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
vii - DIR/NINDS
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73
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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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
CHECK APPROPRIATE BOX(ES)
QTj (a) Human subjects j ] (b) Human tissues I I (c) Neither
] (a1) Minors
I | (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Understanding of neuromuscular diseases is founded on careful clinical observation,
electrodiaqnostic studies and pathology. This protocol has been developed to learn
more about established diseases, to characterize new diseases, to assess current
methodologies and technologies, and to refine old methods and develop new ones.
Studies initiated underthis protocol include:
a. The effect of varying the detection threshold on interference pattern analysis in
healthy subjects patients with primary muscle disease.
b. The use of mixed nerve action potentials (MNAPs) in patients with chronic
inflammatory demyelinatinq neuropathy (CIDP), including the use of monopolar
recording to detect temporal dispersion.
c. The use of medial plantar nerve action potentials (NAPs) in patients with axonal
neuropathies.
PHS 6040 (Rev. 184)
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ANNUAL REPORT
October 1, 1989 - September 30, 1990
Instrumentation and Computer Section
Office of Director, Basic Neurosciences Program, DIR
National Institute of Neurological Disorders and Stroke
Table of Contents Page
ORGANIZATIONAL STRUCTURE AND SERVICES 1
INSTR. VENTATION 2-5
COMPUTER SUPPORT 5-10
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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
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published methodological papers on edge detection, calculation of fractal dimension, and the
application of these to the form of neuronal cells. Software has been produced for PDP-1 1 and
Macintosh computers. A paper using fractal analysis to study the development of glial cells has been
submitted to Neuroscience. Fourier Analysis: In collaboration with LNP and LNC, a method of
analyzing cell shape has been developed using a Fourier transform of the oudine of cells produced by
one of the edge detecting techniques. This method is now being applied to both neural and glial cell
images.
Analysis of Postural Dynamics. The same techniques described above are being applied to
the analysis of the trajectories of center of gravity in normal young, normal old, and Alzheimer
individuals. This work is in collaboration with a team from Marquette University and the Veterans
Hospital in Milwaukee, Wisconsin. Preliminary results will be reported at the Society for
Neuroscience meetings in 1990. They show that the fractal dimension of the trajectory of the center
of pressure on a plate on which a subject stands is quite different in the three classes of subjects. It
is hoped that this work will lead to a diagnostic tool in the early detection of conditions which
influence the dynamics of posture.
Extensions to Image. The fractal geometry and Fourier analysis techniques used in the
morphological classification of cells are also being incorporated into the Section's Image program.
This effort will enable an object outlined in Image to be analyzed by computing its fractal index or
by the Fourier technique. In addition, the fractal index of any image displayed on the screen may be
computed. A second, unrelated extension to Image involves a collaborative effort with LCB,
NIMH to control the motorized stage of a microscope. The objective is to enable Image to
automatically scan a brain section slide and, either automatically or through manual input, identify
and label cells in the section. The location of the selected cells will be stored for future analysis and a
plot of the image of the section with the cells identified will be generated.
Flow Cytometry Studies. A comprehensive collaborative effort is in progress with LNP,
NINDS which involves the use of voltage-sensitive dyes and flow cytometry techniques to study the
electrical and pharmacological properties of embryonic rat and chick CNS neurons, and various
clonal lines. Section effort is focused on experimental design, statistical and graphical analysis of
data, and the development of custom software. Experimental Design and Data Analysis: ICS has
been intimately involved in day-to-day design of experiments and analysis of data on specific
projects on development in rat spinal cord, in hippocampus and striatum, and in chick spinal cord.
New methods have been developed allowing for quantification of previously qualitative results on
the population responses to neural transmitters and ion channel agents. Software Development:
Programs have been developed for the smoothing, integrating, overlaying and statistical analysis of
distributions of optical data from the flow cytometer. These programs were developed to run on
PDP-11 computers; most of them have now been converted for use on LPN's new MicroVAX.
Publication: ICS is involved in writing and editing papers on this work especially as it applies to
methodology. In fiscal year 1990, papers have been published, or are in press, on rat spinal cord,
hippocampus and striatum, and a paper is in preparation on chick spinal cord. These will all appear
in Brain Research or Developmental Brain Research. They have all been reported at the 1989
Society for Neuroscience meetings and follow-up reports will be given at the 1990 meetings.
9 BNP/DIR (ICS)
Nonlinear Dynamics in Electrophysiology. The Section has begun to collaborate with
members of LNP, NINDS on the application of phase space analysis of the nonlinear dynamics of
cells (commonly referred to as chaos theory) as evidenced by transmembrane voltage measurements.
Programs originally developed at Bryn Mawr College for the IBM PC have been rewritten and are
now in use on the Section's MicroVAX. The application so far has been on research of voltage
recordings in chick cells exposed to agents which modify lipid metabolism. Preliminary results will
be reported this fall at the Society for Neuroscience meetings and at a symposium on mathematical
methods in medicine in Prague.
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ANNUAL REPORT
October 1, 1989 through September 30, 1990
ANIMAL HEALTH AND CARE SECTION, OP, DIR
National Institute of Neurological and Disorders and Stroke
Table of Contents
Summary Report 1
Major Tasks and Accomplishments 4
Major Goals for FY 91 6
ANNUAL REPORT
ANIMAL HEALTH AND CARE SECTION, OP, DIR
National Institute of Neurological Disorders and Stroke
October 1, 1989 through September 30, 1990
E. Christopher Staley, D.V.M., Chief
The Animal Health and Care (AHC) Section is responsible for providing a full-range
of laboratory animal support to investigators in the NINDS, Division of Intramural
Research Included in this responsibility are: (1) animal procurement/supply, (2)
subject conditioning, (3) the provision of routine and emergency veterinary care, (4)
provision of technical support (e.g., monitoring anesthesia, sample collection, etc.),
(5) quarantine of new or infectious animals, and (6) administration of an animal
quality assurance/preventative medicine proqram
The AHCS professional staff also serves in an advisory capacity in planning studies
involving the use of animals. All animal use must be in compliance with the
regulations promulgated by the USDA to enforce the Animal Welfare Act, the NIH
Guide for the Care and Use of Laboratory Animals, PHS Policy on Humane Care and
Use of Laboratory Animals, and the applicable chapters of the NIH Manual.
Guidance and training are provided to investigators and technicians regarding
model and subject selection, and animal handling, as well as inoculation, sampling,
and surgical techniques, etc. In additon, the Section's professional staff performs
collaborative research involving animal model development, refinement of
techniques, and improvement of animal husbandry.
The Chief serves as the Institute Veterinarian and as Executive Secretary of the NINDS
Animal Care and Use Committee (ACUC). In this capacity, he utilizes his expertise to
design and develop the NINDS Animal Care and Use Program in accordance with all
applicable regulations, and to advise and guide the Committee in the
implementation of the Institute's goals for animal care and use practices in
conjunction with the functions of the Committee. The ACUC is responsible for: (1)
review and approval of all Animal Study Proposals; (2) maintenance of protocol files;
(3) investigation of allegations regarding regulation violations involving the care,
use and handling of animals, (4) performance of a semi-annual review of the NINDS
Animal Care and Use Program, and (5) publication of Guidelines and Policies
regarding the use of animals in the DIR.
Within the AHC Section, the veterinarians, laboratory animal technicians, and animal
caretakers are organized as three service units: Primate Unit, Small Animal Unit, and
Building 376 Unit. The Primate Unit is responsible for the support of DIR
investigators utilizing primates on approved research protocols in NINDS facilities in
Buildings 36, 9, and 14on the Bethesda Campus, and at the NIH Animal Center in
Poolesville, MD. The Small Animal Unit provides support to NINDS investigators
utilizing rabbits, rodents, amphibians and othersmall animal species within
Buildings 9,10, and 36 on the Bethesda Campus, and the Park 5 Building in Rockville,
MD. The Small Animal Unit Veterinarian also serves as the Facility Veterinarian of
the Building 36 Combined Animal Facility, which provides housing and care to
investigators from NINDS, NIMH, NHLBI, NICHD, and NIDCD under an intraagency
agreement. The Building 376 Unit is located within the NINDS facility in Building
376, Ft. Detrick, Frederick, MD. Its personnel provide primate care and research
support to NINDS investigators within the facility, and the Facility
Veterinarian serves as the contract monitor for the Program Resources, Inc. small
animal care/technical support contract.
In order to facilitate the supply of animals and animal tissues to investigators within
the DIR, the AHC Section maintains breeding colonies of primates and rodents.
