronic  infusion  of  1-DOPA 
have  been  investigated  in  these  animals  in  conjunction  with  studies  on  the 
behavioral  and  biochemical  effects  of  these  treatments  currently  being 
conducted  in  the  Clinical  Pharmacology  Section.  These  studies  have  shown  that 
large  variations  in  blood  levodopa  levels  leads  to  desensitization  of  D1  and 
GABA  receptor  mediated  mechanisms  of  basal  ganglia  output  through  the 
substantia  nigra  pars  reticulata.  We  have  also  been  investigating  the  changes 
in  dopamine  receptor  function  following  chronic  reserpine  treatment;  a 
strategy  which  is  providing  new  insight  into  the  compensatory  responses  of 
systems  postsynaptic  to  the  dopamine  cells  following  dopamine  depletion. 
Systemic  administration  of  a  D1  agonist  causes  changes  in  activity  at  sites 
downstream  to  the  striatum  which  are  different  from  those  seen  in  control  but 
in  the  opposite  direction  from  those  seen  in  the  more  chronically  lesioned 
rats.  Most  recently  we  find  similar  changes  in  rats  with  relatively  short- 
term  lesions  of  the  striatonigral  pathway.  This  evidence  for  a  biphasic 
response  pattern  in  dopamine  agonist  effects  after  dopamine  depletion 
indicates  a  greater  range  and  complexity  in  the  compensatory  responses  to 
dopamine  loss  than  previously  appreciated.  Mechanisms  will  be  explored  in 
the  coming  year.  Intracellular  recording  studies  may  provide  significant 
insight  into  these  phenomena.  The  findings  are  immediately  relevant  to 
strategies  for  treatment  of  parkinsonism  and  tardive  dyskinesia. 

4)  Excitatory  Amino  Acids  and  the  Pedunculopontine  Tegmental  Nucleus  in 
Regulation  of  Basal  Ganglia  Function 

Another  series  of  questions  involves  the  relationships  of  the  various 
components  of  the  basal  ganglia  to  one  another.  These  "systems"-related 
issues  have  been  receiving  clinical  attention  as  new  strategies  are 
considered  for  compensating  dopamine  cell  loss  by  addressing  "downstream" 
change  induced  by  the  lesion.  More  selective  glutamate  antagonists  have 
become  available  leading  to  concern  about  how  excitatory  amino  acids  affect 
movement  and  regulate  globus  pallidus  and  dopamine  cell  activity,  especially 
via  the  subthalamic  nucleus.  In  addition  to  intracellular  recording 
investigations  into  glutamate' s  role  in  striatal  function,  we  have  initiated 
studies  using  ketamine- induced  anesthesia  and  other  NMDA  receptor  antagonists 
to  examine  the  role  of  NMDA  receptors  in  drug  and  stimulation  induced 
responses  in  various  basal  ganglia  and  substantia  nigra  nuclei. 


10  -  ET/IR 


The  role  of  cholinergic  and  excitatory  amino  acid  outputs  from  the 
pedunculopontine  tegmental  nucleus  in  regulating  basal  ganglia  function  is 
another  newly  appreciated  and  potentially  significant  issue.  The 
pedunculopontine  tegmental  nucleus  is  a  relatively  unexplored  region  only 
recently  emerging  as  an  area  important  for  basal  ganglia  function. 
Neuroanatomic  tracing  studies  have  identified  extensive  connections  between 
the  pedunculopontine  tegmental  nucleus  and  the  basal  ganglia  and  substantia 
nigra.  Implication  of  the  pedunculopontine  tegmental  nucleus  in  certain 
neurologic  disorders  is  derived  from  demonstration  of  pedunculopontine 
tegmental  nucleus  degeneration  in  progressive  supranuclear  palsy,  Parkinson's 
disease,  and  Alzheimer's  disease.  A  further  link  between  the 
pedunculopontine  tegmental  nucleus  and  changes  associated  with  Parkinson's 
disease  has  been  demonstrated  in  2-deoxyglucose  studies  in  MPTP-treated 
monkeys  by  Perino  and  coworkers.  We  have  found  that  the  pedunculopontine 
tegmental  nucleus  appears  to  exert  a  very  significant  tonic  influence  on 
substantia  nigra  dopamine  cell  activity.  No  other  afferent  of  the  region  has 
been  shown  to  have  such  an  effect.  Indications  for  a  functional  link  between 
the  pedunculopontine  tegmental  nucleus  and  the  substantia  nigra  dopamine 
cells  have  raised  questions  about  whether  pedunculopontine  tegmental  nucleus 
(PPN)  degeneration  and  dopamine  cell  loss  in  Parkinson's  disease  are 
independent  phenomena  or  somehow  causally  related.  We  have  successfully 
recruited  a  new  Staff  Fellow  with  an  extensive  research  background  in 
pedunculopontine  tegmental  nucleus  neurophysiology  and  are  initiating  new 
studies  in  this  area.  Preliminary  evidence  suggests  that  both  mono-  and 
polysynaptic  pathways  may  be  involved  in  mediating  the  influence  of  the 
pedunculopontine  tegmental  nucleus  on  the  dopamine  cells.  A  combination  of 
systemic  and  microiontophoretic  studies  are  being  employed  to  address  the 
nature  of  the  neurotransmitter(s)  and  receptor  subtypes  involved  in 
pedunculopontine  tegmental  nucleus  projections  to  midbrain  dopamine  neurons 
to  further  elucidate  the  role  this  nucleus  plays  in  regulation  of  dopamine 
cell  function. 

Clinical  Pharmacology  Section 

Research  conducted  by  the  Clinical  Pharmacology  Section  links  basic 
neuroscience  investigations  carried  out  by  other  Branch  components  with  the 
neurologically-disordered  patient.  Clinical  and  preclinical  studies  mainly 
apply  transmitter  pharmacologic  approaches  to  the  development  of  improved 
symptomatic  therapies.  In  addition,  pathogenetic  mechanisms  are  studied  that 
might  provide  a  basis  for  pharmaceutical  interventions  that  attempt  to  modify 
the  basic  disease  process.  The  Section's  investigative  efforts  remain 
focused  on  Parkinson's  disease  and  related  extrapyramidal  disorders  and  to  a 
lesser  extent  on  Alzheimer's  disease  and  related  degenerative  dementias. 

Extrapyramidal  Disorders 

Studies  of  the  pathogenesis  and  treatment  of  the  peak  dose  dyskinesias 
and  fluctuations  of  the  wearing-off  and  on-off  types  that  ultimately  disable 
most  levodopa-treated  Parkinsonian  patients  continue  as  a  major  target  of 
Section  research.  Our  earlier  clinical  pharmacologic  studies  suggested  that 
wearing-off  phenomena  may  be  attributable  to  a  progressive  shortening  of  the 
antiparkinsonian  action  of  levodopa  due  to  nigrostriatal  dopamine  system 
degeneration.  On-off  fluctuations  and  the  abnormal  involuntary  movements 


11  -  ET/IR 


occurring  at  maximal  drug  levels,  on  the  other  hand,  appear  to  reflect  the 
participation  of  pathogenetic  mechanisms  in  addition  to  dopamine  cell 
degeneration.  Previous  Section  studies  indicated  that  the  levodopa  dose- 
antiparkinsonian  response  relation  becomes  progressively  steeper  as  both  the 
duration  of  symptoms  and  the  duration  of  levodopa  exposure  increase.  As  this 
dose-response  curve  becomes  nearly  vertical,  small  variations  in  dopamine 
levels  at  postsynaptic  receptor  sites  may  become  sufficient  to  abruptly 
switch  patients  between  the  on  and  off  states.  Furthermore,  our  finding  of  a 
progressive  narrowing  of  the  therapeutic  window  for  levodopa  such  that  the 
threshold  dose  for  dyskinesia  induction  ultimately  approximates  the  threshold 
dose  for  parkinsonism  reduction  could  explain  the  increasing  frequency  of 
peak  dose  dyskinesias  that  complicate  levodopa  therapy  in  advanced 
Parkinson's  disease.  Indirect  clinical  evidence  suggests  that  these  changes 
may  reflect  secondary  alterations  at  the  postsynaptic  level  arising  as  a 
consequence  of  chronic  intermittent  stimulation.  Work  during  the  past  year 
has  sought  to  both  extend  and  explicate  these  observations. 

Consistent  with  the  view  that  wearing-off  fluctuations  reflect  the  loss 
of  natural  storage  sites  within  dopamine  terminals,  we  were  the  first  to 
report  these  phenomena  promptly  disappear  when  optimal  dose  levels  of 
levodopa  are  administered  by  continuous  intravenous  infusion.  Results  from 
studies  of  continuous  parenterally  administered  levodopa  conducted  during  the 
past  3  years  in  more  than  100  Parkinsonian  patients  have  recently  been 
analyzed.  The  variability  in  plasma  Dopa  levels  attending  standard  oral 
dosing  schedules  could  immediately  be  reduced  by  more  than  80%  by  converting 
to  intravenous  administration.  Although  wearing  off  fluctuations  disappeared 
during  the  first  day  of  levodopa  infusion,  fluctuation  of  the  on-off  type 
only  gradually  improved  over  the  ensuing  days  and  weeks.  Thus,  when  patients 
were  examined  at  the  end  of  their  first  day  of  continuous  infusion,  those 
Judged  clinically  to  exhibit  only  wearing  off  phenomena  on  oral  levodopa  had 
a  52%  decrease  in  motor  response  variability,  while  those  considered  to  also 
manifest  on-off  fluctuations  had  only  a  12%  decrease.  Most  patients  judged  to 
have  either  wearing-off  or  on-off  phenomena  actually  have  a  combination  of 
both  responses,  although  in  widely  differing  ratios.  The  extent  to  which 
their  motor  fluctuations  diminish  with  conversion  to  infusion  therapy 
depended  on  the  degree  to  which  on-off  phenomena  contribute  to  their  overall 
response  instability.  These  observations  support  the  view  that  since  wearing 
off  fluctuations  clear  promptly  upon  stabilization  of  circulating  levodopa 
levels,  they  are  most  likely  due  to  the  loss  of  natural  storage  sites  for 
dopamine  as  a  consequence  of  the  progressive  loss  of  dopaminergic  neurons. 
With  advancing  disease,  a  diminishing  proportion  of  intrasynaptic  dopamine 
originates  in  residual  dopaminergic  terminals  that  are  able  to  store  and 
release  the  transmitter  amine  under  appropriate  neuronal  control,  and  more 
derives  from  nondopaminergic  cells  lacking  these  capacities.  Under  such 
circumstances,  swings  in  dopamine  precursor  levels  that  attend  oral  dosing 
produce  concomitant  swings  in  dopamine  receptor  stimulation. 

Recent  findings  have  also  clarified  the  pathogenesis  of  the  putative 
postsynaptic  changes  underlying  on-off  fluctuations  and  peak  dose 
dyskinesias.  In  response  to  10  days  of  around-the-clock  intravenous  levodopa 
infusions,  motor  fluctuations  gradually  diminished.  Clinical  improvement  was 
associated  with  a  shift  of  the  levodopa  dose-antiparkinsonian  response  curve 
to  the  right  and  the  therapeutic  index  improved  substantially  as  patients 
evidenced  a  marked  increase  in  their  antiparkinsonian  response  at  a  dose  that 


12  -  ET/IR 


produced  no  significant  change  in  the  severity  of  their  dyskinetic  response. 
These  results  indicate  that  alterations  in  central  dopaminergic  mechanisms 
contributing  to  motor  complications  in  advanced  Parkinson's  disease  can  be 
modified  by  procedures  that  provide  continuous  dopamine  replacement  and 
further  that  on-off  phenomena  relate  to  postsynaptic  changes,  possibly 
occurring  as  a  consequence  of  chronic  intermittent  stimulation.  Conceivably, 
interactions  between  mechanisms  mediated  by  the  Dl  and  D2  subclasses  of 
dopamine  receptors  or  alterations  in  other  striatal  transmitter  systems  may 
explain  both  the  improvement  in  clinical  fluctuations  and  the  restoration  in 
neuropharmacology  indices  with  continuous  levodopa  therapy.  The 
stabilization  of  motor  fluctuations  over  several  days  of  continuous  levodopa 
administration  reassures  that  virtually  all  patients  who  have  already 
developed  motor  complications  will  respond  to  steady-state  infusions  of 
levodopa  of  sufficient  duration.  Hence,  we  continue  to  develop  practical 
means  for  providing  stable  level  dopaminergic  replacement  for  ambulatory 
Parkinsonian  patients. 

Investigations  conducted  in  the  experimental  animal  appear  to  have 
provided  additional  insight  into  the  pathogenesis  of  the  postsynaptic  changes 
postulated  to  underlie  on-off  fluctuations  and  peak  dose  dyskinesias.  Our 
earlier  studies  indicated  that  twice  daily  injections  of  levodopa  for  30  days 
enhanced  the  rotational  response  to  the  mixed  dopamine  agonist,  apomorphine, 
in  rats  with  unilateral  6-hydroxydopamine-induced  lesions  of  the 
nigrostriatal  dopaminergic  pathway;  no  such  effect  was  obtained  when  the  same 
daily  dose  of  levodopa  was  infused  continuously  around-the-clock.  Now  we  have 
found  that  the  chronic  administration  of  levodopa  by  continuous  infusion 
enhanced  the  rotational  response  to  the  D2  dopamine  receptor  agonist 
quinpirole,  but  had  no  effect  on  rotation  induced  by  the  Dl  agonist  SKF 
38393.  The  rotational  responses  to  the  selective  dopamine  agonists  differed 
dramatically  in  rats  given  levodopa  by  intermittent  injection,  a  treatment 
schedule  resembling  that  ordinarily  used  in  Parkinsonian  patients; 
intermittently  treated  rats  evidenced  a  substantially  increased  response  to 
quinpirole,  a  greatly  diminished  response  to  SKF  38393,  and  a  modestly 
enhanced  response  to  apomorphine.  These  findings  suggest  that  the 
intermittence  of  central  dopamine  receptor  stimulation  may  be  an  important 
factor  in  determining  the  subsequent  responses  of  the  dopamine  system. 
Specifically,  our  data  appear  to  indicate  that  intermittent  treatment  with 
the  dopamine  precursor  increases  the  sensitivity  of  D2  dopamine  receptor 
mechanisms  and  decreases  the  sensitivity  of  mechanisms  associated  with  D1 
receptors.  The  dissociation  between  the  effects  of  both  continuous  and 
intermittent  levodopa  on  Dl  and  D2  agonist-induced  rotation  indicates  that  Dl 
and  D2  dopamine  receptor-mediated  mechanisms  respond  differently  to  chronic 
levodopa  treatment.  We  hypothesize  that  chronic  intermittent  stimulation  of 
the  normally  tonically  active  dopamine  system  may  produce  alterations  at  the 
level  of  the  postsynaptic  receptor  or  in  more  distal  neural  elements  that 
give  rise  to  on-off  fluctuations  and  peak  dose  dyskinesias. 

Additional  insight  into  these  matters  were  derived  from  studies  of  the 
effects  of  striatal  lesions  on  both  turning  behavior  and  globus  pallidus 
single  unit  responses  to  the  administration  of  a  dopamine  agonist.  Pallidal 
neurons  in  normal  rats  are  excited  by  the  systemic  administration  of 
postsynaptically  active  doses  of  apomorphine.  The  role  of  the  striatum  in 
mediating  this  phenomenon  was  examined  by  investigating  the  effects  of 
apomorphine  on  neuronal  activity  in  the  globus  pallidus  and  on  turning 


13  -  ET/IR 


behavior  in  rats  with  unilateral  quinolinic  acid  lesions  of  the  striatum.  The 
lesion,  which  reduced  striatal  choline  acetyltransferase  activity  by  over  65% 
and  GABA  content  by  more  than  60%,  markedly  attenuated  the  effect  of 
systemically  administered  apomorphine  on  pallidal  neuron  activity.  In 
lesioned  animals,  both  the  degree  of  attenuation  of  the  pallidal 
electrophysiologic  response  and  the  neurotoxin-induced  decreases  in  striatal 
choline  acetyltransferase  activity  and  GABA  content  correlated  with  the 
degree  of  apomorphine- induced  turning.  These  findings  indicate  that  the 
stimulatory  effect  of  apomorphine  on  pallidal  neuron  firing  in  normal  rats  is 
probably  mediated  indirectly  via  striatopallidal  neurons.  Because  a  striatal 
lesion  attenuated  the  response  of  pallidal  neurons  to  apomorphine,  the 
present  data  suggest  that  at  least  one  of  the  two  dopamine  receptor  subtypes 
involved  in  mediating  these  phenomena  are  localized  on,  or  acting  through, 
the  striatal  efferent  pathway. 

The  contribution  of  D1  and  D2  receptor-mediated  mechanisms  to  the 
pathogenesis  of  disorders  other  than  those  primarily  involving  extrapyramidal 
motor  function  have  received  increased  investigative  attention  during  the 
past  year.  In  one  study,  we  sought  to  evaluate  the  effects  of  selective  D1 
and  D2  receptor  stimulation  and/or  blockade  on  the  propagation  and  motor 
expression  of  pilocarpine-induced  seizures.  At  moderate  doses,  pilocarpine 
produced  limbic  stereotypies  but  no  convulsions.  Following  pretreatment  with 
the  D1  agonist,  SKF  38393,  but  not  its  (S)-  enantiomer,  the  same  dose  of 
pilocarpine-induced  convulsive  activity  as  revealed  by  behavioral, 
electroencephalographic,  and  histologic  alterations.  These  features  were 
identical  to  those  produced  by  a  higher,  convulsant  dose  of  pilocarpine.  On 
the  other  hand,  pretreatment  with  the  D2  agonist,  LY  171555,  failed  to  induce 
seizures.  Furthermore,  the  D1  receptor  antagonist  SCH  23390,  prevented 
convulsive  activity  induced  by  either  SKF  38393  plus  pilocarpine  or  by  higher 
dose  pilocarpine  given  alone.  However,  neither  dopamine  agonists  nor 
antagonists  altered  the  limbic  stereotypies  induced  by  pilocarpine, 
apparently  indicating  dopamine  system  involvement  primarily  in  the  mechanisms 
of  seizure  generalization.  These  results  suggest  the  participation  of  D1 
dopamine  receptors  in  the  propagation  of  convulsive  activity,  possibly  via  D1 
receptors  on  terminals  of  striatonigral  GABAergic  projections. 

Increasing  evidence  suggests  that  striatal  dopaminoceptive  cells  may  be 
affected  in  Parkinsonian  patients  both  by  the  basic  disease  process  as  well 
as  by  chronic  dopaminomimetic  treatment.  As  one  early  result  of  our  efforts 
to  focus  both  clinical  and  preclinical  studies  on  peptides  found  in  GABA 
containing  striatal  efferents  we  have  recently  reported  on  measurements  of  a 
proenkephalin  derived  peptide,  met5-enkephalin-arg-gly-leu  in  the  lumbar 
spinal  fluid  of  patients  with  Huntington's  disease  and  progressive 
supranuclear  palsy  (PSP).  Decreases  averaging  37?  in  Huntington  patients  and 
47%  in  PSP  patients  were  found  in  comparison  to  healthy,  age-matched  control 
subjects.  No  significant  correlation  could  be  established  between  CSF  levels 
of  the  proenkephalin  precursor  derivative  and  the  age  or  symptom  duration  of 
patients  in  either  group.  The  reduction  found  in  spinal  fluid  from 
Huntington's  disease  patients  likely  reflects  the  loss  of  proenkephalin- 
containing  neurons.  In  PSP,  on  the  other  hand,  deficits  in  enkephalinergic 
neurons  are  not  reported  and  the  observed  spinal  fluid  abnormality  may 
reflect  a  functional  decline  in  the  activity  of  this  system  secondary  to  the 
striatal  cholinergic  deficit.  These  studies  are  now  being  extended  to 
patients  with  Parkinson's  disease  both  in  the  untreated  and  levodopa  treated 


14  -  ET/IR 


states  and  could  have  important  implications  for  the  design  and  monitoring  of 
future  therapeutic  interventions. 

Cognitive  disorders 

Widely  disparate  rates  of  symptom  progression  have  long  been  recognized 
in  patients  with  Alzheimer's  disease.  We  ranked  U6  patients  who  met  NINCDS- 
ADRDA  and  DMS-III-R  criteria  for  Alzheimer's  disease  and  who  had  received  a 
complete  neuropsychologic  evaluation  by  an  index  of  progression  rate.  The 
index  for  13  of  the  patients  exceeded  the  median  value  for  the  entire  group, 
and  these  individuals  were  considered  rapid  progressors;  remaining  patients 
were  classified  as  slow  progressors.  Comparison  of  the  rapid  and  slow 
progressing  groups  revealed  no  significant  differences  in  age  at  symptom 
onset,  sex,  education  or  global  intellectual  function.  There  were,  however, 
characteristic  differences  in  neuropsychologic  profile:  those  with  rapidly 
progressing  symptoms  had  significantly  lower  scores  on  the  Mental  Control 
subtest  of  the  Wechsler  Memory  Scale,  on  the  Initiation  and  Perseveration  as 
well  as  Conceptualization  subtests  of  the  Mattis  Dementia  Scale  and  the 
Vocabulary  subtest  of  the  WAIS-R.  Interestingly,  all  these  tests,  with  the 
exception  of  Vocabulary,  which  may  capture  a  more  general  level  of 
intellectual  functioning,  are  considered  primarily  as  a  measure  of  frontal 
lobe  function.  Performance  on  neuropsychologic  tests  of  memory  as  well  as  of 
parietotemporal  lobe  function  were  indistinguishable  between  the  two  groups. 
These  results  suggest  that  the  diagnostic  rubric  of  Alzheimer's  disease  may 
subsume  two  distinct  subgroups:  one  characterized  by  relatively  slow 
clinical  progression  and  neuropsychologic  evidence  of  mainly  parietal  lobe 
dysfunction,  the  other  with  faster  progression  of  dementia  symptoms 
associated  with  functional  abnormalities  in  both  parietal  and  frontal  lobes. 

The  pathogenesis  of  Alzheimer's  disease  remains  unclear,  although  there 
is  some  evidence  to  suggest  that  excitatory  amino  acids  might  contribute  to 
the  pathogenetic  cascade  culminating  in  selective  neurodegeneration.  Among 
the  various  excitotoxins  studied,  quinolinic  acid  is  the  only  endogenous 
compound  known  to  be  present  together  with  its  metabolic  machinery  in  human 
brain.  We  analyzed  postmortem  brain  tissues  and  spinal  fluid  samples  for 
quinolinic  acid  in  Alzheimer  patients  and  in  age-matched  controls.  In  the 
control  group,  frontal  cortex  and  caudate  nucleus  had  higher  concentrations 
of  quinolinic  acid  than  the  other  regions  studied.  No  significant  differences 
were  found  between  Alzheimer  brains  and  controls  in  any  region  analyzed. 
Studies  in  lumbar  spinal  fluid  showed  no  gradient  for  quinolinic  acid  along 
the  neuraxis,  a  trend  for  increasing  levels  with  normal  aging,  and  no 
difference  between  Alzheimer  patients  and  age-matched  control  subjects.  Since 
quinolinic  acid  does  not  appear  to  cross  the  blood-brain  barrier,  levels  in 
brain  and  spinal  fluid  most  likely  reflect  local  production.  The  lack  of 
increased  central  quinolinic  acid  levels  does  not  necessarily  negate  the 
possibility  of  this  or  other  excitotoxins  contributing  to  cell  death:  levels 
in  the  lumbar  sac  may  not  reflect  changes  in  regional  brain  levels;  moreover, 
a  similarity  in  levels  hardly  excludes  the  possibility  of  altered  metabolic 
turnover  of  quinolinic  acid. 

The  cognitive  impairment  occurring  in  patients  with  progressive 
supranuclear  palsy  (PSP)  has  been  the  subject  of  two  recent  neuropsychologic 
studies.  In  one,  which  sought  to  evaluate  verbal  memory,  significant 
abnormalities   in   learning,   consolidation,   and   retrieval  were   found   in 


15  -  ET/IR 


comparison  with  age-,  sex-  and  education-matched  controls.  Information 
scanning,  which  requires  the  use  of  short-term  memory  processes,  remained 
intact.  The  duration  of  symptoms  and  degree  of  motor  dysfunction  correlated 
with  intrusions  during  learning.  Mo  relation  between  cerebral  dopamine 
metabolism,  as  assessed  by  spinal  fluid  levels  of  dopamine  and  of  its  major 
metabolite,  and  memory  dysfunction  could  be  established.  The  observed  memory 
deficits  may  at  least  be  partially  explained  by  an  inability  to  initiate  and 
maintain  a  systematic  and  strategic  search  of  stored  information.  This 
inability  could  be  related  to  a  disturbance  in  the  organizational  and 
temporal  aspects  of  encoding  and  retrieval  subserved  by  the  frontal  lobe. 
Clearly,  memory  dysfunction  contributes  significantly  to  disability  in  this 
disorder  and  appears  to  be  operationally  independent  of,  yet  correlated  with, 
motor  dysfunction. 

To  test  the  hypothesis  that  PSP  patients  may  be  impaired  on  tasks  used 
to  assess  "executive  and  attentional"  processes  presumably  subserved  by  the 
prefrontal  cortex,  a  second  neuropsychologic  investigation  evaluated  patients 
with  this  disorder  during  baseline,  placebo,  and  cholinesterase  inhibitor 
treatment  periods.  The  results  indicate  that  patients  with  PSP  are 
particularly  impaired  in  tasks  requiring  sequential  movements,  conceptual 
shifting,  monitoring  the  frequency  with  which  stimuli  are  presented,  and  the 
rapid  retrieval  of  verbal  knowledge.  These  deficits  could  not  be  accounted 
for  by  slowed  information  processing  or  by  problems  with  representational 
knowledge.  Conceivably,  "weak  activation"  of  frontal  lobe  representational 
knowledge  characterized  by  an  observed  attentional  deficit  results  in  the 
neuropsychological  impairments  noted  in  these  patients.  The  oral 
administration  of  physostigmine  failed  to  facilitate  executive  or  attentional 
performance  as  evaluated  by  our  tasks.  Our  results  indicate  that  above  and 
beyond  a  general  slowing  of  cognitive  processes,  PSP  patients  have  difficulty 
performing  some  tasks  traditionally  associated  with  the  prefrontal  lobe.  The 
contribution  of  basal  ganglia  dysfunction  to  this  impairment  remains  to  be 
elucidated. 


16  -  ET/IR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02578-08  ET 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (tOcnaraclarlOr  >«lf.    f'tlamutl  ttt  on  ona  tin*  oatwaan  Pfea  boroart.) 

Pharmacology  and  Cellular  Biology  of  Peptidergic  Neurons 


PRINCIPAL  INVESTIGATOR  dill  olnar  protatvonat  oartofinal  batom  tha  Prm<iaat  Invasttgator.)  (Nama.  titla.  laboratory,  ana  .nutluta  attthaiionl 

PI:  M.  Maral  Mouradian,  M.D.  Head,  Genetic  Pharmacology     ETB/NINDS 


Others:         Takashi  Minowa,  Ph.D.         Visiting  Fellow 


ETB/NINDS 


COOPERATING  UNITS  fVmyj 

Laboratory  of  Cellular  Biology,  NIMH;  Department  of  Physiology,  Uniformed  Services  University  of  the 
Health  Sciences 


LAB/BRANCH 

Experimental  Therapeutics  Branch 


SECTION 

Genetic  Pharmacology  Unit 


INSTITUTE  AND  LOCATION 

NINDS.  NIH,  Bethesda.  Maryland  20892 


TOTAL  MAN-YEARS: 


2.3 


PROFESSIONAL: 


1.3 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I      I   (a)  Human  subjects 
(an)  Minors 

(a2)  Interviews 


I      I  (b)  Human  tissues  I      I  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

In  FY90,  the  Genetic  Pharmacology  Unit  concentrated  on  the  molecular  regulation  of  neurotransmitter 
and  transmitter  receptor  genes.  Two  model  systems  were  studied: 

1)  Regulation  of  POMC  gene  transcription 

Studies  done  in  FY90  were  based  on  earlier  findings  in  this  laboratory  about  several  exonuclease  stop 
sites  in  the  1Kb  5'  flanking  region  of  the  mouse  POMC  gene  suggesting  the  presence  of  a  number  of 
potential  sites  for  DNA-protem  interaction.  Two  of  these  have  been  characterized  in  detail.  One  located 
between  -119  and  -106  bp  upstream  from  transcription  start  site  is  homologous  to  the  transcription 
factor  AP-2  thought  to  be  involved  in  signal  transduction,  and  the  other  located  between  -137  and  -131 
has  70%  homology  to,  but  is  distinct  from,  AP-1 . 

2)  Regulation  of  striatal  dopamine  receptor  genes 

This  project  was  initiated  in  FY90.  For  the  D-2  receptor,  a  full  length  cDNA  clone  was  characterized 
and  the  5'  region  sequenced  Screening  a  rat  genomic  library  has  yielded  several  candidate  clones  that 
are  currently  being  characterized.  Similarly  for  the  D-1  receptor,  screening  a  human  genomic  library  with 
the  recently  cloned  rat  D-1  receptor  cDNA  that  is  coupled  to  adenylate  cyclase  has  yielded  13  candidate 
clones.  Current  studies  focus  on  characterizing  the  promoter  region  of  these  genes  and  study  their 
transcriptional  regulation. 


17-ET/IR 


PHStO«0(R«».  I  Ml 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PERIOOCOVEREO 

October  I,  1989  through  September  30,  1990 


PROJECT  NUMBER 


Z01  NS  02263-14  ET 


TITLE  OF  PROJECT  (90  ihtraamrt  or  fit.    r;rf«musf  tit  on  or>9  imm  b*t**—n  thmborOmrt.) 

Biochemical  and  Pharmacological  Studies  of  Dopamine  Receptors 


PRINCIPAL  INVESTIGATOR  (tin  othmr  profmutonsi  ptrionnrnt  miow  thm Printipsi  tnvtt>gstor.)  (N»mm.  titlm.  laboratory,  andmstitutm  atiiiiationf 

PI:  David  R.  Sibley,  PhD      Head,  Molecular  Pharmacology  Unit  ETB/NINDS 

Others:         Frederick  J.  Monsma,  Jr.,  Ph.D.  IRTA  Fellow  ETB/NINDS 

LorisD.  McVittie,  Ph.D.  IRTA  Fellow  ETB/NINDS 

Anne  C.  Barton,  Ph.D.  IRTA  Fellow  ETB/NINDS 

Lauren  E.  Black,  Ph.D.  IRTA  Fellow  ETB/NINDS 

Mario  Rinaudo,  M.D.  Visiting  Fellow  ETB/NINDS 

Elizabeth  L.  Webster,  Ph.D.  Staff  Fellow  ETB/NINDS 


COOPERATING  UNITS  (lt»n,) 

Lab  Cell  Biology,  NIMH;  Genetic  Pharmacol  Unit,  ETB/NINDS;  Dept  Anatomy  &  Neurobiology,  U. 
Vermont;  Ctr  for  Molecular  &  Behavioral  Neurosci,  Rutgers  U;  Molecular  Probes,  Inc.;  Nova  Pharm  Corp. 


LAB/BRANCH 


Experimental  Therapeutics  Branch 


SECTION 

Molecular  Pharmacology  Unit 


INSTITUTE  AND  LOCATION 

NINDS.  NIH,  Bethesda.MD  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  5  75 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)Human  subjects  □  (b)  Human  tissues  HH  (c)  Neither 

(a  1)  Minors 

| ]  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  long-term  goal  of  this  project  is  the  biochemical  and  molecular  characterization  of  dopaminergic 
receptor  mediated  information  transduction,  and  its  regulation,  across  neuronal  membranes.  Two  major 
interrelated  areas  of  research  on  D1  and  D2  dopamine  receptors  are  currently  under  investigation: 

1.  Cell  Biology  and  Regulation  of  Dopamine  Receptors.  The  functional  and  regulatory  properties  of  D1 
and  D2  dopamine  receptors  on  various  neuroblastoma  and  retinoblastoma  cell  lines  were  characterized. 
The  D1  receptors  were  shown  to  undergo  homologous  desensitization  in  response  to  agonist  stimulation 
while  a  heterologous  desensitization  was  observed  in  response  to  cAMP.  D2  receptors  were  also  shown 
to  undergo  desensitization  and  down-regulation  in  response  to  agonist  treatment.  A  variety  of 
fluorescent,  affinity,  and  antibody  probes  for  dopamine  receptors  were  developed.  Utilizing  these 
probes,  it  was  shown  that  D1  receptors  were  localized  to  most  of  the  cells  in  the  striatum  as  well  as 
numerous  fibers  while  the  D2  receptors  appeared  to  be  confined  to  a  smaller  population  of  neurons. 

2.  Molecular  Cloning  of  Dopamine  Receptors.  Investigations  concerning  the  RNA  splice  variants  of  the 
D-2  dopamine  receptor  were  continued  in  FY  90.  A  series  of  stably  transfected  cell  lines  were  constructed 
which  express  either  the  short  (D-2S)  or  long  (D-2L)  isoforms  of  the  D2  receptor.  Both  isoforms  were 
shown  to  exhibit  identical  pharmacologic  and  functional  properties  with  respect  to  adenylyl  cyclase 
inhibition.  Both  isoforms  were  also  found  to  augment  arachidonic  acid  release.  A  cDNA  encoding  the 
D1  dopamine  receptor  linked  to  the  stimulation  of  adenylyl  cyclase  was  cloned  and  expressed.  A  novel 
subtype  of  the  D-1  receptor  was  discovered  by  expressing  rat  striatal  mRNA  in  Xenopus  oocytes.  This 
new  D-1  receptor  subtype  is  linked  to  the  stimulation  of  phosphatidylinositol  turnover  and  calcium 
mobilization. 


18-ET-IR 


PH5  6040  («».  I. Ml 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS02139-16ET 


PERIOO  COVERED 

October  1,  1989  through  September  31,  1990 


TITLE  OF  PROJECT  (lt<iu'i(t«i«i)ii  TW*  »»uii  W  on  on*  ihw  mi«hi  rn«  oo'wi ) 

Pharmacology  and  Physiology  of  the  Substantia  Nigra  and  Basal  Ganglia 


PRINCIPAL  INVESTIGATOR  a'nor/l«rsroh>iiion<'Mrionn«lo«la»fn««rlncis«/lnv«IIi9<Cor.>  (Ate/n*.  till:  ImbOflQty.  tntt  HllflfllM  tMillBlton) 

PI:  Judith  R.Walters      Chief,  Neurophysiological  Pharmacology  Section      ETB/NINDS 


Others:        Debra  Bergstrom 
Michael  Twery 
Kai-Xing  Huang 
MarkKelland 
Lisa  Thompson 


Pharmacologist 
Senior  Staff  Fellow 
Special  Volunteer 
Senior  Staff  Fellow 
Staff  Fellow 


ETB/NINDS 
ETB/NINDS 
ETB/NINDS 
ETB/NINDS 
ETB/NINDS 


COOPERATING  UNITS  (iftnr) 

Clinical  Pharmacology  Section,  Experimental  Therapeutics  Branch 


LAB/BRANCH 

Experimental  Therapeutics  Branch,  CNP 


SECTION 

Neurophysiological  Pharmacology  Section 


INSTITUTE  AND  LOCATION 

NINDS.NIH.Bethesda^MD  20892 


TOTAL  MAN-YEARS: 


4.3 


PROFESSIONAL: 


3.3 


OTHER: 


1.0 


CHECK  APPROPRIATE  BOX(ES) 

I      I   (a)  Human  subjects 

(a  1)  Minors 

I      !  (a2)  Interviews 


]  (b)  Human  tissues  [x]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

1)  Roles  of  D1  and  D2  Dopamine  Receptors  in  Basal  Ganglia.  Mechanisms  underlying  the  synergistic 
interactions  of  D1  and  D2  receptors  are  being  studied  in  striatal  slices  via  intracellular  recording 
techniques,  an  approach  new  to  the  Section  this  year.  Cathodal  pulses  induce  at  least  two  types  of 
response  in  the  striatal  neurons;  relevance  to  striatal  mechanisms  will  be  studied.  Focal  stimulation 
elicits  EPSP's  composed  of  at  least  three  potentials;  the  fastest  appears  to  involve  a  non-NMDA  receptor, 
the  slower  potentials  involve  NMDA  receptors.  Focal  electrical  stimulation  also  appears  to  release  a 
modulatory  factor  which  induces  changes  in  current-voltage  relationships.  First  results  show  that 
dopamine  is  not  this  modulatory  factor  but  dopamine  does  appear  to  be  a  modulator  of  the  voltage- 
dependent  conductances  and  may  regulate  repetitive  firing  in  striatal  neurons. 

2)  D2  Autoreceptor  /  D2  Postsynaptic  Receptor  Studies.  N-0923  and  N-0924  are  two  stereoisomers  of 
current  clinical  interest;  we  find  N-0923  is  a  potent  and  efficacious  D2  agonist  and  N-0924  acts  as  a  partial 
D2  agonist.  Differences  in  spare  receptor  number  at  pre-  and  post-synaptic  D2  receptor  sites  account  for 
why  a  partial  dopamine  agonist  can  be  fully  efficacious  at  D2  autoreceptors  while  providing  a  "clamp"  at 
postsynaptic  D2  receptors,  stimulating  weakly  while  blocking  further  stimulation.  In  fact,  maintaining 
therapeutic  levels  of  postsynaptic  dopamine  receptor  stimulation  or  blockade  may  be  more  effectively 
done  with  partial  dopamine  agonists  than  by  adjustment  of  blood  levels  of  more  efficacious  agents. 

3)  Conseguences  of  Dopamine  Cell  Degeneration  in  the  Basal  Ganglia.  Changes  in  dopamine  receptor 
function  following  chronic  reserpine  treatment  are  different  from  those  seen  in  control  but  in  the 
opposite  direction  from  those  seen  in  long  term  6-hydroxydopamine  lesioned  rats.  Similar  changes  are 
being  found  in  rats  with  relatively  short-term  lesions  of  the  striatonigral  pathway.  This  evidence  for  a 
biphasic  pattern  in  dopamine  receptor-mediated  effects  after  dopamine  depletion  indicates  a  greater 
range  and  complexity  in  the  compensatory  responses  to  dopamine  loss  than  previously  appreciated. 

4)  Role  of  the  Pedunculopontine  Tegmental  Nucleus  (PPN).  We  have  found  the  PPN  exerts  effects  on 
substantia  nigra  dopamine  cell  activity  which  appear  mediated  by  both  indirect  and  direct  mechanisms. 
This  raises  questions  about  the  role  of  PPN  degeneration  shown  occurring  in  certain  neurological 
disorders  including  progressive  supranuclear  palsy,  Parkinson's  disease  and  Alzheimer's  disease. 

19-ET/IR 


PHS  6O40  !■.•».  I  Ml 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02265-14   ET 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (SOchatmcffS  or  f«I.    T<ttm  mutt  t>t  on  on*  hnm  brntwrnn  thm  bor<*rt.) 

Pharmacology,  Biochemistry  and  Physiology  of  Central  Neurotransmitters 


PRINCIPAL  INVESTIGATOR  (Lift  othtr  protosuontt  pmrsonnml  b*io*t  th9  FrtMpal  inwstigstof.)  (Nam*,  tittm.  laboratory,  and  mstttutm  stiillation) 

PI:  Thomas  N.  Chase,  M.D.         Chief  ETB/NINDS 

Others:         Fabio  Baronti,  Visiting  Fellow;  Jerome  Blin,  Visiting  Associate;  Thomas  Davis,  Special 

Volunteer;  Thomas  M.  Engber,  Senior  Staff  Fellow;  Marianne  Giuffra,  Clinical  Associate; 
Ulrike  Mann,  Visiting  Fellow;  Concepcion  Marin,  Visiting  Fellow;  M.  Maral  Mouradian,  Senior 
Staff  Fellow;  John  H.  Ownby,  Clinical  Associate;  Zvi  Susel,  Visiting  Fellow,  ETB/NINDS 


COOPERATING  UNITS  ut.ny) 

NIMH  Clinical  Brain  Disorders  Branch,  NIDR  Neurobiology  and  Anesthesiology  Branch,  NIMH  Laboratory 
of  Clinical  Science 


LAB/BRANCH 

Experimental  Therapeutics  Branch 


SECTION 


Clinical  Pharmacology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda.  MP  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  1Q 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

[xj  (aHHuman  subjects  |~x~l  (b)  Human  tissues  I      I  (c)  Neither 

|X      (a1)  Minors 

I  x  I  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  goal  of  this  project  is  to  develop  improved  pharmacotherapies  for  selected  neurodegenerative 
disorders,  especially  Parkinson's  disease  and  Alzheimer's  disease. 

In  Parkinson's  disease,  on-off  fluctuations  declined  by  nearly  50%  during  a  10-day  around-the-clock 
infusion  of  optimal  dose  levodopa.  Clinical  improvement  was  associated  with  a  marked  shift  of  the 
levodopa  dose-antiparkinsonian  response  curve  towards  desensitization,  a  substantial  improvement  in 
the  therapeutic  index  for  the  dopamine  precursor,  and  a  significant  prolongation  in  its  duration  of 
antiparkinsonian  action.  On-off  phenomena,  which  ultimately  disable  most  parkinsonian  patients,  now 
appear  to  reflect  relatively  plastic  postsynaptic  changes,  occurring  as  a  consequence  of  nonphysiological 
intermittent  stimulation.  Findings  in  an  animal  model  of  parkinsonism  indicate  that  Dl  and  D2  dopamine 
receptor-mediated  mechanisms  respond  differently  to  chronic  levodopa  treatment,  and  suggest  that 
intermittence  of  central  dopamine  receptor  stimulation,  which  increases  sensitivity  of  D2  dopamine 
receptor  mechanisms  and  decreases  sensitivity  of  mechanisms  associated  with  the  D1  receptor,  may  be  an 
important  factor  in  determining  subsequent  responses  of  the  dopamine  system. 

In  Alzheimer's  disease,  recent  results  indicate  that  the  diagnostic  rubric  may  subsume  two  distinct 
subgroups:  one  characterized  by  relatively  slow  clinical  progression  and  neuropsychological  evidence  of 
mainly  parietal  lobe  dysfunction,  the  other  with  faster  progression  of  dementia  symptoms  associated 
with  functional  abnormalities  in  both  parietal  and  frontal  lobes,  a  distinction  having  obvious 
implications  relative  to  the  pathogenesis  and  treatment  of  this  disorder.  Neuropsychologic  evaluations 
of  patients  with  progressive  supranuclear  palsy  (PSP)  found  significant  abnormalities  in  learning, 
consolidation,  and  retrieval  in  comparison  with  matched  controls.  PSP  patients  were  also  observed  to  be 
particularly  impaired  in  tasks  requiring  sequential  movements,  conceptual  shifting,  and  the  rapid 
retrieval  of  verbal  knowledge.  Orally  administered  physostigmine  failed  to  improve  performance  on  any 
of  the  measures  tested. 

20-ET/IR 


PHS  4040  («•».  IWI 


Annual  Bibliography 
Experimental  Therapeutics  Branch,  DIR,  NINDS 

Ariano  MA,  Monsma  FJ,  Jr,  Barton  AC,  Kang  HC,  Haugland  RP,  Sibley  DR.  Direct 
visualization  and  cellular  localization  of  D1  and  D2  dopamine  receptors  in 
rat  forebrain  using  novel  fluorescent  ligands.  Proc  Natl  Acad  Sci  USA 
1989;86:8570-4. 

Barone  P,  Palma  V,  Parashos  SA,  Marin  C,  Chase  TN,  Carapanella  G.  D-1  dopamine 
agonist  administration  reduces  the  threshold  for  convulsions  produced  by 
pilocarpine.  Boll  Soc  Ital  Biol  Sper  1 989; 65: 337-4 1 . 

Barone  P,  Parashos  SA,  Palma  V,  Marin  C,  Campanella  G,  Chase  TN.  Dopamine  D-1 
receptor  modulation  of  pilocaropine-induced  convulsions.  Neuroscience 
1990;34:209-17. 

Bishop  JF,  Rinaudo  MS,  Ritter  JK,  Chang  ACY,  Conant  K,  Gehlert  DR.  A  putative 
AP-2  binding  site  in  the  5U  flanking  region  of  the  mouse  POMC  gene.  FEBS  Lett 
1990;264:125-9. 

Braun  A,  Mouradlan  MM,  Mohr  E,  Fabbrini  G,  Chase  TN.  Selective  D-1  dopamine 
receptor  agonist  effects  in  hyperkinetic  extrapyramidal  disorders.  J  Neurol 
Neurosurg  Psychiat  1989;52:631-5. 

Carlson,  J.H.,  Bergstrom,  D.A.,  Demo,  S.D.  and  Walters,  J.R.:  Nigrostriatal 
lesion  alters  neurophysiological  responses  to  selective  and  nonselective  D-1 
and  D-2  dopamine  agonists  in  globus  pallidus.  Synapse  1990;5:83-93. 

Chase  TN,  Baronti  F,  Fabbrini  G,  Heuser  IJ,  Juncos  JL,  Mouradian  MM. 
Rationale  for  continuous  dopaminomimetic  therapy  of  Parkinson's  disease. 
Neurology  1989;39(Supl  2) :7- 10. 

Engber  TM,  Susel  Z,  Juncos  JL,  Chase  TN.  Continuous  and  intermittent  levodopa 
differentially  affect  rotation  induced  by  D-1  and  D-2  dopamine  agonists.  Eur 
J  Pharmacol  1989;168:291-8. 

Fagarasan  M0,  Bishop  JF,  Rinaudo  MS,  Axelrod  J.  Interleukin  1  induces  early 
protein  phosphorylation  and  requires  only  a  short  exposure  for  late  induced 
secretion  of  b-endorphin  in  a  mouse  pituitary  cell  line.  Proc  Natl  Acad  Sci 
USA  1990;87:2555-9. 

Grafman  J,  Litvan  I,  Gomez  C,  Chase  TN.  Frontal  lobe  function  in  progressive 
supranuclear  palsy.  Arch  Neurol  1990;47:553-8. 

Juncos  JL,  Mouradian  MM,  Fabbrini  G,  Chase  TN.  Levodopa  infusion  therapy.  In: 
Koller  WC,  Paulson  GW,  eds.  Therapy  of  Parkinson's  disease.  New  York:  M. 
Dekker,  1990; 185-203. 


Litvan  I,  Gomez  C,  Atack  JR,  Gillespie  M,  Kask  AM,  Mouradian  MM,  Chase  TN. 
Physostigmine  treatment  of  progressive  supranuclear  palsy.  Ann  Neurol 
1989;26:404-7. 

Litvan  I,  Grafman  J,  Gomez  C,  Chase  TN.  Memory  impairment  in  patients  with 
progressive  supranuclear  palsy.  Arch  Neurol  1989;46:765-7. 

Mahan  LC,  Monsma  FJ,  Jr,  Burch  R,  Sibley  DR.  Expression  of  rat  mRNA  coding 
for  a  D1  dopamine  receptor  coupled  to  Ca2+  mobilization  in  Xenopus  oocytes, 
Proc  Natl  Acad  Sci  USA  1990;87:2196-200. 

Mann  UM,  Mohr  E,  Chase  TN.  Rapidly  progressive  Alzheimer's  disease.  Lancet 
1989;2:799. 

Monsma  FJ,  Jr,  Barton  AC,  Sibley  DR.  Expression  of  functional  D2  dopamine 
receptors  following  differentiation  of  human  retinoblastoma  cells.  J 
Neurochem  1989;52:1641-4. 

Monsma  FJ,  Jr,  McVlttie  LD,  Gerfen  CR,  Mahan  LC,  Sibley  DR.  Multiple  D2 
dopamine  receptors  produced  by  alternative  RNA  splicing.  Nature  1989;342:926- 
9. 

Monsma  FJ,  Jr,  Sibley  DR.  Identification  and  characterization  of  D1  and  D2 
dopamine  receptor  subtypes  expressed  in  cultured  neuroblastoma  and 
retinoblastoma  cell  lines.  Brain  Res  1989;492:314-24. 

Mouradian  MM,  Chase  TN.  Clinical  perspectives  in  neuropeptides  in  the  central 
nervous  system.  Curr  Opin  Neurol  Neurosurg  1989;2:517-9. 

Mouradian  MM,  Chase  TN.  Parkinson's  Disease:  therapeutic  aspects.  Current 
Opin  Neurol  Neurosurg  1989;2:309-13. 

Mouradian  MM,  Heuser  IJE,  Baronti  F,  Chase  TN.  Modification  of  central 
dopaminergic  mechanisms  by  continuous  levodopa  therapy  of  advanced 
Parkinson's  disease.  Ann  Neurol  1990;27:18-23. 

Mouradian  MM,  Heyes  MP,  Pan  J-B,  Heuser  IJE,  Markey  SP, Chase  TN.  No  changes 
in  central  quinolinic  acid  levels  in  Alzheimer's  disease.  Neurosci  Lett 
1989;105:233-8. 

Pan  HS,  Engber  TM,  Chase  TN,  Walters  JR.  The  effects  of  striatal  lesion  on 
turning  behavior  and  globus  pallidus  single  unit  response  to  dopamine  agonist 
administration.  Life  Sci  1989;46:73-80. 

Vyas  S,  Bishop  JF,  Gehlert  DR,  Patel  J.  Effects  of  protein  kinase  C  (PKC) 
down  regulation  on  secretory  events  and  POMC  gene  expression  in  anterior 
pituitary  tumor  (AtT-20)  cells.  J  Neurochem  1990;54:248-55. 

Weinberger  DR,  Mann  U,  Gibson  RE,  Coppola  R,  Jones  DW,  Braun  AR,  Berman  KF, 

Suderland  T,  Reba  RC,  Chase  TN.  Cerebral  muscarinic  receptors  in  primary 

dementia  as  evaluated  by  SPECT  with  iodine-123  labeled  QNB.  Adv  Neurol 
1990;51:147-9. 


Experimental  Therapeutics  Branch 

"Thomas  N.  Chase,  M.D.,  Chief 

Senior  Staff 

•Judith  R.  Walters,  Ph.D. 

Fellows 

Michael  Twery,  Ph.D. 
Mark  Kelland,  Ph.D. 
Lisa  Thompson,  Ph.D. 
Thomas  M.  Engber,  Ph.D. 
Marianne  Giuffra,  M.D. 
M.  Maral  Mouradian,  M.D. 
John  H.  Ownby,  M.D. 
Frederick  J.  Monsma,  Jr.,  Ph.D. 
Loris  D.  McVittie,  Ph.D. 
Anne  C.  Barton,  Ph.D. 
Lauren  E.  Black,  Ph.D. 
Elizabeth  L.  Webster,  Ph.D. 
David  R.  Sibley,  Ph.D. 

Visiting  Program 

Fabio  Baronti,  M.D. 
Jerome  Blin,  M.D. 
Concepcion  Marin,  M.D. 
Zvi  Susel,  M.D. 
Ulrike  Mann,  M.D. 
Takashi  Minowa,  Ph.D. 
Mario  Rinaudo,  M.D. 

Special  Volunteers 

Kai-Xing  Huang,  Ph.D. 
Rafael  G.  Blesa,  M.D. 
Thomas  L.  Davis,  M.D. 


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ANNUAL  REPORT 

October  1, 1989  through  September  30, 1990 

Medical  Neurology  Branch 

Clinical  Neurosciences  Program,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 

Table  of  Contents 

RESEARCH  SUMMARY  1-36 

PROJECT  REPORTS 

Clinical  Pharmacology  of  Antiepileptic  Drugs  37 

Z01  NS  02318-13  MNB 

Diagnostic  and  Therapeutic  Reevaluation  of  Patients  With  Intractable  Epilepsy  38 

Z01  NS  02236-1 5  MNB 

Physiological  Analysis  of  Involuntary  Movements  39 

Z01  .V  02667-06  MNB 

Trial  of  Isoniazid  for  Action  Tremor  40 

Z01  NS  02668-06  MNB 

Physiological  Analysis  of  Voluntary  Movement  41 

Z01  NS  02669-06  MNB 

Utility  and  Physiology  of  Botulinum  Toxin  for  Involuntary  Movement  Disorders  42 

Z01  NS  02711-05  MNB 

Non-invasive  Stimulation  of  Human  Central  Nervous  System  43 

Z01  NS  0271 2-05  MNB 

Hemispheric  Development  and  Specialization  of  the  Intellectual  Functions  44 

Z01  NS  01658-23  MNB 

Behavioral  Modulation  by  the  Limbic  System  in  Man  45 

Z01  NS  01424-24  MNB 

EEG  Learning  Correlates  Using  Scalp  and  Intracranial  Depth  Electrodes  46 

Z01  NS  01245-25  MNB 

Cognitive  and  Emotional  Profile  of  Neuropsychiatric  Disorders  47 

Z01  NS  00200-36  MNB 

Pharmacological  Studies  of  Ion  Channels  in  Cultured  Cells  48 

Z01  NS  02732-04  MNB 


i-MNB/DIR 


Excitability  Properties  of  Enzymatically  Dissociated  CNS  Neurons  49 

Z01  NS  02733-04  MNB 

Development  of  Uncompetitive  NMDA  Antagonists  as  Anticonvulsants  50 

Z01  NS  02772-03  MNB 

Neuropsychological  Investigations  of  Human  Cognition  and  Mood  State  51 

Z01  NS  02792-02  MNB 

Cognitive  Neuroscience  52 

Z01  NS  02793-02  MNB 

Event-Related  Potential  Studies  of  Normal  and  Abnormal  Cognitive  Processing  53 

Z01  NS  02794-02  MNB 

Combined  Clinical,  Viral  and  Immunological  Studies  of  Neuromuscular  Diseases  54 

Z01  NS  02038-18  MNB 

Studies  in  Neuromuscular  and  CNS  Diseases  and  Their  Experimental  Models  55 

Z01  NS  02531-09  MNB 

Radiographic,  Radioisotopic,  CT,  and  MR  Angiography  of  the  Spinal  Cord  56 

Z01  NS  01 195-26  MNB 

Nuclear  Magnetic  Resonance  and  Computed  Tomography  (Transmission)  57 

Z01  NS  02073-17  MNB 

Positron  Emission  Tomography  58 

Z01  NS  0231 5-13  MNB 


ii-MNB/DIR 


ANNUAL  REPORT 
October  1,  1989  through  September  30,  1990 

Medical  Neurology  Branch 

Clinical  Neurosciences  Program,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 

Mark  Hallett,  M.D.,  Chief 

The  Medical  Neurology  Branch  conducts  research  on  human  epilepsy,  including  new 
approaches  to  diagnosis  and  treatment;  investigates  basic  questions  related  to 
normal  and  abnormal  neuronal  excitability;  performs  studies  on  human  motor 
control;  investigates  cognitive  and  emotional  processes  in  man;  conducts  research 
on  memory,  perception,  language,  and  problem  solving  in  neurological  patients; 
investigates  immunological  and  virological  aspects  of  neuromuscular  disorders;  and 
investigates  the  central  nervous  system  (CNS)  using  various  research  imaging 
techniques. 

The  Branch  is  divided  into  seven  sections  in  addition  to  the  Office  of  the  Chief.  On 
June  8,  1990,  the  Neuroimaging  Section  of  the  Clinical  Neurosciences  Program  was 
transferred  to  the  Medical  Neurology  Branch.  Giovanni  Di  Chiro,  M.D.,  serves  as 
Chief,  Neuroimaging  Section.  The  Section  investigates  the  CNS  in  health  and 
diseases  using  as  research  tools  X-rays,  radionuclides,  and  magnetic  resonance 
imaging.  The  Section  also  studies  brain  tumors,  movement  disorders,  intracerebral 
bleedings,  iron  distribution  in  the  aging  and  pathological  brain,  cerebrospinal  fluid 
circulation,  vascular  malformations  andtumors  of  the  spinal  cord,  pituitary  gland 
adenomas,  and  certain  aspects  of  cerebral  ischemia.  In  addition,  the  Neuromuscular 
Diseases  Unit  and  the  Cognitive  Neuroscience  Unit  became  sections  on  June  8,  1990. 

William  H.Theodore,  M.D.,  Deputy  Chief,  MNB,  also  serves  as  Chief  of  the  Clinical 
Epilepsy  Section.  The  Clinical  Epilepsy  Section  includes  the  Unit  on  Cerebral  Blood 
Flow  and  Metabolism  headed  by  Dr.  Theodore,  and  the  Unit  on  Neurophysiology 
headed  bySusumu  Sato,  M.D.  Mark  Hallett,  M.D.,  is  Chief  of  the  Human  Motor 
Control  Section.  The  Human  Motor  Control  Section  includes  the  Cortical  Physiology 
Unit  headed  by  Dr.  Leonardo  G.  Cohen.  Paul  Fedio,  Jr.,  Ph.D.,  is  Chief  of  the  Clinical 
Neuropsychology  Section.  Marinos  Dalakas,  M.D.,  is  Chief  of  the  Neuromuscular 
Diseases  Section.  Jordan  Grafman,  Ph.D.,  is  Chief  of  the  Cognitive  Neuroscience 
Section.  In  1990,  Michael  Rogawski,  M.D.,  Ph.D.,  was  named  as  Chief,  Neuronal 
Excitability  Section. 

CLINICAL  EPILEPSY  SECTION 

The  Clinical  Epilepsy  Section  is  undertaking  a  series  of  studies  in  patients  with  severe 
uncontrolled  seizures  using  new  techniques  in  order  to  improve  clinical  control,  as 
well  as  to  elucidate  the  pathophysiology  of  epilepsy.  The  studies  being  carried  out 
at  this  time  focus  on  two  main  groups  of  patients:  adults  or  children  with  complex 
partial  seizures;  and  children  with  atonic  /  myoclonic  seizures  (the  Lennox-Gastaut 
syndrome).  After  seizure  frequency  and  type  are  characterized  by  intensive 
monitoring  techniques,  the  patients  are  placed  in  an  appropriate  research  protocol. 
Before  patients  are  offered  surgical  therapy  or  trial  of  an  experimental  antiepileptic 
drug,  the  therapeutic  regimen  is  adjusted  to  obtain  optimal  seizure  control  using 
conventional  agents.  Several  investigations  are  performed  in  collaboration  with  the 


1  -MNB/DIR 


Clinical  Neuropsychology  Section,  the  EEG  Laboratory,  the  Neuronal  Excitability 
Section,  and  the  Surgical  Neurology  Branch. 

Neuroimaging  techniques  may  be  used  to  obtain  both  physiologic  and  anatomic 
data  and  assist  in  the  clinical  evaluation  of  patients  with  severe  seizures.  Positron 
emission  tomography  (PET)  is  a  technique  using  the  intravenous  injection  of  a 
radioactive  isotope  to  determine  regional  rates  of  cerebral  metabolism,  blood  flow, 
or  tracer  distribution.  Magnetic  resonance  imaging  (MRI)  is  being  used  to  help 
elucidate  the  anatomical  substrates  of  altered  physiologic  patterns  revealed  by  PET. 
Initial  attempts  have  been  made  to  use  nuclear  magnetic  resonance  (NMR) 
spectroscopy  to  study  biochemical  parameters  of  human  epileptic  tissue  in  vivo. 
Single  photon  emission  computed  tomography  (SPECT)  is  being  used  as  an 
additional  tool  to  study  cerebral  blood  flow.  Clinical,  electroencephalographic 
(EEG),  and  neuropsychological  data,  as  well  as  ultrastructural  and  biochemical 
investigations  of  epileptic  tissue  removed  at  surgery,  are  correlated  with  function 
and  structural  results  of  imaging  studies. 

One  of  the  major  interests  of  the  Clinical  Epilepsy  Section  has  been  the  use  of 
neuroimaging  techniques  to  study  neuropharmacology.  In  clinical  pharmacology 
research,  it  has  always  been  much  easier  to  derive  pharmacokinetic  than 
pharmacodynamic  data.  We  have  been  taking  advantage  of  the  ability  of  PET  to 
provide  quantitative  information  to  study  the  effect  of  drugs  on  cerebral  blood  flow 
and  metabolism,  as  well  as  the  distribution  of  putative  neuroreceptor  liqands  in  vivo. 

Recent  and  ongoing  studies: 

Estimation  of  cerebral  glucose  metabolism  using  18-flouro  2-deoxyglucose  PET 
showed  that  patients  with  uncontrolled  complex  partial  seizures  have  regions  of 
focal  hypometabolism  which  correspond  to  electrographic  localization  of  epileptic 
foci.  In  patients  with  structural  lesions  on  computed  tomography  (CT)  or  MRI, 
hypometabolism  was  often  much  more  extensive  than  suggested  by  the  lesion  itself. 
In  the  Lennox-Gastaut  Syndrome,  a  severe  childhood  epileptic  encephalopathy,  we 
found  diffuse  neocortical  hypometabolism  with  relative  preservation  of  function  in 
the  basal  ganglia  and  diencephalon,  even  when  CT  and  MRI  are  normal.  Further 
studies  are  in  progress  to  study  the  relation  between  metabolic  dysfunction,  age  of 
seizure  onset,  severity  and  duration  of  seizures.  The  results  were  in  marked  contrast 
to  those  obtained  in  patients  with  absence  seizures,  who  had  normal  glucose 
metabolism.  Distinct  metabolic  pathophysiological  processes  were  present  in 
patients  with  distinct  electroclinical  epileptic  syndromes.  We  have  used  150  water  to 
study  cerebral  blood  flow  in  patients  with  uncontrolled  partial  seizures.  With  this 
tracer  it  is  possible  to  compare  ictal  and  interictal  states,  as  well  as  to  compare  blood 
flow  with  FDG  scans  performed  in  the  same  patients.  Preliminary  results  indicate 
that  there  may  be  mismatches  between  blood  flow  and  metabolism  in  epileptic 
tissue,  and  that  glucose  metabolism  is  reduced  to  a  greater  degree  than  blood  flow. 
This  may  be  an  important  marker  for  altered  physiology  in  epileptic  foci.  Further 
studies  are  in  progress,  using  improved  PET  techniques.  Moreover,  focal  increases  in 
blood  flow  have  been  found  in  patients  who  have  had  secondary  generalized 
seizures  during  I5fj  PET  scans.  These  studies  may  help  to  provide  a  means  of 
localizing  seizure  onset. 

In  patients  with  complex  partial  seizures,  a  speech  discrimination  task  was  used  to 
"activate"  cerebral  glucose  metabolism,  and  investigate  the  possibility  of  functional 
reorganization  due  to  unilateral  temporal  lobe  damage.  Patients  with  complex 
partial  seizures  and  a  unilateral  EEG  focus  were  compared  to  normal  controls. 


2-MNB/DIR 


Preliminary  results  suggested  that  metabolic  activation  during  speech  performance 
may  have  a  broader  distribution  than  suspected  on  the  basis  of  purely  anatomic 
studies,  and  that  hypometabolism  in  patients  with  left  temporal  foci  became 
relatively  more  severe  during  the  task.  Using  ^O  water,  we  have  investigated  the 
effect  OT  verbal  learning  and  recall  on  cerebral  blood  flow.  Further  studies  of  word 
and  object  recognition,  and  verbal  and  nonverbal  memory  are  planned. 

We  studied  the  effect  of  antiepileptic  drugs  on  cerebral  glucose  metabolism. 
Phenobarbital  depressed  global  LCMRglu  by  30-40%  while  carbamazepine  and 
phenytoin  lowered  glucose  utilization  by  only  10-15%.  These  results  are  consistent 
with  a  possible  difference  between  the  mechanisms  of  action  of  the  compounds. 
Phenobarbital  interacts  with  the  GABA-benzodiazepine  receptor  complex  while 
phenytoin  and  carbamazepine  appear  to  affect  currents  in  active  sodium  channels. 
Moreover,  other  studies  have  shown  that  GABA  agonists  reduce  glucose  metabolism 
in  experimental  animals.  Thus,  the  PET  results  may  be  of  particular  relevance  to 
investigations  into  the  mechanism  of  action  of  antiepileptic  drugs.  Moreover,  they 
may  provide  evidence  for  explaining  the  difference  in  neuropsychological  toxicity, 
whicn  is  much  greater  for  phenobarbital  than  for  phenytoin  or  carbamazebine. 
Subsequently,  valproic  acid  was  found  to  reduce  LCMRglc  by  20-25%,  suggesting 
that  it  may  have  a  GABAerqic  effect  at  therapeutic  doses  in  vivo. 

Two  approaches  have  been  used  to  evaluate  the  opiate  receptor  sytem,  which  has 
been  implicated  in  the  pathophysiology  of  epilepsy.  [18F]  cyclofoxy,  a  naltrexone 
analogue,  was  used  to  image  Mu  and  Kappa  receptors.  We  did  not  find  any 
alterations  in  activity  in  the  region  of  the  epileptic  focus.  We  administered  1  mg/kg 
naloxone  in  order  to  see  whether  blocking  opiate  receptors  would  alter  blood  flow 
in  epileptic  foci.  Although  a  transient  decrease  in  blood  flow  occurred,  there 
appeared  to  be  no  specific  effect.  These  studies  suggest  that  opiate  receptors  may 
not  play  a  dramatic  role  in  the  pathophysiology  of  human  epilepsy. 

Recent  investigations  in  the  clinical  pharmacology  of  antiepileptic  drugs  include 
studies  of  carbamazepine  metabolism  and  phenytoin  protein  binding.  We  have 
been  evaluating  the  effect  of  drug  withdrawal  on  seizure  frequency,  in  order  to 
assess  the  presence  or  absence  of  transient  exacerbations  which  could  be 
distinguished  from  a  simple  loss  of  drug  effect.  This  was  clearly  present  in  the  case  of 
phenobarbital,  but  absent  for  phenytoin.  Data  on  carbamazepine  are  being 
collected.  These  data  are  important  for  clinical  practice.  A  physician  wishing  to 
withdraw  a  drug  known  to  cause  a  transient  exacerbation  during  taper  may  be  more 
likely  to  perservere  when  seizures  increase. 

The  Clinical  Epilepsy  Section  has  completed  a  trial  of  a  promising  antiepileptic  drug 
which  was  identified  through  the  NIH  Antiepileptic  Drug  Development  program. 
The  drug,  known  asfelbamate,  is2-phenyl-1,  3-propanediol  dicarbamate.  In  pre- 
clinical testing,  the  drug  is  effective  in  animal  models  which  correlate  with  partial 
seizures  in  man  and  is  quite  nontoxic  in  animals.  Evaluation  of  the  protective  indices 
indicate  that  felbamate  has  a  significant  and  adequate  margin  of  safety.  A 
randomized  placebo-controlleddouble-blind  study  was  performed.  The  purpose  of 
this  study  was  to  obtain  definitive  information  regarding  the  efficacy  and  safety  of 
felbamate  in  patients  with  uncontrolled  partial  seizures  who  are  receiving 
concomitant  carbamazepine  therapy.  The  study  used  a  unique  three-period  cross- 
over design  in  order  to  eliminate  carry-over  effects  from  drug  to  placebo  periods. 
The  results  suggested  that  felbamate  can  reduce  the  frequency  of  partial  seizures.  A 
trial  of  felbamate  monotherapy  for  complex  partial  seizures,  and  of  felbamate  in 
addition  to  valproic  acid  in  the  Lennox-Gastaut  syndrome  is  being  planned. 


3-MNB/DIR 


Sphenoidal,  and  in  some  cases,  subdural  electrodes  are  used  in  the  evaluation  of 
potential  surgical  candidates,  coupled  with  long-term  video-EEG  recording 
techniques.  These  techniques  allow  the  acquisition  of  EEG  data  not  available  via 
surface  recordings.  These  data  are  correlated  with  PET  and  MRI  to  obtain  the  best 
possible  presurgical  localization  of  epileptic  foci.  A  prospective  evaluation  of  the 
relative  value  of  invasive  (subdural)  and  noninvasive  methods  of  presurgical 
evaluation  is  in  progress.  Twenty-six  patients  with  medically  refractory  complex 
partial  seizures  had  temporal  lobectomy  after  evaluation  which  included  prolonged 
scalp  EEG  recordings,  PET,  MRI,  and  X-ray  computed  tomography.  PET  showed  a 
region  of  focal  interictal  temporal  hypometabolism  corresponding  to  electrographic 
localization  of  seizure  onset  in  twenty-one  patients.  Five  patients  had  a  region  of 
increased  MR  signal  intensity  on  the  spin  echo  image  in  the  region  of  the  EEG  focus; 
two,  an  abnormality  ipsilateral  to  but  distinct  from  the  EEG  focus;  and  one  had 
bilateral  findings.  CTscan  was  abnormal  in  three  cases;  two  had  tumors.  Three 
patients  had  low-grade  tumors  (one  with  a  normal  PET  scan).  PET  can  detect 
metabolic  dysfunction  associated  with  mild  pathologic  changes  in  epileptic  foci,  but 
increased  signal  intensity  on  MRI  does  not  necessarily  correlate  with  the  degree  of 
pathologic  abnormality.  Tumors  may  be  less  likely  when  both  CT  and  MRI  are 
normal. 

During  surgery,  direct  electrocorticographic  recordings  are  made  before  and  after 
tissue  is  resected,  in  order  to  guide  the  surgical  approach.  Tissue  from  both 
epileptogenic  and  nonepileptogenic  regions  is  obtained  for  biochemical  study. 
Preliminary  results  indicate  that  glycine,  glutamate,  and  aspartate  were  significantly 
higher  in  spiking  than  nonspiking  regions,  while  GABA,  taurine,  and  alanine  were 
unchanged.  These  data  suggest  that  alterations  in  excitatory  rather  than  inhibitory 
amino  acids  may  occur  in  epileptic  foci. 

Magnetoencephalography  (MEG)  is  a  new  approach  to  the  problem  of  localizing 
abnormal  cerebral  potentials  which  may  represent  an  epileptic  focus.  Initial  studies 
suggest  that  MEG  may  provide  more  precise  three-dimensional  information  than 
EEG,  allowing  detection  and  localization  of  epileptic  foci  in  the  depths  of  the  brain, 
without  the  need  for  invasive  procedures. 

HUMAN  MOTOR  CONTROL  SECTION 

In  the  past  year,  Dr.  Leonardo  G.  Cohen  received  Intent  for  Tenure  and  was 
appointed  Head  of  the  Cortical  Physiology  Unit  in  the  Section.  Research  was  pursued 
in  a  variety  of  areas  related  to  human  movement  and  its  disorders. 

VOLUNTARY  LIMB  MOVEMENTS. 

Further  data  analysis  has  been  done  on  data  collected  in  previous  years  in  relation  to 
(a)  the  silent  period  in  the  antagonist  muscle  prior  to  a  ballistic  movement  in  normal 
subjects;  (b)  adaptation  to  movement  with  mirror-vision  in  patients  with  Parkinson's 
disease  (PD);  and  (c)  reaction  time  studies  in  patients  with  cerebellar  disease  and  PD. 
Major  effects  have  been  directed  to  the  role  of  the  cerebellum  in  coordination  and 
motor  skill  learning. 

(1)  The  contribution  of  the  cerebellum  to  coordination:  There  exist  long-standing 
questions  about  the  precise  role  of  the  cerebellum  in  coordination.  Much 
theoretical  and  experimental  work  has  emphasized  the  action  of  the  cerebellum  in 


4-MNB/DIR 


calculating  the  appropriate  distribution  of  neural  activity  to  various  muscle  groups 
involved  in  a  movement.  More  recently,  work  by  Keele  and  others  has  focused  on 
the  possibility  that  the  cerebellum's  fundamental  computation  is  that  of  clocking 
neural  motor  control  signals.  To  evaluate  the  validity  of  these  and  other  viewpoints, 
the  detailed  kinematics  of  upper  extremity  movements  in  two  dimensions  in  human 
cerebellar  patients  and  in  normal  controls  are  being  examined.  In  addition, 
electromyography  (EMG)  signals  are  obtained  from  involved  muscle  groups.  These 
data  are  then  used  in  conjunction  with  a  biomechanical  model  of  the  upper 
extremity  to  infer  the  joint  torques  which  are  developed  during  the  movement.  A 
comparison  is  then  made  between  the  force  patterns  in  normals  and  cerebellar 
patients  with  regard  to  magnitude,  distribution  and  timing.  This  is  essentially  an 
extension  to  two-joint  movements  of  the  work  on  EMG  patterns  in  single-joint 
movements  performed  by  our  group  earlier.  The  goal  is  to  identify,  if  possible,  the 
fundamental  motor  control  derangement  in  cerebellar  patients  which  leads  to  ataxic 
movements.  Studies  performed  this  year  comparing  hand  trajectories  executed 
under  either  fast  or  accurate  movement  constraints  have  revealed  intriquing 
differences  between  patients  with  cerebellar  disease  and  controls.  Under  the  "move 
accurately"  constraint,  the  cerebellar  patient's  trajectories  had  normal  paths  and 
final  position  accuracy.  In  contrast,  in  the  "move  fast"  condition,  the  cerebellar 
patients  exhibited  deficits  in  final  position  control  and  significant  deviations  from 
straight-line  trajectories,  consistent  with  the  notion  that  functions  subserved  by  the 
cerebellum  are  critical  to  the  portion  of  the  motor  execution  process  involving 
compensation  for  limb  dynamic  changes  during  high-speed  movements. 

A  detailed  biomechanical  analysis  of  cerebellar  movements  is  being  undertaken  .  To 
date,  an  extensive  computer  program  has  been  developed  to  first  filter  and  then 
analyze  trajectories  individually  and  as  a  group  with  regard  to  several  parameters 
including  degree  of  deviation  from  linearity,  maximum  velocity,  movement  time, 
linear  and  angular  acceleration,  and  jerk.  A  submodule  then  simulates  the 
movement  using  a  two-link  rotary-joint  biomechanical  arm  model  and  thereby 
predicts  the  torque  and  torque-change  at  each  joint.  The  output  is  presented 
graphically  and  can  be  exported  in  data  files  for  further  analysis.  Preliminary 
evaluation  of  data  from  several  cerebellar  patients  reveals  that  the  patients  make 
systematic  errors  when  attempting  to  rapidly  produce  a  straight  line.  These  errors 
appear  to  differ  primarily  in  magnitude,  rather  than  in  character,  from  the  trajectory 
errors  of  normal  volunteers.  Work  is  now  being  done  to  quantify  the  degree  of 
kinematic  abnormality  and  to  attempt  to  relate  this,  if  possible,  to  a  consistent 
underlying  abnormality  in  the  dynamics  and  EMG  findings. 

(2)  The  contribution  of  the  cerebellum  to  learning:  It  has  long  been  postulated  that 
the  cerebellum  and  its  input  and  output  pathways  play  a  significant  role  in  long-  and 
short-term  changes  in  motor  behavior  and  quality  of  motor  performance  in  response 
to  an  alteration  of  either  the  sensory  inputs  or  task  demands.  Studies  in  the  past 
mainly  focusing  on  modifications  ot  the  vestibulo-ocular  reflex  (VOR)  have 
demonstrated  in  various  species  that  the  integrity  of  cerebello-olivary  pathways  is 
required  in  order  to  be  able  to  modify  the  gain  of  pre-existing  reflex  pathways  in 
response  to  an  alteration  of  the  normal  input-output  pattern.  Based  on  these 
findings,  it  has  been  proposed  that  the  modification  of  Purkinje  cell  firing  pattern 
caused  by  an  alteration  of  climbing  fiber  input  might  represent  one  part  of  the 
neuronal  substrate  of  learning  ana  memory  in  humans. 

To  evaluate  the  role  of  the  cerebellum  in  the  acquisition  of  motor  skills  in  humans, 
we  have  initiated  a  series  of  experiments  that  study  the  time  course  and  mechanisms 
of  skill  aquisition  in  a  paradigm  involving  complex  two-dimensional  multijoint  arm 


5-MNB/DIR 


movements.  Subjects  are  instructed  to  track  a  visually-guided  path  on  a  digitizing 
tablet  while  attempting  to  increase  speed  and  accuracy  of  their  movements. 
Initially,  evaluation  of  kinematic  movement  parameters  (trajectory  variability, 
tangential  velocity  and  acceleration  profiles)  will  be  emphasized.  Operating 
characteristics  in  trie  speed-accuracy  domain  shall  be  assessed  before  and  after 
various  practice  trials.  Shifts  in  the  speed-accuracy  operating  characteristic  will  be 
used  to  quantify  improvements  of  motor  performance.  Short-term  skill  acquisition, 
i.e.,  skill  improvement  within  practice  sessions  and  long-term  retention  over  periods 
of  several  days,  will  be  compared  in  normal  subjects  and  in  patients  with  cerebellar 
deficits.  In  a  second  stage,  the  dynamic  aspects  (torque  and  torque-change)  of  the 
employed  optimization  strategies  during  practice  of  multijoint  arm  movements  as  a 
model  of  motor  learning  shall  De  investigated. 

PET  STUDIES. 

