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Full text of "Annual report : National Institute of Neurological Disorders and Stroke"

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 


02820-01 LMB 


5 


9 


ZOl NS 


02821-01 LNNS 


10 


30 


ZOl NS 


02822-01 LNNS 


10 


31 


ZOl NS 


02823-01 SN 


19 


35 



PROJECT NUMBER 



TAB PAGE 



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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 



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24 (6) 5 
LABORATORY OF 
VIRAL AND MOLECULAR 
PATHOGENESIS 

Monique Dubois-Dalcq (Acting) 
4 5 (1) 




NEURAL AND MOLECULAR 
BIOLOGY SECTION 
Monique Dubois-Dalcq 
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VIRAL PATHOGENESIS 
SECTION 

Heinz Arnheiter (Acting) 
3 7 (1) 1 




VIRAL REPLICATION 
SECTION 
Manfred Schubert (Arting) 
3 4 (1) 1 



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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 



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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 



CHEC K 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 . 



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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 Imag e. 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: 



CHEC K 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: 



CHEC K APPROPRIATE BOX(ES) 

] (a) H uman 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: 



CHEC K 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: 



CHEC K 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 



CHEC K 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 ch annel, 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 



CHEC K APPROPRIATE BOX(ES) 

] (a) H uman 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 



CHEC K APPROPRIATE BOX(ES) 

i ] (a) H uman 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: 



CHEC K 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 the stereocilia , 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 

1 - LCNSS 



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 

2 - LCNSS 



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. 



3 - LCNSS 



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. 

4 - LCNSS 



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 
A g in g. 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. 



6 - LCNSS 



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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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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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. 



Hi gh 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 

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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 

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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. 



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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: 



CHEC K APPROPRIATE BOX(ES) 

I x | (a) H uman 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. 

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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 

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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 



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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 

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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. 



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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: 



CHEC K APPROPRIATE BOX(ES) 

I x | (a) H uman 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 (x10 6 ) 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. 

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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 



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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 



24 - LCNSS 



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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. 



27 - LCNSS 



Z01 NS 00969-26 CNSS 

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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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 LENP 1 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 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

I | (a) H uman 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) H uman 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 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

I I (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.) 

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 (,tan y ) 

None 



LAB/BRANCH 

Laboratory of Experimental Neuropathology 



SECTION 

Cellular Neuropathology Section 



INSTITUTE AND LOCATION 

NINDS, NIH, Bethesda, MP 20892 



TOTAL MAN YEARS. 



PROFESSIONAL: 



OTHER: 



CHEC K 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 



CHEC K 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 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

J (a) H uman 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 



CHEC K APPROPRIATE BOX(ES) 

I | (a) H uman 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: 



CHEC K 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) 



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00 



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03 
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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 Mg 2 + 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 Mg 2+ is 
removed from the incubation medium. With Mg 2 + 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 Mg 2+ 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 Mg 2 + 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 Mg 2+ and glucose. Corticosterone appears to potentiate NMDA agonist 
toxicity by relieving the NMDA channel Mg 2+ 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 



CHEC K APPROPRIATE BOX(ES) 

J (a) H uman subjects 
] (a1) Minors 

J (a2) Interviews 



] (b) Human tissues (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 



CHEC K 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 



CHEC K 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: 



CHEC K 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 



CHEC K 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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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: 



CHEC K APPROPRIATE BOX(ES) 

I | (a) H uman 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 



CHEC K APPROPRIATE BOX(ES) 

O (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 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 transcriptiona l 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 ^ 



CHEC K 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 
i nterferon 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 



CHEC K APPROPRIATE BOX(ES) 

[ | (a) H uman 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 



CHEC K 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 AD8 7( M) a n d HIV-1 BaL ) or brain (HIV-1jr_fl). 
whereas T-cell adapted and HIV-2 strains did not replicate. Peak levels of RT activity (1-2x10 5 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 



CHEC K 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 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

] (a) H uman 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: 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

J (a) H uman 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 



CHEC K APPROPRIATE BOX(ES) 

I I (a) H uman 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 



CHEC K APPROPRIATE BOX(ES) 

] (a) H uman subjects 
] (a1) Minors 

J (a2) Interviews 



] (b) Human tissues (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 



CHEC K APPROPRIATE BOX(ES) 

I x | (a) H uman 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 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

1 1 (a) H uman 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 



CHEC K APPROPRIATE BOX(ES) 

I | (a) H uman 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 



CHEC K APPROPRIATE BOX(ES) 

J (a) H uman 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 



CHEC K 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) 



> 

w 

00 



CO 

o 

30 

> 

o 

30 

-< 



c 

30 

o 

03 



O 

-< 



ANNUAL REPORT 



October 1, 1989 through September 30, 1990 

Laboratory of Neurobiolog y 

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 (<10 3 electron/ nm 2 ), 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: 



CHEC K APPROPRIATE BOX(ES) 

] (a) H uman 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 



CHEC K 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: 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

LJ (a) Human subjects ] (b) Human tissues (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 



CHEC K APPROPRIATE BOX(ES) 

J (a) H uman 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 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

I I (a) H uman 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 



CHEC K 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 



CHEC K 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 (,fan f ) 

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 



CHEC K 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 



CHEC K 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 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

I (a) H uman subjects 
] (a1) Minors 

J (a2) Interviews 



] (b) Human tissues (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 



CHEC K 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: 



CHEC K 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). a ncl 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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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 45 Ca 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 45 Ca 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 4 5Ca 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 4 5Ca 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 4 5Ca 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 4 5Ca 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 45 Ca, 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 3 H-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 4 5Ca 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 



CHEC K 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.) 