These colonies are located within NINDS facilities in Bethesda, Frederick, and
Poolesville, as well as at off-campus contract sites. The Chief, AHCS serves as the
Project Officer for the breeding contracts as well as a co-Project Officer for all
intramural contracts involving the care, housing, and use of intramurally-owned
animals.
The Institute Veterinarian and the AHCS also provide veterinary support to the
National Institute of Deafness and Other Communication Disorders (NIDCD) under
an intraagency agreementsigned in October, 1989, following the formation of the
NIDCD, primarily from elements of the NINDS.
Major tasks undertaken by the Animal Health and Care Section this Fiscal Year:
I. Assumption of responsibility for animal care/research support in Building 376.
Prior to this fiscal year, the caretakers and attending veterinarian in Building 376
were assigned to the LCNSS. Effective October 1, 1989, all animal care personnel
were reassigned to the AHCS.
II. Renovation of NINDS animal area in Building 10 (ACRF Tower).
The NINDS animal facility in the ACRF Tower was vacated for three months, and
extensive repairs/renovations were made to the floors, air handling system, and
lights. The renovations were successfully completed in August 1990, and animals
have been returned to the facility.
III. Redesign and renovation of Building 36/5C containment area.
The Building 36/5C infectious disease containment area was redesigned to
eliminate the use of Horsfall isolation units. The new design involves the use of
state-of-the-art Lexan cubicles and incorporates many environmental enrichment
features. This renovation will continue into FY91.
IV. Establishment of a clinical diagnostic program with quality control.
A program to support investigators with clinical pathology/diagnostic laboratory
services was developed and initiated. Included in the capabilities of the Section's
laboratory are performance of routine and emergency complete blood counts
(CBC) with or without differential cell counts, a complete panel of serum
chemistry tests, blood gas determinations, etc.
V. Establishment of a recycling program for non-human primates.
In an effort to conserve NINDS primates, and ensure their availability to NINDS
investigators, a central service has been established to recycle primates from non-
terminal studies to other investigators within the NINDS and other Institutes.
VI. Poolesville AIDS Facility Task Force
The Chief, AHCS serves as the Chairman of the Poolesville AIDS Facility Task Force,
responsible for establishing programmatic requirements, evaluating design
alternatives, etc., for a facility that will house NINDS and N I AID non-human
primate on AIDS or AIDS - related studies.
VII. Collaborations Undertaken
A. BMAA Neurotoxin evaluation
Dr. Barton G. Weick (AHCS) collaboration
with Mark Duncan (CNB) February 1990 to July 1990
B. Refinement of Audiogenic seizure model
Dr. Lowrey Rhodes Jr., and Dr. Lisa T. Pegues (AHCS)
collaboration with Dr. Yamaguchi (MNB). This
collaboration is expected to extend into FY91 .
C. Dietary Control of Cholesterol in Lowland Gorillas
Dr. E. C. Staley and Dr. Lowrey Rhodes (AHCS)
collaboration with Dr. C. J. Gibbs (LCNSS) and
Dr. Scott Citano (Miami Metrozoo)
This collaboration is expected to continue into FY91
D. Evaluation of immune response in Mycobacterium Tuberculosis sensitized
monkeys (Macaca mulatta) following exposure to each three measles vaccines.
Dr. E. C. Staley (AHCS) collaboration with Dr. Janice
Southers (VRP, NCRR) and Dr C. O. Thoen (Iowa State Univ.).
This collaboration is expected to continue into FY91
Major Goals for FY91
I. Attain AAALAC accreditation for NINDS Animal Care and Use Program
Included in this goal are the completion of all facility renovations/repairs,
program evaluation and updating, etc.
II. Continue collaborative efforts and emphasize Animal Health and Care Section
involvement in animal model development.
III. Initiation of NINDS wide program for the provision of environmental
enrichment/exercise for non human primates utilized by NINDS investigators.
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ANNUAL REPORT
October 1, 1989 through September 30. 1990
Laboratory of Biophysics
Basic Neurosciences Program. DIR
National Institute of Neurological Disorders and Stroke
Gerald Ehrenstein. Ph.D., Acting Chief
Table of Contents
RESEARCH SUMMARY 1
PROJECT REPORTS
Function and Structure of Membrane Ionic Channels 4
Z01 NS 02088-16 LB
The Physiological Role of Microglia in the Brain 5
Z01 NS 02218-15 LB
Gated Ionic Channels in Membranes 6
Z01 NS 02526-09 LB
Calcium Channels in Vertebrate Nerve Terminals 7
Z01 NS 02606-07 LB
Comparative Aspects of Ionic Conductances in 8
in Nerve and Heart Cell Membranes
Z01 NS 02608-07 LB
Mechanism of Egg Activation Following Fertilization 9
Z01 NS 02609-07 LB
Secretion of Neurotransmitters and Hormones 10
Z01 NS 02709-05 LB
Electro-Mechanical Transduction Mechanism in 1 1
Outer Hair Cells
Z01 NS 02799-02 LB
i - LB/DIR
Annual Report
October 1 , 1 989 through September 30, 1 990
Laboratory of Biophysics
National Institute of Neurological Disorders and Stroke
Gerald Ehrenstein, Ph.D., Acting Chief
The Laboratory of Biophysics pioneered the study of ionic channels, their voltage
dependence, and their relation to experimentally observed ionic currents. Our present
research emphasizes the way in which channel properties relate to important physio-
logical processes. Of particular interest in this effort are calcium channels, calcium-
activated channels, and pressure-sensitive channels. Among the physiological processes
we are studying are transmitter release by exocytosis, control of the firing pattern in
heart cells, the increase in intracellular calcium concentration in eggs following
fertilization, and the fine-tuning of audio reception.
The small size and inaccessibility of vertebrate presynaptic nerve terminals has
posed a major problem for the study of transmitter release. In order to examine with
direct recording techniques the physiological events that underlie this process in a
vertebrate system, we have developed a calyx-type preparation from the chick ciliary
ganglion. In this ganglion, the presynaptic terminal is apposed to the postsynaptic cell
over a relatively large area. If this terminal were dissociated from the surrounding
tissue, a large area of the presynaptic cell would be exposed. We have found an enzyme
treatment that allows us to dissociate the ganglia and to obtain single ciliary neurons
with attached calyces. With this treatment, standard patch-clamp techniques can be used
to study the channels present in the calyces.
Using this approach, we have characterized the presynaptic calcium channels. The
channels activate and deactivate very rapidly; they have little inactivation, even after
prolonged depolarization; and they are blocked by cadmium and by w-conotoxin, but not
by nifedipine. The pharmacology of the channels is consistent with the pharmacology of
synaptic transmission through the calyx-synapse, as evaluated by extracellular
recording from the intact ganglion. Thus, the presynaptic calcium channels that we have
observed in this fast-transmitting vertebrate synapse have both physiological and
pharmacological properties consistent with their putative role as triggers of acetyl-
choline release. Interestingly, they do not fit neatly into any of the previously described
categories.
Fertilization is another process where an increase in the cellular calcium
concentration causes a number of important reactions. A lot is known about the
biochemistry of these reactions, but the primary messenger that triggers activation
(the increase in the internal calcium concentration of the fertilized egg) is unknown.
1 - LB/DIR
We have used sea urchin gametes, standard preparations for studying fertilization, to
determine the identity of the primary messenger in sperm that triggers activation of the
egg. We now have strong evidence that the primary messenger is inositol trisphosphate
and that it acts on the internal surface of the egg. It had previously been known that the
injection of inositol trisphosphate into the egg would activate it, but it was generally
thought that the inositol trisphosphate acted as a second messenger. Our measurement of
inositol trisphosphate in sea urchin sperm, however, indicates an exceptionally high
concentration (about a hundred-fold higher than in eggs). Furthermore, there is
evidence that during the acrosome reaction, the amount of inositol trisphosphate in
sperm increases several-fold. Taken together, these results indicate that the quantity of
inositol trisphosphate that is brought into the egg by a single spermatozoon is compar-
able to the quantity that has been shown to trigger activation by injection. The use of
inositol trisphosphate as a primary messenger in fertilization may be very general, as
evidenced by our observation that the concentration of inositol trisphosphate in goat
sperm is comparable to that in sea urchin sperm.
We also have independent evidence that activation occurs at the internal surface of
the egg. Eggs were placed in a solution containing an extract of sperm, and no activation
was observed. When the eggs were electrically permeabilized so that the extract could
enter the egg, activation was observed.