Using  [18F]-2-deoxyglucose  for  PET  scans,  we  have  investigated  regional  cerebral 
metabolism  of  glucose  with  voluntary  movement  and  with  sensory  activation.  We 
have  failed  to  detect  any  increase  in  regional  cerebral  cortical  metabolism  with 
simple  hand  movements.  Using  H2O15  as  a  marker  for  cerebral  blood  flow,  we  have 
also  looked  for  changes  in  the  sensorimotor  cortex  with  simple  hand  movements  and 
with  sensory  stimulation.  We  have  found  an  increase  in  the  cerebral  blood  flow  in 
the  sensorimotor  cortex  contralateral  to  voluntary  wrist  movement.  Also,  there  was 
increased  blood  flow  in  the  supplementary  motor  cortex  and  in  the  ipsilateral 
sensorimotor  cortex. 

With  this  as  a  background,  we  have  gone  on  to  study  changes  in  regional  cerebral 
blood  flow  (rCBF)  during  various  sensorimotor  tasks.  The  studies  this  year  have 
included:  (1)  comparisons  of  regional  activation  in  self-paced  movements  and 
passive  vibration  of  the  same  body  part;  (2)  comparisons  of  self-paced  and  visually- 
triggered  hand  movements;  (3)  comparisons  of  activation  associated  with  eye 
movements  of  different  kinds  ( saccades,  pursuit,  and  fixation  );  and  (4)  rCBF  changes 
associated  with  acquisition  of  a  manual  motor  skill.  Approximately  six  to  ten  normal 
volunteers  have  been  studied  in  each  of  these  experiments. 

A  major  problem  in  the  interpretation  of  our  data  has  been  the  neuroanatomical 
localization  of  the  rCBF  changes  observed  in  particular  behavioral  states.  Existing 
techniques  that  utilize  either  a  neuroanatomical  brain  atlas  or  generic  stereotaxic 
coordinates  are  subject  to  large  errors  in  spatial  localization  due  to  the  large 
numbers  of  degrees  of  freedom  involved  in  the  process  of  determining  a  conformal 
mapping  between  two  imaging  modalities.  For  this  reason  we  have  mounted  a 
major  effort,  in  collaboration  with  Dr.  Pellizari  of  the  University  of  Chicago,  to 
develop  a  computational  technique  that  allows  rapid  and  accurate  spatial 
registration  of  pairs  of  three-dimensional  data  structures.  Using  this  approach,  it  is 
possible  to  mark  regions  of  interest  (ROI)  in  data  obtained  with  one  imaging 
modality  and  then,  through  the  mapping  matrix,  retrieve  values  from  the 
corresponding  locations  in  a  dataset  obtained  with  a  different  imaging  modality. 
This  allows  determination  of  ROI's  using  a  subject's  MRI  scan  and  then  retrieval  of 
the  corresponding  changes  in  rCBF  from  the  corresponding  PET  study. 

Preliminary  analysis  of  the  self-paced  wrist  movements  has  provided  information 
relevant  to  the  understanding  of  functional  organization  of  cortical  motor  areas. 
The  largest  increases  in  rCBF  were  observed  in  cortical  motor  areas,  including:  the 
contralateral  sensorimotor  cortex  (mean  -42%,  S.D.  -  13%),  the  ipsilateral 
sensorimotor  cortex  (mean  -  19%,  S.D.  -  8%),  and  the  medial  frontal  cortex  (mean  - 


6-MNB/DIR 


30%,  S.D.  -  14%).  There  was  a  strong  relationship  between  the  contralateral  and 
ipsilateral  increase  in  each  subject,  such  that  the  ipsilateral  increase  was  30%  to  40% 
of  the  corresponding  contralateral  change.  It  was  not  possible  to  determine  the 
laterality  of  the  observed  medial  frontal  increases.  In  seven  of  the  eleven  subjects,  a 
contralateral  activation  in  the  superior  parietal  lobule  was  noted.  These  results 
demonstrate  that  the  involvement  of  ipsilateral  cortical  motor  areas  in  the  planning 
or  execution  of  simple,  alternating  limb  movements  may  be  more  extensive  than 
previously  thought. 

EVOKED  POTENTIAL  STUDIES. 

(1)  Studies  in  patients  with  subdural  electrodes:  Three  additional  epileptic  patients 
who  were  going  to  be  operated  on,  with  a  grid  of  subdural  electrodes  positioned 
over  sensorimotor  areas,  were  studied  before  and  after  implantation  this  year. 
Before  implantation,  somatosensory  evoked  potentials  (SEP)  from  scalp  electrodes 
were  recorded  to  finger,  nerves  ana  skin  stimulation  of  the  arm.  After  implantation, 
SEPswere  recorded  to  the  same  modalities  of  stimulation  from  the  subdural 
electrodes.  Comparison  of  both  noninvasive  and  invasive  procedures  are  under  way. 
Mild  electric  stimuli  were  delivered  through  pairs  of  subdural  electrodes  precisely 
localized  by  X-ray  and  MRI  scans  and  the  signal  recorded  from  scalp  electrodes  with 
the  purpose  of  determining  the  precision  of  noninvasive  techniques  for  dipole 
localization.  SEP  studies  provided  information  on  the  precise  localization  of  sensory 
representation  areas.  These  results  were  correlated  with  those  from  magnetic 
stimulation  studies  and  attenuation  of  somatosensory  perception  by  magnetic 
stimulation.  In  this  manner,  we  were  able  to  define  precise  maps  of  cortical 
functional  anatomy.  Using  this  information,  we  have  also  developed  an  improved 
method  for  evaluating  electrical  brain  dipoles. 

(2)  Dystonia:  Short  latency  SEPs  to  median  nerve  stimulation  were  evaluated  in  ten 
dystonic  patients  and  ten  normal  volunteers.  The  amplitude  of  the  N30  component 
was  larger  in  patients  than  in  controls.  Previous  work  has  shown  a  decreased  N30  in 
patients  with  Parkinson's  disease.  Changes  in  N30  amplitudes  may  be  indicative  of 
abnormal  excitatory  effects  upon  cortex  resulting  from  diseases  of  the  basal  ganglia. 

(3)  Parkinson's  disease  (PD):  We  have  evaluated  seven  patients  with  PD  to  measure 
the  N30  component  of  median  nerve  SEPs.  The  amplitude  of  this  component  has 
been  reported  as  attenuated  in  PD,  and  we  wish  to  repeat  this  to  compare  to  our 
results  in  dystonia.  In  the  seven  patients  tested  so  far,  N30  was  not  obviously  smaller 
than  controls,  but  the  project  is  still  ongoing  and  statistical  analysis  is  not  yet 
appropriate. 

(4)  Hemispherectomy:  Studies  are  under  way  to  determine  the  scalp  distribution  of 
short  latency  SEPs  in  patients  with  hemispherectomy  performed  for  intractable 
seizures.  We  have  so  far  evaluated  short  latency  SEPs  in  two  patients.  Our 
preliminary  results  indicate  that  stimulation  of  the  arm  contralateral  to  the  excised 
hemisphere  evoked  subcortical  P15  and  N18  components  but  failed  to  evoke  clear 
postcentral  N20-P27  and  precentral  P22-N30  complexes.  For  this  reason,  we  have 
been  unable  to  detect  reorganization  in  sensory  pathways  in  these  patients.  Further 
studies  are  under  way  looking  at  later  SEP  components. 

(5)  Stroke:  We  have  studied  two  patients  with  stroke  (both  with  small  left 
subcortical  infarcts)  with  the  purpose  of  detecting  changes  in  the  scalp  distribution 
of  cortical  components  of  SEPs  to  median  nerve  stimulation  following  these  lesions. 
In  both  cases,  cortical  components  of  SEPs  were  absent  and  the  last  detectable 


7-MNB/DIR 


component  on  the  affected  side  was  the  subcortical  N18,  which  had  a  widespread 
scalp  distribution  similar  to  controls.  In  the  future,  we  plan  to  evaluate  long-latency 
SEP  components  and  patients  with  more  focal  lesions. 

(6)  Congenital  mirror  movements:  Short  latency  SEPs  to  right  median  nerve 
stimulation  were  recorded  in  two  patients.  The  scalp  distribution  of  the  postcentral 
N20  and  precentral  P22  was  similar  to  that  observed  in  normal  volunteers  (100) 
indicating  that  bilateral  representation  observed  in  motor  systems  is  not  present  in 
somatosensory  systems. 

(7)  Amputees:  Two  patients  with  unilateral  upper  limb  amputation  were  studied. 
The  goal  was  to  determine  side  to  side  differences  in  the  scalp  distribution  and 
amplitudes  of  SEPs  to  stimulation  of  symmetric  body  parts  immediately  proximal  to 
the  stump.  In  these  two  cases,  we  have  found  no  differences. 

(8)  Spinal  cord  injury:  We  have  finished  collecting  data  from  ten  patients  with 
thoracic  lesions  leading  to  paraplegia.  Results  have  not  yet  been  analyzed. 

MOVEMENT  RELATED  POTENTIALS. 

We  have  used  the  standard,  widely-spaced  electrode  montage  in  topographic 
electroencephalography  (EEG)  mapping  to  study  the  scalp  distribution  of  the 
terminal  phase  of  the  Bereitschaftspotential  using  a  collection  window  of  499  ms. 
After  establishing  normative  data  on  the  subpotentials  preceding  the  voluntary 
index  finger  movements  in  a  normal  population,  we  have  been  able  to  do  detailed 
analysis  on  the  generators  of  the  different  subpotentials  using  a  tightly  spaced 
electrode  montage  over  the  sensorimotor  area  of  the  scalp.  Four  distinct  negative 
events  were  identified:  the  peak  of  the  negative  slope  (NS1),  the  initial  slope  of 
motor  potential  (isMP),  the  parietal  peak  of  motor  potential  (ppMP),  and  the  frontal 
peak  of  motor  potential  (fpMP).  For  self-paced  movements,  NS'  and  isMP  occurred 
before  the  onset  of  electromyographic  (EMG)  activity,  and  ppMP  and  fpMP  occurred 
afterthe  onset  of  EMG  activity.  NS'  had  a  wide  distribution,  covering  the  parietal 
region  with  slight  contralateral  predominance.  The  isMP  mapped  focally  over  the 
contralateral  hand  motor  area  on  the  scalp.  The  location  of  ppMP  was  similar  to  that 
of  isMP.  The  fpMP  was  localized  anterior  and  medial  to  motor  cortex  with  a 
contralateral  preponderance  and  possible  location  over  the  supplementary  motor 
area  (SMA).  The  isMP  appears  to  reflect  activation  of  the  cortical  cells  in  the  hand 
area  of  motor  cortex  for  the  execution  of  voluntary  movement,  and  the  fpMP 
appears  to  reflect  proprioceptive  feedback  from  the  periphery. 

We  studied  the  scalp-recorded,  movement-related  cortical  potentials  occurring 
immediately  before  and  after  the  onset  of  movement  in  five  patients  with 
asymmetric  Parkinson's  disease.  The  topographic  distribution  of  the  isMP  was 
diffuse  for  voluntary  finger  movements  of  the  more  affected  hand  but  normal  for 
movements  of  the  less  affected  hand.  The  fpMP  was  located  more  posterior  in 
patients  than  in  normal  subjects.  The  peak  amplitudes  of  the  potentials  were 
normal  in  all  patients.  The  topographic  abnormalities  might  reflect  inadequate 
excitatory  activity  from  the  basal  ganglia  to  the  primary  motor  cortex  and  the  SMA. 

We  studied  six  patients  with  cerebellar  degenerative  disease.  All  patients  had  an 
abnormal  topographic  pattern  of  potentials  compared  with  normal  subjects.  The 
amplitudes  and  latencies  of  the  potentials  were  normal  in  all  patients  except  two,  in 
whom  NS'  was  absent.   The  isMP  was  diffuse  and  bilateral  in  the  patients.  The 
topography  of  the  fpMP  was  more  posterior  in  the  patients  than  in  normal  subjects. 


8-MNB/DIR 


These  patterns  indicate  a  derangement  of  sensorimotor  cortex  activity  in  voluntary 
movement  as  a  consequence  of  cerebellar  dysfunction. 

Eight  patients  with  dystonia  were  evaluated  with  the  particular  goal  of  measuring 
the  FPmp  which  might  reflect  SMA  function.  Preliminary  evaluation  shows  that  the 
scalp  distribution  and  the  magnitude  of  this  component  was  similar  in  both  groups. 

BALANCE  AND  GAIT. 

The  focus  of  studies  in  human  balance  has  been  the  maintenance  of  upright  posture. 
Detailed  quantification  of  postural  sway  has  been  applied  to  normal  subjects  at 
various  ages.  Our  studies  snow  that  acceleration  of  individual  body  segments 
characterizes  normal  upright  stance;  older  normal  subjects  maintain  a  more  rigid 
posture,  utilizing  large  and  inconsistent  adjustments  to  maintain  stability.  This  may 
relate  to  the  instability  of  stance  in  the  elderly.  Additionally,  we  have  begun  an 
evaluation  of  EMG  phase  relationships  to  descriptors  of  sway.  Gastrocnemius,  for 
example,  appears  to  act  in  a  feedforward  mode  as  a  brake  to  forward  sway. 

We  have  completed  studies  of  standing  balance  in  patients  with  PD  and  patients  on 
gentamycin  who  might  be  developing  vestibular  deficits.  The  data  have  not  yet 
been  analyzed  in  detail,  but  it  appears  that  postural  studies  might  be  a  sensitive 
method  for  detecting  vestibular  toxicity. 

Our  previous  studies  have  demonstrated  that  normal  subjects  make  postural 
adjustments  in  advance  of  voluntary  arm  movements.  This  has  been  studied  in  detail 
in  self  paced  and  reaction  time  movements.  Postural  adjustments  precede  arm 
movement  more  in  the  self  paced  situation  (when  there  is  more  time).  However,  we 
have  not  been  able  to  detect  any  increased  instability  in  the  reaction  time 
circumstance.  The  linkage  of  postural  adjustments  to  limb  movements  is  not  fixed, 
but  varies  with  the  circumstance. 

We  have  begun  an  analysis  of  gait  in  patients  with  autism  and  cerebellar  ataxia. 
Cerebellar  gait  is  characterizedby  marked  variability  compared  to  normal  and 
disorganization  of  interlimb  coordination.  Patients  with  autism  are  said  to  have 
cerebellar  lesions,  but  our  analysis  does  not  show  any  cerebellar  features  in  their 
gait. 

REFLEXES. 

In  past  years  the  laboratory  has  done  extensive  studies  with  reciprocal  inhibition  in 
patients  with  dystonia  ana  PD.  We  conclude  that  there  is  an  important  deficit  in 
reciprocal  inhibition  in  these  disorders.  There  are  a  number  of  phases  in  the 
reciprocal  inhibition  curves  and  the  physiology  of  each  is  not  clear.  During  further 
studies  of  this  project,  it  became  clear  that  the  method  can  be  improved  and  better 
standardized.  Therefore,  a  study  of  different  parameters  that  influence  the 
reciprocal  inhibition  of  the  H-reflex  in  wrist  flexors  was  undertaken.  The  most 
important  parameter  forthe  inhibition  in  all  three  periods  (with  peaks  at  or  around 
0  ms,  10  ms,  and  75  ms  of  delay  between  the  shock  to  the  radial  and  median  nerves) 
is  the  intensity  of  the  radial  nerve  shock.  The  same  parameter  also  strongly 
influences  the  relative  excitation  of  the  H-reflex  around  30  ms  of  delay  between  the 
shock  to  the  radial  and  median  nerves.  The  duration  of  the  stimulus  delivered  to  the 
radial  nerve  is  comparatively  less  important;  however,  there  is  a  trend  for  more 
inhibition  when  the  duration  of  the  stimulus  to  the  radial  nerve  is  0.5  ms  instead  of 
longer  or  shorter  durations.  Moreover,  preliminary  data  suggest  that  another 


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important  parameter  is  the  amplitude  of  the  H-reflex,  and  that  smaller  H-reflexes 
can  be  inhibited  more  effectively  by  a  radial  nerve  shock  than  larger  ones. 

The  physiology  of  rigidity  in  PD  can  be  investigated  by  studying  reflexes.  Cutaneous 
reflexes  were  measured  in  ten  patients  with  PD,  and  in  ten  age-  and  sex-matched 
normal  volunteers.  EMG  activity  was  recorded  from  the  first  dorsal  interosseous 
muscle  with  surface  electrodes,  rectified  and  averaged.  Stimuli  of  0.2  ms  duration 
were  delivered  at  a  rate  of  3  Hz  through  ring  electrodes  to  the  index  finger. 
Stimulus  intensity  was  four  times  sensory  threshold  or  at  the  threshold  of  pain, 
whichever  was  lower.  The  subjects  abducted  the  index  finger  with  20%  of  maximal 
force.  The  reflex  is  composed  of  successive  excitatory  and  inhibitory  events.  While 
the  latencies  of  the  different  reflex  components  ana  the  amplitudes  of  the 
excitatory  peaks  were  not  different  in  the  two  groups,  the  first  inhibitory  peak, 
occurring  at  a  mean  latency  of  51  ms,  was  less  pronounced  in  patients  with  PD  as 
compared  to  normals  (t-test,  p  <  0.05).  The  result  is  compatible  with  the  loss  of  a 
spinal  inhibitory  mechanism  elicited  by  cutaneous  afferents  and  can  be  a  partial 
explanation  for  increased  tone  in  PD. 

Spasmodic  dysphonia  and  orofacial  dyskinesia  (Meige  syndrome)  are  focal  dystonias. 
A  common  clinical  sign  in  these  disorders  is  the  absence  of  habituation  of  trigemino- 
facial  reflexes,  such  as  the  blink  reflex  or  perioral  reflexes,  which  can  be  evoked  by 
tapping  either  the  glabella  or  the  skin  in  the  vicinity  of  the  lips.  Recent  studies  on 
the  recovery  cycle  of  the  R2  response  of  the  blink  reflex  in  patients  with  spasmodic 
dysphonia  have  demonstrated  a  loss  of  inhibitory  influence  on  brainstem 
interneurons  which  are  conveying  the  afferent  impulse  from  the  trigeminal  to  the 
facial  nucleus.  However,  in  some  patients  with  spasmodic  dysphonia  the  blink  reflex 
and  the  blink  reflex  recovery  cycle  may  be  normal,  whereas  a  tapping  to  the  perioral 
skin  evokes  increased  reflex  responses  in  the  perioral  muscles  indicating  an 
inhomogeneous  influence  of  the  basal  ganglia  on  different  parts  of  the 
somatotopical  organized  facial  nerve  nucleus.  Thus,  a  precise  electrophysiological 
assessment  of  trigemino-facial  reflexes  of  lower  face  muscles  may  provide  further 
information  on  the  functional  integrity  of  the  trigemino-facial  pathways  in  addition 
to  blink  reflex  studies. 

We  developed  a  technique  to  evoke  perioral  reflexes  by  stimulating  branches  of  the 
trigeminal  nerve  (infraorbital  and  mental  nerve)  either  electrically  or  indirectly  via 
stimulation  of  cutaneous  receptors  by  deliverinq  well-defined  taps  to  the  skin  in  the 
vicinity  of  the  lips,  mimicking  the  clinical  test.  The  reflex  responses  consisting  of  an 
ipsilateral  early  R1  response,  a  late  bilateral  R2  response,  and  occasionally  a  rare 
third  response  are  recorded  using  surface  electrodes.  This  reflex  pattern  is  very 
similiarto  the  blink  reflex. 

We  have  established  normative  data  and  studied  patients  suffering  from  orofacial 
dyskinesias  and  spasmodic  dysphonia.  In  both  groups  of  patients,  thresholds  for 
eliciting  R2  components  of  facial  reflexes  were  lower  and  recruitment  curves  were 
steeper  as  compared  to  normals.  Hyperexcitable  reflex  responses  could  be  observed 
to  a  similar  extent  after  applying  mechanical  as  well  as  electrical  stimuli  and  were 
most  easily  seen  in  the  upper  facial  muscles.  Reflex  responses  recorded  from 
periorbital  muscles  appeared  to  be  the  most  sensitive  parameter  to  indicate 
hyperexcitability  of  facial  reflexes.  This  was  true  not  only  for  stimulation  of  the 
supraorbital  nerve,  but  also  for  stimulation  of  the  infraorbital  and  the  mental  nerve. 
In  a  subgroup  of  spasmodic  dysphonia  patients,  there  was  a  dissociation  of  responses 
in  upper  and  lower  facial  muscles  with  isolated  hyperexcitable  responses  to 


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mechanical  stimulation  of  the  receptive  skin  area  of  the  mental  nerve  or  direct 
electrical  stimulation  of  the  mental  nerve. 

R2  responses  obtained  by  electrical  stimulation  of  infraorbital  nerve  and  mental 
nerve  showed  a  larger  degree  of  intertnal  variability  with  respect  to  amplitude  and 
duration  in  normal  subjects  as  well  as  in  patients,  as  compared  to  a  rather  stable 
responses  pattern  following  the  stimulation  of  the  supraorbital  nerve.  This 
phenomenon  is  most  likely  to  be  attributed  to  biological  differences  in  anatomical 
and  physiological  substrates  of  the  investigated  polysynaptic  reflex  arc.  The 
evaluation  of  the  recovery  cycle  itself  using  paired-shock  techniques  similar  to  blink 
reflex  studies  therefore  proved  not  to  be  appropiate,  since  the  inconvenience  of 
averaging  large  numbers  of  stimuli  at  various  stimulus  intervals  would  have  been 
required  in  order  to  obtain  reliable  data. 

HYPERPLEXIA. 

We  have  studied  several  patients  in  a  family  with  this  condition.  The  relationship 
between  the  normal  startle  reflex  and  the  involuntary  movement  in  this  disorder  has 
not  been  clear.  There  has  been  previously  only  limited  physiological  data,  and  that 
has  been  ambiguous.  Our  results  showed  that  the  movement  in  these  patients  is  an 
exaggerated  startle,  abnormal  by  virtue  of  its  ease  in  being  elicited,  its  rapid 
recruitment,  and  its  lack  of  habituation.  Our  work  helps  clarify  and  classify  this 
interesting  type  of  myoclonus. 

CORTICAL  STIMULATION. 

(1)  Conduction  velocities  in  motor  pathways:  We  have  developed  our  own  set  of 
control  data  for  measurement  of  central  conduction  velocities  in  motor  pathways.  A 
few  patients  from  different  groups  in  the  Clinical  Center  have  been  studied  for 
clinical  purposes.  The  first  five  normal  volunteers  and  five  patients  had  normal  EEGs 
recorded  before  and  afterthe  procedure  indicating  the  absence  of  epileptogenic 
activity  following  stimulation. 

(2)  Evaluation  of  motor  representation  areas:  We  have  looked  at  the  effects  of 
different  magnetic  coil  designs  on  the  characteristics  of  the  magnetic  and  electric 
fields  induced.  The  localization  of  effects  from  magnetic  coil  stimulation  is  not 
immediately  obvious.  We  measured  the  magnetic  fields  produced  by  several 
different  coils  and  compared  the  results  with  theoretical  calculations.  Magnetic 
stimuli  were  delivered  from  a  Cadwell  MES-10  magnetic  stimulator  using  three 
circular  coils  (one  9  cm  in  diameter;  two  rounded  to  a  point,  5  and  9  cm  in  diameter) 
and  twin  oval  coils  arranged  in  a  butterfly  shape  (each  coil  approximately  4  cm  in 
diameter)  and  from  a  Novametrix  Magstim  200  using  two  circular  flat-spiral  coils  (6.7 
and  14  cm  in  diameter).  We  found  that  for  all  coils  except  the  butterfly-shaped  coil, 
the  largest  electric  field  was  at  the  circumference  of  the  coils.  The  butterfly-shaped 
coil  induced  the  largest  currents  under  its  center,  where  the  circumferences  of  the 
two  component  coils  come  together.  We  determined  that  the  butterfly  coil  is  the 
most  focal  and  consequently,  we  used  it  for  our  mapping  studies.  Models  have  been 
detailed  for  the  electric  fields  induced  within  the  arm  and  skull. 

In  mapping  studies  of  normal  subjects,  we  observed  that  arm  muscles  could  be 
activated  with  stimulation  of  scalp  regions  several  centimeters  wide.  These  motor 
representation  areas  overlapped  extensively  when  muscles  studied  were  from  the 
same  limb  and  shifted  positions  abruptly  when  muscles  were  from  different  limbs. 


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Distal  hand  muscles  were  easier  to  activate  than  proximal  muscles  and  showed 
exclusively  a  contralateral  representation. 

We  evaluated  latencies  of  motor  evoked  potentials  (MEP)  obtained  with  different 
methods.  Progressively  longer  MEP  latencies  were  obtained  in  five  groups 
depending  on  the  type  and  position  of  stimulation.  The  shortest  latencies  were 
obtained  with  (a)  electric  stimulation  during  muscle  contraction,  and  (b)  nonfocal 
magnetic  stimulation  during  muscle  contraction;  magnetic  stimulation  at  rest 
produced  longer  latencies  with  stimulation  of  (c)  an  optimal  position,  (d)  a 
suboptimal  position,  and  (e)  a  nonoptimal  position.  Mean  latency  differences 
between  successive  groups  were  1.9,  2.0,  1.6,  and  2.6  msec  for  the  abductor  pollicis 
brevis  (APB);  similar  latency  differences  were  found  for  the  other  arm  muscles.  The 
results  are  compatible  with  the  hypothesis  that  the  different  latencies  evoked  by 
stimulation  at  different  scalp  locations  are  determined  by  the  summation  at  spinal 
motoneurons  of  excitatory  postsynaptic  potentials  generated  by  successive  numbers 
of  I  waves. 

(3)  Plastic  reorganization  in  the  brain  and  motor  pathways  following  a  variety  of 
lesions: 

(a)  Amputees:  To  evaluate  reorganization  in  motor  pathways  following 
amputation,  we  studied  MEPs  to  transcranial  magnetic  stimulation  in  seven  patients 
with  unilateral  upper  limb  amputations,  a  patient  with  congenital  absence  of  a 
hand,  and  ten  normal  subjects.  EMG  recordings  were  made  from  biceps  and  deltoid 
muscles  immediately  proximal  to  the  stump  and  the  same  contralateral  muscles.  The 
amplitude  of  MEPs  was  expressed  both  as  absolute  values  and  as  a  percentage  of 
maximal  responses  to  peripheral  nerve  stimulation.  Threshold  for  activation  of 
muscles  ipsilateral  and  contralateral  to  the  stump  and  the  region  of  excitable  scalp 
positions  were  also  determined  in  seven  patients. 

Magnetic  scalp  stimulation  induced  a  sensation  of  movement  in  the  missing  hand 
or  fingers  in  the  patients  with  acguired  amputation,  but  failed  to  do  so  in  the 
patient  with  congenital  absence  of  a  limb.  It  evoked  larger  MEPs,  recruited  a  larger 
percentage  of  the  motoneuron  pool,  and  elicited  MEPs  at  lower  intensities  of 
stimulation  in  muscles  ipsilateral  to  the  stump  than  in  contralateral  muscles. 
Muscles  ipsilateral  to  the  stump  could  be  activated  from  a  larger  area  than  those 
contralateral  to  the  stump.  These  results  are  compatible  with  cortical  or  spinal 
reorganization  in  adult  human  motor  pathways  targeting  muscles  proximal  to  the 
stump  after  amputations. 

We  studied  the  locus  of  motor  reorganization  after  amputation  in  five  subjects 
who  had  loss  of  a  lower  limb.  A  Cadwell  MES-10  stimulator  was  used  to  deliver  a 
magnetic  stimulus  through  a  round  coil  centered  on  the  sagittal  line  4  cm  anterior  to 
Cz  and  through  an  8-shaped  coil  positioned  over  locations  1  cm  apart  along  the 
coronal  axis.  Surface  EMG  was  recorded  bilaterally  from  guadriceps  femoris. 
Excitability  of  the  spinal  alpha-motoneuron  pool  to  la  afferents  was  evaluated  by 
comparing  the  maximal  H-reflex  with  the  maximal  M  wave  (H:M  ratio).  In  all  five 
subjects,  stimuli  of  egual  intensity  in  both  hemispheres  recruited  a  larger  percentage 
of  trie  alpha-motoneuron  pool  in  muscles  ipsilateral  to  the  stump  than  in 
contralateral  muscles  (46.8%  vs.  7.7%,  p<0.05)  and  mean  onset  latencies  were  2  ms 
shorter  (p<0. 01).  Muscles  ipsilateral  to  the  stump  were  activated  from  a  larger 
number  of  scalp  positions  than  contralateral  muscles  (p<0. 05).  There  was  no 
difference  in  the  H:M  ratio  between  the  legs  (8.0%  ipsilateral  vs.  1 1.0  % 
contralateral).  The  apparent  absence  of  a  change  in  the  excitability  of  the  alpha- 


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motoneuron  pool  after  amputation  suggests  that  reorganization  occurs  at  a 
suprasegmental  level. 

(b)  Spinal  cord  injury:  We  studied  seven  patients  with  severe  thoracic  T9-T1 2 
spinal  cord  injury  occurring  between  two  and  five  years  before  testing.  EMG 
recordings  were  made  from  three  levels  (T6to  T12)  on  the  right  external  oblique 
abdominal  muscle  and  from  one  level  on  the  left.  Magnetic  stimulation  evoked 
larger  MEPs  with  shorter  latencies  from  a  larger  number  of  scalp  positions  in  muscles 
immediately  proximal  to  the  level  of  a  spinal  cord  injury  than  in  corresponding 
muscles  in  controls.  These  phenomena  were  present  at  rest  but  not  during  voluntary 
activation  of  the  target  muscles.  Our  findings  suggest  an  enhanced  excitability  of 
motor  representation  areas  targeting  muscles  proximal  to  the  level  of  a  spinal 
transection.  These  findings  are  compatible  with  the  notion  of  a  limited  flexible 
relationship  between  primary  motor  cortex  and  its  target  muscles  following 
alterations  of  normal  input-output  patterns. 

Patients  were  asked  to  report  sensations  felt  after  each  stimulus.  Magnetic 
stimulation  evoked  tingling  and  numbness  sensations  lasting  for  up  to  ten  seconds 
referred  to  different  parts  of  legs  and  toes  in  three  patients.  Sensations  were  felt 
more  distally  the  closer  the  site  of  stimulation  was  to  the  midline.  These  results 
indicate  that  at  least  portions  of  the  cortical  representation  areas  for  body  parts 
deafferented  by  a  complete  spinal  cord  injury  can  remain  related  to  those  body  parts 
for  up  to  several  years  following  the  lesion. 

(c)  Congenital  mirror  movements:  We  studied  two  patients  with  congenital 
mirror  movements  by  means  of  various  neurophysiological  and  metabolic 
techniques,  including  mapping  of  MEPs  to  transcranial  electric  and  magnetic 
stimulation,  premovement  and  SEPs,  kinematics  of  voluntary  movements,  muscle 
reflexes,  and  PET.  Abnormalities  in  maps  of  motor  and  premovement  potentials  and 
in  PET  scans  were  consistent  with  a  bilateral  representation  of  hand  muscles  in  the 
motor  cortex,  the  existence  of  physiologically  active  connections  capable  of 
conducting  fast  efferent  volleys  from  the  motor  cortex  to  ipsilateral  muscles,  the 
presence  of  mirror  EMG  activity  in  either  hand  with  intended  voluntary  movement 
of  the  other  hand,  the  absence  of  mirrored  EMG  responses  from  wrist  flexors  and 
extensors  to  mechanical  perturbation  of  the  contralateral  wrist,  and  normal  scalp 
distribution  of  SEPs  to  right  and  left  median  nerve  stimulation.  Our  findings  are 
consistent  with  aberrant  organization  of  motor  representation  areas  and 
corticospinal  pathways  with  ipsilateral  as  well  as  contralateral  control  of  voluntary 
movement. 

(d)  Hemispherectomy:  We  have  so  far  studied  two  patients  with 
hemispherectomy  performed  for  treatment  of  unresponsive  seizure  disorder. 
Stimulation  of  the  remaining  hemisphere  evoked  bilateral  muscle  responses  in 
proximal  and  distal  muscles  at  similar  latencies  indicating  a  bilateral  representation 
of  arm  muscles  and  the  existence  of  physiologically  active  ipsilateral  pathways. 

(e)  Stroke:  At  this  time  we  are  studying  changes  in  sensorimotor  maps  in  patients 
with  stroke. 

(4)  Timing,  locus,  topographical  extension:  So  far  we  have  observed  reorganization 
in  motor  pathways  as  early  as  four  to  six  months  following  amputations  and  spinal 
cord  injuries  leading  to  quadriplegia.  These  changes  are  not  present  in  every  patient 
after  such  a  short  time.  Part  of  our  future  efforts  will  be  dedicated  to  determining 


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the  factors  that  influence  the  development  of  reorganization  and  its  timing 
characteristics. 

(5)  Epilepsy:  In  this  area,  we  have  studied  three  patients  with  intractable  epilepsy 
who  had  a  grid  of  subdural  electrodes  positioned  over  sensorimotor  areas  for 
evaluation  of  surgical  intervention.  The  purpose  of  this  study  was  to  determine:  (a) 
focality  of  noninvasive  magnetic  stimulation  in  activating  hand  motor 
representation  areas;  (b)  development  of  seizures  or  afterdischarges  in  epileptic 
patients  after  magnetic  stimulation  (60).  Magnetic  stimulation  with  an  8-  shaped 
coil  was  accurate  in  localizing  the  peak  of  the  hand  motor  representation  area  for 
abductor  pollicis  brevis  within  1  cm  in  the  coronal  axis  in  the  three  patients. 
Magnetic  stimulation  was  unable  to  elicit  afterdischarges  or  other  EEG  changes  in 
any  of  the  patients  with  subdural  electrodes. 

(6)  Test  of  language  laterality:  A  goal  of  our  work  is  the  development  of  a  test  of 
language  laterality  using  magnetictranscranial  stimulation  which  is  less  invasive  and 
egually  accurate  to  sodium  amytal  testing  (Wada  test).  Pilot  studies  have  been 
carried  out  on  one  subject.  Naming  was  maximally  disturbed  by  stimulation  over  the 
right  or  left  superior  marginal  gyrus  (SMG)  with  a  latency  of  magnetic  stimulation  of 
200  ms  after  the  target.  Rhyming,  a  phonological  processing  task,  was  interfered 
with  to  a  greater  degree  by  stimulation  to  the  left  SMG  than  the  right  SMG. 

(7)  Evaluation  of  motor  and  sensory  physiology:  In  a  reaction  time  situation,  it  is 
possible  to  delay  the  expected  voluntary  response  by  a  magnetic  stimulus  delivered 
over  sensorimotor  cortex  given  just  before  the  movement.  Using  a  relatively  weak 
magnetic  stimulus  that  does  not  produce  a  motor  response  (MEP)  when  the  body 
part  is  at  rest,  but  will  produce  a  response  when  the  body  part  is  activated,  it  is 
possible  to  divide  the  reaction  time  period  into  two  phases.  In  the  first  phase  there  is 
no  MEP  and  the  motor  cortex  remains  "unexcitable. '  In  the  second  phase  there  is  a 
gradual  increase  in  MEP  amplitude  even  though  the  voluntary  EMG  activity  has  not 
yet  appeared.  This  "excitable"  phase  demonstrates  an  activation  of  motor  cortex 
before  the  motor  command  isdelivered.  Applying  the  lasttechnigue  to  the  analysis 
of  prolonged  reaction  time  (akinesia)  in  patients  with  PD,  it  can  be  demonstrated 
that  the  excitable  phase  is  prolonged.  This  demonstrates  a  mechanism  for  difficulty 
in  generating  a  motor  command  in  these  patients. 

We  studied  the  effects  of  focal  transcranial  magnetic  stimulation  on  ipsilateral 
muscles  in  six  normal  volunteers.  An  8-shaped  coil  was  used  to  deliver  stimuli  to 
different  scalp  positions  over  one  hemisphere.  EMG  recordings  were  made  from 
muscles  of  both  upper  extremities,  at  rest  and  at  several  degrees  of  voluntary 
ipsilateral  muscle  activation.  No  ipsilateral  responses  were  observed  with  the  arm  at 
rest.  During  muscle  activation,  stimuli  evoked  a  transient  attenuation  of  ongoing 
voluntary  EMG  activity  (ipsilateral  silent  period,  ISP).  ISPs  were  highly  reproducible 
and  could  be  evoked  in  scalp  locations  which  activated  corresponding  contralateral 
muscles.  The  ISP  in  abductor  pollicis  brevis  had  an  onset  latency  of  35  +  4  msec  (1 2 
msec  longerthan  the  onset  of  the  contralateral  excitatory  response),  and  a  duration 
of  46  +  15  msec.  At  50%  muscle  activation,  the  peak  attenuation  was  71  +  18%.  In 
wrist  flexors,  biceps  and  deltoid,  ISPs  tended  to  be  less  prominent.  On  some 
occasions,  excitatory  responses  preceded  the  ISPs.  This  study  demonstrates  the 
existence  of  a  powerful  inhibitory  influence  of  sensorimotor  cortex  on  ipsilateral 
upper  extremity  muscles  and  on  distal  muscles  in  particular. 

Particular  attention  is  being  paid  now  to  the  evaluation  of  interhemispheric 
right/left  differences  in  motor  representation  areas  targeting  different  arm  and  leg 


14-MNB/DIR 


muscles.  We  have  completed  a  pilot  study  in  ten  normal  volunteers,  and  have 
detected  somatotopic  organization  of  the  arm  motor  representation  area.  Distal 
muscles  were  significantly  more  excitable  than  proximal  muscles.  Motor 
representations  targeting  proximal  arm  muscles  were  significantly  more  excitable  in 
the  rightthan  in  the  left  hemisphere. 

Magnetic  stimulation  of  the  brain  can  be  used  to  investigate  sensory  and  motor 
physiology  and  pathophysiology  in  intact  humans.  Although  uncommon,  it  is 
possible  for  magnetic  stimulation  over  the  sensorimotor  cortex  to  produce 
paresthesias.  It  is  possible  in  all  subjects  to  block  the  conscious  sensation  of  an 
electric  shock  delivered  to  the  index  finger  with  magnetic  stimulation.  The  magnetic 
stimulus  must  be  delivered  in  the  interval  from  300  ms  before  to  200  ms  after  the 
cutaneous  shock,  and  must  be  positioned  over  the  contralateral  hand  region  of 
sensorimotor  cortex. 

During  tonic  voluntary  contraction,  a  period  of  EMG  silence  follows  the  motor 
evoked  potential  produced  by  transcranial  stimulation  of  the  contralateral  motor 
cortex.  We  studied  the  silent  period  in  the  wrist  flexors  of  three  normal  volunteers 
during  20%  of  maximal  contraction.  To  test  the  excitability  of  the  spinal  motor 
neuron  pool  during  the  period  of  silence,  the  H-reflex  was  evoked  at  different 
intervals  after  cortical  stimulation.  The  amplitude  of  the  H-reflex  in  the  silent  period 
was  expressed  as  a  percentage  of  the  amplitude  during  complete  relaxation.  The  H- 
reflex  was  profoundly  depressed  at  the  beginning  of  the  silent  period  (13.5-27%  of 
the  control  measurement).  Despite  continuing  complete  suppression  of  voluntary 
activity,  the  H-reflex  showed  a  clear  tendency  to  recover  towards  the  end  of  the 
silent  period.  There,  the  H-reflex  amplitude  was  71-84%  of  the  control.  This  implies, 
that  at  least  in  the  late  part  of  the  silent  period,  a  reduction  in  the  excitability  ofthe 
spinal  motor  neuron  pool  does  not  play  a  major  role  in  determining  the 
phenomenon,  but  that  it  is  probably  caused  by  lack  of  central  drive. 

BOTULINUM  TOXIN  THERAPY. 

(1)  Focal  hand  dystonia    Open  trial:  We  have  studied  54  patients  with  writer's 
cramp.  Out  of  this  number,  15  experienced  major  improvement,  13  improved 
moderately,  12  experienced  minor  improvement  and  14  had  very  little  or  no 
improvement.   Finding  the  appropriate  dose  is  tricky  and  extensor  cramps  seem 
more  easily  treated  than  flexor  cramps.  The  EMG  appearance  of  the  spasm  is  the 
same  both  before  and  after  therapy  suggesting  that  the  major  mode  of  action  is  to 
weaken  the  muscle  and  not  to  reduce  the  amount  of  cramping.  Botulinum  toxin  has 
proven  to  be  both  safe  and  effective. 

(2)  Focal  hand  dystonia.  Double-blind:  More  definitive  results  on  the  effects  of 
botulinum  toxin  on  writer  s  cramp  require  a  double-blind  study.  We  are  now 
engaged  in  performing  this  double-blind  study  which  so  far  includes  nine  patients 
with  hand  cramps.  Patients  coming  for  a  first  time  are  started  on  the  open  trial  until 
making  sure  that  a  certain  dose  was  associated  with  subjective  improvement.  After 
the  beneficial  effects  disappear,  the  patient  returns  to  clinic  and  is  started  on  the 
double-blind  study  and  then  returns  to  the  open  trial.  Results  of  this  study  have  not 
yet  been  analyzed. 

(3)  Spasmodic  torticollis-Botox  type  A:  Two  patients  have  been  entered.  One 
experienced  major  and  the  other  moderate  improvement  after  the  initial  injections. 
Evaluation  of  swallowing  has  not  yet  been  done. 


15-MNB/DIR 


CLINICAL  NEUROPSYCHOLOGY  SECTION 

Members  of  the  Clinical  Neuropsychology  Section  have  eagerly  accepted  the 
investigative  challenges  of  the  "Decade  of  the  Brain"  and  proposed  a  series  of 
studies  to  codify  brain  mechanisms  that  service  cognitive  and  emotional  behavior, 
drawing  on  different  cadres  of  neurological  patients.  To  meet  this  challenge, 
program  priorities  have  been  redirected  from  the  progressive  neurological  and 
dementing  disorders  to  assess  the  effects  of  selective  and  focal  brain  disorders, 
primarily  temporal  lobe  epilepsy.  The  staff  is  comprised  of  new  members  who  retain 
expertise  in  cognitive  and  clinical  neuropsychology  and  are  utilizing  a  broad 
spectrum  of  clinical  and  experimental  tools  to  study  cerebral  localization  of  complex 
mental  functions.  These  procedures  include  studies  of  pre-  and  post-temporal 
lobectomy  changes,  functional  mapping  with  electrical  brain  stimulation, 
intracarotid  amytal  procedures,  electroencephalographic  (EEG)  and  electrodermal 
techniques,  and  new  and  exciting  initiatives  with  positron  emission  tomography 
(PET).  The  primary  and  specific  questions  examine  the  contributions  of  lateral  and 
mesial  temporal  structures  to  perception  and  memory,  and  how  these  limbic  systems 
guide  cognitive  and  emotional  behavior. 

One  line  of  investigations  under  the  direction  of  Dr.  Blaxton  examines  mnemonic 
processes  in  patients  before  and  after  unilateral  temporal  lobectomy  for  the  relief  of 
pharmacologically  intractable  seizures.  This  work  intends  to  clarify  the  role  of  mesial 
temporal  structures  for  learning  and  emotional  behavior.  Since  these  structures  are 
removed  surgically,  in  whole  or  part,  to  relieve  seizures,  temporal  lobectomy 
patients  constitute  an  ideal  study  group  to  identify  the  neuroanatomical  substrate 
underlying  different  types  of  memory  dysfunctions  exhibited  by  patients  with 
neurological  disorders. 

One  theoretical  line  focuses  on  implicit  and  explicit  memory  paradigms  and 
examines  differences  in  memory  performance  between  tasks  in  which  patients  are 
explicitly  instructed  to  remember  information  from  a  particular  study  episode,  and 
tasks  in  which  memory  is  assessed  more  implicitly  without  reference  to  prior  study. 
Of  particular  interest  is  the  question  of  whether  performance  on  these  two  types  of 
tests  reflects  the  operation  of  separate  underlying  memory  and  brain  stores. 

Memory  researchers  have  shown  that  some  amnesic  subjects  are  grossly  impaired  on 
explicit  tasks  (e.g.,  free  recall)  but  they  perform  as  well  as  normals  on  implicit  tasks 
such  as  perceptual  identification.  Our  research  has  been  aimed  at  two  main 
questions:  (1)  Do  temporal  lobe  patients  exhibit  patterns  on  these  memory  tests  as 
other  populations  of  patients  with  memory  impairments?  and  (2)  Do  physically 
separate  memory  systems  exist  or  can  the  amnestic  effects  be  better  explained  in 
other  ways? 

Studies  dedicated  to  these  processes  have  utilized  temporal  lobe  epileptic  patients 
and  matched  normal  controls  who  studied  pictures,  words,  or  abstract  designs. 
Depending  upon  the  experiment,  subjects  participated  in  four  study/test  cycles  in 
which  they  received  two  explicit  and  two  implicit  memory  tests.  These  tests  were 
constructed  such  that  one  in  each  of  these  categories  required  primarily  data-driven 
processing,  and  the  other,  conceptually-driven  processing.  Explicit  tasks  included 
free  recall,  semantic  cued  recall,  graphemic  cued  recall,  and  yes/no  recognition. 
Implicit  tests  included  word  fragment  completion,  word  stem  completion,  general 
knowledge  questions,  and  spelling  of  auditorily  presented  homophones.  Study 


16-MNB/DIR 


conditions  and  types  of  tests  were  usually  manipulated  as  within  subjects  factors  and 
materials  were  counterbalanced  across  subjects  and  tests. 

Three  studies  have  been  completed  which  strongly  suggest  that  the  pattern  of 
dissociations  among  memory  measures  observed  in  botn  patients  and  normals  is 
better  predicted  by  a  processing  account  of  memory  than  by  a  system  account. 
Results  for  normals  and  right  hemisphere  damaged  patients  showed  higher 
performance  levels  on  all  memory  tasks  under  conditions  in  which  the  type  of 
processing  engaged  at  study  (either  data-driven  or  conceptually-driven)  matched 
that  required  at  test  than  wnen  the  types  of  processing  differed  at  study  and  test. 
This  pattern  persisted  regardless  of  whether  the  tests  were  implicit  or  explicit. 
Results  for  patients  with  left  hemisphere  lesions,  in  contrast,  showed  that 
performance  levels  were  normal  on  data-driven  tests  but  impaired  on  both  implicit 
and  explicit  conceptually-driven  tests.  Thus,  the  fact  that  these  subjects  often 
perform  poorly  on  explicit  tests  of  memory  may  be  due  in  part  to  the  conceptually- 
driven  nature  of  those  particular  tests,  and  not  to  the  fact  that  they  are  explicit  tests, 
perse. 

Several  studies  dealing  with  explicit  memory  have  been  completed  which  compared 
recognition  memory  in  patients  with  left  or  right  hemisphere  lesions  with  matched 
controls.  These  experiments  attempted  to  tease  apart  data  vs.  conceptually  driven 
components  of  recognition  using  a  method  described  by  Gardiner  (1989).  Although 
normal  controls  and  left  lesion  patients  performed  normally  on  this  task,  patients 
with  right  hemisphere  lesions  showed  deficits  in  data-driven  processing  when 
learning  nonverbal  materials. 

Using  PET  activation  procedures  to  identify  brain  mechanisms  responsible  for 
storage  and  retrieval  for  both  short-  and  long-term  memory  stores,  Dr.  Fedio  has 
studied  four  patients,  one  of  whom  presented  a  severe  amnestic  disorder  related  to 
encephalitis.  Preliminary  data  indicate  that  the  left  inferior  and  lateral  temporal 
cortex  was  activated  with  each  task,  more  so  with  learning  new  episodic  materials 
(word  lists).  Left  frontal  regions  were  hypermetabolic  when  the  patients  were 
required  to  select  (recognition)  words  tnat  they  had  memorized  a  few  minutes 
(short-term)  or  several  days  before  (long-term).  There  was  evidence  of  frontal 
activation  during  recall  from  long-term  memory  stores,  much  less  when  short-term 
recall  was  activated  which  in  turn  provoked  parietal  regions  and  right  frontal  areas. 
The  left  thalamus  was  more  active  for  short-  than  for  long-term  memory.  Finally, 
regional  activation  for  the  amnestic  patient  showed  little  involvement  of  the  left 
brain  and  more  activation  from  the  right,  notably  the  frontal  lobe.  These  data 
indicate  that  frontal  mechanisms  are  brought  into  play  with  recognition  memory, 
whereas  the  inferior  and  lateral  temporal  lobe  and  parietal  lobe  are  engaged  when 
learning  and  recall  tap  into  short-term  memory  stores.  These  data  will  be  reanalyzed 
with  more  attention  to  quantitate  mesial  temporal  regions  and  the  cingulum. 

Neuroanatomic  correlates  of  implicit  and  explicit  memory,  conceptually-  and  data- 
driven,  will  be  explored  more  directly  with  brain  invasive  techniques.    In  a  series  of 
experiments  using  PET  imaging,  normal  subjects  will  be  scanned  while  performing 
different  memory  tasks  on  separate  scans  within  a  session.  These  tasks  may  be 
divided  along  implicit/explicit  lines,  or  in  terms  of  type  of  processing  they  require. 
Another  line  of  research  will  involve  electrical  stimulation  of  critical  brain  sites  via 
subdural  electrodes  in  temporal  lobe  patients,  and  will  employ  a  continuous  explicit 
memory  paradigm  during  stimulation  coupled  with  an  implicit  test  to  be  given 
following  stimulation.  A  separate  line  of  investigations  under  the  direction  of  Dr. 
Bookheimer  examines  the  contributions  of  the  temporal  lobe  to  perception  through 


17-MNB/DIR 


the  manipulation  of  spatial  attributes.  Previous  work  has  suggested  that  the  left 
and  right  brain  assume  a  dominant  role  in  utilizing  high  or  low  spatial  frequencies  to 
guide  visual  behavior,  but  what  remains  to  be  confirmed  is  whether  or  not  the  left 
hemisphere  is  more  efficient  in  dealing  with  high  frequencies  and  conversely,  the 
right,  with  low-frequency  information.  This  hypothesis  was  challenged  in  a  series  of 
studies,  and  examines  task-specific  processing  demands  on  high  ana  low  spatial 
frequency  analysis  in  normal  subjects  and  patients  with  unilateral  brain  lesions. 

Several  perceptual  paradigms  have  been  developed.  One  task  used  photographs  of 
faces  wherein  higher  or  lower  50%  spatial  frequencies  were  filtered.  Another  task 
applied  spatial  frequency  grids  with  a  normal  face  stimulus  in  a  masking  paradigm. 
Critical  band  masking  procedures  were  also  used  in  several  studies,  involving 
discrimination,  priming,  and  visual  persistence  of  high-  versus  low-frequency  images. 
This  technique  determines  the  range  of  frequencies  that  are  important  for  particular 
tasks  and  fields  of  presentation.  The  results  showed  that  the  efficiency  with  which 
high  or  low  spatial  frequency  filtered  faces  were  processed  depended  on  the 
cognitive  operations  necessary  for  the  task.  For  example,  when  the  stimuli  were 
identical,  high  spatial  frequency  information  was  preferred,  but  when  stimuli 
differed,  low-frequency  information  was  preferred  by  normal  subjects.  This  was 
interpreted  as  supporting  the  importance  of  strategic  variables  or  processing 
strategy  in  analyzing  visual  information.  Different  task  demands  may  selectively  call 
for  different  ranges  of  spatial  frequencies.  In  exploring  this  hypothesis  with  patients 
presenting  unilateral  epileptogenic  foci,  it  was  found  that  right  temporal  patients, 
while  performing  worse  than  normal  controls,  showed  the  same  interaction 
between  task  demands  and  spatial  frequency  as  the  controls.  In  contrast,  left 
hemisphere  damaged  patients  showed  no  interaction.  These  data  suggest  that  the 
left  hemisphere  is  important  in  strategic  contributions  to  information  processing,  a 
finding  consistent  with  other  research  on  strategic  processing  in  the  left  hemisphere. 

In  a  separate  study,  filtered  faces  were  presented  in  an  upright  or  inverted  position, 
and  while  patients  performed  worse  than  controls  in  processing  upright  faces,  they 
actually  performed  better  than  controls  with  the  inverted  faces.  This  was 
interpreted  as  suggesting  that  strategic  variables  which  aid  in  processing  upright 
faces  may  inhibit  processing  of  atypically  presented  visual  information.  Patients  who 
showed  a  deficit  in  strategic  processing  did  not  suffer  from  the  atypical  presentation 
and  performed  exceptionally  well. 

Studies  of  critical  band  masking  confirmed  the  importance  of  high  spatial 
frequencies  for  material  perceived  in  the  right  visual  field,  and  low  frequencies  for 
stimuli  in  the  left  visual  field.  Further,  the  presence  of  a  mask  inhibits  perception  by 
the  right  hemisphere  but  enhances  left  hemisphere  performance  in  critical 
frequency  ranges.  These  data  confirm  the  hypothesis  that  the  hemispheres  differ  in 
mode  of  information  processing.  Specifically,  perceptual  asymmetries  are  less 
pronounced  between  the  hemispheres  than  processing  asymmetries,  and  modes  of 
processing  interact  with  perceptual  characteristics  of  stimuli  to  bias  perception 
toward  high  or  low  frequencies. 

Additional  studies  are  currently  in  progress  to  further  explain  lateral  differences  in 
spatial  frequency  and  mode  of  visual  information  processing.  These  studies  measure 
critical  frequency  bands  when  performance  demands  are  altered.  Other  studies 
intend  to  examine  the  relationship  between  spatial  frequency  and  attentional 
processes  to  determine  how  directed  attention  influences  the  processing  of  specific 
ranges  of  spatial  frequencies.  Also,  we  are  examining  the  effects  of  simple  visual 
information  to  prime  attention  toward  a  subset  of  larger  sets  of  visual  information. 


18-MNB/DIR 


In  a  separate  series  of  studies  developed  by  Dr.  Fedio  and  Ms.  August,  several  tasks  of 
working  memory  were  developed  and  used  to  identify  critical  language  zones  via 
indwelling  electrodes  for  the  purpose  of  guiding  intended  resections.  There  was 
special  interest  to  functionally  map  the  basal  temporal  zones  which  are  inaccessible 
with  standard  intraoperative  stimulation  procedures.  Stimulation  of  Broca's  zone 
produced  speech  arrest;  in  traditional  Wernicke's  area,  anomia  was  elicited  and  was 
followed  by  amnesia  forthose  items  that  were  not  named  during  stimulation.  In 
contrast,  paraphasic  and  anomic  errors  were  evoked  with  low  level  stimulation  (3-5 
mA)  of  basal  temporal  sites  in  a  small  number  of  patients.  The  common  feature  of 
these  patients  was  that  each  had  presented  a  history  of  interictal  dysnomia, 
confirmed  by  neuropsychometric  tests.  Moreover,  anomia  with  basal  stimulation 
was  not  accompanied  by  anterograde  memory  errors.  That  is,  after  stimulation, 
these  patients  correctly  recalled  the  names  of  all  objects  which  they  did  not  name  or 
misnamed  during  stimulation.  Finally,  whereas  omissions  are  the  most  typical 
anomic  errors  with  stimulation  of  the  more  classic  language  area,  phonemic  errors 
were  observed  with  basal  temporal  stimulation  (e.g.,  'hammer"  for  "hat").  Finally, 
this  work  assumes  special  diagnostic  significance  for  select  patients  who  are  at  risk  to 
become  dysphasic  following  standard  resections  that  encroach  4  to  6  cm  along  the 
inferiortemporal  gyrus. 

The  preliminary  data  extend  the  findings  of  Luders  et  al.,  and  indicate  that  the  basal 
temporal  region  may  mediate  language  in  select  patients  with  mesial  and  inferior 
temporal  epileptogenic  lesions.  That  these  patients  were  clearly  anomic  prior  to 
surgery  encourages  interpretations  that  some  language  skills  are  displaced  in  these 
patients  and  that  linguistic  dysfunctioning  extends  to  systems  that  manage  word 
retrieval  and  access  to  semantic  categories.  Past  studies  with  thalamic  stimulation 
have  yielded  similar  anomic  errors  and  intact  memory.  Thus,  a  corollary  hypothesis 
may  argue  that  this  zone  functionally  links  the  thalamus  and  mesial  temporal 
structures  with  the  posterior  temporoparietal  language  area  and  mediates  access  to 
select  categories  in  semantic  memory.  Additional  studies  are  being  designed  to 
probe  the  basal  temporal  region  more  extensively,  and  will  employ  tasks  where  the 
patient  generates  semantic  and  phonemic  associates,  and  category  names.  Another 
approach  will  attempt  to  assess  the  importance  of  recall  vs.  recognition  memory 
from  this  region.  At  the  same  time,  a  match-to-sample  and  delayed  match  task  with 
random  patterns  will  be  used  during  stimulation  via  right  brain  electrodes. 

In  separate  studies,  Dr.  Fedio  has  modified  the  intracarotid  amytal  (Wada)  test  which 
has  taken  on  bolder  experimental  significance  to  determine  hemispheric  dominance 
for  perception  and  memory,  and  semantic  and  lexical  processes  in  pre-surgical 
epileptic  patients.  The  study  was  designed  to  examine  basic  semantic  processes  and 
how  the  left  and  right  brain  deal  with  spatial,  phonological  and  color  attributes  to 
perceive  and  remember  objects  and  words  cast  in  normal  and  unusual  format.  One 
hypothesis  was  dedicated  to  exploring  the  perceptual  rules  imposed  by  the  right 
hemisphere  for  naming  and  recognizing  pictorial  objects  appearing  in  black/white, 
natural  or  idiosyncratic  coloration.  The  same  neural  network  was  challenged  by 
lexical  disembedding.  Conversely,  these  functions  could  be  compared  in  the  same 
patient  after  a  left  injection,  and  extended  to  analyze  how  the  left  brain  applies 
linguistic  and  phonological  templates  to  name  ana  read  stimuli. 

The  paradigm  utilized  object  (achromatic)  naming  and  reading,  each  with  three 
stimuli  of  a  single  category,  and  post-distractional,  recognition  memory.  The 
intervening  distractors  were  two  chromatic  objects,  one  depicted  in  natural,  and  the 
other  in  incongruous,  coloration  which  simply  had  to  be  named  (e.g.,  [red]  rose  and 


19-MNB/DIR 


[pink]  zebra).  A  pair  of  unrelated  words  were  used  as  distractors  with  the  reading 
test:  a  pronounceable  nonsense  word  and  the  other  contained  two  words  which 
could  be  disembedded,  e.g.,  BAGel  was  read  as  "bag. .bagel"  and  buSINess  as 
"sin. .business";  the  foreground  word  was  printed  in  bold,  upper  case  format.  After 
a  baseline  of  naming  and  reading  was  established,  the  injection  was  made  with  75 
mg  of  sodium  amytal  via  a  transfemoral  catheter  threaded  into  the  internal  carotid 
artery.  The  first  injection  was  made  in  the  side  ipsilateral  to  the  lesion  and  the 
second  followed  about  45  minutes  later.  EEG  was  monitored  for  contralateral 
hemispheric  irrigation,  and  the  session  was  recorded  audiovisually  for  analysis. 

Dysnomia  and  dyslexia  persisted  several  minutes  after  the  left  injection;  there  was 
appreciably  less  disruption  after  the  right  injection,  and  the  errors  were  immediate 
and  short-lived  with  amytal.  With  the  left  injection,  the  error  rate  was  higher  with 
black/white  objects  than  for  those  colored  in  a  natural  or  incongruous  hue.  After 
the  right  injection,  in  contrast,  patients  were  not  anomic,  but  were  more  likely  to 
misname  (semantically)  incongruously  colored  objects.  These  preliminary  data 
indicate  that  coloration  is  a  critical  identifying  attribute  in  certain  stimuli,  and  is  as 
important  as  other  visuospatial  attributes  which  are  dependent  upon  right 
hemisphere  processing.  Overall,  omission  errors  were  most  prevalent,  but  phonetic 
errors  were  more  common  after  left,  and  semantic  errors  after  right,  injection. 

Reading  errors  were  more  prominent  with  left  than  right  injection.  After  the  left 
injection,  the  patient  read  embedded  words  correctly  but  tended  to  use  the  same 
phonological  pattern  and  to  mispronounce  the  carrier  word;  this  was  so  even 
though  he/she  correctly  read  the  mate  (script)  or  nonsense  word.  With  the  right 
injection,  the  reverse  pattern  was  recorded;  that  is,  the  patient  correctly  read  the 
carrier  word  while  failing  to  read  (and  perceive)  the  embedded  word.  These  results 
confirmed  that  agnosia  and  dyslexia  may  arise  from  spatial  errors  and  right 
hemisphere  disturbances,  whereas  kindred  disorders  in  naming  and  reading  may 
result  from  violation  of  phonetic  rules  with  left  hemisphere  changes. 

Recognition  memory  for  objects  and  words  was  more  impaired  after  amytal  was 
injected  into  the  left,  than  trie  right,  carotid,  due  in  part  to  dysphasia.  This  deficit 
was  more  severe  with  memory  for  words  than  objects.  The  most  effective  type  of 
entrapment  was  that  object  or  word  which  was  spatially  similar  to  one  of  the 
original  stimuli;  memory  for  words  was  better  preserved  after  the  right  injection. 
The  study  established  that  the  processing  of  form  and  color  attributes  are  mediated 
by  divergent  systems  in  the  left  and  right  brain.  Object  naming  and  reading 
processes  rely  on  well  established  rules  which  are  primarily  phonological  with  left, 
and  spatial  with  right,  brain  mechanisms.  Following  amytal  injection  into  the  left, 
perseveration  of  phonological  features  dominated  lexical  processing  separate  from 
dysnomia;  the  patients  were  unable  to  inhibit  phonological  rules  in  moving  from 
embedded  to  carrier  words,  while  applying  correct  lexical  rules. 

Delayed  recall  and  recognition  for  pictures  and  words  were  assessed  after  each 
injection;  recognition  was  superior  to  recall  and  pictures  were  far  better  recalled 
than  words.  Overall,  left  temporal  lobe  epilepsy  (TLE)  patients  did  not  do  as  well  as 
right  TLE  patients  with  recall  and  recognition  tasks.  Right  TLE  patients  were  three 
times  more  likely  than  left  TLE  patients  to  make  false  positive  recognition  errors 
which  may  reflect  in  part,  indiscriminate  verbal  processing  by  the  left  brain;  on  a 
speculative  note,  it  is  suggestive  of  a  positive  emotional  style  and  high  incidence  of 
euphoria  displayed  by  right  TLE  and  dysphoria  by  left  TLE  patients  following  right 
and  left  injection,  respectively,  in  this  study. 


20-MNB/DIR 


The  amytal  diagnostic  procedure  is  commonly  used  at  many  facilities  but  lacks 
standardization,  and  employs  very  few  items  to  reliably  assess  retrograde  and 
anterograde  amnesia  in  patients  at  risk  for  postsurgical  amnesia.  However,  the 
middle  cerebral  artery  may  not  irrigate  the  hippocampus,  which  together  with  the 
aforementioned  limitation  increases  the  chances  for  false  positive  errors  and 
exclusion  of  surgical  candidates.  The  technique  developed  at  NIH  is  unique  in  this 
regard  because  it  monitors  memory  continuously  for  several  classes  of  stimuli,  and  by 
using  a  smaller  dosage  of  amytal  and  a  serial  naming  task,  there  is  less  risk  of 
confounding  interpretations  due  to  aphasia.  The  present  series  will  be  expanded  to 
analyze  these  clinical  questions  and  examine  the  relative  patient  differences  in  terms 
of  early  vs.  late  onset,  clinical  history  of  anomia,  and  right  hemisphere  language 
dominance;  correlational  analysis  will  include  MRI  and  PET  data. 

Members  of  the  Section  are  currently  developing  several  procedures  that  may 
differentiate  attentional  and  mnemonic  operations  in  the  mesial  and  lateral  cortex 
of  the  temporal  lobe.  This  will  include  a  collaborative  project  with  Dr.  Marianne 
Regard,  University  Hospital,  Zurich,  who  has  the  opportunity  to  study  patients 
following  selective  amygdala-hippocampectomy.  The  current  obstacle  of 
developing  transcultural  test  stimuli  is  slowly  resolving;  this  work  will  also  be 
extended  to  compare  Nl.    and  Zurich  patients  with  experimental  personality  tests 
and  procedures. 

Neuropsychological  research  interests  in  dementia  were  directed  by  Dr.  Eric  Mohr 
who  achieved  a  distinguished  career  at  NIH  and  recently  accepted  a  position  with 
the  Royal  Ottawa  Hospital,  Ottawa,  Canada.  Dr.  Mohr  earned  distinction  by 
developing  protocols  to  study  neuropsychological  deficits  in  patients  with 
deteriorative  disorders,  primarily  Alzheimer's  (AD)  and  Parkinson's  (PD)  disorders. 
This  work  examined  the  specificity  and  magnitude  of  cognitive  deficits  in  PD.  To 
address  this  question,  PD  patients  with  exceptional  professional  and  educational 
distinction  who  continue  to  function  in  leadership  positions  were  compared  with 
normal  individuals.  The  patients  maintained  excellent  verbal  skills  and  executive 
functions,  but  experienced  decrements  in  episodic  memory  and  visuospatial 
integration  and  performed  poorly  on  perceptuomotor  tasks.  Although  losses  on 
tasks  requiring  speed  and  manual  manipulation  were  likely  reflective  of  motoric 
problems,  perception  errors  were  more  likely  reflective  of  cognitive  losses.  This 
observation  gives  further  credence  to  the  assertion  that  dopamine  function  and 
possibly  other  neurotransmitter  systems  are  implicated  in  the  pathophysiology  of 
PD. 

Visuospatial  deficits  are  commonly  cited  in  PD.  To  quantitate  differences  between 
PD  and  AD  dementia,  patients  matched  for  overall  dementia  severity,  age  and 
education,  were  evaluated.  Achievements  with  visuospatial  and  memory  tasks, 
especially  those  demanding  executive  functions,  were  defective  in  both  patient 
groups  but  tended  to  be  more  impaired  in  PD.  PD  dementia  appears  to  inflict 
relatively  more  aggravated  deficits  in  "executive  function"  and  less  severe 
decrements  in  visuospatial  tasks  with  generic  memory  components,  suggestive  of 
frontal  neuropathogenic  involvement. 

Response  fluctuations  in  motor  function,  complicating  long-term  dopaminomimetic 
therapy  of  PD,  may  extend  to  the  cognitive  realm.  To  evaluate  levodopa  treatment 
effects  on  attention  as  well  as  acquisition  and  retrieval  (memory),  PD  patients  were 
examined  while  medicated  with  levodopa/carbidopa  ("on")  and  when  the 
medication's  antiparkinsonian  effect  had  worn  off  ("off").  Significant  cognitive 
differences  emerged  only  on  the  delayed  recall  of  complex  verbal  materials  where 


21  -MNB/DIR 


patients  when  "on"  performed  better  than  in  the  "off"  state.  Comparison  of 
change  scores  across  states  (administration  or  withholding  of  levodopa/carbidopa 
between  acquisition  and  retrieval,  "off"  to  "on"  or  "on"  to  "off")  revealed  no 
substantial  differences  as  a  function  of  dopaminomimetic  therapy.  Thus,  slight 
changes  in  cognition  are  associated  with  dopaminomimetic  therapy  of  Parkinson's 
disease,  but  may  be  task-specific. 

Perception  and  attention,  assessed  by  dichotic  listening  (DL)  tasks  were  compared  in 
AD  patients  and  normal  subjects,  manipulating  list  length,  stimulus  matching  and 
order  of  recall.  AD  patients  tended  to  show  qualitatively  similar,  but  significantly 
worse  performance  with  increasing  list  length  as  well  as  specific  types  of  items 
(semantic  versus  phonemic  and  unrelated).  Furthermore,  these  patients  were 
unable  to  selectively  attend  to  one  or  another  auditory  channel  and  could  not 
increase  a  right  or  left  ear  advantage,  a  task  easily  mastered  by  normal  subjects.  As 
such,  Alzheimer's  disease  disrupts  brain  mechanisms  involved  in  the  selective 
allocation  of  attention,  which  may  provide  a  basis  for  the  pervasive  amnestic  defects 
associated  with  the  disease. 

The  functional  role  of  the  noradrenergic  system  in  learning  and  memory  in  the 
experimental  animal,  together  with  reports  of  relative  sparing  of  cortical 
noradrenergic  receptors  in  AD,  encouraged  double-blind,  placebo-controlled  clinical 
trials  of  two  noradrenomimetic  agents,  clonidine  and  guanfacine.  As  cortical 
noradrenergic  receptors  appear  relatively  spared  in  AD,  we  attempted  a  double- 
blind  therapeutic  trial  with  clonidine,  a  centrally  active  alpha-2  receptor  agonist. 
Verbal  and  visuospatial  memory  as  well  as  attention  were  assessed,  and  small 
improvement  was  seen  in  verbal  memory  with  low  dose  treatment;  clinically 
observable  effects  were  limited  to  decreased  blood  pressure  and  mild  sedation  at 
higher  doses.  These  disappointing  results  might  reflect  dose-limiting  side  effects  as 
well  as  the  need  for  other  approaches  to  noradrenomimetictherapy;  clondine 
confers  modest  therapeutic  benefits. 

The  character  of  visuospatial  dysfunction  in  Huntington's  disease  (HD)  was  evaluated 
in  relation  to  actuarial  indices,  such  as  symptom  duration,  age  at  onset,  and  severity. 
Factor  analytic  procedures  indicated  that  perceptuomotor  capacity  (Factor  1)  as  well 
as  the  ability  to  manipulate  spatial  information  (Factor  3)  were  markedly  affected. 
In  contrast,  spatial  discrimination  (Factor  2)  appeared  to  remain  relatively  intact. 
Age  at  onset  had  no  relationship  with  these  variables,  while  severity  of  dementia 
was  related  to  overall  impairment  of  visuospatial  processing.  Most  importantly, 
duration  of  symptoms  was  associated  with  the  declining  ability  of  patients  to 
mentally  manipulate  spatial  memoranda.  That  circumscribed  visuospatial 
impairment  was  present  in  HD  patients  may  have  important  consequences  for  the 
evaluation  of  efficacy  of  experimental  therapeutic  interventions.  Currently,  the  PET 
profile  of  each  patient  is  being  computed  with  an  intent  to  examine  the  relations 
between  neuropsychological  patterns  and  frontostriatal  changes. 

NEURAL  BASIS  OF  EMOTIONALITY 

Studies  of  emotional  changes  in  neurological  patients  have  postulated  different 
hypotheses  to  account  for  the  expression  of  depression  or  euphoria  after  left  or  right 
brain  injury.  In  brief,  the  right  hemisphere  may  assume  a  dominant  role  to  regulate 
mood  and  affect  and  is  specialized  in  dealing  with  emotional  perception  and 
modulation,  more  so  with  negative  emotionality.  The  right  hemisphere  does  better 
than  the  left  to  recognize  emotional  nuances.  In  general,  denial,  imperception  and 


22-MNB/DIR 


hypomania  are  more  likely  to  follow  riqht  brain  injury  whereas  catastrophic 
reactions  and  depression  are  associated  with  left  brain  lesions. 

Patients  with  temporal  lobe  lesions  provide  members  of  the  Section  with  exciting 
opportunities  to  probe  both  orthogonal  and  interactive  cognitive  and  affective 
components  of  limbic  mechanisms.  More  specifically,  there  has  been  much  debate 
about  whether  patients  with  temporal  lobe  epilepsy  develop  distinct  personality 
traits  and  are  at  greater  risk  for  maladaptive  behaviors  than  are  patients  with  other 
types  of  epilepsy  or  neurological  injury.  This  controversy  has  kindled  questions 
about  the  effects  of  limbic  discharges  during  the  interictal  period  and  sensory- 
affective  associations,  and  other  contributory  factors  such  as  site  of  lesion, 
neuropsychological  status  and  actuarial  variables  (sex,  age  at  onset),  socioeconomic 
status,  anticonvulsant  medications,  and  psychosocial  events.  To  study  these 
parameters,  personality  inventories  and  questionnaires  as  well  as  mood-inducing 
experimental  conditions  will  be  used  and  supplemented  by  techniques  to  monitor 
psychophysiological  and  behavioral  responsivity. 

Although  the  intracarotid  amytal  (Wada)  test  is  used  primarily  as  a  clinical 
investigative  procedure,  there  have  been  scattered  reports  that  a  small  cohort  of 
patients  exhibit  mood  changes,  specifically,  dysphoria  with  left,  and  euphoria,  with 
right  injections.  The  present  cadre  of  NIH  investigators  was  concerned  that  cognitive 
and  mood  changes  induced  by  amytal  may  be  enveloped  by  extensive  dysphasia  and 
confusional  states  which  normally  accompany  injection  of  125-175  mg  of  amytal 
(usual  dosage).  By  modifying  the  clinical  tests  of  speech/language  and  memory,  we 
were  able  to  successfully  use  smaller  dosages  of  amytal  (75  mg)  without 
compromising  the  diagnostic  utility  of  the  Wada  procedure.  At  the  same  time,  we 
observed  a  very  high  incidence  of  dramatic  mood  shifts  in  left  and  right  temporal 
epileptic  patients. 

The  most  consistent  finding  was  the  emergence  of  euphoria  with  right  and 
dysphoria  with  left  injection,  and  that  right  temporal  patients  were  more  likely  than 
left  temporal  patients  to  display  mood  changes  following  amytal  injection.  The 
patients  displayed  only  one  mood  state  which  was  usually  with  injection  to  the  side 
with  the  lesion;  changes  were  not  observed  following  injection  of  the  hemisphere 
opposite  the  lesion.  Moreover,  patients  who  experienced  late  age  onset  of  epilepsy 
were  more  prone  to  react  emotionally  after  amytal  injection.  Female  patients  were 
more  likely  than  male  patients  to  display  emotions,  and  unexpectedly, 
ambidexterous  and  left-handed  patients  failed  to  evince  affective  reactions.  The 
mood  shifts  were  induced  immediately  after  injection,  persisted  for  several  minutes, 
and  in  most  cases,  were  abrupt,  easily  perceived  and  usually  reported  by  the  patients 
who  described  their  reactions  as  "high. ..pleasurable. ..exhilarating"  or  as 
"depressing. ..sad. ..tearful". 

A  caveat  needs  to  be  affixed  to  the  interpretation  that  right  TLE  patients  are  more 
likely  to  respond  emotionally  after  amytal  injection.  Whereas  the  dissociation  of 
euphoria  with  right,  and  dysphoria  with  left,  injection  can  not  be  challenged,  it  is 
possible  that  the  basic  anhedonia  and  depression  common  to  left  TLE  patients  was 
exacerbated  with  left  injection,  and  difficult  to  disentangle  from  aphasia.  In  an 
attempt  to  correct  for  this,  we  have  developed  an  emotional  rating  scale  whereby 
each  patient  rates  him/herself  with  an  adjective  checklist  before  and  after  each 
injection.  The  preliminary  results,  however,  are  unrevealing  because  the  right 
temporal  patients  present  a  positive  and  inflated  profile  before  the  procedure, 
establishing  a  ceiling  effect;  the  left  temporal  patients  show  a  reverse  pattern. 


23-MNB/DIR 


Currently,  there  is  a  full-scale  effort  to  draw  evidence  from  MRI  and  PET  studies  to 
examine  the  integrity  of  the  amygdala  and  frontal  cortex  as  possible  explanations 
for  amytal  induced  dysinhibition.  In  a  bid  to  characterize  the  emotional  "responders 
vs.  non-responders,"  these  TLE  patients  were  also  examined  by  standard  tests: 
Minnesota  Multiphasic  Personality  Inventory  (MMPI);  Buss-Durkee  Inventory  of 
Hostility;  Spielberger  Anxiety  Scale;  and  Bear-FedioTL  Behavioral  Inventory.  The 
findings  supported  basic  left/right  temporal  lobe  personality  characteristics  with  the 
right  patients  more  often  being  "responders." 

Coupled  with  the  preliminary  Wada  study  of  a  patient  with  secondary  mania,  these 
data  suggest  that  positive  mood  may  be  supported  by  the  right  and  negative  affect 
by  the  left  brain,  and  that  amytal  injection  produces  dysinhibition  and  release  of 
emotions  regulated  by  brain  mechanisms  ipsilateral  to  the  injection.  Together  with 
data  derivedfrom  intracranial  stimulation  and  post-operative  evaluations  of  these 
patients,  the  present  results  may  challenge  the  popular  model  that  positive  and 
negative  emotions  are  regulated  by  the  left  and  right  hemispheres,  respectively. 