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: 



CHEC K 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: 



CHEC K APPROPRIATE BOX(ES) 

I [ (a) H uman 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: 



CHEC K 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: 



CHEC K 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: _ 



CHEC K 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 



CHEC K 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 



CHEC K 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 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

I 1 (a) H uman 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 



CHEC K APPROPRIATE BOX(ES) 

] (a) H uman subjects 
] (a1) Minors 
] (a2) Interviews 



] (b) Human tissues (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 



CHEC K 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 
4 5uptake 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: 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

I 1 (a) H uman 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: 



CHEC K APPROPRIATE BOX(ES) 

1 | (a) H uman 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 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

] (a) H uman subjects 
] (a1) Minors 
J (a2) Interviews 



] (b) Human tissues (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 



CHEC K 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 



CHEC K 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: 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

I | (a) H uman 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 



CHEC K APPROPRIATE BOX(ES) 

J (a) H uman 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: 



CHEC K 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) 



> 

CO 



> 
o 

30 

> 

H 

O 

30 

< 



c 

O 

I 
-< 
V/1 



O 
O 

-< 



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 Ca 2 + -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 Ca 2+ 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 



CHEC K 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\ T P, 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: 



CHEC K 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: 



CHEC K 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: 



CHEC K 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: 



CHEC K 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: 



CHEC K 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 chi ck 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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CO 



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03 
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30 
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H 

o 

30 

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2 

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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, G s , 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-Cys 7 -Aoc 817 - 
Cys 20 ]pNPY, both competed for [ 125 I]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 G s and Gj 
whereas SMS-MSN cells contained G s , Gj and G - Whereas Gj is involved in coupling 
receptors to the inhibition of adenylate cyclase, G 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 G s 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 G s . 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 Ca 2 + -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 



CHEC K 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 G s 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 Y 2 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 



CHEC K 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) H uman 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 



CHEC K 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 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

[ I (a) H uman 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 GP120 will 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 



CHEC K 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 (lfi V ) 

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 



CHEC K 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 



CHEC K 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 



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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 Ceredase ra 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 

B F S B, nTR, NTN DS 



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. 



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 o f the EEG i n Febril e S e izur es 



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 



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 
afebril e 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.n ma 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 



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. NIH T 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 

Biome try 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 5 



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 
pregna ncy 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 , 19 89 th r o ugh S ept ember 30, 199 



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 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

| J (a) H uman 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 G m i 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: 



CHEC K APPROPRIATE BOX(ES) 

I I (a) H uman 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 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 



CHEC K APPROPRIATE BOX(ES) 

I I (a) H uman 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 



CHEC K APPROPRIATE BOX(ES) 

I x | (a) H uman 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 



CHEC K APPROPRIATE BOX(ES) 

1 x | (a) H uman 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 



CHEC K APPROPRIATE BOX(ES) 

J (a) H uman 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 (.( *n Y > 



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 



CHEC K APPROPRIATE BOX(ES) 

I « ] (a) H uman 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: 



CHEC K APPROPRIATE BOX(ES) 

] (a) H uman 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: 



CHEC K APPROPRIATE BOX(ES) 

I x I (a) H uman 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 



CHEC K APPROPRIATE BOX(ES) 

] (a) H uman 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: 



CHEC K APPROPRIATE BOX(ES) 

I [ (a) H uman 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 10 8 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 



CHEC K APPROPRIATE BOX(ES) 

] (a) H uman 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: 



CHEC K APPROPRIATE BOX(ES) 

] (a) H uman 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 p60 src and the gag 
polyproteins of a number of retroviruses including HIV. Interference with myristoylation has been shown 
to greatly reduce the transforming potential of p60 src 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 5 1 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 5 1 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 



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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 B m ax- * 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 B ma x 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 45ca 2+ 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: 



CHEC K APPROPRIATE BOX(ES) 

I I (a) H uman 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: 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

I I (a) H uman 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: 



CHEC K 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 [ 1 8F]-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 (NS 1 ), 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. 



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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 
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 



9-MNB/DIR 



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 



10-MNB/DIR 



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. 



11 -MNB/DIR 



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- 



12-MNB/DIR 



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 



13-MNB/DIR 



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 



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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. 



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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 



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[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. 



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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 



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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, 



26-MNB/DIR 



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 



27-MNB/DIR 



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 



28-MNB/DIR 



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 



29-MNB/DIR 



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 



30-MNB/DIR 



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 1 H 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-[ 1 8F]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-[ 1 8F]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: 



CHEC K APPROPRIATE BOX(ES) 

| x | (a) H uman 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: 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

I x | (a) H uman 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: 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

I x [ (a) H uman 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 



CHEC K 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 



CHEC K APPROPRIATE BOX(ES) 

I x | (a) H uman 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 Spe cialization 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 e pileptic 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 ) 

Co gnitive 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: 



CHEC K APPROPRIATE BOX(ES) 

1 | (a) H uman 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 anticonv