A key issue involving internal calcium is determination of the mechanism for
calcium-dependent exocytosis. We have previously addressed this issue both experi-
mentally and theoretically, and have now obtained additional support for a model that
assigns a central role to calcium-activated ion channels. In our model, a calcium-
activated cation or anion channel and a calcium-independent channel that is permeable to
ions of the opposite sign are located in the membrane of a secretory vesicle in the region
where the vesicle is apposed to the plasma membrane. An increase in intracellular
calcium causes the calcium-activated channel to open, resulting in a flow of both cations
and anions into the vesicle. This ionic flow reduces the salt concentration in the space
between the vesicle membrane and the plasma membrane, leading to a decrease in the
osmolarity of the space. The decreased osmolarity causes a flow of water out of the
space, allowing the two apposed membranes to move closer together. It is this moving
together of the two apposed membranes that triggers membrane fusion and exocytosis.
Previous experiments in several types of cells have shown a very good correlation
between the calcium dose-response curve for opening of calcium-activated ion channels
and the calcium dose-response curve for secretion, consistent with our model for
exocytosis. In addition, the model requires that secretory vesicles containing calcium-
activated cation channels also contain calcium-independent anion channels and that
secretory vesicles containing calcium-activated anion channels also contain calcium-
independent cation channels. We are measuring the properties of channels in secretory
vesicles of the bovine neurohypophysis by reconstituting the channels into lipid
bilayers; our preliminary measurements are consistent with this requirement of the
model.
2-LB/DIR
We are studying the secretory process from a different point of view in microglia,
where we are particularly interested in comparing microglia secretion in trisomy 16
mice with microglia secretion in normal littermates. (Trisomy 16 mice serve as an
animal model for Down's syndrome, which is caused by trisomy 21 in humans.) We
previously found that secretion of the superoxide radical from microglia of trisomy 16
mice is very much larger than that from microglia of normal littermates subjected to
the same chemical stimuli. Since neurons and synapses can be functionally damaged by
oxyradicals, it is possible that overproduction of these radicals in trisomy 16 mice is
responsible for the observed impairment of their central nervous system. We are now
investigating possible causes of the enhanced secretion of superoxide in the trisomy 16
mice. We found that alpha interferon increases the production of interleukin-1 in both
normal and trisomy 16 mice, but that the effect is significantly larger for trisomy 16
mice. This raises the possibility that enhanced secretion of superoxide might be caused,
at least in part, by enhanced production of interleukin-1. Alternatively, enhanced
production of interleukin-1 might stimulate other reactions, such as gliosis, that could
lead to impairment of the central nervous system.
Both L-type and T-type calcium channels have been found in heart cells. The L-type
calcium channel, which is activated in the -40 to -30 mV range, underlies the plateau
phase of the cardiac action potential in the myocardium. The T-type calcium channel,
which is activated in the -70 to -60 mV range, is involved in diastolic depolarization of
the s-a node, the primary pacemaker of the heart. We have found both components in
embryonic chick heart cells during the first week of embryonic development. During
the second week of development, however, the T-type channels are no longer found in
atrial or ventricular heart cells. This suggests that both types of channels are present
in precursor cells, but that T-type channels are needed only in pacemaker cells. These
channels may be unnecessary or even undesirable in other heart cells, and hence may be
lost during development.
In our study of electromechanical transduction in outer hair cells from the organ of
Corti, we have previously shown that the membrane potential of these cells regulates
cellular movement. Also, it is known that hyperpolarization of these cells results in
elongation. We have now used the patch-clamp method to search for ion channels in
outer hair cells, and have found stretch-activated potassium channels in the lateral wall.
Opening of these channels would hyperpolarize the cells, causing elongation, and elonga-
tion, in turn, would stretch the membranes, opening more stretch-activated channels.
Thus, the stretch-activated potassium channels may be important elements in a positive
feedback system providing efficient electromechanical transduction.
3-LB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02088- 17 LB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (flO characters or less. Title must fit on one line between the borders.)
Function and Structure of Membrane Ionic Channels
PRINCIPAL INVESTIGATOR iUst other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: G. Ehrenstein, Ph.D. Research Physicist LB, NINDS
Others. K Iwasa, Ph.D. Special Expert LB, NINDS
N. Moran, Ph.D. Visiting Associate LB, NINDS
COOPERATING UNITS (if any)
Weed Science Laboratory- AEQI, Dept. of Agriculture, Beltsville, MD(C. Baireand C. Mischke)
University of Connecticut (R. Satter)
LAB/BRANCH
Laboratory of Biophysics, BNP, DlR
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [x~l (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
*This project has been terminated.
4-LB/DIR
PHSbMO(Hev I/V4)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
201 NS 02218-15 LB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (fiO characters or less title must fit on one line between the borders.)
The Physiological Role of Microglia in the Brain*
PRINCIPAL INVESTIGATOR (IJSf other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
PI: Daniel L.Gilbert, Ph.D. Research Physiologist NINDS, LB
Others: Liana Harvath, PhD
Microbiologist
CBER, DBBP
COOPERATING UNITS Visny)
Georgetown University, Washington, DC (C. Colton, J. Yao, J. Keri)
John Hopkins University, Baltimore, MD (ML. Oster-Granite)
LAB/BRANCH
Laboratory of Biophysics, BNP, DIR
SECTION
INSTITUTE AND LOCATION
NINDS, NlH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.2
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
] (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues [IT] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Experiments have been performed on microglia, the resident macrophage in the cen-
tral nervous system cultured from cerebral cortices of normal and trisomy 16 (Ts16)
mice. The Ts16 mouse serves as an animal model of humans afflicted with Down's syn-
drome (trisomy 21). We found that interferon (alfa IFN), a cytokine, increases the sensi-
tivity of phorbol myristate acetate (PMA) on microglial production of the superoxide
radical anion and that IFN also increases the production of interleukm-1 (IL-1), another
cytokine. The increase in production of IL-1 is larger for the Ts1 6 mouse than for nor-
mal controls.
*Formerly: "Effect of Drugs on Voltage-Dependent Ionic Conductance in Membranes."
5-LB/DIR
PHS6O40(R*v. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02526-09 LB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (fitKharacrtrs or less. Title must fit on one line between the borders.)
Gated Ionic Channels in Membranes
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: E.F.Stanley, Ph.D. Staff Physiologist LB, NINDS
Other: A. Atrakchi, Ph.D. Visiting Fellow LB, NINDS
COOPERATING UNITS Of an,)
LNN, NICHD
LAB/BRANCH
Laboratory of Biophysics, BNP, DIR
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects ] (b) Human tissues QT] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
*This project has been terminated.
6-LB/DIR
PHi 6040 (Rev. l 1)1)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02606-07 LB
PERIODCOVEREO
Octoberl, 1989 through September 30, 1990
TITLE OF PROJECT (SO characters or less. Iitle must fit on one line between the border! )
Calcium Channels in Vertebrate Nerve Terminals*
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
PI: E.F.Stanley, PhD Staff Physiologist LB, NINDS
Other: G Goping Technician LCBG, NIDDK
COOPERATING UNITS (H any)
LAB/BRANCH
Laboratory of Biophysics, BNP, DIR
SECTION
INSTITUTE AND LOCATION
NINDS.NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL: 1 1
OTHER:
CHECK APPROPRIATE BOX(ES)
Q (a) Human subjects □ (b) Human tissues [T] (c) Neither
] (a1) Minors
] (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Calcium-dependent transmitter release from presynaptic nerve terminals is involved in
virtually all functions of neurons. We have used the cholinergic calyx-type synapse of
the chick ciliary ganglion to develop a unique experimental preparation in which it is
possible to apply voltage clamp techniques to the presynaptic nerve terminal. We have
used this technique to characterize presynaptic calcium channels by recording the
voltage-dependent calcium current The presynaptic calcium channels are
dihydropyridine blocker-insensitive, u-conotoxin-sensitive but voltage-dependent-
inactivation resistant. These channels may represent a type that is unique to fast
transmitting nerve terminals.
*Formerly: "Chemical Transmission at the Squid Giant Synapse"
7 - LB/DIR
PHSt040(Kev. I M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02608-07 LB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. ritle must fit on one line between the borders.)