In  an  independent  study  of  personality  traits,  Ms.  Ryan  administered  the  Millon 
Clinical  Multiaxial  Inventory  and  Tennessee  Self-Concept  Scale  to  TLE  patients.  The 
test  profiles  of  the  left  and  right  TLE  patients  differed  but  each  was  not 
pathognomic.  Overall,  the  positive  image  endorsed  by  right  temporal  patients 
contrasted  with  the  negative  self-perception  of  left  temporal  patients.  Left  TLE 
patients  admitted  greater  maladjustment  and  personality  defects,  and  poor  self- 
satisfaction  enveloped  their  ratings  of  moral-ethical  self;  they  reported  less  self- 
esteem  in  social  relationships.  Self-ratings  by  right  TLE  patients  were  much  less 
harsh  and  critical,  and  emphasized  self-assurance.  Left  TLE  patients  endorsed  items 
emphasizing  dependency,  schizoid  and  avoidant  behavior,  heightened  anxiety  and 
somatic  complaints/preoccupation.  In  comparison,  the  profile  of  right  TLE  patients 
was  more  normal,  albeit  showing  elevated  histrionic  and  narcissistic  features. 

These  preliminary  results  affirm  that  patients  with  unilateral  left  or  right  TLE  display 
different  organic  psychosyndromes.  The  personality  style  of  left  TLE  patients 
projected  a  reflective,  detached  and  inward  orientation  that  was  denigrating  and 
dysphoric.  In  contrast,  right  TLE  patients  assumed  a  self-enhancing  posture  that  was 
optimistic  and  extrapersonally  oriented. 

Temporal  lobe  lesions  invading  the  amygdala  and  adjacent  mesial  temporal 
structures  diminish  emotional  arousal  and  reactions.  The  asymmetry  in  the  alerting 
capacity  of  the  left  and  right  brain  suggests  that  right  hemisphere  injury  is  more 
likely  to  produce  inattention  and  hypoarousal.  This  model  was  tested  in 
collaboration  with  Dr.  Alex  Martin  (NIMH)  and  Dr.  Robyn  Davidson  in  studies  of 
patients  following  unilateral  temporal  lobectomy  while  monitoring  effector  limbic 
activity  via  electrodermal  (EDR)  measures.  Skin  conductance  levelsTsCL)  and 
responses  (SCR)  were  monitored  while  the  patients  performed  habituation  and 
discrimination  tasks  and  viewed  evocative  stimuli.  The  investigation  dealt  with  how 
the  left  and  right  temporal  lobes  modulate  arousal  and  affect,  and  behavioral 
adaptation.  The  results  may  also  help  to  resolve  the  debate  about  the  role  of  each 
hemisphere  in  perceiving  and  expressing  negative  and  positive  emotions,  and  the 
ipsi-contralateral  relation  between  EDR  responses  and  unilateral  brain  lesions. 

While  monitoring  EDR  activity,  affective  chromatic  slides  portraying  positive, 
negative  or  neutral  scenes  were  viewed  and  rated  each  along  a  seven-point  scale  of 
very  positive  to  very  negative,  and  was  followed  by  tasks  of  habituation  and 
discrimination  to  auditory  tones  (1000  Hz  and  500  Hz).  Bilateral  skin  conductance 


24-MNB/DIR 


was  recorded  from  electrodes  affixed  to  the  volar  surface  of  the  middle  and  ring 
digit  of  the  left  and  right  hand.  EDR  activity  was  measured  with  a  constant  voltage 
as  units  of  skin  conductance  and  expressed  in  standard  formats  of  SCL  and  SCR;  data 
were  range  corrected  to  normalize  variability  for  each  subject.  There  were  no 
differences  between  the  normal  and  RTL  lobectomy  patients  in  rating  the  emotional 
scenes,  whereas  the  LTL  patients  assigned  lower  and  more  neutral  and  fewer 
positive  emotional  ratings.  In  contrast,  RTL  patients  appeared  to  be  less  responsive 
on  EDR  measures  while  viewing  negative  scenes.  All  normal  and  lobectomy  subjects 
demonstrated  diminished  responsivity  and  habituation  over  1 5  trials.  The  greatest 
SCR  changes  occurred  during  the  initial  four  trials  with  the  LTL  showing  the  slowest 
rate  of  habituation,  or  conversely,  a  very  high  level  of  arousability  that  was 
maintained  across  most  trials.  The  RTL  tended  to  extinguish  very  rapidly  and  were 
unresponsive  by  the  third  trial,  whereas  normal  subjects  assumed  an  intermediate 
rate  of  change.  The  LTL  patients  also  showed  higher  rates  of  nonspecific  fluctuations 
(NSF),  suggestive  of  increased  arousal  and  anxiety. 

There  were  no  group  differences  in  SCR  measures  during  tonal  discrimination 
although  the  high  tone  (target)  evoked  greater  responsivity  than  the  low  tone. 
When  the  analysis  was  limited  to  the  first  habituation  trial,  the  group  effect  was  due 
primarily  to  the  elevated  SCR  amplitude  maintained  by  LTL  patients.  The  rapid 
extinction  evinced  by  RTL  patients  during  the  initial  habituation  trials  persisted  and 
later  playback  of  the  same  tone  was  not  evocative;  the  groups  did  not  differ  in 
discriminating  the  tones. 

Analysis  of  left  vs.  right  hand  EDR  differences  during  the  habituation  and 
discrimination  conditions  uncovered  larger  SCR  from  the  right  hand  and  there  were 
no  LTL  vs.  RTL  differences.  Although  the  basal  levels  of  the  right  hand  for  the  left 
and  right  temporal  lobe  patients  and  normals  were  markedly  similar,  RTL  patients 
showed  lower  SCR  levels  from  the  left  hand  (contralateral  to  surgery);  these 
differences,  however,  failed  to  achieve  statistical  difference. 

The  present  study  showed  that  unilateral  temporal  lobectomy,  including  the 
amygdala  and  adjacent  limbic  structures  did  not  produce  general  hypoarousal,  per 
se.  Rather,  the  decrease  was  evident  only  for  right  temporal  lobectomy  patients 
who  showed  relatively  intact  EDR  resting  levels.  When  provoked,  however,  RTL 
patients  exhibited  extremely  rapid  habituation  whereas  LTL  patients  tended  to 
remain  overly  responsive  and  hypervigilant.  In  the  context  of  theories  of 
emotionality,  these  preliminary  data  suggest  that  injury  to  the  left  temporal  lobe 
may  predispose  the  individual  to  hypervigilance  ana  anxiety,  and  an  overinclusive 
orientation.  In  contrast,  injury  to  the  right  temporal  lobe  may  produce  inattention 
and  hypoarousal  that  may  be  overcome  and  activated  by  novelty  and  trenchant 
emotional  changes.  This  study  will  be  extended  to  examine  stimulus  generalizations 
byTLE  patients. 

Studies  of  normal  subjects  tackled  a  similar  question,  examining  left  and  right  brain 
based  judgments  of  emotional  verbal  stimuli.  Neurobiological  studies  of  affective 
changes  following  left  and  right  brain  injury  have  suggested  that  the  right 
hemisphere  assumes  a  dominant  role  in  the  perception  and  expression  of  emotions, 
or  conversely,  that  positive  emotions  are  mediated  by  the  left,  and  negative 
emotions  by  the  right,  brain.  The  debate  is  fueled  by  concern  that  the  investigations 
may  have  favored  the  visuospatial  domain  native  to  the  right  hemisphere  by  using 
faces  and  pictorial  stimuli.  Trie  few  studies  using  "emotio'nal"  words  have 
lateralized  emotional  perception  to  the  left  hemisphere  along  with  verbal  functions; 
some  specific  priming  effects  have  also  been  found  by  visual  field  and  emotion. 


25-MNB/DIR 


In  an  attempt  to  improve  upon  some  of  the  methodological  flaws  of  past  research, 
we  presented  a  large  number  of  words  to  subjects  at  sub-  and  suprathreshold  levels. 
The  significance  of  affective  vs.  cognitive  processing  of  emotional  words  was  also 
explored  by  requiring  subjects  to  read  each  word  and/or  identify  emotional  valences, 
and  to  perform  implicit  memory  tasks.  Data  analysis  derived  from  normal  subjects 
confirmed  the  superiority  of  perceiving  material  delivered  to  the  central  field  such 
that  reaction  times  were  fastest,  responses  were  most  accurate,  and  response  bias 
was  minimal.    Materials  projected  at  the  longer  durations  yielded  faster  responses 
during  the  label  than  label/read  condition  and  faster  and  more  accurate  responses  to 
right  visual  field  (RVF)  compared  with  left  visual  field  (LVF)  words.  Reading  accuracy 
was  superior  for  stimuli  projected  to  the  right,  compared  with  the  left,  visual  field. 
Responses  were  faster  to  emotional  (positive  and  negative)  than  neutral  stimuli,  and 
emotional  stimuli  were  read  more  accurately  than  neutral  stimuli.  Stimuli  appearing 
in  the  LVF  generally  elicited  a  greater  response  bias  than  those  appearing  in  the  RVF. 

At  subthreshold  levels,  neutral  stimuli  were  reported  most  accurately,  owing  in  part 
to  a  response  bias  to  classify  material  conservatively  as  neutral.  Subjects  accuracy  to 
label  words  projected  to  the  lateral  fields  at  subliminal  speeds  did  not  significantly 
exceed  chance  level,  and  at  the  briefest  delivery  rate,  no  accurate  reading  responses 
were  recorded  from  the  lateral  fields.  Reaction  times  at  10  msec  were  fastest  to  label 
words  displayed  in  the  LVF,  followed  by  RVF  and  then  the  central  visual  field  (CVF). 
Parenthetically,  at  suprathreshold  levels  the  usual  pattern  was  for  fastest  responses 
to  occur  from  CVF,  RVF,  and  LVF,  which  corresponded  to  accuracy  levels. 

Increased  accuracies  and  longer  reaction  times  occurred  at  suprathreshold  levels 
where  the  reading  condition  led  to  greater  accuracy  of  labeling  negative  stimuli  in 
the  RVF.  Lateral  field  projection  elicited  different  biases;  there  was  a  greater 
negative  than  positive  emotional  bias  for  stimuli  in  the  RVF  and  a  LVF  bias  toward 
positive  rather  than  negative  emotions.  Also,  more  negative  than  positive  or  neutral 
words  were  correctly  read  in  the  RVF,  while  more  positive  than  negative  or  neutral 
words  were  read  in  the  LVF.  Accuracy  of  labeling  scores  corrected  for  guessing 
showed  the  reverse  pattern  under  the  label  condition :  the  RVF  perceived  positive 
and  the  LVF,  negative,  stimuli  more  accurately. 

Analysis  of  word  stems  completed  after  the  10  msec  level  showed  no  field  or 
emotion  advantage.  At  120  msec,  the  expected  superiority  of  central  field 
perception  was  followed  by  right  and  then  left  field  accuracy.  More  word  stems 
were  completed  following  read/label  than  just  label  command.  In  the  reading 
condition,  more  positive  word  stems  were  completed  from  the  LVF  and  negative 
words  from  the  RVF,  although  overall  completion  rate  was  minimal. 

That  positive  words  were  labeled  faster  than  negative,  which  were  labeled  faster 
than  neutral,  may  reflect  a  strategy  whereby  subjects  tended  initially  to  evaluate 
emotional  coloration  of  stimuli.  The  neutral  bias  at  subthreshold  levels,  in  turn, 
suggests  that  subjects  were  cautious  to  classify  positive  or  negative  stimuli  when 
insufficient  material  was  presented  from  which  to  make  a  confident  judgment.  The 
reversal  of  field  pattern  (LVF  <  RVF  <  CVF)  for  reaction  time  at  the  briefest 
presentation  suggests  that  syncretic  cognition  within  the  right  hemisphere  takes 
precedence  at  this  brief  exposure. 

A  three-way  interaction  involving  gender,  condition,  and  visual  fields  emerged  such 
that  the  labeling  accuracy  scores  for  males  were  highest  in  the  RVF  under  the 
reading  condition  and  in  the  LVF  under  the  labeling  condition.  Females,  however, 


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scored  most  highly  with  the  labeling  instructions  in  both  visual  fields.  Such  findings 
are  suggestive  of  a  more  strict  lateralization  of  verbal  skills  in  males  and  a  more 
diffuse  and  integrated  verbal  organization  in  females. 

The  present  findings  are  at  variance  with  tachistoscopic  studies  reporting  a  right 
hemisphere  (left  visual  field)  advantage  in  perceiving  emotions.  One  explanation 
may  be  that  the  majority  of  former  studies  used  "facial  expressions",  advantaging 
the  native  processes  of  the  right  brain.  In  the  same  vein,  by  using  verbal  stimuli,  trie 
present  study  tended  to  favor  left  brain  functions,  reflected  by  the  overall  right 
visual  field  (left  brain)  superiority.  This  functional  advantage,  however,  does  not 
account  for  the  finding  that  subjects  dissociated  positive  and  negative  emotions  by 
hemisphere  and  type  of  cognitive  processing  required.  Results  of  the  implicit  stem 
completion  task  also  support  this  interpretation  which  assigns  negative  emotions  to 
the  left,  and  positive  to  the  right,  hemisphere.  These  data  suggest  that  positive  and 
negative  mood  reactions  following  right  and  left  brain  injury,  respectively,  do  not 
reflect  contralateral  disinhibition,  but  rather,  each  hemisphere's  basic  emotional  and 
cognitive  style.  Under  more  highly  verbal  conditions,  the  left  hemisphere  is  biased 
toward  negative,  and  the  right,  toward  positive,  emotions.  This  protocol  will  be 
extended  to  study  how  accurately  patients  with  right  or  left  TLE  perceive 
emotionally  tinted  material  projected  into  lateral  visual  fields. 

Studies  of  lateral  brain  differences  in  emotionality  have  utilized  EEG  while  patients 
view  and  rate  pleasant,  neutral,  and  horrific  materials.  This  preliminary  project  uses 
both  standard  scalp  electrodes  and  recordings  from  chronic  indwelling  flap 
electrodes  in  TLE  patients.  The  paradigm  demands  either  cognitive  judgments  about 
emotionally  evocative  material  or  "feelings"  and  attempts  to  examine  the  role  of 
the  left  and  right  temporal  lobe  in  this  differential  processing.  Finally,  EEG  indexing 
(power  spectrum)  of  attentional  and  linguistic  processes  will  be  derived  from  EEG 
recordings  taken  during  the  Wada  procedure  in  which  sodium  amytal  is  injected  into 
each  hemisphere.  The  analysis  may  provide  anatomical  references  to  patients  who 
allegedly  display  bilateral  language,  amnesia,  and  euphoria/dysphoria. 

These  procedures  have  been  recently  supplemented  by  electrophysiological  studies 
in  collaboration  with  Dr.  Barry  Smith  (University  of  Maryland).  In  one  study,  normal 
subjects  were  selected  for  "hemisphericity  dominance";  that  is,  reliance  on  a  primary 
cognitive  strategy  that  emphasizes  analytical  (left)  or  syncretic  (right)  processing. 
The  subjects  were  required  to  view  a  series  of  photographs  of  individuals  portraying 
different  emotions,  and  to  perform  either  a  cognitive  or  affective  chore;  spectral 
analysis  of  alpha  band  activity  was  performed  off-line  from  leads  positioned  over  the 
left  and  right  temporal  and  parietal  regions. 

The  preliminary  results  indicate  greater  diminished  alpha  with  cognitive  vs. 
emotional  processing.  Moreover,  left  hemisphericity  subjects  showed  increased 
activity  from  left  recording  sites,  especially  during  cognitive  processing.  The 
converse  was  seen  with  right  hemisphericity  subjects  who  showed  increased  activity 
from  right  temporoparietal  leads,  especially  while  performing  an  emotional 
processing  task. 

NEURONAL  EXCITABILITY  SECTION 

During  the  past  year,  the  Neuronal  Excitability  Section  under  the  direction  of 

Dr.  Rogawski  continued  pharmacological  studies  of  voltage-dependent  K*  channels 

and  N-methyl-D-aspartate  (NMDA)-gated  cation  channels  in  cultured  mammalian 


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CNS  neurons  using  whole  cell  voltage-clamp  and  single  channel  recording 
techniques.  The  aim  of  these  studies  was  to  explore  new  strategies  for  the  rational 
development  of  antiepileptic  drugs  based  upon  their  interaction  with  ion  channel 
systems  that  are  critical  to  the  regulation  of  CNS  neuronal  excitability  and 
epileptogenesis.  Work  was  focused  in  three  areas:  (1)  drugs  that  block  the 
ionophoreof  the  NMDA  receptor-channel  complex,  (2)  K+  channel  activator  drugs, 
and  (3)  cloned  K+  channel  genes  expressed  in  fibroblasts.  In  addition,  a  theoretical 
analysis  of  T-type  voltage-dependent  Ca2  +  channels  and  their  role  in  the  generation 
of  the  low  threshold  spike  (LTS)  was  completed  in  collaboration  with  researchers  in 
the  Mathematical  Biology  Branch,  NIDDK.  Finally,  Drs.  Rogawski  and  Yamaguchi  in 
conjunction  with  the  Laboratory  of  Medicinal  Chemistry,  NIDDK,  continued  the 
evaluation  of  a  series  of  novel  NMDA  antagonists  for  their  activity  as 
anticonvulsants. 

Cellular  Electropharmacoloqy  of  Uncompetitive  NMDA  Antagonists 

Excitatory  neurotransmission  mediated  by  NMDA  receptors  plays  a  critical  role  in 
epileptogenesis.  Blockers  of  the  NMDA  receptor-associated  ion  channel,  such  as  the 
dissociative  anesthetics  phencyclidine  (PCP)  and  MK-801,  are  powerful 
anticonvulsants.  However,  side  effects,  including  ataxia  and  cognitive  disturbances, 
limit  the  practical  usefulness  of  these  drugs  in  the  treatment  of  seizure  disorders. 
Researchers  in  the  Section  have  identifiedseveral  dissociative  anesthetic  analogs 
that  are  effective  anticonvulsants  in  animal  seizure  models  but  which  fail  to  cause 
motor  toxicity  at  anticonvulsant  doses,  suggesting  that  they  may  provide  clues  to  the 
development  of  NMDA  antagonists  that  are  clinically  useful  to  treat  seizure 
disorders.  During  the  present  reporting  period,  Drs.  Jones  and  Rogawski 
investigated  the  interaction  of  two  of  the  compounds,  PCA  (an  analog  of  PCP)  and 
ADCI  (an  analog  of  MK-801)  with  the  NMDA  receptor  channel  complex  using  cellular 
electrophysiological  techniques.  They  found  that  both  drugs  are  able  to  block 
NMDA  responses  and  that  they  do  so  in  an  uncompetitive  (use-  and  voltage- 
dependent)  fashion.  However,  a  kinetic  analysis  revealed  that  the  novel  analogs 
effect  their  block  much  more  rapidly  than  do  the  parent  compounds.  It  has 
therefore  been  proposed  that  fast  NMDA  receptor  blockade,  at  least  in  part, 
accounts  for  the  more  favorable  therapeutic  indices  of  the  novel  compounds. 

Synthesis  and  Evaluation  of  Novel  Uncompetitive  NMDA  Antagonists 

During  the  past  year,  the  collaborative  program  with  the  Laboratory  of  Medicinal 
Chemistry,  NIDDK,  for  the  synthesis  and  evaluation  of  novel  anticonvulsants 
targeted  at  the  NMDA  receptor-channel  complex  was  continued.  Compounds 
provided  were  screened  in  several  animal  seizure  models  and  in  a  motor  toxicity  test. 
A  study  was  completed  of  38  PCA  analogs.  Many  of  these  analogs  protected  against 
maximal  electroshock  seizures  in  mice  and  several  showed  a  substantial 
improvement  in  therapeutic  index  for  seizure  protection  compared  with  the  parent 
compound.  Of  particular  interest  were  3-fluro-PCA  and  1-phenylcyclopentylamine 
which  had  exceptionally  high  therapeutic  indices  when  administered  orally.  For 
example,  the  therapeutic  index  of  3-fluro-PCA  in  the  rat  was  >  62.  In  addition, 
studies  were  continued  on  the  MK-801/carbamazepine  analog  5-aminocarbonyl-5H- 
dibenzo[a,d]cyclohepten-5,10-imine  (ADCI).  ADCI  was  found  to  have  a  broad 
spectrum  of  anticonvulsant  activity  in  various  animal  seizure  models.  In  addition  to 
high  potency  in  the  maximal  electroshock  test,  ADCI  was  effective  against 
pentylenetetrazol  seizures  and  at  relatively  low  doses  also  against  NMDA  induced 
seizures.  This  latter  result  suggested  that  ADCI  is  an  NMDA  antagonist  and 
prompted  the  confirmatory  electrophysiological  studies  discussed  above.  ADCI  has 


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the  most  favorable  parenteral  therapeutic  index  of  any  NMDA  antagonist  described 
to  date  (but  is  also  active  orally),  and  is  currently  being  considered  for  clinical  trials. 

K  *  Channel  Activator  Drugs 

Voltage-dependent  K*  channels  regulate  neuronal  excitability  by  acting  to 
repolarize  the  neuronal  membrane.  Recently,  several  different  antihypertensive 
drugs  have  been  shown  to  stimulate  the  opening  of  K*  channels  in  muscle  cells. 
Drs.  Politi  and  Rogawski  have  completed  studies  showing  that  one  of  these  drugs, 
cromakalim,  promotes  the  opening  of  ATP-sensitive  K +  channels  in  cultured 
hippocampal  neurons.  These  channels,  which  have  not  previously  been  identified  in 
neuronal  cells,  could  play  a  role  in  protecting  against  brain  ischemia,  including  that 
which  may  occur  during  prolonged  seizures.  Moreover,  there  is  evidence  that 
cromakalim  is  an  effective  anticonvulsant  in  several  animal  seizure  models  when 
injected  intracerebroventricularly.  Thus,  K+  channel  activator  drugs  could  be  of  use 
in  the  treatment  of  seizure  disorders  (and  possibly  also  in  protecting  against  seizure- 
induced  brain  damage)  if  blood-brain  barrier  permeable  analogs  can  be  developed. 

Cloned  K*  Channel  Genes  Expressed  in  Fibroblasts 

In  view  of  the  recently  discovered  molecular  hetergeneity  of  voltage-dependent  ion 
channel  proteins,  it  has  become  apparent  that  pharmacological  studies  of  ion 
channels  will  be  much  more  useful  if  attention  is  focused  on  molecularly  defined 
populations  of  channels.  Cells  bearing  homogeneous  populations  of  channels  can 
be  obtained  by  expressing  cloned  channel  genes  in  cells  that  lack  voltage-dependent 
ion  channels,  such  as  fibroblasts.  The  Neuronal  Excitability  Section  has  undertaken  a 
long-range  program  to  explore  the  physiological  and  pharmacological  properties  of 
molecularly  defined  channel  proteins  with  the  aim  of  discovering  agents  that 
selectively  affect  the  activity  of  specific  molecular  forms  of  the  channels.  During  the 
present  reporting  period,  studies  were  completed  on  one  isoform  of  a  delayed 
rectifier  type  K+  channel  expressed  in  a  fibroblast  cell  line.  The  results  indicate  that 
the  channel  has  a  specific  pharmacological  sensitivity  that  is  different  from  that  of 
other  delayed  rectifier  type  channels,  and  support  the  concept  that  specific 
molecular  forms  of  the  channel  can  be  selectively  targeted  with  drugs. 

Theoretical  Model  of  the  Low  Threshold  Spike  in  Thalamic  Neurons 

Based  upon  data  obtained  from  electrophysiological  studies,  a  theoretical  model  of 
the  low  threshold  spike  (LTS)  in  thalamic  neurons  was  formulated  in  collaboration 
with  Drs.  Xiao-Jing  Wang  and  John  Rinzel  of  the  Mathematical  Research  Branch, 
NIDDK.  The  LTS  isa  Ca2  +  -dependent  potential  mediated  primarily  by  T-type  (low 
voltage  activated)  Ca2  +  channels  that  is  responsible  for  the  transition  between  tonic 
and  burst  firing  in  thalamic  neurons  and  may  play  a  critical  role  in  the  generation  of 
absence  (petit  mal)  seizures.  T-type  Ca2  +  channels  may  also  be  an  important  target 
for  anti-absence  drugs  (such  as  ethosuximide  and  dimethadione).  This  study 
demonstrated  that  the  shape  of  the  LTS  can  be  accounted  for  almost  entirely  by  the 
intrinsic  properties  of  T-type  Ca2  +  channels,  and  provides  support  for  the  role  of  the 
LTS  in  rodent  absence  seizure  models. 


COGNITIVE  NEUROSCIENCE  SECTION 

The  primary  objectives  of  the  Cognitive  Neuroscience  Section  are  to  identify  and 
model  the  components  of  information  processing,  the  cognitive  computations  that 


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underlie  each  component,  and  the  categories  and  architecture  of  knowledge 
representation  systems.  Furthermore,  we  make  an  effort  to  map  cognitive  processes 
onto  human  brain  physiology,  structures,  and  systems. 

Investigators  in  the  Cognitive  Neuroscience  Section  are  currently  studying  a  wide 
range  of  cognitive  processes  including  problem  solving  and  reasoning;  memory  and 
knowledge  representation;  number  processing  and  calculation;  reading,  writing, 
and  naming;  visual  perception;  object  recognition;  and  the  relationship  of  mood 
state  and  emotions  to  stored  knowledge.  Although  many  of  these  studies  utilize 
young  and  old  normal  subjects,  the  majority  of  our  studies  are  conducted  with 
central  nervous  system  (CNS)-impaired  patients.  CNS-impaired  patients  are  studied 
because  their  cognitive  deficit  pattern  often  implies  dissociations  between  types  of 
information  processing  components,  cognitive  computational  properties,  or 
knowledge  domains.  Thus,  not  only  can  such  patients  teach  us  about  the  structure 
of  cognition  on  the  basis  of  their  dissociations  (and  associations  too),  but  the  nature 
and  direction  of  the  dissociations  may  lead  to  inferences  regarding  the  contribution 
of  anatomical  and  neurotransmitter  systems  to  cognitive  processing.  Both  single- 
case  within-subject  and  group  study  designs  are  utilized. 

The  methodological  appropriateness  of  studying  the  components  of  information 
processing  is  supported  by  several  lines  of  research.  For  example,  the  study  of 
memory  in  multiple  sclerosis  patients  has  narrowed  their  memory  processing  deficits 
to  two  components:  the  articulatory  rehearsal  loop  in  working  memory  which 
temporarily  stores  information  in  a  buffer  when  it  cannot  be  processed  on-line,  and 
a  post-representation  retrieval  pathway.  Dr.  Ray  Johnson,  Jr.  has  demonstrated  that 
event-related  potentials  (ERP)  provide  a  physiological  index  of  the  temporal  course 
of  information  processing  with  specific  wave-forms  and  their  topography  associated 
with  particular  information  processing  components.  His  recent  work  has 
demonstrated  that  ERPs  can  reflect  both  the  information  load,  and  the  time  course, 
of  rehearsal  processes  in  working  memory.  Additional  work  in  his  Cognitive 
Psychophysiology  Unit  by  Dr.  Marten  Scheffers  is  examining  the  role  of  visual 
attention  in  controlled  and  automatic  processing.  Error  analysis  in  CNS-impaired 
patients  has  provided  clues  as  to  the  nature  of  the  computational  processes.  For 
example,  Dr.  Rhonda  Friedman  is  currently  analyzing  different  forms  of  acquired 
reading,  writing,  and  naming  disorders  using  error  analyses  to  identify  both  the 
disordered  component  (e.g.,  semantic  lexicon)  as  well  as  the  probable  impaired 
computational  property  (e.g.,  activation  of  associational  links  between  stored  lexical 
items).  Dr.  Angela  Sirigu  is  studying  the  effects  of  damage  to  an  early  visual 
information  extraction  system  that  aids  in  object  recognition.    Ms.  Marian  Stewart  is 
examining  the  effects  of  visual  spatial  contrast  sensitivity  function  on  later  visual 
information  processing  components  (i.e.,  form  and  object  discrimination  and 
recognition).  If  patients  have  a  problem  in  visual-spatial  contrast  sensitivity,  Stewart 
will  attempt  to  identify  which  computational  property  (i.e.,  specific  contrast 
sensitivity  channel)  is  responsible.  Finally,  Drs.  Rhonda  Friedman  and  Jeffrey  Hadley 
using  priming  tasks,  and  Dr.  Jordan  Grafman,  using  tasks  requiring  retrieval  of  items 
from  different  domains  of  knowledge,  are  mapping  out  the  structure  and  categories 
of  knowledge  representation. 

As  a  result  of  these  studies,  we  have  been  able  to  tentatively  assign  cognitive 
components  and  knowledge  representation  systems  to  brain  locations.  For  example, 
structural  analysis  of  visual  stimuli  takes  place  in  the  posterior  cortex  in  regions 
distinct  from  where  meaningful  analysis  of  stimuli  takes  place.  In  addition,  the  more 
complex  the  nature  of  stimulus  representation  (  e.g.,  schemas),  the  more  anterior  in 
the  brain  is  its  representation.  Representational  systems  are  organized  both  serially 


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and  in  parallel,  can  be  activated  in  parallel,  are  partially  information  redundant,  and 
while  informationally  hierarchical,  can  be  activated  selectively  via  attentional 
mechanisms.  Basal  ganglia  structures  appear  to  aid  in  the  execution  of 
representations  (e.g.,  via  motor  procedures  or  cognitive  planning).  The  involvement 
of  many  different  neural  structures  and  neurotransmitter  systems  in  cognition  are 
currently  being  studied.  While  we  are  concerned  with  specifying  the  cognitive 
components  of  specific  neural  systems  and  structures,  we  also  expect  in  the  next  few 
years  to  describe  generic  principles  of  cognitive  processing  and  representational 
knowledge  and  to  map  these  broad  principles  to  brain  chemo-  and  neuroanatomy. 
As  alluded  to  above,  specific  projects  carried  out  in  the  Cognitive  Neuroscience 
Section  have  supported  the  conceptual  distinction  between  cognitive  components, 
computational  properties,  and  knowledge  domains  that  are  at  the  heart  of  the 
models  of  cognition  developed  by  Section  members. 

NEUROMUSCULAR  DISEASES  SECTION 

The  Neuromuscular  Diseases  Section  conducts  clinical  studies  and  laboratory 
investigations  to  determine  etiology  (infection/immunity  and/or  genetics)  of 
patients  with  neuromuscular  disorders  and  explore  new  therapeutic  modalities. 
Current  studies  include:  (1)  motor  neuron  disease  syndromes  such  as  amyotrophic 
lateral  sclerosis  (ALS)  and  post-polio  syndrome;  (2)  Demyelinatinq  polyneuropathies; 
(3)  neuromuscular  diseases  associated^  with  HIV  infection;  (4)  Duchenne's  muscular 
dystrophy;  (5)  experimental  models  of  retroviruses-induced  polymyositis;  (6)  studies 
on  muscle  regeneration;  (7)  studies  involving  the  interactions  of  the  lymphoid 
system  with  the  central  or  peripheral  nervous  system;  (8)  studies  involving  the 
infection  of  muscle  or  Schwann  cells  in  tissue  culture  with  various  viruses,  especially 
HIV  and  enteroviruses;  and  (9)  clinical  experimental  therapeutic  studies  in  patients 
with  post-polio  syndrome,  polymyositis,  and  HIV-related  neurological  diseases. 

We  have  defined  the  clinical  symptomatology  of  the  post-polio  syndrome  and 
provided  evidence  that  the  site  of  pathologic  involvement  is  in  the  distal  nerve 
terminals.  We  have  found  changes  in  all  the  muscles  regardless  of  the  presence  of 
new  symptoms  suggestive  of  an  ongoing  neuronal  dysfunction  which  appears  to 
continue  slowly  since  the  original  polio  attack.  Abnormal  immunoregulatory 
mechanisms  may  also  play  a  role  in  the  manifestation  of  these  patients'  symptoms 
based  on  the  presence  of  inflammation  in  the  histological  specimens  of  muscle 
biopsies  and  spinal  cords  and  abnormal  lymphocyte  subsets  in  the  circulation. 

We  have  studied  the  metabolic  activity  of  the  cortex  in  ALS  patients  and  correlated 
the  glucose  utilization  of  the  cortical  neurons  with  the  histopathological  findings  of 
the  same  brains  at  autopsy.  We  found  that  ALS  is  a  complex  disorder  affecting 
multiple  cortical  regions  tnat  extend  beyond  the  resolution  of  routine 
histopathology. 

In  an  effort  to  determine  if  in  Duchenne's  muscular  dystrophy  the  weakness  is  due  to 
absent  dystrophin  or  to  loss  of  muscle  fibers,  we  measured  the  force  generated  by 
skinned  muscle  fibers  and  correlated  it  with  the  composition  of  muscle  proteins  in 
the  same  fibers  separated  electrophoretically.  We  found  that  in  Duchenne's 
dystrophy  the  remaining  muscle  fibers  generate  normal  force  in  spite  of  the  absence 
of  dystrophin. 

We  have  defined  the  spectrum  of  neuromuscular  diseases  associated  with  HIV 
infection  and  investigated  the  immunopathogenesis  of  these  disorders  in  an  effort 


31  -MNB/DIR 


to  find  effective  therapies.  We  found  that  two  antiretroviral  drugs,  AZT  and  DDC, 
currently  used  in  the  treatment  of  AIDS,  can  cause  either  a  painful  destructive 
mitochondrial  myopathy  (AZT)  or  a  painful  axonal  neuropathy  (DDC). 

Using  antibodies  to  thymosin  hormones,  thymosin  alpha  1  and  beta  4,  we  have 
demonstrated  that  the  myelin  producing  cells  in  the  CNS  (oligodendrocytes)  and  PNS 
(Schwann  cells)  share  common  antigenic  determinants  with  cells  of  the  lymphoid 
system.  These  observations  are  helpful  to  understand  the  immune  mechanisms  of 
demyelination. 

The  IgM  paraprotein  associated  with  demyelinating  polyneuropathy  was  found  to 
be  an  antibody  against  peripheral  nerve  glycolipids.  When  injected  intraneurally  in 
the  sciatic  nerve  of  the  cat,  the  IgM  induced  demyelination  suggesting  that  this 
protein  is  responsible  for  the  cause  of  the  demyelinating  neuropathy. 

The  cellular  events  of  muscle  fiber  regeneration  are  being  examined  using 
monoclonal  antibodies  that  recognize  satellite  muscle  cells.  Adhesion  molecules  N- 
CAM,  l-CAM,  Leu-19  and  other  common  antigens  shared  between  regenerating 
muscle  fibers,  satellite  cells  and  lymphoid  cells  are  examined  in  the  muscle  biopsies 
of  patients  with  various  neuromuscular  diseases  and  their  experimental  models. 

We  have  been  exploring  a  series  of  new  therapies  in  patients  with  post-polio 
syndrome  and  polymyositis  refractory  to  available  immunotherapies.  Studies  are 
conducted  with  combination  of  intravenous  methotrexate,  plasmaphereses  and 
azathioprine.  The  role  of  intravenous  immunoglobulin  is  also  studied  in  a  double- 
blind  placebo-controlled  trial.  Preliminary  findings  indicate  that  gamma  globulin  is 
effective  in  the  treatment  of  patients  with  paraproteinemic  polyneuropathies.  A 
double-blind  placebo-control  trial  using  prednisone  for  the  treatment  of  post-polio 
syndrome  has  just  began. 

In  collaboration  with  the  National  Naval  Medical  Center,  we  are  ready  to  begin, 
under  an  approved  clinical  protocol,  a  dose  comparison  study  of  high-  versus  low- 
dose  AZT  in  the  treatment  of  HIV-related  neurological  diseases  and  assess  the 
appropriate  dose  required  for  the  management  of  such  patients. 

NEUROIMAGING  SECTION 

HONORS,  AWARDS. 

"Best  paper":  Southern  Neurosurgical  Society  Annual  Meeting,  April  30,  1990,  Key 
West,  Florida,  "Positron  emission  tomography  of  pituitary  Macroadenomas: 
Hormone  production  and  effects  of  therapies." 

RESEARCH  SUMMARY. 

Following  is  a  summary  of  the  major  findings  for  the  research  protocols  of  the 
Neuroimaging  Section  in  the  fiscal  year  October  1,  1989  through  September  30, 
1990: 

(1)  Radiographic  and  Radioisotopic,  CT,  and  MR  Angiography  of  the  Spinal  Cord. 

We  have  continued  our  attempts  aiming  at  a  non-invasive  demonstration  of 
magnetic  resonance  angiography  (MRA)  of  arteriovenous  malformations  (AVMs)  of 


32-MNB/DIR 


the  spinal  cord.  The  technical  challenge  of  this  project  has  proven  harder  than 
anticipated. 

(2)  Nuclear  Magnetic  Resonance  (Imaging  and  Spectroscopy)  and  Computed 
Tomography  (Transmission). 

The  NMR  imaging  research  has  developed  along  several  lines  in  both  imaging  and 
spectroscopy: 

Continuing  MRI  assessment  of  patients  with  spinal  cord  pathology.  We  have 
been  able  to  confirm,  by  repeat  MRI  studies,  that  cord  cavities  associated  with 
hemangioblastomas  will  decrease  in  size  (or  disappear)  after  surgical  removal  of  the 
tumoral  nodule(s).  Recently  we  have  initiated  work  with  MR  angiography  (flow 
based  studies)  in  cord  AVMs  and  cine-MRI  in  patients  with  syringomyelia  and  other 
cord  cavities. 

Studies  of  pulsatile  CSF  flow  (head  and  spine  applications)  using  longitudinal 
imaging  (flow  direction  in  the  image  plane)  and  phase  reconstruction.  This  method 
is  advantageous  for  its  speed  ard  simplicity;  pulsatile  flow  direction  and  velocity  may 
be  easily  assessed,  and  reliable  information  about  CSF  bulk  flow  is  obtained. 

Continuing  our  assessment  by  phase  imaging  of  the  normal  spinal  cord 
pulsation-motility,  and  its  disappearance  in  cases  of  "fixed"  cord  (due  to  tethering, 
scarring,  tumor  compression).  Emphasis  has  been  placed  on  follow-up  studies  after 
corrective  surgery  for  the  condition  responsible  for  the  "fixed  cord". 

Imaging  the  spinal  epidural  veins.  We  are  particularly  interested  in  flow  within 
these  vessels  and  its  modifications  (Valsalva's  maneuver,  intraspinal  tumors, 
intervertebral  disc  herniations). 

Imaging  the  pars  compacta  of  the  substantia  nigra  in  normal  subjects  and  in 
patients  affected  by  Parkinson's  disease  (PD),  (hemiparkinsonian  subjects  are  of 
particular  interest). 

Analysis  of  the  signal  intensity  changes  in  MRI  of  patients  affected  by  a  variety 
of  movement  disorders  with  emphasis  on  the  significance  of  concomitant  changes  in 
the  cerebral  iron  distribution  (accumulation). 

Continuing  in  vivo  and  in  vitro  investigation  of  the  relaxation  times  (Ti,T2)  of 
extravasated  intracranial  blood.  Changes  in  Ti  and  T2  are,  in  great  part,  related  to 
the  chemical  modifications  and  progressive  denaturation  of  hemoglobin. 

Comparing  clinical  MRI  imaging  results  with  those  of  CT  and  particularly  PET  in 
a  variety  of  abnormal  conditions,  with  emphasis  on  CNS  tumors. 

Imaging  with  experimental,  small-bore  (animal  studies)  2  Tesla  and  4.7  Tesla 
imaging-spectroscopy  NMR  devices. 

Imaging  (2T)  of  PML-affected  (experimental  model)  monkeys:  Our  goal  is  the 
early  recognition  of  demyelinating  processes. 

Imaging  of  monkeys  of  various  ages  at  0.5,  1.5,  2.0  and  4.7  Tesla  to  assess  the 
iron  content  in  the  basal  ganglia,  its  increase  with  age  and  its  possible  modifications 


33-MNB/DIR 


in  pathological  conditions  (experimental  parkinsonism).  Iron  distribution  is  verified 
by  Perls'  stain  of  brain  specimens. 

Successful  demonstration  by  MRI  of  selective  basal  ganglia  damage  following 
intracarotid  injection  of  MPTP. 

The  design  and  fabrication  (under  contract)  of  a  variable  field  T1-T2  analyzer 
that  will  enable  rapid  measurement  of  relaxation  times  in  tissue  samples  at  fields 
from  0.05  to  1 .5  Tesla  is  well  underway. 

Probably  our  most  important  NMR  contribution  this  year  is  represented  by  the 
successful  initiation  of  localized  proton  spectroscopy  (1 H-MRS)  at  1 .5  Tesla  in  human 
patients.  For  the  moment,  we  have  concentrated  our  attention  on  brain  tumors 
(some  50  patients  studied  already).  Our  emphasis  has  been  on  correlations  between 
MRS,  results,  particularly  lactate  recognition,  FDG-PET  findings,  and  clinical 
evaluations.  Cats  and  other  animals  were  studied  at  2.0  T  and  4.7  T.  Prior  to  1989, 
31 P  and  1H  MRS  data  were  obtained  with  surface  coil  localization.  This  technique 
samples  mostly  cortical,  but  poorly  defined  tissue  volumes,  and  requires  surgical 
retraction  of  overlying  scalp.  Methods  of  achieving  reliable  water  suppression  using 
these  techinques  have  been  evaluated. 

Post-ischemic  metabolism  in  a  cat  model  of  transient  cerebral  ischemia  was 
studied  to  determine  whether  MRS-detectable  abnormalities  correlated  with  long- 
term  functional  deficiencies.  Despite  prominent  alterations  of  31 P  and  1 H  spectra 
(i.e.,  lactate  production,  energy  failure  and  acidification)  during  10  minute  global 
ischemia,  the  spectra  renormalized  within  an  hour  of  reestablishing  blood  flow. 
Repetition  of  transient  ischemia  produced  only  minor  changes  in  the  metabolic 
recovery  rate.  MR  spectra  appeared  highly  sensitive  to  flow,  and  high  time 
resolution  monitoring  is  likely  to  be  useful  for  evaluating  the  completeness  of 
ischemia.  Localized  spectroscopic  measurements  in  the  MCAO  model  revealed 
lactate  elevations  that  were  stable  for  many  hours. 

(3)  Positron  Emission  Tomography. 

A  follow-up  FDG-PET  study  has  been  completed  of  a  series  of  patients  initially 
harboring  low  grade  gliomas  which  later  changed  grade.  The  FDG-PET  method  has 
been  found  optimal  for  monitoring  the  grade  change  (malignant  degeneration). 
Confirmation  has  been  obtained  that  the  FDG-PET  method  is  the  optimal  procedure 
for  differentiating  tumor  recurrence  from  postradiation  and/or  post  chemotherapy 
cerebral  necrosis. 

The  FDG-PET  method  of  tumor  evaluation  has  been  extended  from  the  gliomas 
to  other  intracranial  tumors,  particularly  meningiomas.  We  have  clear  indications 
that  FDG-PET  is  an  excellent  method  to  predict '  post-removal"  recurrence  of 
meningiomas.  We  have  completed  a  study  of  pituitary  microadenomas  using  the 
FDG-PET  method.  This  technique  has  proven  to  be  a  valuable  adjunct  to  the  other 
imaging  methods  (CT,  MRI). 

We  have  continued  our  PET  research  on  the  dopaminergicsystem  using  FDG 
and  6-[18F]fluoro-L-dopa  (6FD)  in  both  animals  (primate  hemiparkinson  level)  and 
human  hemiparkinsonian  patients.  Our  results  follow: 

(a)  Primate  Hemiparkinson  Model:  Unilateral  intracarotid  administration  of 
MPTP  results  in  a  striking  decrease  of  the  6FD-derived  activity  retention  in  the 


34-MNB/DIR 


exposed  putamen,  whereby  60-1 50  minutes  after  6FD  administration  there  is 
approximately  a  67%  decrease.  The  decrease  in  the  caudate  is  much  less.  A 
differential  involvement  of  these  structures  is  also  found  in  idiopathic  PD.  MRI  with 
Gd-DTPA  revealed  a  striking  increase  of  signal  intensity  on  T2-weighted  sequences 
and  decreased  intensity  on  T1  -weighted  sequences  beginning  3-6  days  after 
exposure.  This  change  most  likely  reflects  cytotoxic  (rather  than  vasogenic)  edema 
associated  with  dopaminergic  neuronal  injury.  The  MRI  findings  persist  for  2-3 
weeks.  The  absence  of  any  basal  ganglia  MRI  abnormalities  in  the  chronic  animals  is 
similarto  findings  in  idiopathic  PD.  In  the  MPTP-exposed  putamen,  rCBF  was 
decreased  up  to  25%  while  exposed  caudate  rCBF  was  decreased  by  less  than  10%. 
These  findings  indicate  thatdopaminergicdenervation  produces  quantifiable 
regional  physiologic  changes. 

(b)  Cerebral  Metabolism  in  Human  Hemiparkinson's  Disease:  In  the 
hemiparkinsonian  patients,  no  statistically  significant  metabolic  abnormalities  were 
encountered  either  in  cortical  structures,  or  in  the  caudate,  thalamus  and 
cerebellum.  However,  hypermetabolism  was  present  in  the  lenticular  nuclei 
contralateral  to  the  most  affected  body  side.  Lenticular  metabolic  asymmetry 
correlated  both  with  degree  of  clinical  akinesia  and  a  scale  of  overall  disease  severity 
(modified  Columbia  University  Rating  Scale). 

(c)  Cerebral  Metabolism  after  Intravenous  L-Dopa  Infusion  in  Human  PD:  The 
L-dopa  infusion  increased  glucose  metabolism  globally,  except  in  the  basal  ganglia 
where  the  relative  glucose  utilization  was  actually  decreased.  The  L-dopa  infusion 
significantly  improved  the  patients'  PD  signs. 

(d)  Caudate  Grafts  in  Primates:  MRI  with  Gd-DTPA  revealed  that  the  blood- 
brain  barrier  disruption  (BBBD)  was  only  present  in  the  first  three  months  after 
surgery.  Hence,  BBBD  is  an  unlikely  explanation  forthe  long-term  improvement 
seen  in  some  of  the  subjects.  Among  the  surgically  grafted  primates,  the  time  course 
and  extent  of  clinical  improvement  was  much  greater  for  the  fetal  mesencephalic 
grafts  as  compared  to  the  other  groups.  6FD-derived  activity  was  elevated  only  in 
areas  containing  viable  fetal  mesencephalic  graft.  6FD-PET  revealed  a  diffuse 
elevation  of  regional  activity  at  or  near  the  surgical  target  area  in  primates  which 
displayed  some  clinical  improvement.  This  type  of  change  was  seen  in  the  control 
grafted  subjects  and  in  those  receiving  cavitation  alone.  Histology  revealed  the 
presence  of  tyrosine  hydroxylaseimmun-oreactive  fibers  coursing  toward  the  graft 
bed.  It  is  proposed  that  these  (sprouting)  fibers  are  being  detected  by  6FD-PET  and 
are  possibly  responsible  for  the  clinical  improvement. 

(e)  Adrenal  Medullary  Autografts  in  Human  PD:  In  the  four  grafted  subjects, 
the  post-operative  clinical  improvement  was  judged  to  be  mild.  Enhancement  with 
Gd-DTPA,  consistent  with  BBBD  was  seen  in  at  least  two  of  the  operated  subjects. 
However,  there  was  no  detectable  difference  on  PET  scanning  between  patients 
who  received  grafts  and  those  who  did  not. 

(f)  Peripheral  Metabolism  of  6FD  and  PET  Image  Quality:  The  main  metabolite 
of  6FD,  3-methoxy-6-[18F]fluorotyrosine,  crosses  the  blood  brain  barrier  and 
contributes  to  the  background  signal.  In  preliminary  work,  we  have  been  studying 
the  effect  of  carbidopa  and  OR-462,  inhibitors  of  the  enzymes  dopa  decarboxylase 
and  catechol-O-methyl-transferase,  respectively.  The  best  improvement  in  image 
quality  is  obtained  using  both  drugs  simultaneously. 


35-MNB/DIR 


Finally  we  have  carried  out  the  critical  experiments  necessary  to  proceed  with  the 
investigation  by  PET  of  the  NMDA  receptors.  An  analog  of  PCP  bearing  the  fluorine 
atom,  FTCP,  has  been  recently  synthesized  (Kieswetter  et  al.,  1989).  PCP  and  its 
analogs  bind  with  high  affinity  to  an  allosteric  site  of  the  NMDA  receptors  only  when 
glutamate  is  present  to  open  the  cationic  channels.  In  other  words,  the  PCP-like 
ligands  are  tools  to  investigate  functionally  active  NMDA  receptors,  and  also  to 
measure  indirectly  glutamate  concentrations  in  different  brain  areas. 

We  have  at  present  shown  that  FTCP  retains  the  "in  vitro"  binding  characteristics 
(affinity,  specificity)  of  the  related  compounds  PCP,  TCP,  and  MK801 .  Moreover, 
3H-FTCP  binding  is  subjected  to  regulation  by  glutamate  and  glycine.  We  have  also 
shown  brain  uptake  and  "in  vivo"  pharmacological  actions  after  intravenous 
injection  of  the  drug  in  mice,  rats,  and  one  monkey. 


36-MNB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02318-13  MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (ao  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Clinical  Pharmacology  of  Antiepileptic  Drugs 


PRINCIPAL  IN  VE  STIGATOR  (List  other  professional  personnel  below  (he  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  William  H.Theodore,  M.D.         Chief,  CES  MNB      NINDS 

Others:        Susumu  Sato,  M.D.  Chief,  EEG  LAB  OCD       NINDS 

Paul  Fedio,  Ph.D.  Chief,  CNS  MNB      NINDS 

Frank  Nice,  M.S.,  M.P.A.  Pharmacist  MNB      NINDS 


COOPERATING  UNITS  (if  any) 


Office  of  The  Clinical  Director,  NINDS 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Clinical  Epilepsy  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


1.2 


PROFESSIONAL: 


1.2 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

|  x  |  (a)  Human  subjects 
|~x~|  (a1)  Minors 

J   (a2)  Interviews 


]  (b)  Human  tissues  L]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Current  studies  include  a  double-blind  randomized  placebo  add-on  controlled  trial  of  felbamate.  The 
unique  three  period  cross-over  design  allows  unbiased  estimates  of  drug  effects  even  in  the  presence  of  a 
carry-over  effect  from  one  period  to  the  next 

The  first  study  has  been  completed,  and  the  results  suggest  the  drug  may  be  effective  against  partial 
seizures.  A  study  of  felbamate  monotherapy  is  planned,  as  well  as  an  add-on  to  valproic  acid  in  the 
Lennox-Gastaut  syndrome.  We  are  studying  carbamazepine  withdrawal,  to  establish  the  presence  or 
absence  of  transient  seizure  exacerbation.  We  have  evaluated  the  effect  of  valproic  acid  on  cerebral 
glucose  metabolism,  using  positron  emission  tomography  (PET).  This  drug  is  of  particular  interest  due  to 
its  possible  inhibition  of  GABA  degradative  enzymes.  We  compared  its  effect  to  that  of  phenytoin, 
carbamazepine,  and  phenobarbital.  Valproic  acid  reduced  cerebral  metabolism  by  20%,  suggesting  an 
interaction  with  the  GABA-benzodiazepine  receptor  complex.  PET  is  also  being  used  to  study  the  opiate 
receptor  system  in  patients  with  epilepsy.  18  F-cyclofoxy  binding  was  assessed  in  patients  with  complex 
partial  seizures,  and  naloxone  administered  to  study  its  effect  on  blood  flow  and  metabolism. 


37-MNB/DIR 


PHS  6040  (Rev   MA) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02236-15  MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJ  ECT  (SO  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Diagnostic  and  Therapeutic  Reevaluation  of  Patients  With  Intractable  Epilepsy 


PRINCIPAL  IN  VE  STIG  ATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  William  Theodore,  M.D.       Chief,  CES  MNB      NINDS 

Others:        Susumu  Sato,  M.D.  Chief,  EEG  Lab  OCD       NINDS 

Paul  Fedio,  PhD  Chief,  CNS  MNB      NINDS 


COORPERATING  UNITS  (if  any) 

Office  of  The  Clinical  Director,  NINDS 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Clinical  Epilepsy  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  1  q 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

[xj   (a)  Human  subjects  [7~|  (b)  Human  tissues  I      I  (c)  Neither 

|  x  [  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  Clinical  Epilepsy  Section  has  been  developing  and  testing  new  techniques  to  improve  seizure  control, 
medication  tolerance,  and  rehabilitation  in  patients  with  severe  epilepsy.  Patients  with  uncontrolled 
seizures  are  admitted  for  a  complete  evaluation,  including  simultaneous  video  and  telemetered 
electroencephaloqraphic  (EEG)  recording  of  seizures,  daily  determinations  of  antiepileptic  drug  serum 
concentrations,  positron  emission  tomography  (PET),  magnetic  resonance  imaging  (MRI).  and 
maqnetoencephaloqraphy  (MEG).  A  specific  seizure  diagnosis  is  established  allowing  each  patient  to  be 
assigned  to  an  appropriate  research  protocol  and  therapy.  PET  in  patients  with  localized  brain  lesions 
has  demonstrated  focal  hypometabolic  cerebral  areas  corresponding  to  the  interictal  seizure  EEG  focus. 
In  some  patients,  PET  has  been  able  to  detect  a  focus  when  other  methods  have  failed.  Studies  of 
patients  during  partial  seizures  have  shown  a  change  from  hypo-  to  hypermetabolism  at  the  site  of  the 
focus.  In  the  Lennox-Gastaut  syndrome,  PET  has  revealed  the  existence  of  two  separate  metabolic 
patterns  despite  clinical  seizure  similarity.  PET  studies  allow  more  definitive  identification  of  the 
epileptic  lesion  and  suggest  new  avenues  of  investigation  into  the  basic  mechanisms  of  the  epilepsies. 
MRI  may  show  small  structural  lesions  underlying  PET  hypometabolism  even  when  computed 
tomography  (CT)  is  normal.  Further  studies  will  elucidate  the  relation  of  metabolic  and  pathologic 
changes.  MEG  may  have  the  potential  to  accurately  localize  the  subsurface  origin  of  spikes.  EEG 
provides  little  information  on  the  spatial  distribution  of  epileptiform  discharges  in  cortical  depths;  MEG 
may  be  superior.  Comparison  of  invasive  localization  of  epileptic  foci  using  subdural  electrodes  and  non- 
invasive evaluation  is  being  performed.  After  surgery,  patients  are  followed  with  serial  clinical, 
neuropsychological,  and  electroencephalographs  evaluation. 


38-MNB/DIR 


PHS  6040  (Rev.  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02667-06  MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJ  ECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Physiological  Analysis  of  Involuntary  Movements 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.:  Mark  Hallett,  M.D. 

Others:        Leo  Cohen,  M.D 
Peter  Fuhr,  M.D. 
HelgeTopka,  M.D. 
Joseph  Matsumoto,  M.D. 


Clinical  Director 

OCD 

ODIR 

DIR 

NINDS 

Chief 

HMCS 

MNB 

DIR 

NINDS 

Visiting  Scientist 

HMCS 

MNB 

DIR 

NINDS 

Visiting  Fellow 

HMCS 

MNB 

DIR 

NINDS 

Special  Volunteer 

HMCS 

MNB 

DIR 

NINDS 

Special  Volunteer 

HMCS 

MNB 

DIR 

NINDS 

COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Human  Motor  Control  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.5 


PROFESSIONAL: 


1.0 


OTHER: 


0.5 


CHECK  APPROPRIATE  BOX(ES) 

I  x  |   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


J  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Involuntary  movements  have  often  been  difficult  to  classify  clinically.  Clinical  and  physiological  analysis 
of  a  continuing  series  of  patients  has  led  to  new  classifications  and  pathophysiological  insights. 
Dystonias  and  Parkinson's  disease  (PD)  have  been  the  focus  of  our  recent  work. 

The  laboratory  has  done  extensive  studies  with  reciprocal  inhibition  studies.  The  conclusion  of  these 
studies  is  that  there  is  an  important  deficit  in  reciprocal  inhibition  in  dystonia  and  PD.  There  are  a 
number  of  phases  in  the  reciprocal  inhibition  curves  and  the  physiology  of  each  is  not  clear.  During 
further  studies  of  this  project,  it  became  clear  that  the  method  can  be  improved  and  better  standardized. 
Therefore,  a  study  of  different  parameters  that  influence  the  reciprocal  inhibition  of  the  H-reflex  in  wrist 
flexors  was  done. 

Cutaneous  reflexes  were  measured  in  patients  with  PD.  The  reflex  is  composed  of  successive  excitatory 
and  inhibitory  events.  While  the  latencies  of  the  different  reflex  components  and  the  amplitudes  of  the 
excitatory  peaks  were  not  different  from  normal,  the  first  inhibitory  peak,  occurring  at  a  mean  latency  of 
51  ms,  was  less  pronounced  in  patients  .  The  result  is  compatible  with  the  loss  of  a  spinal  inhibitory 
mechanism  elicited  by  cutaneous  afferents  and  can  be  a  partial  explanation  for  increased  tone  in  PD. 

We  developed  a  technique  to  evoke  perioral  reflexes  by  stimulating  branches  of  the  trigeminal  nerve 
either  electrically  or  indirectly  via  stimulation  of  cutaneous  receptors  by  delivering  well-defined  taps  to 
the  skin  in  the  vicinity  of  the  lips,  mimicking  the  clinical  test.  We  have  established  normative  data  and 
studied  patients  suffering  from  orofacial  dyskinesias  and  spasmodic  dysphonia.  In  both  groups  of 
patients,  thresholds  for  eliciting  R2  components  of  facial  reflexes  were  lower  and  recruitment  curves 
were  steeper  as  compared  to  normals. 


We  have  studied  several  patients  in  a  family  with  hyperekplexia. 
in  these  patients  is  an  exaggerated  startle  reflex. 

39-MNB/DIR 


Our  results  showed  that  the  movement 


PHS  6040  (Rev   184) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02668-06  MNB 


PERIOD  COVERED 

October  1 ,  1 989  through  September  30,  1 990* 


TITLE  OF  PROJ  ECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Trial  of  Isoniazid  for  Action  Tremor 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.:  MarkHallett,  M.D.  Clinical  Director  OCD       ODIR      DIR    NINDS 

Chief  HMCS    MNB      DIR    NINDS 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Human  Motor  Control  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  - 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

H   (a)  Human  subjects  ]  (b)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 
*This  project  was  terminated  9/89. 


40-MNB/DIR 


PHSfcMOOttv.  1/M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02669-06  MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  toss.   Title  must  fit  on  on*  line  between  the  borders.) 

Physiological  Analysis  of  Voluntary  Movement 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.: 
Others: 


Mark  Hallett,  M.D. 

Clinical  Director 

OCD 

ODIR 

DIR 

NINDS 

Chief 

HMCS 

MNB 

DIR 

NINDS 

Leo  Cohen,  M.D. 

Visiting  Associate 

HMCS 

MNB 

DIR 

NINDS 

Victoria  Panzer,  PhD 

Staff  Fellow 

HMCS 

MNB 

DIR 

NINDS 

Rocco  Agostino,  M.D. 

Visiting  Fellow 

HMCS 

MNB 

DIR 

NINDS 

InaTarkka,  Ph.D. 

Volunteer 

HMCS 

MNB 

DIR 

NINDS 

COOPERATING  UNITS  at  <«,) 

Department  of  Rehabilitation  Medicine,  Clinical  Center 
Department  of  Nuclear  Medicine,  Clinical  Center 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Human  Motor  Control  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


5.5 


PROFESSIONAL: 


4.0 


OTHER: 


1.5 


CHECK  APPROPRIATE  BOX(ES) 

I  x  [  (a)  Human  subjects 
]  (a1)  Minors 

J   (a2)  Interviews 


J  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Studies  of  voluntary  movement  focused  on  the  role  of  the  cerebellum.  One  issue  was  the  contribution  of 
the  cerebellum  to  coordination.  The  results  seem  to  indicate  that  the  cerebellum  is  critical  to  the  portion 
of  the  motor  execution  process  involving  compensation  for  limb  dynamic  changes  during  high-speed 
movements.  A  second  issue  is  the  role  of  the  cerebellum  in  motor  learning.  Tasks  are  being  developed  to 
assess  motor  skill  learning.  A  study  was  done  of  the  role  of  the  silent  period  in  the  antagonist  muscle 
prior  to  a  ballistic  movement. 

Using  0-15  labelled  water  as  a  marker  for  cerebral  blood  flow  in  positron  emission  tomography  (  PET) 
studies ,  we  have  been  working  on  methods  for  improved  anatomical  correlation  of  regions  of  metabolic 
change  by  superimposing  the  PET  image  onto  an  MRI  image.  Studies  include  analysis  of  the  role  of  the 
cerebellum  in  motor  learning  and  the  supplementary  motor  area  in  self-paced  movement. 

In  evoked  potential  studies,  we  have  been  assessing  changes  in  topography  of  sensory  potentials  with 
lesions  of  the  central  and  peripheral  nervous  system  looking  for  evidence  of  plasticity.  Studies  of  the  N30 
component  of  the  median  nerve  sensory  evoked  potential  was  found  to  be  enhanced  in  patients  with 
dystonia. 


In  studies  of  movement  related  potentials,  we  have  studied  patients  with  Parkinson's  disease,  cerebellar 
degenerations  and  dystonia.  Abnormalities  in  the  first  two  groups  suggest  disturbances  in  the  cortical 
control  of  movement  in  these  patients. 

Studies  in  the  Biomechanics  Laboratory  of  the  Department  of  Rehabilitation  Medicine  have  focused  on 
the  control  of  balance  and  gait.  In  a  study  of  balance  in  aging,  we  found  that  older  normal  subjects 
maintain  a  more  rigid  posture,  utilizing  large  and  inconsistent  adjustments  to  maintain  stability.  This 
may  relate  to  the  instability  of  stance  in  the  elderly. 

41 -MNB/ DIR 


PHS  6040  (Rev    1  84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  0271 1-05  MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJ  ECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Utility  and  Physiology  of  Botulinum  Toxin  for  Involuntary  Movement  Disorders 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.:  Mark  Hallett,  M.D. 

Others:    Leo  Cohen,  M.D. 

Reginald  Cole,  M.D. 


Clinical  Director 

OCD 

ODIR 

DIR 

NINDS 

Chief 

HMCS 

MNB 

DIR 

NINDS 

Visiting  Scientist 

HMCS 

MNB 

DIR 

NINDS 

Clinical  Associate 

HMCS 

MNB 

DIR 

NINDS 

COOPERATING  UNITS  (if  any) 

Speech  Pathology  Unit,  NIDCD 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Human  Motor  Control  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.0 


PROFESSIONAL: 


0.5 


OTHER: 


0.5 


CHECK  APPROPRIATE  BOX(ES) 

I  x  |   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


J  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Botulinum  toxin  injected  in  small  doses  directly  into  muscle  binds  to  the  neuromuscular  junction  and 
inactivates  it  for  approximately  three  months. 

Studies  of  utility  of  botulinum  toxin  are  been  carried  out  in  writer's  cramp  (and  its  varients  such  as 
pianist's  cramp)  in  open  label  and  double-blind  trials.  Treatment  appears  effective. 

We  have  begun  a  trial  of  botulinum  toxin  in  spasmodic  torticollis  with  the  purpose  of  analyzing  the 
dysphagia  that  some  of  these  patients  have  following  injection. 


42-MNB/DIR 


PHS  6040  (Rev   1/841 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02712-05  MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJ  ECT  (80  characters  or  lass.  Title  must  fit  on  one  line  betvreen  the  borders.) 

Non-invasive  Stimulation  of  Human  Central  Nervous  System 


PRINCIPAL  IN  VESTIGATORUist  other  professional  personnel  belom  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


P.I.:  MarkHallett,  M.D. 

Others:         Leo  Cohen,  M.D. 
Peter  Fuhr,  M.D. 
Rocco  Agostino,  M.D. 
HelgeTopka,  M.D. 
Eric  Wassermann,  M.D. 


Clinical  Director 

Chief 

Visiting  Associate 

Visiting  Fellow 

Visiting  Fellow 

Special  Volunteer 

Clinical  Associate 


OCD 

ODIR 

DIR 

NINDS 

HMCS 

MNB 

DIR 

NINDS 

HMCS 

MNB 

DIR 

NINDS 

HMCS 

MNB 

DIR 

NINDS 

HMCS 

MNB 

DIR 

NINDS 

HMCS 

MNB 

DIR 

NINDS 

OCD 

ODIR 

DIR 

NINDS 

COOPERATING  UNITS  (if  any) 

Speech  and  Voice  Pathology  Unit,  IRP,  NIDCD 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 
SECTION 

Human  Motor  Control  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


4.0 


PROFESSIONAL: 


3.5 


OTHER: 


0.5 


CHECK  APPROPRIATE  BOX(ES) 

I  x  |   (a)  Human  subjects 
]  (a1)  Minors 

]  (a2)  Interviews 


3  (b)  Human  tissues  Q  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Recently  techniques  have  become  available  for  the  non-invasive  stimulation  of  the  human  cortex  and 
deep  proximal  peripheral  nerves.  Stimulation  can  be  with  a  high  voltage,  extremely  brief  electrical  pulse 
or  with  magnetic  stimulation.  One  purpose  is  to  use  these  methods  for  noninvasive  localization  of 
different  parts  of  the  human  cortex  including  motor  cortex,  sensory  cortex  and  language  cortex. 
Another  purpose  is  to  study  cortical  physiology  in  different  disease  states. 

We  have  made  a  number  of  advances  in  understanding  the  technical  aspects  of  magnetic  stimulation, 
trying  to  define  the  optimal  method  to  map  different  body  part  representations  in  motor  cortex.  In 
detailed  mapping  studies,  distal  muscles  were  significantly  more  excitable  than  proximal  muscles  and 
motor  representations  targeting  proximal  arm  muscles  were  significantly  more  excitable  in  the  right 
than  in  the  left  hemisphere.  Plastic  reorganization  of  the  brain  has  been  demonstrated  in  a  number  of 
circumstances.  In  amputees,  muscles  ipsilateral  to  the  stump  could  be  activated  from  a  larger  area  than 
those  contralateral  to  the  stump.    In  patients  with  spinal  cord  injury,  magnetic  stimulation  evoked  larger 
motor  evoked  potentials  with  shorter  latencies  in  muscles  immediately  proximal  to  the  level  of  a  spinal 
cord  injury  than  in  corresponding  muscles  in  controls.   In  patients  with  hemispherectomy,  stimulation  of 
the  remaining  hemisphere  evoked  bilateral  muscle  responses  in  proximal  and  distal  muscles  at  similar 
latencies  indicating  a  bilateral  representation  of  arm  muscles  and  the  existence  of  physiologically  active 
ipsilateral  pathways.  Detailed  mapping  studies  have  also  been  done  with  paresthesias  produced  by 
magnetic  stimulation. 

Akinesia  in  Parkinson's  disease  was  studied  with  motor  evoked  potentials  elicited  prior  to  a  reaction  time 
movement.  Results  showed  that  it  took  longer  than  normal  for  patients  to  bri  ng  the  cortex  to  a 
sufficient  level  of  excitation  to  produce  a  voluntary  movement. 


43-MNB/DIR 


PHS  6040  (Rev    l  84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS   01658-23  MNB 


PERIOD  COVERED 


Ort-nher    1,     1989    through    September    30,     1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Ht-misphpr^    DggeJ  opmenr.   and   Specialization   of   the    Intellectual   Functions 

PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI: 

P. 

Fedio,  Ph.D 

E. 

Mohr,  Ph.D. 

Others : 

T. 

Blaxton,  Ph 

D. 

S. 

Bookhe  imer , 

Ph.D 

L. 

Ryan ,   M . A . 

Chief 

Psychologist 
Senior  Staff  Fellow 
IRTA  Fellow 
Psychologist 


CNS,  MNB,  NINDS 
Ottawa,  Canada 
CNS,  MNB,  NINDS 
CNS,  MNB,  NINDS 
CNS,  MNB,  NINDS 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 


Medical  Neurology,    CNP.   DIR 


SECTION 


Clinical   Neuropsychology 


INSTITUTE  AND  LOCATION 


NTNDS,    NTH,     Bethesda,    MP    20892 


TOTAL  MAN-YEARS: 


1    o 


PROFESSIONAL: 


0.5 


OTHER: 


-Q.J. 


CHECK  APPROPRIATE  BOX(ES) 

S  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  effects  of  chronic  progressive  neurological  disorders  in  adults  and  children 
were  evaluated  by  a  broad  range  of  neuropsychological  tests  evaluating 
bra  in - behavior  relations.   A  neuropsychological  profile  was  plotted  for  patients 
with  Alzheimer's  (AD).  Huntington's  (HP),  or  Parkinson's  (PD)  disorder.   The 
evaluations  extended  into  memory .  learning  and  perception,  applying  standard  and 
experimental  tasks  to  identify  functional  changes  accompanying  aging  processes. 

The  results  implicated  dopamine  deficiencies  and  frontal  pathophysiology  in  PD, 
most  notably,  losses  in  executive  capabilities  and  visuospatial  and  generic  memory 
functions.   With  HD  patients,  perceptuomotor  capacity  and  the  abilility  to 
manipulate  spatial  information  were  affected  whereas  spatial  discrimination  was 
relatively  intact.   With  a  dichotic  task,  AD  patients  did  poorer  and  were  unable 
to  selectively  attend  to  serial  information.   The  behavioral  data  extend 
neuropathologic  impressions  of  degeneration  of  the  frontal  striatal  system  in  HD 
and  temporoparietal .  cortical  Involvement  in  AD. 


44-MNB/DIR 


PHS  6040  (Rev.  1/84) 


OPO  814-918 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01    NS    01424-24   MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (BO  characters  or  less   Title  must  fit  on  one  line  between  the  borders ) 

Behavioral  Modulation  by  the  Limbic  System  in  Man 


PRINCIPAL  INVESTIGATOR  (List  other  protessional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 

Chief 

Senior  Staff  Fellow 
IRTA  Fellow 
Psychologist 
Medical  Officer 
Medical  Officer 
Medical  Officer 
Psychologist Catholic  University 


PI: 

P. 

Fedio,  Ph.D 

Others : 

T. 

Blaxton,  Ph 

D. 

S. 

Bookheimer , 

Ph.D 

L. 

Ryan,  M.A. 

C. 

Kufta,  M.D. 

S. 

Sato,  M.D. 

W. 

Theodore,  M 

D. 

CNS, 

MNB, 

NINDS 

CNS, 

MNB, 

NINDS 

CNS, 

MNB, 

NINDS 

CNS, 

MNB, 

NINDS 

SNB, 

NINDS 

CES, 

MNB, 

NINDS 

CES, 

MNB, 

NINDS 

A.  August,  M.A. 


COOPERATING  UNITS  (it  any) 

Surgical  Neurology  Branch,  DIR,  NINDS 

Department  of  Medical  Psychology,  John  Hopkins  University,  Baltimore,  MD 

Department  of  Psychology,  Catholic  University,  Washington,  D.C. 


LAB/BRANCH 

Medical  Neurology,  CNP,  DIR 


SECTION 

Clinical  Neuropsychology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS 


1.5 


PROFESSIONAL 


1.0 


OTHER 


0.5 


CHECK  APPROPRIATE  BOX(ES) 

D3  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type  Do  not  exceed  the  space  provided) 

Emotional  and  cognitive  characteristics  were  studied  in  epileptic  patients  before 
and  following  unilateral  left  or  right  temporal  lobe  resection,  and  during  brain 
stimulation  and  intracarotid  amvtal  -injection  (Wada ) .   Physiological  events  (skin 
conductance;  and  EEG  measures  were  also  monitored  during  select  test  performance. 
The  research  examined  the  role  of  the  temporal  lobe  in  establishing  limbic  sensory 
associations  as  a  basis  for  cognition  and  emotion. 

In  affective  spheres,  dysphoria  followed  pharmacological  deactivation  of  the  left 
hemisphere  whereas  euphoria  accompanied  amytal  injection  into  the  right  internal 
carotid.   The  transient  mood  state  was  more  common  for  patients  with  right  temporal 
lesions,  with  late  age  at  onset  of  seizure  disorder.   With  neuropsychometric  proce- 
dures, the  patients  differed  along  an  introver s ion- extrover s ion  continuum,  the  left 
being  more  inclined  to  rate  themselves  as  avoidant,  depressed  and  overly  anxious. 
In  contrast,  the  right  temporal  patients  presented  themselves  in  a  more  favorable 
light,  but  the  test  profile  showed  histrionic  and  aggressive  features.  Patients 
presenting  an  aura  of  fear  are  more  likely  to  exhibit  maladaptive  behaviors.   These 
data  suggest  that  unilateral  temporal  lobe  injury  disrupts  the  normal  linkage  of 
cognitive-affective  associations  mediated  by  frontal -limbic  interaction. 

Following  unilateral  temporal  lobectomy,  physiological  hypoarousal  was  more  common 
with  left,  and  hyperarousal,  with  right,  resections.   Changes  in  activation  level 
may  account  for  the  clinical  signs  of  dysphoria  and  euphoria  with  left  and  right 
brain  lesions,  respectively. 


45-MNB/DIR 


PHS  6040  (Rev.  1/84) 


GPO   914-018 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  01245-25  MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (BO  characters  or  less.  Title  must  lit  on  one  line  between  the  borders.) 

EEG  Learning  Correlates  Using  Scalp  and  Intracranial  Depth  Electrodes 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI: 

P. 

Fedio,  Ph.D. 

Others : 

S. 

Sato,  M.D. 

M. 

Balish,  M.D. 

B. 

Smith,  M.D. 

C. 

Kufta,  M.D. 

Chief 

Medical  Officer 

Medical  Officer 

Psychologist 

Medical  Officer 


CNS,  MNB   NINDS 
CES,  MNB,  NINDS 
CES,  MNB,  NINDS 
University  of  MD 
SNB,  NINDS 


COOPERATING  UNITS  (it  any) 

Surgical  Neurology  Branch,  DIR,  NINDS 
Department  of  Psychology,  University  of  MD. 


College  Park,  MD 


LAB7BRANCH 

Medical  Neurology,  CNP,  DIR 


SECTION 

Clinical  Neuropsychology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS 


1.0 


PROFESSIONAL: 


0.5 


OTHER: 


0.5 


CHECK  APPROPRIATE  BOX(ES) 

(S  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type  Do  not  exceed  the  space  provided.) 

Perception  and  judgment  with  cognitive  and  emotional  tasks  were  monitored  by 
electroencephalographic  (EEG)  activity  recorded  from  left  and  right  brain  regions, 
of  patients  following  unilateral  temporal  lobectomy.   EEG  disturbances  in 
bra in -behavior  relations  in  neuropsychiatric  patients  were  also  evaluated, 
relating  left  and  right  brain  dysfunctioning  to  maladaptive  ideative  and  emotional 
reactions,  respectively. 

With  temporal  lobectomy  patients,  preliminary  results  indicate  that  electrographic 
shifts  in  frequency-amplitude  differed  in  that  right  temporal  patients  showed 
greater  responsivity  to  pleasant  and  horrific  materials,  and  less  so  while 
applying  cognitive  stategies  to  deal  with  imaginary  emotionally  charged 
situations.   The  converse  was  true  with  left  temporal  patients  who  generated 
greater  activity  while  intellectually  resolving  emotional  tasks.   These  data 
underscore  the  dual  cognitive  and  emotional  roles  of  the  limbic  system  in 
modulating  human  behavior. 

Left  brain  stimulation  (indwelling  flap  electrodes)  of  posterior  sites,  produced 
storage  and  retrieval  memory  errors  with  anterior  and  posterior,  temporal  sites, 
respectively.   Stimulation  of  frontal  cortex  produced  defects  suggestive  of  mechan- 
isms that  collate  immediate  and  long-term  plans.   There  was  a  dissociation  between 
aphasia  and  amne s ia  with  inferior,  posterior  temporal  stimulation,  emphasizing  the 
importance  of  this  region  to  retrieval  from  episodic  memory  registers.   With  right 
brain  stimulation,  paralinguistic  disturbances  were  produced  in  prosody,  and  there 
were  errors  in  interpreting  ambiguous  statements,  and  in  pattern  discrimination 
and  recognition. 

46-MNB/DIR  

spo  »i  4-an 


PHS  6040  (Rev.  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01    NS    00200-36    MNB 


PERIOD  COVERED 

October  1.  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less   Title  must  fit  on  one  line  between  the  borders  ) 

Cognitive  and  Emotional  Profile  of  Neuropsychlatrlc  Disorders 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:      P.  Fedlo,  Ph.D. 

T.  Blaxton,  Ph.D. 

S.  Bookheimer,  Ph.D. 

Others:  L.  Ryan,  M.A. 

D.  Ronsaville,  Ph.D. 
T.  Chase,  M.D. 

E.  Mohr,  Ph.D. 
A.  August,  M.A 


Chief 

Senior  Staff  Fellow 

IRTA  Fellow 

Psychologist 

Psychologist 

Neurologist 

Psychologist 

Psychologist 


CNS,  MNB,  NINDS 
CNS,  MNB,  NINDS 
CNS,  MNB,  NINDS 
CNS,  MNB,  NINDS 
CNS,  MNB,  NINDS 
Chief,  ETB,  NINDS 
Ottawa,  Canada 
Catholic  University 


COOPERATING  UNITS  (if  any) 

Experimental  Therapeutics  Branch,  DIR,  NINDS 


LAB/BRANCH 

Medical  Neurology,  CNP,  DIR 


SECTION 

Clinical  Neuropsychology 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN- YEARS 


2.0 


PROFESSIONAL 


1.0 


OTHER: 


1.0 


CHECK  APPROPRIATE  BOX(ES) 

B  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□  (b)  Human  tissues  D  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type  Do  not  exceed  the  space  provided.) 

Experiments  were  initiated  to  identify  the  neuroanatomical  basis  underlying 
different  types  of  memory  and  perceptual  dysfunctions  exhibited  by  patients  with 
neurological  disorders.   One  theoretical  line  focused  on  using  implicit  and 
explicit  memory  tasks  within  the  framework  of  data- versus  conceptua 1 1 y - dr iven ; 
other  investigations  examined  the  role  of  the  temporal  lobe  in  perceiving 
information  containing  high  and  low  spatial  frequencies. 

Normals  and  right  brain-damaged  patients  did  better  when  memory  processing  engaged 
at  study  (data-or  conceptually-driven)  matched  that  required  at  test.   Patients 
with  left  hemisphere  lesions  showed  normal  performance  levels  on  data-driven  tests 
but  were  impaired  on  both  implicit  and  explicit  conceptually  driven  tests. 
Although  controls  and  left  lesion  patients  performed  normally,  patients  with  right 
hemisphere  lesions  showed  deficits  with  data-driven,  nonverbal  materials. 

When  sets  of  perceptual  stimuli  were  identical,  high  spatial  frequency  information 
was  preferred,  but  when  stimuli  differed,  low  frequency  information  was  perceived 
better.   This  pattern  did  not  hold  for  left  brain  damage  subjects  who  were 
impaired  in  strategic  processing.   Also,  patients  performed  worse  than  controls  in 
processing  upright  faces  but  actually  performed  better  with  the  inverted  faces. 
Perceptual  asymmetries  are  less  pronounced  between  the  hemispheres  than  processing 
asymmetries,  and  the  mode  of  processing  interacts  with  perceptual  characteristics 
of  incoming  stimuli  to  bias  perception  toward  high  or  low  spatial  frequencies. 


47-MNB/DIR 


PHS  6040  (Rev.  1/84) 


GPO   914-819 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02732-04  MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJ  ECT  (SO  chtrtctan  or  toss.  Tltfe  must  lit  on  ona  Una  barman  !/>«  borders) 

Pharmacological  Studies  of  Ion  Channels  in  Cultured  Cells 


PRINCIPAL  INVESTIGATOR  (Uft  olhar  prohtiStonml pononntl  balow  tht  rrinaotl  Inwstigttor.)  (Utmo.  t/lta.  iMborttory.  tnd institute  tffilittion) 

PI:  Michael  A.  Rogawski,  M.D.,  Ph.D.  Chief,  NEXS,  MNB,  NINDS 


Others:         Dora  M.T.  Politi,  M.D. 

Norman  Hershkowitz,  M.D. 
Taco  R.  Werkman,  Ph.D. 
Susan  M.  Jones,  Ph.D. 
Karen  Wayns 


Visiting  Fellow,  NEXS,  MNB,  NINDS 
NRC  Fellow,  NEXS,  MNB,  NINDS 
Visiting  Fellow,  MNB,  NINDS 
NRC  Fellow,  NEXS,  MNB,  NINDS 
Bio.  Lab.  Tech.,  NEXS,  MNB,  NINDS 


COOPERATING  UNITS  Vltny) 

Bruce  Smith,  Ph.D.,  Research  Services  Branch,  NIMH;  Haruhiro  Higashida,  M.D.,  Kanazawa  University, 
Japan;  J.M.H.  ffrench-Mullen,  Ph.D.,  ICI  Americas,  Wilmington,  DE. 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Neuronal  Excitability  Section 


INSTITUTE  AND  LOCATION 
NINDS,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

1      |   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


]  (b)  Human  tissues  [x]  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Drug  interactions  with  voltage-dependent  K+  channels  and  N-methyl-D-aspartate  (NMDA)  receptor 
coupled-cation  channels  were  studied  in  cultured  hippocampal  neurons  and  in  fibroblasts  transfected 
with  K  +  channel  genes  using  whole-cell  voltage-clamp  and  single  channel  recording  techniques.  The 
aim  of  this  work  was  to  explore  new  strategies  for  the  rational  development  of  antiepileptic  drugs  based 
upon  their  interaction  with  neuronal  ion  channel  systems.  Work  was  focused  in  four  areas:  (1)  kinetic 
analysis  of  NMDA  antagonism  by  novel  dissociative-anesthetic-like  anticonvulsant  drugs;  (2)  tetrahydro- 
aminoacridine  (THA)  block  of  NMDA-activated  cation  channels;  (3)  mechanism  of  action  of  K>  channel 
activator  drugs;  and  (4)  drug  effects  on  K  +  channel  currents  carried  by  molecularly  defined  K  +  channel 
proteins  in  fibroblast  cells  transfected  with  K  +  channel  genes.  The  novel  anticonvulsants  phenylcvclo- 
hexylamine  (PCA)  and  5-aminocarbonvl-5H-dibenzo[a,dkvclohepten-5,10-imine  (ADCI)  are  structurally 
related  to  the  dissociative  anesthetics  phencyclidine  and  MK-801 .  However,  unlike  their  parents  which 
cause  motor  toxicity  at  low  doses,  PCA  and  ADCI  protect  against  seizures  in  animal  models  at  doses  that 
fail  to  cause  motor  impairment.  We  have  determined  that  the  more  favorable  toxicity  profile  of  PCA  and 
ADCI  may  relate  to  their  ability  to  block  NMDA  responses  more  rapidly  than  do  PCP  and  MK-801 .  Tetra- 
hydroaminoacridine  (THA),  a  centrally  active  cholinesterase  inhibitor  that  may  provide  symptomatic 
benefit  in  Alzheimer's  disease,  was  found  to  produce  a  voltage-dependent  block  of  NMDA  responses  in 
cultured  hippocampal  neurons  and  also  to  reduce  the  frequency  and  duration  of  NMDA  evoked  single 
channel  currents  in  outside-out  membrane  patches.  The  antihypertensive  cromakalim  has  been  prev- 
iously found  to  activate  a  K  +  current  in  cultured  hippocampal  neurons.  We  have  demonstrated  that 
metabolic  inhibitors  can  activate  a  similar  K  +  current.  We  have  also  obtained  evidence  for  the  existence 
of  ATP-sensitive  K  +  channels  in  hippocampal  neurons,  and  our  data  indicate  that  cromakalim  and  met- 
abolic inhibitors  can  activate  these  channels.  These  channels  may  play  a  role  in  protecting  central  neur- 
ons from  brain  ischemia.  Drugs  like  cromakalim  that  activate  K  +  channels  in  CNS  neurons  have  potential 
as  anticonvulsants,  and  perhaps  also  in  the  treatment  of  brain  ischemia.  The  effects  of  various  K  + 
channel  blockers  and  activators  are  being  studied  on  K  +  channels  expressed  in  fibroblast  cells  transfect- 
ed with  K  +  channel  genes.  We  observed  that  the  NGK1  K+  channel  is  insensitive  to  tetraethylammon- 
ium.  but  is  effectively  blocked  by  4-aminopyridine,  PCP,  and  histrionicotoxin.  an  alkaloid  from  frog  skin. 

48-MfiE7DIR  


PHSSOMOUv.  1/M) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02733-04  MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  lau.  Tula  must  fit  on  ono  Una  bodveon  the  borders.) 

Excitability  Properties  of  Enzymatically  Dissociated  CNS  Neurons 


PRINCIPAL  INVESTIGATOR  (List  othar  professional  personnel  Mm  (ho  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  Michael  A.  Rogawski,  M.D.,  Ph.D.        Chief,  NEXS,  MNB,  NINDS 

Others:      Xiao-Jing  Wang,  Ph.D.  Visiting  Fellow,  Mathematical  Research  Branch,  NIDDK 

John  Rinzel,  M.D.  Chief,  Mathematical  Research  Branch,  NIDDK 


COOPERATING  UNITS  (if  an,) 

Mathematical  Research  Branch,  NIDDK 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Neuronal  Excitability  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN-YEARS: 


0.5 


PROFESSIONAL: 


0.5 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

]   (a)  Human  subjects 
]  (a1)  Minors 
]  (a2)  Interviews 


]  (b)  Human  tissues  (jT]  W  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Electrophysiological  techniques  were  used  to  characterize  the  ionic  channels  of  neurons  isolated  from 
slices  of  the  adult  guinea  pig  thalamus.  Thalamic  neurons  undergo  a  shift  from  tonic  to  phasic  (burst) 
firing  upon  hyperpolarization.  This  state  transition  results  from  deinactivation  of  a  regenerative 
depolarizing  event  referred  to  as  the  low-threshold  spike  (LTS).  We  have  previously  shown  that  LTS-like 
events  can  be  evoked  in  isolated  thalamic  (dorsal  lateral  geniculate)  neurons  and  that  these  potentials 
are  mediated  by  a  low-threshold,  rapidly  inactivating  (T-type)  calcium  current.  Hodgkin-Huxley 
modeling  of  the  T-type  calcium  current  indicated  that  the  shape  of  the  LTS  can  be  accounted  for  almost 
entirely  by  the  intrinsic  properties  of  T-type  calcium  channels.  Our  model  is  novel  in  that  we  used  two 
inactivation  gates  to  account  for  the  slow  recovery  from  inactivation  of  the  T-type  calcium  current.  Burst 
firing  in  thalamic  neurons  mediated  by  the  LTS  is  believed  to  be  critical  to  the  generation  of  absence 
seizures  since  drugs  which  specifically  block  the  LTS  (T-type  calcium  channels)  prevent  absence  seizures. 
Our  theoretical  model  of  the  LTS  is  compatible  with  this  idea. 


49-MNB/DIR 


PHSWHMRev   1441 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02772-03  MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (SO  characters  or  less,  rule  must  fit  on  one  line  between  the  borders.) 

Development  of  Uncompetitive  NMDA  Antagonists  as  Anticonvulsants  * 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI: 


Michael  A.  Rogawski,  M.D.,  Ph.D.    Chief,  NEXS,  MNB,  NINDS 


Others: 


Shun-lchi  Yamaguchi,  Ph.D. 
Andrew  Thurkoff,  Ph.D. 
Brian  de  Costa,  Ph.D. 
KennerC.  Rice,  Ph.D. 
Arthur  E.  Jacobson,  Ph.D. 


Psychologist,  NEXS,  MNB,  NINDS 

NRSA  Fellow,  Laboratory  of  Medicinal  Chemistry,  NIDDK 
Fogarty  Fellow,  Laboratory  of  Medicinal  Chemistry,  NIDDK 
Chief,  Laboratory  of  Medicinal  Chemistry,  NIDDK 
Senior  Research  Scientist,  Laboratory  of  Medicinal 
Chemistry,  NIDDK 


COOPERATING  UNITS  (if any) 

Laboratory  of  Medicinal  Chemistry,  NIDDK 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Neuronal  Excitability  Section 


INSTITUTE  AND  LOCATION 

NINDS,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

J   (a)  Human  subjects 
]  (a1)  Minors 

J   (a2)  Interviews 


]  (b)  Human  tissues  \j~\  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  dissociative  anesthetics  PCP  and  MK-801  are  powerful  anticonvulsants  in  a  wide  variety  of  animal 
seizure  models.  However,  undesirable  side  effects  that  occur  in  the  same  dosage  range  as  seizure 
protection  limit  their  practical  usefulness  in  the  treatment  of  seizure  disorders.  Despite  their 
unfavorable  therapeutic  indices,  PCP  and  MK-801  can  be  considered  prototypes  upon  which  to  base  the 
design  of  less  toxic  and  potentially  more  clinically  useful  antiepileptic  drugs.  We  have  examined  the 
anticonvulsant  activity  of  more  than  40  PCP  analogs  in  an  attempt  to  obtain  compounds  with  enhanced 
anticonvulsant  activity  relative  to  their  neurotoxic  side  effects.  Drugs  were  screened  for  anticonvulsant 
activity  in  mice  with  the  maximal  electroshock  (MES)  test  and  by  administration  of  the  chemoconvulsants 
pentylenetetrazol  (PTZ)  and  N-methyl-D-asparate  (NMDA).  PCP  had  approximately  equal  potency  in  the 
MES  test  and  in  a  motor  toxicity  test  so  that  its  "therapeutic  index"  (Tl;  ratio  of  dose  causing  toxicity  in 
50%  of  animals  to  dose  causing  seizure  protection  in  50%  of  animals)  was  about  1.  Certain  of  the 
analogs  had  Tl  values  higher  than  that  of  PCP.  The  most  favorable  of  these  were  derivatives  of  1- 
phenylcyclohexylamine  modified  by  (1)  certain  stereochemically  orientated  cyclohexane  ring  methyl 
substituents,  (2)  ortho  substituents  on  the  phenyl  ring,  and  (3)  contraction  of  the  cyclohexane  ring. 

Studies  were  also  conducted  on  compounds  related  to  the  dibenzocyclohepteneimine  MK-801.  Of 
particular  interest  is  5-aminocarbonyl-5H-dibenzo[a,d]cyclohepten-5,10-imine(ADG)  which  is 
structurally  related  to  the  commonly  used  antiepileptic  carbamazepine.  ADCI  was  a  potent 
anticonvulsant  in  the  MES  test  (ED50, 8.9  mg/kg)  and  showed  a  six-fold  higher  Tl  than  PCP.  Unlike 
carbamazepine,  ADCI  was  able  to  protect  against  PTZ  (ED50,  37  mg/kg)  and  NMDA-induced  seizures 
(ED50, 15  mg/kg).  The  ability  of  ADCI  to  block  NMDA  receptor-mediated  responses  was  confirmed  in 
cellular  electrophysiological  studies  (see  project  Z01  NS  02732-04  MNB).  We  conclude  that  ADCI  may 
offer  advantages  over  carbamazepine  in  the  treatment  of  resistant  seizure  disorders  because  of  its  ability 
to  block  NMDA  receptor-mediated  responses. 

*  (Formerly,  "Development  of  Phencyclidine  Analogs  as  Anticonvulsants") 

50-MNB/DIR 


PHS  6040  (R«v    1  84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02792-02  MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  /ess.   Title  must  fit  on  one  Una  between  the  borders.) 

Neuropsychological  Investigations  of  Human  Cognition  and  Mood  State 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  Jordan  Grafman,  Ph.D.  Chief  CNS        MNB       NINDS 


Others 


Ray  Johnson,  Jr.,  Ph.D. 

Special  Expert 

CNS 

MNB 

NINDS 

Rhonda  Friedman,  Ph.D. 

Special  Expert 

CNS 

MNB 

NINDS 

MarkHallett,  M.D. 

Chief 

MNB 

NINDS 

COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Cognitive  Neuroscience  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN-YEARS: 


0.4 


PROFESSIONAL: 


0.2 


OTHER: 


0.2 


CHECK  APPROPRIATE  BOX(ES) 

I  x  |   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


J  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Current  studies  in  the  Cognitive  Neuroscience  Section  focus  on  amnesia,  thinking,  neurolinquistics, 
event-related  evoked  potentials,  social  cognition,  and  visual  processing.  Both  single-case  and  group 
design  studies  are  used.  Normal  controls,  inpatients  and  outpatients  are  evaluated.  Memory  is 
studied  in  experiments  focusing  on  implicit  and  explicit  retrieval,  priming,  autobiographical  recall, 
discourse  processing,  naming  and  word  retrieval,  and  categorization  tasks.  Reasoning  and  problem- 
solving  are  studied  in  experiments  focusing  on  planning,  syllogisms,  analogical  thinking,  and  schema 
organization.  Dyslexia,  dvsgraphia,  and  dysnomia,  are  studied  in  experiments  focusing  on  single 
word  reading  and  writing,  lexical  decision,  associative  and  semantic  priming,  and  similar  tasks. 
Event-related  evoked  potentials  are  measured  for  latency,  amplitude,  and  distribution  and  used  as  a 
physiological  indice  of  information-processing  stages,  working  memory,  visual  attention,  and 
automatic  processing.  Emotions,  impression  and  preference  formation,  and  social  judgment  are 
studied  in  experiments  focusing  on  judgment  of  interpersonal  behavior,  word  association,  and  mood 
state.  Finally,  visual  information  processing  is  studied  beginning  with  experiments  examining  spatial 
frequency  contrast-sensitivity,  object  recognition,  and  visual  categorization.  Although  developing 
theoretically  valid  and  testable  models  of  cognitive  processing  is  the  primary  aim  of  the  Section, 
there  is  also  a  strong  effort  to  relate  the  profile  of  cognitive  deficits  in  patients  to  lesion  location  in 
order  to  topographically  map  the  components  of  cognitive  processing  to  brain  regions  and  systems. 
Pharmacologic  challenge  and  infusion  studies  are  planned  to  evaluate  the  dissociability  of 
hypothesized  components  of  memory  processing.  PET  scan  studies  are  planned  to  examine  whether 
familiar  and  unfamiliar  faces  are  processed  in  a  different  brain  location  from  real  and  non-objects 
and  forms. 


51-MNB/DIR 


PHS  6040  (Rev   V84| 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02793-02  MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJ  ECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Cognitive  Neuroscience*** 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:  Jordan  Grafman,  Ph.D. 

Others:        Ray  Johnson,  Jr.,  Ph.D. 

Rhonda  Friedman,  Ph.D 

Jeffrey  Hadley,  Ph.D. 

Angela  Sirigu,  Ph.D. 

Marten  Scheffers,  Ph.D. 


Chief 
Special  Expert 
Special  Expert 
IRTA 

Visiting  Fellow 
Visiting  Fellow 


CNS 

MNB 

NINDS 

CNS 

MNB 

NINDS 

CNS 

MNB 

NINDS 

CNS 

MNB 

NINDS 

CNS 

MNB 

NINDS 

CNS 

MNB 

NINDS 

COOPERATING  UNITS  (if  any) 

Walter  Reed  Army  Medical  Center,  Wash,  DC;  National  Naval  Medical  Center,  Bethesda,  MD;  Centre  Paul 
Broca,  Paris,  France;  Hopital  Salpetriere,  Paris,  France;  Hospital  Clinicas,  Montevideo,  Uruguay;  ** 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Cognitive  Neuroscience  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD  20892 


TOTAL  MAN-YEARS: 


0.4 


PROFESSIONAL: 


0.2 


OTHER: 


0.2 


CHECK  APPROPRIATE  BOX(ES) 

I  x  |   (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


J  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Memory  and  cognition  are  studied  in  experiments  focusing  on  representational  knowledge,  working 
memory,  priming,  procedural  learning,  number  processing  and  calculation,  autobiographical  memory, 
visual  attention,  naming,  and  categorization.  Normal  subjects  and  patients  with  progressive  dementia, 
focal  lesions,  and  psychiatric  disorders  are  studied.  New  studies  focusing  on  the  composition  of  mental 
structures  in  the  frontal  lobes  have  just  begun. 


*Continued: 

A.  Salazar,  M.D. 
S.Rao,  Ph.D. 
F.  Boiler,  Ph.D. 
Y.Agid.M.D. 
J.Sergent,  Ph.D. 

C.  Chouza,  M.D. 

D.  Hermann,  Ph.D. 
J.  Hallenbeck,  M.D. 
A.  Newell,  Ph.D. 

R.  Desimone,  Ph.D. 
C.  Chaviox,  Ph.D. 
J.  Duncan,  Ph.D. 
I.  Biederman,  Ph.D. 
J.  Rapoport,  M.D. 
S.  Swedo,  M.D. 
E.Zaidel.Ph.D. 


Department  of  Neurology,  Walter  Reed  Army  Med.  Ctr. 

Dept.  of  Neurology,  Medical  College  of  Wisconsin 

INSERM  U.  324  Centre  Paul  Broca,  Paris,  France 

INSERM  U.  289  Hopital  Salpetriere,  Paris,  France 

Department  of  Neurology,  Montreal  Neurological  Institute 

Neurology  Institute,  Hospital  Clinicas,  Montevideo,  Uruguay 

Department  of  Labor,  Washington,  DC 

Department  of  Neurology,  National  Naval  Medical  Center 

Computer  Science  Dept.,  Carnegie-Mellon  Univ.,  Pittsburgh,  PA 

National  Institute  of  Mental  Health 

INSERM  Unit  320,  Caen,  France 

Applied  Psychology  Unit,  Cambridge,  England 

Dept.  of  Psychology,  USC,  Los  Angeles,  CA 

National  Institute  of  Mental  Health 

National  Institute  of  Mental  Health 

Dept.  of  Psychology,  UCLA,  Los  Angeles,  CA 


**    Medical  College  of  Wisconsin,  Milwaukee,  Wisconsin;  Montreal  Neurological  Institute,  Montreal, 
Canada;  National  Institute  of  Mental  Health,  NIH. 

**    (Formerly,  "Amnesia  and  Cognitive  Neuroscience") 

52-MNB/DIR 


PHS  6040  (Rev  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02794-02  MNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJ  ECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Event-Related  Potential  Studies  of  Normal  and  Abnormal  Cognitive  Processing 


PRINCIPAL  INVESTIGATOR  (list  other  professional  personnel  belojv  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:  Ray  Johnson,  Jr.,  Ph.D.         Special  Expert  CNU       MNB      DIR         NINDS 


Others:        Marten  Scheffers,  Ph.D. 
Jordan  Grafman,  Ph.D. 
Daniel  Ruchkin,  Ph.D. 
Wolfgang  Miltner,  Ph.D. 


Visiting  Fellow  CNU       MNB      DIR         NINDS 

Chief  CNU       MNB      DIR         NINDS 

Elec.  Engineer  U.  of  Maryland  School  of  Medicine 

Psychologist  U.  of  Tuebingen,  West  Germany 


COOPERATING  UNITS  (if  an,) 

University  of  Maryland  School  of  Medicine,  University  of  Tuebingen,  West  Germany 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Cognitive  Neuroscience  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN-YEARS: 


20 


PROFESSIONAL: 


2.0 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I  x   |   (a)  Human  subjects 
]  (a1)  Minors 

]  (a2)  Interviews 


I      I  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Event-related  brain  potentials  (ERP)  were  used  to  study  cognitive  processes  such  as  short-  and  long-term 
memory,  spatial  attention  and  visual  search,  mental  rotation,  mental  arithmetic,  and  language  compre- 
hension. ERP  studies  of  normal  subjects  were  intended  to  reveal  the  brain  mechanisms  underlying  cogni- 
tion. Studies  of  patients  with  neuropsychiatry  disorders  were  intended  to  provide  information  on  the 
physiological  sources  of  these  cognitive  processes  while  allowing  us  to  characterize  better  the  patients' 
information  processing  deficits.  Data  analysis  continues  on  studies  of  temporal  lobectomy  patients. 
Turner's  patients,  and  the  maturation  of  cognitive  processes.  ERP  studies  of  dementia  are  continuing  for 
patients  with  Alzheimer's  disease  while  data  collection  has  been  completed  for  patients  with  HIV  disease 
and  progressive  supranuclear  palsy.  The  results  from  the  HIV  and  PSP  studies  indicate  that,  in  the  earliest 
stages  of  subcortical  disease,  processing  at  the  cortical  level  is  more  affected  than  processing  at  the  sub- 
cortical level  (i.e.,  resembling  a  cortical  dementia).  This  pattern  reverses  as  the  subcortical  disease  pro- 
gresses. Studies  of  the  mechanisms  underlying  and  affecting  attentional  processes  continue.  Data  col- 
lection was  completed  recently  in  two  studies,  one  on  how  normal  controls  visually  search  a  spatial  array 
for  items  previously  stored  in  short-term  memory  and  one  on  the  effects  of  fatigue  on  attention  in  pa- 
tients with  chronic  fatigue  syndrome  and  normal  controls.  Studies  with  Daniel  Ruchkin  have  been  aimed 
at  understanding  a  newly  emerging  class  of  ERP  components  known  as  slow  waves.   Completed  studies 
have  investigated  the  processes  of  mental  arithmetic,  mental  rotation,  and  short-term  memory.  Data 
collection  is  nearly  complete  in  an  experiment  on  the  differences  between  rehearsal  processes  for  verbal 
and  spatial  material.  Studies  with  Wolfgang  Miltner  have  been  aimed  at  providing  additional  data  on 
the  neural  sources  of  ERP  components.  Completed  studies  have  provided  a  comparison  of  the  ERP  com- 
ponents elicited  by  auditory  and  somatosensory  stimuli  using  large  electrode  montages.   A  new  experi- 
ment was  recently  finished  so  that  the  neural  source  localization  program,  BESA,  can  be  applied  to  the 
data.  Patient  and  control  data  have  been  used  to  validate  the  predictions  of  Johnson's  model  of  the  vari- 
ables controlling  P300  amplitude.  These  data  revealed  that,  contrary  to  the  widely  accepted  notion,  the 
P300  is  a  modality-dependent  component  whose  amplitude  represents  the  sum  of  a  number  of  distinct 
neural  generators  which  we  are  attempting  to  characterize  and  localize. 

53-MNB/DIR 


PHS  6040  (Rev   1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02038-18  MNB 


PERIOD  COVERED 

October  1,1989  through  September  30,1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Combined  Clinical,  Viral  and  Immunological  Studies  of  Neuromuscular  Diseases 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:  M.C.  Dalakas,  M.D. 

Other:  M.  Hallett,  M.D. 

B.  Sonies,  Ph.D. 

R.  Quarles,  Ph.D. 

P.  Plotz,  M.D. 

G.  DiChiro,  M.D. 

R.  Podolsky,  Ph.D. 

W.A.  Gahl.M.D. 


Medical  Officer 
Clinical  Director 
Speech  Pathologist 
Biochemist 
Rheumatologist 
Neuroradiologist 
Biochemist 
Medical  Officer 


MNB,  DIR,  NINDS 
OCD,  DIR,  NINDS 
CC,  DIR,  NINDS 
DMN,  DIR,  NINDS 
ARB,  DIR,  NIMSD 
OD,  DIR,  NINDS 
ARB,  DIR,  NIMSD 
HGB,  DIR,NICHD 


COORPERATING  UNITS  (if  any) 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Neuromuscular  Diseases 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.5 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I  x  I   (a)  Human  subjects 
|     ]  (a1)  Minors 
J  (a2)  Interviews 


J  (b)  Human  tissues  j  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Clinical  and  laboratory  studies  are  conducted  to  determine  etiology  (infection,  immunity  and/or 
genetics)  of  chronic  diseases  of  the  neuromuscular  system.  Current  studies  include  polymyositis/derm- 
atomyositis.  post-polio  syndrome,  amyotrophic  lateral  sclerosis  (ALS),  demyelinatinq  polyneuropathies. 
neuromuscular  diseases  associated  with  HIV  infection,  and  Duchenne's  muscular  dystrophy.  The  post- 
polio  syndrome,  a  neuromuscular  disease  that  occurs  in  patients  who  have  had  poliomyelitis  at  an  early 
age,  has  been  clinically  defined.  A  longitudinal  study  is  now  in  progress  to  define  the  rate  of  progression 
by  quantifying  the  muscle  strength  with  a  series  of  dynamometric  measurements.  The  pathogenesis  of 
this  syndrome  has  been  also  investigated  with  a  series  of  electrophysiological,  virological,  immunological 
and  histological  studies.  Its  relevance  to  other  motor  neuron  diseases  is  now  being  examined  and 
possible  persistence  of  mutated  poliomyelitis  virus  is  sought  using  tissue  cultures,  PCR,  and  in  situ 
hybridization.  Because  abnormal  immunoregulation  was  found  in  some  patients  with  post-polio 
syndrome,  a  double-blind  placebo-controlled  trial  using  prednisone  has  begun.  The  spectrum  of 
neuromuscular  disorders  associated  with  H]V  infection  has  been  studied  and  the  role  of  the  virus  as  the 
cause  of  neuropathies  or  myopathies  in  HIV-positive  patients  was  investigated  with  a  variety  of 
immunocytochemical  studies  and  in  situ  hybridization.  The  neuromuscular  complications  of  antiretro- 
viral  drugs  AZT  and  DDC  were  studied  and  factors  that  could  limit  their  neurotoxicity  were  identified. 
Patients  with  polymyositis  are  studied  and  the  muscle  changes  before  and  after  a  randomized  double- 
blind  controlled  study  with  intravenous  methotrexate,  azathioprine  or  plasmapheresis/lymphocyto- 
pheresis  are  investigated.  The  force  of  individual  (skinned)  muscle  fibers  from  the  muscle  biopsies  of 
patients  with  Duchenne's  muscular  dystrophy  was  measured  and  correlated  with  the  biochemical 
composition  of  the  contractile  muscle  proteins.  We  have  found  that  in  spite  of  the  absence  of 
dystrophin,  muscle  fibers  in  Duchenne's  dystrophy  generate  normal  force.  The  metabolic  activity  of  the 
cortex  in  ALS  patients  was  studied  using  the  PET  scan  and  18FDG  and  the  metabolic  abnormalities  were 
correlated  with  the  morphological  changes  in  the  neurons  on  brain  tissues  obtained  at  autopsy.  A 
variety  of  neuromuscular  diseases  associated  with  nephropathic  cvstinosis  and  renal  Fanconi  syndrome 
were  studied  morphologically  and  biochemically.  A  myopathy  with  lipid  or  cystine  storage  in  the  muscle 
was  found  prompting  a  therapeutic  trial  with  carnitine  or  cysteamine. 

54-MNB/DIR       


PHS  6040  (Rev.  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02531-09  MNB 


PERIOD  COVERED 

October  I,  1 989  through  September  30, 1 990 


TITLE  OF  PROltCJ  (SO  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Studies  in  Neuromuscular  and  CNS  Diseases  and  Their  Experimental  Models 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  belo»  the  Principal  Investigator.)  (Name,  tit/e.  laboratory,  and  institute  affiliation) 


PI:  M.C.  Dalakas,  M.D. 

OTHERS:  R.  Quarles,  Ph.D. 

G.H.  Pezeshkpour,  M.D. 

M.Mutchnich,  M.D. 

I.  Ilia,  M.D,  Ph.D. 

M.  Monzon,  Ph.D. 

L.  Lamperth,  M.D. 


Medical  Officer 

Biochemist 

Neuropathologist 

Gastroenterologist 

Neurologist 

Special  Expert 

Visiting  Associate 


MNB,  DIR,  NINDS 
DMN,  DIR,  NINDS 
AFIP,  Washington,  D.C. 
Detroit,  Ml 
MNB,  DIR,  NINDS 
MNB,  DIR,  NINDS 
MNB,  DIR,  NINDS 


COORPERATING  UNITS  (if  any) 

Department  of  Gastroenterology,  Wayne  State  University,  Detroit,  Ml 
AFIP,  Washington  D.C. 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 

Neuromuscular  Diseases 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.5 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

I  X  |   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


[xl  (b)  Human  tissues  O  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Immunocytochemical  studies  were  conducted  using  specific  antibodies  to  thymic  peptides  to  investigate 
the  interaction  of  the  immune  system  with  the  central  and  peripheral  nervous  systems.  Thymosin  beta  4, 
an  immunomodulating  thymic  polypeptide,  was  found  to  be  a  common  antigen  shared  by  macrophages, 
dendritic  lymphoid  cells  and  the  myelin  producing  cells  in  the  CNS  (oligodendrocytes)  and  the  PNS 
(Schwann  cells).  Prothymosin,  a  nuclear  protein,  and  thymosin-a1,  were  found  present  in  astrocytes  of 
normal  human  brain  and  could  play  a  role  in  cell  proliferation  and  gliosis.  The  IgM  of  certain  patients 
with  paraproteinemic  polyneuropathies  has  been  identified  as  a  specific  antibody  to  acidic  qlycolipids; 
intraneural  injection  of  IgM  in  the  sciatic  nerve  of  the  cat  induced  demyelination  suggesting  a  direct  role 
in  the  pathogenesis  of  the  neuropathy.  The  nature  of  amyloid  protein  in  patients  with  "sporadic" 
amyloid  polyneuropathy  was  identified  using  specific  antibodies  to  amyloid  proteins;  point  mutations 
and  direct  sequencing  of  prealbumin  genes,  the  precursor  protein,  were  studied  in  the  amyloid  tissue 
using  the  polymerase  chain  reaction.  The  mechanism  of  inflammatory  myopathy  in  monkeys  with 
immunodeficiency  (Simian  AIDS)  cause  by  SRV-1  andSIV-1  retroviruses,  was  studied.  Antibodies  to  SRV-1 
immunoreacted  with  inflammatory  cells  invading  muscle  fibers;  SRV-1  was  capable  of  infecting 
myoblasts  in  tissue  culture  without  exerting  a  cytopathic  effect  in  the  muscle.  The  role  of  SIV-1  is 
similarly  studied.  The  effect  of  aging  on  the  neuromuscular  system  of  monkeys  from  age  5  to  25  is  being 
studied  with  a  detailed  morphological  and  morphometrical  analysis  of  their  muscle  and  nerve  biopsies. 
The  mechanism  of  muscle  regeneration  is  studied  examining  markers  on  satellite  cells  including  the  role 
of  adhesion  on  molecules  such  as  laminin,  N-CAM,and  ICAM.  The  monoclonal  antibody  Leu-19(NKH) 
that  identifies  natural  killer  cells  was  found  to  share  common  antigenic  determinants  with  the  satellite 
muscle  cells.  NKH  also  stains  regenerating  muscle  fibers  and  could  play  a  role  in  muscle  regeneration. 
The  induction  of  abnormal  mitochondria  in  the  muscle  by  several  nucleoside  analogues  is  investigated  in 
the  muscle  fibers  in  tissue  culture. 


55-MNB/DIR 


PHS  6040  (Rev   184) 


PROJECT  NUMBER 
DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT  z01    NS   01195-26  MNb- 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  tit  on  one  line  Between  me  Borders  I 

Radiographic,    Radioisotopic,    CT,    and  MR  Angiography   of    the    Spinal   Cord 


PRINCIPAL  INVESTIGATOR  (List  other  orotessional  personnel  Below  the  Principal  Investigator.)  (Name,  title.  laBoratory.  ana  institute  affiliation) 

Giovanni  Di  Chiro,  M.D.,  Chief,  NIS,  MNB,  CNP ,  DIR,  NINDS  Others: 

NIS,  NINDS 
DRD,  CC 
SN,  NINDS 
NMD,  CC 

Neuroradiology,  GTU 
Neurosurgery,  Childrens 


L. 
J. 

M.  Levy,  M.D. ,  Ph 
L.  Doppman,  M.D. 

D. 

Guest 
Chief 

Researcher 

E. 

H.  Oldfield,  M.D. 

Chief 

R. 

D.  Neumann,  M.D. 

Chief 

D. 

Schellinger,  M.D. 

Chief 

D. 

C.  McCullough 

Chief 

COOPERATING  UNITS  (it  any) 

Diagnostic  Radiology  and  Nuclear  Medicine  Departments, 
Clinical  Center,  NIH;  Surgical  Neurology  Branch,  NINDS;  Medical  Examiner's 
Office,  Department  of  Public  Health  Philadelphia,  PA. 


LAB/BRANCH 


Medical  Neurology  Branch, CNP,  DIR 


SECTION 


Neuroimaging 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD   20892 


TOTAL  MAN-YEARS: 

0.3 


PROFESSIONAL: 

0.3 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

H  (a)  Human  subjects  □  (b)  Human  tissues  □  (c)  Neither 

Q  (a1)  Minors 
□   (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Selective  arteriography  (radiographic)  of  the  spinal  cord  is  a 
diagnostic  technique  which  has  proven  to  be  informative  in  cases  of 
arteriovenous  malformation  (AVM),  tumor,  obstructive  vascular  disease, 
trauma,  and  postradiation  damage  of  the  spinal  cord. 

Radioisotope  angiography  of  the  spinal  cord  offers  some  advantages  as  a 
screening  method,  and  in  certain  types  of  intraspinal  pathology  may  £ive 
information  not  available  by  any  other  diagnostic  test. 

Experience  with  the  techniques  of  dynamic  computed  tomography,  digital 
subtraction  angiography  (DSA),  positron  emission  tomography  (PET)  using 
lbp_2-deoxyglucose  and  nuclear  magnetic  resonance  imaging  (MRI)  of  the 
spine  (without  and  with  a  contrast  agent  such  as  Gd-DTPA)  indicates  that 
these  methods  may  be  useful  screening  and  follow-up  procedures  in  the 
evaluation  of  certain  vascular  lesions  and  tumors  of  the  spinal  cord. 


*  Formerly  in  OCD ;  transrerred  to  MNB  on  6/8/90. 


56-MNB/DIR 


PHS  6040  (Rev.  1/84)  OPO  >I44II 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  ■  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01   NS   02073-17  MNB* 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  tit  on  one  line  Between  the  borders  ) 

Nuclear  Magnetic  Resonance  and  Computed  Tomography  (Transmission) 

PRINCIPAL  INVESTIGATOR  (List  other  prolessional  personnel  belo*  the  Principal  Investigator  I  (Name   title,  laboratory,  and  institute  affiliation) 

Giovanni  Di  Chiro,  M.D.,  Chief,  NIS,  MNB,  CNP,  DIR,  NINDS  Others  (NIS) :  J.  R. 
Alger,  Ph.D.,  Special  Expert;  R.  A.  Brooks,  Ph.D.,  Staff  Physicist;  L.  M.  Levy, 
M.D.,  Ph.D.,  Guest  Researcher;  A.  Bizzi,  M.D.,  Visiting  Fellow;  A.  Brunetti,  M.D. , 
Visiting  Fellow;  T.  Francavilla,  M.D.,  Staff  Fellow;  D.  LeBihan,  M.D. ,  Ph.D., 
R.  Turner,  Ph.D.;  C.  Moonen,  Ph.D.;  B.  DeSouza,  M.D.,  Staff  Fellow;  R.  S. 
Miletich,  M.D.,  Ph.D.,  Senior  Staff  Fellow;  M.  J.  Fulham,  M.D.,  Visiting 
Associate,  N.  Patronas,  M.D.,  DRD ,  CC;  J.  Frank  M.D.,  DRD,  CC;  J.  L.  Doppman,  M.D. 
Chief,  DRD,  CC;  Joanna  M.  Hill,  M.D.,  Senior  Staff  Fellow,  NSB,  NIMH 


cooperating  units  (,t  any,   Diagnostic  Radiology,  Clinical  Center,  NIH;  "In  Vivo  NMR 
Research  Center,  BEIB,  NIH;  Division  of  Neuropathology  of  Case  Western  Reserve 
University  Medical  School,  Cleveland. 


LAB/BRANCH 

Medical   Neurology   Branch,    CNP,    DIR 


section 
Neuroimaging 


INSTITUTE  ANO  LOCATION 

NINDS,  NIH,  Bethesda,  MD 


20892 


TOTAL  MAN-YEARS 
2.0 


PROFESSIONAL 
2.0 


OTHER 


CHECK  APPROPRIATE  BOX(ES) 

Cid  (a)  Human  subjects 
Q  (a1)  Minors 
D   (a2)  Interviews 


D  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

NMR:  Our  NMR  imaging  research  is  developing  along  eleven  main  lines:  1) 
study  of  patients  with  spinal  cord  pathology,  particularly  arteriovenous 
malformations  (AVM);  2)  recognition  of  artifactual  linear  regions  of 
abnormal  signal  intensity  along  the  length  of  the  spinal  cord;  3) 
assessment  of  pulsatile  CSF  flow  using  longitudinal  imaging;  4)  studies 
of  the  "mobile"  (normal)  and  "fixed"  (pathologic)  spinal  cord;  5)  con- 
tinuing the  in  vivo  and  i_n  vitro  investigation  of  extravasated 
intracranial  blood;  6)  analysis  of  the  signal  intensity  from  critical 
areas  (basal  ganglia)  in  patients  affected  by  a  variety  of  movement 
disorders;  7)  trying  to  learn  more  about  the  NMR  signal  of  various 
abnormal  tissues;  8)  comparing  clinical  MRI  imaging  results  with  those 
of  CT  and  particularly  PET;  9)  Analysis  of  iron  accumulation  in  the 
basal  ganglia  of  normal  primates  of  various  ages  as  well  as  in 
parkinsonian  (MPTP)  animals;  10)  MRI  of  MPTP  induced  lesions  in  the 
basal  ganglia  of  primates;  11)  animal  CNS  imaging  and  spectroscopy  with 
experimental,  small  bore  devices  (2.0  and  4.7  Tesla).   Finally,  we  have 
initiated  A)  investigation  of  NMR  spectroscopy  (proton)  in  patients  with 
brain  tumors;  B)  cine-MRI  applications  Lo  the  assessment  of  patients 
with  syringomyelia  and  other  cord  cavities;  C)  MR  angiography  (flow 
based)  in  cord  AVMs ;  and  D)  comparison  of  diffusion  (Intra-Voxel 
Incoherent  Motion  Analysis)  with  PET  in  brain  tumors. 

CT:  Ongoing  research  studies  of  tumoral,  degenerative,  demyelinating  and 
atrophic  processes  of  the  brain,  plus  hydrocephalus,  brain  edema,  post- 
radiation  necrosis  and  epilepsy. 

*Formerly  in  OCD;  transferred  to  MNB  on  6/8/90. 

57-MNB/DIR 


I 

PHS  6040  (Rev    1/84) 


SCO  114-tll 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02315-13  MNB* 


PERIOD  COVERED 


October  1,  1Q8Q  through  September  30,  1 QQO 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  tit  on  one  line  between  the  borders.) 

Positron  Emission  Tomography 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Giovanni  Di  Chiro,  M.D.,  Chief,  NIS,  MNB,  CNP,  DIR,  NINDS.   OTHERS: 


R.  A.  Brooks,  Ph.D. 

R.  S.  Miletich,  M.D., 

M.  J.  Fulham,  M.D. 

C.  F.  Ferrarese.  M.D. 

J.  W.  Melisi,  M.D. 

M.  0.  Duhaney,  M.D. 

A.  Brunetti ,  M.D. 


Staff  Physicist 
Ph.D.  Sr.  Staff  fellow 

Visiting  Associate 
Visiting  Associate 
Special  Volunteer 
Irta  Fellow 
Visiting  Fellow 


NIS,  NINDS 

NIS,  NINDS 

NIS,  NINDS 

NIS,  NINDS 

NIS,  NINDS 

NIS,  NINDS 

NTS,  NTNuS** 


COOPERATING  UNITS  (it  any) 

NM,  CC;  MN,  NINDS,  SN,  NINDS;  DIR,  NINDS;  BEIB,  DRS;  DRD,  CC;  LCM,  NIMH; 
or.n,  NTNDS,  r.NR,  ntnds 


LAB/BRANCH 

Medical  Neurology  Branch,  CNP,  DIR 


SECTION 


Neuroimaging 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  MD   20892 


TOTAL  MAN- YEARS: 

2.0 


PROFESSIONAL: 

2.0 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

0   (a)   Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□   (b)  Human  tissues  □   (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 


Positron  emission  tomography  (PET)  allows  us  to  obtain  anatomical  data 
(e.g.,  axial  transverse,  coronal  or  sagittal  images  of  the  brain)  as 
well  as  dynamic  functional  data  (such  as  regional  cerebral  glucose 
consumption  rate,  using  1 ^F-2-deoxyglucose  (FDG).   Besides  FDG,  other 
radiopharmaceuticals  (tagged  with  1°F,  15q,  11c,  13n)  can  be  used-with 
PET  to  study  the  blood-brain  barrier  (BBB),  oxygen  metabolism,  protein 
synthesis,  and  neuroreceptors.  The  unique  property  of  PET  is  that  it 
provides  physiologic  and  pathophysiologic  information  not  available  with 
any  other  imaging  procedure. 


**Continued: 

T.  Francavilla 

G.  Comi 

B.  DeSouza 
I.  J.  Kopin 

E.  H.  Oldfield 

C.  V.  Kufta,  M.D. 
M.  Hallett,  M.D. 

R.  J.  Polinsky,  M.D. 

L.  Sokoloff,  M.D. 


Staff  Fellow 

Visiting  Fellow 

Staff  Fellow 

Director 

Chief 

Staff  Physician 

Clinical  Director 

Chief,  CNS 

Chief 


NIS,  NINDS 
NIS,  NINDS 
NIS,  NINDS 
DIR,  NINDS 
SN,  NINDS 
SN,  NINDS 
DIR,  NINDS 
CN,  NINDS 
LCM,  NIMH 


*Formerly  in  OCD;  transferred  to  MNB  on  6/8/90 


58-MNB/DIR 


PHS  6040  (Rev.  1/84) 


FPO  fit  4-018 


> 

00 


C 

30 

O 

m 

O 


o 
o 

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03 
30 
> 

z 
n 

x 


z 

m 
03 


ANNUAL  REPORT 

October  1,  1989  through  September  30,  1990 

Neuroepidemioloqy  Branch 

Clinical  Neurosciences  Program,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 

Table  of  Contents 
RESEARCH  SUMMARY  1-9 

PROJECT  REPORTS 

Clinical,  Genetic,  Pathophysiologic  Study  of  10 

Hereditary  Movement  Disorders 
Z01  NS  01924-20  NEB 

Clinical,  Genetic,  Pathophysiologic  Study  of  11 

Hereditary  Nervous  System  Tumors 
Z01  NS  01927-20  NEB 

denetic  Epidemiology  Studies  in  MS  and  Other  12 

Multifactorial  Neurologic  Disorders 
Z01  NS  02167-16  NEB 

Epidemiology  of  Dementia  13 

Z01  NS  02240-14  NEB 

Pediatric  Neuroepidemiology  14 

Z01  NS  02243-14  NEB 

Mortality  from  Neurologic  Disorders:   National  15 

and  International  Comparisons 
Z01  NS  02297-14  NEB 

Reviews  of  Epidemiologic  Aspects  of  Neurologic  16 

Disease 
Z01  NS  02299-14  NEB 

Clinical  Course  and  Medical  Care  for  Neurologic  17 

Disorders 
Z01  NS  02300-14  NEB 

Collaborative  Studies  of  Less  Common  or  Less  18 

Debilitating  Neurologic  Disorders 
Z01  NS  02301-14  NEB 

Educational  Resources  in  Neurological  Epidemiology        19 
Z01  NS  02307-14  NEB 

i  NFB/DIR 


Table  of  Contents  (cont'd) 

Racial  and  Geographic  Differences  in  Occurrence  20 

of  Neurologic  Disease 
Z01  NS  02370-12  NEB 

Development  of  Data  Resources  for  Neuroepidemiology       21 
Z01  NS  02423-11  NEB 

National  History  of  ALS-PD  in  Guam  22 

Z01  NS  02570-08  NEB 

Epilepsy  Neuroepidemiology  23 

Z01  NS  02715-05  NEB 

Phenobarbital  Clinical  Trial  in  Children  with  24 

Febrile  Seizures 
Z01  NS  02746-04  NEB 

Dental  Markers  of  Maldevelopment  25 

Z01  NS  02747-04  NEB 

Dermatoglyphic  Markers  of  Maldevelopment  2  6 

Z01  NS  02748-04  NEB 

California  Cerebral  Palsy  Registry  27 

Z01  NS  02819-01  NEB 


ii  NEB/DIR 


Annual  Report 
October  1,  1989  through  September  30,  1990 

Neuroepidemiology  Branch 

Division  of  Intramural  Research 

Clinical  Neurosciences  Program 

National  Institute  of  Neurological  Disorders  and  Stroke 


Jonas  H.  Ellenberg,  Ph.D.,  Acting  Chief 
Gustavo  C.  Roman,  M.D.  Chief  Designate 


The  Neuroepidemiology  Branch  (NEB)  is  responsible  for  the  development  and 
implementation  of  epidemiologic  and  genetic  programs  to  investigate  the  cause, 
prevention,  and  treatment  of  neurologic  disorders  in  human  populations.  Emphasis 
has  been  placed  on  major  neurologic  diseases  in  which  the  diagnoses  can  be 
clinically  verified  to  the  satisfaction  of  skilled  neurologists. 

On  July  14,  1987,  Bruce  S.  Schoenberg,  M.D.,  Dr.P.H.,  Chief  of  the 
Neuroepidemiology  Branch  since  its  inception,  passed  away  at  a  very  early  age. 
Dr.  Gustavo  C.  Roman,  Professor  of  Neurology  and  Acting  Chairman  of  the 
Departs  it  of  Medical  and  Surgical  Neurology  at  Texas  Tech  University  School  of 
Medicine  in  Lubbock,  Texas,  was  appointed  as  Chief  Designate  of  the  NEB  as  of 
January,  1990.  Dr.  Roman,  a  long  time  collaborator  of  Dr.  Schoenberg,  will  be 
joining  the  NIH  on  a  full-time  basis  as  of  Sept.  1,  1990. 

Neuroepidemiologic  research  studies  require  collaboration  of  many  individuals. 
However,  since  there  is  a  severe  shortage  of  available  manpower  in 
neuroepidemiology,  the  Branch  has  developed  an  information  program  for  current 
and  future  collaborative  investigators.  A  series  of  six  videotapes  on  aspects 
of  neuroepidemiologic  methods  produced  by  the  Branch  is  available.  A  monograph 
entitled  Neurological  Epidemiology:  Principles  and  Clinical  Applications  was 
published  in  1978.  A  new  monograph  which  will  cover  current  methodology  and 
disease  specific  overviews  of  etiology,  incidence,  prevalence  and  intervention 
studies  is  in  press. 

Another  important  problem  for  the  neuroepidemiologist  is  the  enormous  cost  of 
maintaining  neurologic  surveillance  on  a  large  number  of  patients.  Therefore, 
we  have  attempted  to  utilize  existing  registries  of  neurologic  diseases.  We 
collaborate,  for  example,  with  the  Mayo  Clinic  in  Rochester  and  utilize  their 
records-linkage  system  to  study  neurologic  diseases  in  the  population  of 
Rochester,  MN;  the  California  Birth  Defects  Monitoring  Program  registry;  the 
Israeli  National  Disease  Registry;  and  the  Israeli  Cancer  Registry. 

Epilepsy 

Epilepsy  is  a  major  cause  of  morbidity  and  mortality  on  a  world-wide  basis.  A 
considerable  research  effort  has  been  devoted  by  personnel  of  the  Branch  to 
studying  this  disease.  A  protocol  is  in  place  for  a  clinical  study  of  the 
Lennox-Gastaut  syndrome (LGS) ,  a  severe  childhood  epileptic  encephalopathy  with 
significant  morbidity  and  mortality,  characterized  by  uncontrolled  seizures, 

1  -  NFB/DIR 


mental  retardation,  and  possible  mental  deterioration.  Evaluations  include  sleep 
studies,  auditory  evoked  potentials,  EEGs  and  videotape-EEG  monitoring, 
psychologic  testing,  and  a  battery  of  immunologic  tests.  Neuroimaging  studies 
obtained  under  an  ongoing  protocol  will  be  evaluated  with  the  clinical  data. 
The  project  is  being  undertaken  in  collaboration  with  the  Clinical  Epilepsy 
Section,  DIR,  NINDS. 

In  collaboration  with  the  University  of  Skopje  in  Yugoslavia,  we  are  examining 
the  utility  of  the  electroencephalogram  as  a  predictor  of  recurrence  of  febrile 
seizures  in  a  defined  population  in  the  region  of  Macedonia;  676  children  have 
been  examined  clinically  and  by  EEC  An  initial  manuscript  has  been  submitted 
concerning  clinical  and  EEG  findings  at  study  entry. 

The  international  literature  indicates  that  many  persons  who  experience  a  first 
convulsion  do  not  have  a  recurrence  in  the  subsequent  two  to  five  years,  while 
many  others  develop  chronic  epilepsy.  It  is  unknown  whether  treatment  early 
after  an  initial  seizure  can  reduce  the  likelihood  of  developing  chronic 
epilepsy.  NEB  is  collaborating  with  the  Biometry  and  Field  Studies  Branch  in 
examining  the  feasibility  of  randomized  controlled  trials  of  treatment  with 
anticonvulsant  medication  following  a  first  convulsion  in  subjects  ages  seven 
to  60  years  who  present  for  care  to  the  Beijing  Tiantan  Hospital,  a  municipal 
hospital  affiliated  with  the  Beijing  Neurosurgical  Institute  and  to  hospitals 
in  the  Western  Galilee  Region  of  Israel.  The  initial  efforts  are  focusing  on 
testing  the  feasibility  of  recruitment  and  standardization  of  procedures  and 
piloting  of  protocols.  The  number  of  patients  meeting  study  criteria,  the  ease 
of  identification  of  eligibility,  the  proportion  of  patients  to  be  excluded  for 
each  indication,  the  establishment  and  testing  of  rules  for  initiation  of 
treatment  in  the  placebo  arm  (e.g.,  would  patients  continue  to  accept  placebo 
after  a  first  recurrence),  the  willingness  of  patients  to  participate  and  return 
for  scheduled  visits,  the  time  from  seizure  onset  to  study  entry,  will  all  be 
evaluated.  Pharmacy  procedures  in  obtaining  placebo,  and  establishing  and 
maintaining  the  blind  will  also  be  tested.  Procedures  for  the  informed  consent 
will  be  developed  by  staff. 

Alzheimer's  disease 

Another  neurologic  disease  of  increasing  importance  in  most  of  the  developed 
countries,  including  the  U.S.,  is  Alzheimer's  disease.  This  is  another  area  of 
major  research  interest  to  Branch  personnel.  Uniform  diagnostic  criteria  are 
applied  to  all  studies  of  Alzheimer's  disease  being  conducted  in  the  Branch. 
The  clinical  diagnosis  is  based  on  the  criteria  proposed  by  the  NINDS- 
Alzheimer's  Disease  and  Related  Disorders  Association,  Inc.  work  group.  Where 
possible,  pathological  verification  of  the  diagnosis  is  obtained. 

Alzheimer's  disease/senile  dementia,  despite  its  high  apparent  clinical  frequency 
among  the  elderly,  has  not  been  well  studied  in  a  U.S.  population.  Descriptive 
studies  are  yielding  etiologic  hypotheses  which  can  be  further  investigated  using 
the  case-control  approach,  and  will  provide  the  opportunity  to  investigate 
dementia  occurring  with  other  neurologic  disorders,  such  as  Parkinson's  disease. 
These  studies  should  also  provide  evidence  whether  Alzheimer's  disease  occurring 
in  the  very  elderly  and  that  in  the  presenium  represent  the  same  disease  process. 
Utilizing  a  common  protocol  will  permit  international  comparisons. 

In  addition,  three  case-control  studies  to  determine  risk  factors  for  Alzheimer's 

2 ,-  NEB/DIR 


disease  have  been  completed  or  are  in  progress.  The  first  uses  cases  and 
controls  selected  from  the  Rochester,  MN  population.  Past  medical  records  have 
been  utilized  to  obtain  information  concerning  possible  associations  between 
Alzheimer's  disease  and  other  medical  conditions  or  surgical  procedures.  This 
study  has  the  advantage  that  recall  bias  cannot  affect  the  results  of  the  study 
since  data  are  being  abstracted  from  medical  records.  Information  on  the 
occurrence  of  head  trauma  with  loss  of  consciousness  (LOC)  was  abstracted  from 
the  medical  records  of  274  cases  and  controls.  A  statistically  significant 
difference  in  LOC  between  the  two  groups  was  not  detected. 

A  second  case-control  study  of  factors  associated  with  Alzheimer's  disease  in 
collaboration  with  the  Burke  Rehabilitation  Center,  White  Plains,  New  York,  has 
been  completed  and  analysis  is  in  progress.  A  third  case-control  study  of 
possible  environmental  etiology  is  underway  at  Mt.  Sinai  (New  York)  Department 
of  Community  Medicine  and  Neurology.  The  study,  using  interviews  for  deriving 
past  environmental  exposures,  has  enrolled  approximately  65  patients  and  an 
equal  number  of  hospital  based  controls.  Data  collection  was  completed  by  the 
end  of  FY  1989. 

Vascular  Dementia 

Dementia  due  to  cerebrovascular  disease  (also  known  as  multi-inf arct  dementia) 
is  the  second-most  common  cause  of  dementia  in  the  elderly.  However,  diagnostic 
criteria  for  this  condition  are  deficient,  and  this  has  hampered  the  development 
of  comparable  international  data.  New  concepts  in  the  pathogenesis  of  vascular 
dementia  include  the  possible  role  of  white  matter  hypoperfusion  as  playing  a 
major  role  in  this  condition.  The  term  Binswanger's  disease  has  been  suggested 
as  a  better  option  for  this  form  of  dementia.  Dr.  Roman  has  championed  this  idea 
in  several  reviews  (JAMA  248:1782-1788,  1987)  and  international  meetings 
("Cerebral  Ischemia  and  Dementia"  International  Workshop,  June  25-28,  1990  Prien 
Am  Chiemsee/Bavaria,  Germany,  and  "Study  Group  on  Neuroepidemiology"  AIREN/NINDS- 
NIH/WHO  Geneva,  Switzerland,  July  9-11,  1990). 

A  number  of  studies  on  the  neuroepidemiology  of  vascular  dementia  are  in  the 
planning  stage  and  a  workshop  for  the  definition  of  diagnostic  criteria  of 
vascular  dementia  has  been  scheduled. 

Analysis  of  death  certificate  data 

Although  death  certificate  data  are  limited  by  possible  misdiagnosis,  incomplete 
case  ascertainment,  errors  in  coding,  etc.,  detailed  mortality  information  on 
some  neurologic  diseases  for  the  entire  U.S.  is  not  available.  Analysis  of 
mortality  data  can  be  particularly  useful  for  some  neurologic  diseases  because, 
indirectly,  these  may  indirectly  contribute  to  death.  Since  there  are  no  uniform 
criteria  for  what  constitutes  the  underlying  cause  of  death  in  patients,  it  is 
important  to  examine  all  deaths  in  which  a  disease  is  listed  as  an  underlying, 
immediate,  associated,  or  a  contributory  cause  of  death  to  get  more  complete 
information  about  the  relationship  between  the  disease  and  death.  Association 
of  diseases  occurring  at  the  time  of  death  was  studied  for  all  deaths  occurring 
in  the  U.S.  for  many  neurologic  diseases.  Diseases  occurring  together  may 
provide  important  information  in  the  search  for  their  etiology.  Such  detailed 
analysis  of  mortality  data  have  been  done  for  Huntington's  disease,  Alzheimer's 
disease  and  related  diagnoses,  Friedreich's  disease,  motor  neuron  disease, 
Parkinson's  disease,  etc.,  for  1971-78.  The  overall  patterns  which  have  emerged 

3  -  NEB/DIR 


have  been  useful  in  evaluating  trends  over  time  and  in  formulating  etiologic 
hypotheses . 

All  death  certificates  for  the  entire  U.S.  for  the  years  1971,  and  1973  through 
1978  were  searched  for  the  diagnosis  of  Huntington's  disease.  Age-,  race-,  and 
sex-specific  mortality  rates  for  deaths  due  to  and  deaths  with  Huntington's 
disease  were  calculated.  Time  trends  in  the  age-adjusted  mortality  rates  between 
1971  and  1978  were  also  calculated.  To  determine  which  conditions  may  be 
associated  with  reduced  survival  in  patients  with  Huntington's  disease,  all  death 
certificates  in  the  United  States  for  1978  on  which  Huntington's  disease  was 
mentioned  have  been  studied.  Each  case  was  compared  with  two  control  deaths 
which  were  matched  for  age,  race,  sex,  county  and  year  of  death.  In  the  case- 
control  study,  pneumonia,  choking,  nutritional  deficiencies,  and  chronic  skin 
ulcers  were  increased  in  cases  relative  to  controls.  The  overall  mortality  rate 
was  2.27  per  million  population  per  year. 

All  cases  of  Friedreich's  disease  (FD)  diagnosed  between  1945  through  1984  among 
residents  of  a  defined  area  of  northwestern  Italy  were  ascertained  (N=58).  These 
patients  were  followed  to  death  or  to  December  31,  1984  (whichever  came  first) 
to  determine  the  patterns  of  survival.  The  10-,  20-,  and  30-year  survival  rates 
were  respectively  96%,  80%,  and  61%,  suggesting  a  better  prognosis  than 
previously  reported.  Survival  of  FD  patients  was  poorer  than  expected  from  the 
general  population.  Survival  for  males  was  poorer  than  females  even  after 
adjustment  for  expected  survival.  Age  of  onset  was  not  a  significant  prognostic 
factor.  Survival  for  patients  diagnosed  in  1960  or  later  was  better  than  for 
those  diagnosed  before  1960;  however,  the  difference  was  not  statistically 
significant. 

A  study  of  reported  international  average  annual  age-adjusted  mortality  rates 
for  primary  tumors  of  the  nervous  system  was  undertaken  for  the  periods  1951-58 
and  1967-73.  International  comparisons  of  average  annual  age-adjusted  mortality 
rates  for  primary  tumors  of  the  nervous  system  showed  marked  geographical 
variation  for  both  study  periods.  For  the  majority  of  countries,  the  mortality 
rates  increased  by  at  least  40%  in  the  intervening  15-year  period,  while  in  20% 
of  the  countries  the  rates  increased  by  over  100%.  The  percentage  increases 
varied  from  12.2  to  345.5.  The  improvement  in  the  diagnosis  of  these  tumors, 
particularly  among  elderly  individuals  (who  have  the  highest  age-specific 
incidence  rates  for  these  neoplasms),  presumably  accounts  for  most  of  this 
change. 

Potential  risk  factors  for  various  types  of  stroke  were  studied  using  a  case- 
control  study  design.  All  the  U.S.  death  certificates  in  1978  which  had  the 
registered  underlying  cause  of  death  as  subarachnoid  hemorrhage  (SAH),  cerebral 
hemorrhage  (CH) ,  or  cerebral  infarction  (CI)  were  identified.  In  a  case-control 
Btudy  for  potential  risk  factors  for  various  types  of  stroke,  the  data  provided 
new  information,  such  as  the  occurrence  of  peripheral  vascular  disease  in 
association  with  SAH,  the  risk  of  CH  in  epileptic  and  cirrhotic  patients,  and 
the  association  of  benign  neoplasms  of  the  nervous  system,  motor  neuron  disease, 
and  'paralysis  agitans'  with  CI. 

Prevalence  surveys 

There  is  continuing  controversy  regarding  racial  differences  in  the  occurrence 
of  major  neurologic  diseases.   Thus,  a  study  was  conducted  in  Copiah  County, 

4  -  NER/DIR 


Mississippi  to  specifically  address  this  question.  A  strategy  was  developed 
which  eliminated  the  requirement  that  persons  must  have  entered  the  health  care 
system  for  detection  of  disease.  The  basis  of  the  investigation  was  a 
door-to-door  survey  which  utilized  a  detailed  questionnaire  inquiring  not  only 
about  diagnoses,  but  also  about  signs  and  symptoms  suggestive  of  neurologic 
dysfunction.  Over  99%  of  the  households  agreed  to  the  interview.  Those 
household  members  suspected  of  having  one  of  the  disorders  of  interest  were  then 
asked  to  have  a  neurologic  examination  conducted  by  a  senior,  board-certified 
neurologist.  This  two-stage  procedure  has  proven  most  effective,  and  the 
strategy  for  this  type  of  survey  is  being  utilized  in  other  areas  of  the  world. 

Prevalence  and  incidence  data  from  different  parts  of  the  world  are  often  not 
directly  comparable  because  of  the  use  of  different  definitions  of  disease,  and 
results  affected  by  the  availability  and  accessibility  to  experts  in  neurology. 
A  uniform  protocol  developed  by  the  Branch  in  collaboration  with  the  World  Health 
Organization  has  helped  diminish  these  differences.  The  protocol  which  is  being 
used  to  estimate  the  prevalence  of  major  neurologic  disorders  in  many  parts  of 
the  world,  will  enable  international  comparisons. 

The  prevalence  of  a  wide  spectrum  of  neurologic  disorders  using  the  World  Health 
Organization  protocol  has  been  reported,  based  on  the  Copiah  County  door-to-door 
model.  Research  protocols  for  these  neurologic  studies  have  been  completed  in 
India  and  Spain.  The  prevalence  study  of  the  Parsi  Community  of  Bombay,  India 
provided  point  prevalence  estimates  of  328.3/100,000  for  Parkinson's  disease  and 
1591.7/100,000  for  essential  tremor.  In  the  pilot  prevalence  study  in  an  urban 
area  of  Madrid,  Spain,  migraine  and  epilepsy  were  the  most  frequent  disorders 
with  point  prevalence  ratios  of  163.3/1000  and  9.3/1000,  respectively. 

A  large  prevalence  survey  for  Amyotrophic  Lateral  Sclerosis  -  Parkinsonism 
Dementia  Complex  of  Guam  (ALS-PDC)  was  undertaken  in  three  rural  villages  in 
southern  Guam.  The  survey,  utilizing  a  door-to-door  approach,  screened  all 
individuals  40  years  and  over  for  ALS-PDC.  Only  rare  cases  of  ALS  were 
encountered,  although  PDC  was  found  to  be  still  common  in  the  population.  Data 
editing  is  in  progress  and  comprehensive  analysis  and  publication  of  results 
will  follow  completion  of  the  final  data  file. 

Neurogenetics 

The  Clinical  Neurogenetics  Unit  of  the  Neuroepidemiology  Branch  seeks  the  cause, 
cure  and  prevention  of  specific  neurologenetic  diseases.  For  those  diseases  of 
apparent  single  gene  origin  etiologic  efforts  focus  on  mapping  and  then  isolating 
the  mutant  gene.  For  those  disorders  of  more  complex  origins,  a  balanced  search 
is  made  for  both  genetic  and  environmental  factors.  Specific  activities  and 
responsibilities  of  the  unit  include  clinical  care,  clinical  and  laboratory 
research,  teaching,  and  liaison  with  several  patient  organizations. 

Over  1000  families,  some  consisting  of  over  500  individuals,  have  been  studied 
in  the  course  of  research  on  single  neurogenetic  disorders  such  as  the  dystonias, 
the  neurofibromatoses,  the  hereditary  leukodystrophies  and  the  hereditary 
ataxias . 

More  than  200  twin  pairs  and  their  families  have  been  evaluated  in  the  course 
of  study  of  complex  neurological  disorders  such  as  Alzheimer's  disease, 
Parkinson's  disease,  multiple  sclerosis,  and  post  polio   syndrome. 

5  -  NEB/DIR 


Clinical  care  is  delivered  through  the  NIH  Interinstitutes  Genetics  Clinic. 
Presently,  we  and  a  team  of  8  specialists  participate  in  the  screening, 
diagnosis,  evaluation  and  management  of  persons  at  high  risk  for  selected 
neurologic  disorders,  particularly  neurofibromatosis  1  (NF1)  which  formerly  was 
known  as  von  Recklinghausen's  disease,  and  NF2  which  was  formerly  known  as 
bilateral  acoustic  neurofibromatosis.  Earlier,  we  pointed  out  that  the 
neurofibromatoses  together  constitute  the  most  common  of  all  neurogenetic 
conditions.  NF2  has  been  shown  to  be  due  to  a  mutation  of  a  single  gene  in  the 
region  of  chromosome  22  in  one  family.  Subsequently,  the  gene  for  NF1  was 
localized  to  chromosome  17  suggesting  there  are  two  distinct  forms  of  NF. 
Recently  the  NF1  gene  and  its  product  have  been  identified. 

Since  NF2  is  often  associated  with  multiple  brain  and  spinal  cord  tumors  in 
addition  to  acoustic  neuromas,  with  over  10  meningiomas,  neurofibromas  and 
astrocytomas  in  some  cases,  management  of  an  individual  case  requires  careful 
coordination.  However,  we  now  consider  NF2  a  preventable  cause  of  deafness, 
facial  disfigurement  and  even  death,  with  optimum  management. 

An  approach  has  evolved  involving  two  other  neurosurgical  centers,  and  of  over 
50  cases  followed  during  the  past  three  years  only  one  individual  has  died  (a 
man  in  his  30 's  whose  first  tumor  was  noted  at  age  18)  and  only  two  other  have 
become  completely  deaf  (both  females  with  multiple  brain  tumors  and  deteriorating 
neurological  status).  We  are  gratified  with  this  record  for  a  condition  that 
generally  has  been  associated  with  severe  morbidity  and  premature  death. 

Clinical  research  has  consisted  of:  description  of  the  nosology  and  natural 
history  and  inheritance  pattern  of  a  number  of  neurogenetic  diseases  based  on 
family  study;  genetic  epidemiologic  studies  seeking  either  factors  influencing 
the  severity  of  the  disorder  often  using  sibs  or  co-twins  as  one  of  the  control 
groups;  formal  descriptive  neuroepidemiologic  studies  of  selected,  defined 
populations;  and  evaluation  of  treatment  programs  using  our  extensive  patient 
series. 

Laboratory  studies  have  been  carried  out  with  collaborators  in  a  number  of  fields 
including:  brain  imaging  with  CT  scan,  MRI  or  PET;  cell  biology  looking  for 
factors  influencing  growth  of  NF2  tumor  cells;  molecular  genetics  for  gene 
mapping  or  gene  isolation  and  characterization;  and  neuropathology  for  detailed 
anatomic  and  neurochemical  analysis  of  brain  tissue. 

Teaching  responsibilities  begin  with  students  and  fellows  in  the  NIH 
Interinstitutes  Genetics  Program  and  the  clinical  staff  at  NIH  and  other  area 
hospitals,  and  extends  to  professional  and  lay  groups  interested  in  the  combined 
genetic-epidemiologic  approach  we  employ  to  understand  specific  neurologic 
disease. 

An  especially  active  and  successful  area  of  responsibility  for  the  Neurogenetics 
Unit  involves  liaison  with  a  number  of  voluntary  lay  organizations.  Over  the 
years  we  have  assisted  in  the  formation  of  eight  local  or  national  foundations, 
including  the  National  Neurofibromatosis  Foundation  and  the  Washington,  D.C. 
chapter;  the  Dystonia  Medical  Research  Foundation  and  the  Washington,  D.C. 
chapter;  the  National  Jewish  Genetic  Disease  Foundation,  the  Washington,  D.C. 
chapter  of  the  National  Ataxia  Foundation,  Neurofibromatosis,  Inc.  and  its  local 
chapter;  and  most  recently  iNFormer  2  which  is  a  network  for  NF2  families.   We 

6  -  NEB/DIR 


have  tried  to  help  these  organizations  with  advice.  They  in  turn  have  enabled 
us  to  perform  three  recent  clinical  studies  by  enlisting  the  cooperation  of  their 
membership. 

Sinole  gene  disorders;  Recent  work  of  the  Neurogenetics  Unit  include  the 
following: 

NF2  -  The  natural  history  of  NF2  appears  different  in  males  with  the  gene 
than  it  does  in  females.  Affected  men  tend  to  have  fewer  tumors,  require  less 
surgical  intervention  and,  when  they  pass  on  the  gene,  have  it  expressed  later 
in  their  children  than  their  affected  sisters  do.  A  pathognomonic  sign  has  been 
described  by  our  team.  It  is  the  presence  at  a  young  age  of  one  or  more  discreet 
opacities  in  the  posterior  portion  of  the  lens  of  the  eye  which  can  appear  before 
any  other  signs  or  symptoms.  With  hand-held  ophthalmoscope  it  is  seen  as  a  black 
fleck  and  is  known  locally  as  the  'Kaiser-Kupfer  Cinder'.  A  review  has  been 
prepared  on  the  practical  management  of  NF2,  which  makes  the  following  points: 
delineates  5  groups  at  high  risk  for  NF2;  emphasizes  that  no  one  should  be 
operated  on  for  a  presumed  solitary,  sporadic  acoustic  neuroma  unless  this 
syndrome  with  bilateral  acoustic  neuromas  is  first  ruled  out;  and  that  watchful 
waiting  rather  than  aggressive  surgical  intervention  is  the  proper  course  in  the 
majority  of  cases.  A  consensus  conference  dealing  with  these  issues  is  planned 
for  June  of  1991. 

NF1  -  A  study  has  been  completed  of  an  appraisal  of  attitudes  of  over 
300  affected  individuals  and  their  relatives  toward  NF1  itself  and  toward 
predictive  testing  for  NF1. 

Hereditary  Leukodystrophy  -  We  have  described  an  adult  form  which  has  been 
often  misdiagnosed  as  chronic  progressive  multiple  sclerosis.  Four  families  in 
the  U.S.  have  now  been  ascertained.  A  high  concentration  of  a  previously 
undescribed  familial  leukodystrophy  of  early  onset  has  been  identified  by 
colleagues  in  Saudi  Arabia. 

Complex,  or  multifactorial,  neurologic  disorders 

Alzheimer's  disease  -  demonstration  in  the  first  and  only  nationwide  twin 
study  that  most  monozygotic  twins  are  discordant  and  remain  so  for  over  12  years, 
indicating  that  environmental  factors  are  important  in  many  cases. 

Multiple  sclerosis  -  Noted  the  similarity  between  MS  and  paralytic  polio 
concordance  rates  in  monozygotic  and  dizygotic  twins,  indicating  that 
environmental  and  genetic  factors  both  must  play  a  role  in  each  condition. 

Parkinson's  disease  -  Demonstrated  in  the  first  nationwide  twin  study  in 
the  U.S.  low  concordance  rates,  a  finding  since  confirmed  in  English  and  Finnish 
studies.  In  the  U.S.  study  there  were  far  more  full  sibs  and  parents  affected 
than  co-twins,  suggesting  a  familial  tendency  for  Parkinson's  and  a  possible 
'protective  effect'  on  the  co-twin.  A  characteristic  life-long  personality 
difference  suggested  an  early  environmental  event  'set'  a  twin's  risk.  These 
observations  plus  mouse  data  indicating  genetic  influence  on  dopamine  neuron 
number  led  us  to  offer  the  'Initial  Neuron  Number',  or  INN,  theory  as  one  of  the 
processes  contributing  to  Parkinson's  disease.  If  true  it  would  mean  that  there 
is  a  humoral  factor  capable  of  reducing  one's  risk  or  of  treating  one's  symptoms. 

7  -  NEB/DIR 


Pediatric  neuroepidemioloqy 

In  the  area  of  pediatric  neuroepidemiology,  the  efficacy  and  side  effects  of 
phenobarbital  in  young  children  with  febrile  seizures,  randomized  to 
phenobarbital  or  placebo  is  being  studied.  A  number  of  endpoints  have  been  or 
will  be  assessed:  IQ,  seizure  recurrence,  sleep  pattern,  temperament  scale,  and 
a  number  of  behavioral  and  cognitive  measures.  The  design  of  this  study  permits 
comparison  of  measures  of  tested  intelligence  and  of  behavior  in  children  with 
febrile  seizures  who  were  treated  with  phenobarbital,  in  those  randomized  to 
placebo,  and  in  a  group  of  seizure-free  control  children.  It  was  presumed  that 
the  group  randomized  to  placebo  would  experience  more  recurrences  of  seizures, 
while  the  group  assigned  to  phenobarbital  might  be  subject  to  unfavorable  side 
effects  of  medication.  This  comparison  allows  assessment  of  benefit  and  risk 
of  treatment  in  a  common  childhood  neurologic  problem.  Children  in  this  study 
were  also  tested  six  months  after  completion  of  receipt  of  phenobarbital,  so  that 
the  reversibility  of  any  observed  effects  could  be  judged.  All  subjects  have 
been  recruited,  follow-up  testing  has  been  completed.  The  initial  manuscript 
describing  the  primary  results  has  been  published  and  two  others  submitted.  Work 
has  begun  on  other  manuscripts  from  this  data. 

Studies  of  dental  and  dermatoglyphic  markers  of  maldevelopment  have  been 
initiated  in  cooperation  with  the  National  Institute  of  Dental  Research  and  the 
Institute  of  Aging.  These  studies  of  enamel  defects  and  characteristics  of 
dermatoglyphics  and  palmar  creases  in  children  with  cerebral  palsy  or  mental 
retardation  will  attempt  to  recognize  the  occurrence  and  timing  of  events  adverse 
to  neurologic  development. 