Comparative Aspects of Ionic Conductances in Nerve and Heart Cell Membranes
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: J.R.Clay, Ph.D. Research Physicist LB, NINDS
Others: V. Kowtha, Ph.D. NRC Fellow LB, NINDS
COOPERATING UNITS (if an,)
McGill University (A. Shrier)
LAB/BRANCH
Laboratory of Biophysics, BNP, DIR
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL: y Q
OTHER: q 1
CHECK APPROPRIATE BOX(ES)
□ (a)Human subjects ] (b) Human tissues [x~| (c) Neither
J (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project is concerned with a comparative analysis of ionic current channels in nerve
and heart cell membranes and the relationship of these channelsto electrical excitabil-
ity in both preparations. During the past year the primary experimental preparations
have been squid giant axons and embryonic chick heart cells. One major finding has
been the discovery of Wenkebach firing patterns in the squid axon in response to peri-
odic current pulse stimulation. This phenomenon, which has not previously been char-
acterized in this preparation, may be relevant to cardiac arrhythmias. In the chick heart
cell preparation, we have discovered a developmental loss of a particular type of cal-
cium ion channel, the It component, during the first week of embryonic development
from atrial and ventricular heart cells. This project also has a major emphasis on com-
puter simulations of electrical activity in nerve and heart cells. A major advance in this
part of the project is the development of a model of the action potential in guinea-pig
ventricular myocytes based on experimental measurements of underlying ion current
components in the literature
8-LB/DIR
PHi 6040 (Rev 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02609-07 LB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 character* or /ess. Title must fit on one line between the borders.)
Mechanism of Egg Activation Following Fertilization
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
PI: K H. Iwasa, Ph.D. Special Expert LB, NINDS
Others: G. Ehrenstein, PhD
J.T.Russell, Ph.D.
Research Physicist
Research Chemist
LB, NINDS
LNN, NICHD
COOPERATING UNITS litany)
Emory University, Atlanta, GA ( L. J. DeFelice)
LAB/BRANCH
Laboratory of Biophysics, BNP, DIR
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 10892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER
CHECK APPROPRIATE BOX(ES)
] (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues [x] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
We have previously demonstrated that a fertilization membrane forms around a sea
urchin egg when it is injected with a soluble spermatozoa fraction isosmotic with
seawater. We found that a spermatozoon contains inositol trisphosphate at such a
high level as to trigger calcium elevation leading to to the exocytosis of the cortical
granules and the elevation of the fertilization membrane. We observed a similar
amount of inositol triphosphate in goat sperm. Thus, inositol triphosphate plays the
role not only of a second messenger within eggs as in other cells but of the primary
messenger from spermatozoa to eggs at fertilization.
9-LB/DIR
PHS 6040 (Rev. 184)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02709-05 LB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (BO characters or less. Title must fit on one line between the borders.)
Secretion of Neurotransmitters and Hormones
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: G. Ehrenstein, Ph.D. Research Physicist LB, NINDS
Others: K. Krebs, Ph.D.
S. Pocotte, Ph.D.
E. F. Stanley, Ph. D.
Staff Fellow
Staff Fellow
Research Physiologist
LB, NINDS
LB, NINDS
LB, NINDS
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Biophysics, BNP, DIR
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
3.2
PROFESSIONAL:
2.9
OTHER:
0.3
CHECK APPROPRIATE BOX(ES)
i ] (a) Human subjects
; ] (a1) Minors
J (a2) Interviews
J (b) Human tissues j J (c) Neither
Summary OF WORK (use standard unreduced type. Do not exceed the space provided.)
In our model for calcium-dependent secretion of hormones and neu rotransm itters, an important
function of calcium is to open calcium-activated ion channels present in secretory vesicles. The opening
of these channels would allow the transport of ions from the intermembrane space between the plasma
membrane and the secretory vesicle membrane into the secretory vesicle. The resultant osmotic imbal-
ance would remove water from the intermembrane space, allowing the two membranes to move closer
together and fuse.
According to the model, every secretory granule for calcium-dependent secretion should contain
some type of calcium-activated channel. In order to test this, we are currently reconstituting channels
from vesicles of the bovine neurohypophysis into lipid bilayers. Preliminary results indicate that the vesi-
cles contain calcium-activated anion channels.
Another approach involves measurement of the secretion properties of bovine parathyroid cells.
These cells have a unique calcium dose-response curve for secretion: secretion decreases when calcium
concentration increases. We have previously examined the vesicle channel properties and the secretion
properties of these cells, and found a good correlation, as predicted by our hypothesis. As a further test,
we are now trying to block ionic flow through these channels and to determine whether this will block
secretion.
10-LB/DIR
PHS6M0(Rev. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02799-02 LB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (so characters or less Title must fit on one line between the borders.)
Electro-Mechanical Transduction Mechanism in Outer Hair Cells
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
PI: K. H. Iwasa, Ph.D. Special Expert LB, NINDS
Others: M.-XLI, MD
M.Jia,MD
B. Kachar, MD
Visiting Scientist
Visiting Scientist
Visiting Scientist
LB, NINDS
LB, NINDS
LMO, NIDCD
COOPERATING UNITS Of an,)
LAB/BRANCH
Laboratory of Biophysics, BNP, DlR
SECTION
INSTITUTE AND LOCATION
NINDS.Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
J (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues QT] (c) Neither
Summary OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The fast mechanical response is believed to be the cellular basis of the positive
feedback mechanism required for the fine tuning process of the hearing organ. Using
an externally applied electric field with and without membrane permeabilizing agents,
we demonstrated that the fast mechanical response of the outer hair cell is membrane-
potential dependent. To clarify further how a change in the membrane potential
causes mechanical displacement of the cell, we examined the role of the calcium ion in
the fast response. The result indicated that the calcium ion is a modifier and not a
messenger. We found stretch-activated potassium channels in the lateral wall,
indicating that the lateral wall, as well as thestereocilia, can act as a mechanoreceptor.
11-LB/DIR
PHSM40 (Rev. 1/84)
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ANNUAL REPORT
October 1, 1989 through September 30, 1990
Laboratory of Central Nervous System Studies
National Institute of Neurological Disorders and Stroke
Table of Contents
RESEARCH SUMMARY 1 - 13
PROJECT REPORTS
Neurobiology of Population Isolates: Study of Child Growth, 14- 18
Development, Behavior and Learning, and Disease Patterns in
Isolated and Primitive Groups
Z01 NS 01282-26 CNSS (5 Subprojects)
Chronic Central Nervous System Disease Studies: Slow, 19 - 28
Latent and Temperate Virus Infections
Z01 NS 00969-26 CNSS (16 Subprojects)
PUBLICATIONS 29 - 33
CONTRACTS 34
Z01 NS 01282-26 and Z01 NS 00969-26
ANNUAL REPORT
Laboratory of Central Nervous System Studies
October 1, 1989 - September 30, 1990
D. Carleton Gajdusek, M.D.
The elucidation of the pathogenesis of dementing brain amyloidoses, both the transmissible
type (such as Kuru-Creutzfeldt-Jakob Disease (CJD)-Gerstmann-Straussler syndrome (GSS)-Scrapie,
and bovine spongiform encephalopathy (BSE), caused by slow, unconventional viruses) and the
nontransmissible type (such as normal aging, Alzheimer's disease, and Down's syndrome) continues to
be our primary area of inquiry. Our program now revolves around the heretic pardigm of genetic
controls of de novo generation of infectious amyloid proteins from host precursors in kuru, CJD, GSS,
scrapie and BSE.
Our repeated assertions that the unconventional viruses contain no nonhost proteins and were
replicating polypeptides now appear to be vindicated by much more work from our own and many
other laboratories. It also appears that the scrapie amyloid precursor protein is converted to an
infectious form by configurational changes in the secondary and tertiary structure of the normal scrapie
precursor protein (SPP). On inoculation of susceptible hosts, the scrapie monomer autonucleates and
autopatterns this conversion of the normal, noninfectious host SPP to the infectious form. We also
believe that most sporadic cases of CJD arise by de novo spontaneous conversion of the normal SPP to
the infectious form, a rare event occurring at the frequency of one per million persons per year (the
annual incidence of CJD throughout the world). In the familial forms of CJD and GSS where the
occurrence is an autosomal dominant trait, we have found that each family has one of four different
mutations, three causing a single amino acid change and the other an insertion of an octapeptide
repeat (6 x 8 = 48 amino acids). Each mutation causes a million-fold increased probability of the
spontaneous configurational change to an infectious polypeptide, and appears as an autosomal
dominant trait. Thus, the behavior of the transmissible cerebral amyloidosis parallels completely
that of the transthretin amyloidoses causing familial amyloidotic polyneuropathy, in which there
are 15 different point mutations, each one of which increases enormously the likelihood of
configurational change of pre-albumin to an amyloid in several dozen different families.