NEB  staff  were  involved  in  the  analysis  of  a  randomized  controlled  clinical  trial 
undertaken  in  the  NICHD  to  evaluate  the  safety  and  efficacy  of  phototherapy  in 
the  prevention/reduction  of  hyperbilirubinemia  in  the  neonate.  An  earlier  study 
indicated  the  efficacy  of  the  treatment  in  preventing  hyperbilirubinemia.  This 
phase  investigated  the  frequency  of  adverse  outcomes,  most  of  them  neurological, 
in  this  sample  at  follow-up  examination  at  six  years  of  age.  Infants  randomized 
to  treatment  with  phototherapy  experienced  lower  ranges  of  bilirubin  as  neonates 
but  did  not  experience  increased  frequency  of  adverse  neurologic  outcomes.  One 
manuscript  has  been  published,  one  is  in  press,  and  another  is  in  preparation. 

NEB  staff  in  collaboration  with  the  California  Birth  Defects  Monitoring  Program, 
and  the  Health  Officers  Association  of  California,  is  establishing  a 
population-based  registry  of  children  with  cerebral  palsy  (CP)  in  five  counties 
of  the  San  Francisco  Bay  Area  born  between  1983-1985.  Children  are  ascertained 
through  client  records  at  California  Children  Services  and  the  Department  of 
Development  Services  and  their  Regional  Centers  for  the  Developmentally  Disabled. 
CP  cases  meeting  the  registry  criteria  will  receive  a  clinical  examination  to 
establish  a  research  diagnosis,  and  data  will  be  obtained  on  associated  features. 
Obstetric  and  pediatric  records  will  be  reviewed  by  the  staff.  Information  will 
be  used  to  establish  trends  in  incidence,  to  examine  for  time-space  clustering, 
and  to  formulate  hypotheses  for  testing  suspected  etiologic  factors,  medical  and 
environmental,  in  case-  control  studies.  Ecological  studies  will  also  be 
conducted  comparing  rates  of  CP  among  births  in  Santa  Clara  County  census  tracts 
which  contain  EPA  superfund  waste  disposal  sites,  to  rates  in  the  county  as  a 
whole  and  to  rates  in  the  remainder  of  the  Bay  area;  the  purpose  of  this  aspect 
of  the  project  is  to  examine  the  possibility  that  toxic  exposures  may  contribute 

8  -  NEB/DIR 


to  some  forms  of  neurologic  morbidity  as  well  as  to  other  birth  defects. 
Examinations  for  case  classification  have  been  completed  and  birth  record 
abstraction  is  underway. 

Rare  disorders  of  the  nervous  system 

Emphasis  is  placed  on  research  on  the  major  diseases  of  the  nervous  system,  and 
the  Branch  also  investigates  other  diseases  which  may  be  less  frequent  or  less 
debilitating,  but  important  in  terms  of  pathogenesis  or  clues  to  disease 
etiology. 

In  collaboration  with  other  governmental  agencies  (Centers  for  Disease  Control), 
other  governments  (Colombia,  Republic  of  Seychelles) ,  international  organizations 
(World  Health  Organization),  and  universities  (Mayo  Medical  School),  methods  have 
been  developed  to  investigate  lees  common  neurologic  disorders.  Several  such 
studies  have  been  completed  as:  a  prevalence  and  case-control  study  of 
progressive  supranuclear  palsy  (PSP);  and  a  door-to-door  survey  of  21  provinces 
of  the  People's  Republic  of  China  to  determine  the  prevalence  of  migraine. 

The  prevalence  and  natural  history  survey  of  PSP  in  two  counties  in  New  Jersey 
provided  a  prevalence  ratio  of  1.39/100,000.  The  median  intervals  to  onset  of 
requiring  assistance  was  3.1  years;  of  visual  symptoms,  3.9  years;  dysarthria, 
3.4  years;  dysphasia,  4.4  years;  requiring  wheel  chair,  8.2  years;  and  death 
9.7  years.  In  the  case-control  study  of  PSP  with  two  matched  controls,  as 
adults,  PSP  cases  lived  in  areas  with  low  population  significantly  more 
frequently  than  controls.  The  study  identified  no  other  factors  associated  with 
PSP  including  a  history  of  stroke,  hypertension,  or  smoking.  The  prevalence 
survey  of  migraine  in  the  People's  Republic  of  China  yielded  a  point  prevalence 
ratio  of  690/100,000.  The  prevalence  ratio  of  migraine  for  females  was  higher 
than  that  for  males. 

HTLV-I  Infections  of  the  Nervous  System 

The  first  human  retrovirus,  HTLV-I,  has  been  confirmed  as  the  causal  agent  of 
tropical  spastic  paraparesis  (TSP)  and  HTLV-I-associated  myelopathy  (HAM),  as 
well  as  some  forms  of  polymyositis.  Neuroepidemiologic  studies  have  been  carried 
out  in  Tumaco( Colombia)  and  the  Seychelles  Islands. 

The  NEB  will  begin,  in  collaboration  with  the  Pan  American  Health  Organization, 
a  Registry  of  HTLV-I  Infections  of  the  Nervous  System,  in  order  to  obtain  data 
on  the  magnitude  of  this  problem  in  the  Americas. 


9  -  NEB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  •  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 
Z01    NS    01924-20    NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  fit  on  one  line  between  the  borders.) 

Clinical,  Genetic,  Pathophysiologic  Study  of  Hereditary  Movement  Disorders 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.:     Roswell  Eldridge,  M.D.         Medical  Geneticist     NEB,  DIR,  NINDS 


Others:   Robert  Cohen,  M.D. 

Elizabeth  Mathew,  M.D. 


Chief 
Neurologist 


LBI,     IRP,    NIMH 
LBI,    IRP,    NIMH 


COOPERATING  UNITS  (il  any) 

CNB,  DIR,  NINDS;  LCS,  DCBR,  NIMH;  Dept.  of  Neurology,  University  of  Mississippi 
School  of  Medicine 


LAB/BRANCH 

Neuroepidemiology   Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland   20892 


TOTAL  MAN-YEARS 

0.35 


PROFESSIONAL: 

0.3 


OTHER 


0.05 


CHECK  APPROPRIATE  BOX(ES) 

[X  (a)  Human  subjects 
.   (al)  Minors 
QLj   (a2)  Interviews 


LS  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided ) 

In  this  project,  we  seek  to   1)  clarify  and  expand  the  nosology  of  the 
hereditary  movement  disorders;  2)  contribute  to  the  understanding  of  the 
underlying  biochemical  basis;  3)  determine  the  most  effective  treatment  and 
predictive  testing  for  each  disorder;  and  4)  suggest  guidelines  for  counseling 
individuals  at  risk.   General  syndromes  under  study  include  the  dystonias,  tic 
disorders,  the  ataxias  and  myoclonus.   Approaches  include  standard  epidemiologic 
and  clinical  genetic  studies,  and  collaborative  efforts  in  evaluating  the  role 
of  neurotransmitters  such  as  dopamine,  and  PET  Studies. 


We  are  performing  PET  studies  of  at  risk  members  of  two  large  kindreds  with 
hereditary  ataxia.   Preliminary  results  suggest:  a  more  generalized  involvement 
than  previously  recognized;  presymptomatic  changes  by  PET;  and  evidence  for 
genetic  heterogeneity. 


10   -    NEB/DIR 


PHS  6040  (Rev    1/84) 


SPO   S14*91« 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 
Z01    NS    01927-20    NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  tit  on  one  line  between  the  borders  j 

Clinical,  Genetic,  Pathophysiologic  Study  of  Hereditary  Nervous  System  Tumors 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  ana  institute  affiliation i 
P.I.t  Roswell    Eldridge,    M.D  Medical    Geneticist  NEB,    DIR,    NINDS 


Others: 


Bracie  Watson,  M.S. 
Murial  Kaiser-Kupfer, 
Anita  Pikus 
Wesley  McBride,  M.D. 
Dilys  Parry,  Ph.D. 


Geneticist 
M.D.  Chief 

Audiologist 
Molecular  Geneticist 
Acting  Chief 


Judith  Schaeffer,  Ph.D.JD  Vestibulographer 


NEB,  DIR,  NINDS 

OGB,  IP,   NEI 

CA,  IP,   NIDCD 

LBC,  IP,  NCI 

CEB,  IP,  NCI 

CA,  IP,  NIDCD 


cooperating  units  (if  any)       OP,  CC :  SN ,  DIR,  NINDS;  Division  of  Medical  Genetics,  Dept.  of 
Pediatrics,  Children's  Hospital  National  Medical  Center;  Dept.  of  Neurosurgery, 
Massachusetts  General  Hospital,  Boston,  MA;  Uniformed  Services  University  of 
Health  Sciences 


LAB/BRANCH 

Neuroepidemiology  Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland   20892 


TOTAL  MAN-YEARS 

3.5 


PROFESSIONAL 

3.0 


OTHER 


0.5 


CHECK  APPROPRIATE  BOX(ES) 

E  (a)  Human  subjects 
Lx   (a1)  Minors 
LE   (a2)  Interviews 


B  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

In  this  project  we  seek  to  define  and  classify  hereditary  tumors  of  the  nervous 
system  such  as  occur  in  the  neurofibromatoses ;  to  add  to  the  clinical 
description  and  natural  history  of  these  diseases;  to  suggest  methods  for  early 
diagnosis;  to  evaluate  present  modes  of  treatment:  and  to  develop  methods  for 
preclinical  detection  and  screening. 

Our  studies  have  led  to  the  recognition  of  a  preventable  cause  of  deafness, 
visual  loss  or  even  death:   neurofibromatosis  2  or  bilateral  acoustic 
neurofibromatosis .   The  genes  for  two  distinct  forms  of  neurofibromatosis  have 
now  been  mapped  to  specific  chromosomes:   1)  the  classical  form  as  described  by 
von  Recklinghausen  (neurofibromatosis  1)  recently  isolated  on  #17,  and  2)  a  form 
in  which  bilateral  acoustic  neuromas  are  the  hallmark  (neurofibromatosis  2)    may 
be  on  #22.   Efforts  in  the  latter  have  been  directed  at  improving  and 
simplifying  screening  of  high-risk  individuals,  confirming  diagnosis, 
establishing  criteria  for  intervention,  and  isolating  and  characterizing  the 
gene.   A  consensus  conference  dealing  with  these  issues  is  planned  for  June, 
1991. 


Our  first  major  study  involving  neurofibromatosis  1,  a  multi-disciplinary 
project,  demonstrated  mild  but  consistent  impairment  of  neurologic  and  cognitive 
status  in  these  patients  compared  to  their  unaffected  6ibs.   A  second  study 
assessing  the  burden  of  NFI  and  attitudes  towards  predictive  testing  has  been 
completed.   There  is  great  interest  in  such  testing  but  most  would  not  terminate 
a  positive  pregnancy. 

11  -  NEB/DIR 


PHS  6040  (Rev  1/84) 


spo  i  i«-»  ■■ 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02167-16  NEB 


P6c€olDerEF¥,D  1989   through   September   30,    1990 


TITLE  OF  PROJECT,  (80  characters  or  tess_  Title,  must  fit  on  age  line  between  Jhe  bordersj.    .  ,       ,  .     ,     .,  ,         .        _  .  , 

Genetic  Epidemiology  Studies  in  MS  ana  other  Multifactorial  Neurologic  Disorders 


>£INCIPAL  INVF^TIGAT^^ 


ridge 

Others:  Henry  F.  McFarland,  M.D. 
Halter  A.  Rocca,  M.D. 
Gustavo  Roman,  M.D. 
Marinos  Dalakas,  M.D. 
Linda  Nee,  MSW 


title,  laboratoi 


Assistant  Chief 
Epidemiologist 
Chief 

Neurologist 
Research  Associate 


NI,  DIR,  NINDS 
NEB,  DIR,  NINDS 
NEB,  DIR,  NINDS 
N,  DIR,  NINDS 
CNB,  DIR,  NINDS 


cWPllR,AT!filiBJ,NI'Ii§f,antiNB,  NINDS;  M;  National  Rehabilitation  Hospital,  D.C.  Italian 
Multicenter  Study  on  Dementia,  S.M.I.D.  Centers,  Florence,  Italy;  Departments 
Pediatrics  and  Neurology,  King  Faisal  Specialty  Hospital,  Riyadh,  Saudi  Arabia 


l^euroepidemiology  Branch 


SECTION 


l'MsE,AW,CABmhe6da,    Maryland      20892 


TOTAL  MAN-YEARS- 


PROFESSIONAL 


0.3 


OTHER 


CHECK  APPROPRIATE  BOX(ES) 

LXJ  (a)  Human  subjects 
E   (a1)  Minors 
E   (a2)  Interviews 


DO  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided  I 

In  this  project  we  are  coupling  genetic  and  environmental  studies  in  selected 
families,  twin  pairs  £  populations  with  disorders  such  as  multiple  sclerosis, 
Parkinson's  disease,  and  Alzheimer's  disease,  in  an  effort  to  distinguish 
specific  contributing  factors. 

Multi-disciplinary  twin  studies  of  Parkinson's  disease,  multiple  sclerosis  and 
Alzheimer's  disease  indicate  each  is  complex  and  involves  an  interaction  of 
I  environmental  and  genetic  factors. 

1  A  study  similar  in  design  involving  twins  with  documented  polio  over  30  years 
I  ago  is  designed  to  look  at  factors  contributing  to  'post  polio'  syndrome. 

:  An  autosomal  dominant,  hereditary  leukoencephalopathy  simulating  MS  with  onset 

!  at  about  age  35  is  under  study  in  a  kindred  with  over  20  affected.   Derangement 

of  the  autonomic  nervous  system  is  often  seen  early  in  the  course  and  when 

recognized  clinically,  serves  to  distinguish  this  single  gene  disorder  from 

I  multiple  sclerosis. 

i 

i 

In  Saudi  Arabia  a  multidisciplinary  study  of  an  extended  kindred  with  a  novel 

form  of  leukodystrophy  is  planned.   Epidemiologists  and  geneticists  working 

together  will  assess  the  relative  burden  of  environmental  and  genetic  factors  in 

thiB  inbred  population. 


12    -   NEB/DIR 


PHS  6040  (Rev    1/84) 


GPO   fit  4-G1  e 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01    NS    02240-14    NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  tit  on  one  line  between  the  borders  ) 

Epidemiology  of  Dementia 


PRINCIPAL  INVESTIGATOR  (Ust  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 

Acting  P.I.J  Jonas  H.  Ellenberg,  Ph.D.  Acting  Chief  NEB,  DIR,  NINDS 

Gustavo  C.  Roman,  M.D.     Chief  Designate        NEB, DIR, NINDS 

(1/1/90-9/30/90) 


COOPERATING  UNITS  (it  any) 

Vijay  Chandra,  M.D.,  Ph.D.  New  Dehli,  India;  Francis  M.  Baker,  M.D.,  University 
of  Texas  Health  Science  Center 


LAB/BRANCH 

Neuroepidemiology  Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland   20892 


TOTAL  MAN-YEARS 

0.05 


PROFESSIONAL 

0.05 


OTHER 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

B  (a)  Human  subjects 
C   (a1)  Minors 
SJ   (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

Analytic  studies  to  determine  risk  factors  for  Alzheimer's  disease  (AD)  are 
planned  or  being  conducted.   A  large  case-control  study  of  head  trauma  with  loss 
of  consciousness  as  a  risk  factor  for  AD  has  been  completed  in  Rochester,  MN. 
There  was  no  detected  increase  in  risk  of  AD  following  head  trauma.   A 
case-control  study  of  Alzheimer's  disease  is  in  progress  in  White  Plains,  New 
York.   An  attempt  has  been  made  to  evaluate  and  improve  instruments  used  in 
case-control  studies  of  dementia. 


13    -    NEB/DIR 


PHS  6040  (Rev    1/84) 


SPO   81  4-*l» 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  •  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01    NS    02243-14   NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less   Title  must  fit  on  one  line  between  the  borders.) 

Pediatric   Neuroepidemiology 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.:     Karin  B.  Nelson,  M.D.      Medical  Officer  NUB,   DIR,  NINDS 


Others:   Jonas  H.  Ellenberg,  Ph.D. 
Sherrie  Emoto,  Ph.D. 


Acting  Chief 
Staff  Fellow 


NEB,   DIR,  NINDS 
BFSB,  DIR,  NINDS 


COOPERATING  UNITS  (if  any) 

Peter  Scheidt,  M.D.  Medical  Officer,  NICHD 


LAB/BRANCH 

Neuroepidemiology   Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland 


20892 


TOTAL  MAN-YEARS 

0.4 


PROFESSIONAL. 

0.3 


OTHER 


0.1 


CHECK  APPROPRIATE  BOX(ES) 

Lx  (a)  Human  subjects 
LX   (a1)  Minors 
Lj   (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

Several  studies  in  pediatric  neuroepidemiology  are  ongoing:   1)  analysis  of 
case-control  data  for  putative  predictors  of  febrile  seizures,  based  on  a  data 
set  from  six  cities  from  the  People's  Republic  of  China  has  been  completed;   2) 
The  prenatal  and  perinatal  antecedents  of  febrile  seizures  were  examined  in 
NINDS  data;   3)  With  Yugoslav  colleagues,  we  are  examining  the  utility  of  the 
electroencephalogram  as  a  predictor  of  recurrence  of  febrile  seizures  in  a 
defined  population  in  Yugoslavia;   4)  NINDS  participants  were  involved  in  the 
analysis  of  a  randomized  controlled  clinical  trial  undertaken  in  the  NICHD  to 
evaluate  the  safety  and  efficacy  of  phototherapy  in  the  prevention/reduction  of 
hyperbilirubinemia  in  the  neonate.   We  investigated  the  frequency  of  adverse 
neurologic  outcomes  at  six  years  of  age. 


14    -    NEB/DIR 


PHS  6040  (Rev    1/84) 


cpo  si4-«ia 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01    NS    02297-14    NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  tit  on  one  line  between  the  borders  ) 

Mortality  from  Neurologic  Disorders:   National  and  International  Comparisons 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 

Acting  P.I.:  Jonas  H.  Ellenberg,  Ph.D.  Acting  Chief  NEB,  DIR,  NINDS 

Gustavo  C.  Roman,  M.D.     Chief  Designate   NEB, DIR, NINDS 
(1/1/90-9/30/90) 


COOPERATING  UNITS  (it  any) 

VJ  Chandra  M.D.,  India;  W.A.  Rocca  M.D. 


Firenze,  Italy 


LAB/BRANCH 

Neuroepidemiology  Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland 


20892 


TOTAL  MAN-YEARS 

0.05 


PROFESSIONAL 

0.05 


OTHER 

0.0 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)  Human  subjects 
D   (a1)  Minors 
□   (a2)  Interviews 


□  (b)  Human  tissues  L5   (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

Although  death  certificate  data  are  limited  by  possible  misdiagnosis,  incomplete 
case  ascertainment,  errors  in  coding,  etc.,  detailed  mortality  information  on 
some  neurologic  diseases  for  the  entire  U.S.  is  not  available.   Analysis  of 
mortality  data  can  be  particularly  useful  for  some  neurologic  diseases  because 
these  may  contribute  to  death  indirectly.   Since  there  are  no  uniform  criteria 
for  what  constitutes  the  underlying  cause  of  death  in  patients,  it  is  important 
to  examine  all  deaths  in  which  a  disease  is  listed  as  an  underlying,  immediate, 
associated,  or  contributory  cause  of  death  to  get  more  complete  information 
about  the  relationship  between  the  disease  and  death.   Association  of  diseases 
occurring  at  the  time  of  death  are  also  being  studied  for  all  deaths  occurring 
in  the  U.S.  for  many  neurologic  diseases.   Diseases  occurring  together  may 
provide  important  information  in  the  search  for  etiology  of  diseases. 

Detailed  analyses  of  mortality  data  have  been  accomplished  for  Huntington ' s 
disease  (comorbidity  at  time  of  death),  Friedreich's  disease  (survival 

I  analysis),  international  comparisons  of  average  annual  age-adjusted  mortality 
rates  for  primary  tumors  of  the  nervous  system,  and  stroke  (case-control  study 

j  of  risk  factors). 


15  -  NER/DIR 


PHS  6040  (Rev  1/84) 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01   NS   02299-14   NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  tit  on  one  line  between  the  borders.) 

Reviews  of  Epidemiologic  Aspects  of  Neurologic  Disease 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Acting  P.I.:  Jonas  H.  Ellenberg,  Ph.D.  Acting  Chief  NEB,  DIR,  NINDS 

Gustavo  C.  Roman,  M.D.     Chief  Designate       NEB, DIR, NINDS 

(1/1/90-9/30/90) 


COOPERATING  UNITS  (if  any) 

N.  Bharucha,  M.D.,  Bombay,  India;  William  Koller,  M.D.,  Ph.D.,  University  of 
Kansas;  Ian  Irwin,  Ph.D.,  Institute  for  Medical  Research,  San  Jose 


LAB/BRANCH 

Neuroepidemiology  Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland   20892 


TOTAL  MAN-YEARS 

0.05 


PROFESSIONAL: 

0.05 


I  OTHER 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□  (b)  Human  tissues  GO  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

Development  of  new  neurologic  studies  requires  thorough  historic  and 
methodologic  reviews  of  prior  investigations.   These  yield  important  unexplored 
etiologic  clues  that  may  be  investigated  using  current  technology.   Major 
emphasis  has  been  given  to:  cerebrovascular  disease;  otitis  media;  inherited 
ataxias;  Huntington's  disease;  febrile  seizures;  Tourette's  syndrome;  peripheral 
neuropathy;  neurologic  disease  in  the  elderly;  controlled  therapeutic  trials  of 
motor  neuron  disease;  epilepsy;  descriptive,  analytic,  and  experimental  methods 
in  neuroepidemiology;  procedures  for  neuroepidemiologic  investigations  in 
developing  countries;  and  epidemiologic  studies  of  Alzheimer's  disease; 
myasthenia  gravis;  cerebral  malaria;  and  autism. 


16   -   NEB/DIR 


PHS  6040  (Rev    1/84) 


6PO  914416 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01    NS    02300-14    NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30, 


1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  lit  on  one  line  between  the  boroers ) 

Clinical  Course  and  Medical  Care  for  Neurologic  Disorders 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory  ana  institute  affiliation) 
Acting    P.I.:    Jonas    H.    Ellenberg,    Ph.D.    Acting   Chief  NEB,    DIR,    NINDS 

Gustavo   C.    Roman,    M.D.  Chief    Designate  NEB, DIR, NINDS 

(1/1/90-9/30/90) 


COOPERATING  UNITS  (it  any) 

Shi-Chuo  Li,  M.D. 


Beijing  Neurosurgical  Institute,  PRC 


LAB/BRANCH 

Neuroepidemiology  Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland   20892 


TOTAL  MAN-YEARS 

0.0 


PROFESSIONAL 

0.0 


OTHER 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□  (b)  Human  tissues  LS  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

The  study  uses  a  review  and  abstraction  of  data  from  records  for  a  selected 
group  of  neurological  disorders.   It  obtains  the  items  of  data  necessary  to 
determine  onset  of  the  disorder,  comorbid  conditions,  duration,  date  and  cause 
of  death,  or  current  status.   These  data  will  be  used  to  construct  modified  life 
tables  to  estimate  the  expectation  of  life  after  diagnosis,  the  survival  curve, 
and  morbidity  and  severity  estimates.   It  will  also  include  analysis  of  type  and 
duration  of  medical  care  received  by  patients  with  neurologic  disorders  derived 
from  a  well-defined  population. 


17    -    NEB/DIR 


PHS  6040  (Rev    1/84) 


SPO   8  1  4-9  1  » 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 
Z01    NS    02301-14    NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30, 


1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  lit  on  one  line  between  the  borders.) 

Collaborative  Studies  of  Less  Common  or  Less  Debilitating  Neurologic  Disorders 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  atliliation) 

Acting  P.I.:  Jonas  H.  Ellenberg,  Ph.D.  Acting  Chief  NEB,  DIR,  NINDS 

Gustavo  C.  Roman,  M.D.     Chief  Designate        NEB, DIR, NINDS 

(1/1/90-9/30/90) 


COOPERATING  UNITS  (it  any) 

B.  Chen,  M.D.,  Beijing,  China;  G.  Roman,  M.D.,  Lubbock,  Texas;  L.  Golbe,  M.D., 
New  Brunswick,  New  Jersey;  C.  Tanner,  M.D.,  Chicago,  111 


LAB/BRANCH 

Neuroepidemiology   Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland   20892 


TOTAL  MAN-YEARS 

0.05 


PROFESSIONAL: 

0.05 


OTHER 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□  (b)  Human  tissues  ffl  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided) 

A  number  of  collaborative  efforts  involve  the  investigation  of  the 
characteristics  of  unusual  or  less  debilitating  (e.g.,  headache)  neurologic 
disease  phenomena.   Unusual  associations  or  space/time  clusters  of  neurologic 
disorders  may  provide  leads  to  etiology  or  therapy.   These  may  be  tested  through 
more  formal  approaches. 

Surveys  of  prevalence  and  natural  history  and  a  case  control  study  of 
progressive  supranuclear  palsy  (PSP)  have  been  completed  in  two  counties  in  New 
Jersey.   The  point  prevalence  was  1.39/100,000.   The  median  intervals  to  onset 
of  requiring  assistance  was  3.1  years  and  to  death  was  9.7  years.   The  case 
control  study  did  not  implicate  a  history  of  stroke,  hypertension  or  smoking  as 
risk  factors  for  PSP.   A  prevalence  survey  of  migraine  in  the  People's  Republic 
of  China  yielded  a  point  prevalence  ratio  of  690/100,000,  and  indicated  that 
the  prevalence  ratio  for  females  was  higher  than  that  for  males. 


18  -  NEB/DIR 


PHS  6040  (Rev  1/84) 


GPO  9\4-»IB 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 
Z01    NS    02307-14    NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30, 


1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  tit  on  one  line  between  the  borders. ) 

Educational  Resources  in  Neurological  Epidemiology 


PRINCIPAL  INVESTIGATOR  (List  other  protessional  personnel  below  the  Principal  Investigator  I  (Name,  title,  laboratory,  and  institute  artiHation) 
Acting    P.I.:    Jonas    H.    Ellenberg,    Ph.D. Acting   Chief  NEB,       DIR,    NINDS 

Gustavo   C.    Roman,    M.D.  Chief    Designate  NEB,       DIR,    NINDS 

(1/1/90-9/30/90) 


Other: 


Dallas  W.Anderson,  Ph.D.  Mathematical  Statistician  BFSB, DIR, NINDS 


COOPERATING  UNITS  (it  any) 


LAB/BRANCH 

Neuroepidemiology  Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland   20892 


TOTAL  MAN-YEARS 

0.05 


PROFESSIONAL 


0.05 


OTHER 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□  (b)  Human  tissues  LH  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type  Do  not  exceed  the  space  provided ) 

Because  there  is  severe  shortage  of  available  manpower  in  neuroepidemiology,  the 
Branch  has  developed  an  active  teaching  program  for  current  and  future 
collaborative  investigators.   A  series  of  six  video  tapes  produced  by  the  Branch 
are  distributed  on  a  loan  basis  without  charge.   A  monograph,  entitled 
NEUROLOGICAL  EPIDEMIOLOGY:   PRINCIPLES  AND  CLINICAL  APPLICATIONS  was  published 
in  1978.   A  new  monograph  which  will  cover  current  methodology  and  disease 
specific  overviews  of  etiology,  incidence,  prevalence  and  intervention  studies 
has  been  completed. 

A  set  of  video  tapes  have  been  produced  for  training  interviewers  in  the 
methodology  of  interviewing  for  case-control  studies.   This  has  been  done  in 
both  Italian  and  English. 


19   -   NEB/DIR 


PHS  6040  (Rev   1/84) 


spo  814-aia 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02370-12  NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Racial  and  Geographic  Differences  in  Occurrence  of  Neurologic  Disease 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  attiliation) 
Acting   P.I.:    Jonas   H.    Ellenberg,    Ph.D.    Acting   Chief  NEB,       DIR,    NINDS 

Gustavo   C.    Roman,    M.D.  Chief   Designate  NEB,       DIR,    NINDS 

(1/1/90-9/30/90) 


Other: 


Dallas  Anderson,  Ph.D.  Mathematical  Statistician  BFSB,DIR,  NINDS 


COOPERATING  UNITS  (if  any) 

OBFS,  OD,  NINDS:  A.  Haerer,  M.D.,  Univ.  of  Mississippi;  U.S.  Bureau  of  the 
Census;  N.  Bharucha,  M.D.  (India) 


LAB/BRANCH 

Neuroepidemiology   Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland   20892 


TOTAL  MAN-YEARS 

0.05 


PROFESSIONAL 

0.05 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

E  (a)  Human  subjects 
13  (al)  Minors 
SI   (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided.) 

The  purpose  of  these  studies  is  to  accurately  document  possible  racial, 
environmental  and  geographic  differentials  in  the  prevalence  of  major  neurologic 
disorders  by  surveying  an  entire  geographically  defined  population.  The 
disorders  investigated  include  cerebral  palsy,  dementia,  psychomotor  delay, 
epilepsy,  Parkinson's  disease,  essential  tremor,  multiple  sclerosis  and 
cerebrovascular  disease. 

Research  protocols  for  these  neurologic  studies  have  been  completed  in  the 
United  States  (Copiah  County),  India  and  Spain.   The  prevalence  study  of  the 
Parsi  Community  of  Bombay,  India  provided  point  prevalence  estimates  of 
328.3/100,000  for  Parkinson's  disease  and  1591.7/100,000  for  essential  tremor. 
In  the  pilot  prevalence  study  in  an  urban  area  of  Madrid,  Spain,  migraine  and 
epilepsy  were  the  most  frequent  disorders  with  point  prevalence  ratios  of 
163.3/1000  and  9.3/1000,  respectively. 


20    -  NEB/DIR 


PHS  6040  (Rev    1/84) 


SPO  914-916 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 
Z01    NS    02423-11    NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  tit  on  one  tine  between  the  borders ) 

Development  of  Data  Resources  for  Neuroepidemiology 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  {Name,  title,  laboratory,  and  institute  atliliation) 

Acting  9.1. i      Lawrence  Lavine,  D.O.,  M.P.H.  Medical  Officer   NEB,  DIR,  NINDS 


COOPERATING  UNITS  lit  anyl 


LAB/BRANCH 

Neuroepidemiology  Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland   20892 


TOTAL  MAN-YEARS 

0.00 


i  PROFESSIONAL 

0.00 


OTHER 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

E  (a)  Human  subjects 
□   (a1)  Minors 
G   (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

To  develop  1)  a  registry  of  hospitalized  patients  with  neurologic  disease  in  a 
geographically  well-defined  population;  2)  resources  for  case-control  studies  of 
neurologic  diseases  using  uniform  methods  of  data  collection;  and  3)  a  registry 
of  neurologic  disease  in  the  well-defined  population  of  the  United  States 
military. 


This  project  is  completed, 
other  project  areas. 


Future  projects  in  this  area  will  be  subsumed  under 


21    -   NEB/DIR 


PHS  6040  (Rev    1/84) 


SPO  II  44  II 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 
Z01    NS    02570-08    NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30, 


1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Natural  History  of  ALS-PD  in  Guam 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.:     Lawrence  Lavine,  D.O.,  M.P.H.  Medical  Officer        NEB,  DIR,  NINDS 

Other:    Zhen-xin  Zhang,  M.D.      Guest  Researcher  NEB,  DIR,  NINDS 

Dalas  W.  Anderson,  Ph.D.  Mathematical  Statistician  BFSB,DIR,  NINDS 

Karin  B.  Nelson,  M.D.     Pediatric  Neurologist       NEB,  DIR,  NINDS 


cooperating  units  (it  any)         Leonard  Kurland,  M.D.  ,  Mayo  Clinic,  Rochester,  MN;  John 
Steele,  M.D.,  Veterans  Administration,  Guam;  Xhen-xin  Zhang,  M.D.,  People's 
Republic  of  China;  Donald  Calne,  M.D.,  Univ.  of  British  Columbia,  Vancouver,  BC; 
Peter  Spencer,  Ph.D.,  Albert  Einstein  College  of  Medicine,  NY. 


uvb/branch 
Neuroepidemiology  Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland   20892 


TOTAL  MAN-YEARS 

1.0 


PROFESSIONAL: 

0.9 


OTHER: 


0.1 


CHECK  APPROPRIATE  BOX(ES) 

IE  (a)  Human  subjects 
□   (a1)  Minors 
LH   (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided.) 

As  a  continuation  of  previous  projects  on  clinical,  pathological,  and 
epidemiologic  surveillance  of  Guamanian  amyotrophic  lateral  sclerosis  (ALS)  and 
Parkinsonism-dementia  (PD)  in  the  Marianas  Islands,  identified  survivors, 
including  suspects,  in  the  NINDS  Registry  as  of  January  1,  1983,  were  followed 
at  intervals  of  six  months  for  detailed  clinical  descriptions  of  patterns  of 
progression,  by  a  qualified  neurologist.    The  patients  still  alive  have  entered 
a  chronic  state  and  final  exams  were  completed  in  the  fall  of  1988.   As  these 
patients  expire,  attempts  will  be  made  to  obtain  the  appropriate  pathologic 
material  for  examination  by  various  neuropathologists  around  the  world.   All 
attempts  to  examine  this  material  should  be  made  because  of  the  uniqueness  of 
these  patients. 

The  prevalence  survey  of  three  southern  Guamanian  villages  to  determine  the 
current  prevalence  of  ALS/PDC  was  completed  in  1988.   As  this  is  a  transitional 
society,  dietarily  and  culturally  since  WW-II,  a  simultaneous  survey  for  other 
major  neurologic  diseases  and  major  diseases  of  aging  populations  was  also 
performed.   A  chart  review  study  using  the  NINDS  Guam  registry  for  ALS/PDC  for 
the  period  1944-85  was  performed.   Downward  trends  in  age-adjusted  incidence 
rates  and  upward  trends  in  age  of  onset  were  seen  for  both  sexes.   Since  1980, 
new  cases  occurred  only  among  persons  over  50  years  of  age,  whereas  younger  age 
at  onset  had  been  noted  in  the  past. 

The  NINDS  no  longer  is  supporting  this  program.   It  is  now  being  operated  under 
a  grant  from  NIA,  with  Dr.  Leonard  T.  Kurland  as  principal  investigator.   This 
project  is  completed. 

22   -   NEB/DIR 


PHS  6040  (Rev    1/84) 


GPOII  4-0  is 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  •  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 
Z01    NS    02715-05    NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  lit  on  one  line  between  the  borders .) 

Epilepsy  Neuroepidemiology 


PRINCIPAL  INVESTIGATOR  (List  other  protessional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory  ana  institute  attiiiation/ 
P.I.:  Karin    B.    Nelson,    M.D.  Medical   Officer  NEB,       DIR,    NINDS 


Others:   Jonas  H.  Ellenberg,  Ph.D. 
William  Theodore,  M.D. 


Chief 

Medical  Officer 


BFSB,  DIR,  NINDS 
MNB,   DIR,  NINDS 


COOPERATING  UNITS  (it  any) 

Shi-chuo  Li,  M.D.,  Beijing  Neurosurgical  Institute,  PRC;  Judith  Manelis,  M.D., 
Western  Galilee  Regional  Hospital,  Israel 


LAB/BRANCH 

Neuroepidemiology  Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland 


20892 


TOTAL  MAN-YEARS 

.35 


PROFESSIONAL 

0.3 


OTHER 


0.5 


CHECK  APPROPRIATE  BOX(ES) 

Lx!  (a)  Human  subjects 
H   (a1)  Minors 
S   (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

Several  studies  on  convulsive  disorders  are  being  planned  and  tested  for 
feasibility,  or  in  progress.   A  protocol  is  in  effect  for  a  clinical  study  of 
the  Lennox-Gastaut  syndrome  (LGS),  a  severe  childhood  epileptic  encephalopathy 
with  significant  morbidity,  characterized  by  uncontrolled  seizures,  mental 
retardation,  and  possible  mental  deterioration,  to  define  the  pathophysiology 
and  anatomic  locus  of  disturbance  in  LGS.   We  are  evaluating  the  feasibility  of 
performing  randomized  and  placebo  controlled  clinical  trials  of  treatment  after 
an  initial  convulsion  in  subjects  presenting  for  care  to  the  Beijing  Tiantan 
Hospital  and  to  a  consortium  of  hospitals  in  Jerusalem.   With  Yugoslav 
colleagues,  we  are  examining  the  utility  of  the  electroencephalogram  as  a 
predictor  of  recurrence  of  febrile  seizures  in  a  defined  population  in 
Yugoslavia. 


23   -   NEB/DIR 


PHS  6040  (Rev    1/84) 


SPO  SI  4411 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 
Z01    NS    02746-04    NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30, 


1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  lit  on  one  line  between  the  borders.) 

Phenobarbital  Clinical  Trial  in  Children  with  Febrile  Seizures* 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 
P.I.:  Karin   B.    Nelson,    M.D.  Medical    Officer  NEB,       DIR,         NINDS 


Others:   Deborah  Hirtz,  M.D. 

Young  Jack  Lee,  Ph.D. 
Jonas  H.  Ellenberg,  Ph.D. 


Pediatric  Neurologist  DNB,  DCDND,  NINDS 
Mathematical  Statistician  NEB,  DIR,  NINDS 
Chief  BFSB,  DIR,    NINDS 


COOPERATING  UNITS  (it  any) 

Jacqueline  Farewell, 


M.D.,  Dept.  of  Neurosurgery,  Univ.  of  Washington,  Seattle 


LAB/BRANCH 

Neuroepidemiology  Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland   20892 


TOTAL  MAN-YEARS 

0.1 


PROFESSIONAL 


0.1 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

[X  (a)  Human  subjects 
S   (a1)  Minors 
□   (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

The  objectives  of  the  study  are:   To  assess  the  effects  on  tests  of  intelligence 
and  behavior  of  phenobarbital ,  a  commonly  prescribed  anticonvulsant  in  children. 

The  design  of  this  study  permited  comparison  of  measures  of  tested  intelligence 
and  of  behavior  in  children  with  febrile  seizures  who  had  been  treated  with 
phenobarbital,  and  in  a  group  of  seizure-free  control  children.   A  comparison  of 
the  groups  allowed  assessment  of  benefit  and  risk  of  treatment  for  a  common 
childhood  neurologic  problem. 

*[This  study  supports  the  DNB/DCDND/NINDS  contract  study  entitled:  "Behavioral 
and  cognitive  side  effects  of  phenobarbital  used  for  prevention  of  febrile 
seizure  recurrence."   The  project  officer  is  Dr.  Deborah  G.  Hirtz,  DNB,  DCDND, 
NINDS,  and  the  contractor  of  the  study  is  the  University  of  Washington. ] 


24   -   NEB/DIR 


PHS  6040  (Rev    1/84) 


GPO  914-918 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 
Z01    NS    02747-04    NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30, 


1990 


TITLE  OF  PROJECT  (BO  characters  or  less    Title  must  lit  on  one  line  between  the  borders  ) 

Dental  Markers  of  Maldevelopment 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 
P.I.:       Karin    B.    Nelson,    M.D.  Medical    Officer  NEB,    DIR,    NINDS 


COOPERATING  UNITS  (if  any) 

Mohandas  Bhat,  D.D.S. 


D.P.H.,    EODPP,    NIDR 


LAB/BRANCH 

Neuroepidemiology  Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland 


20892 


TOTAL  MAN-YEARS 

0.1 


PROFESSIONAL 


0.1 


OTHER 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

LH  (a)  Human  subjects 
13   (a1)  Minors 
□   (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type   Do  not  exceed  the  space  provided ) 

This  is  an  exploratory  effort  to  examine,  in  a  group  of  children  with  chronic 
motor  disability  of  early  onset  and  nonprogressive  course  (cerebral  palsy,  CP), 
potential  markers  of  maldevelopment.   It  will  focus  on  the  frequency  and  nature 
of  dental  abnormalities  in  affected  children,  in  comparison  with  the  frequency 
of  similar  characteristics  among  healthy  children. 

The  objectives  are  to  examine  whether  dental  abnormalities,  especially  enamel 
defects,  can  serve  as  markers  of  maldevelopment,  and  whether  such  findings  can 
provide  information  concerning  timing  of  adverse  events  or  exposures. 

The  significance  of  the  research  is  that  enamel  hypoplasias  and  other  dental 
anomalies  can  offer  clues  as  to  the  timing  of  insults  or  exposures  that  occur 
from  the  fourth  month  of  gestation  to  the  age  of  about  12  months  of  extrauterine 
life.  Correlation  of  dental  with  clinical  data  may  offer  a  means  to  explore  the 
timing  of  departure  from  the  normal  course  of  development  in  a  group  of  children 
with  chronic  motor  disability  of  early  onset  and  nonprogressive  course  (cerebral 
palsy,  or  mental  retardation) . 

This  study  of  local  populations  is  in  abeyance  while  investigators  concentrate 
on  the  dental  findings  in  the  California  CP  registry  study  (q.v.) 


25  -  NEB/DIR 


PHS  6040  (Rev  1/84) 


CPO  IU4II 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTtCE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02748-04  NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30, 


1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders  ) 

Dermatoglyphic  Markers  of  Maldevelopment 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.:   Karin  B.  Nelson,  M.D.     Medical  Officer  NEB,  DIR,  NINDS 


COOPERATING  UNITS  (if  any) 

Chris  Plato,  Ph.D.,  GRC,  NIA;  Mrs.  Cathy  Fox,  University  of  Maryland 


LAB/BRANCH 

Neuroepidemiology  Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland 


20892 


TOTAL  MAN-YEARS 

0.1 


PROFESSIONAL 


0.1 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

S  (a)  Human  subjects 
Lxl   (a1)  Minors 
n   (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  CF  WORK  (Use  standard  unreduced  type   Do  no!  exceed  the  space  provided.) 

Fingerprints  are  in  place  by  nine  to  1€  weeks  of  intrauterine  life,  dermal 
creases  by  19  weeks.   The  objective  of  this  pilot  study  is  to  examine  whether 
dermatoglyphic  abnormalities  and  dermal  creases  can  serve  as  markers  of 
maldevelopment,  and  whether  such  findings  can  provide  information  concerning 
timing  of  adverse  events  or  exposures  in  children  with  abnormal  neurologic 
development.   This  is  an  exploratory  effort  to  examine,  in  a  group  of  persons 
with  chronic  motor  disability  of  early  onset  and  nonprogressive  course  (cerebral 
palsy,  CP),or  with  mental  retardation,  potential  markers  of  maldevelopment.   It 
will  focus  on  the  frequency  and  nature  of  dermatoglyphic  abnormalities  in 
affected  individuals,  in  comparison  with  the  frequency  of  similar 
characteristics  among  healthy  persons. 

The  significance  of  the  research  is  that  a  time  course  of  establishment  of 
dermatoglyphics  and  palmar  creases  has  been  developed.   The  hypothesis  has  been 
offered,  and  some  data  accrued  to  indicate,  that  dermal  findings  may  offer  clues 
as  to  the  timing  of  insults  or  exposures  during  early  prenatal  life,  providing  a 
means  to  explore  the  timing  of  departure  from  the  normal  course  of  development 
in  a  group  of  neurologically  handicapped  children. 

This  project  is  completed.  Future  projects  in  this  area  will  be  subsumed  under 
other  project  areas. 


26   -   NEB/ DIP. 


PHS  6040  (Rev.  1/84) 


opo  ei  4-bib 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 
Z01    NS    02819-01    NEB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  tit  on  one  line  between  the  borders  ) 

California  Cerebral  Palsy  Registry 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory   and  institute  affiliation) 

P.I.J     Karin  B.  Nelson,  M.D.       Medical  Officer  NEB, IRP,NINCDS 


COOPERATING  UNITS  (H  any) 

Judith  Grether,  Ph.D.,  Susan  Cummins,  M.D.,  Birth  Defects  Monitoring  Group, 
Environmental  Epidemiology  and  Toxicology  Branch,  Department  of  Health  Services, 
California;  Health  Officers  Association  of  California. 


LAB/BRANCH 

Neuroepidemiology  Branch 


SECTION 


INSTITUTE  AND  LOCATION 

NINCDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS 

0. 


PROFESSIONAL 

0.1 


OTHER 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

B  (a)  Human  subjects 
Lxi   (a1)  Minors 
S   (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided ) 

In  cooperation  with  the  California  Birth  Defects  Monitoring  Program  of  the 
California  Department  of  Health  and  the  March  of  Dimes,  NEB  is  participating  in 
the  establishment  and  utilization  of  a  population-based  registry  of  children 
with  cerebral  palsy  in  four  counties  of  the  San  Francisco  Bay  Area  for  births  in 
1983-1985.   Cases  were  ascertained  through  client  records  of  the  California 
Childrens'  Services  and  the  Regional  Centers  for  the  Developmentally  Disabled. 
Cases  meeting  CP  registry  criteria  have  received  a  standardized  examination  by  a 
single  study  pediatrician,  including  videotaping  of  all  children,  photographs  of 
teeth  and  hands  and  systematic  scrutiny  and  measurement  for  dysmorphologic 
features. 

All  children  meeting  registry  criteria  have  been  examined.   In  this  birth  cohort 
of  approximately  165,000  children,  230-290  cases  have  been  identified,  excluding 
those  with  abnormalities  apparently  related  to  events  after  the  first  month  of 
life.   Analyses  using  the  examinations,  birth  certificates,  initial  interview, 
and  linkage  with  birth  defects  registry  information  are  imminent. 

The  information  will  permit  descriptive  studies  of  prevalence  and  distribution 
of  cerebral  palsy,  evaluation  of  markers  of  maldevelopment ,  and  exploration  of 
risk  factors,  medical  and  environmental.   Ecologic  studies  will  compare  rates  of 
CP  among  births  in  Santa  Clara  County  census  tracts  containing  EPA  superfund 
waste  disposal  sites,  to  rates  in  the  county  as  a  whole  and  to  rates  in  the 
remainder  of  the  Bay  area.   Trends  in  incidence  and  time-space  clustering  will 
be  sought  and  hypotheses  formulated  for  testing  in  case-control  studies. 

27   -   NEB/DIR 


PHS  6040  (Rev    1/84) 


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ANNUAL  REPORT 

October  1,  1989  through  September  30,  1990 

Clinical  Neurosciences  Program,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 

Neuro immunology  Branch 

Table  of  Contents 

RESEARCH  SUMMARY  1-6 

PROJECT  REPORTS 

Immunological  Studies  in  Patients  with  Multiple 

Sclerosis  and  Other  CNS  Diseases 
Z01  NS  02202-15  NI  7 

Membrane  Molecules  Involved  in  Immune  Reactivity 

Z01  NS  02203-15  NI  8 

Immunologic  Mechanisms  Operative  in  Experimental 

Autoimmune  Diseases  of  the  Nervous  System 
Z01  NS  02204-15  NI  9 

Interactions  Between  the  Human  Immune  System  and 

Antigens  in  the  Nervous  System 
Z01  NS  02205-15  NI  10 

Molecular  Mechanisms  of  Lymphoid  Cell-Cell  Interactions 

Z01  NS  02603-07  NI  11 

Immunologic,  Virologic,  and  Molecular  Aspects  of 
Retroviruses  Associated  with  Chronic  Neurologic 
Diseases 

Z01  NS  02817-01  NI  12 


NIB/DIR 


Annual  Report 

October  1,  1989  to  September  30,  1990 

Clinical  Neurosciences  Program,  DIR 

National  Institutes  of  Neurological  and 

Communicative  Disorders  and  Stroke 

Dale  E.  McFarlin,  Chief 

Research  in  the  Neuro immunology  Branch  (NIB)  includes  a  number  of 
interrelated  fundamental,  immunological  studies  and  clinical  investigations  of 
disorders  affecting  the  nervous  system.   This  is  conducted  in  four 
administrative  groups,  the  Office  of  the  Chief,  the  Neurological  Disease 
Section  (NDS),  the  Molecular  Immunology  Section  (MIS),  and  the  Cellular 
Immunology  Section  (CIS).   During  FY  1990,  considerable  emphasis  was  placed  on 
the  genetic  influence  on  immune  reactivity  and  the  application  of  molecular 
biological  strategies  to  both  fundamental  and  clinical  investigations. 

The  clinical  research  remained  focused  on  multiple  sclerosis  (MS).   Recent 
findings  from  the  NIB,  as  well  as  other  institutions,  indicate  that  magnetic 
resonance  imaging  (MRI)  is  valuable  for  investigating  the  natural  history, 
pathogenic  mechanisms,  and  the  possible  effect  of  treatment  in  MS. 
Consequently,  the  use  of  MRI  in  the  investigation  of  MS  has  been  a  major 
clinical  activity  in  the  NIB,  over  the  past  year.   Results  from  our  studies, 
as  well  as  other  institutions,  suggest  that  an  early  event  in  the  development 
of  MS  lesions  is  breakdown  in  the  blood-brain  barrier  (BBB).   This  can  be 
identified  by  the  demonstration  of  enhancing  lesions,  after  the  administration 
of  gadolinium.   In  order  to  determine  the  frequency,  magnitude,  and  natural 
history  of  gadolinium-enhancing  lesions,  a  new  protocol  was  conducted  in  FY 
1990.   In  this  study  a  small  group  of  patients  with  mild  (Kurtzke  disability 
status  scale  of  3.5  or  less)  relapsing-remitting  MS  were  evaluated  at  monthly 
intervals  by  complete  neurological  assessment  and  MRI  scanning,  including  the 
administration  of  gadolinium.   During  the  course  of  this  protocol,  60  clinical 
evaluations  were  conducted  and  3  exacerbations  were  documented.   MRI 
evaluations,  during  the  same  period,  showed  the  appearance  of  91  new 
gadolinium-enhancing  lesions.   This  indicated  that  a  marked  amount  of 
clinically  undetectable  disease  activity  was  occurring.   The  design  of  this 
protocol  enabled  evaluation  of  the  natural  history  of  gadolinium-enhancing 
lesions.   Sixty-three  lesions  persisted  for  less  than  four  weeks,  twenty-four 
for  less  than  eight  weeks,  and  only  four  beyond  eight  weeks.   The  gadolinium 
enhancing  lesions  frequently  evolved  into  nonenhancing  lesions.   During  the 
course  of  the  study,  it  appeared  that  the  amount  of  pathology  detectable  by 
MRI  gradually  increased.   Attempts  to  quantitate  these  changes  by  computer 
technology  are  in  progress.   These  MRI  findings  have  major  implications  for 
the  investigation  of  a  pathological  process  in  MS  and  to  the  evaluation  of 
experimental  therapeutic  agents  in  the  treatment  of  the  disease.   Future 
studies  of  experimental  therapeutic  agents  in  the  NIB  will  use  MRI  technology 
for  assessing  efficacy. 

The  MRI  findings  reinforce  the  importance  of  investigation  of  the  BBB. 
Brain  capillary  endothelial  cells  (EC)  that  have  tight  junctions  are  a  major 
component  of  the  BBB.   For  the  past  five  years,  imnuinological  functions  of  EC 
have  been  studied  in  the  NDS  in  collaboration  with  Drs .  Richard  McCarron  and 


1  -  NIB/DIR 


Maria  Spatz  in  the  LNP.   Isolated  EC  lack  class  II  major  histocompatibility 
complex  (MHC)  molecules,  but  their  expression  can  be  induced  by  the 
administration  of  gamma-interf eron.   EC  that  express  class  II  HLA  molecules 
are  capable  of  presenting  antigen  to  sensitized  T-lymphocytes  in  vitro.   This 
finding  led  to  the  hypothesis  that  antigen  recognition  occurs  at  the  luminal 
surface  of  EC.   A  new  finding  is  that  EC  treated  with  gamma- interferon  and 
pulsed  with  myelin  basic  protein  (MBP)  can  be  lysed  by  MBP-specific  CD4+ 
cytotoxic  T  lymphocytes  (CTL).   The  occurrence  of  this  reactivity  in  vivo 
would  provide  an  immunological  mechanism  for  a  partial  breakdown  of  the  BBB. 
Recent  experiments  have  shown  that  other  cytokines  such  as  tumor  necrosis 
factors  (TNF)  and  interleukin  1  (IL1)  down-regulate  the  induction  of  class  II 
molecules  on  the  surface  of  EC. 

Both  clinical  and  fundamental  studies  in  the  NIB  continued  to  examine 
genetic  components  possibly  related  to  the  pathogenesis  of  MS.   A  major 
conclusion  from  our  studies  of  genetic  factors  in  MS  is  that  multiple  genes 
are  related  to  susceptibility.   An  immunopatho logical  process  has  long  been 
postulated  to  be  operative  in  MS,  and  products  of  genes  on  three  different 
chromosomes  are  involved  in  the  recognition  of  antigen  by  immune  T  cells.   The 
T-cell  receptor  (TCR)  for  antigen  consists  of  an  alpha-  and  beta-chains 
encoded  by  chromosomes  14  and  7,  respectively.   These  receptors  recognize 
antigen  presented  by  HLA  molecules  encoded  on  chromosome  6.   For  some  time,  MS 
in  some  Caucasian  populations  has  been  known  to  be  linked  to  class  II  HLA 
molecules,  namely,  HLA-DR2.   Two  years  ago,  association  between  germline  genes 
encoding  the  beta-chain  of  the  T-cell  receptor  was  identified  by  the  MIS  in 
collaborative  studies  with  Dr.  Leroy  Hood  at  California  Institute  of 
Technology.   Subsequently,  similar  conclusions  were  reached  by  investigators 
at  Harvard  University  and  a  team  of  investigators  at  Stanford  University  have 
described  linkage  between  MS  and  a  gene  that  encodes  a  variable  gene  segment 
of  the  TCR  alpha-chain.   Studies  in  the  MIS  compared  the  TCR  germline 
repertoire  of  40  chronic  MS  patients  with  normals.   In  order  to  characterize 
this  association  in  greater  detail,  germline  TCR  alpha-  and  beta-chain  gene 
haplotypes,  as  well  as  HLA  molecules,  are  being  assessed  in  families  with 
multiple  affected  members  in  a  continuation  of  the  collaboration  with  Dr. 
Leroy  Hood.   In  order  to  identify  the  affected  individuals  in  each  family  as 
precisely  as  possible,  each  of  the  family  members  is  evaluated  both  clinically 
and  by  MR I. 

Extensive  investigations  of  various  aspects  of  HLA  structure  function 
relationships  and  regulation  of  HLA  expression  are  being  conducted.   Studies 
of  the  association  between  MS  and  class  II  HLA  molecules  have  included  a 
comparison  of  the  sequences  of  these  genes  in  MS  and  normal  individuals. 
Although  susceptibility  and  resistance  to  diabetes  has  been  correlated  to  a 
single  base  change  in  the  coding  region  of  the  HLA-DQ  beta-chain,  similar 
sequence  differences  that  are  related  to  susceptibility  have  not  been 
identified  in  MS.   Total  genomic  sequences  for  HLA-DQ  beta  genes  from  a 
homozygous  MS  patient  and  a  normal  homozygous  cell  line  showed  no  sequence 
differences.   The  findings  in  a  single  patient  were  extended  and  focus  was 
placed  on  exon  2  of  the  alpha  and  beta  gene  which  code  for  the  antigen  binding 
regions.   Genes  for  DQ-alpha,  DQ-beta,  and  DR-beta  of  five  MS  patients  have 
been  sequenced  and  no  differences  from  the  reported  sequences  in  normal 
individuals  were  detected.   From  this  series  of  investigations,  it  has  been 
concluded  that  there  is  no  coding  difference  in  class  II  HLA  genes  that  is 

2  -  NIB/DIR 


related  to  susceptibility  or  resistance  of  MS.   Therefore,  the  above-mentioned 
association  of  susceptibility  with  class  II  genes  probably  is  due  to  genetic 
effects  that  do  not  involve  specific  antigen  binding  by  class  II  MHC 
molecules.   Current  efforts  are  designed  to  analyze  the  segregation  of 
noncoding  regions  of  class  II-associated  polymorphic  markers  with  various  HLA 
haplotypes  in  a  series  of  15  multiplex  families. 

A  major  difference  between  the  nervous  system  and  other  tissues  is  that 
MHC  molecules  are  expressed  in  lower  amounts  or  are  undetectable.   Major 
emphasis  is  being  placed  on  the  fundamental  mechanisms  responsible  for  reduced 
expression.   Three  experimental  systems  are  being  studied:   1)  the  regulation 
of  class  I  MHC  molecules  is  being  assessed  in  explanted  murine  fetal  CNS  cells 
in  collaboration  with  Dr.  Keiko  Ozato,  in  the  Laboratory  of  Developmental  and 
Molecular  Immunity,  NICHHD;  2)  the  expression  and  regulation  of  both  class  I 
and  class  II  molecules  on  adult  human  glial  cells  (AHGC)  is  being  studied  in 
vitro,  in  collaboration  with  Dr.  Conrad  V.  Kufta,  SNB;  and  3)  the  molecular 
regulation  of  class  II  molecules  is  being  studied  in  cell  lines  derived  from 
human  gliomas  and  medulloblastomas  which  retain  the  properties  of  neurons  in 
collaboration  with  Dr.  Darell  D.  Bigner  at  Duke  University. 

A  major  conclusion  from  the  studies  of  fetal  CNS  cells  is  that  class  I  MHC 
molecules  are  regulated  differently  in  astrocytes,  oligodendrocytes,  and 
neurons.   An  implication  of  this  finding  is  that  each  of  these  cell  types 
varies  in  the  capacity  to  function  as  targets  for  CD8+  cytotoxic  T 
lymphocytes  that  recognize  antigen  in  association  with  class  I  MHC  molecules. 
Cultured  neonatal  astrocytes  spontaneously  express  class  I  molecules  in  tissue 
culture  and  have  been  used  to  investigate  the  molecular  mechanisms  responsible 
for  regulation  of  these  molecules.   In  non-CNS  cells,  the  transcription  of 
class  I  genes  is  regulated,  in  part,  by  a  series  of  enhancer  regions  including 
the  class  I  regulatory  element  (CRE)  and  the  interferon  consensus  sequence 
(ICS)  that  are  5'  to  the  promoter.   Studies  using  cultures  of  neonatal 
astrocytes  transfected  with  constructs  consisting  of  portions  of  the  5' 
enhancers  coupled  to  a  reporter  gene,  chloramphenicol  acetyl  transferase, 
indicate  that  the  induction  of  class  I  MHC  molecules  is  mediated  through  the 
ICS.   In  contrast  to  all  other  cells  so  far  examined,  the  CRE  in  astrocytes 
has  minimal  enhancer  activity  and  may  even  contain  negative  regulatory 
elements.   Nuclear  binding  proteins  which  react  with  the  5'  enhancer  motifs 
are  being  characterized  using  gel  mobility  shift  assays.   Non-CNS  tissues 
express  high  levels  of  proteins  that  bind  to  the  CRE  region  I  and  the  ICS. 
These  proteins  are  undetectable  in  extracts  of  normal  brain,  but  are 
spontaneously  expressed  in  cultured  astrocytes  at  a  time  that  coincides  with 
the  production  of  class  I  molecules. 

Procedures  for  the  maintenance  of  AHGC  in  tissue  culture  have  been 
developed.   Treatment  of  AHGC  with  gamma-interf eron  leads  to  the  induction  of 
class  I  and  class  II  molecules  and  enables  these  cells  to  function  as 
immunological  targets  for  CD4+  and  CD8+  CTL.   Because  the  T  cell  response 
of  MS  patients  to  MBP  is  almost  entirely  HLA  class  II-restricted  (discussed 
below),  focus  is  being  placed  on  the  molecular  events  responsible  for  the 
regulation  of  these  molecules.   These  experiments  have  been  initiated  with 
established  human  glial  cell  lines  and  will  be  extended  to  AHGC  when 
sufficient  numbers  of  these  cells  become  available.   The  observations,  to 
date,  indicate  that  class  II  molecules  are  up-regulated  by  gamma-interf eron, 

3  -  NIB/DIR 


but  other  cytokines,   particularly  IL1,  down-regulate  the  expression  of  these 
molecules.   As  with  class  I  molecules,  the  regulation  of  class  II  molecules  is 
controlled  by  5'  enhancer  sequences.   The  molecular  sites  of  action  for  the 
various  cytokines  is  being  mapped  with  a  group  of  molecular  constructs  in 
collaboration  with  Dr.  Jeremy  Boss,  at  Emory  University.   The  preliminary 
findings  indicate  that  one  particular  regulatory  segment,  the  "Z  box",  has  a 
major  influence  on  the  regulation  of  these  molecules  by  gamma-interf eron.   The 
negative  influence  of  IL1  also  maps  to  the  same  5'  region.   This  suggests  that 
different  nuclear  binding  proteins  are  responsible  for  opposite  effects  of 
these  cytokines  on  class  II  regulation. 

In  an  elegant  series  of  investigations,  the  MIS  has  obtained  new  insight 
into  the  molecular  basis  of  antigen  presentation  by  class  I  molecules.   In 
these  experiments,  a  functional  peptide  competition  assay  was  used  to  assess 
the  presentation  of  the  influenza  matrix  (Ml)  protein  55-73  by  HLA-2.1 
molecules  to  CD8+  specific  CTL.   Initial  findings  showed  that  approximately 
25  percent  of  a  panel  of  unrelated  peptides  bound  to  HLA-A2.1  and  blocked 
presentation  of  the  Ml  peptide  to  CTL.   Subsequent  studies  examined  the 
capacity  of  these  peptides  to  bind  to  another  class  I  molecule,  HLA-B37. 
Although  hi   percent  of  the  same  panel  of  unrelated  peptides  bound  and 
inhibited  antigen  presentation  by  HLA-B37,  none  of  these  HLA-B37  binding 
peptides  were  in  the  group  initially  shown  to  bind  to  HLA-2.1  molecule.   These 
observations  demonstrate  that  individual  HLA  class  I  molecules  can  bind 
distinct  subsets  of  peptides. 

The  MIS  previously  demonstrated  that  the  fungal  antibiotic  Brefeldin  A 
(BFA)  abolishes  the  capacity  of  a  cell  to  present  endogenously  synthesized 
influenza  viral  peptides  to  class  I-restricted  CTL.   This  indicated  that  in 
virus-infected  cells,  viral  peptides  bind  to  the  class  I  molecules  in  the 
endoplasmic  reticulum  and  are  then  transferred  to  the  cell  surface  where 
recognition  by  CD8+  CTL  occurs.   During  FY  1990,  these  studies  were  extended 
to  class  II  molecules  using  the  influenza  matrix  protein  Ml.   Two  distinct 
pathways  involved  in  the  presentation  of  this  antigen  were  identified.   One  is 
an  exogenous,  chloroquine-sensitive,  BFA-insensitive  pathway  and  the  second  is 
an  endogenous  BFA-sensitive  pathway.   The  conclusion  from  these  studies  is 
that  endogenous  antigenic  peptides  can  associate  with  both  class  I  and  class 
II  molecules  in  the  endoplasmic  reticulum  and  are  subsequently  transferred  to 
the  cell  surface  for  recognition  by  TCR.   These  fundamental  observations  on 
antigen  presentation  and  recognition  have  major  implications  for  the 
assessment  of  immune  function  in  MS  and  HTLV-I-associated  myelopathy/tropical 
spastic  paraplegia  (HAM/TSP). 

The  CIS  previously  demonstrated  that  patients  with  MS  have  impaired 
capacity  to  generate  CTL  against  measles  virus.   The  measles  virus-specific 
CTL  can  be  identified  in  individuals  long  after  infection  and  are  CD'++,  HLA 
class  II-restricted.   Because  impaired  generation  of  measles  virus-specific 
CTL  is  the  only  known  antigen-specific  defect  in  MS,  the  CIS  has  examined  the 
significance  of  this  finding.   In  order  to  understand  the  generation  of 
measles-specific  T  cell  response,  adults  with  acute  measles  infection  have 
been  studied.   The  results  of  these  studies  indicate  that  the  infected 
individuals  who  are  apparent  vaccine  failures  are  fully  capable  of  responding 
to  measles  virus  after  natural  infection.   Thus,  their  failure  to  respond  to 
the  virus  after  vaccination  was  not  related  to  a  genetically  determined 

k   -   NIB/DIR 


impairment  in  immunity.   Immediately  following  acute  infection,  these 
individuals  develop  CD8+  class  I-restricted  CTL;  subsequently,  this  cellular 
response  disappears  and  is  replaced  by  circulating  precursors  of  CD4+  class 
II-restricted  CTL.   The  findings  are  important  in  understanding  mechanisms  of 
vaccine  failure  and  indicate  that  the  CD4+  class  II-restricted  CTL  response 
which  is  defective  in  MS  probably  contributes  to  the  development  of  long-term 
immunity  against  the  virus. 

MS  has  long  been  believed  to  have  an  immunopathological  component,  but  the 
antigen(s)  responsible  for  the  immunopathological  process  has  not  been 
identified.   A  leading  candidate  is  MBP,  and  during  FY  1990,  the  CIS  placed 
major  emphasis  on  characterizing  the  human  cellular  reaction  to  this 
molecule.   T  cell  lines  selected  by  repeated  antigenic  stimulation  with  the 
whole  MBP  molecule  have  been  used  to  identify  the  major  antigenic  epitopes. 
Two  major  immunodominant  regions  have  been  detected.   These  include  an  epitope 
in  residues  87-106  in  the  center  of  the  molecule  and  a  second  epitope  residing 
in  residues  154-172  in  the  C-terminal  portion.   Cell  lines  specific  for  these 
epitopes  have  been  derived  from  patients  with  MS  and  normal  individuals. 
Related  experiments  are  examining  the  HLA  molecules  that  present  these 
epitopes  and  the  TCR  molecules  involved  in  recognition.   The  findings  show 
that  at  least  three  different  HLA-DR  types,  each  of  which  is  over-represented 
in  certain  MS  populations,  can  present  the  epitope  contained  in  residues 
87-106  to  T  cells.   The  data  obtained,  to  date,  indicate  that  multiple  TCRs 
are  used.   The  implication  of  these  observations  is  that  in  humans  a  variety 
of  antigen  presentation  molecules  and  TCRs  are  used  to  react  with  MBP.   This 
is  in  contrast  to  findings  in  inbred  rodents  that  develop  experimental 
allergit  encephalomyelitis  (EAE).   In  both  rats  and  mice,  TCR  usage  has 
recently  been  shown  to  be  restricted  and  has  provided  the  rationale  for 
experimental  treatment  of  EAE  by  the  administration  of  monoclonal  antibodies 
to  the  TCR  or  vaccination  with  peptides  corresponding  to  the  sequence  of  the 
TCR  used  by  encephalitogenic  T  cells.   The  findings  by  the  CIS  indicate  that 
if  a  cellular  immune  response  contributes  to  the  pathogenesis  of  MS, 
experimental  treatment  of  this  disease  would  require  a  more  customized 
strategy  in  individual  patients. 

Recent  experiments  in  the  Autoimmune  Unit  of  the  NDS  are  relevant  to  the 
treatment  of  human  immunological  disorders.   This  group  has  obtained  data 
which  indicate  that  during  chronic-relapsing  EAE,  the  specificity  of  T  cell 
reactivity  shifts.   In  these  studies,  encephalitogenic  T  cells  against  one 
epitope  of  MBP  were  selected  in  vitro  by  repeated  antigenic  stimulation  with 
synthetic  peptides  of  the  encephalitogenic  epitope.   Transfer  of  these  cells 
into  normal  mice  produced  relapsing  EAE,  however,  T  cells  that  recognize  other 
encephalitogenic  epitopes  appeared  during  the  chronic  phase  of  the  disease. 
This  suggests  that  inhibition  of  immune  reactivity  to  a  single  epitope  may  not 
be  effective  for  treatment  of  the  chronic  disease.   An  alternative  would  be  to 
use  an  approach  that  down-regulates  immune  reactivity  without  dramatically 
producing  immunosuppression.   In  other  experiments,  this  group  has  demonstrated 
that  treatment  with  the  cytokine  transforming  growth  factor  beta  can  both 
prevent  EAE  and  when  given  to  mice,  after  an  initial  episode,  reduces  the 
severity  of  chronic-relapsing  disease. 

During  FY  1990,  considerable  research  has  been  conducted  on  human 
retroviruses  and  a  new  project  (Z01  NS  02817-01)  on  the  investigation  of  the 

5  -  NIB/DIR 


immune  reactivity  to  these  viruses  has  been  initiated.   In  previous  studies  of 
HAM/TSP,  patients  with  this  disorder  were  found  to  have  activated  lymphocytes 
in  the  blood  that  undergo  spontaneous  proliferation  in  vitro.   During  FY  1990, 
studies  of  normal  carriers  of  HTLV-I  performed  in  collaboration  with  Dr. 
William  A.  Blattner,  Chief  of  the  Viral  Epidemiology  Section,  EEB,  NCI  have 
shown  that  these  individuals  also  have  circulating  lymphocytes  that  undergo 
spontaneous  proliferation  in  tissue  culture  and  express  surface  molecules 
including  interleukin  2  receptor  consistent  with  lymphocyte  activation.   A  new 
study  has  been  initiated  in  collaboration  with  Dr.  Blattner 's  group  to  examine 
lymphocyte  function  in  individuals  persistently  infected  with  HTLV-II.   Over 
the  past  year,  it  has  become  apparent  that  this  virus  is  prevalent  in  users  of 
intravenous  drugs.   The  findings  indicate  that  blood  lymphocytes  from  carriers 
of  this  virus  spontaneously  also  proliferate  in  vitro.   The  molecular 
mechanisms  for  this  have  not  been  identified,  but  the  HTLV-I  5'  tax  protein  is 
believed  to  be  responsible  and  is  involved  in  the  trans-activation  of  a  number 
of  host  genes. 

Immunological  studies  of  HTLV-I  have  produced  a  number  of  new  findings.   T 
cell  lines  derived  from  patients  with  HAM/TSP  by  repetitive  stimulation  with 
autologous  infected  lymphocytes  react  with  the  envelope  protein  in  association 
with  class  II  HLA  molecules.   The  epitope  recognized  by  CD4+  CTL  has  been 
characterized  in  collaboration  with  Dr.  Thomas  Palker  at  Duke  University  and 
is  identical  to  an  immunodominant  region  of  the  envelope  protein  that  is  the 
major  site  for  antibody  recognition.   During  the  immune  response  to  most 
viruses,  T  cells  and  B  cells  respond  to  different  epitopes.   Consequently,  the 
demonstration  that  antibody  and  T  cells  react  to  the  same  region  may  be  a 
unique  aspect  of  immune  reactivity  to  human  retroviruses  because  similar 
observations  have  been  made  in  HIV  infection. 

A  second  group  of  experiments  conducted  in  collaboration  with  Drs.  Scott 
Koenig  and  Anthony  Fauci  in  the  Laboratory  of  Immunoregulation,  NIAID,  have 
identified  the  presence  of  circulating  CD8+  class  I-restricted  CTL  in 
patients  with  HAM/TSP.   The  presence  of  these  CTL  in  circulating  blood 
indicate  that  these  cells  must  persist  in  high  concentrations  in  this 
disease.   Of  particular  interest  is  that  circulating  CTL  have  not  been  found 
in  the  blood  of  normal  carriers  of  HTLV-I  or  patients  with  ATL  associated  with 
HTLV-I.   The  peptide  specificity  of  the  class  I  restricted  CD8+  CTL  has  been 
investigated  in  transfection  experiments  using  vaccinia  constructs  coupled  to 
genes  for  individuals  HTLV-I  peptides.   A  second  striking  finding  is  that 
circulating  CTL  are  largely  directed  to  the  tax  protein  specified  by  3' 
regulatory  regions  of  the  virus.   This  series  of  observations  has  led  to  the 
hypothesis  that  HAM/TSP  has  an  immunopathological  component  consisting  of  CTL 
reactivity  to  regulatory  proteins  encoded  by  the  virus. 


NIB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS   02202-15  NI 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  '80  characters  or  less    Title  must  tit  on  one  line  between  the  borders.) 

Immunological  Studies  in  Patients  with  Multiple  Sclerosis  and  Other  CNS  Diseases 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  ana  institute  affiliation) 


PI:   Dale  E.  McFarlin,  M.D. 
Others: 

Henry  F.  McFarland,  M.D. 

Ajay  Gupta,  M.D. 

David  Mattson,  M.D.,  Ph.D. 

Jonathan  Harris,  M.D. 

Joseph  A.  Frank,  M.D. 

Elliot  Cowan,  Ph.D. 


Chief 

Deputy  Chief 
Visiting  Associate 
Medical  Staff  Fellow 
Clinical  Associate 

Radiologist 
Special  Expert 


NI 


DIR 


NINDS 


NI 

DIR 

NINDS 

NI 

DIR 

NINDS 

NI 

DIR 

NINDS 

NI 

DIR 

NINDS 

DR 

CC 

NI 

DIR 

NINDS 

COOPERATING  UNITS  (if  any) 

Leroy  Hood,  M.D.,  Chairman,  Dept.  of  Biology,  Cal.  Institute  of  Technology;  Steven 
Beall,  M.D.,  Cal.  Institute  of  Technology;  Nicholas  Patronas,  M.D.,  Dept. 
of  Diagnostic  Radiology,  Georgetown  University. 


LAB/BRANCH 

Neuroimmunology,    CNP 


SECTION 

Office   of    the   Chief 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS 

12.0 


PROFESSIONAL: 
8.0 


OTHER 
4.0 


CHECK  APPROPRIATE  BOX(ES) 

53   (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


E  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  is  designed  to  evaluate  multiple  factors  related,  either  singly  or 
in  combination,  to  the  pathogenesis  of  neurological  diseases.   Focus  is  currently 
placed  on  multiple  sclerosis  (MS).   Magnetic  resonance  imaging  (MRI )  is  important 
in  the  assessment  of  MS  and  the  recent  detection  of  gadolinium-enhancing  lesions 
by  this  technology  provides  evidence  that  an  early  event  in  the  pathogenesis  is 
breakdown  of  the  blood-brain  barrier.   To  determine  the  frequency  and  natural 
history  of  such  lesions,  a  group  of  patients  with  mild,  early  MS  are  being  studied 
monthly.   MRI  studies  are  being  conducted  in  familial  leukodystrophy. 

Particular  attention  is  being  given  to  genetic  and  immunological  factors  which 
contribute  to  pathogenesis  of  MS.   Genes  encoding  for  HLA  molecules  and  the  T-cell 
receptors  are  being  evaluated  in  patients  with  sporadic  disease  and  families  witn 
multiple  affected  members. 

Trials  of  experimental  therapeutic  agents  for  MS  are  conducted.   The  findings  from 
MRI  investigations  with  gadolinium  enhancement  are  being  used  to  develop 
strategies  for  the  future  evaluation  of  new  treatments.   A  recent  double-blind 
study  of  cyclosporine  A  showed  that  patients  treated  with  this  agent  progress  less 
rapidly  than  patients  who  received  placebo;  however,  renal  toxicity  was  a 
significant  side  effect.   The  relationship  between  cyclosporine  A  treatment  and 
nephrotoxicity  is  currently  being  assessed  in  an  open-label  study. 

The  chronic  myelopathy,  HTLV-I-associated  myelopathy/t ropical  spastic  paraplegia 
( HAM/ T SP )  is  being  investigated.   Immunological,  clinical,  and  MRI  findings  in 
HAM/TSP  are  being  compared  to  normal  carrier  of  HTLV-I. 

7   -   NIB/DIR 


PHS  6040  (Rev.  1/84) 


GPO  91  4-9K 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS   02203-15  NI 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  tit  on  one  line  between  the  borders.) 

Membrane  Molecules  Involved  in  Immune  Reactivity 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

PI:   Dale  E.  McFarlin,  M.D.         Chief  NI   DIR   NINDS 


COOPERATING  UNITS  (it  any) 


LAB/BRANCH 

Neuroimmunology,  CNP 


SECTION 

Office   of    the   Chief 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 
0 


PROFESSIONAL: 


0 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


□  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

This  project  was  terminated  10/89. 


8   -  NIB/DIR 


PHS  6040  (Rev.  1/84) 


GPO   Bl  4-918 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  •  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01   NS   02204-15  NI 


PERIOD  COVERED 

lOctober   1,    1989   through   September   30,    1990 


j  title  of  project  ao  characters  or  lass  Tula  must  in  on  on»  iir*  Datwten  tnt  borders  i   Immunologic  Mechanisms 
Operative    in   Experimental  Autoimmune   Diseases  of    the   Nervous   System 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator  )  (Same   Me.  laboratory,  ana  institute  affiliation) 


PI:      Dale    £.   McFarlin,    M.D. 
I    Others: 

Michael    R.    Racke,    M.D. 
Robert    Fallis,    M.D. 
Paul   rtassa,    Ph.D. 
Mary   E.    Smith,    M.D. 


Chief 

Special  Volunteer 
Senior  Staff  Fellow 
Senior  Staff  Fellow 
Clinical  Associate 


NI 


DIR 


NINDS 


NI 

DIR 

NINDS 

NI 

DIR 

NINDS 

NI 

DIR 

NINDS 

NI 

DIR 

NINDS 

8eTrEfcT'^UR'aVnen,WPh.D.,  Professor,  Albert  Einstein  U.;  Maria  Spatz,  M.D. ,  Section 
Chief,  LNNS,  DIR,  NINDS;  Richard  McCarron,  Ph.D.,  Senior  Staff  Fellow,  LNNS,  DIR, 
NINDS;  Keiko  Ozato,  Ph.D.,  Section  Chief,  LDMI,  DIR,  NICHHD 


|  LAB/BRANCH 

iNeuroimmunology,    CNP 


SECTION 

Neurological  Diseases  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS 
3.5 


PROFESSIONAL 
2.0 


OTHER: 

1.5 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)  Human  subjects 
D  (a1)  Minors 
n  (a2)  Interviews 


D  (b)  Human  tissues     LXJ  (c)  Neither 


SUMMARY  OF  WORK  (Use  stanaera  unreduced  type  Do  not  exceed  the  space  provided) 
Experimental  allergic  encephalomyelitis  (EAE),  a  model  of  autoimmune  disease 
is  being  used  to  study  immune  reactivity  and  inflammation  in  the  central 
nervous  system  (CNS).   Current  research  is  focused  on  a  model  produced  in 
syngeneic  animals  by  the  transfer  of  lymphocytes  sensitized  against  myelin 
basic  protein.   Under  optimal  conditions,  neurological  dysfunction  occurs; 
this  is  characterized  pathologically  by  inflammation  and  primary 
demyelinat ion.   Many  mice  recover  and  develop  chronic-relapsing  disease.   The 
immunological  mechanisms  responsible  for  both  the  initial  and  the  chronic 
disease  are  being  investigated.   An  early  event  is  the  migration  of  immune 
cells  across  the  blood-brain  barrier  into  the  CNS.   Initially,  there  is 
interaction  with  the  capillary  endothelial  cells,  but  astrocytes  and  microglia 
are  in  close  proximity.   Antigen  presentation  by  microglia,  brain  capillary 
endothelial ,  and  astrocytes  are  being  compared.   The  regulation  and  expression 
of  MHC  molecules  in  these  cells  is  also  being  evaluated.   The  studies  are 
Deing  conducted  with  cultures  of  murine  fetal  CNS  cells,  adult  human  glia 
cells,  and  human  cell  lines  derived  from  glioblastomas.   The  expression  and 
regulation  of  MHC  molecules  by  neurons  is  also  being  studied  in  fetal  murine 
cultures  and  human  medul loblastoma  cell  lines.   The  effect  of  various 
cytokines  on  chronic-relapsing  EA£  is  being  studied  in  order  to  gain  insight 
to  the  pathogenic  mechanisms  involved  and  to  provide  rationale  for 
experimental  treatment  of  human  demyelinat ing  disorders. 


9    -   NIB/DIR 


PHS  6040  (Rev    t(84i 


SPO   >I44H 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02205-15  NI 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  tit  on  one  line  between  the  borders.) 

Interactions  Between  the  Human  Immune  System  and  Antigens  in  the  Nervous  System  * 


PRINCIPAL  INVESTIGATOR  (List  other  protessional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory. 


PI:   Henry  F.  McFarland,  M.D. 
Others: 

Dale  E.  McFarlin,  M.D. 

William  E.  Biddison,  Ph.D. 

Suhayl  Dhib-Jalbut,  M.D. 

Shari  de  Silva,  M.D. 

Rhonda  Voskuhl,  M.D. 

Roland  Martin,  M.D. 


Section  Chief 

Chief 

Section  Chief 
Senior  Staff  Fellow 
Clinical  Associate 
Clinical  Associate 
Special  Volunteer 


NI 


and  institute  affiliation) 
DIR  NINDS 


NI 

DIR 

NINDS 

NI 

DIR 

NINDS 

NI 

DIR 

NINDS 

NI 

DIR 

NINDS 

NI 

DIR 

NINDS 

NI 

DIR 

NINDS 

COOPERATING  UNITS  (if  any)  .  _  _  .  n     -cc 

John  R.  Richert,  M.D. ,  Assist.  Prof.,  Georgetown  U. ;  Diane  Griff 
Prof.,  Dept.  Neurology,  Johns  Hopkins  U. ;  Richard  Johnson,  M.D 
Neurology,  Johns  Hopkins  U. ;  Eric  Long,  Ph.D.  Visiting  Scientist 


in,  M.D.,  Ph.D. , 
Chairman,  Dept. 
BRB,  DIR,  NIAID 


LAB/BRANCH 

Neuro immunology,    CNP 


SECTION 

Cellular  Immunology  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 
4.5 


PROFESSIONAL: 
4.0 


OTHER: 
0.5 


CHECK  APPROPRIATE  BOX(ES) 

□  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


HI   (b)  Human  tissues  □   (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  purpose  of  this  project  is  to  examine  the  manner  in  which  immunological 
mechanisms  may  contribute  to  diseases  of  the  central  nervous  system  (CNS)  in 
humans.   These  studies  included  examination  of  the  cellular  immune  response  to 
viruses  which  can  commonly  effect  the  CNS  and  which  may  be  related  to  the 
induction  of  autoimmune  processes  of  the  nervous  system.   In  addition,  these 
studies  have  examined  the  immune  response,  and  in  particular,  the  cellular 
immune  response  to  antigens  of  the  nervous  system  such  as  myelin  basic  protein 
and  proteolipid  protein.   The  emphasis  in  these  studies  has  been  on 
identifying  differences  which  may  occur  in  patients  with  diseases  of  the  CNS 
such  as  multiple  sclerosis  as  compared  to  healthy  individuals.   Particular 
attention  has  been  paid  to  the  influence  of  genetic  makeup  on  both  the 
induction  and  effector  phases  of  the  immune  response  to  viruses  as  well  as 
antigens  of  the  CNS.   Focus  has  been  placed  on  identification  of  cytotoxic  T 
cells  specific  for  both  viruses  or  antigens  of  the  nervous  system.   In 
addition,  the  capacity  of  cells  of  the  CNS  to  process  and  present  antigens  to 
immune  T  cells  has  been  examined  in  order  to  help  understand  how  immunological 
mechanisms  are  induced  within  the  nervous  system. 

*  (Formerly,  "Interaction  Between  Viruses  and  the  Host  Immune  System") 


10   -  NIB/DIR 


PHS  6040  (Rev   1/84) 


GPO  914-918 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS    02603-07   NI 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  tit  on  one  line  between  the  borders  ) 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  ana  institute  affiliation) 


PI:   William  E.  Biddison,  Ph.D. 
Others: 

Beatriz  Carreno,  Ph.D. 

Ursula  Utz,  Ph.D. 

Lael  A.  Stone,  M.D. 


Section  Chief 

Visiting  Fellow 
Special  Volunteer 
Special  Volunteer 


NI    DIR 


NINDS 


NI 

DIR 

NINDS 

NI 

DIR 

NINDS 

NI 

DIR 

NINDS 

COOPERATING  UNITS  (it  any)  ,  „  ,  „,_        „,.__        „  .     ,  ,    _ 

John  E.  Coligan,  Ph.D.,  Chief,  Biological  Resources  Branch,  DIR,  NIAID;  Richard  D. 
Klausner,  M.D.,  Chief,  Cell  Biology  and  Metabolism  Branch,  NICHD;  Leroy  Hood,  M.D., 
Ph.D.,  Professor,  Department  of  Biology,  California  Institute  of  Technology 


LAB/BRANCH 

Neuro immunology,    CNP 


SECTION 

Molecular  Immunology  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 
6.0 


PROFESSIONAL: 

3.0 


OTHER: 
3.0 


CHECK  APPROPRIATE  BOX(ES) 

EX  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


£3  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type  Do  not  exceed  the  space  provided ) 

The  general  objective  of  this  project  is  to  define  the  mechanisms  by  which 
human  lymphoid  cells  interact  with  antigen-presenting  cells  in  order  to 
produce  and  regulace  immune  responses.   Over  the  past  year,  there  have  been 
three  major  efforts  underway  that  are  targeted  on  this  objective:   1) 
dissection  of  tne  molecular  basis  tor  T  cell  recognition  of  antigens  presented 
by  riLA  class  I  molecules;  2)  determination  of  endogenous  versus  exogenous 
antigen  presentation  pathways  for  class  I  and  II  HLA  molecules;  and  3) 
analysis  of  the  potential  roles  of  human  T  cell  receptor  (TCR)  alpha  and  beta 
cnain  germline  genes  and  HLA  genes  in  susceptibility  to  multiple  sclerosis 
(MS).   The  principle  findings  are  as  follows:  1)  two  different  class  I 
molecules,  HLA-A2 . 1  and  HLA-B37,  bind  largely  nonover lapping  sets  of  peptides; 
2)  the  kinetics  of  peptide  binding  to  RL.A-A2  and  the  conformation  of  the 
peptide-A2  complex  can  be  determined  Dy  amino  acid  side  chains  on  the  floor  of 
the  peptide  binding  groove;  3)  class  II  HLA  molecules  can  use  the  endogenous 
pathway  of  antigen  presentation;  and  4)  in  certain  families  that  have  multiple 
members  with  MS,  TCR  and/or  HLA  genes  can  be  shown  to  be  associated  with 
susceptibility  to  MS. 


11   -  NIB/DIR 


PHS  6040  (Rev    1/84) 


GPO  914-018 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS   02817-01  NI 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  tit  on  one  line  between  the  borders- )     Immunologic,     Virologic,     and 

Molecular  Aspects  of  Retroviruses  Associated  with  Chronic  Neurologic  Diseases 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator)  (Name,  title,  laboratory,  and  institute  affiliation) 


PI:   Steven  Jacobson,  Ph.D. 
Others: 

Dale  £.  McFarlin,  M.D. 

Masataka  Nishimura,  M.D. 

Scott  Koenig,  M.D. 

Suhayl  Dhib-Jalbut,  M.D. 

Thomas  Palker,  Ph.D. 

Cedric  S.  Raine,  Ph.D. 


Senior  Staff  Fellow 


NI 


DIR 


NINDS 


Chief 

NI 

DIR 

NINDS 

Visiting  Fellow 

NI 

DIR 

NINDS 

Senior  Staff  Fellow 

I 

DIR 

NIAID 

Senior  Staff  Fellow 

NI 

DIR 

NINDS 

Assoc.  Professor 

Duke 

University 

Professor 

Albert  Einstein  U. 

COOPERATING  UNITS  (if  any) 

Dr.  William  Blattner,  Chief,  VES,  NCI;  Dr.  Ashley  Haase,  Chairman,  Dept. 


of 


Microbiology,  University  of  Minnesota;  Dr.  Thomas  Waldmann,  Chief,  MET  Branch, 
NCI,  Dr.  Anthony  Fauci,  Director,  NIAID 


LAB/BRANCH 

Neuroimmunology,    CNP 


SECTION 

Neurological  Disease  Section 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 

5.0 


PROFESSIONAL: 
2.5 


OTHER: 
2.5 


CHECK  APPROPRIATE  BOX(ES) 

E  (a)  Human  subjects 

□  (a1)  Minors 

□  (a2)  Interviews 


Q  (b)  Human  tissues  □  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  objectives  of  this  project  are  to  assess  the  involvement  of  human 
retroviruses  in  chronic-progressive  rayeloneuropathy  termed  HTLV-I-related 
myelopathy /tropical  spastic  paraparesis  (HAM/ISP)  and  multiple  sclerosis. 
These  investigations  have  two  major  components:  1)  the  molecular/viro logical 
identification  of  the  virus(es)  that  are  associated  with  the  pathogenesis  of 
these  disorders,  and  2)  the  immunological  response  of  the  host  to  the 
identified  agent(s).   Parallel  studies  are  being  conducted  in  normal  carriers 
of  HTLV-I. 

Human  retroviruses  are  isolated  from  cells  in  the  peripheral  blood  and 
cerebrospinal  fluid.   The  polymerase  chain  reaction  is  used  to  detect  and 
characterize  viral  genes  in  lymphocytes  and  in  the  central  nervous  system. 
These  viral  genomes  are  being  cloned  and  sequenced.   Tissue  sections  from 
spinal  cord  and  brain  from  individuals  with  neurological  diseases  also  are 
analyzed  for  the  presence  of  retroviral  DNA  by  in  situ  hybridization.   Immune 
reactivity  to  these  agents  are  characterized  by  demonstrating  virus-specific 
cytotoxic  T  cell  (CTL)  responses  in  these  patients,  as  well  as 
lymphoproliferative  responses.   CTL  are  generated  from  long-term  cultures, 
addition,  the  presence  of  CTL  in  the  peripheral  blood  of  individuals  with 
active  infection  is  being  examined.   The  responding  cells  and  the  antigenic 
specificity  of  the  response  are  being  characterized. 


In 


12   -  NIB/DIR 


PHS  6040  (Rev.  1/84) 


GPO   9  1  4-9)  S 


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ANNUAL  REPORT 

October  1,  1989  through  September  30,  1990 

Surgical  Neurology  Branch  -  Clinical  Neurosciences  Program,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 

Edward  H  Oldfield,  M.D.,  Chief 

Table  of  Contents 

Summary  of  Studies 1 

I.  Clinical  Neurosurgery  Section 6 

II.  Biochemistry  Section 14 

III.  Central  Nervous  System  Implantation  Unit 18 

IV.  Tumor  Biology  Unit 21 

V.  Molecular  Biology  Unit 22 

VI.  Brain  Imaging  Unit 25 

PROJECT  REPORTS 

Studies  of  Human  Pituitary  Tumors 

201  NS02454-010SN-PI:  Oldfield  EH 27 

Pharmacokinetics  of  Direct  Brain  Infusion 

Z01  NS  02813-01  SN-PI:  Bobo  RH 28 

Pentobarbital  Effects  on  Damage  of  the  Primate  Brain  by  Fractionated  Whole  Brain  Radiation 

Z01  NS  02812-01  SN-PI:  Bobo  RH 29 

Phase  II  Clinical  Trials  of  Suramin  and  Hydrocortisone  in  the  Therapy  of  Current  Malignant  Gliomas 

201  NS  0281 1-01  -SN-PI:  Bobo  RH 30 

Protection  of  the  Brain  Against  Injury  by  Ionizing  Radiation  with  Pentobarbital 

Z01  NS02697-06SN-PI:  Olson  JJ 31 

Studies  on  the  Heterogeneity  of  Drug  Delivery  During  Intracarotid  Chemotherapy 

Z01  NS  02728-04  SN-PI:  Saris  S 32 

Adoptive  Immunotherapy  of  Brain  Tumors 

Z01  NS  02778-03  SN-PI:  Saris  S 33 

Monoclonal  Antibodies  Linked  to  Ricin  for  Use  in  Human  Bone  Marrow  Transplantation 

Z01  NS02673-06SN-PI:  Youle  RJ        34 

Antibody-Toxin  Conjugates  for  the  Treatment  of  Human  Brain  Tumors 

Z01  NS  02823-01  SN-PI:  Youle  RJ   35 

Monoclonal  Antibody-Toxin  Conjugates  for  Tumor  Therapy  in  vivo 

Z01  NS  02674-06  SN-PI:  Youle  RJ  36 

Tissue  Implantation  in  Parkinsonian  Models 

Z01  NS  02781-03  SN-PI:  Plunkett  RJ 37 

Adrenal  Medullary  Autografts  in  Parkinsonian  Patients 

Z01  NS  02729-04  SN-PI:  Plunkett  RJ 38 

Vascular  Permeability  Factor  Produced  by  Human  Glioma  Cells 

Z01  NS  02708-05  SN-PI:  Merrill  M 39 

The  Role  of  Insulin  and  Insulin-like  Growth  Factors  in  Glioma  Cells 

Z01  NS  02707-05  SN-PI:  Merrill  M 40 

Genetic  Abnormalities  in  Primary  Glial  Tumors 

Z01  NS  02814-01  SN-PI:  AM  1 41 

Molecular  Genetics  of  Pituitary  Conticotroph  Adenomas 

Z01  NS  02815-01  SN-PI:  Alii 42 

Metabolic  Mapping  of  the  Brain  During  Rewarding  Brain  Stimulation 

Z01  NS  02760-03  SN  -  PI:  Porrino  L 43 

Quantitative  Autoradiographic  Determination  of  Dopamine  Receptor  Distribution  in  Primates 

Z01  NS  02762-03  SN-PI:  Porrino  L 44 

Metabolic  Mapping  of  Animal  Models  of  Parkinsonism 

Z01  NS  02761-03  SN-PI:  Porrino  L 45 

Clinical  and  Laboratory  Investigation  of  Central  Nervous  System  Vascular  Disorders 

Z01  NS  02739-04  SN-PI:  Oldfield  EH 46 


i    SNB/DIR 


October  1,  1989  through  September  30,  1990 

Surgical  Neurology  Branch,  Division  of  Intramural  Research 

Clinical  Neurosciences  Program 

National  Institute  of  Neurological  Disorders  and  Stroke 

Edward  H.  Oldfield,  M.D.  -  Chief 

SUMMARY  OF  STUDIES 

The  Surgical  Neurology  Branch  (SNB)  principal  activities  are  conducted  in  the 
following:  I)  Clinical  Neurosurgery  Section;  II)  Biochemistry  Section;  III)  Central 
Nervous  System  Implantation  Unit;  IV)  Tumor  Biology  Unit;  V)  Molecular  Biology 
Unit;  VI)  Brain  Imaging  Unit. 

The  Surgical  Neurology  Branch  has  as  its  major  research  functions:  (1)  the  study 
of  the  biology  and  therapeutic  approaches  to  malignant  and  benign  tumors  of  the 
brain  and  pituitary  gland;  (2)  investigation  of  the  capability  of  implanted  tissues  into 
the  brain  of  animals  and  humans  to  survive  and  integrate  anatomically  or 
biochemically  with  the  host  brain  to  influence  brain  function;  (3)  the  investigation 
of  certain  vascular  disorders  of  the  Central  Nervous  System  (CNS);  and  (4)  the 
investigation  and  surgical  treatment  of  patients  with  epilepsy.  Its  clinical  function  is 
to  provide  the  neurosurgical  services  to  its  own  research  protocol  patients  and  to 
patients  seen  in  consultation  in  the  NIH,  Clinical  Center.  The  SNB  is  presently  located 
in  Buildings  9  and  10.  Its  staff  includes  14  clinical  neurosurgeons  at  various  levels  of 
training  and  experience,  as  well  as  2  senior  scientist,  3  junior  scientists  and  a  support 
staff  of  scientific,  technical  and  administrative  individuals. 

In  addition  to  its  primary  functions  of  clinical  and  basic  research,  the  SNB 
provides  young  neurosurgeons  with  experience  in  clinical  and  laboratory 
investigation  in  a  combined  clinical  and  neuroscience  environment.  Of  those  who 
have  participated  in  the  SNB  program  as  Medical  Staff  Fellows  and  Senior  Staff 
Fellows,  many  have  entered  academic  positions  in  neurosurgery. 

The  following  significant  observations  have  been  made  during  the  past  24 
months.  In  the  area  of  brain  tumors  we  have  made  several  observations.  Using 
Positron  Emission  Tomography  (PET)  scanning  and  positron-labeled  fluoro-2-deoxy- 
glucose,  we  previously  demonstrated  that  thiopental  selectively  suppresses  brain 
metabolism  while  leaving  tumor  metabolism  unaffected.  Similar,  but  less 
suppression  was  demonstrated  with  sedative  doses  of  pentobarbital.  Means  to 
exploit  this  capacity  to  selectively  influence  brain  metabolism  without  effecting 
tumor  metabolism  for  therapy  were  then  investigated. 

Early  and  delayed  cerebral  radiation  toxicity  in  rats  was  prevented  by 
pretreatment  with  pentobarbital.  Survival  of  animals  receiving  whole  brain 
irradiation  was  increased  from  0-20%  to  45-70%  by  sedation  with  pentobarbital  just 
before  irradiation  exposure.  The  mechanism  of  this  protection  was  investigated  and 
shown  to  be  linked  to  interaction  of  barbiturates  with  the  GABA  receptor.  Other 
agents  which  functionally  bind  the  GABA  receptor  were  shown  to  be  equally 
effective.  Since  tumors  lack  functional  GABA  receptors,  and  tumor  metabolism  had 
been  shown  not  to  be  influenced  by  barbiturates,  animals  with  implanted  tumors 
were  investigated.  No  reduction  of  tumor  sensitivity  was  demonstrated.  Hence, 
pentobarbital  suppressed  metabolism  of  normal  brain  and  protected  against 


1  SNB/DIR 


radiation  toxicity,  but  tumors  had  no  metabolic  suppression  and  were  not  protected 
against  irradiation  injury. 

Further  progress  was  made  in  the  development  of  immunotoxins  for  treatment 
of  CNS  tumors.  The  major  goal  of  this  project  is  to  develop  immunotoxins  which  will 
selectively  kill  tumor  cells  in  vivo.  An  antitransferrin  receptor  antibody-toxic  peptide 
conjugate  was  created  against  the  transferrin  receptor  which  specifically  kills  tumor 
cells  in  vitro  and  also  when  injected  into  the  cerebrospinal  fluid  (CSF).  A  cloned 
diphtheria  toxin  was  created  in  which  the  C-terminal  1 5  kD  region  was  deleted.  This 
eliminated  much  of  the  nonspecific  binding  of  the  diphtheria  toxin,  but  retained  the 
entry  function  of  the  toxin  into  the  cytosol  of  cells  and  retained  toxicity.  Recent 
results  indicate  that  point  mutations  can  increase  selective  cytotoxicity  of  antibody- 
toxin  conjugates  up  to  10,000-fold.  Animal  studies  indicated  a  5-log  kill  of  tumor 
cells  inoculated  into  the  peritoneal  cavity  in  vivo.  Utility  of  the  immunotoxins  in 
animal  models  of  leptomeningeal  tumor  was  demonstrated.  Treatment  of  tumor  in 
a  guinea  pig  model  of  leptomeningeal  neoplasia  demonstrated  a  2-  to  3-log,  and  in 
some  animals  a  5-log  kill  of  tumor  cells  in  the  CNS  with  this  conjugate.  Clinical  trials 
in  patients  with  carcinomatous  meningitis  began  this  year  with  a  monoclonal 
antibody  against  the  human  transferrin  receptor  linked  to  recombinant  ricin  A 
chain.  Five  patients  have  been  treated  in  a  phase  I  trial  without  toxicity. 

Several  investigations  of  adoptive  immunotherapy  for  treatment  of  malignant 
brain  tumors  were  completed.  Techniques  were  perfected  to  successfully  implant 
reproducible  lymphokine  activated  killer  (LAK)  cells  into  the  brain  of  rodents  and 
primates.  Clinical  investigations  of  adoptive  immunotherapy  using  interleukin-2 
(IL2)and  LAK  against  cerebral  gliomas  indicated  that  repeated  injections  of  IL2  and 
LAK  into  tumor  cavities  or  directly  into  brain  tumors  produced  increased  cerebral 
edema  and  transient  or  permanent  new  neurologic  deficits  in  all  patients. 
Investigation  of  intravenous  IL2  therapy  in  patients  with  glioblastomas 
demonstrated  increased  edema  from  the  tumor-bearing  region.  The  results  of  these 
studies  indicate  that  adoptive  immunotherapy  using  IL2  and  LAK  cells  produces 
significant  toxicity  and  rarely  elicits  a  measurable  tumor  response.  In  separate 
clinical  studies,  IL2  was  (1)  demonstrated  to  penetrate  the  blood-CSF  barrier  in 
patients  without  CNS  tumor  involvement  in  sufficient  concentration  to  maintain 
activity  of  LAK  cells  in  the  CSF,  and  (2)  using  magnetic  resonance  imaging  (MRI) 
scanning,  IL2  was  shown  to  increase  the  water  content  of  the  normal  brain, 
suggesting  alteration  of  the  normal  blood-brain-barrier  by  IL2.  Animal  experiments 
indicated  that  the  cerebral  edema  results  from  IL2-induced  increased  vascular 
permeability  in  the  vessels  of  the  tumor  and  the  surrounding  brain.  It  was  shown  in 
an  animal  model  of  cerebral  metastases  that,  although  lung  metastases  responded 
to  I L2/LAK  treatment,  brain  deposits  of  the  same  tumor  in  the  same  animal  had  no 
response  to  treatment,  indicating  that  the  blood-brain-tumor  barrier  restricts 
effectiveness  of  immunotherapy  of  this  type.  The  requirement  of  continued 
exposure  of  LAK  cells  to  IL2  for  maintenance  of  optimal  cytotoxicity  was 
demonstrated  in  an  in  vitro  analysis  of  LAK  cytotoxicity  against  glioma  cells. 
Intraarterial  infusion  of  IL2  was  shown  to  increase  delivery  of  IL2  to  brain  by  20-fold 
compared  to  intravenous  infusion. 

In  addition,  the  importance  of  aminoacid  309  of  the  B  chain  of  diphtheria  toxin 
for  translocation  of  the  toxin  across  cell  membranes  was  demonstrated;  studies  were 
initiated  to  examine  the  pharmacokinetics  of  immunotoxins  after  direct  infusion 
into  the  brain  and  tumors;  and  ways  to  block  the  immune  response  to  immunotoxins 
were  found.  That  drug  streaming,  which  may  cause  toxicity  and  limit  efficacy  after 
intraarterial  infusion  of  chemotherapy,  can  be  eliminated  in  humans  by  phased 


2SNB/DIR 


diastolic  delivery  was  demonstrated  in  humans  using  PET  scanning  and  positron- 
labeled  water  to  demonstrate  the  distribution  and  to  quantify  the  delivery  of 
infusate  to  the  brain  after  intracarotid  delivery.  Patients  treated  with  intracarotid 
cisplatin  using  a  prototype  pump  which  delivers  drug  into  the  internal  carotid  artery 
only  during  a  brief  period  of  diastole,  so  that  drug  streaming  is  eliminated,  had  no 
retinal  or  neural  toxicity  related  to  drug  streaming.  However,  a  trial  of  intraarterial 
high  dose  BCNU  via  phased  diastolic  delivery  in  patients  with  malignant  glioma 
demonstrated  retinal  and  brain  toxicity  despite  the  elimination  of  drug  streaming. 

A  protein  that  is  secreted  by  malignant  brain  tumors  and  increases  vascular 
permeability  to  macromolecules  was  isolated  from  the  incubation  media  from 
malignant  brain  tumors.  We  have  demonstrated  that  this  material  is  a  protein  of 
approximately  45,000  daltons  molecular  weight  and  that  its  secretion  is  eliminated 
by  incubation  of  the  tumor  cells  with  dexamethasone.  Further  experiments 
indicated  that  it  binds  a  heparin-like  moiety  on  the  cell  surface  of  endothelial  cells 
and  that  influx  of  Ca2  +  into  the  endothelial  cell  occurs  in  response  to  this 
interaction.  These  observations  explain  the  origin  of  brain  edema  in  patients  with 
brain  tumors  and  may  permit  development  of  therapy  against  tumor-associated 
cerebral  edema,  which  now  requires  high  dose  glucocorticoid  therapy  and  is 
associated  with  significant  complications.  Recent  advances  have  shown  that  this 
protein  is  the  same  molecule  as  pituitary-derived  vascular  endothelial  growth  factor, 
a  potent  mitogen  selective  for  endothelial  cells.  These  observations  indicate  that 
vascular  permeability  factor  (VPF)  secreted  by  malignant  brain  tumors  may  be 
responsible  for  brain  edema  and  tumor  angiogenesis. 

Insulin  and  the  insulin-like  growth  factors  1  and  2  (IGF-1  and  IGF-2)  were 
investigated  for  a  role  in  the  growth  of  malignant  gliomas.  High  levels  of  IGF-1 
receptors  were  demonstrated  on  several  gliomas  and  shown  to  be  functional  as 
determined  by  autophosphorylation  of  the  receptor  and  DNA  synthesis. 
Additionally,  high  levels  of  high  affinity  IGFI-binding  proteins  were  shown  to  be 
produced  by  human  glioma  cells  in  vitro  and  in  vivo.  These  results  suggest  a  role  of 
IGF-1  and  its  receptor  in  the  regulation  of  glioma  growth.  The  clinical,  genetic  and 
radiographic  features  of  patients  with  symptomatic  von  Hippel-Lindau  disease  (VHL) 
were  investigated.  In  collaboration  with  Drs.  Bert  Zbar  and  Marston  Linehan  of  NCI 
and  Dr.  Michelle  Filling-Katz  of  NINDS,  we  investigated  the  genetics  of  9  tumors 
from  5  patients  with  VHL  disease  and  hemangioblastomas  of  the  brain  and  spinal 
cord.  The  tumors  were  shown  to  have  absence  of  the  wild  type  (normal)  allele  and 
were  shown  to  arise  by  a  mechanism  analogous  to  that  of  familial  retinoblastomas, 
i.e.,  by  homozygous  absence  of  a  tumor-suppressing  gene.  In  addition,  the  same 
genetic  abnormality  was  shown  in  sporadic  hemangioblastomas  of  the  CNS.  MRI 
scanning  was  used  to  demonstrate  the  incidence  of  syrhingomyelia  and  the  extent  of 
the  syrhingomyelia  cavity  in  patients  with  hemangioblastomas  of  the  spinal  cord. 
MRI  with  Gadolinium  Ga-EDTA  contrast  enhancement  was  used  to  investigate  the 
brain  and  spinal  cord  in  VHL  disease  and  shown  to  the  more  sensitive  than  MRI 
without  Gd-EDTA  and  CT  scanning,  and  to  have  similar  sensitivity  as  arteriography, 
for  tumor  detection.  The  syrhingomyelia  that  is  associated  with  most 
hemangioblastomas  of  the  spinal  cord,  and  which  is  responsible  forthe  neurologic 
deficit  in  many  patients,  was  shown  to  resolve  with  treatment  directed  only  at  tumor 
removal  and  not  to  require  separate  consideration  for  treatment.  A  previously 
unrecognized  distribution  of  hemangioblastomas  of  the  CNS  to  preferentially 
involve  the  cervical  segments  of  spinal  cord  was  demonstrated. 

Further  experience  with  selective  pituitary  venous  sampling  for  Cushing's 
syndrome  indicated  that  selective  bilateral  simultaneous  inferior  petrosal  sinus 


3SNB/DIR 


sampling  successfully  (100%  accuracy)  distinguishes  patients  with  Cushing's  disease 
from  those  with  ectopic  ACTH  syndrome.  This  technique  permits  tumor  localization 
within  the  pituitary  gland  in  patients  with  Cushing's  disease  with  about  75% 
accuracy.  It  was  also  demonstrated  that  repeat  surgery  could  be  used  to  successfully 
treat  (70%  cure  rate)  patients  with  persistent  or  recurrent  Cushing's  disease  after 
prior  pituitary  surgery  or  pituitary  irradiation.  Predictive  features  of  successful 
outcome  and  guidelines  for  intraoperative  management  were  established  by  a  study 
of  48  patients  with  repeat  surgery.  Gadolinium-EDTA  enhancement  with  MRI 
scanning  was  shown  to  be  superior  to  current  imaging  techniques  for  demonstration 
of  the  site  of  pituitary  microadenomas  in  Cushing's  disease  pre-operatively.  The 
critical  aspect  of  timing  the  image  procurement  within  5-10  minutes  after  the 
injection  of  Gd-EDTA  was  demonstrated.  However,  in  a  study  of  65  patients  with 
surgically  proven  Cushing's  disease,  MRI  pituitary  scanning  was  normal  in  45%.  In 
addition,  100  normal  subjects  were  scanned  to  estimate  the  incidence  of  pituitary 
adenomas  in  the  normal  population  and  1 5%  were  found  to  have  pituitary  tumors. 

Encouraging  progress  was  made  in  the  area  of  implantation  of  tissues  from  the 
CNS  into  the  host  brain  in  primates.  Initially,  a  model  of  parkinsonism  which  affects 
only  one-half  of  the  brain  was  developed  by  infusing  MPTP  into  the  internal  carotid 
artery.  Using  this  model  of  hemiparkinsonism  and  the  previously  available  model  of 
MPTP-induced  full  parkinsonism  in  the  primate,  we  investigated  functional  recovery 
and  graft  survival  after  implanting  dopaminergic  tissues  into  the  caudate  nucleus  of 
lesioned  animals.  Both  adrenal  and  fetal  substantia  nigra  allografts  to  induced 
partial  functional  recovery  in  the  host  animals.  However,  the  recovery  was  much 
more  pronounced  using  fetal  substantia  nigra  for  implantation.  Immunohistological 
assessment  demonstrated  functional  survival  of  the  fetal  grafts  and  prominent 
sprouting  of  dopaminergic  fibers  from  resident  fibers  in  the  host  brain  surrounding 
the  area  of  implantation.  By  screening  monkey  and  human  fetal  tissues  for  evidence 
of  neurite-promoting  activity,  using  the  chick  dorsal  root  ganglion  in  vitro  assay,  the 
fetal  amnion  was  identified  as  having  potent  activity.  Primates  implanted  with  fetal 
amnion  were  then  shown  to  induce  sprouting  of  dopaminergic  fibers  and  functional 
recovery  in  the  hemiparkinsonian  primate  model  that  was  comparable  to  that  which 
occurred  after  fetal  mesencephalic  implant.  These  results  indicate  that  sprouting  of 
the  intrinsic  dopaminergic  pathways  of  the  host  brain  is  induced  by  either  surgical 
cavitation  or  fetal  implants.  If  the  basic  mechanisms  responsible  for  this  induced 
sprouting  can  be  identified  and  used  for  other  neuronal  systems,  practical 
application  of  CNS  implants  for  Parkinson's  disease  and  other  CNS  disorders  may  be 
possible.  The  sprouting  in  response  to  cavitation  and  implantation  which  appeared 
to  be  related  to  functional  recovery  of  the  animals  appeared  to  derive  from  the 
ventral  tegmental  tract,  a  dopaminergic  neural  tract  which  is  separate  from  the 
nigrostriatal  tract  and  which  is  not  affected  by  MPTP  to  the  same  extent  as  the 
substantia  nigra.  To  study  this  phenomenon  in  greater  detail,  and  without  requiring 
of  large  numbers  of  subhuman  primates,  a  rat  model  of  parkinsonism  was  developed 
with  preservation  of  the  ventral  tegmental  tract.  In  addition,  accumulation  of  a 
material  which  induces  neural  sprouting  in  an  in  vitro  assay  was  found  in  the  cavities 
of  monkeys  and  humans  after  surgical  cavitation.  Finally,  a  technique  was 
developed  to  assess  the  biochemistry  of  the  basal  ganglia  of  implanted  primates  in 
vivo  by  implanting  fine  dialysis  fibers  into  the  basal  ganglia.  This  technique  should 
not  only  be  rewarding  for  investigating  of  implantation  to  tissues  to  reverse 
parkinsonism,  but  should  have  wide  application  clinically. 

Finally,  the  investigation  of  the  mechanism  and  physiologic  role  of 
programmed  cell  death,  which  occurs  in  both  the  CNS  and  the  immune  system 
during  normal  development,  demonstrated  that  thymocytes  can  be  induced  to  die 


4SNB/DIR 


by  the  same  signal  that  stimulates  mature  T-cells  to  proliferate.  RNA  and  protein 
synthesis  inhibitors  which  block  new  gene  expression  interrupted  the  programmed 
cell  death.  This  is  consistent  with  a  new  model  of  immune  system  self-tolerance  that 
states  that  potentially  autoreactive  cells  are  induced  to  die  by  crosslinking  the  T-cell 
receptor  early  in  development,  the  same  signal  that  induces  mature  T-cells  to  react 
against  foreign  antigens.  Studies  are  being  extended  into  the  nervous  system  by 
examining  the  mechanism  of  glutamate-induced  neuron  death  in  vitro  and  in  vivo. 
Immunotoxin-induced  selective  Purkinje  cell  death  in  an  animal  model  has  been 
observed.  This  model  is  being  used  to  examine  the  possible  mechanism(s)  of  Purkinje 
cell  death  in  a  variety  of  neurologic  disorders. 

A  Molecular  Biology  Unit  was  established  to  study  genetic  abnormalities 
associated  with  various  nervous  system  disorders,  especially  brain  and  pituitary 
tumors.  The  clonality  of  ACTH-secreting  pituitary  tumors  was  examined  using  the  X- 
chromosomal  inactivation  technique.  Nine  of  27  tumors  proved  to  be  evaluable  with 
this  technique.  The  results  demonstrated  that  these  adenomas  may  be  monoclonal 
(6  patients)  or  polyclonal  (3  patients,  one  of  whom  had  Nelson's  syndrome),  and 
indicate  two  mechanisms  of  oncogenesis  are  possible.  The  Brain  Imaging  Unit  was 
developed  in  the  Surgical  Neurology  Branch  this  year  to  integrate  quantitative 
autoradiographic  studies  with  our  studies  of  parkinsonism  neural  implantation, 
physiology  of  brain  tumors,  and  certain  aspects  of  pharmacology  associated  with 
these  problems.  Using  [3H]naloxone  an  increased  density  of  opiate  receptor  sites  in 
MPTP-induced  hemiparkinsonism  monkeys  was  observed  in  the  striatum  of  the 
MPTP-treated  side.  The  distribution  of  which  was  similar  to  the  distribution  of 
dopaminergic  Di  receptor  binding  sites.  This  suggests  potential  interactions  of 
dopamine  and  opiate  peptides  in  the  striatum.  Using  the  2-[Ci4]deoxyglucose 
method  and  selective  lesioning  of  the  substantia  nigra  behavioral  effects  were 
correlated  with  precise  loci  of  damage  along  the  medial-lateral  axis  of  the  substantia 
nigra. 


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I.  CLINICAL  NEUROSURGERY  SECTION 

Edward  H.  Oldfield,  M.D.,  Chief 

The  clinical  activities  of  the  Surgical  Neurology  Branch  are  primarily  directed  at 
investigating  the  biologic  behavior  and  mechanisms  of  pathophysiology  of 
malignant  primary  brain  tumors,  pituitary  tumors,  certain  vascular  disorders  of  the 
CNS,  surgical  management  of  epilepsy  refractive  to  medical  therapy,  and 
investigation  of  tissue  implantation  in  the  CNS  to  reverse  neurological  disorders. 

A.    Brain  Tumors 

1.  Barbiturate  Sedation  in  the  Management  of  Brain  Tumors 

Study  of  the  radioprotective  effect  of  alternative  barbiturates  to 
pentobarbital.  pPentobarbital  has  been  shown  to  have  significant  radioprotective 
effect  on  the  brains  of  rats  who  undergo  high-dose,  single-fraction,  whole-brain  X- 
irradiation.  As  pentobarbital  is  a  radioprotectant,  further  study  of  alternative 
barbiturates  was  carried  out  to  evaluate  their  potential  radioprotective  effect.  This 
was  performed  in  the  standard  rat  model,  utilizing  Fisher  344  rats  receiving  70  Gy  of 
whole-brain  X-irradiation  in  a  single  fraction.  Neither  phenobarbital  nor 
methohexital  delivered  any  radioprotection  as  measured  in  this  model.  Thiopental 
enhanced  survival  to  an  extent  equal  to  pentobarbital.  As  thiopental  is  a  more 
commonly  utilized  barbiturate  in  human  anesthesia,  this  implies  greater  potential 
applicability  clinically. 

Investigation  of  the  mechanism  of  the  radioprotective  effect  of  pentobarbital 
in  the  rodent  model.  In  spite  of  continued  investigation  of  this  phenomenon  and 
documentation  of  its  reproducibility,  the  mechanism  of  the  effect  remains  to  be 
delineated.  An  investigation  utilizing  simultaneous  quantitative  autoradiographic 
analysis  of  cerebral  blood  flow  and  glucose  utilization  in  the  irradiated  rat  is 
currently  underway. 

Investigation  of  the  radioprotective  effect  of  pentobarbital  in  a  primate  model 
of  cerebral  radiation  toxicity.  A  concern  remains  as  to  the  applicability  of  a  model  of 
acute  radiation  toxicity  in  a  rodent  to  the  radiation  toxicity  seen  in  humans  with 
cerebral  radiation  injury  due  to  treatment  of  brain  tumors.  A  primate  model  was 
designed  to  better  approximate  the  injury  seen  in  humans,  which  is  more  delayed  in 
onset.  Two  groups  of  animals  were  utilized.  One  received  15  Gy  of  whole-brain,  X- 
irradiation  while  lightly  sedated  with  ketamine,  an  anesthetic  having 
radioprotective  effect  in  the  rat  model.  The  second  group  underwent  radiation 
after  a  dose  of  pentobarbital  which  results  in  30  to  60  second  EEG  burst  suppression. 
Baseline  studies  priorto  X-irradiation  included  anatomic  studies  utilizing  MRI 
without  and  with  gadolinium,  physiologic  studies  utilizing  position  emission 
tomography  with  i8f|uoro-deoxyglucose  (to  evaluate  neuronal  and  glial  integrity) 
and  68Ga-EDTA  (to  evaluate  blood  brain  barrier  and  microvascular  integrity),  and 
neuroendocrinologic  studies  utilizing  insulin,  L-dopa,  arginine,  thyroid-releasing 
hormone,  and  gonadotropin-releasing  hormone  as  stimulants  to  measure  glucose, 
Cortisol,  growth  hormone,  prolactin,  thyroid-stimulating  hormone,  follicle- 
stimulating  hormone,  and  luteinizing  hormone  responses.  MRI  studies  were 
subsequently  planned  for  1  week,  1  month,  6  months,  12  months,  18  months  and  24 
months  after  X-irradiation.  PET  studies  were  at  1  week,  1  month,  1  year  and  2  years 


6SNB/DIR 


after  X-irradiation.  Endocrinologic  studies  are  carried  out  1  week,  1  month,  3 
months,  6  months,  12  months,  18  monthsand  2  yearsafter  irradiation.  The  animals 
are  sacrificed  2  years  after  X-irradiation  and  histologic  exam  performed. 

The  primate  study  is  long-term  and  is  ongoing  at  this  time.  Four  animals  have 
been  enrolled  in  each  group.  All  animals  surviving  beyond  6  months  have 
demonstrated  white  matter  abnormalities  to  varying  degrees,  regardless  of  the 
group  to  which  they  belong.  The  supports  the  efficacy  of  the  method  of  radiation 
injury.  Intergroup  differences  in  white  matter  injury  as  assessed  by  MRI  await 
quantitation  at  the  completion  of  the  study. 

Endocrinologic  studies  reveal  that  animals  irradiated  under  the  influence  of 
ketamine  have  evidence  of  early  hypothalamic/pituitary  functional  abnormalities. 
After  3  months,  the  peak  luteinizing  hormone  response  in  the  control  group  was 
significantly  less  (mean,  23.1  ng/ml  [SD,  3.9  ng/ml])  than  in  the  pentobarbital  group 
(mean,  31.7  ng/ml  [SD,  4.4  ng/ml]).  The  prolactin  level  30  minutes  after  L-dopa 
administration  rose  progressively  after  3  and  6  months  in  control  monkeys  as 
compared  with  those  treated  with  pentobarbital.  Peak  thyroid-stimulating 
hormone  response  to  thyrotropin-releasing  hormone  in  the  control  group  (mean, 
13.7iiU/ml  [SD,  4.1  iiU/ml])  was  less  than  that  of  the  pentobarbital  group  (22.6  uU/ml 
[SD  5.0  pU/ml]).  No  difference  was  noted  between  the  treated  and  control  groups 
when  measuring  growth  hormone,  follicle-stimulating  hormone,  or  Cortisol 
responses. 

Two  of  the  four  animals  radiated  with  ketamine  for  anesthesia  succumbed  to 
radiation  toxicity  as  of  6  months  following  treatment.  The  other  two  animals  remain 
intact  1  to  2  years  following  this  treatment.  One  of  the  four  animals  radiated  under 
deep  pentobarbital  anesthesia  succumbed  7  months  after  treatment.  The  other 
three  animals  remain  alive,  well  and  growing,  12  to  18  months  after  treatment.  This 
substantiates  the  degree  of  toxicity  imparted  by  the  radiation  dose  utilized.  The 
protective  effect  imparted  by  pentobarbital  as  seen  in  the  original  rat  model  of 
cerebral  injury  is  supported  in  the  findings  with  the  primate  model  thus  far.  As  seen 
in  the  rat  model,  this  protection  can  be  overwhelmed  by  high  doses  of  radiation. 
Importantly,  there  appears  to  be  protection  of  the  hypothalamic/pituitary  axis,  an 
area  frequently  injured  in  children  undergoing  brain  radiation. 

In  an  additional  primate  study  the  potential  radiation  protective  effect  of 
pentobarbital  is  being  examined  with  multiple  fractions  of  radiotherapy, 
fractionation  analogous  to  that  used  clinically.  Information  on  the  basic 
mechanism(s)  of  radiation  damage  of  the  brain  and  hypothalamic-pituitary  axis  will 
also  be  obtained. 


2.       Clinical  use  of  immunotoxins  to  treat  CNS  tumors 

A  Phase  I  clinical  trial  was  begun  to  investigate  the  clinical  use  of  immunotoxins 
in  the  treatment  of  leptomeningeal  carcinomatosis.  Immunotoxin,  a  conjugate  of  a 
human  antitransferrin  receptor  antibody  with  ricin  A  chain,  is  being  administered  in 
single  injections  in  patients  who  have  leptomeningeal  carcinomatosis  refractory  to 
current  therapies.  Five  patients  have  been  treated,  no  toxicity  has  been  seen. 
Efficacy  remains  to  be  established. 

Methods  are  being  investigated  for  direct  injection  into  the  brain  and  into  CNS 
tumors,  to  achieve  a  homogeneous  distribution  of  antiglioma  agents  throughout 


7SNB/DIR 


the  tumor  volume  by  direct  parenchymal  infusion.  At  present  this  study  is  limited  to 
studing  the  spatial  and  temporal  distribution,  using  quantitative  autoradiography, 
of  representative  agents  infused  into  the  feline  cerebrum. 

3.  Phase  II  trial  of  Suramin  to  treat  patients  with  malignant  gliomas 

The  efficacy  of  suramin  combined  with  hydrocortisone  therapy  in  the 
treatment  of  patients  with  malignant  gliomas  which  occur  after  radiation  therapy  is 
being  performed.  In  vitro  data  indicate  that  this  combination  inhibits  angiogenesis 
and  interrupts  growth  of  glioma  cells  by  binding  certain  growth  factors.  This  new 
type  of  systemic  chemotherapeutic  agent  has  been  demonstrated  to  be  effective  in 
patients  with  adrenal  cortical  cancer  but  has  not  previously  been  evaluated  in 
patients  with  brain  tumors. 

4.  Intraarterial  chemotherapy 

One  of  the  basic  tenets  of  anticancer  chemotherapy  is  that  increased  tumor 
exposure  to  a  drug  should  result  in  increased  tumor  response.  One  method  currently 
being  used  to  increase  drug  exposure  of  malignant  brain  tumors  is  by  intracarotid 
infusion.  Our  research  results  have  demonstrated  the  following : 

Drug  streaming  during  intracarotid  delivery  results  in  maldistribution  of  the 
infused  agent  with  the  potential  of  delivering  very  high  (toxic)  concentrations  of 
drug  to  some  regions  of  the  brain,  while  other  areas  receive  minimal  drug. 

The  presence  of  streaming,  and  its  elimination  by  phased,  diastolic  infusion, 
was  investigated  in  9  glioma  patients  in  vivo  using  PET.  We  demonstrated  that 
standard  infusion  techniques  (continuous  infusion  of  1-4  cc/min)  of  H215  O-labeled 
water  into  the  supra-ophthalmic  segment  of  the  internal  carotid  artery  caused 
extremely  heterogeneous  delivery  of  the  inf  usate  to  the  brain  in  the  distribution  of 
the  internal  carotid  artery.  When  the  infusions  were  done  with  a  phased,  diastolic 
infusion,  heterogeneous  delivery  was  eliminated.  These  findings  confirm  our  data  in 
other  species,  and  will  lead  to  further  clinical  studies  with  intracarotid  drug  infusions 
to  improve  drug  delivery,  limit  toxicity,  and  potentially  improve  survival  of  glioma 
patients. 

We  completed  two  projects  which  quantified  permeability  of  the  blood- 
brainbarrier  in  the  primate  brain  before  and  after  barrier  disruption  by  intracarotid 
mannitol  using  PET  scanning.  In  a  study  of  baboons  the  permeability  across  normal 
brain  vessels  in  the  gray  and  white  matter  was  measured  repeatedly  and  the 
duration  and  extent  of  attention  in  permeability  was  determined.  A  similar  study  in 
humans  permitted  quantification  of  vascular  permeability  in  primary  brain  tumors, 
brain  around  tumor  and  normal  brain.  The  results  of  these  studies  will  permit 
rational  designs  of  approaches  to  deliver  drugs  of  various  types  to  the  brain  using 
this  technique. 

5.  Studies  of  patients  with  von  Hippel-Lindau  Disease 

Von  Hippel-Lindau's  (VHL)  disease  is  an  inherited  disorder  in  which  patients 
suffer  from  hemangioblastomasof  the  retina,  cerebellum  and  spinal  cord  as  well  as 
renal  cell  carcinomas,  pheochromocytomas,  and  visceral  cysts.  The  disease  is  passed 


8SNB/DIR 


in  an  autosomal  dominant  pattern  which  has  been  linked  by  recombinant  fragment 
length  polymorphism  analysis  to  the  short  arm  of  the  third  chromosome.  The 
defective  gene  is  in  the  3p  14  region. 

In  collaboration  with  Drs.  Berton  Zbar  and  Marston  Linehan  of  the  NCI,  we 
studied  1 3  separate  tumors  from  5  patients,  analyzing  them  for  loss  of  alleles  on  the 
third  chromosome.  These  tumors  demonstrate  loss  of  one  copy  of  the  vHL  allele. 
However,  in  the  tumor  cells,  the  loss  is  of  the  normal,  wild  type  allele  inherited  from 
the  normal  parent.  This  loss  of  the  balancing  wild  type  allele  leaves  the  tumor  with 
only  the  abnormal  allele  inherited  from  the  parent  carrying  von  Hippel-Lindau 
disease.  This  supports  the  theory  that  the  tumors  in  VHL  disease  are  caused  by  the 
loss  of  a  tumor  suppressor  gene  similar  to  that  seen  with  retinoblastoma  and  type  II 
neurofibromatosis;  the  affected  parent  contributes  a  defective  copy  of  the  gene 
allowing  cells  at  risk  to  develop  tumors  when  the  other  copy,  the  balancing  wild- 
type  gene  from  the  normal  parent,  is  lost  or  rendered  nonfunctional. 


9SNB/DIR 


B.    Pituitary  Tumors 

Venous  sampling  to  establish  the  diagnosis  and  location  of  hormone-secreting 
pituitary  microadenomas 

We  have  now  performed  bilateral  and  simultaneous  inferior  petrosal  sinus 
sampling  in  over  320  patients  with  Cushing's  syndrome.  The  results  are  used  (1)  to 
confirm  the  diagnosis  of  Cushing's  disease  preoperatively,  and  (2)  to  determine  the 
half  of  the  pituitary  gland  in  which  a  microadenoma  resides.  The  study  has  been 
particularly  rewarding  and  has  demonstrated  the  following:  (1)  sampling  from  both 
inferior  petrosal  sinuses  simultaneously  with  CRH  injection  consistently  distinguishes 
patients  with  Cushing's  disease  from  those  with  ectopic  ACTH  syndrome  (100% 
accuracy);  (2)  sampling  from  a  single  inferior  petrosal  sinus,  as  has  previously  been 
general  practice,  to  establish  the  diagnosis  of  Cushing's  syndrome,  may  be 
misleading  and  could  result  in  an  incorrect  assumption  of  the  source  of  excess  ACTH 
secretion  in  as  many  as  43%  of  patients  with  Cushing's  syndrome;  and  (3) 
preoperative  sampling  for  ACTH  concentrations  in  the  inferior  petrosal  sinuses 
determines  the  site  of  ACTH-  secreting  microadenomas  within  the  pituitary  gland 
with  about  85%  accuracy.  Therefore,  bilateral  sampling  permits  the  surgeons'  search 
for  small  microadenomas  to  be  focused  to  one  side  of  the  gland,  which  should  be 
helpful  in  finding  smallertumors.  If  no  tumor  is  found,  the  half  of  the  gland 
containing  the  microadenoma  can  be  removed.  This  technique  is  now  being  widely 
employed  in  the  evaluation  of  patients  with  Cushing's  syndrome. 

Radiologic  imaging  and  Cushing's  syndrome 

To  evaluate  the  MRI  scan  as  a  means  of  aiding  the  differential  diagnosis  of 
patients  with  Cushing's  syndrome  and  as  a  mechanism  for  tumor  localization  pre- 
operatively, we  prospectively  evaluated  the  results  of  CT  scanning  in  67  patients  with 
Cushing's  syndrome.  The  diagnostic  accuracy  of  MRI  scanning  with  gadolinium  - 
DTPA  contrast  was  only  55%.  The  conclusion  of  this  study  was  that  MRI  scanning  is 
only  occasionally  helpful  in  locating,  pre-operatively,  the  site  of  an  ACTH-secreting 
pituitary  microadenoma,  and  is  therefore  only  rarely  beneficial  as  a  diagnostic  aid  in 
the  differential  diagnosis  of  patients  with  Cushing's  syndrome. 

Autopsy  studies  have  indicated  an  incidence  of  pituitary  adenomas  in 
asymptomatic  patients  to  be  7-33%.  To  establish  the  incidence  of  MRI  abnormalities 
in  the  general  population  consistent  with  a  diagnosis  of  a  pituitary  adenoma  we 
studied  100  normal  subjects  with  and  without  gadolinium  enhanced  MRI  scans.  The 
results  of  this  study  indicate  that  15%  of  the  normal  population  have  pituitary 
lesions  that  appearto  be  pituitary  adenomas  by  MRI  scanning.  These  results  indicate 
that  an  appreciable  percentage  of  normal  adults  have  pituitary  adenomas  and 
indicate  that  positive  findings  on  an  MRI  in  a  patient  with  endocrinopathy  do  not 
necessarily  establish  that  the  source  of  the  endocrinopathy  is  in  the  pituitary  gland. 

Repeat  pituitary  surgery  in  patients  with  Cushing's  disease  despite  previous 
treatment 

Since  the  ACTH-secreting  pituitary  tumors  that  cause  Cushing's  disease  respond 
to  negative  feedback  by  glucocorticoids,  but  have  a  higher  threshold  for 
glucocorticoid  suppression  than  normal  corticotrophs,  a  potential  abnormality  in  the 


10SNB/DIR 


tumor  cells  accounting  for  hypercortisolism  in  these  patients  is  that  the 
glucocorticoid  receptor  is  abnormal.  Pituitary  adenomas  from  8  patients  with 
Cushing's  disease  were  investigated  to  determine  if  the  glucocorticoid  receptor  in 
the  adenomas  in  Cushing's  disease  was  normal  or  abnormal  as  determined  by 
restriction  fragment  length  polymorphism  analysis.  Results  of  this  study  indicated 
no  abnormality  in  the  adenomas  compared  to  normal  tissues.  It  appears  unlikely 
that  a  genetic  abnormality  in  the  glucocorticoid  receptor  is  the  sole  explanation  of 
the  resistances  to  negative  feedback  by  steroids  in  these  tumors. 

Origin  of  ACTH-secretinq  pituitary  microadenomas  in  Cushing's  disease 

Because  of  the  female  preponderance  (90%)  in  Cushing's  disease,  it  has  been 
thought  that  these  tumors  may  be  induced  by  endocrine  factors.  In  experimental 
models  in  animals  induction  of  pituitary  tumors  by  hormonal  manipulation 
(thyroidectomy  or  treatment  with  high-dose  estrogen)  initially  produces  areas  of 
pituitary  hyperplasia.  Small  focal  pituitary  adenomas  arise  several  weeks  later.  To 
assess  whether  ACTH-secreting  tumors  in  Cushing's  disease  originated  from  a  single 
cell  or  from  multiple  cells  we  used  the  X-chromosomal  inactivation  scheme.  The 
results  indicated  that  3  of  the  tumors  originated  from  multiple  cells  whereas  6 
originated  from  a  single  cell  (see  Molecular  Biology  Units  of  this  portion  of  this 
annual  report  for  more  detail  presentation). 


11  SNB/DIR 


C.  Surgical  Treatment  of  Medically  Intractable  Epilepsy 

The  aim  of  the  surgical  arm  of  the  NINDS  epilepsy  program  is  to  develop 
surgical  techniques  that  allow  more  accurate  localization  and  safer  resection  of 
epileptogenic  foci  than  can  be  achieved  with  methods  now  available.  The 
development  and  implementation  of  surgical  treatments  for  patients  whose  seizures 
are  intractable  to  currently  available  medical  and  surgical  therapies  is  the  immediate 
and  long-term  goal  of  this  program. 

Special  subdural  surface  electrodes  designed  and  built  at  NIH  in  collaboration 
with  the  BEI  Branch  of  DRS  are  now  being  implanted  in  selected  patients  so  that  EEG 
recordings  can  be  obtained  directly  from  the  cortical  surface  for  much  longer  periods 
than  is  possible  during  intraoperative  electrocorticography.  During  the  period  of 
implantation  these  electrodes  are  also  utilized  for  focal  cortical  stimulation  to 
discriminate  areas  that  can  be  safely  resected  from  areas  critical  for  motor,  sensory, 
language,  memory-related  and  other  functions.  Such  discrimination  is  crucial  in  the 
topographic  identification  of  overlap  between  critical  cortical  areas  and  epileptic 
foci  during  surgical  procedures. 

A  subset  of  patients  with  epileptogenic  foci  originating  in  the  language- 
dominate  hemisphere  are  undergoing  implantation  of  a  new  type  of  subdural 
electrode  designed  and  built  here  at  NIH.  Recordings  and  functional  stimulation 
mapping  utilizing  these  electrodes  during  a  period  preceding  resective  surgery 
should  allow  maximum,  and  safer,  surgical  resection  to  be  performed  under  general 
anesthesia  in  these  patients  who  are  difficult  or  impossible  to  cure  using  current 
approaches.  During  the  past  year  a  novel  language  area  has  been  identified  and 
located  within  the  cortex  of  the  basal  temporal  lobe.  To  our  knowledge,  we  are  the 
first  group  to  confirm  this  observation  and  are  working  to  anatomically  and 
functionally  define  this  basal  temporal  language  area. 

Analysis  of  data  from  several  recently  developed  methods  for  localizing 
epileptic  foci  is  being  performed  to  determine  comparative  sensitivity  and  reliability 
of  the  various  techniques.  During  surgery  for  focal  epilepsy,  depth  and  special 
subdural  (surface  cortical)  electrodes  are  being  used  to  record  from  deep  structures 
inaccessible  by  routine  electrocorticography  to  identify  and  confirm  areas  of 
potential  epileptogenic  activity  suggested  by  the  preoperative  investigational 
methods  of  PET  scanning,  magnetoencephalography,  MRI  and  magnetic  resonance 
spectroscopy  (MRS). 

MEG  offers  the  possibility  for  localization  of  abnormal  seizure-related 
electromagnetic  phenomena  in  3  dimensions.  No  other  methodology  currently 
available  can  noninvasively  acquire  this  type  of  information,  which  is  highly 
desirable  for  identifying  epileptogenic  foci  for  surgical  excision  and  to  study  basic 
mechanisms  in  epilepsy.  In  several  patients  we  were  able  to  predict  correctly  the 
localization  of  foci  causing  complex  partial  seizures  using  MEG  data  in  patients 
whose  preoperative  EEG  (in  retrospect)  gave  misleading  and/or  false  localizing 
information.  These  foci,  which  were  successfully  extirpated,  would  have  been 
missed  if  a  standard  temporal  lobectomy  had  been  performed.  The  new  7-channel 
MEG,  now  in  place,  promises  even  greater  3-dimensional  localizing  capability,  and 
we  are  now  studying  focal  motor  seizures  as  well  as  complex  partial  and  generalized 
tonic-clonic  seizures  with  this  technique. 


12SNB/DIR 


In  selected  patients,  subdural  electrodes  are  modified  so  that  in  addition  to 
their  recording  capability,  they  can  be  utilized  to  create  a  current  dipole.  This  dipole 
can  then  be  detected  by  the  MEG  technique,  allowing,  for  the  first  time,  direct  proof 
of  the  ability  of  the  MEG  to  accurately  and  precisely  predict  the  location  of  a  current 
dipole  source  within  the  three-dimensional  space  of  the  human  cranium. 

Electrocorticography  (ELOG)  under  general  anesthesia  at  resective  surgery 
often  generates  data  of  uncertain  significance.  An  ongoing  study  is  carefully 
evaluating  the  effect  of  different  anesthetic  agents  on  interictal  spikes.  Background 
data  gathered  from  the  first  group  of  patients  studied  under  this  protocol  reveal 
that  isoflurane  suppresses  the  number  of  abnormal  spikes  during  surgical  ELOG 
when  compared  to  recordings  immediately  before  and  after  its  use  in  the  same 
patient.  Enflurane  under  similar  conditions  increases  the  number  of  abnormal  spikes 
and  produces  paroxysms  of  synchronous  high-voltage  spikes.  Isoflurane  can 
suppresses  epileptogenic  foci  and  that  both  isoflurane  and  enflurane  can  distort  the 
ECOG,  confounding  accurate  identification  of  the  seizure  foci  during  surgery.  When 
used  judiciously,  however,  enflurane  may  be  a  potent  synchronizer  and  activator  of 
the  epileptogenic  focus,  permitting  easier  identification. 

An  approach  that  should  yield  information  about  the  chemical  changes  that 
occur  in  tissues  in  which  seizures  arise  is  magnetic  resonance  spectroscopy  (in  vivo 
NMR  spectroscopy).  Under  a  new  protocol,  pH  and  lactate  measurements  are  taken 
from  specific  regions  of  interest  in  seizure  patients  and  are  compared  with 
measurements  from  similar  regions  in  the  opposite  hemisphere  and  with 
measurements  in  normal  volunteers.  This  study  is  being  done  in  collaboration  with 
Drs.  Giovanni  Di  Chiro  and  Jeffrey  Alger,  NIS. 

One  of  the  most  severe  postoperative  deficits  suffered  by  patients  undergoing 
temporal  lobectomy  is  unpredictable  recent  memory  loss.  It  is  presumed  that  this 
memory  deficit  results  from  poor  memory  reserve  in  the  temporal  lobe  contralateral 
to  the  resection.  Routine  preoperative  amytal  (WADA)  testing  has  been  only 
minimally  successful  in  predicting  post-operative  memory  deficits  in  patients  who 
receive  unilateral  hippocampectomy  for  seizures.  Therefore,  a  study  of  super 
selective  posterior  cerebral  artery  amytal  testing  began  in  collaboration  with  the 
Radiology  Department,  CC.  This  test  as  proposed  should  selectively  block 
hippocampal  function  unilaterally  in  the  awake  patient  allowing  predictions  for  the 
state  of  postoperative  memory  function  after  surgery. 


13SNB/DIR 


II.  BIOCHEMISTRY  SECTION 

Richard  J.  Youle,  Ph.D.  -  Chief 

A.  Monoclonal  Antibody  Mediated  Killing  of  Tumor  Cells 

The  Section  of  Biochemistry,  headed  by  Dr.  Richard  Youle,  is  studying  the  use  of 
monoclonal  antibodies  to  kill  disease-causing  cells.  Monoclonal  antibodies  which 
selectively  bind  tumor  cells  can  be  generated,  but  alone  are  usually  not  cytotoxic  to 
the  tumor.  Toxic  proteins  such  as  ricin  and  diphtheria  toxin  can  be  chemically  linked 
to  monoclonal  antibodies.   The  new  hybrid  molecules  bind  tumor  cells  via  the 
antibody  moiety  and  then  kill  the  cells  via  the  toxin  moiety.  The  toxins  used  are 
enzymes  that  catalytically  inactivate  protein  synthesis  in  target  cells  with  only  one  or 
two  molecules  in  the  cytoplasm  killing  a  cell.  However,  the  non-target  cell  toxicity  of 
the  toxins  must  be  blocked  with  excess  lactose  to  prevent  toxin  binding  and  this 
currently  limits  this  approach  to  in  vitro  applications.  The  cell-type-specific  reagents 
have  immediate  clinical  application  in  vitro  in  bone  marrow  transplantation  where 
T-cell  depletion  improves  allogeneic  transplantation.  The  Section  of  Biochemistry  is 
collaborating  in  clinical  trials  in  bone  marrow  transplantation  at  the  University  of 
Minnesota.  Twenty-four  patients  have  now  been  treated  with  immunotoxin  purged 
bone  marrow  as  the  sole  prophylaxis  for  graft-versus-host  disease.  Comparing  the 
outcomes  with  historic  controls  treated  post-transplant  with  methotrexate,  several 
conclusions  can  be  drawn.  The  patients  had  a  milder  course  as  evidenced  by  a 
significantly  shorter  hospitalization.  Engraftment  of  donor  marrow  occurred  within 
a  shor    r  time  until  leucocyte  generation  and  no  severe  graft-versus-host  disease 
was  seen. 

The  major  goal  of  the  laboratory  is  to  develop  immunotoxins  which  will 
selectively  kill  tumor  cells  in  vivo.  Currently  the  limiting  steps  for  antibody-toxin 
hybrids  are  (1)  the  entry  of  the  toxin  molecule  into  the  cell;  and  (2)  in  vivo  access  of 
the  drug  to  the  tumor  cells.  To  promote  access  of  monoclonal  antibody-toxin 
conjugates  to  tumors  we  have  focused  on  tumors  localized  in  body  compartments 
such  as  the  brain  and  the  peritoneal  cavity.  The  brain  may  be  an  optimal 
compartment  for  antibody  modulation  of  cell  function. 

The  discovery  of  point  mutants  of  diphtheri  toxin  (DT)  that  increase 
immunotoxin  selectivity  in  FY  88  and  the  demonstration  of  immunotoxin  activity  in 
animal  models  of  leptomeningeal  cancer  in  FY88  have  been  moved  into  clinical  trials 
in  FY  89.  Extensive  toxicity  testing  has  shown  that  a  100-10000fold  therapeutic 
window  exists  between  the  concentration  toxic  to  tumor  cells  and  the  dose  tolerable 
in  rats,  guinea  pigs  and  monkeys.  Based  upon  our  thorough  preclinical  testing  we 
determined  that  immunotoxins  may  offer  a  therapeutic  benefit  to  patients  with 
leptomeningeal  carcinomatsis  and  medulloblastoma  and  we  have  begun  clinical 
trials.  We  have  begun  trials  with  a  monoclonal  antibody  against  the  human 
transferrin  receptor  linked  to  recombinant  ricin  A  chain  supplied  by  Cetus  Corp.  We 
have  treated  5  patients  so  far  in  a  dose  escalation  that  has  spanned  a  100-fold 
increase  in  drug  injected.  As  yet  we  have  not  reached  dose  limiting  toxicity.  We 
have  examined  the  immunotoxin  pharmacology,  and  immunogenecity.  At  this  early 
stage  we  cannot  establish  efficacy  of  the  drug  to  the  patients.  We  will  continue  our 
dose  escalation  with  this  reagent  as  well  as  prepare  second  generation 
immunotoxins  that  may  offer  greater  potency  for  clinical  trials. 


14SNB/DIR 


To  improve  entry  of  immunotoxins  into  cells  we  have  modified  toxins 
chemically,  molecular  biologically  and  with  monoclonal  antibodies.  We  are  studying 
the  structure-function  relations  of  the  toxin  molecule  and  the  cell  biology  of  toxin 
internalization  and  membrane  penetration. 

Toxins  may  be  best  adapted  for  tumor  specific  toxicity  by  alterations 
of  amino  acid  sequence  at  the  gene  level.  To  begin  improving  immunotoxins  at  the 
gene  level  we  have  worked  with  the  prokaryotic  toxin,  diphtheria  toxin.  Intact 
diphtheria  toxin  was  linked  to  a  monoclonal  antibody  specific  for  human  T  cells  and 
was  found  to  specifically  kill  target  cells  at  10-12M.  The  rate  of  specific  killing  was  10- 
fold  faster  than  previously  reported  immunotoxins.  This  model  system  was  then 
used  to  study  cloned  fragments  of  diphtheria  toxin.  In  collaboration  with  Dr.  Larry 
Greenfield,  who  has  cloned  DT,  we  deleted  the  C-terminal  1 5kD  region  shown  to 
include  the  cell  surface  binding  site.  This  left  the  toxin  Asubunit  plus  a  17  kD 
fragment  of  DT  B-chain  thought  to  facilitate  transmembrane  transport.  When 
linked  to  monoclonal  antibodies,  this  truncated  DT  was  100-fold  more  toxic  than  DT 
A-chain  linked  to  antibody,  and  the  toxicity  was  blocked  by  excess  antibody.  The 
cloned  DT  fragment  was  1000  times  less  toxic  to  guinea  pigs  than  the  native  toxin. 
Therefore,  the  fragment  of  DT  B-chain  included  by  cloning  increased  target  cell 
toxicity  more  than  non-target  cell  toxicity  indicating  that  separation  of  B-chain  entry 
functions  from  binding  was  accomplished  to  some  degree.  We  compared  intact  DT 
linked  to  monoclonal  antibody  with  the  cloned  fragment  of  DT,  which  showed  the 
C-terminal  fragment  of  DT  further  increased  the  antibody  mediated  toxicity  100- 
fold. 

We  have  identified  point  mutations  in  the  receptor  binding  site  of  diphtheria 
toxin.  CR.M103  has  a  single  point  mutation  at  amino  acid  508  and  is  1000  times  less 
toxic  to  cells  than  native  diphtheria  toxin.  CRM107  has  a  single  point  mutation  at 
amino  acid  525  and  is  10,000  times  less  toxic  than  native  diphtheria  toxin.  Upon 
linking  either  of  these  two  mutants  to  monoclonal  antibodies,  full  potency, 
indistinguishable  from  that  of  the  10  12M  potency  of  the  native  toxin  linked  to 
antibodies,  was  achieved.  These  mutations  offer  an  improved  therapeutic  window 
from  other  immunotoxins.  Most  recently  we  have  combined  these  two  beneficial 
mutants  into  one  new  toxin  called  CTM 1 .  We  are  currently  studying  whether  or  not 
this  mutant  will  have  an  even  greater  therapeutic  window  than  either  of  the  single 
point  mutants. 

In  collaboration  with  the  biomedical  engineering  branch  we  have  studied 
immunotoxin  pharmacology.  We  have  found  that  immunotoxins  specifically  bind 
tumors  in  vivo  and  have  developed  a  new  model  that  incorporates  the  number  of 
receptor  binding  sites  per  cell  times  the  affinity  constant.  We  have  also  initiated 
studies  of  direct  infusion  of  immunotoxins  into  brain  and  solid  brain  tumors. 

We  have  recently  identified  point  mutations  in  a  hydrophobic  domain  of 
diphtheria  toxin  B-chain  that  inhibit  membrane  translocation  activity  90%.  This 
results  shows  a  role  of  protein  309  in  the  translocation  activity  of  diphtheria  toxin. 

A  recent  paper  in  Science  reports  that  diphtheria  toxin  is  a  DNase.  We  have 
found  that,  although  DNase  activity  was  detectable  in  commercial  samples  of 
diphtheria  toxin,  recombinant  diphtheria  toxin  from  Cetusand  the  CRM107  mutant 
purified  by  collaborator  at  the  NIH  had  very  little  DNase  activity.  Upon  HPLC 
purification  of  the  commercial  DT,  the  DNase  activity  was  separated  from  the  DT 
activity  indicating  that  the  initial  report  was  wrong. 


15SNB/DIR 


We  have  found  ways  to  block  the  immune  response  animals  generate  against 
immunotoxins.  Injection  of  a  monoclonal  antibody  against  the  helperT-cell  CD4 
antigen  completely  prevent  the  primary  immune  response  to  weekly  injections  of 
immunotoxins  for  3  months  or  more. 

In  an  effort  to  develop  immunotoxins  to  treat  AIDS  we  have  collaborated  with 
Biogen  to  create  CD4-toxin  conjugates  that  specifically  kill  HIV-infected  cells.  This 
project  has  just  begun  and  the  bioactivity  of  these  new  toxins  is  not  yet  known. 

During  investigation  of  the  clinical  potential  for  immunotoxins  injected 
intrathecal^  we  noticed  that  animals  became  atoxic.  Thorough  analysis  of  this 
revealed  that  immunotoxins  specifically  kill  up  to  70%  of  Purkinje  cells  in  animals 
with  no  detectable  toxicity  to  other  neurons  or  cells.  This  affords  a  new  animal 
model  of  Purkinje  cell  loss,  yields  a  valuable  clue  to  the  potential  dose  limiting 
toxicity  of  immunotoxins  in  clinical  trials,  and  points  to  a  new  receptor-mediated 
uptake  pathway  in  the  CNS. 

B.  Programmed  Cell  Death  in  the  Nervous  and  Immune  System 

Our  laboratory  has  begun  a  project  to  study  the  mechanism  and  physiologic 
role  of  programmed  cell  death.  In  both  the  nervous  system  and  the  immune  system, 
massive  numbers  of  cells  die  during  normal  development.  In  the  spinal  cord,  for 
example,  over  half  the  neuronsdie  during  fetal  development.  The  immune  system 
also  has  large  cell  loss  at  precise  stages  in  development  such  as  thymocyte  death  and 
thymus  involution  at  puberty.  The  physiologic  role  and  the  biochemical  mechanism 
of  programmed  cell  deaths  is  unknown.  Understanding  the  mechanism  and 
regulation  of  such  physiologic  cell  deaths  may  shed  light  on  neurodegenerative 
diseases  and  immunodeficiency  disorders. 

In  our  initial  studies  we  found  that  thymocytes  could  be  induced  to  die  by  the 
same  signal  that  stimulates  mature  T-cel Is  to  proliferate.  RNA  and  protein  synthesis 
inhibitors  block  new  gene  expression  as  well  as  the  programmed  cell  death.  In 
addition,  we  found  that  prolactin  can  block  the  glucocorticoid-induced  cell  death  of 
thymocytes.  This  result  has  significance  for  clinical  immunosuppression  and 
autoimmune  disorders. 

In  conjunction  with  our  studies  studies  on  immunotoxins  we  have  made  now 
animal  models  of  Purkinje  cell  death.  Injection  of  immunotoxins  can  lead  to  70-80% 
loss  of  Purkinje  cells  without  affecting  other  neurons.  This  discovery  not  only 
permits  generation  of  a  new  animal  model  but  also  points  toward  the  possible 
mechanism  of  Purkinje  cell  loss  in  a  variety  of  neurological  disorders.  Purkinje  cell 
loss  in  inherited  metabolic  diseases  may  be  a  direct  consequence  of  the  high 
endocytosis  rate  expressed  by  these  cells. 

We  are  extending  our  studies  into  the  nervous  system  by  examining  the 
mechanism  of  thymocyte  death  in  various  ataxic  mutant  mice  and  by  examining  the 
mechanism  of  glutamate-induced  neuron  death  in  vitro  and  in  vivo.    We  have 
found  that  thymocyte-programmed  cell  death  can  be  followed  morphologically  with 
Nromarski  optics  and  that  the  thymocyte  death  resembles  the  neuronal  cell  death. 
Morphologic  analysis  of  nuclear  disintegration  has  allowed  us  to  test  whether  cell 
death  is  due  to  production  of  a  toxic  factor  or  due  to  the  loss  of  a  protective  factor. 
We  found  that  the  cell  death  mechanism  is  a  dominant,  trans-acting  death  induction 
in  cell-cell  hybrids.  We  are  now  testing  the  transacting  death  signal  in  neuronal  cells. 


16SNB/DIR 


III.  Central  Nervous  System  Implantation  Unit 

Robert  J.  Plunkett,  M.D.,  Head 

1.  Tissue  implantation  in  Parkinsonian  animal  models 

The  effect  of  tissue  implants  into  the  caudate  nucleus  of  Parkinsonian  animals 
is  being  studied  from  behavioral,  biochemical,  and  histological  viewpoints.  The 
models  utilized  are  the  hemiparkinsonian  monkey  and  rat  model  developed  in  our 
lab.  Our  previous  work  showed  that  grafts  of  fetal  mesencephalon  lead  to  rapid, 
near  complete  recovery  from  the  motor  deficits  in  the  monkeys,  while 
nondopaminergic  fetal  grafts  lead  to  moderate  motor  recovery.  In  contrast,  adrenal 
allografts,  adrenal  autografts,  nondopaminergic  adult  tissues,  and  surgical 
cavitation  alone  all  lead  to  modest  behavioral  recovery  which  occurs  over  many 
months.  One  phenomenon  which  we  have  observed  to  varying  degrees  after  all 
implants  and  after  cavitation  alone  is  sprouting  of  intact  host  dopaminergic  fibers, 
presumably  from  the  mesolimbic  dopamine  system.  Efforts  to  trace  the  sprouted 
fibers  using  retrograde  and  anterograde  tracers  have  thus  far  not  been  successful, 
but  further  work  continues.  We  have  performed  cell  counts  of  dopaminergic 
neurons  in  the  midbrain  of  normal  and  hemiparkinsonian  monkeys;  the  pattern  of 
cell  loss  after  MPTP  suggests  that  some  of  the  nuclei  included  in  the  mesolimbic 
system  are  susceptible  to  MPTP  and  are  functionally  related  to  the  substantia  nigra. 