The remarkable swift discoveries, all in our laboratory, that a point mutation in codon 200
causing a glutamic acid to lysine replacement is associated with all cases of CJD in the two high
incidence foci of CJD in the Orava and Lucenec regions of Slovakia has been followed by our
demonstration that this same point mutation is linked to CJD cases in Eastern Europe from Poland
through Greece, and even in cases in the United States and in South Americans of Eastern European
Slavic origin. This has led to our quickly discovering that this same point mutation underlies the
high incidence of CJD in Sephardic Jews of Libyan origin, both in immigrants to Israel and Israeli-
born. Furthermore, we have found this same mutation in Sephardic Jews with CJD from Tunis and
Greece and thus, it is a circum-Mediterranean Jewish trait as well as in Eastern European Slavs. Since
the Sephardic Jews moved across Northern Africa to the Iberian Peninsula, and, in the 15th century,
from Spain to Greece, this codon 200 mutation may prove to be a marker of the "Wandering Jew of the
Diaspora". We are investigating CJD cases from Spain.
After six years of incubation, we have finally had transmission of one of these Sephardic
Jewish CJD cases to a chimpanzee. Obviously the chimpanzee scrapie precursor gene and protein do
not carry this point mutation into their infectious isoform.
The possibility that synthetic polypeptide of the sequence of the SPP with the codon 200
glutamic acid replacement by lysine might itself be infectious and serve to nucleate the autopatterned
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configurational change in the host precursor protein to its infectious form is being investigated by
inoculation of many species with such synthetic polypeptide. The codon 102 mutation replacing a
proline with a leucine in synthetic polypeptides is also being tested for infectivity as is also the 48
amino acid insert mutation which augments the 5 copies of the octapeptide repeat in the normal
scrapie procurser protein to 11 copies. This perhaps naive oversimplification is nevertheless so
important if it were true that it is worth the years of waiting for long incubation periods in the
animals and certainly a justified experiment.
We are also investigating the possible infectiousness of cloned copies of the human scrapie
amyloid containing these mutation in Baculovirus.
Our concept that the formation of brain amyloid from a normal host precursor protein underlies
the pathogenesis of Alzheimer's disease (AD) has provoked considerable molecular study of brain
amyloid during the past three years. We were the first to characterize the gene for this aging brain
amyloid precursor protein (fj-protein; A4 protein), to locate it on chromosome 21 of man and 16 of mouse,
and to show its high evolutionary conservation. This gene is identical to that for an excreted rapidly-
turned-over protein which specifically binds to the gamma-subunit of nerve growth factor and many
other serine proteases and cell modulators which contain such serine protease as binding regions. This
important negative feedback control loop may explain the rapid synthesis, short half-life and wide
distribution in neurons of this brain amyloid precursor. Our work showing that the gene was expressed
in several alternatively spliced forms, with and without a 57 bp or a 76 bp insert which specify a
serine protease inhibitor, now fits well with the identity of protease nexin II and the alternatively
spliced forms of our amyloid precursor protein containing the serine protease inserts.
Our studies of mRNA expression in different brain cells of normal juveniles, aging brain and
AD brains have revealed that all neurons which develop neurofibrillary tangles (NFT) and are most
vulnerable to loss in aging and in AD carry a very high level of turned-on message. Not all cells with
high levels of amyloid fi-protein mRNA develop NFT, and thus its high expression appears to be a
necessary, but not a sufficient, condition for NFT formation. Interestingly, in Guamanian amyotrophic
lateral sclerosis and Parkinsionsim-dementia (ALS/PD) typical NFT appear in large motor neurons
which contain a high, up-regulated message. We have also studied the regulation of mRNA and the
precursor protein expression in hippocampal neurons and in endothelial cells in vitro. Thus, continued
molecular and cell biological studies of brain amyloid p-protein biosynthesis, processing and
regulation will surely continue to dominate research on AD and aging brain for some time.
The discovery that the subunit of aging brain amyloid in AD and Down's syndrome had no
amino acid homology with the much larger subunit of scrapie-kuru-CJD amyloid in scrapie-associated
fibrils (SAFs) or kuru-plaques led to the clear differentiation of transmissible from non-transmissible
cerebral amyloidoses. Scrapie and the transmissible dementias (kuru, CJD and its GSS variant) form
the amyloid of SAF and scrapie or kuru plaques from a proteolytically cleaved portion of the larger
infectious form of the scrapie amyloid precursor protein. The genes for aging brain amyloid precursor
and for the scrapie amyloid precursor show no sequence homology and the amyloidogenic subunits
cleaved from the full-length precursors are extremely different. The subunit protein for the
nontransmissible brain amyloidoses of aging and AD is a polypeptide of 4.1 kDa (42 amino acids) in
size and is nonglycosylated; that from scrapie-kuru-CJD is 27 kDa in size and has two glycosylated
sites. The genes for aging brain amyloid precursor is on chromosome 21 in man and for the scrapie
amyloid precursor on chromosome 20. (In mice they are on chromosome 16 and 2, respectively).
We have established a multifaceted research program to investigate the pathogenetic
mechanisms underlying the brain amyloidoses. These studies include in vitro study of amyloid fibrils
formation from synthetic polypeptides. Using the unique resource of our tissue bank of specimens from
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experimental animals we have initiated studies on the structure of the amyloid protein in different
strains of virus from the same host. Virus properties of pathogenesis, incubation time and host range
must depend on secondary and tertiary corifigurational changes in a host precursor if no pleomorphism
in amino acid sequence can be shown between virus strains from a single breed of host.
Recent experimental data from our laboratory confirms our hypothesis that the ALS/PD in
high-incidence foci in the Western Pacific is caused by a pathogenetic elemental deposition in the
central nervous system (CNS)which results from severe calcium and magnesium deficiency. ALS-like
neurofibrillary tangles have been produced in cynomolgus monkeys and rabbits and in vitro in motor
neurons by chronic aluminum toxicity. This confirmation of the mineral deposit etiology in Guamanian
ALS/PD has stimulated a renewed worldwide interest in these high incidence foci in the Western
Pacific. Interestingly, we have never been able to give serious credence to extensive claims that cycad
toxicity was involved in the high incidence ALS/PD foci. This skepticism is now vindicated.
Research work on acquired immunodeficiency syndrome (AIDS) and related topics continues to
occupy a significant portion of our resources. This includes studies on pediatric and adult encephalitis
caused by human immunodeficiency virus (HIV), a major cause of encephalitic death in the United
States today. We also are conducting studies in nonhuman primates to develop an experimental
animal model for AIDS, and to evaluate antibody response produced by potential vaccines against
HIV infection.
Our research on other human retroviruses, particularly human T-cell leukemia /lymphoma
virus type I (HTLV-I), increases in depth and scope. This includes intensive investigation of an
encephalomyelopathic syndrome called, variously, tropical spastic paraparesis, Jamaican
neuropathy, Pacific spastic paraparesis, and HTLV-I-associated myelopathy. Our virologic,
immunologic, clinical, and epidemiologic research points to HTLV-I as the primary cause of this
syndrome, which occurs in high-incidence foci of HTLV-I infection worldwide.
Our work on the hantaviruses, causative agents of hemorrhagic fever with renal syndrome,
continues as a worldwide collaborative effort, involving groups in Europe and Asia. This year,
however, we have concentrated on finding domestic manifestations of hantavirus infection, and on
developing experimental models of Prospect Hill and Puumala virus infection in primates.
We continue our studies on the mechanism of language acquisition, including the naturalistic
observation of extreme polylinguality. Our comparative inquiries on widely divergent styles of
psychosexual development in children from diverse cultural milieus continue to yield new data on
neurological programming which departs from "normal" behavior much further than previously
imagined by most psychiatrists, psychologists, sociologists, and anthropologists.
We have always studied not only the clinical and laboratory aspects of neurologic syndromes,
but also the social and public health implications of these syndromes. Our long-term data gathering
and analysis activities concerning kuru in Papua New Guinea, ALS/PD in Guam, and Viliuisk
encephalomyelitis in the Soviet Union, as well as others, continue to provide valuable insights on
cultural reaction to ongoing epidemic and endemic diseases, and to suggest available social
alternatives. Much that we learn is applicable, by extrapolation, to contemporary problems we face
in the United States, such as Alzheimer's disease and senile dementia, the AIDS epidemic, and the
seemingly uncontrollable illicit use of drugs.
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Slow Unconventional Viruses Causing Transmissible Brain Amyloidoses
Our laboratory has concentrated its main effort on elucidating the relationship between the
viruses of kuru, Creutzfeldt-Jakob disease (CJD), and scrapie and their host-specified precursor proteins.