To  further  investigate  this  sprouting,  we  implanted  fetal  monkey  amnion 
(which  produces  a  neurite-promoting  factor  in  vitro)  into  hemiparkinsonian 
monkeys.  We  observed  significant  behavioral  improvement  (almost  as  good  as  seen 
after  fetal  dopaminergic  implants).  We  are  studying  these  animals  with  a  variety  of 
techniques  including  6-fluorodopa  PET,  receptor  autoradiography,  and 
immunohistochemistry.  Term  amnion,  which  is  much  more  readily  accessible  than 
fetal  amnion,  has  also  been  implanted,  but  the  behavioral  response  has  been 
minimal.  We  have  also  developed  an  in  vivo  microdialysis  system  to  study 
biochemical  changes  in  the  implanted  monkeys.  The  system  is  useful  to  compare  the 
normal  and  hemiparkinsonian  sides  and  to  detect  increased  dopaminergic  activity 
after  implantation. 

Since  many  of  the  cells  or  tissues  which  may  be  used  for  implantation  are 
allografts,  we  have  established  the  appropriate  immunohistochemical  techniques  to 
characterize  the  immune  or  inflammatory  response  to  grafting.  Using  these 
techniques,  we  have  determined  that  rejection  did  occur  in  one  of  the  animals 
receiving  a  fetal  dopaminergic  implant.  In  addition,  in  adrenal  autografts  or  at  the 
site  of  cavitation  alone,  there  are  macrophages  and  T  lymphocytes  found  from 
weeks  to  more  than  a  year  after  grafting.  This  persistence  of  inflammatory  cells 
(some  expressing  the  IL-2  receptor,  a  sign  of  activation)  suggests  a  role  for  these  cells 
in  the  host  regenerative  sprouting  response.  We  have  performed  two  experiments 
in  which  an  allograft  or  a  xenograft  was  placed  in  monkeys,  and  peripheral  blood 
examined  for  the  IL-2  receptor  and  the  lymphocytic  response.  This  system  is  not  yet 
sensitive  enough  to  detect  rejection  peripherally. 


17SNB/DIR 


2.  Neurite-promotinq  activity  from  damaged  central  nervous  system  and  other 
tissues 

We  have  previously  established  that  there  is  neurite-promoting  activity  at  the 
site  of  tissue  injury  in  the  cortex  of  rats  or  monkeys,  and  in  the  caudate  of  monkeys. 
The  bioassay  used  is  based  on  the  chick  dorsal  root  ganglion  and  is  qualitative,  with 
nerve  growth  factor  (NGF)  used  as  the  standard.  In  addition,  two  other  assays  have 
been  developed :  the  rat  superior  cervical  ganglion  and  the  fetal  mesencephalon  of 
rat.  These  are  closer  to  the  in  vivo  situation  in  Parkinsonian  models.  We  have 
studied  several  other  rat,  monkey,  and  human  tissues  using  these  assays  and  have 
found  significant  neurite-promoting  activity  in  fetal  kidney  and  amnion  and  less 
activity  in  term  amnion.  Current  efforts  are  directed  at  trying  to  fractionate  the 
proteins  in  buffer  from  term  amnion  and  to  assay  the  fractions,  and  then  to  purify 
the  protein  with  neurite-promoting  activity.  We  are  also  studying  the  RNA  from 
amnion  with  molecular  probes  for  known  growth  factors. 

3.  Cellular  implantation  in  a  new  rat  model  of  parkinsonism 

In  order  to  have  a  small  animal  model  to  perform  implantation  experiments, 
we  modified  the  standard  hemiparkinsonian  rat  model.  The  changes  we  made  allow 
destruction  of  the  substantia  nigra  while  sparing  the  ventral  tegmental  area  (this 
model  is  analogous  to  the  MPTP-hemiparkinsonian  monkey  model).  In  this  model 
there  is  some  tyrosine  hydroxylase  activity  in  the  medial  caudate,  but  very  consistent 
and  stable  turning  in  response  to  amphetamine.  We  chose  to  examine  cellular 
implants  delivered  stereotaxically  to  see  if  they  would  influence  the  turning 
behavior. 

Three  cell  populations  have  been  examined  in  this  model.  Activated 
inflammatory  cells  from  the  peritoneal  cavity  of  the  rat  implanted  into  the 
denervated  caudate  lead  to  significant  motor  improvement  which  is  not  seen  in 
sham-implanted  or  inactivated  cell  implanted  hemiparkinsonian  rats.  There  is  some 
evidence  that  the  improvement  is  mediated  through  a  dopaminergic  mechanism. 
Implantation  of  cultured  rat  microglial  also  leads  to  improvement  in  this  model. 
Finally,  cell  suspension  grafts  of  amnion  lead  to  moderate  recovery  which  is  not  seen 
when  killed  amnion  cells  are  implanted.  We  are  currently  looking  at 
autoradiography  for  the  blood-brain  barrier,  dopamine  uptake,  and  dopamine 
receptors  to  try  and  determine  the  mechanism(s)  of  improvement.  The  results  with 
inflammatory  cells  and  microglia  may  reflect  a  cytokine/glia/neuronal  interaction 
which  leads  to  increased  turnover  of  dopamine  or  to  sprouting  of  new  dopaminergic 
fibers.  It  is  possible  that  direct  implantation  of  fetal  astrocytes  would  also  lead  to 
recovery  and  this  experiment  is  planned. 

4.  Fetal  pituitary  and  pituitarv/hypothalamic  transplants 

We  have  developed  a  successful  model  of  transplantation  of  fetal  rat  pituitary 
+  hypothalamus  into  hypophysectomized  rats.  We  use  small  tissue  fragments 
delivered  stereotaxically  to  the  region  of  the  median  eminence.  There  are  peripheral 
plasma  levels  of  some  anterior  pituitary  hormones  post-transplantation,  and 
immunohistochemical  evidence  of  surviving  anterior  pituitary  cells.  There  is  some 
cellular  infiltrate  in  the  implants;  we  are  currently  characterizing  these  cells  using 
specific  monoclonal  antibodies.  Grafted  animals  have  been  tested  for  evidence  of 
integration  of  the  graft  into  the  hypothalamic/pituitary/peripheral  organ  feedback 


18SNB/DIR 


loop   The  grafts  do  respond  to  direct  administration  of  releasing  factors,  e.g.  CRF, 
but  not  to  indirect  stimulation,  e.g.  insulin  stress.  Further  work  is  underway  to 
determine  if  cell  suspension  grafts  of  pituitary,  or  earlier  grafting  after 
hypophysectomy  can  lead  to  better  integration  into  the  host.  We  are  also 
collaborating  in  efforts  to  transfect  fibroblasts  with  releasing  factor  genes. 


19SNB/DIR 


IV.  Tumor  Biology  Unit 

Marsha  J.  Merrill,  Ph.D.,  Head 

A.  Vascular  Permeability  Factor  (VPF)  Produced  by  Glioma  Cells 

Cerebral  edema  is  a  significant  cause  of  the  neurologic  deficits  and  elevated 
intracranial  pressure  associated  with  malignant  brain  tumors,  and  is  an  important 
challenge  in  the  clinical  management  of  patients  with  this  disease.  We  have 
determined  that  medium  conditioned  by  cultured  human  glioma  cells  contains  a 
substance  capable  of  increasing  vascular  permeability.  This  substance  is  also  found 
in  cyst  fluid  from  brain  tumor  patients.  This  VPF  is  probably  at  least  partially 
responsible  for  the  cerebral  edema  associated  with  brain  tumors.  VPF  is  an 
angiogenic  endothelial  cell  mitogen  which  also  increases  capillary  permeability.  It  is 
a  40  kDa  cationic  polypeptide  with  some  homology  to  PDGF.  Recent  advances  in  the 
field  have  determined  that  VPF  is  the  same  molecule  as  pituitary-derived  vascular 
endothelial  growth  factor,  a  potent  mitogen  selective  for  endothelial  cells.  Evidence 
to  date  suggests  that  the  mechanism  of  action  of  VPF  involves  binding  to  endothelial 
cells  through  a  specific  receptor  on  the  cell  surface  and  subsequent  influx  of  Ca^  + . 
Glucocorticoids  are  a  useful  tool  in  the  clinical  management  of  brain  tumor- 
associated  edema.  Our  studies  include  investigations  into  the  mechanisms  of  action 
of  glucocorticoids  both  at  the  level  of  regulation  of  expression  of  VPF  in  brain  tumor 
cells,  and  at  the  level  of  the  effects  of  VPF  on  the  target  endothelial  cells.  Through 
these  studies  we  hope  to  gain  further  insight  into  the  mechanism  of  action  of  VPF, 
the  role  of  this  factor  in  brain  tumor-associted  edema  and  angiogenesis,  and  the 
possibility  of  improved  therapeutic  intervention. 

B.  The  Role  of  Insulin  and  Insulin-Like  Growth  Factors  in  Glioma  Cells 

Insulin  and  insulin-like  growth  factor  (IGF)  receptors  have  been  detected  in  the 
human  central  nervous  system.  The  role  of  IGFs  in  the  nervous  system  is  not  well 
understood  although  IGF-1  may  be  involved  in  the  growth  of  the  peripheral  nervous 
system,  and  IGF-2  may  play  a  role  in  glial  cell  growth  and  maturation  in  the  brain. 
We  initiated  studies  to  determine  the  nature  of  insulin  and  IGF  receptors  on  human 
glioma  cellsderived  from  surgical  specimens.  Our  results  demonstrate  (1)  high  levels 
of  IGF-1  receptors  on  some  gliomas;  (2)  the  presence  of  receptors  for  IGF-1  and  IGF- 
2,  but  not  for  insulin  on  cultured  cellsderived  from  human  gliomas;  and  (3)  IGF-1 
receptors  on  cultured  glioma  cells  are  functional,  as  determined  by  the  ability  of  IGF- 
1  to  stimulate  autophosphorylation  of  the  receptor  and  DNA  synthesis.  In  addition, 
high  levels  of  high  affinity  IGF-binding  proteins  (IGF  BP)  are  produced  by  glioma  cells 
and  associated  with  some  glioma  surgical  specimens.  Certain  characteristics  of  this 
IGF  BP  suggest  it  may  be  distinct  from  previously  described  IGF  BPs  and  thus  may 
have  a  function  specific  for  glioma  cells.  These  results  suggest  a  role  for  IGF-1 ,  its 
receptor  and  binding  protein  in  the  regulating  growth  in  some  gliomas. 


20SNB/DIR 


V.  Molecular  Biology  Unit 

Iqbal  U.  AM,  Ph.D.,  Head 

The  Molecular  Biology  Unit  of  the  Surgical  Neurology  Branch  is  studying  the 
genetic  abnormalities  of  various  brain  disorders,  especially  brain  cancers.  During  the 
past  12-15  months  we  have  focused  on  molecular  genetic  analyses  of  two  CNS 
tumors,  glioblastomas  and  pituitary  adenomas. 

Glioblastomas 

Extensive  studies  of  the  molecular  origins  of  various  human  cancers  done  over 
the  past  several  years  have  implicated  dominantly  acting  proto-oncogenes  in  various 
malignancies.  These  proto-oncogenes  appear  to  have  essential  roles  in  normal 
cellular  physiology  and  are  probably  involved  in  regulatory  functions.  Several 
mechanisms,  such  as  point  mutations,  rearrangement,  amplification  and/or  elevated 
expression,  are  responsible  for  the  conversion  of  these  normal  genes  into 
oncogenes.  Recently,  yet  another  mechanism,  homozygous  loss  of  gene  function,  is 
believed  to  be  one  of  the  genetic  events  involved  in  the  development  of  certain 
neoplasms. 

Glial  tumors,  which  account  for  the  vast  majority  of  primary  tumors  of  the  CNS, 
are  exceptionally  diverse  in  origin  location,  histology  and  biologic  behavior. 
Malignant  gliomas,  with  a  whole  spectrum  of  increasing  anaplasia  starting  from 
well-differentiated  astrocytoma  to  anaplastic  glioblastoma  multiforme,  offer  an 
excellent  model  system  to  study  molecular  mechanisms  that  confer  upon  these  cells 
an  increased  ability  to  grow,  interact  with  the  environment  and  eventually 
metastasize.  A  variety  of  genetic  lesions  may  therefore  be  expected  to  contribute  to 
the  expression  of  neoplastic  phenotype  of  gliomas.  We  are  studying  primary  glial 
tumors  as  well  as  tissue  culture  cell  lines  derived  from  glioblastomas.  We  have  taken 
the  following  approaches  to  understand  the  genetic  aberrations  responsible  for  the 
expression  of  malignant  phenotype  in  glial  tumors,  which  probably  evolve  through  a 
series  of  genetic  lesions. 

1.  We  have  analyzed  the  genomic  integrity  and  organization  of  several  genes 
that  are  related  to  growth  factors  or  growth  factor  receptors  for  e.g.  bFGF, 
TGFa,  TGF{3,  c-sis,  c-erbB2,  c-erbA2,  and  int-2  in  a  panel  of  20  primary  glial 
tumors  and  eight  glioblastoma  cell  lines.  In  addition,  we  have  also  screened 
these  tumors  and  cell  lines  for  possible  abnormalities  of  those  proto-oncogenes 
that  have  been  found  to  be  frequently  altered  in  various  other  human 
malignancies  for  ras  and  myc.  Our  results  did  not  show  any  gross 
rearrangements  and/or  amplifications  of  these  genes  in  primary  tumors  or 
tumor-derived  cell  lines. 

2.  One  of  the  characteristic  features  of  glioblastoma  is  vascular  proliferation 
suggesting  a  response  to  angiogenic  factors.  A  wide  variety  of  factors  that 
share  structure  characteristic  of  signal-transmitting  molecules  seem  to  have 
both  growth  promoting  the  angiogenic  activities.  These  include  acidic  and 
basic  FGFs,  FGF-5,  int-2,  hst-1,  c-sea,  TGFs,  and  int-1.  We  analyzed  several 
glioblastoma  cell  lines  for  the  expression  of  various  growth  factor  genes  and 
proto-oncogenes.  These  include  bFGF,  TFGa,  TFG(J,  c-sis  (which  encodes  for  the 
P  chain  of  platelet  derived  growth  factor),  c-erbB2  (which  is  related  to  the  EGF 


21  SNB/DIR 


receptor),  N-ras,  H-ras  and  c-myc  genes.  Elevated  expression  of  bFGF,  TFGa, 
TGFJ3,  and  N-ras  genes  was  observed  in  most  glioblastoma  cell  lines  compared 
to  the  normal  brain  and  other  human  tumor  cell  lines. 

3.       The  loss  of  heterozygosity  of  specific  genes,  as  detected  by  restriction  fragment 
length  polymorphism  analysis  (RFLP),  suggests  the  presence  of  oncogenetic 
mutations.  However,  it  may  also  represent  somatic  events  associated  with 
tumor  progression.  Molecular  genetic  analyses  have  identified  allelic  losses  on 
several  chromosomes  in  human  lung  cancers,  colorectal  tumors,  and  primary 
breast  carcinoma.  We  have  so  far  analyzed  30  astrocytomas  and  glioblastomas 
and  their  matched  lymphocytes  for  possible  allelic  deletions  of  genes  on 
various  chromosomes  and  have  identified  loss  of  heterozygosity  of  genes  on 
chromosomes  10  and  17  in  a  significant  number  of  tumors.  These  deletions 
identify  the  map  positions  of  genes  with  possible  roles  in  the  developmental 
and  regulatory  processes  of  glial  cells. 

Pituitary  Adenomas 

Corticotroph  adenomas  are  ACTH  (adrenocorticotropic  hormone)  producing 
tumors  that  generally  originate  in  the  anterior  lobe  corticotrophs  of  the  pituitary 
gland.  These  tumors  cause  increased  ACTH  secretion  and  consequent  hypersecretion 
of  Cortisol,  a  condition  first  described  by  Cushing  half  a  century  ago  and  therefore 
named  after  him.  Corticotroph  adenomas  in  association  with  Cushing's  disease  are 
often  microadenomas  which  do  not  grow  over  several  years.  However,  in  some 
patients,  with  a  condition  called  Nelson's  syndrome,  adrenalectomy  gives  rise  to 
hypersecretion  of  ACTH  by  pituitary  adenomas  which  are  usually  large,  aggressive, 
and  rapidly  growing  neoplasms.  Hypersecretion  of  ACTH  can  some  times  also  be 
caused  by  ectopic  nonpituitary  tumors. 

We  have  initially  taken  two  approaches  to  study  different  kinds  of  ACTH- 
secretion  adenomas:  1)  analysis  of  the  clonal  composition,  and  2)  mutations  of  the 
ras  gene  family. 

Analysis  of  the  Clonal  Composition 

Somatic  mutation  theory  of  carcinogenesis  implies  that  most  cancers  develop 
as  a  consequence  of  an  extremely  rare  somatic  mutation  in  a  single  cell  giving  rise  to 
tumors  of  monoclonal  origin.  Polyclonal  composition,  on  the  other  hand,  may  be 
expected  in  hereditary  tumors  or  endocrine  tumors  where  the  stimulus  can  act  on 
large  population  of  cells  in  the  target  tissue.  We  have  studied  the  clonal 
composition  of  ACTH-producing  adenomas  by  using  X-chromosome  inactivation 
analysis.  The  method  exploits  RFLPs  of  X-chromosome  linked  genes  in  female  cells 
which  undergo  random  inactivation  of  one  X-chromosome  during  embryogenesis 
and  stably  transfer  the  pattern  to  the  progeny.  Since  active  genes  undergo  changes 
in  methylation  patterns,  methylation-sensitive  enzymes  are  used  to  distinguish 
between  active  and  inactive  genes.  Out  of  27  tumors  analyzed,  nine  showed 
heterozygosity  at  hypoxanthine  phosphoribosyl  transferase  (HPRT)  or 
phosphoglycerate  kinase  (PGK)  genes.  Six  tumors  showed  a  single  active  allele  of  the 
X  chromosome  linked  genes  and  were  monoclonal  in  nature.  The  other  three 
tumors,  including  one  microadenoma  from  a  patient  with  Nelson's  syndrome, 
revealed  a  polyclonal  pattern.  Our  results  demonstrate  that  pituitary  corticotroph 


22  SNB/DIR 


adenomas  can  be  monoclonal  or  polyclonal,  thus  emphasizing  the  complexity  of 
genetic  events  with  fundamentally  different  mechanisms  involved  in  oncogenesis. 

Mutation  of  the  ras  genes 

Mutations  leading  to  the  activation  of  the  transforming  potential  of  cellular 
ras  genes  have  been  implicated  in  the  development  of  many  human  tumors  of 
diverse  origin.  Mostly  ras  gene  mutation  is  associated  with  the  progression  of  the 
disease,  but  it  can  also  be  an  early  event,  possibly  even  an  initiating  event  such  as  in 
colorectal  adenomas.  Analysis  of  a  large  number  of  activated  ras  proto-oncogenes 
derived  from  a  variety  of  tumors  showed  that  there  are  three  mutational  hot  spots 
(codons  12,  13,  and  61)  in  all  three  ras  genes  i.e.,  H-ras,  K-ras,  and  N-ras. 

We  have  amplified  the  regions  encompassing  codons  12  and  13  in  exon  1  and 
codon  61  in  exon  2  of  all  three  ras  genes  by  the  polymerase  chain  reaction  (PCR)  from 
several  pituitary  corticoph  adenomas.  We  are  now  sequencing  these  amplified 
regions  for  the  presence  of  possible  mutations. 


23SNB/DIR 


VI.  Brain  Imaging  Unit 

Linda  J.  Porrino,  Ph.D.  -  Head 

The  Unit  on  Brain  Imaging  employs  autoradiographic  methods  to  study  the 
effects  of  dopaminergic  denervation  in  primates  and  rodents  as  as  model  of 
parkinsonism.  These  methods  are  also  used  to  study  the  effects  of  the  acute  and 
chronic  administration  of  a  variety  of  pharmacological  agents.  A  variety  of  methods 
are  utilized  including  measurement  of  rates  of  local  cerebral  glucose  utilization, 
determination  of  the  topographic  distribution  and  the  density  of  receptors, 
measurement  of  local  rates  of  protein  synthesis,  and  autoradiographic 
determination  of  alterations  in  the  blood  brain  barrier. 


Studies  of  animal  models  of  parkinsonism 

In  our  previous  work  we  have  characterized  the  alterations  in  the  anatomic 
distribution  of  both  Di  and  D2  receptor  binding  sites  in  the  primate  model  of 
hemiparkinsonism  provided  by  the  intracarotidfinfusion  of  the  neurotoxin,  1- 
methyl-4-phenyl-1 ,2,3,6-tetrahydropyridine  (MPTP)  which  produces  destruction  of 
the  dopaminergic  cells  of  the  substantia  nigra  parscompacta.  We  have  extended 
these  studies  to  include  other  receptor  populations.  Quantitative  autoradiography 
after  in  vitro  radioligand  binding  with  [3H]naloxone  (opiate  antagonist)  was  used  to 
determine  the  distribution  of  opiate  receptor  sites  in  MPTP-induced 
hemiparkinsonian  monkeys.  An  increased  density  of  opiate  sites  was  observed  in  the 
striatum  on  the  side  of  dopaminergic  denervation.  The  anatomic  distribution  of 
these  sites  resembled  the  distribution  of  dopaminergic  D1  binding  sites  in  the 
striatum,  suggesting  potential  interactions  between  dopamine  and  opiate  peptides 
in  the  striatum. 

In  other  studies  the  2-p4c]deoxyglucose  (2-DG)  method  is  being  applied  to 
animals  in  which  unilateral  lesions  of  the  substantia  nigra  pars  compacta  have  been 
made  in  order  to  map  the  neural  circuits  involved  in  the  symptoms  of  parkinsonism 
and  its  treatment  with  dopaminergic  agonist  drugs.  We  have  completed  our  studies 
of  glucose  utilization  in  hemiparkinsonian  monkeys  following  treatment  with 
apomorphine  and  L-DOPA,  and  have  now  begun  studies  of  the  effects  of  lesions  of 
the  substantia  nigra  pars  compacts  produced  by  the  local  infusion  of  6- 
hydroxydopamine.  Our  lesions  have  been  relatively  small  and  localized  to  either  the 
medial  or  lateral  portions  of  the  substantia  nigra.  We  have  shown  that  the  loci  of 
the  damage  to  dopaminergic  cells  along  the  medial-lateral  axis  of  the  substantia 
nigra  is  important  in  determining  the  resulting  pattern  of  behavioral  effects. 
Furthermore,  the  level  of  functional  activity  in  the  substantia  nigra  pars  reticulata 
determines  the  direction  of  turning  behavior  elicited  by  the  administration  of 
dopaminergic  drugs  such  as  apomorphine. 

Pharmacologic  Studies 

In  these  experiments  we  have  used  metabolic  mapping  methods  to  examine 
different  aspects  of  the  behavioral  and  pharmacologic  actions  of  abused  drugs,  i.e., 
cocaine,  methamphetamine,  and  morphine.  In  one  set  of  experiments  the 
quantitative  2-DG  method  was  used  to  map  the  distribution  of  alterations  in  local 
rates  of  cerebral  glucose  metabolism  associated  with  the  self-administration  of 


24SNB/DIR 


cocaine  in  rats.  Triads  of  male  littermate  rats  were  studied.  One  rat  of  each  triad 
was  allowed  to  self-administer  cocaine,  another  received  identical  yoked  infusions  of 
cocaine,  while  the  third  received  yoked  saline  infusions.  We  demonstrated  that  the 
metabolic  effects  of  the  contingent  presentation  of  cocaine  are  different  from  the 
effects  of  noncontingent  presentation.  These  differences  may  be  directly  related  to 
the  reinforcement  associated  with  cocaine  self-administration.  Contingent  self- 
administration  was  associated  with  increased  rates  of  local  cerebral  glucose 
utilization  in  the  mesocorticolimbic  system  including  the  prefrontal  cortex,  nucleus 
accumbens,  olfactory  tubercle,  and  amygdala,  when  compared  to  yoked  cocaine  or 
saline  controls.  These  neural  circuits  appear  to  be  central  to  the  mediation  of 
positive  reinforcement. 

Experiments  to  compare  the  sites  of  action  of  the  reward-enhancing  effects  of 
morphine  as  measured  in  self-stimulation  paradigms  with  the  sites  of  action  of  the 
antinociceptive  properties  of  morphine  have  also  been  completed  this  year.  Our 
data  have  identified  the  dorsal  raphe  and  lateral  habenula  as  important  substrates 
of  the  analgesic  effects  of  morphine,  whereas  the  euphorigenic  properties  of 
morphine  appear  to  be  mediated  by  portions  of  the  mesocorticolimbic  system.  The 
brain  regions  involved  in  the  rewarding  effects  of  morphine  appear  to  be  quite 
different  from  those  involved  in  analgesia. 


25SNB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02454-10  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Studies  of  Human  Pituitary  Tumors 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 

Edward  Oldfield,  M.D.  Chief,  SNB,  Principal  Investigator,  NINDS 


COOPERATING  UNITS  Of  an,) 

Developmental  Endocrinology  Branch,  NINDS 
Diagnostic  Radiology,  CC 


LAB/BRANCH 

Surgical  Neurology  Branch 


SECTION 

Clinical  Neurosurgery  Section, CNP 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  Q  g 


OTHER:  QQ 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  [T]  (b)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

]  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

We  continue  to  investigate  venous  samplings  of  the  pituitary  venous  drainage  to  aid  in  the  diagnosis  and 
treatment  of  patients  with  Cushinq's  syndrome    Over  300  patients  have  now  received  bilateral 
simultaneous  inferior  petrosal  sinus  (IPS)  sampling    The  results  indicate  that  1)  the  procedure  can  be 
performed  successfully  in  all  patients  with  Cushing's  syndrome  (successful  sampling  has  been  performed 
in  over  99%  of  the  patients  in  whom  it  has  been  attempted);  2)  the  procedure  distinguishes  patients 
with  ectopic  ACTH  secretion  from  those  with  pituitary  adenomas  with  100%  accuracy;  3)  IPS  sampling 
successfully  determines  which  side  of  the  pituitary  gland  microadenomas  reside  in  patients  with 
Cushing's  disease  with  75%  accuracy;  and  4)  unilateral  inferior  petrosal  sinus  sampling,  which  is 
commonly  used  clinically,  is  frequently  misleading. 

Repeat  transsphenoidal  surgery  is  successful  in  elimmating  the  hypercortisolism  of  Cushing's 
disease  in  about  70%  of  patients   This  therapy  for  patients  with  Cushing's  disease  after  previous 
pituitary  surgery  had  not  previously  been  examined. 

The  CRF  stimulation  test  and  the  dexamethasone  suppression  test  are  equally  accurate  in 
determining  and  distinguishing  patients  with  Cushing's  disease  from  those  with  ectopic  ACTH  secretion 
in  Cushing's  syndrome    Both  have  approximately  10-15%  diagnostic  inaccuracy,  when  used  alone,  in  our 
experience 

MRI  scanning  with  and  without  gadolinium-EDTA  was  used  to  evaluate  patients  with  Cushing's 
disease  preoperatively.  This  technique  permitted  identification  of  the  adenoma  in  about  55%  of  those 
patients  with  surgically  proven  microadenomas    Proper  timing  of  the  MRI  after  the  administration  of 
gadolinium  EDTA  was  critical  in  the  optimal  use  of  the  technigue.  Pituitary  adenomas  were  detected  in 
15%  of  100  normal  subjects  with  MRI  scanning  with  contrast. 


PHHMOHr.   1  sj  27  SNB  DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02813-01  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Pharmacokinetics  of  Direct  Brain  Infusion 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

R.  Hunt  Bobo,  M.D.  Senior  Staff  Fellow,  NINDS 

Edward  Oldfield,  M.D.  Chief,  Surgical  Neurology  Branch,  NINDS 

Aytac  Akbasak,  M.D.  Visiting  Associate,  NINDS 


COOPERATING  UNITS  (it any) 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Clinical  Neurosurgery  Section 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS:  -  , 


PROFESSIONAL:  Q  3 


OTHER:  QQ 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  ]  (b)  Human  tissues  [V]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  temporal  and  spatial  distribution  (pharmacokinetics)  of  potential  anti-glioma  agents  administered 
into  the  feline  brain  by  direct  infusion  are  being  studied.  Reasons  for  direct  infusion  are  avoidance  of 
the  blood  brain  barrier,  decreased  systemic  exposure,  and  avoidance  of  systemic  metabolic  and 
immunologic  interaction  with  the  agent.  The  aim  of  the  project  is  to  develop  the  methods  to  administer 
antiglioma  agents  by  direct  infusion,  and  achieve  an  homogeneous  distribution  throughout  the  tumor 
volume,  in  a  clinical  setting.  Agents  for  study  will  be  analogous  to  immunotoxins,  1-125  labeled 
iododeoxyuridine,  and  commercially  available  antineoplastic  agents. 


PHS&MOIRe.    114  ?8  SNB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02812-01  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PRO  J  E  CT  (80  characters  or  loss.    Titlo  musl  fit  on  one  line  between  the  borders.) 

Pentobarbital  Effects  on  Damage  of  the  Primate  Brain  by  Fractionated  Whole  Brain  Radiation 


PRINCIPAL  IN  VE  STIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (htame.  title,  laboratory,  and  institute  affiliation) 

R.  Hunt  Bobo,  M.D.  Senior  Staff  Fellow,  SNB 


Tom  Goffman,  M.D 
KathrynOrr,  R.N. 
Edward  Oldfield,  MD 


Radiation  Oncology  Branch,  NCI 
Radiation  Oncology  Branch,  NCI 
Surgical  Neurology  Branch,  NINDS 


COOPERATING  UNITS  of  an,) 

Radiation  Oncology  Branch,  NCI 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Clinical  Neurosurgery  Section 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS. 


0.4 


PROFESSIONAL: 


0.4 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

1      |   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


J  (b)  Human  tissues  |  «    |  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

After  baseline  MRI  scans  of  the  brain  and  neuroendocrine  testing,  primates  undergo  whole  brain 
radiotherapy  in  10  daily  fractions,  340  rads  each.  Six  of  these  primates  will  be  anesthetized  with 
pentobarbital  during  the  irradiation,  six  will  not  receive  any  pentobarbital.  Neuroendocrine  testing  and 
MRI  scans  will  be  done  at  3,  6,  12,  18  and  24  months  after  radiation.  Quantitative  histology  will  be  done 
on  the  capillary  bed,  glial,  and  neuronal  populations  after  sacrifice.  The  purpose  of  this  study  is  to 
determine  if  pentobarbital  has  protective  effects  on  multiple  fractionated  whole  brain  irradiation  and  to 
further  elucidate  the  microanatomic  substrate  of  cerebral  radiation  damage. 


PHSMMOfRev    1  M 


29  SNB  DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  0281 1-01  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJ  ECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Phase  II  Clinical  Trial  of  Suramin  and  Hydrocortisone  in  the  Therapy  of  Current  Malignant  Gliomas 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator .)  (Name,  title,  laboratory,  and  institute  affiliation) 

Edward  H.  Oldfield,  M.D.  Chief,  SNB,  Principal  Investigator,  NINDS 


R.  HuntBobo,  M.D. 

Hetty  DeVroom,  R.N. 

RV  LaRocca,  M.D.,  M.  Cooper, 

David  Kohler,  Pharm.D. 

Barry  Goldspiel,  Pharm.D. 


M.D..CE  Myers,  M.D. 


Senior  Staff  Fellow,  Associate  Investigator,  NINDS 
Clinical  Nurse,  NINDS 
National  Cancer  Institute 
Pharmacy  Department,  CC 
Pharmacy  Department,  CC 


COOPERATING  UNITS  (if  any) 

Radiation  Oncology 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Clinical  Neurosurgery  Section 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


0.4 


PROFESSIONAL: 


0.4 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

I  x   |   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


J  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 


This  is  a  phase  II  study  of  the  combination  of  suramin  (a  polysulfonated  naphthylurea)  and 
hydrocortisone  in  the  treatment  of  malignant  gliomas,  recurrent  after  radiation  therapy.  Both  efficacy 
and  toxicity  will  be  monitored.  Tumor  volumes  are  measured  on  MRI  scans  to  judge  radiographic 
respone  or  progression.  Fourteen  patients  will  be  treated  the  first  year,  followed  by  10  more  the 
following  year,  if  at  least  one  partial  response  is  obtain  during  the  first  year. 


PHS  6040  (Rtv.  1/84 


30  SNB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02697-06  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Protection  of  the  Brain  Against  Injury  by  Ionizing  Radiation  with  Pentobarbital 


PRINCIPAL  INVESTIGATOR  (tut  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Jeffrey  J.  Olson,  M.D.  Principal  Investigator,  SNB,  NINDS 

Edward  H.  Oldfield,  M.D.  Chief,  SNB  ,  NINDS 

Craig  Shelley,  M.D.  Medical  Staff  Fellow,  SNB 

Calvin  Hawkins  Bio  Lab  Technician,  SNB 


COOPERATING  UNITS  Of  any) 

National  Cancer  Institute,  Radiation  Oncology  Branch 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Clinical  Neurosurgery  Section 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

LJ   (a)  Human  subjects  [7]  (b)  Human  tissues  |      |  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  efficacy  of  radiation  therapy  in  the  treatment  of  brain  tumors  is  limited  by  the  toxicity  of  ionizing 
radiation  to  the  surrounding  normal  tissue 

In  the  rat  model  of  cerebral  radiation  injury,  pentobarbital  has  been  shown  to  enhance  overall 
survival  in  a  dose-dependent  manner.  Evaluation  of  alternative  barbiturates  reveals  that  thiopental  is  of 
equivalent  radioprotective  value  to  pentobarbital. 

The  rodent  model  of  radiation  injury  does  not  parallel  that  of  human  injury.  A  primate  model  was 
designed  to  assess  the  role  of  pentobarbital  in  circumstances  more  applicable  to  the  human  situation 
This  ongoing  study  has  thus  far  revealed  the  ability  of  pentobarbital  to  limit  the  toxicity  of  the  radiation 
utilized.  Neuroendocrinoloqic  evaluation  has  revealed  early  dysfunction  of  thyroid-stimulating 
hormone,  luteinizing  hormone,  and  prolactin  responses  to  stimulatory  testing  in  the  animals  irradiated 
while  anesthetized  with  ketamine.  Significantly  less  abnormalities  have  occurred  in  the  pentobarbital 
group. 


PHS  6M0  (Rev   1  84  31  SNB'DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02728-04  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJ  ECT  (80  characters  or  less,  rule  must  fit  on  one  line  between  the  borders.) 

Studies  on  the  Heterogeneity  of  Drug  Delivery  During  Intracarotid  Chemotherapy 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Stephen  Saris,  M.D.  Senior  Staff  Fellow,  Principal  Investigator,  SNB 


Robert  Lutz,  Ph.D. 
Ron  Blasberg,  M.D. 
Donald  Wright,  M.D. 
Edward  H.OIdfield,  M.D. 


Staff  Fellow 

Senior  Investigator,  Department  of  Nuclear  Medicine 

Medical  Officer,  SNB,  NINDS 

Chief,  SNB,  NINDS 


COOPERATING  UNITS  (if  any) 

Department  of  Nuclear  Medicine 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP  

SECTION 

Clinical  Neurosurgery  Section 

INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.0 


PROFESSIONAL: 


1.0 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

|  x   |   (a)  Human  subjects 
!     ]  (a1)  Minors 
]  (a2)  Interviews 


]  (b)  Human  tissues  Q  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 
Project  was  completed  in  July,  1990. 


PHS  6040  (H»v   I'M 


32  SNBTJIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02778-03  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.    Title  must  fit  on  one  line  between  the  borders  t 

Adoptive  Immunotherapy  of  Brain  Tumors 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  atiiliation) 

Stephen  Saris,  M.D. 


Aytac  Akbasak,  M.D. 
Ikejiri,  Barbara 
Edward  Oldfield,  MD. 


Principal  Investigator,  SNB,  NINDS 

Special  Volunteer,  SNB,  NINDS 
Biologist,  SNB,  NINDS 
Chief,  SNB,  NINDS 


COOPERATING  UNITS  (it any) 

Surgical  Oncology  Branch,  NCI 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Clinical  Neurosurgery  Section 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.0 


PROFESSIONAL: 


10 


OTHER: 


00 


CHECK  APPROPRIATE  BOX(ES) 

I  x   |    (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


j  (b)  Human  tissues  J  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

There  are  10-15,000  malignant  brain  tumors  diagnosed  each  year   The  most  common  of  these  are 
glioblastomas.  Numerous  treatments  involving  surgery,  chemotherapy,  and  radiation  are  of  palliative 
benefit  only.  We  study  the  adoptive  immunotherapy  of  malignant  brain  tumors  with  interleukin-2  (IL-2), 
lymphokine-activated  killer  (LAK)  cells,  and  tumor  infiltrating  lymphocytes  (TILs)  in  animal  models  and  in 
patients.  In  the  9L  gliosarcoma  rat  model,  we  studied  the  efficacy  of  parenteral  IL-2  and  its  effect  on  the 
blood-brain  barrier  of  normal  and  neoplastic  tissue,  and  in  the  C3H  mouse  we  studied  the  cytolytic 
activity  of  LAK  against  metastases  in  the  brain.  In  patients  with  extracranial  cancer,  we  examined  the 
kinetics  of  IL-2  in  the  cerebrospinal  fluid  as  compared  to  serum,  and  in  patients  with  gliomas  we 
investigated  the  cerebral  toxicity  of  parenteral  IL-2.  We  are  currently  completing  our  studies  with  IL-2 
and  LAK  cells,  and  are  expanding  our  efforts  with  TILs    In  animals  models  of  primary  and  metastatic 
brain  tumors,  we  have  raised  TILs  against  tumors  in  the  subcutaneous  space;  these  cells  will  then  be  used 
to  treat  animals  with  intracerebral  tumors  and  to  study  trafficking  across  the  blood-brain  barrier  with 
111lndium  labeled  cells.  In  patients,  we  are  raising  TILs  from  brain  tumors  grown  in  the  subcutaneous 
space.  These  have  been  expanded  in  vitro  with  IL-2,  and  injected  parenterally  into  mice  to  study 
lymphocyte  trafficking  and  efficacy  against  intracerebral  tumors    In  patients  we  have  raised  TILs  from 
fresh  specimens  sent  from  the  operating  room    This  creates  the  possibility  of  proceeding  with  Phase  I 
clinical  trials  based  on  the  final  outcome  of  our  laboratory  efforts.  Lastly,  we  are  investigating  the 
regulation  of  major  histocompatability  antigens  of  brain  tumors  in  vitro  by  interleukins,  interferons,  and 
tumor  factor. 


PHSMMOISe.   1  8J 


33SNBT5IR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02673-06  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Monoclonal  Antibodies  Linked  to  Ricin  for  Use  in  Human  Bone  Marrow  Transplantation 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Richard  J.  Youle,  Ph.D.  Principal  Investigator,  SNB,  NINDS 


Pat  Johnson 

Virginia  Johnson,  Ph.D. 


Bio  Laboratory  Technician,  SNB,  NINDS 
Senior  Staff  Fellow,  SNB,  NINDS 


COOPERATING  UNITS  Of  any) 

American  Red  Cross;  Washington  Children's  Hospital;  National  Cancer  Institute,  Immunology  Branch, 
DCBD. 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Biochemistry  Section 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.5 


PROFESSIONAL: 


1.5 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

[  x   |   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


I  x   I  (b)  Human  tissues  j  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 
This  project  was  completed  July,  1990. 


f>HS6M0(R*».  VM 


34SNBT)IR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02823-01  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Antibody-Toxin  Conjugates  for  the  Treatment  of  Human  Brain  Tumors 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Richard  J.  Youle,  Ph.D.  Chief,  Biochemistry  Section,  SNB,  NINDS 

Deborah  Wilson,  B.S  Biologist,  SNB,  NINDS 

Karin  M.  Muraszko,  M.D.  University  of  Michigan,  Section  of  Neurosurgery 

Edward  H.  Oldfield,  M.D.  Chief,  Surgical  Neurology  Branch,  NINDS 

David  Katz,  M.D.  NINDS 

Cynthia  Sung,  Ph.D.  BEIB 

Robert  Dedrick,  Ph.D.  BEIB 


COOPERATING  UNITS  Of  *ny) 

Diagnostic  Radiology;  Nuclear  medicine  Department;  National  Cancer  Institute 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Biochemistry  Section 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  ..   r 


OTHER: 


00 


CHECK  APPROPRIATE  BOX(ES) 

[7]   (a)  Human  subjects  ]  (b)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  purpose  of  this  study  is  to  investigate  the  use  of  immunotoxins  in  the  treatment  of  primary  brain 
tumors  and  meningeal  carcinomatosis    Immunotoxins  are  molecular  hybrids  composed  of  monoclonal 
antibodies  covalently  attached  to  peptide  toxins.  The  monoclonal  antibody  targets  the  conjugate  to  the 
surface  of  tumor  cells  bearing  the  appropriate  antigen    The  toxin  moiety  then  penetrates  the  cell 
membrane  and  inactivates  protein  synthesis.  The  compound  454A12MAB-rRA  is  a  conjugate  of  an  anti- 
human  transferrin  receptor  antibody  (454A12)  and  the  recombinant  A  chain  portion  of  the  toxin  ricin 
We  have  shown  that  454A1 2MAB-rRA  kills  various  cell  lines  of  tumor  that  spread  through  the  CSF 
pathways.  In  an  animal  model  of  intrathecal  leukemia,  immunotoxin  therapy  killed  90-99  9%  of  the 
tumor  cells  in  the  CSF  and  a  single  injection  of  immunotoxins  prolonged  survival  of  the  guinea  pigs   At 
present  there  is  a  Phase  l/ll  trial  with  454A12MAB-rRAforthe  treatment  of  refractory  carcinomatous 
meningitis.  It  is  ongoing  in  the  Surgical  Neurology  Branch.  The  purpose  of  the  study  is  to  determine  the 
toxicity  of  454A12MAB-rRA  administered  intrathecal  I  y  in  a  limited  escalation  dosage  schedule    The  CSF 
pharmacokinetics  of  intraventricular^  administered  454A12MAB-rRA  are  determined  and  will  be 
compared  to  the  pharmacokinetics  that  have  been  previously  obtained  from  primate  studies  using  the 
same  immunotoxin.  In  addition,  we  will  determine  the  formation  of  antibodies  to  454A1 2MAB-rRA 
after  intrathecal  administration.  Finally  we  will  examine  the  potential  therapeutic  efficacy  of  this 
intrathecal  immunotoxin  in  ieptomeninqeai  meningitis 

To  date,  5  patients  have  been  treated  with  immunotoxin  injections  into  the  intrathecal  space  and 
pharmacokinetic  studies  have  been  performed  on  each  of  these  patients.  No  toxicity  has  been  found  at 
this  time  related  to  the  immunotoxin.  The  maximum  dose  administered  to  date  is  120  mg  of  immuno- 
toxin into  the  intrathecal  space    There  have  been  3  deaths  within  the  study  group,  none  of  which  was 
related  to  the  administration  of  immunotoxin.  Two  of  these  patients  have  undergone  autopsy  and  no 
toxicity  has  been  appreciated  on  histologic  analysis  of  their  tissues  after  immunotoxin  injection 

Pharmacokinetic  data  of  the  immunotoxin  suggests  that  it  circulates  well  within  the  CSF,  that  the 
immunotoxin  is  stable  within  the  CSF  and  that  further  dose  escalation  will  be  performed  We  plan  to 
proceed  with  further  dose  escalation  in  these  patients  and  to  continue  to  monitor  them  both  for  the 
pharmacokinetic  data  within  the  CSF  as  well  as  for  efficacy  as  judged  both  clinically  and  by  CSF  cytology 


PHS6M0{Rev  184  35  SNB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02674-06  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Monoclonal  Antibody-Toxin  Conjugates  for  Tumor  Therapy  In  Vivo 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Richard  J.  Youle,  Ph.D.  Principal  Investigator,  SNB,  NINDS 

Virginia  Johnson,  Ph.D.  Senior  Staff  Fellow,  SNB,  NINDS 

Debbie  Wilson  Bio  Lab  Technician,  SNB,  NINDS 

Dianne  Newton  Special  Volunteer,  SNB,  NINDS 

Susanna  Rybak,  Ph.D.  Special  Expert,  SNB,  NINDS 

Peter  Nicholls,  Ph.D.  Visiting  Fellow,  SNB,  NINDS 


COOPERATING  UNITS  (if  any) 

Biogen 

Cetus  Corporation 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Biochemistry  Section 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

I  x  I   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


I  x   |  (b)  Human  tissues 


(c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Monoclonal  antibodies  selectively  bind  tumor  cell  differentiating  antigens  in  vitro  and  in  vivo.  Natural 
effector  mechanisms  often  do  not  mediate  killing  of  monoclonal  antibody  bound  cells  so  we  have 
devised  methods  of  linking  extremely  toxic  proteins  to  the  antibodies  to  selectively  kill  tumor  cells. 

Two  methods  of  coupling  toxic  proteins,  like  ricin  to  antibodies,  have  been  used  to  kill  antigen  positive 
cells  in  vitro.  Ricin  has  two  subunits,  the  A  subunit  blocks  protein  synthesis  when  in  the  cytosol  and  the  B 
subunit  binds  galactose  groups  on  all  cell  surfaces  but  also  facilitates  the  transport  of  ricin  A  chain  to  the 
cytosol.  1)  Linkage  of  the  ricin  A  chain  to  antibodies  yields  reagents  with  low  non-target  toxicity  but 
target  cell  toxicity  too  slow  for  in  vivo  applications;  2)  Linkage  of  intact  ricin  to  antibodies  results  in  very 
potent  target  cell  toxicity  but  the  non-target  cell  killing  must  be  prevented  by  a  ligand  which  blocks  ricin 
B  chain  binding  to  cells.  This  has  limited  its  application  to  in  vitro  situations  where  100  mM  lactose  can 
block  ricin  binding. 

We  have  succeeded  in  developing  several  new  approaches  to  apply  immunotoxins  in  vivo.  1)  Cloning  of 
toxins  then  altering  their  structure  at  the  gene  level  to  decrease  non-target  cell  toxicity;  2)  intrathecal 
administration  of  immunotoxins  for  therapy  of  brain  tumors  that  kill  2-5  logs  of  tumor  cells  in  animal 
models;  3)  preparation  of  genetically  engineered  immunotoxins  for  clinical  trials  of  human  brain  tumor 
patients;  4)  prevention  of  immune  response  against  immunotoxin  with  anti-CD4  antibodies;  5) 
construction  of  HIV  infected  cell  immunotoxins;  and  6)  specific  deletion  of  Purkinje  cells  in  rats,  guinea 
pigs  and  rhesus  monkeys. 


CHS  6040  (Rev   1/84 


36  SNB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02781-03  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders  t 

Tissue  Implantation  in  Parkinsonian  Models 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Wame.  title,  laboratory,  and  institute  affiliation) 

Robert  Plunkett,  M.D.  Principal  Investigator,  SNB 

Kris  Bankiewicz,  M.D.  -  Visiting  Fellow,  SNB  JG  Sheng,  M.D.  -  Visiting  Fellow,  SNB 

Mark  Luciano,  M.D.  -  Medical  Staff  Fellow,  SNB  Bernhard  Zunkeler,  M.D.  -  Visiting  Fellow,  SNB 

Ian  McCutcheon,  M.D.  -  Medical  Staff  Fellow,  SNB  Jin  Wang,  M.D  -  Visiting  Fellow,  SNB 

Scott  Ewing,  M.D.  -  Visiting  Medical  Student,  SNB  Kap  Holloway,  M.D.  -  Special  Volunteer,  SNB 

Jim  Kim,  M.D.  -  Visiting  Fellow,  SNB 


COOPERATING  UNITS  (,f  an,) 

Meg  Palmatier,  Ph.D.  -  Staff  Fellow,  Clinical  Neuroscience  Branch;  R.  Gress,  M.D.,  Scientist,  NCI 
RJ  Weber,  Ph.D.,  Scientist,  NIDDK;  Peter  Basser,  Ph.D.,  Biomedical  Engineering,  NIH 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Clinical  Neurosurgery  Section,  Central  Nervous  System  Implant  Unit 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health  Bethesda,  Maryland 


TOTAL  MAN-YEARS:  ,  c 

0.3 


PROFESSIONAL:  fi  5 


OTHER:  QQ 


CHECK  APPROPRIATE  BOX(ES) 

[xj   (a)  Human  subjects  [7]  (b)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  type.  Do  not  exceed  the  space  provided.) 

The  behavioral,  biochemical,  and  histological  effects  of  tissue  implants  in  rodent  and  primate  models  of 
parkinsonism  is  being  studied  The  grafts  which  have  been  examined  include  fetal  and  adult 
dopaminergic  and  nondopaminergic  tissues.  There  is  some  behavioral  improvement  with  any  operative 
trauma  to  the  caudate,  whether  a  graft  is  placed  or  not    Generally,  fetal  tissue  grafts  (dopaminergic  or 
nondopaminergic)  lead  to  a  much  greater  degree  of  recovery  than  adult  tissue  grafts  or  trauma  alone. 
The  histologic  observation  of  dopaminergic  fiber  ingrowth  (sprouting)  in  all  these  animals  suggests  that 
the  improvement  is  mediated  through  a  neurotrophic  interaction.  We  are  trying  to  determine  the  cell- 
to-cell  interaction  which  leads  to  new  growth  of  fibers  from  an  adult  neuron,  using  in  vivo  and  in  vitro 
methods.  Two  major  areas  of  emphasis  are:  what  cascade  of  events  in  the  host  after  trauma  leads  to 
sprouting  and  why  does  fetal  tissue  enhance  the  recovery  (even  nondopaminergic  tissue) 

The  current  experiments  include  implantation  of  term  amnion  into  hemiparkinsoman  monkeys  (solid 
tissue  into  preformed  cavities),  cell  suspension  implants  of  term  amnion  into  rats,  and  biochemical  and 
molecular  analysis  of  the  neurotrophic  factor(s)  produced  by  amnion  cells   We  are  also  incorporating 
laminin  into  a  slow-release  polymer  and  implanting  this  in  hemiparkinsonian  rats    To  further  investigate 
the  host  response  to  tissue  trauma,  we  are  implanting  inflammatory  cells  in  the  denervated  caudate. 
Macrophages,  T-cells,  and  microglia,  alone  or  combined,  and  their  secretory  products  such  as  IL-1  are 
being  studied.  We  are  now  using  autoradiographic  techniques  to  study  the  blood  brain  barrier,  receptor 
binding  and  dopamine  uptake  in  the  inflammatory  cell  implant  model 


PHS  6040  <Re«.  I  M  37  SNB  DIP 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02729-04      SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (30 characters  or  less.  Title  must  fit  on  one  line  between  the  borders.) 

Adrenal  Medullary  Autografts  in  Parkinsonian  Patients 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Robert  J.  Plunkett,  M.D.  Principal  Investigator,  SNB 

Kris  Bankiewicz,  M.D.  Visiting  Associate,  SNB 

Jeff  Norton,  M.D.  Senior  Staff  Fellow,  NCI 

Hetty  DeVroom,  R.N.  Clinical  Nurse,  SNB,  NINDS 

Robert  Miletich,  M.D.,  Ph.D.  Senior  Staff  Fellow,  NIS,  NINDS 

Edward  Oldfield,  M.D.  Chief,  SNB 


COOPERATING  UNITS  (if  any) 

Surgery  Branch,  National  Cancer  Institute 
Neuroimaging  Section,  NINDS 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Clinical  Neurosurgery  Section,  Central  Nervous  System  Implantation  Unit 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  .   c 


OTHER:  00 


CHECK  APPROPRIATE  BOX(ES) 

|T]   (a)Human  subjects  □  (b)  Human  tissues  □  (c)  Neither 

J  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  type.  Do  not  exceed  the  space  provided.) 

This  study  is  designed  to  assess  the  safety  and  efficacy  of  adrenal  medullary  autografts  and  fetal 
mesencephalic  grafts  in  patients  with  severe  Parkinson's  disease   The  patients  selected  will  have  Stage  IV 
disease,  but  still  show  some  response  to  oral  dopamine  replacement  therapy.  The  patient  will  undergo 
extensive  testing  pre-operatively  including  gait,  posture,  reaction  time,  speech,  and  neuropsychiatric 
assessment.  Cavities  will  be  created  in  the  right  caudate  nucleus  via  a  transcallosal  approach,  and  a 
ventricular  reservoir  placed.  Two  weeks  later  one  adrenal  gland  will  be  removed,  and  placed  into  the 
preformed  cavity  (or  fetal  mesencephalic  tissue  will  be  placed  in  the  cavity  and  no  adrenalectomy 
performed).  The  preoperative  testing  will  be  repeated  at  regular  intervals  after  implantation.  CSF 
biochemistry  and  6-fluorodopa  PET  studies  will  also  be  part  of  the  testing  carried  out. 

One  patient  has  received  an  adrenal  medullary  autograft.  He  has  shown  mild  improvement  with 
reduction  of  the  severity  of  his  on/off  fluctuations  and  improved  mobility  during  his  "off"  time.  In 
addition,  he  has  had  a  lessening  of  the  dyskinesias  which  limit  his  tolerance  for  oral  therapy.  The  CSF 
studies  have  revealed  an  elevation  of  enkephalin  in  the  first  few  months  compared  to  pre-operatively, 
but  no  long-lasting  elevation  of  enkephalin  or  elevation  of  catecholamines.  No  patients  have  received 
fetal  mesencephalic  grafts  due  to  a  ban  on  such  work  by  the  Department  of  Health  and  Human  Services. 


PHS6M0  (Rev  1,84  38  SN8/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02708-05  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less    Title  must  fit  on  one  line  between  the  borders.) 

Vascular  Permeability  Factor  Produced  by  Human  Glioma  Cells 


PRINCIPAL  INVESTIGATOR  tList  other  professional  personnel  below  the  Principal  Investigator  I  (Name,  title,  laboratory,  and  institute  affiliation) 

Marsha  Merrill,  Ph.D.  Principal  Investigator,  SNB,  NINDS 

Nancy  Edwards,  B. A  Biologist,  SNB 

Calvin  Hawkins  Technician,  SNB 

Lou  Rosa,  M.D  Senior  Staff  Fellow,  SNB 

Edward  Oldfield,  M.D.  Chief,  SNB 


COOPERATING  UNITS  (.» any) 

Jack  Knightly,  M.D.,  National  Naval  Medical  Center,  Bethesda,  Maryland 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Clinical  Neurosurgery  Section,  Tumor  Biology  Unit 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland 


TOTAL  MAN-YEARS:  ? 


PROFESSIONAL:  .   c 


OTHER.  1   q 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  [7]  (b)  Human  tissues  |      |  (c)  Neither 

]  (a1)  Minors 

]  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  cerebral  edema  associated  with  malignant  brain  tumors  causes  neurologic  deficits  and  increased 
intracranial  pressure,  and  contributes  to  the  morbidity  and  mortality  associated  with  the  neoplasm.  We 
have  determined  that  the  conditioned  medium  of  glioblastoma-derived  cell  cultures  contains  a 
substance  capable  of  increasing  the  vascular  permeability  in  a  bioassay  which  measures  the  induction  of 
capillary  permeability  in  normal  skin.  This  substance  has  been  identified  as  vascular  permeability  factor 
(VPF)  and  may  be  partially  responsible  for  the  cerebral  edema  associated  with  brain  tumors.  VPF  is  a  40 
kDa  cationic  polypeptide  with  some  homology  to  PDGF.  In  addition  to  increasing  capillary  permeability, 
VPF  is  also  an  angiogenic,  endothelial  cell  mitogen.  Evidence  to  date  suggests  that  the  mechanism  of 
action  of  VPF  involves  binding  to  endothelial  cells  through  a  specific  receptor  on  the  cell  surface  and 
subsequent  influx  of  Ca2  + .  Glucocorticoids  are  a  useful  tool  in  the  clinical  management  of  brain  tumor- 
associated  edema.  Ou  studies  indicate  that  glucocorticoids  act  both  at  the  level  of  the  regulation  of 
expression  of  VPF  in  brain  tumor  cells  and  on  the  target  endothelial  cells   Through  these  studies  we  hope 
to  gain  further  insight  into  the  mechanism  of  action  of  VPF,  the  role  of  this  factor  in  brain  tumor- 
associated  edema  and  angiogenesis,  and  the  possibility  of  improved  therapeutic  intervention. 


pms  two  («*»  i  8«  39SNBDIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02707-05  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

The  Role  of  Insulin  and  Insulin-like  Growth  Factors  in  Glioma  Cells 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Marsha  Merrill,  Ph.D.  Principal  Investigator,  SNB 

Nancy  Edwards  Biologist,  SNB 

Jeffrey  Olson,  M.D.  Senior  Staff  Fellow,  SNB 


COOPERATING  UNITS  (if  any) 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Clinical  Neurosurgery  Section,  Tumor  Biology  Unit 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  ..  n 


OTHER: 


0.5 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)  Human  subjects  [7]  (b)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Insulin  and  insulin-like  growth  factors  (IGF-1  and  -2  or  somatomedins)  are  anabolic  effectors  in  many 


tissues  and  cultured  cells  including  astrocytes  and  neurons.  Receptors  for  insulin  and  IGFsare  found 


throughout  the  human  brain.  Receptors  for  several  growth  factors  are  increased  in  tumor  vs.  normal 
tissue  and  this  discrepancy  may  have  therapeutic  value  in  targeting  toxic  agents  to  tumor  cells.  To 
evaluate  the  potential  role  of  IGFs  in  human  CNS  tumors,  we  examined  the  level  of  insulin  and  IGF 
receptors  in  tumors  (astrocytomas  and  glioblastomas)  and  in  normal  brain.  Although  all  surgical 


specimens  contain  receptors  for  all  three  growth  factors,  the  highest  values  were  observed  with  IGF-1 
binding  to  glioma  specimens.  The  IGF-1  receptor  in  tumor  is  the  same  size  (1 18  kDa  a-subunit)  as  the 
receptor  in  normal  brain,  confirming  the  neural  origin  of  the  tumor  cells  expressing  the  IGF-1  receptor. 
Cultured  cells  derived  from  glioma  specimens  also  express  IGF-1  receptors,  and  many  of  these  lines 
demonstrate  a  functional  receptor  as  indicated  by  stimulation  of  DNA  synthesis  and  receptor 
autophosphorylation  in  response  to  IGF-1.  This  demonstration  of  functional  IGF-1  receptors  in  glioma 
cells  suggests  a  role  for  this  receptor  in  the  regulation  of  glioma  cell  growth.  In  addition,  high  levels  of 
high  affinity  IGF  binding  proteins  (IGF  BP)  are  produced  by  glioma  cells.  Certain  characteristics  of  this  IGF 
BP  suggest  it  may  be  distinct  from  previously  described  IGF  BPs,  and  thus  may  have  a  function  specific  for 
glioma  cells. 


phs  M40  (Rev  i  84  40  SNB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02814-01  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less-   Title  must  fit  on  one  line  between  the  borders.) 

Genetic  Abnormalities  in  Primary  Glial  Tumors 


(jenetic  ADnormanties  in  primary  unai  I  umors 

PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Iqbal  U.  Ali,  Ph.  D.  Principal  Investigator,  SNB,  NINDS 

Abha  Saxena,  Ph.D.  Visiting  Fellow,  SNB,  NINDS 

Craig  Shelley,  M.D.  Medical  Staff  Fellow,  SNB,  NINDS 

William  C.  Reinhold  Molecular  Biologist,  SNB,  NINDS 

Edward  H.  Oldfield,  M.D.  Chief,  SNB,  NINDS 


COOPERATING  UNITS  Of  any) 

St.  Judes  Hospital,  Memphis,  Tennessee 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 


Molecular  Biology  Unit 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health  Bethesda,  Maryland 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER:  nn 


CHECK  APPROPRIATE  BOX(ES) 

□   (aJHuman  subjects  [7]  (D)  Human  tissues  H  (c)  Neither 

]  (a1)  Minors 

J   (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Gliomas  are  the  most  common  primary  brain  tumors  ranging  from  benign  low-grade  astrocytomas  to 
highly  malignant  glioblastoma  multiforme.  The  classification  of  primary  glial  tumors  reflecting  their 
biological  aggressiveness  is  based  upon  the  histopathologic  characteristics   We  have  taken  three 
approaches  to  understand  and  identify,  at  a  molecular  level,  the  underlying  mechanisms  that  translate 
into  the  phenotypic  differences  of  various  grades  of  gliomas. 

1 .  The  genomic  organization  of  H-ras,  N-ras,  c-myc,  bFGF,  TGFa,  TGFfJ,  c-sis,  c-erbB2,  c-erbA2,  and  int-2 
was  analyzed  and  found  to  be  normal  in  20  primary  tumors  and  eight  glioblastoma  cell  lines. 

2.  The  transcription  of  bGFG,  TGFa,  TGF0,  and  N-ras  was  generally  elevated  in  most  of  the  glioblastoma 
cell  lines  tested  as  compared  to  normal  brain,  an  SVuo  transformed  human  astroglial  cell  line,  human 
breast  cancer  cell  lines,  a  bladder  carcinoma  cell  line,  T24,  and  a  hematopoetic  cell  line  K562.  An 
increased  level  of  N-ras  and  c-myc  protooncogenes  was  observed  in  all  human  tumor  cell  lines 
examined,  whereas  c-erbB2  expression  was  relatively  increased  in  breast  cancer  cell  lines. 

3.  Recessive  mutations  that  predispose  to  cancer  are  unmasked  in  several  human  cancers  by  the  loss  of 
normal  alleles.  Restriction  fragment  length  polymorphism  analysis  (RFLP)  was  used  to  compare  the 
constitutional  and  tumor  genotypes  in  a  panel  of  30  astrocytomas  and  glioblastomas.  Loss  of 
heterozygosity  of  several  markers  on  chromosomes  17  and  10  was  detected  in  a  significant  number 
of  astrocytomas  and  glioblastoma  multiforme  respectively.  Deletion  mapping  studies  are  being 
carried  out  to  more  precisely  define  the  position  of  the  gene(s)  whose  deletion  is  probably  important 
in  the  genesis  of  these  tumors 


PHS  6040  (Kev   1/84  41  SNB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02815-01  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders-) 

Molecular  Genetics  of  Pituitary  Conticotroph  Adenomas 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator  )  (Name,  title,  laboratory,  and  institute  affiliation) 

Iqbal  U.  AM,  Ph.D.  Principal  Investigator,  SNB,  NINDS 

HeinrichSchulte,  M.D.  Visiting  Associate,  SNB,  NINDS 

Salvatore  Aguanno,  Ph.D.  Visiting  Fellow,  SNB,  NINDS 

David  Katz,  M.D.  Special  Expert,  Neuropathologist,  NINDS 

Barbara  Ikejiri  Biologist,  SNB,  NINDS 

Edward  Oldfield,  M.D.  Chief,  SNB,  NINDS 


COOPERATING  UNITS  {if  any) 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 


Molecular  Biology  Unit 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health  Bethesda,  Maryland 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER:  0Q 


CHECK  APPROPRIATE  BOX(ES) 

\*~\   (a)  Human  subjects  [T]  (b)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Cushinq's  disease  is  caused  by  the  pituitary  hypersecretion  of  ACTH  and  occurs  predominately  in  women. 


Patients  are  cured  by  surgical  removal  of  an  ACTH-producing  adenoma,  suggesting  evolution  and 
expansion  of  a  genetically  aberrant  cell.  However,  hypothalamic  dysfunction  and  excessive  stimulation 
of  anterior  pituitary  corticotrophs  by  one  or  more  neurotransmitter  substances  may  also  lead  to  the 
development  of  corticortropic  adenomas. 

To  study  the  clonal  composition  of  ACTH-producing  pituitary  adenomas,  we  used  restriction 
fragment  length  polymorphism  (RFLP)  of  two  X-chromosome  linked  genes,  hypoxanthine 
phosphoribosyl  transferase  (HPRT)  and  phosphoglycerate  kinase  (PGK),  in  conjunction  with  their 
methylation  patterns.  Tumors  from  female  patients  only  were  used  because  in  all  female  cells  only  one 


X-chromosome  remains  active  which  can  e  detected  by  the  methylation  pattern.  Out  of  27  tumors 
analyzed,  nine  were  heterozygous  at  the  HPRT  or  PGK  loci    The  clonal  analysis  demonstrated  a 
monoclonal  pattern  in  six  of  these  tumors,  whereas  a  polyclonal  pattern  was  observed  in  three  tumors 
including  a  pituitary  adenoma  from  a  patient  with  the  Nelson's  syndrome.  There  was  no  correlation 
between  the  clonal  composition  and  the  clinical  features  of  the  tumors. 

Studies  are  also  in  progress  to  search  for  possible  mutations  in  the  members  of  the  rasqene  family. 
Specific  regions  of  the  three  ras  genes,  H-,  K-,  and  N-ras,  including  the  mutational  hot  spots  in  codons  12, 
13,  and  61  were  amplified  by  the  polymerase  chain  reaction  chain  reaction,  PCR,  and  will  be  directly 
sequenced  using  the  dideoxynucleotide  chain  termination  method. 


PHS  6040  (Rev   184  42  SNB  DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02760-03  SN' 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  /ess.   Title  must  fit  on  one  line  between  the  borders  ) 

Metabolic  Mapping  of  the  Brain  During  Rewarding  Brain  Stimulation 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator )  (Name,  title,  laboratory,  and  institute  affiliation) 

Linda  Porrino,  PhD  Principal  Investigator,  Research  Psychologist,  SNB,  NINDS 


Francesco  Pontieri,  M.D. 
Conan  Kornetsky,  Ph.D. 
Lewis  Seiden,  Ph  D 
Mark  Kleven,  Ph.D. 


Visiting  Fellow,  NIMH 
Boston  University 
University  of  Chicago 
University  of  Chicago 


COOPERATING  UNITS  Of  any) 

Laboratory  of  Cerebral  Metabolism,  NIMH;  Behavioral  Pharmacology  Laboratory,  Boston  University 
School  of  Medicine;  Department  of  Pharmacology,  University  of  Chicago 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Brain  Imaging  Unit 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


1.1 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

J   (a)  Human  subjects 
]  (a1)  Minors 
J  (a2)  Interviews 


J  (b)  Human  tissues  |  x   |  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  deoxyglucose  method  is  being  used  to  study  the  alterations  in  local  cerebral  metabolic  activity 
resulting  from  both  rewarding  and  aversive  electrical  brain  stimulation  to  discrete  brain  sites  as  well  as 
resulting  from  the  administration  of  drugs  of  abuse    By  mapping  metabolic  activity  in  rats  under  these 
conditions,  information  can  be  obtained  about  these  areas  of  the  brain  involved  in  motivation  and 
reinforcement.  Furthermore,  the  functional  consequences  of  chronic  drug  used  can  be  assessed. 


'Formally  CNB  -  transferred  to  SNB  1/14/90 


PHS&M0(N«»    1  M 


43  SNB  DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02762-03  SNB* 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (BO  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Quantitative  Autoradiographic  Determination  of  Dopamine  Receptor  Distribution  in  Primates 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Linda  Porrino,  Ph.D.  Principal  Investigator,  Research  Psychologist,  SNB,  NINDS 

Francesco  Pontieri,  Ph.D.  Visiting  Fellow,  NIMH 

Massako  Kadekaro,  Ph.D.  University  of  Texas  Medical  Branch 


COOPERATING  UNITS  (if  any) 

Laboratory  of  Cerebral  Metabolism,  NIMH 

Department  of  Neurosurgery,  University  of  Texas  Medical  Branch  -  Gal venston 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Brain  Imaging  Unit 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


.2 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

]  (a)  Human  subjects 
]  (a1)  Minors 

J  (a2)  Interviews 


j  (b)  Human  tissues  (Vj  (c)  Neither 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  primate  model  of  hemiparkinsonism  produced  by  theintracarotid  infusion  of  the  neurotoxin,^ 
methyl-4-phenvl-1,2,3,6-tetrahvdropyridine  (MPTP),  allows  the  study  of  neurochemical  changes 
associated  with  lesions  of  the  substantia  nigra  parscompacta    Quantitative  autoradiography  after  in 
vitro  radioligand  binding  with  PH]naloxone  (opiate  antagonist)  was  used  to  determine  the  distribution 
of  opiate  receptor  sites  in  MPTP-induced  hemiparkinsonian  moneys.  An  increased  density  of  opiate  sites 
was  observed  in  the  striatum  on  the  side  of  dopaminergic  denervation.  The  anatomic  distribution  of 
these  sites  resembled  the  distribution  of  dopaminergic  Di  binding  sites  in  the  striatum,  suggesting 
potential  interactions  between  dopamine  and  opioid  peptides  in  the  striatum. 


•Formally  CNB  -  transferred  to  SNB  1/14/90 


PHS  6040  (Rev   1  M 


44  SNB  D1R 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02761-03  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJ  ECT  (BO  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Metabolic  Mapping  of  Animal  Models  of  Parkinsonism 


PRINCIPAL  INVESTIGATOR  ttist  other  professional  personnel  below  the  Principal  Investigator  )  (Name,  title,  laboratory,  and  institute  affiliation) 

Linda  Porrino,  Ph.D.  Principal  Investigator,  Research  Psychologist,  SNB,  NINDS 

Francesco  Pontieri  Visiting  Fellow,  NIMH 


COOPERATING  UNITS  i.tany) 

Laboratory  of  Cerebral  Metabolism,  NIMH 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Brain  Imaging  Unit 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health,  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL: 


OTHER: 


CHECK  APPROPRIATE  BOX(ES) 

Q   (a)  Human  subjects  ]  (b)  Human  tissues  \~*~\  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Neurotoxic  destruction  of  the  dopaminergic  cells  of  the  substantia  nigra  parscompacta  of  primates  and 
rodents  produces  useful  models  of  parkinsonism    The  2-[14C]deoxyqlucose  method  is  being  applied  to 
animals  in  which  unilateral  lesions  of  the  substantia  nigra  pars  compacta  have  been  made  in  order  to 
map  the  neural  circuits  involved  in  the  symptoms  of  parkinsonism  and  its  treatment  with  dopaminergic 
agonist  drugs.  Metabolic  studies  in  rats  with  anatomically  specific  lesions  of  the  substantia  nigra  pars 
compacta  have  demonstrated  the  significance  of  the  loci  of  the  damage  to  dopaminergic  cells  along  the 
medial-lateral  axis  of  the  nigra  in  determining  the  resulting  pattern  of  behavioral  effects.  Furthermore, 
the  level  of  functional  activity  in  the  substantia  nigra  pars  reticulata  has  been  shown  to  be  the 
determinant  of  the  directions  of  turning  behavior  elicited  by  the  administration  of  dopaminergic  drugs 


*  Formal  ly  CNB  -  Transferred  to  SNB  1-1 4-90 


PHS6M0(Rev   1/84  45   SNB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 


Z01  NS  02739-04  SN 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (80  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

Clinical  and  Laboratory  Investigation  of  Central  Nervous  System  Vascular  Disorders 


PRINCIPAL  INVESTIGATOR  (List  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

Edward  H.  Oldfield,  M.D.  Chief,  SNB,  Principal  Investigator,  NINDS 

David  Barba,  M.D.  Senior  Staff  Fellow,  SNB 

Michele  Filing-Katz,  M.D.  SeniorStaff  Fellow,  ETB,  NINDS 

Marston  Linehan,  M.D.  Surgical  Branch,  NCI 

Berton  Zbar,  M.D.  Senior  Investigator,  NCI 


COOPERATING  UNITS  (if  any) 

Diagnostic  Radiology  Department,  CC,  Experimental  Therapeutics  Branch,  NINDS 
Surgery  Branch,  National  Cancer  Institute 


LAB/BRANCH 

Surgical  Neurology  Branch,  CNP 


SECTION 

Clinical  Neurosurgery  Section 


INSTITUTE  AND  LOCATION 

NINDS,  National  Institutes  of  Health  Bethesda,  Maryland  20892 


TOTAL  MAN-YEARS: 


PROFESSIONAL:  y  5 


OTHER: 


0.0 


CHECK  APPROPRIATE  BOX(ES) 

[V]   (a)  Human  subjects  [T]  (b)  Human  tissues  ]  (c)  Neither 

]  (a1)  Minors 

J  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  clinical  features,  arteriographic  findings,  and  treatment  of  81  patients  with  spinal  arteriovenous 
malformations  (AVSs)  demonstrated  that  there  are  distinguishing  clinical  features  in  patients  with 
arteriovenous  malformations  of  the  spinal  cord  compared  to  those  of  patients  with  arteriovenous  (AV) 
fistulas  of  the  spinal  dura.  The  findings  indicate  that  spinal  arteriovenous  fistulas  are  acquired  lesions, 
and  not  congenital,  as  was  previously  thought,  and  support  arteriovenous  malformations  of  the  spinal 
cord  as  congenital  lesions.  The  findings  also  indicate  that  AVMs  of  the  spinal  cord  produce  myelopathy 
as  a  result  of  high  blood  flow,  but  that  patients  with  spinal  dural  AV  fistulas  develop  myelopathy  as  a 
result  of  increased  venous  pressure  in  the  spinal  cord. 

Magnetic  resonance  imaging  permits  demonstration  of  the  presence  and  site  of  AVMs  of  the  spinal 
cord  and  therefore  is  a  valuable  and  safe,  noninvasive  technique  to  investigate  patients  suspected  of 
having  spinal  AVMs. 

Foix-Alajounine  syndrome  was  demonstrated  to  be  due  to  venous  congestion,  and  not  venous 
thombosis,  and  therefore,  amenable  to  reversal  by  treatment  of  the  spinal  AVM.  Spinal  AVMs  were 
shown  to  recanalize  consistently  after  embolic  occlusion. 

Patients  with  Von  Hippel-Lindau  disease  were  investigated  and  the  following  were  shown: 
investigation  of  the  molecular  biology  of  the  hemanqioblastomas  of  the  central  nervous  system  revealed 
a  homozygous  deletion  of  a  portion  of  the  short  arm  of  the  third  chromosome,  demonstrating  that  these 
tumors  are  probably  caused  by  the  absence  of  a  tumor  suppressing  gene,  as  are  familial  retinoblastomas. 
MRI  with  gadolinium  EDTA  contrast  enhancement  was  shown  to  be  a  sensitive  technique  of  detection 
for  small  hemangioblastomas  of  the  central  nervous  system  Excision  of  the  tumors  alone  was  shown  to 
result  in  resolution  of  syringomyelia  associated  with  spinal  cord  hemangioblastomas.  Therefore,  the 
tumor-associated  syrinx  does  not  need  separate  treatment 


PHS  6040  (Rev  184  46SNBOIR 


> 

00 

ISJ 


z 
n 
> 


c 

30 

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30 

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z 

00 


ANNUAL  REPORT 

October  1,  1989  through  September  30,  1990 

Clinical  Neuroscience  Branch 

National  Institute  of  Neurological  Disorders  and  Stroke 


Table  of  Contents 

RESEARCH  SUMMARY  1-12 

RESEARCH  REPORTS 

Biochemical  Indices  of  Adrenergic  Function  in  Humans 

Z01  NS02115-17CNB  13 

Clinical,  Genetic  and  Biochemical  Studies  of  Familial 
Alzheimer's  Disease 

Z01  NS  02630-07  CNB  14 

Neurotoxins  and  Animal  Models  of  Neurological  Diseases 

Z01  NS  02716-04  CNB  15 

Biochemical  Evaluation  of  Aminergic  Function  During 
Responses  to  Stress  and  in  Disease  States 

Z01  NS  02717-05  CNB  16 

Regulation  of  Synthesis  and  Expression  of  Neurotrophic 
Agents  and  Neuropeptides 

Z01  NS  02752-03  CNB  17 


ANNUAL  REPORT 

October  1,  1989  through  September  30,  1990 

Clinical  Neuroscience  Branch 

Clinical  Neurosciences  Program,  DIR 

National  Institute  of  Neurological  Disorders  and  Stroke 

Irwin  J.  Kopin,  M.D.,  Chief 

The  Clinical  Neuroscience  Branch  (CNB)  conducts  research  on  the  role  of 
neurotransmitters  in  brain  and  in  peripheral  autonomic  function;  the  effects  of 
abnormalities  in  neurotransmitter  metabolism  and  receptor  activation  and  their 
occurrence  in  neurologic  disorders;  and  the  effects  of  drugs  and  other  therapies  on 
regulation  of  neurotransmitter  release  and  actions.  At  present  the  Branch  is  divided 
into  two  Sections,  with  a  third  section  planned.  Investigators  in  the  Section  on 
Clinical  Neuropharmacology  examine  neurotransmitter  function  in  patients  with 
autonomic  insufficiency  or  Alzheimer's  disease.  In  the  Section  on  Aminergic 
Mechanisms  (SAM),  studies  in  animals  and  animal  models  of  human  neurologic 
disorders  are  designed  to  examine  the  role  of  neurotransmitters  and  their  receptors 
in  pathogenesis  and  treatment  of  neurologic  diseases,  with  particular  emphasis  on 
biogenic  amines.  A  new  Section  on  Sympathetic  Function  under  the  leadership  of 
Dr.  David  Goldstein  will  be  developed  during  the  next  year.  The  clinical  research 
efforts  of  this  Section  will  complement  the  studies  in  animals  performed  in  the  SAM; 
studies  with  new  18F-Iabelled  compounds  appropriate  for  Positron  Emission 
Tomography  (PET)  imaging  are  planned  and  will  be  performed  in  collaboration  with 
investigators  in  the  Neuroimaging  Branch. 

There  is  also  a  Unit  on  Growth  Factors  in  CNB  which,  in  addition  to  independent 
research  on  regulation  of  growth  factors,  provides  support  for  studies  related  to  the 
development  or  regeneration  and  the  function  in  vivo  of  specific  neuronal  systems. 
Much  of  this  work  is  performed  in  collaboration  with  investigators  in  the  Surgical 
Neurology  Branch. 


CLINICAL  NEUROPHARMACOLOGY  SECTION 

The  Clinical  Neuropharmacology  Section  continues  to  develop  biochemical  and 
pharmacologic  methods  to  assess  neurotransmitter  function  and  metabolism  in  man. 
These  strategies  have  been  applied  in  the  investigation  and  treatment  of  patients 
with  autonomic  nervous  systems  disorders.  Investigation  of  patients  with  lesion(s)  of 
the  autonomic  nervous  system  provides  an  opportunity  to  examine  the  interaction 
between  autonomic  and  other  hormonal/peptide  control  systems.  Two  distinct 
disorders  remain  the  main  focus  of  these  efforts:  autonomic  failure  may  occur  alone 
(pure  autonomic  failure,  PAF)  or  in  association  with  (central  nervous  system,  CNS) 
degeneration  (multiple  system  atrophy,  MSA). 

Our  studies  primarily  focus  on  the  noradrenergic  system  because  norepinephrine 
(NE)  is  released  by  most  postganglionic  sympathetic  neurons  and  also  is  important  in 
central  pathways  concerned  with  blood  pressure  regulation.  High  performance 
liquid  chromatography,  liquid  scintillation  spectrometry,  and  gas  chromatography- 


1  -CNB/DIR 


mass  spectroscopy  are  used  to  measure  neurotransmitter  and  metabolite  levels  in 
various  biologic  fluids.  Radioenzymatic  methods  are  employed  to  determine  the 
activities  of  synthetic  and  degradation  enzymes  related  to  neurotransmitter 
metabolism.  Peptide  and  hormonal  levels  are  analyzed  by  radioimmunoassay. 
These  substances  are  measured  under  basal  conditions  and  after  a  variety  of  stimuli 
have  been  applied  to  evaluate  the  responsiveness  of  the  particular  system  under 
investigation. 

Previously,  we  pharmacologically  distinguished  PAF  and  MSA  patients  based  on  their 
blood  pressure  responses  to  NE.  Patients  with  PAF  manifest  low  basal  plasma  NE 
levels  and  a  shift-to-the-left  of  their  blood  pressure  dose-response  curve  to  NE, 
consistent  with  noradrenergic  receptor  supersensitivity.  The  increase  in  slope 
without  a  shift  of  the  dose-response  curve  in  MSA  suggests  a  preganglionic  lesion. 
Since  the  intermediolateral  column  (ILC)  innervates  sympathetic  ganglia  and  the 
number  of  ILC  neurons  is  substantially  lower  than  normal  in  MSA,  we  measured  the 
plasma  NE  response  to  intravenous  acetylcholine  (Ach)  in  patients  with  autonomic 
failure  to  assess  ganglionic  responsiveness.  All  subjects  were  pretreated  with 
glycopyrrolate  to  block  peripheral  muscarinic  effects  of  Ach.  Mean  blood  pressure 
and  heart  rate  did  not  change  during  the  infusion  in  any  group.  Normal  subjects 
increased  plasma  NE  proportional  to  the  rate  of  Ach  infusion.  Patients  with  PAF 
failed  to  significantly  increase  plasma  NE,  consistent  with  post-ganglionic 
sympathetic  neuronal  dysfunction.  Patients  with  MSA  can  be  subdivided  into 
nonresponders  and  responders  based  on  their  plasma  NE  response  at  low  doses  of 
Ach.  The  nonresponders  do  not  increase  their  plasma  NE,  similar  to  PAF  patients. 
The  remaining  MSA  patients  increase  their  plasma  NE  significantly  greater  than 
normal  at  low  doses,  followed  by  a  return  to  basal  levels  as  the  infusion  rate  was 
increased.  This  biphasic  response  suggests  that  some  patients  with  MSA  may  have 
cholinergic  supersensitivity  since  the  increase  in  plasma  NE  levels  at  low  doses  is 
more  pronounced  than  in  normal  subjects.  The  subsequent  reduction  in  plasma  NE 
could  result  from  increased  removal  from  plasma,  enhanced  neuronal  uptake,  or 
decreased  NE  release.  Increased  clearance  from  plasma  is  not  likely  since  blood 
pressure  and  heart  rate  did  not  change.  The  most  plausible  mechanism  is  the 
development  of  depolarization  blockade  at  higher  Ach  infusion  rates,  consistent 
with  ganglionic  supersensitivity.  Those  MSA  patients  who  lacked  a  NE  response 
might  have  postganglionic  involvement  although  it  is  possible  that  extreme 
supersensitivity  might  have  produced  blockade  at  even  the  lowest  doses  employed 
in  this  study.  We  plan  to  use  other  neurochemical  (co-release  of  peptides, 
measurement  of  NE  metabolites)  and  electrophysiologic  methods  to  further  clarify 
the  mechanism  of  this  abnormality.  When  it  becomes  technically  feasible,  the  use  of 
i8F-dopamine  PET  scanning  might  be  employed  to  examine  NE  turnover  in  these 
patients. 

Much  of  the  research  on  patients  with  autonomic  failure  is  concerned  with  a- 
adrenergic  function.  However,  our  previous  work  demonstrating  abnormal 
circadian  patterns  of  melatonin  secretion  and  alterations  in  the  release  of  gastrin 
during  hypoglycemia  suggest  that  ^-adrenergic  function  is  also  impaired  in  patients 
with  PAF  and  MSA.  We  examined  cardiovascular,  plasma  NE,  and  renin  (PRA) 
responses  to  isoproterenol  infusion  in  patients  with  autonomic  failure  and  in  normal 
subjects.  Increased  slopes  of  the  blood  pressure  dose-responses  curves  in  the 
patients  are  consistent  with  impaired  baroreflex  modulation.  A  leftward  shift  in 
patients  with  PAF  suggests  ^-adrenergic  receptor  supersensitivity.  Plasma  NE  and 
PRA  increased  proportional  to  the  log  of  the  plasma  isoproterenol  level  in  normal 
subjects.  These  normal  responses  may  be  due  to  reflexive  cardiovascular  and  renal 


2-CNB/DIR 


mechanisms  since  neither  patient  group  manifested  increases  in  NE  or  PRA  during 
isoproterenol  infusion  despite  a  reduction  in  mean  blood  pressure. 

In  addition  to  abnormal  cardiovascular  control,  patients  with  autonomic  dysfunction 
also  develop  symptoms  related  to  disruption  of  the  innervation  to  other  organs. 
Although  diminished  or  absent  sweating  occurs  in  most  patients,  there  have  not 
been  any  systematic  pharmacologic  investigations  of  sudomotor  function.  We 
measured  sweat  production  to  direct  gland  stimulation  with  intradermal 
methacholine  and  also  assessed  the  sympathetic  skin  response  to  electrical 
stimulation.  Sweat  production  was  significantly  less  than  normal  in  PAF  and  MSA. 
There  was  no  correlation  between  the  sympathetic  skin  response  and  sweat  induced 
by  methacholine.  The  diminished  sweat  production  in  response  to  intradermal 
methacholine  suggests  that  human  sweat  glands  do  not  develop  denervation 
supersensitivity. 

Clinical  distinction  between  MSA  and  PAF  may  be  difficult  in  the  early  stages  prior  to 
the  development  of  central  neurologic  signs.  Our  previous  experience  indicates  that 
it  is  necessary  to  wait  at  least  5  years  from  the  onset  before  making  a  definitive 
diagnosis  of  PAF.  We  have  analyzed  the  results  of  neuroimaging  studies  in  43 
patients  using  CT,  MRI,  and  PET  scanning.  In  contrast  to  normal  findings  in  PAF,  the 
main  abnormalities  in  patients  with  MSA  were  atrophy  of  the  cerebellar 
hemispheres  and  vermis,  MRI  signal  hypointensity  in  the  posterolateral  putamen, 
and  decreased  glucose  utilization  in  the  cerebellum  and  most  supratentorial 
structures.  Thus,  neuroimaging  studies  facilitate  distinction  between  MSA  and  PAF. 

Neuropathology  examination  of  post-mortem  tissue  in  MSA  reveals  neuronal  loss 
and  gliosis  in  a  number  of  central  nervous  system  areas.  The  nonspecific  nature  of 
these  changes  does  not  yield  any  clues  to  trie  underlying  cause  of  MSA  Immune 
processes  have  been  described  in  a  variety  of  neurologic  disease  states.  We  used 
immunocytochemical  methods  to  search  for  CSF  antibodies  against  specific  regions 
in  the  rat  brain  that  parallel  the  known  areas  affected  by  degeneration  in  MSA.  CSF 
immunoreactivity  to  rat  locus  coeruleus  occurred  in  a  significantly  greater  number  of 
samples  from  MSA  patients  compared  to  control  subjects  or  patients  with  PAF. 
Other  brain  regions  infrequently  showed  immunoreactivity.  Degeneration  in  MSA 
may  release  antigen(s)  that  induce  antibodies  against  locus  coeruleus  neurons.  The 
specificity  of  this  abnormality  is  particularly  interesting  since  immunoreactivity 
towards  other  brain  regions  known  to  degenerate  was  not  observed.  Furthermore, 
patients  with  Parkinson's  disease  have  a  CSF  antibody  that  reacts  with  the  substantia 
nigra  but  not  the  locus  coeruleus.  This  finding  increases  the  diagnostic  potential  of 
the  immunoreactivity  in  patients  with  MSA.  Further  characterization  of  this 
immunologic  abnormality  is  in  progress  to  determine  whether  it  has  pathogenetic 
significance.  Lack  of  CSF  immunoreactivity  in  PAF  is  consistent  with  primarily 
peripheral  involvement. 

We  also  continue  to  collect  psychosocial  histories  and  pedigrees  on  all  autonomic 
failure  patients  to  determine  whether  social,  environmental,  or  familial  factors 
contribute  to  the  development  and  progression  of  these  disorders.  A  more  detailed 
analysis  of  our  preliminary  observation  of  increased  exposures  to  toxic  substances 
has  been  completed  in  collaboration  with  the  Environmental  Epidemiology  Branch, 
NCI.  MSA  patients  had  significantly  more  exposures  to  metal  dusts  and  fumes, 
plastic  monomers  and  additives,  organic  solvents,  and  pesticides  than  the  control 
population.  The  exposures  were  determined  in  most  subjects  by  their  reported  usual 
occupations.  These  toxic  exposures  in  the  patients  and  the  increased  incidence  of 
autonomic  and    neurologic  symptoms  in  their  first-degree   relatives  suggests  a 


3-CNB/DIR 


multifactorial  etiology.  There  may  be  a  genetically  determined,  selective 
vulnerability  that  provides  targets  for  the  toxic  insults  resulting  in  neuronal  death 
within  specific  systems.  These  studies  will  be  expanded  to  further  address  the 
questions  raised  by  these  data. 

Treatment  and  emotional  support  are  provided  to  all  patients  with  MSA  and  PAF. 
Orthostatic  hypotension  is  the  primary  focus  for  therapeutic  trials.  Fludrocortisone 
and  ibuprofen  remain  the  most  effective  pharmacologic  agents  for  raising  blood 
pressure.  Although  we  are  using  guanfacine  to  assess  peptide  responses  mediated 
by  central  noradrenergic  pathways,  a  treatment  phase  has  been  initiated. 
Preliminary  therapeutic  efforts  with  guanfacine  have  not  been  encouraging.  We 
plan  to  re-evaluate  the  use  of  L-threo-DOPS,  an  unnatural  NE  precursor,  for  treating 
patients  with  MSA  and  PAF.  It  has  now  been  shown  that  the  drug  increases  brain  NE, 
even  when  the  normal  synthetic  pathway  is  available.  This  agent  may  be  useful  for 
treating  the  peripheral  and  central  noradrenergic  deficiencies  we  have  observed  in 
these  patients.  Use  of  a  somatostatin  analog  may  also  help  patients  who  fail  more 
conventional  approaches.  During  the  past  6  months  new  noninvasive  technology 
has  become  commercially  available  and  may  be  adapted  as  the  sensor  arm  of  the 
sympathetic  neural  prosthesis.  This  would  allow  accurate  control  of  blood  pressure 
without  invasive  monitoring.  Pending  availability  of  funds,  we  hope  to  extend  the 
contract  for  an  additional  4  months  in  order  to  realize  the  desired  outcome  from  this 
project.  It  should  be  possible  to  test  the  device  within  the  next  year  pending 
approval  by  the  U.S.  Food  and  Drug  Administration. 

Many  adjustments  in  lifestyle  are  necessary  for  patients  and  their  families  to 
successfully  cope  with  a  progressive  degenerative  disorder.  Of  major  concern  to  this 
patient  population  are  the  increasing  cost  of  chronic  care,  lack  of  resources  for  care 
(home  care  nursing),  and  quality  of  life  decisions  as  the  disease  progresses 
(tracheostomy,  gastric  tubes).  In  an  effort  to  increase  support,  a  meeting  was  held  to 
organize  a  national  support  group.  This  facilitated  dissemination  of  information  on 
research  resources  and  provided  opportunity  to  share  our  experience  in  dealing  with 
these  chronic  disorders. 

A  number  of  other  studies  are  currently  in  progress: 

1.  Peptide  pathways  in  sympathetic  and  dorsal  root  ganglia  as  well  as  spinal  cord 
are  being  studied  in  collaboration  with  Dr.  Margaret  Matthews,  Oxford 
University.  Preliminary  results  differentiate  PAF  and  MSA. 

2.  The  radioimmunoassay  for  neuropeptide  Y  has  not  been  satisfactory.  We  are 
currently  evaluating  a  new  antibody.  We  hope  to  use  colocalization  of  peptides 
as  a  strategy  for  evaluating  mechanisms  involved  with  release  of 
neurotransmitters. 

3.  Modification  of  several  peptide  assays  for  cerebrospinal  fluid  CSF  are  in  progress. 
These  include  CRF,  substance  P,  calcitonin  gene-related  peptide,  brain  natriuretic 
peptide,  and  endothelin. 

4.  Several  protocols  have  been  initiated  commensurate  with  the  arrival  of  a  new 
Medical  Staff  Fellow.  It  was  necessary  to  delay  these  projects  in  order  to  facilitate 
completion  of  other  protocols.  These  studies  include:  (1)  evaluation  of  the 
sympathetic  response  to  hypercarbia,  (2)  measurement  of  physiologic  and 
neurochemical  effects  of  ANP  infusion,  and  (3)  sleep  studies. 

5.  Detailed  studies  of  excitatory  amino  acid  receptors  and  subtypes  of  GABA 
receptors  in  the  cerebellum  of  post-mortem  brain  specimens  from  MSA  patients  is 
being   carried   out  in   collaboration  with   Dr.  Anne   B.   Young,   University   of 


4-CNB/DIR 


Michigan.  Four  amino  acid  receptors  will  be  measured  by  autoradiography:  (1) 
NMDA,  (2)  AMPA,  (3)  kainate,  and  (4)  metabotropic  receptor.  The  high  and  low 
affinity  forms  of  the  GABAAand  GABAb  receptors  will  also  be  evaluated. 

6.  We  have  studied  p-adrenergic  responses  and  ACTH  responses  to  arecoline  in  two 
patients  with  multiple  sclerosis.  The  data  will  be  analyzed  after  5  patients  have 
been  investigated.  It  has  been  suggested  that  autonomic  lesions  may  play  a  role 
in  the  progression  of  this  demyelinating  disease. 

7.  A  follow-up  study  was  initiated  to  evaluate  all  twins  in  North  Carolina  who 
developed  paralytic  polio  between  1940-1948.  Detailed  family  studies  will  be 
obtained.  This  work  has  relevance  to  our  study  of  MSA  patients  since  4  of  the 
MSA  cases  had  a  history  of  paralytic  polio. 

The  Clinical  Neuropharmacology  Section  has  continued  the  study  of  familial 
Alzheimer's  disease  (AD)  as  a  major  priority  within  the  scope  of  its  research  efforts. 
AD  remains  a  major  medical  and  social  problem  since  it  is  the  most  common  cause  of 
irreversible,  chronic  dementia.  Clinical  and  therapeutic  studies  in  AD  are 
significantly  limited  by  accuracy  and  timing  of  diagnosis.  Thus,  the  main  justification 
for  studying  the  more  uncommon,  autosomal  dominant  subgroup  of  familial  cases 
lies   in   the   accuracy   of  diagnosis  that   may   be   inferred   through    post-mortem 


examination  of  other  affected  family  members.   Our  two  major  research  directions 

the  primary,  underlyi 
of  AD,  and  (2)  longitudinal  investigation  of  clinical  and  biochemical  characteristics  of 


are:  (1)  genetic  linkage  to  identify  the  primary,  underlying  molecular  pathogenesis 


the  disease  to  provide  clues  for  earlier,  accurate  diagnosis  and  more  rational 
therapeutic  strategies. 

Additional  samples  have  been  submitted  to  the  Coriell  Institute  for  Medical 
Research,  Camden,  New  Jersey,  for  the  establishment  of  skin  fibroblast  and 
peripheral  blood  lymphoblast  cultures  under  an  intra-agency  agreement  between 
the  NINDS  and  NIA.  Currently,  we  are  working  with  20  families  that  have  an 
autosomal  dominant  inheritance  pattern.  Samples  have  been  submitted  from  four 
categories  of  family  members  who  undergo  neurologic  examination:  affected,  at- 
risk  (first-degree),  escapees,  spouses.  This  is  also  part  of  an  international 
collaboration  organized  and  coordinated  by  our  Section.  Families  from  the  U.S., 
Canada,  Italy,  France,  and  Germany  are  participating  in  this  effort.  Identification  of 
new  branches  of  these  families  and  continued  follow-up  with  the  families  to  verify 
clinical  status  of  at-risk  members  increases  their  value  for  genetic  linkage  studies. 
The  cultures  continue  to  serve  as  a  renewable  source  of  DNA  and  cells  for  basic 
research  in  AD. 

Further  investigation  of  clinical  genetics  and  linkage  analysis  support  the  concept  of 
heterogeneity.  Only  early  onset  families  appear  to  be  linked  to  markers  on 
chromosome  21,  although  a  significant  lod  score  has  still  not  been  obtained  using  a 
single  marker  in  an  individual  family.  We  plan  to  compare  phenotypic  characteristics 
between  various  pedigrees  to  identify  parameters  that  may  allow  selection  of  more 
homogeneous  subgroups.  In  addition,  we  continue  to  ascertain  new  pedigrees, 
expand  the  number  of  participating  members  of  the  current  families,  and  update 
the  clinical  status  to  provide  the  maximum  amount  of  accurate  data  for  the  linkage 
analysis.  Attempts  to  identify  additional  branches  of  the  large  Canadian  pedigree 
have  not  been  successful.  Efforts  have  also  continued  to  search  for  additional 
informative  markers.  Emphasis  will  be  given  to  VNTR  (variable  number  tandem 
repeat)  probes,  probes  containing  mini-satellite  polymorphisms,  and  probes 
showing  sequence  variability.  A  search  will  also  be  made  to  identify  coding 
sequences  in  AD  brain.  Long-range  physical  mapping  will  be  used  to  search  for  and 

5-CNB/DIR 


clone  structural  chromosomal  rearrangements  in  familial  AD  cases  or  in  other  AD 
cases  that  have  chromosome  21  translocations  since  these  could  be  the  sight  of  the 
gene  defect.  We  will  also  search  for  additional  loci  on  other  chromosomes, 
especially  19  which  was  recently  found  linked  in  late  onset  cases.  In  collaboration 
with  several  other  institutions,  we  participated  in  an  investigation  of  transmission 
and  age-at-onset  patterns  in  70  kindreds  with  familial  AD.  The  age-at-onset  among 
the  families  showed  a  bimodal  distribution;  a  mean  onset  age  of  58  years 
distinguished  early  from  late  onset  families.  The  lifetime  AD  risk  for  at-risk  offspring 
in  early  onset  cases  was  53%,  significantly  less  than  the  risk  of  86%  in  late  onset 
cases.  The  results  in  early  onset  families  are  consistent  with  an  autosomal  dominant 
transmission  model  whereas  the  late  onset  form  appears  to  have  at  least  2 
etiologies. 

Our  longitudinal  investigations  have  continued  and  psychologicsupport  and  genetic 
counseling  have  been  given.  Measurement  of  monoamine  metabolites, 
acetylcholinesterase,  and  somatostatin  provide  further  confirmation  of  our 
preliminary  observations.  A  third  at-risk  subject,  among  the  subgroup  who  have  low 
CSF  MHPG  and  somatostatin,  has  developed  dementia.  These  newly  affected  AD 
patients  highlight  the  significance  of  these  neurochemical  changes.  Studies  of  other 
neuropeptides  including  corticotropin  releasing  factor  are  in  progress.  We  have  also 
used  our  longitudinal  collection  of  CSF  samples  in  a  collaborative  search  for  anti- 
neuronal  antibodies.  Previous  investigations  demonstrated  the  presence  of 
antibodies  in  the  CSF  from  AD  patients;  these  antibodies  recognize  specific  neuronal 
populations  in  the  adult  rat  CNS.  In  the  developing  rat  CNS  different  types  of 
antigens  were  recognized.  One  of  the  most  remarkable  differences  was  the 
recognition  of  amoeboid  microglial  cells.  Electron  microscopy  revealed  that  the  AD- 
CSF  antibody  recognizes  specific  membrane  receptors  in  the  macrophagic  microglia. 
These  results  further  support  the  concept  that  inflammation  and  similar  immune 
mechanisms  may  play  a  role  in  AD. 

Cerebral  glucose  metabolism  has  been  studied  in  8  affected  patients  and  20  first- 
degree  at-risk  subjects.  The  demented  patients  had  significant  hypometabolism  in 
the  parietotemporal  regions,  with  some  involvement  of  the  occipital  and  temporal 
lobes.  Overall,  metabolism  in  the  at-risk  group  did  not  differ  from  normal  subjects. 
Repeat  scans  were  performed  on  three  at-risk  subjects  who  subsequently  developed 
dementia;  these  studies  showed  a  10-17%  decline  in  relative  parietal  lobe 
metabolism.  In  one  of  these  subjects,  it  appears  that  an  isolated  regional  change  in 
cerebral  glucose  metabolism  may  have  preceded  clinical  expression  of  the  disease.  A 
more  detailed  analysis  of  these  preliminary  findings  is  in  progress. 

Additional  studies  that  have  been  continued  or  in  progress  include: 

1.  A  study  of  ACTH  responses  to  arecoline  is  currently  being  conducted.  This  may 
serve  as  a  neuroendocrine  assessment  of  a  central  cholinergic  pathway.  More 
patients  will  be  studied  before  the  results  are  analyzed. 

2.  Two  approaches  are  being  employed  to  investigate  noradrenergic  function: 
evaluation  of  blood  pressure  responses  to  intravenous  NE  and  measurement  of 
urinary  NE  metabolite  patterns.  We  anticipate  that  abnormalities  may  reflect 
locus  coeruleus  involvement  which  occurs  in  most  AD  cases.  The  results  are 
currently  being  analyzed. 

3.  In  collaboration  with  Dr.  Jesse  Sisken,  University  of  Kentucky,  fibroblast  cultures 
are  being  used  to  determine  whether  alterations  in  calcium  metabolism  are 
present   in   fibroblasts  from   patients  with   familial   AD.     The   photoprotein, 


6-CNB/DIR 


aequorin,  is  used  to  probe  for  transient  changes  in  free  intracellular  calcium 
levels  in  cells  that  have  been  serum  deprived  and  then  stimulated  with  either 
serum  or  bradykinin.  Preliminary  results  indicate  that  all  FAD  cells  respond  to  the 
stimulation  but  the  size  of  the  calcium  transient  appears  to  be  smaller  in  the 
patients  compared  to  controls.  It  is  necessary  to  confirm  these  findings  in  a  larger 
series  prior  to  investigating  at-risk  subjects. 

4.  A  collaborative  study  of  DNA  repair  is  being  conducted  with  Dr.  W.  Clark 
Lambert,  UMDNJ.  An  attempt  is  being  made  to  further  elucidate  the  mechanisms 
involved  in  producing  the  defect  in  DNA  repair  which  was  observed  previously  by 
other  collaborators.  Preliminary  results  reveal  decreased  viability  of  lymphoblasts 
following  exposure  to  methyl-methanesulfonate.  Unscheduled  DNA  synthesis  is 
reduced  in  a  sporadic  AD  case;  investigation  of  familial  AD  lines  is  currently  in 
progress. 

5.  Quantitative  assessment  of  smell  is  being  carried  out  in  affected  and  at-risk 
members  of  these  families  with  dominantly  inherited  AD.  This  study  will  attempt 
to  identify  a  clinical  correlate  of  the  neuropathology  abnormality  recently 
described  in  olfactory  neurons. 

SECTION  ON  AMINERGIC  MECHANISMS 

Studies  in  this  Section  have  focussed  on  biochemical  evaluation  of  aminergic 
mechanisms  and  in  part  continue  to  be  collaborative  efforts  with  investigators  in  the 
NHLBI  for  examination  of  peripheral  autonomic  function  and  in  the  Surgical 
Neurology  Branch,  NINDS,  for  evaluation  of  brain  aminergic  function. 

The  studies  on  the  autonomic  nervous  system  have  used  measurements  of  levels  of 
NE,  dopamine  (DA),  3,4-dihydroxyphenylalanine  (DOPA)  and  their  metabolites  in 
body  fluids  to  assess  sympathetic  neuronal  function.  The  results  of  these  studies  have 
shown  that  DOPA,  the  amino  acid  precursor  of  catecholamines,  is  derived  mainly 
from  peripheral  sympathetic  nerves,  but  is  taken  up  and  retained  in  skeletal  muscle 
and  released  into  blood  during  muscular  contraction.  The  amounts  of  DOPA  in 
plasma  reflect  catecholamine  synthesis  in  peripheral  sympathetic  nerves  and  levels 
of  the  amino  acid  increase  during  acute  stress.  There  is  a  dissociation,  however, 
between  the  tissue  levels  of  tyrosine  hydroxylase  and  the  rate  of  tyrosine 
hydroxylation.  During  acute  stress,  tyrosine  hydroxylation  increases  without  any 
significant  alteration  in  the  enzyme  levels,  whereas  after  repeated  stress,  levels  of 
the  enzyme  are  increased  in  some  tissues,  but  without  stress-induced  sympathetic 
activation,  the  rate  of  tyrosine  hydroxylation  does  not  appear  to  be  increased. 
Although  DOPA  is  also  the  precursor  of  melanin,  this  source  does  not  contribute 
significantly  to  plasma  DOPA.  The  intraneuronal  metabolite  of  norepinephrine,  3,4- 
dfhydroxyphenylglycol  (DHPG),  is  an  index  of  sympathetic  neuronal  integrity  which 
in  combination  with  plasma  NE  levels  provides  information  about  sympathetic 
neuronal  function.  DHPG  is  formed  mainly  from  NE  which  "leaks'  into  the 
cytoplasm  from  storage  vesicles,  but  some  is  formed  from  NE  which  is  recaptured 
after  release  into  the  synapse. 

The  venous-arterial  differences  in  radiolabeled  and  unlabelled  NE  and  blood  flow 
provide  a  means  of  assessing  the  rate  of  overflow  of  NE  from  the  sympathetic  nerves 
in  a  tissue.  Overflow  of  this  amine  generally  correlates  with  sympathetic  nerve 
activity,  as  does  release  of  DOPA  and  DHPG  into  the  circulation.  DHPG  clearance, 


7-CNB/DIR 


however,  may  be  altered  by  changes  in  the  distribution  of  blood  flow  so  that  its 
plasma  leveis  may  be  elevated  even  when  net  release  is  diminished. 

Simultaneous  estimation  of  urinary  excretion  rates  on  plasma  levels  of  homovanillic 
acid  (HVA)  (the  major  metabolite  of  dopamine)  and  of  MHPG  and  VMA  (the  major 
metabolites  of  NE)  before  and  during  administration  of  debrisoquin  are  used  to 
estimate  the  rate  of  brain  DA  metabolism.  This  method,  which  had  been  verified 
using  animals  treated  with  MPTP  to  eliminate  brain  dopaminergic  neurons,  is  now 
being  used  to  study  DA  metabolism  in  humans.  Patients  with  early,  untreated 
Parkinson's  disease  have  been  found  to  have  low  levels  of  HVA  formation  in  brain, 
consistent  with  a  deficit  in  available  DA.  Although  in  has  been  reported  that  brains 
of  elderly  persons  brains  have  low  levels  of  DA,  the  rate  of  formation  of  brain  HVA 
does  not  appear  to  differ  with  age,  suggesting  that  the  turnover  rate  of  brain  DA 
increases  with  age  to  compensate  for  the  diminished  DA  content.  A  surprising 
preliminary  finding  of  a  sex  difference  in  brain  DA  is  awaiting  confirmation  in  a 
larger  number  of  subjects. 

18F-labelled  DA  has  been  shown  to  be  a  useful  compound  for  imaging  sympathetic 
nerves  and  their  activity  in  vivo  in  experimental  animals.  In  dogs,  denervation  of  the 
salivary  gland  by  removal  of  one  superior  cervical  ganglion  results  in  loss  of  uptake 
and  retention  of  the  18F-Iabel.  Furthermore,  the  18F  accumulated  in  the  heart 
declines  more  slowly  when  sympathetic  nerve  traffic  is  diminished  and  more  rapidly 
during  pharmacolgically  induced  NE  release.  Thus,  18F-DA  appears  to  be  converted 
to  18F-NE  in  sympathetic  nerves  and,  as  we  had  previously  shown  in  rats,  can  be  used 
to  reflect  NE  turnover. 

Another  major  research  endeavor  is  centered  on  neurotoxins  which  may  produce 
neurologic  disorders.  Current  investigations  centered  on  two  toxins,  MPTP  (1- 
methyl-4-phenyl-1,2,3,6-tetrahydropyridine)  and  BMAA  (2-amino-3-(methylamino)- 
propanoic  acid).  MPTP  toxicity  results  in  a  movement  disorder  in  primates,  including 
humans,  which  closely  resembles  Parkinson's  disease.  When  administered  into  one 
carotid  artery,  this  toxin  produces  in  monkeys  a  hemiparkinsonian  syndrome  which 
we  have  used  to  examine  biochemical  and  metabolic  changes,  behavioral  effects  and 
therapeutic  potential  of  drugs  and  tissue  implants. 

Local  cerebral  glucose  utilization  (LCGU)  in  monkeys  with  MPTP  hemiparkinsonism 
was  examined  using  quantitative  2-[i4C]deoxyglucose  autoradiography.  These 
monkeys  display  rigidity,  bradykinesia,  and  tremor  of  the  limbs  contralateral  to  the 
side  of  MPTP  infusion;  during  spontaneous  activity  they  turn  toward  the  side  of  the 
lesion.  Of  82  brain  areas  examined,  statistically  significant  metabolic  changes  were 
confined  mainly  to  basal  ganglia  structures  ipsilateral  to  the  side  of  the  lesion.  LCGU 
was  reduced  in  the  substantia  nigra  pars  compacta  and  ventral  tegmental  area,  i.e., 
in  the  areas  of  cell  loss,  where  as  increases  in  LCGU  were  found  in  regions  normally 
innervated  by  the  lesioned  area,  e.g.,  the  post-commissural  portions  of  the  putamen 
and  dorsolateral  caudate.  Other  structures  showing  statistically  significant  metabolic 
changes  were  the  external  segment  of  the  globus  pallidus  (  +  40%),  subthalamic 
nucleus  (-17%),  and  pedunculopontine  nucleus  (+15%).  There  were  also  smaller 
changes  in  portions  of  the  thalamus  (ventral  anterior  nucleus,  parafascicular 
nucleus)  and  premotor  cortex.  All  significant  metabolic  changes  were  confined  to 
the  side  of  the  substantia  nigra  lesion  and  were  essentially  restricted  to  regions 
involved  in  the  production  of  movement  or  maintenance  of  posture. 

We  have  previously  reported  that  ethanol  and  acetaldehyde  potentiate  MPTP 
neurotoxicity  in  mice,  enhancing  DA  depletion  in  the  striatum.  This  enhancement  of 


8-CNB/DIR 


neurotoxicity  was  found  to  be  specific  for  the  nigrostriatal  DA  pathway,  but  there 
were  differences  in  vulnerability  to  the  toxin  with  age.  In  5-week-old  mice 
acetaldehyde  treatment  did  not  enhance  DA  depletion,  whereas  in  8-week-old 
animals,  acetaldehyde  or  ethanol  given  with  MPTP  decreased  striatal  DA  content  to 
about  10%  of  controls  in  contrast  to  43%  of  controls  when  MPTP  was  given  alone.  In 
acetaldehyde  or  ethanol  and  MPTP-treated  mice,  changes  in  DA  levels  were 
observed  only  in  the  striatum.  DA  contents  in  the  hypothalamus,  olfactory  bulb  and 
frontal  cortex  were  similar  to  that  in  controls.  Contents  of  NE  and  serotonin  in 
striatum,  hypothalamus,  olfactory  bulb  and  cerebral  cortex  were  not  affected  by  any 
of  the  treatments.  Three  months  after  MPTP  alone,  striatal  DA  recovered  to  74%  of 
controls  in  8-week-old  mice,  whereas  no  recovery  occurred  in  acetaldehyde-  MPTP- 
treated  mice.  Moreover,  both  tyrosine  hydroxylase  immunocytochemistry  and  Cresyl 
violet  staining  showed  an  extensive  and  selective  cell  loss  in  the  parscompacta  of  the 
substantia  nigra  of  the  mice  treated  with  acetaldehyde  or  ethanol  and  MPTP, 
whereas  MPTP  alone  caused  only  a  limited  cell  degeneration. 

Accumulation  of  the  MPTP  toxic  metabolite,  1-methyl-4-phenylpyridinium  ion 
(MPP  +  )  in  both  striatum  and  whole  brain  after  MPTP  alone  or  after  combined 
treatments  with  ethanol  or  acetaldehyde  did  not  affect  MPTP  metabolism.  The 
results  from  the  in  vivo  experiments  indicated  that  relatively  low  doses  of  ethanol  or 
acetaldehyde  directly  potentiate  MPP+  toxicity,  apparently  without  interferring 
with  its  pharmacokinetics.  However,  higher  doses  of  these  drugs  decreased  MPP  + 
clearance  from  the  striatum.  Acetaldehyde  also  increased  initial  MPTP  accumulation 
in  the  whole  brain  but  failed  to  enhance  MPP+  levels,  indicating  that  MPTP 
metabolism  was  unaffected.  In  vitro  studies  confirmed  that  acetaldehyde  does  not 
modify  MPTP  metabolism  in  striatal  or  mesencephalic  astrocytes  in  culture.  In 
mesencephalic  neuronal  cultures,  acetaldehyde  does  not  interfere  with  the 
conversion  of  MPTP  to  MPP+  by  glial  cells  and  does  not  change  the  levels  of  MPP  + 
uptake  nor  its  spontaneous  release.  These  results  indicate  that  the  slower  MPP  + 
clearance  from  the  stratum  after  acetaldehyde  is  not  related  to  a  direct  effect  of 
acetaldehyde  on  DA  neurons  or  astrocytes. 

Cerebellar  granule  cells  in  enriched  primary  culture  are  also  susceptible  to  the 
neurotoxic  effects  of  MPP  +  .  Relatively  high  MPP+  concentrations  are  required  to 
elicit  neurotoxic  effects  at  early  culture  times,  but  lower  concentrations  of  MPP  + 
produce  comparable  neurotoxic  effects  at  later  culture  times.  Under  identical 
culture  conditions,  MPTP  is  not  neurotoxic.  Preincubation  with  the  glutamate 
uptake  blockers,  DL-threo-3-hydroxyaspartic  acid  or  dihydrokainate,  or  the 
dopaminergic  uptake  blocker  mazindol,  protects  the  granule  cells  from  the  cytotoxic 
effects  of  MPP  +  .  Although  MPTP  is  not  neurotoxic  in  an  enriched  granule  cell 
culture,  in  coculture  with  cerebellar  astrocytes  MPTP  is  toxic  to  granule  cells, 
presumably  because  it  is  converted  in  astrocytes  to  MPP  +  .  Cerebellar  astrocytes 
remain  confluent  and  viable.  The  addition  of  pargyline  to  the  coculture  abolishes 
the  neurotoxicity  consistent  with  a  role  of  MAO  B  in  bioactivation  of  MPTP.  The 
concentration  of  MPP+  in  the  coculture  medium  (13  uM)  was  less  than  that  required 
for  the  toxic  effect  in  enriched  neuronal  cultures  at  earlier  culture  times,  suggesting 
that  an  astroglial-neuronal  interaction,  perhaps  by  proximity,  enhances  the 
neurotoxicity  of  MPP  +  .  These  results  might  explain  the  reported  effects  of  MPTP  on 
some  cerebellar  cells  in  mice. 

The  second  toxin,  BMAA,  was  selected  for  study  because  it  had  been  implicated  in 
the  unusually  high  incidence  of  amyotrophic  lateral  sclerosis-parkinsonism-dementia 
(ALS-PD)  disorder  among  the  Chamorros  native  to  the  island  of  Guam.  The  disease 
had  been  attributed  to  consumption  of  flour  prepared  from  the  seeds  of  Cycas 


9-CNB/DIR 


circinalis  L,  the  false  sago  palm.  The  disease  occurs  also  in  other  regions  of  the  South 
Pacific  where  cycad  seeds  have  been  implicated  because  of  their  medicinal  use  on 
open  wounds  or  sores.  In  support  of  this  relationship  it  was  recently  shown  that, 
when  given  in  large  doses  to  primates,  BMAA,  a  minor  cycad  component,  can  cause 
selective  degeneration  of  upper  and  lower  motor  neurons  in  the  spinal  cord  and 
clinical  features  similar  to  those  of  ALS.  In  order  to  test  the  relationship  between  ALS 
and  cycads,  we  developed  a  sensitive  and  precise  gas  chromatography/mass 
spectrometric  (GC/MS)  assay  for  BMAA  which  allows  direct  assessment  of  the  BMAA 
content  in  foods  and  is  directly  applicable  to  the  assay  of  BMAA  in  biologic  tissues 
and  fluids.  BMAA  was  found  in  the  endosperm  of  a  range  of  cycad  seeds  collected 
from  Guam  at  levels  of  approximately  1  g/g  (dry  weight). 

The  levels  of  the  neurotoxin  in  cycad  flour,  however,  indicated  that  over  90%  of  the 
total  BMAA  content  was  removed  during  processing.  Furthermore,  in  one-half  the 
samples  almost  all  (>99%)  of  the  total  BMAA  was  removed.  There  were  no 
significant  regional  differences  in  the  BMAA  content  of  flour  prepared  from  cycad 
seeds  collected  from  several  villages  on  Guam.  The  processed  cycad  flour  as  prepared 
by  the  Chamorros  of  Guam  and  Rota  contains  extremely  low  levels  of  BMAA,  which 
are  in  the  order  of  only  0.005%  by  weight  (mean  values  for  all  samples).  Thus,  even 
when  cycad  flour  is  a  dietary  staple  andeaten  regularly,  it  seems  unlikely  that  these 
low  levels  could  cause  the  delayed  and  widespread  neurofibrillary  degeneration  of 
nerve  cells  observed  in  ALS-PD  of  Guam. 

Another  possibility,  however,  is  that  the  toxin  is  accumulated  selectively  in  specific 
sites  whereafter  chronic  exposure,  concentrations  of  BMAA  become  sufficiently  high 
to  cause  toxic  effects.  To  examine  this  possibility,  BMAA  bioavailability,  uptake, 
storage  and  penetration  into  brain  were  studied  in  rats.  The  results  of  this  study 
showed  that  only  a  small  fraction  of  injected  BMAA,  whether  adminstered  as  a 
single  acute  i.v.  injection  or  infused  s.c.  continuously  over  a  2-week  interval,  reach 
the  brain  and  only  low  concentrations  (average  <300  uM)  are  found.  The  cortex  and 
spinal  cord,  which  are  most  affected  in  ALS-PD,  have  among  the  lowest  levels  of  the 
toxin. 

When  BMAA  toxicity  to  cerebellar  granule  cells  in  tissue  culture  was  compared  to  the 
toxicity  of  cycad  flour  obtained  from  various  sources  in  Guam,  there  was  no 
relationship  between  toxic  effects  and  BMAA  content.  Although  BMAA  was 
nontoxic  in  concentrations  greater  than  found  in  the  flour  samples,  some  of  the 
samples  were  toxic  to  these  cultured  cells.  This  led  us  to  characterize  the  substance 
which  appeared  to  be  responsible  for  the  toxicity.  Because  of  its  physical  properties 
and  stability,  metals  were  implicated;  analysis  for  metals  revealed  high 
concentrations  of  zinc  in  only  the  few  toxic  flour  samples.  The  concentrations  found 
were  sufficient  to  account  for  toxicity  in  the  tissue  culture  and  would  result  in 
dietary  intake  of  zinc  in  excess  of  one  hundred-fold  greater  than  normal.  The  source 
of  the  zinc  may  have  been  galvanized  containers  used  for  soaking  the  cycad  seeds 
during  the  preparation  of  the  flour.  This  observation  provides  a  link  between  the 
two  major  alternative  hypotheses  (cycad  and  trace  metal  imbalance)  for  the  etiology 
of  ALS-PD. 

Investigators  in  the  unit  on  growth  factors  have  been  examining  the  regulation  of 
synthesis  and  expression  of  neurotrophic  agents  and  neuropeptides.  Nerve  growth 
factor  (NGF)  isolated  from  the  mouse  submaxillary  gland  bv  Levi-Montalcini,  was  the 
first  discovered  of  a  number  of  such  protein  factors  which  are  required  by  neurons 
for  survival  and  maintenance  of  function.  Recent  evidence  suggests  that 
many"nerve  growth  factors"  may  exist,  each  specific  for  a  particular  population  of 


10-CNB/DIR 


neurons  in  either  the  CNS  or  the  the  peripheral  autonomic  nervous  systems.    In 
addition,  neuropeptides  may  play  a  role  as  neurotrophic  factors. 

Several  animal  models  of  neurologic  disease  changes  in  the  synthesis  or  tissue 
content  of  factors  appear  to  be  associated  with  the  response  to  CNS  injury  or 
involved  in  recovery.  The  Parkinsonian  disorder  with  destruction  of  dopaminergic 
neurons  of  the  nigrostiatal  pathway  which  appears  in  mice  or  monkeys  after 
systemic  treatment  with  the  neurotoxin  MPTP,  partial  ablation  of  cortex  in  rats, 
"injury"  genetically  programmed  death  of  cerebellar  Purkinje  cells  between  20-50 
days  after  birth  in  an  inbred  mouse  strain,  the  pcd  mutant,  are  all  models  which  can 
be  used  to  determine  whether  proliferating  astrocytes  produce  trophic  factors.  The 
reactive  astrocytes  can  be  cultured  and  growth  factor  activity  sought  in  the  medium. 
Neurotrophic  activity  is  assessed  by  bioassays  which  utilize  chick  dorsal  root  ganglia 
neurons,  rat  superior  cervical  ganglion  or  CNS  neurons,  as  well  as  by  a  2-site  assay  for 
NGF.  In  addition,  mRNA  is  prepared  and  analyzed  for  NGF  mRNA  as  well  as  cross- 
hybridizing  species  (under  low  stringency).  These  blots  are  also  being  examined  for 
changes  in  neuropeptide  and  neurotransmitter  synthetic  enzyme  mRNAs.  Some  of 
the  mRNA  has  been  used  in  the  preparation  of  cDNA  libraries.  Molecular 
mechanisms  for  regulating  the  synthesis  of  NGF  and  other  neurotrophic  factors, 
primary  cultures  of  astrocytes  are  being  studied  as  model  systems.  NGF  mRNA  has 
been  detected  in  primary  cultures  of  rat  cortical,  cerebellar  and  striatal  astrocytes, 
and  Schwann  cells.  NGF  synthesis  can  be  increased  in  astrocytes  by  both  forskolin 
and  isoproterenol,  supporting  the  idea  that  in  these  cells,  cyclic  AMP  regulates 
transcription  of  the  NGF  gene.  Isoproterenol  has  been  shown  to  work  through  the 
classic  ^-adrenergic  receptor:  the  effect  is  blocked  by  ^-antagonists.  The  presence  of 
the  ^-receptor  on  astrocytes  suggests  a  mode  for  neuronal-glial  communications.  In 
addition,  VIP  (vasoactive  intestinal  peptide)  can  stimulate  NGF  synthesis  in 
astrocytes:  whether  this  occurs  via  activation  of  adenylate  cyclase  has  yet  to  be 
determined.  This  finding  is  of  particular  interest  since  in  spinal  cord  cultures,  VIP  acts 
on  astrocytes  to  release  neurotrophic  factors. 

Astrocytes  have  recently  been  found  to  express  certain  neuropeptide  genes,  with 
both  gene-  and  brain  region-specificity.  Astrocytes  from  cortex,  cerebellum  and 
striatum  all  express  the  proenkephalin  (PE)  gene.  The  cells  synthesize  and  process 
the  precursor  to  free  enkephalin  peptides,  although  the  pattern  of  processing 
appears  to  vary  with  the  brain  region  and  cerebellar  cells  contain  a  C-terminally 
extended  form  of  enkephalin.  Only  cerebellar  astrocytes  express  somatostatin  (SS) 
mRNA  and  peptide:  none  express  cholecystokinin,  substance  P  or  the  other  opioid 
peptide  precursors.  Neuropeptide  expression  is  developmentally  regulated,  with 
peak  expression  in  astrocytes  prepared  from  embryonic  day  20  to  postnatal  day  3 
animals;  levels  decrease  sharply  in  cells  prepared  from  8-day-old  animals  and  are 
almost  undetectable  in  cells  prepared  from  adult  animals.  These  results  suggest  the 
possibility  that  enkephalins  or  SS  may  act  as  astrocyte-derived  trophic  factors  for 
neurons  early  in  development,  consistent  with  results  previously  obtained  on  PE 
gene  expression  in  striatum,  as  well  as  analyses  of  proenkephalin  (PE)  and  SS  gene 
expression  in  cortex  and  cerebellum  during  development.  The  developmental  time 
course  of  expression  of  PE  mRNA  in  striatum  is  of  particular  interest,  since  it  shows  a 
biphasic  curve.  The  first  peak  before  synapses  have  yet  formed,  but  when  astrocyte 
expression  is  maximal,  suggesting  that  enkephalins  may  serve  a  trophic-like  function 
early  in  development.  Similar  observations  have  been  made  regarding  other 
neuropeptides  including  vasoactive  intestinal  peptide  (VIP).  The  second  peak  in  PE 
mRNA  occurs  at  the  time  of  active  synapse  formation  and  presumably  represents  the 
expression  of  enkephalins  as  neurotransmitters/neuromodulators.  These  studies  are 
being  complemented  by  situ  hybridization  analysis  of  changes  in  neuropeptide  and 


11  -CNB/DIR 


neurotransmitter  mRNAs  at  the  single  cell  level.  This  information  will  be  used  to 
determine  if  in  vivo  PE  and  SS  gene  expression  occurs  in  astrocytes  early  in 
development.  Current  studies  are  directed  toward  determining  if  expression  of  the 
neuropeptide  genes  turn  on  again  in  reactive  astrocytes  and  if  exposure  of  either 
neurons  or  glia  in  culture  to  peptides  alter  either  morphology  or  specific 
neurochemical  markers. 

Using  a  method  previously  developed  in  our  laboratory,  changes  in  the  turnover  rate 
of  neuropeptides  are  being  examined  in  pharmacologic  studies  on  rat  brain.  The 
method  is  based  on  measurement  of  precursor  mRNA,  precursor  proteins,  and  the 
biologically  active  peptides:  the  combination  of  these  measurements  is  used  to 
determine  whether  a  drug  affects  neuropeptide  levels  by  changing  the  transcription 
of  the  gene,  the  rate  of  translation  to  or  processing  of  the  precursor,  or  the 
utilization  of  the  peptide  itself.  The  results  have  demonstrated  that  certain 
neuroactive  drugs,  such  as  morphine,  can  affect  gene  expression  and  thereby  alter 
peptide  levels,  whereas  others  affect  peptide  levels  independent  of  an  action  on  the 
rate  of  biosynthesis.  We  have  now  analyzed  changes  in  enkephalin  biosynthesis  and 
turnover  following  MPTP  administration  to  mice,  and  find  that  depletion  of  DA 
affects  enkephalinergic  neurons  differently  in  striatum  and  olfactory  tubercle  or 
cortex.  These  studies  contribute  to  understanding  the  role  which  neurotrophic 
factors  may  play  in  neurologic  disease,  the  effect  of  their  absence  on 
neuropeptide/neurotransmitter  function,  the  molecular  mechanisms  by  which 
synthesis  of  these  agents  may  be  regulated,  and  the  possible  neurotrophic  functions 
of  neuropeptidesearly  in  CNS  development. 


12-CNB/DIR 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECTNUMBER 


Z01  NS  02115-  17CNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  ($0  characters  or  less.   Title  must  fit  on  one  line  between  the  borders.) 

BIOCHEMICAL  INDICES  OF  ADRENERGIC  FUNCTION  IN  HUMANS 


PRINCIPAL  INVESTIGATOR  (Ust  other  professional  personnel  below  the  Principal  Investigator.)  (Name,  title,  laboratory,  and  institute  affiliation) 

P.I.:     R.  J.  POUNSKY,  M.D., CHIEF,  CNS,  CNB,  NINDS 

OTHERS:  (CNS)  -  S.M.  BASER,  M.D.,  MEDICAL  STAFF  FELLOW;  A.M.  MARINI,  M.D.,  SENIOR  STAFF 
FELLOW;  (OCD)  -  LE.  NEE,  M.S.W.,  SOCIAL  SCIENCE  ANALYST;  (MNB)  -  M.  HALLETT,  M.D.,  CHIEF; 
(NIS)  -  M.  FULHAM,  M.D.,  VISITING  ASSOCIATE;  G.  DICHIRO,  M.D.,  CHIEF;  (NIB)  -  H.  McFARLAND,  M.D., 
DEPUTY  CHIEF;  (CNB)  -  I.J.  KOPIN,  M.D.,  CHIEF 


COOPERATING  UNITS  (if  an,) 

DEPT.  OF  HISTOLOGY,  UNIV  OF  GOTEBORG,  SWEDEN  (A.  McRAE,  A.  DAHLSTR6M  );  NATIONAL  CANCER  INSTITUTE  (M.  GOMEZ, 
S  BALE);  DEPT.  OF  HUMAN  ANATOMY,  UNIV  OF  OXFORD  (M.  MATTHEWS);  UNIV  OF  MICHIGAN  (A.  YOUNG) 


LAB/BRANCH 

CLINICAL  NEUROSCIENCE  BRANCH,  CNP,  DIR,  NINDS 


SECTION 

CLINICAL  NEUROPHARMACOLOGY  SECTION 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  BETHESDA,  MP  20892 


TOTAL  MAN-YEARS:  ,  j. 


PROFESSIONAL:  2  5 


OTHER: 


1.0 


CHECK  APPROPRIATE  BOX(ES) 

|T]   (a)  Human  subjects  Q  (b)  Human  tissues  I      I  (c)  Neither 

]  (a1)  Minors 

]  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Autonomic  nervous  system  activity  is  essential  for  maintaining  circulatory  and  metabolic  homeostasis.  In 
order  to  study  sympathetic  nervous  system  function  and  its  relationship  to  other  neuroendocrine 
systems,  it  is  necessary  to  measure  neurotransmitter,  hormonal,  and  peptide  levels  in  response  to  various 
stimuli.  The  levels  of  norepinephrine,  epinephrine,  and  dopamine  and  their  metabolites  in  various  body 
fluids  reflect  the  activity  of  the  neurones  from  which  these  neurotransmitters  are  released. 
Cerebrospinal  fluid  levels  of  monoamine  metabolites,  enzymes  related  to  neurotransmitter  synthesis  and 
degradation,  and  peptides  can  be  used  to  assess  central  nervous  system  neurotransmitter/neuropeptide 
function  and  metabolism.  It  is  necessary  to  consider  the  origin  of  these  metabolites  to  make  appropriate 
corrections  for  valid  interpretations  of  the  data.  These  strategies  have  been  used  to  study  patients  with 
neurogenic  orthostatic  hypotension  and  other  disorders  in  which  abnormal  adrenergic  function  is 
suspected.  Plasma  NE  increases  following  intravenously  administered  acetylcholine  in  normal  subjects. 
Lack  of  an  increase  in  patients  with  pure  autonomic  failure  (PAF)  is  consistent  with  post-ganglionic 
sympathetic  neuronal  dysfunction.  Patients  with  multiple  system  atrophy  (MSA)  may  develop 
depolarization  blockade  due  to  cholinergic  receptor  supersensitivity  since  they  manifest  an  exaggerated 
response  at  low  doses  followed  by  a  return  of  plasma  NE  to  baseline  levels.  Only  patients  with  PAF  have 
(3-adrenergic  receptor  supersensitvity.  Increases  in  plasma  norepinephrine  and  renin  activity  following 
isoproterenol  administration  in  normal  subjects  may  result  from  reflex  cardiovascular  and  renal 
mechanisms.  Diminished  sweat  production  to  intradermal  methacholine  in  patients  with  autonomic 
failure  suggests  that  human  sweat  glands  do  not  develop  denervation  supersensitivity.  Cerebrospinal 
fluid  immunoreactivity  to  rat  locus  coeruleus  suggests  that  degeneration  in  MSA  may  release  antigens 
that  induce  antibodies  against  neurons  in  selected  brain  regions   Structural  and  metabolic  brain 
abnormalities  defined  by  neuroimaging  studies  facilitate  clinical  distinction  between  PAF  and  MSA. 
Improved  diagnostic  accuracy  and  a  more  thorough  understanding  of  the  underlying  mechanisms 
involved  in  producing  abnormalities  of  neurotransmitter  metabolism  and  peptide  function  in  these 
clinical  disorders  leads  to  more  rational  therapeutic  approaches. 

13-CND/DIR 


PHS6M0  [Rev    1  84) 


utKAKiMtNT  OF  HEALTH  AND  HUMAN  SERVICES -PUBLIC  HEALTHS,  : 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


PROJECl  NUMBER 
ZOI  NS  02630-07  CNB 


TITLE  OF  PROJECT  (so  chtncfrs  or  ttu.  THIimun  fit  on  on*  lint  bmf¥mtnih»bord»rs.) 

CLINICAL,  GENETIC,  AND  BIOCHEMICAL  STUDIES  OF  FAMILIAL  ALZHEIMER'S  DISEASE 


PRINCIPAL  INVESTIGATOR  (L,sx  othor  pror^suontl  p,ry>n^l  bolow  <h,  Pr.naotl  Inving.ior.)  <Artm«.  Ml:  ItborKory,  tnd  Institul,  tmi*„on) 

PI-:  R.  j.  POUNSKY,  M.D.,  CHIEF,  CNS,  CNB,  NINDS 

OTHERS:             L.E.  NEE,  M.S.W                            SOCIAL  SCIENCE  ANALYST  OCD,  CNP,  NINDS 

S.M.  BASER,  M.D.                          MEDICAL  STAFF  FELLOW  CNS,  CNB,  NINDS 

A.M.  MARIN!,  M.D.                       SENIOR  STAFF  FELLOW  CNS,  CNB,  NINDS 

G.  DICHIRO.M.D.                         CHIEF  N IS,  OCD,' NINDS 

J.  GRAFMAN,  PH.D.                       PSYCHOLOGIST  CNU,  MNB,  NINDS 


COORPERATING  UNITS  (it ,n,) 

GENETICS  UNIT.  MASS.  GEN  HOSP  'J.  GUSELLA.  P.  HYSLOP) ).  5  ILLINOIS  UNIV ..  (  R.  STRUBLE).  SMID-SUD.  ITALY  (A  BRUNI).  KUNIK  BAVARIA  (P  FROMMELT);  UNIV  OF 
KV(j  SISKEN).  UNIV   OFCA(0  COX.  V  SHEFFIELD).  UMONj  (W  C  LAMBERT).  UNIV  OF  GOTEBORG.  SWEDEN  (A  MCRAE.  A.  OAMLSTROM).  CEDARS-SINAI,  CA  (S  PULST) 


LAB/BRANCH 

CLINICAL  NEUROSCIENCE  BRANCH,  CNP,  DIR,  NINDS 


SECTION 

CLINICAL  NEUROPHARMACOLOGY  SECTION 


INSTITUTE  AND  LOCATION 

NINDS.  NIH,  BETHESDA,  MP  20892 


TOTAL  MAN-YEARS: 

4.0 


PROFESSIONAL:  3  Q 


OTHER:  1  q 


CHECK  APPROPRIATE  BOX(ES) 

[I]   (a)Human  subjects  H  (b)  Human  tissues  I      I  (c)  Neither 

I  x  |  (a1)  Minors 

|x|  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Alzheimer's  disease  (AD)  is  the  most  common  cause  of  irreversible,  chronic  dementia. 
Although  AD  may  be  familial  in  only  one-third  of  all  cases,  the  main  justification  for 
studying  autosomal  dominant  cases  lies  in  the  accuracy  of  diagnosis  which  may  be 
inferred  through  post-mortem  examination  of  other  affected  family  members.  More 
than  250  members  of  20  pedigrees  with  an  autosomal  dominant  form  of  AD  have  had 
skin  fibroblast  and  peripheral  blood  lymphoblast  cultures  established.  These  cultures 
serve  as  a  renewable  source  of  DNA  and  cell  lines  for  genetic  linkage,  viability,  and 
biochemical  studies.  Recombinant  DNA  technology  has  been  applied  to  perform 
genetic  linkage  studies  in  these  families  with  inherited  AD.  Additional  families  have 
provided  furtner  confirmation  of  our  earlier  finding  of  a  region  of  chromosome  21  that 
is  linked  to  AD  in  early  onset  cases;  however,  a  single  marker  has  not  been  identified. 
In  a  collaborative  study  of  70  families,  the  age-at-onset  showed  a  bimodal  distribution. 
The  lifetime  AD  risk  for  at-risk  offspring  in  early  onset  cases  was  53%,  significantly 
lower  than  86%  in  late  onset  AD.  Transmission  pattern  and  age-at-onset  provide 
further      evidence      for       heterogeneity.  Longitudinal       investigation       of 

neurotransmitters/neuropeptides  in  crebrospinal  fluid  (CSF)  continues  to  confirm  our 
previous  findings;  three  at-risk  subjects,  among  the  subgroups  with  low  CSF  MHPG  and 
somatostatin,  have  developed  dementia.  Anti-neuronal  antibodies  have  also  been 
detected  in  the  CSF  from  AD  patients.  These  antibodies  recognize  specific  neuronal 
populations  in  the  adult  rat  central  nervous  system.  In  developing  rat  brain  these 
antibodies  recognize  specific  membrane  receptors  ir.  macropnagic  microglia. 
Inflammation  and  similar  immune  mechanisms  may  play  a  pathogenetic  role  in  AD. 
Cerebral  glucose  metabolism  is  significantly  reduced  in  the  parietotemporal  regions 
with  some  occipital  and  temporal  involvement.  A  mild  (10-17%)  reduction  was 
observed  in  three  at-risk  subjects  who  later  developed  AD. 

14-CNB/DIR 


PHS6040IP-X   1/B4I 


ucrMi\  i  men  i  ur  i-icml  in  <ii»^  ■  iui«im«  jtn  ni.u  -  ■  w  —  — 

Z01  NS  02716-05 


NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


CNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (so  c/»  ncimr,oi  mi    Till*  mutt  fit  on  ont  lint  btl-tmn  tnt  bordtrl.) 

Neurotoxins  and  Animal  Models  of  Neurological  Diseases 


PRINCIPAL  INVESTIGATOR  am  oci«<pro»»jwo/M' w'ionn./ixio*  f*.  *inupj/(n««jt.0i(<vj  <*,m«  Mil,  utborttory.  tnd  innttuffftlittion) 

PI:  Irwin  J.  Kopin.M.D.,  Chief,  CNB,  Director,  DIR,  NINDS 

Others  (CNB,  NINDS): 

Virginia  Weise,  Chemist;  AlessandroZuddas,  M.D.,  Visiting  Fellow;  Linda  Porrino,  Ph.D.,  Research 

Psychologist;  Mark  Duncan,  Ph.D.,  Visiting  Fellow;  Ann  M.  Marini,  M.D.,  Sr.  Staff  Fellow:  Margaret 

Palmatier,  Ph.D.,  Staff  Fellow;  Judith  Harvey-White,  B.S.,  Technician. 

Sanford  P.  Markey,  Ph.D.,  Pharmacologist,  LCS,  NIMH 

Krzysztof  S.  Bankiewicz,  M.D., Visiting  Fellow,  SNB 


COORPERATING  UNITS  „!,»,) 

Surgical  Neurology  Branch,  NINDS 
Laboratory  of  Clinical  Science,  NIMH 


LAB/BRANCH 

Clinical  Neuroscience  Branch,  CNP 


SECTION 

Aminerqic  Mechanisms 


INSTITUTE  ANO  LOCATION 

NINDS,  NIH,  Bethesda,  MP  20892 


TOTAL  MAN-YEARS:  -- 


PROFESSIONAL:  2  2 


OTHER:  1  0 


CHECK  APPROPRIATE  BOX(ES) 

LJ  (a)  Human  subjects  Q  (b)  Human  tissues  l"x~1  (c)  Neither 

^  (a1)  Minors 

(a2)  Interviews 


□ 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  objectives  of  this  project  are  to  identify  and  characterize  toxins  which  target  specific  neuronal 
systems,  determine  the  mechanisms  of  action  of  these  toxins,  and  use  them  to  produce  animal  models  of 
human  neurologic  disorders.  At  present  the  main  focus  is  on  two  toxins,  1-methyl-4-phenyl-1, 2,3,6- 
tetrahydropyridme  (MPTP)  and  beta-methylaminoalanine  (BMAA). 

MPTP  is  administered  via  the  internal  carotid  artery  to  produce  hemiparkinsonism  in  monkeys.  These 
animals  are  used  to  evaluate  changes  cerebral  metabolism  (as  indicated  by  autoradiographic 
determination  of  the  uptake  of  14C-2-deoxyglucose)  as  a  result  of  the  toxin-induced  neurologic  disorder 
and  to  examine  the  efficacy  of  tissue  implants  in  alleviating  the  neurologic  deficits.  Biochemical  and 
behavioral  abnormalities  are  examined  to  evaluate  the  efficacy  of  tissue  implants  to  replace  destroyed 
neurons  or  enhance  sprouting  of  surviving  neurons.  In  other  studies,  mice  are  treated  with  various  doses 
of  MPTP  and  with  substances  which  may  potentiate  its  toxic  effects.  Fluid  obtained  from  gelfoam  placed 
in  MPTP  and  surgically  damaged  brain  is  examined  for  substances  which  stimulate  growth  of  neurons  in 
tissue  culture.  Enhancement  of  MPTP  toxicity  by  low  doses  of  ethanol  or  acetaldehyde  is  not  due  to  any 
effect  on  MPTP  metabolism  or  retention  of  its  metabolite,  MPPVThis  enhancement  of  toxicity  has  now 
been  shown  to  be  negligible  in  young  (7-day-old)  mice,  but  marked  in  older  (8-week-old)  animals. 
Furthermore,  ethanol  or  acetaldehyde  appears  to  enhance  the  specificity  of  the  toxin  in  targeting 
specific  dopaminergic  neurons.  Gel  foam  plugs  removed  from  cavities  made  in  the  caudate  nucleus  of 
MPTP  parkinsonian  monkeys  contains  soluble  substance(s)  which  promotes  neurite  outgrowth  in  tissue 
cultured  neurons.  Similar  substances  have  been  found  in  fetal  tissues,  including  amnion. 

BMAA  has  been  implicated  as  responsible  for  the  putative  toxicity  of  flour  produced  from  cycas  circinalis 
and  cited  as  one  etiologic  factor  in  the  Amytrophic  lateral  sclerosis-parkinsonism-dementia  complex  of 
Guam  (Guamanian  ALS-PD).  Samples  of  a  flour  from  Guam  as  well  as  cycad  plants  from  various  sources 
have  been  assayed  (by  GC-MS)  Processing  of  the  cycad  to  produce  the  flour  removes  greater  than  80% 
of  the  BMAA  (usually  over  95%)  and  the  residual  BMAA  content  is  not  sufficient  to  reach  even  a  small 
fraction  of  the  amounts  reported  to  be  toxic  in  monkeys.  The  BMAA  contents  of  flour  from  various 
sources  have  been  compared  with  toxicity  elicited  in  vitro  using  granule  cell  tissue  cultures.  At  the 
concentrations  in  flour,  BMAA  is  not  toxic  to  these  cells,  but  some  of  the  flour  samples  were  toxic  High 
concentrations  of  zinc  were  found  in  only  toxic  samples.  Galvanized  metal  containers  might  have  been 
used  in  preparation  of  these  samples  of  flour,  implicating  zinc  as  a  toxic  metal  related  to  Guamanian 
ALS-PD 

15-CNB/DIR 


Phs(,W0(R«.    I  Ml 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  -  PUBLIC  HtALI  H  btKVILt 

NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


r(\u^v.  i    m  w.' 


Z01  NS  02717-05 
CNB 


PFRI0DC0VERED 

October  1 ,  1 989  through  September  30,  1 990 


TITLE  OF  PROJECT  (SO ctunatrt  or  Itsi-  m*  must  fh  on  on*  lint  Mmm  Itn  bordtrt.) 

Biochemical  Evaluation  of  Aminergic  Function  During  Responses  to  Stress  and  in  Disease  States 


PRINCIPAL  INVESTIGATOR  (Unolhtr  proftsaontl  otrsonntl  btlonr  thi  Frlnctp*!  tnvsiigt'or.)  (hltm*.  tnlt.  KbwctOfy.  tndinstituf  Mffilmlon) 

PI:  Irwin  J.  Kopin,  M.D.,  Chief,  CNB,  Director,  DIR,  NINDS 

Others  (CNB,  NINDS): 

Graeme  Eisenhofer,  Ph.D.,  Visiting  Associate,  CNB,  DIR,  NINDS;  Katalin  Szemeredi,  Ph.D.,  Visiting  Fellow, 

CNB,  DIR,  NINDS;  Anna  Starosta,  M.D.,  Visiting  Fellow,  CNB,  DIR,  NINDS 

Ronald  Polinsky,  M.D.,  Chief,  Clinical  Neuropharmacology  Section 

Virginia  Weise,  Chemist,  CNB,  NINDS;  Judith  Harvey-White,  B.S.,  Technician.,  CNB,  NINDS 

David  Goldstein,  M.D.,  Ph.D.,  Medical  Officer,  HEB,  NHLBI 

foWftWflUKte 

Laboratory  of  Clinical  Science,  NIMH 


LAB/BRANCH 

Clinical  Neuroscience  Branch,  CNP 


SECTION 

Aminerqic  Mechanisms 


INSTITUTE  AND  LOCATION 

NINDS.  NIH.  Bethesda.  MP  20892 


TOTAL  MAN-YEARS:  3J  PROFESSIONAL:  22 


OTHER: 


1.0 


CHECK  APPROPRIATE  BOX(ES) 

□   (a)Human  subjects  ]  (b)  Human  tissues  IT]  (c)  Neither 

J  (a1)  Minors 

II  (a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

The  main  objectives  of  this  project  are  (1)  to  determine  the  physiologic  significance  of  alterations  in 
plasma  levels  and  urinary  excretion  rates  of  the  catecholamines,  their  metabolites  and  their  precursor, 
DOPA;  (2)  to  develop  in  animals  and  humans  clinically  useful  methods  for  assessing  catecholaminergic 
function;  and  (3)  to  apply  these  methods  to  examine  function  of  catecholaminergic  neurons  in  the 
peripheral  sympathetic  nervous  system  and  in  brain. 

Changes  in  tissue  or  plasma  levels  or  urinary  excretion  rates  of  catecholamines  and  their  metabolites, 
and  tissue  tyrosine  hydroxylase  levels  are  evaluated,  during  and  after  stress,  in  response  to 
pharmacologic  treatments,  or  in  disease  states  in  animals,  in  normal  subjects,  and  in  patients  with 
various  neurologic  or  related  disorders  (autonomic  dysfunction,  Parkinson's  disease,  hypertension,  etc). 
Use  of  !8.f  labelled  dopamine  has  been  developed  for  imaging  by  positron  emission  tomography  (PET) 
peripheral  sympathetic  activity  in  vivo  in  dogs  and  is  awaiting  application  to  humans. 

In  rats,  plasma  levels  of  DOPA  parallel  pharmacologic  or  stress-induced  enhancement  of  norepinephrine 
release,  even  after  adrenalmedullectomy.  Tyrosine  hydroxylase  levels  in  tissues  do  not  reflect  the  rate  of 
catecholamine  production,  indicating  that  tyrosine  hydroxylation,  can  be  regulated  independently  of 
the  levels  of  tyrosine  hydroxylase.  Chronic  elevation  of  plasma  glucocorticoid  levels  elevates  rat  blood 
pressure  but  depresses  plasma  norepinephrine  levels.  Baroreceptor  sensitivity  is  unaltered  and  pressor 
responses  to  phenylephrine  are  normal.  There  is,  however,  an  increase  in  the  tachycardia  induced  by 
isoproterenol,  indicating  that  cardiac  beta-ad renoceptors  are  hyperresponsive.  The  mechanism  of 
hypertension  attending  chronic  elevation  of  corticosteriods  differs  from  that  in  other  forms  of 
hypertension. 

Most  patients  with  pure  autonomic  failure  were  found  to  have  low  plasma  levels  of  DOPA  as  well  as  of 
norepinephrine,  DHPG  and  DOPAC,  consistent  with  loss  of  peripheral  sympathetic  neurons,  whereas  in 
most  patients  with  multiple  system  atrophy,  levels  of  DOPA,  DHPG  and  DOPAC  are  generally  normal;  this 
is  consistent  with  normal  catecholamine  biosynthesis  in  these  patients  who  are  believed  to  have  intact 
peripheral  sympathetic  neurons  but  diminished  nerve  impulse  traffic  from  the  central  nervous  system. 

16-CNB/DIR 


PHS  *M0  (Rtv.  1«l 


DEPARTMENT  OF  HEALTH  AND  HUMAN  SERVICES  •  PUBLIC  HEALTH  SERVICE 
NOTICE  OF  INTRAMURAL  RESEARCH  PROJECT 


PROJECT  NUMBER 

Z01  NS  02752-03 
CNB 


PERIOD  COVERED 

October  1,  1989  through  September  30,  1990 


TITLE  OF  PROJECT  (»ch»r»<t«rj  or  ten.   Tit  (•  nvn  fir  on  on*  lint  bttw—rt  tfx  borrtt'l J 

Regulation  of  Synthesis  and  Expression  of  Neurotrophic  Agents  and  Neuropeptides 


PRINCIPAL  INVESTIGATOR  fUn  othtr  provisional  ptrsonr— I  bltow  1h*  Principal  trtvmrtiOHOr.)  fNama.  titlt.  laboratory,  and  inttrtuta  afiltation) 

Joan  P.  Schwartz,  Ph.D.,  Research  Chemist,  CNB,  NINDS 

Others: 

Kunihiko  Mitsuo,  Ph.D.,  Visiting  Fellow,  CNB,  NINDS 
Margaret  Palmatier,  Ph.D.,  Staff  Fellow,  CNB,  NINDS 


COOPERATING  UNITS  (Hany) 


LAB/BRANCH 

Clinical  Neuroscience  Branch,  CNP 


SECTION 

Section  on  Aminerqic  Mechanisms 


INSTITUTE  AND  LOCATION 

NINDS,  NIH,  Bethesda.  MP  20892 


TOTAL  MAN-YEARS:  - 


PROFESSIONAL:  ,  1 


OTHER:  1  q 


CHECK  APPROPRIATE  BOX(ES) 

O  (a)JHuman  subjects  □  (b)  Human  tissues  ["*!  (c)  Neither 

(a1)  Minors 

(a2)  Interviews 


SUMMARY  OF  WORK  (Use  standard  unreduced  type.  Do  not  exceed  the  space  provided.) 

Evidence  suggests  that  parallel  biochemical  and  regulatory  processes  occur  during  normal  development 
and  following  various  forms  of  CNS  injury.  Among  these  areas  of  particular  interest  are:  (1) 
identification  of  CNS  neurotrophic  factors,  and  (2)  the  analysis  of  the  regulation  of  neuropeptide  gene 
expression  during  development  and  in  response  to  injury.  Studies  are  underway  to  identify  trophic 
factors  produced  in  specific  model  systems,  since  recent  evidence  suggests  that  a  family  of  nerve  growth 
factors  (NGF)  exists,  each  specific  for  certain  populations  of  neurons.  An  NGF-like  factor  increases  in  the 
cerebellum  of  the  pcd  mutant  mouse  as  the  Purkinje  cells  die  out  and  astrocytes  proliferate.  In  another 
injury  paradigm,  multiple  cortical  lesions  are  made  in  rat  brain:  one  week  later,  RNA  is  prepared  from 
various  brain  regions  to  be  analyzed  for  any  change  in  NGF-like  mRNAs  relative  to  unlesioned  brain. 
MPTP-lesioned  animals  (both  mice  and  monkeys)  represent  a  Parkinson-like  model  in  which  changes  in 
NGF-like  molecules  are  being  examined.  Since  astrocytes  can  synthesize  NGF,  primary  cultures  of 
astrocytes  are  being  used  to  determine  factors  which  regulate  NGF  gene  transcription  as  well  as  to  assess 
production  of  other  potential  trophic  factors.  Reactive  astrocytes  are  prepared  from  regions  affected  by 
the  various  injuries  and  their  production  of  trophic  factors  compared  to  that  of  control  astrocytes 
Potential  neurotrophic  functions  for  neuropeptides  in  early  CNS  development  are  being  explored  in 
several  model  culture  systems. 

At  the  same  time,  these  injury  models  can  be  evaluated  for  changes  in  neuropeptide  and/or 
neurotransmitter  synthesis  occurring  in  response  to  the  lesions.  One  can  derive  an  estimate  of  peptide 
turnover  by  combining  measurements  of  the  precursor  mRNA,  the  precursor,  and  the  peptide.  Our 
studies  have  demonstrated  that  peptides  are  differentially  regulated  by  such  chronic  drug  treatments  as 
reserpine,  haloperidol,  6-hydroxydopamine  or  5,7-dihydroxytryptamine.  Work  is  in  progress  to 
determine  the  effects  of  CNS  injury  and  recovery,  including  MPTP  treatment,  on  various  neuropeptides 
as  well  as  such  neurotransmitter  synthetic  enzymes  as  tyrosine  hydroxylase  and  GAD,  and  the  dopamine 
D2  receptor. 

17-CNB/DIR 


PHS6W0(R«v    1*4) 


NIH  Library.  Building  10 
Ns:  ; -jt-s  of  Health 

fittftssda,  Md.   20892 


N 


•   LIBRARY 

|H 

|^     Amazing  Research. 
Amazing  Help. 


http://nihlibrary.nih.gov 


10  Center  Drive 

Bethesda,  MD  20892-1150 

301-496-1080 


3  1496  00494  6110 


m