We now know that scrapie virus is the monomeric form of the configurationally changed 35-37 kDa SPP
present in normal brain and in infected brain tissues, but modified by infection to a less soluble protease-
resistant form which is infectious. The 27-30 kDa scrapie amyloid protein (prion protein; PrP27-30)/
which assembles in vitro into congophilic, birefrigent rods resembling the scrapie-associated fibrils
(SAF) of Merz and into kuru-CJD-scrapie plaques is also infectious, even as a monomer. We have now
demonstrated that this normal host protein modified by scrapie virus infection is itself the infectious
agent (an amyloid molecule, autoinducing the modification of host protein precursor into its infectious
form). No nonhost nucleic acid has been demonstrated, even in highly infectious preparations. The
improbable conjecture that the entire infectious process is that of an autonucleated and autopatterned
conformational change of a protein precursor which leads to a crystallization and polymerization
forming amyloid fibers of SAF and kuru plaques is now verified. The host gene specifying the 35-37 kDa
precursor protein has been fully sequenced. It is on chromosome 20 in man, and 2 in mice.
Polyclonal and monoclonal antibodies prepared against synthetic polypeptides of the N-
terminus of the amyloid of scrapie reveal varying distribution and patterns of the epitopes in normal
and infected tissues. Such antibodies have shown reactivity to the scrapie-associated proteins and, to
our surprise, to many purified proteins, including purified natural and synthetic human growth
hormone. However, these antibodies to SAFs or to the synthetic polypeptide specifically label
purified SAFs from kuru-, CJD- and scrapie-infected brains. Such SAFs are not obtainable from brains
of other human neurodegenerative diseases.
These immunocytochemical and molecular biologic studies on the scrapie/kuru/CJD-
associated proteins and their normal precursors are largely aimed at preparing them in high purity
and sufficient amounts for crystallographic study, and investigation at the organic chemical level, of
the fine structural modification involved in the conversion of normal host-protein into amyloid fibers
which appears to be the major pathogenic reaction of these diseases.
Genetic Control of De Novo Generation
of Infectious Amyloid Protein from Host Precursors
For over two decades we have carefully saved at <-70oC frozen tissue from chimpanzees and
other nonhuman primates affected with the human Creuztfeldt-Jakob disease and Gerstmann-
Straussler syndrome viruses, kuru virus, and scrapie, and the frozen tissues collected from cats,
hamsters, guinea pigs and other animals susceptible to these viruses. These were collected and saved
for eventual biochemical study when this would be possible. Newer polymerase chain reaction (PCR)
techniques on long-stored tissues from these collections from crucial cases of familial CJD and GSS
have yielded critical data on the several point mutations underlying the spontaneous generation of
infectious amyloid proteins from host precursors in CJD and GSS. This has led to the association of
this mutation with CJD in Eastern-European Slavs and in Circum-Mediteranean Sephardic Jews from
Tunis, Greece and Isreal. It is now possible to process these tissues for PrP 27-30 protein, SAFs, and the
33-35 kDa scrapie-specific protein and its precursor. As more sophisticated study of the structure of
these proteins is possible, we hope to determine from this material the contribution of the host to
these subacute spongiform encephalopathy viruses or slow unconventional viruses. This we are in a
unique position to do, since it would take from two years to over a decade for other laboratories to
obtain infected brain material from a number of different species each inoculated with the same strain
of virus.
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We have indications that there are many strains of CJD viruses. Using these tissues, it is
possible to answer the critical question of the relative contributions of the host and the virus strain to
the pathogenesis of the disorders and the molecular structure of the virus strains. Since we expect all
strains or passage lines of kuru-CJD-scrapie viruses to replicate by an infectious transformation of the
normal host precursor protein to an infectious configuration, it follows that all virus strains should
produce progeny in a given host which have the identical host amino acid sequence in the infectious
monomer. Strain differences determined by the host precusor gene or its mutations would not "breed
true," i.e. they would not be carried into the progeny. Thus, since we do find strain differences in
viruses from the same host, this would require a different explanation than conservation of genotypic
identity. Rather, we should expect a conservation of secondary and tertiary configurational change by
autonucleation and autopatterning epitaxial crystal replication and growth. The stored frozen brain
passage material, particularly of different viruses (scrapie-kuru-CJD-GSS) passed into the same
breed of host, is being used for resolution of this critical matter.
Non-transmissible Brain Amyloidoses of
Aging. Alzheimer's Disease and Other Dementia
Amino acid sequencing of the 4 kDa polypeptide subunit of the paired helical filaments of
neurofibrillary tangles (NFTs), amyloid plaque cores, and amorphous amyloid in congophilic
angiopathy indicates that all three pathognomonic structures of the aging brain, Alzheimer's disease,
Pick's disease, progressive supranuclear palsy, late Down's syndrome, Guamanian ALS/PD and von
Economo's encephalitis are composed of identical 4 kDa (42 amino acids) subunits. In the preparations
of purified paired helical filaments (PHF) from NFTs of Guamanian ALS and PD, no extracellular
amyloid in the form of amyloid plaques or vascular amyloid deposits were present to produce possible
contamination. This 4 kDa polypeptide subunit which easily associates into dimers, tetramers,
octamers, and hexadecamers, shows no amino acid sequence homology to the infectious scrapie
amyloid subunit of the transmissible cerebral amyloidoses.
Genetically determined familial AD is caused by a mutation in a different gene, not that for
the B-amyloid protein precursor, which must determine a molecule involved in the rapid turn-over of
this protein. In the Dutch families with autosomal dominant hereditary cerebral hemorrhage, with
amyloidotic angiopathy, however, a point mutation in base 1852 produces a replacement at amino
acid 618 of glutamic acid by glutamine, and this is amino acid 22 in the amyloid subunit.
Hippocampal Neuronal and Endothelial Cell Cultures
for In Vitro Study of ft- Amyloid Precursor Gene Regulators
Both vascular endothelial cell and hippocampal neurons in cell culture have been used for
studies of in vitro expression of the amyloid ft-protein precursor and also for the modulation of mRNA
expression of this precursor by growth factors, interleukin-I and other regulatory molecules. This work
obviously has interesting implications for preventative and therapeutic intervention in the over-
expression of the precursor.
The Carbohydrate of Glycosylated Amyloid Subunits
Enzymatic removal of O- and N-linked carbohydrates from the infectious scrapie amyloid or
the infectious form of SPP does not diminish the infectivity titer. Thus, glycosylation is not critical
for virus replication.
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In Vitro Production of Amyloid Fibrils
We have been studying the recoiling of polypeptide chains into different fine structural
configurations which permit beta-pleated sheet stacking into fibrils resembling the amyloid fibrils of
human pathology. Thus, using P-2-microglobulin we have succeeded in producing fibers that have the
congophilic and green birefringent properties of amyloid fibrils, and also the electron microscopic
appearance of such fibers and even paired twisted fiber structures. More specifically, we have
produced amyloid-like fibers from potential precursors of brain amyloids, such as the 200 kDa protein
of neurofilament, and are trying with MAP-tau. The 200 kDa neurofilament protein assembles in vitro
into amyloid-like filaments which are both congophilic and green birefringent, and in morphology
resemble amyloid fibrils; the amyloid-like properties increase on partial cross-linking with
paraformaldehyde fixation.
The possibility that the fine structural changes of the aging or diseased brain may be
reproduced in vitro is obviously intriguing.
Neurofilament Pathology in Human Neurodegenerative Disease
The gene encoding the amyloid P-protein has been shown to be highly conserved in evolution
and is expressed in various human and animal tissues. As a complex transcriptional unit, it utilizes
alternative splicing; alternative spliced forms of the amyloid P-protein precursor cDNAs contain 50%
homology to the Kunitz family of serine protease inhibitors. It may be the absence, inhibition or
overexpression of these alternative forms that modify the host precursor proteins leading to the
production of amyloid p-protein.
These modified forms of the amyloid P-protein in its microfibril or oligomeric forms, like the
fibril amyloid enhancing factor (FAEF) in AA amyloidoses, could act as amyloid enhancing factors, or
as nucleants or niduses that accelerate its own formation by self polymerization and copolymerization
with other molecules like glycosaminoglycans which leads to amyloid deposition.
In Alzheimer's disease and Guamanian parkinsonism-dementia, the 42-amino acid subunits of
the amyloid of the neurofibrillary tangles, amyloid plaque core, and congophilic angiopathy, could
themselves serve, in the form of oligomers or fibril microfragments, as nuclei that enhance their
deposition as amyloid.
Creutzfeldt-Takob Disease and Human Growth Hormone
A further area of intense involvement of our laboratory has been in the problem of CJD in
recipients of human growth hormone (HGH) prepared from pooled autopsy pituitary glands by the
NIH and other programs. Thirteen patients are now known who developed CJD from infected or
contaminated hormone. At least three different batches of pituitary glands have been contaminated,
since two patients occurred in England and one in New Zealand (where none of the American products
were used) and at least one batch was used by all seven American patients. Incubation periods have
ranged from 4 to 20 years. Primates have been inoculated with more than 50 separate batches of
HGH, but incubation periods may be as long as five years. Intense surveillance is under way of some
8000 other young people who received hormone injections, and one further probable case has been
identified. An Australian case has followed use of CJD-contaminated pituitary gonadotropin.
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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
10 - LCNSS
Z01 NS 01282-26 and Z01 NS 00969-26
transmits the infection to his wife about half the time, and much more rarely from infected wife to
uninfected husband.
In Jamaica, the disease occurs islandwide, and new patients are being documented each week.
Household studies have been done and the results are the same as found in Tumaco. A detailed
epidemiologic study is now in progress at the University Hospital, Jamaica, and this includes the
study of patients with other neurological diseases and controls. HLA typing is also being done on TSP
patients and these results will be compared with the findings seen in adult T-cell leukemia patients in
Jamaica. A protocol for treatment with steroids has been established and these trials have been
completed in Jamaica and are in progress in Tumaco. Several isolations of HTLV-I-like virus have
been cultured from the blood and CSF of Jamaican and Colombian patients with TSP and
characterizations of these HTLV-I isolates from blood and CSF samples are underway. Initial results
suggest that there are differences in the isolate from TSP and ATL Jamaican patients. HTLV-I-like
viral particles have been identified in the fixed spinal cord of a Jamaican TSP patient.
In the study of spastic paraparesis in the Seychelles, we have found similar seropositive rates
of IgG antibodies to HTLV-I in serum. At the time of writing last year year, the initial report from
Martinique and our discoveries in Jamaica and Colombia were supported by those from Japan, the
Seychelles and Trinidad. We now have confirmation from twenty-six other countries and the
occurrence of TSP in Caucasians who have not visited other endemic regions has also been reported
from the temperate zones of Chile, France and Italy. These factors serve to emphasize the need for
intensive study of neurologic involvement with HTLV-I infection.
In addition, we discovered that almost 90% of Jamaican patients with polymyositis have IgG
antibodies to HTLV-I, and these findings were confirmed by Western blot. We have now isolated
virus from three patients with polymyositis and have detected HTLV-I viral genomic DNA in the
muscle of one patient. We have also found HTLV-I seropositive polymyositisin Barbados and recently
in Tumaco.
We have investigated a Caucasian patient with mycosis fungoides whom we diagnosed as
having TSP. The patient died 6 months after onset of TSP, and we had autopsy material. Using the
PCR to amplify the pol region of HTLV-I, we demonstrated viral genomic DNA in cerebral cortex,
cerebellum, thoracic spinal cord, lymph node, kidney and spleen of this patient. We were unable to
detect HTLV-I in fixed spinal cord of 3 Jamaican TSP patients, but will repeat this with more
material. In situ hybridization and alkaline phosphatase antiphosphatase studies are now in
progress.
Our previous serologic studies on CSF and serum from many U.S. and European multiple
sclerosis (MS) patients have failed to show any HTLV-I antibody. Frozen MS brain tissues are bein
used to search for the presence of genomic sequences, using in situ hybridization and using PCR. If we
find any positive reactions in the absence of CSF or serum antibody, the significance of such results
will be hard to establish.
High Prevalence of HTLV-I Infection in the Asia-Pacific Basin
In our search for high-prevalence foci of HTLV-I infection in the Western Pacific, we have
tested by ELISA and Western immunoblot, more than 4000 sera collected between 1956 and 1988 from 37
population groups. High prevalences of antibodies against HTLV-I, ranging from 14% among the Touri
to 51% among the Hagahai, were found among the coastal and lowland populations of New Guinea,
the Solomon Islands and Vanuatu. Populations in the more isolated interior of Papua New Guinea
11 -LCNSS
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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
12 - LCNSS
Z01 NS 01282-26 and Z01 NS 00969-26
virus, and have proven that two serotypes of hantaviruses were responsible for severe and mild forms
of HFRS in Yugoslavia.
A severe, fatal hemorrhagic illnesses with renal insufficiency, of suspected hantavirus
etiology in an illegal immigrant in Texas prompted us to investigate the prevalence of hantavirus
infection in wild rodents in designated regions in Texas. A virus anrigenically distinct from other
known hantaviruses has been isolated from Mus musculus captured in Leakey, Texas. Studies are in
progress to further characterize this isolate, to determine its role in human infection and disease, and
to clarify if the seroepidemiology of Leakey virus infection in feral mice resembles that of Seoul virus
infection in urban rats.
We are also continuing to study the mild nephropathy produced by Prospect Hill and Puumala
viruses in cynomolgus monkeys. Tests for glomerular filtration (endogenous creatinine and
phenosulfonophthalein clearances) have been normal but proteinuria suggests an impaired of
glomerular vascular integrity. Serial renal biopsies are being planned in infected monkeys. In
addition, blood and urine specimens are being tested for viral sequencies by the PCR method.
Finally, we are continuing to elucidate the epizootiology of hantavirus infections in the
United States, concentrating on determining what animals other than rodents are important in the
maintenance of the enzootic cycle. Specifically, predatory small mammals (such as weasels and
shrews) and birds (such as hawks and owls) are being tested for evidence of hantavirus infection, and
virus isolation attempts are being made from seropositive animals.
13 - LCNSS
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 01282-26 CNSS
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Neurobiology of Population Isolates: Study of Child Growth, Development, Behavior and Learning, and Disease Patterns in Isolated and Primitive Groups
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: D.C. Gajdusek, M.D. Chief LCNSS
Others: Clarence J. Gibbs, Jr., Ph.D.
David M. Asher, M.D.
Paul Brown, M.D.
Ralph M.Garruto, Ph.D.
Richard Yanagihara, M.D.
Deputy Chief LCNSS
Research Medical Officer LCNSS
Medical Director LCNSS
Senior Research Biologist LCNSS
Medical Director LCNSS
COOPERATING UNITS (if any)
See Sub-Project Summaries
LAB/BRANCH
Laboratory of Central Nervous System Studies, Intramural Reseach
SECTION
INSTITUTE AND LOCATION
NINDS, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
12
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
I x | (a) Human subjects
| x | (a1) Minors
|~x | (a2) Interviews
[x~| (b) Human tissues ] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Studies of human biology of vanishing primitive societies focus on neurological development and
learning patterns in diverse cultural experiments in the human condition found in such isolated groups.
Opportunistic investigation of problems phrased by man in isolation is the basis of approach from which
most of our studies evolved: kuru-CJD, HIV (AIDS), HTLV-I slow virus infections of the CNS, aging and
Alzheimer's disease, dementia, ALS/PD. Techniques of molecular genetics, biochemistry, immunology,
virology, and field epidemiological, clinical, linguistic and behavioral studies in cultural isolates and
genetic and/or geographically isolated primitive bands yield more easily interpretable data than in
cosmopolitan societies. Data and specimens from expeditions to Micronesia, Melanesia, Polynesia, South
America, Asia and Africa proved valuable in recent HIV (AIDS), HTLV-I, Hantavirus, JC virus of PML and
herpesvirus, CMV and EBV studies. Studies on nutrition, reproduction, fertility, age of puberty and
aging, genetic distance and pleomorphisms, unusual and odd use of the higher cortical functions in
language learning, cognitive styles, computation (calculation without words or numbers) and culturally
modified sexual behavior elucidate alternative forms of neurologic functioning for man which we would
be unable to investigate once the natural cultural experiments in primitive human isolates are
amalgamated into the cosmopolitan community of man. Foci of high incidence of kuru, ALS/PD, HTLV-I
myelopathy, epilepsy, familial parkinsonism, Viliuisk encephalopathy, other CNS degenerations,
hysterical disorders, schizophrenia, neoplasms, goiter, cretinism, rheumatoid diseases, diabetes, asthma,
chronic lung disease, malaria, filariasis, leprosy, cysticercosis, and other infections in these isolated
groups have yielded widely significant discoveries. HFRS caused by Hantaviruses in Asia, USSR, Europe
and newly recognized Hantaviruses in the U.S. are studied. Human evolution and adaptability to high
altitude, wet or arid climes, variable food supply, mineral deficiencies, toxic exposures and responses to
severe diseases or social/psychological stress are studied in appropriate population Thus, HTLV-1 and
HIV retroviruses as causes of CNS diseasess in man were first found in isolated or socially segregated
groups, high incidence TSP focus in Tumaco, Colombia; drug using mothers in Newark, New Jersey and
are often best studied in these isolated or socially segregated groups.
14- LCNSS
PHS 6040 (Rev. 1 84)
Z01 NS 01282-26 CNSS
I. Mechanisms of dissemination and transmission of HTLV-I in the Caribbean basin, South America and
Melanesia
PI: Ralph M. Garruto, Ph.D. Senior Research Biologist
Pamela Rodgers-Johnson, M.D. Visiting Scientist
Carlos A. Mora, M.D. Visiting Associate
Richard Yanagihara, M.D. Medical Director
Marta Leon-Monzon, Ph.D. Visiting Scientist
Xueyun Wu, M.P.H. Special Volunteer
Mark A. Miller, M.D. Howard Hughes Fellow
Cooperating Units:
Owen Morgan, University of West Indies, Kingston, Jamaica; Vladimir Zaninovic, Universidad
del Valle, Cali, Colombia; Luis Cartier-Rovirosa, Universidad de Chile, Santiago, Chile; Carol L.
Jenkins, Institute of Medical Research, Goroka, Papua New Guinea; Andrew Ajdukiewicz, Central
Hospital, Honiara, Solomon Islands; Steve Alexander, Biotech Research Laboratories, Rockville,
Maryland
Our seroepidemiologic studies of more than 3000 sera, collected between 1956 and 1988 from 34
Melanesian populations, for antibodies against HTLV-I indicate high prevalences of infection, as
verified by strict Western immunoblot criteria, in several remote population groups having no contact
with Japanese or Africans and minimal contact with Europeans prior to our bleedings. By contrast, some
Micronesian populations having intense contact with Japanese for more than two decades have no
evidence of infection, arguing against the dissemination of HTLV-I in the Pacific basin by the Japanese
and calling into question the venereal spread of HTLV-I. Our recent identification of a case of HTLV-I
myeloneuropathy in a life-long resident of Guadacanal in the Solomon Islands and the successful
isolation of HTLV-I from unrelated individuals from widely separated provinces in the Solomon
Islands and from members of a remote, recently contacted hunter-horticulturist group in Papua New
Guinea adds further credibility to our serologic data. We are continuing to investigate the mechanisms
of transmission of HTLV-I in these remote populations and to determine if the high frequency of
indeterminate HTLV-I Western immunoblots in Melanesia results from the circulation of novel
retroviruses, which are antigenically related to but distinct from HTLV-I.
n. Epidemiology of Creutzfeldt-Jakob disease in recipients of pituitary gland-derived human growth
hormone
PI: Paul Brown, M.D. Medical Director
Lev Goldfarb, M.D. Visiting Scientist
Clarence J. Gibbs, Jr., Ph.D. Research Microbiologist (Deputy Chief)
Cooperating Units:
Judith Fradkin, NIDDKD, DDEM, Bethesda
Continuing surveillance of the outbreak of Creutzfeldt-Jakob disease (CJD) in young people treated
with pituitary gland-derived human growth hormone (hGH) has to date identified 11 subjects (of
whom seven were treated in this country) dying of CJD between 4 and 20 years after their last dose of
hormone. It now appears that, worldwide, the risk of developing CJD in hGH-treated patients is at
least 1 per thousand, and possibly as high as 1 per hundred, compared to the risk of 1 per million in the
general population. Extensive analysis of processing records implicates multiple batches of pituitary
glands as the source of contamination, and all patients so far identified have been treated before 1976.
Molecular genetic analysis of DNA from two patients has so far not identified any mutation in the
15 - LCNSS
Z01 NS 01282-26 CNSS
scrapie amyloid gene as a susceptibility factor in determining which patients among the treated
population develop disease.
Additional cases of iatrogenic CJD due to treatment with pituitary gland-derived gonadotropin, and to
implantation of dura matter grafts, have also been identified, and consultations with the FDA have
assisted in revised policy decisions.
III. Elucidation of the cause and pathogenesis of high-incidence motor neuron disease in different
climatic regions and among diverse ethnic groups
Co-PI: Ralph M. Garruto, Ph.D. Senior Research Biologist
Richard Yanagihara, M.D. Medical Director
Michael J. Strong, M.D. Guest Researcher
Don C. Guiroy, M.D. Visiting Associate
Ikuro Wakayama, M.D. Visiting Fellow
Pamela Rodgers-Johnson, M.D. Visiting Scientist
Pedro Piccardo, M.D. Visiting Associate
Cooperating Units:
Kwang-Ming Chen, Olivia Cruz, Guam Memorial Hospital, Agana, Guam; Chris C. Plato, NIA,
Gerontology Research Center, Baltimore, Maryland
Our multidisciplinary approach to the study of high-incidence motor neuron disease, conducted during
the past three decades among geographically and gentically diverse groups in the Western Pacific,
indicates unequivocally that there is no genetic cause, but rather a defect in mineral metabolism,
provoked by chronic nutritional deficiencies of calcium, leads to increased intestinal absorption of toxic
metals and the intraneuronal co-deposition of calcium, aluminum and silicon, as aluminosilicates and
calcium hydroxyapatites, in affected neurons. This elemental deposition interferes with slow axonal
transport by altering neurofilament biosynthesis and/or catabolism, resulting in excessive
neurofilament accumulation in motor neurons, the ultrastructural hallmark of ALS. Epidemiologic
studies are underway to assess the development of motor neuron disease among migrants from these
high-incidence foci who left during infancy or childhood, and to determine if other ethnic groups in
different grographic regions in Asia and the Pacific are similarly affected. At present the greatly
increased life expectancy of Guamanians has produced the problem of differentiating the expected
cases of Alzheimer's disease in aged Guamanians from the previously more clear-cut cases of
parkinsonism-dementia in younger subjects now no longer seen.
IV. Worldwide epizootiology and epidemiology of hantavirus infection: search for human disease in
the face of a widespread enzootic in the United States
PI: Richard Yanagihara, M.D. Medical Director
David M. Asher, M.D. Research Medical Officer
Bruce K. Johnson, Ph.D. Special Expert
Shuyuan Xiao, M.D. Visiting Fellow
Zayd A. Eldadah Biological Lab Aid
Clarence J. Gibbs, Jr., Ph.D. Research Microbiologist (Deputy Chief)
Mark Godec, M.D. NRC Research Fellow
16 - LCNSS
Z01 NS 01282-26 CNSS
Cooperating Units:
Theodore F. Tsai, CDC, Ft. Collins, Colorado; Patrick Redig, Raptor Center, St. Paul, Minnesota;
Duane Schlitter, Carnegie Museum of Natural History, Pittsburg, Pennsylvania; Robert Traub, Smithsonian
Institution, Washington, D.C.; Ana Gligic, Institute of Virology, Belgrade, Yugoslavia; Yong Kang,
University of Ottawa, Ottawa, Canada; Chin-Ming Hsiang, Hubei Medical College, Hubei, People's
Republic of China
We were first to isolate hantaviruses from meadow voles (Microtus pennsylvanicus) and mice (Mus
musculus) captured in the United States, to demonstrate that the massive epidemics of hemorrhagic fever
with renal syndrome (HFRS) in the People's Republic of China were caused by viruses closely related to
hantaviruses isolated in Korea and Siberia, and to prove that two serotypes of hantaviruses were
responsible for severe and mild forms of HFRS in Yugoslavia. Despite the widespread distribution of
hantaviruses in commensal rats and indigenous wild rodents in the United States, confirmed cases of HFRS
have not been recognized and the overall risk of hantavirus infection in Americans is low, even among
individuals who have frequent exposure to wild rodents. In an effort to further clarify the epizootiology,
ecology and epidemiology of hantavirus infection in the United States, we are currently examining sera
from predatory small mammals (particularly, shrews and weasels) and birds (such as owls and hawks), as
well as sera from patients with non-A, non-B hepatitis and from muskrat trappers for evidence of
hantavirus infection. We are also investigating the distribution of these viruses among indigenous
arvicolid and cricetid rodents in South America and Canada, and the epizootiology of Leakey virus
infection in Mus populations.
V. Studies of high-incidence non-neurologic disorders in specific racial and ethnic groups
PI: Ralph M. Garruto, Ph.D. Senior Research Biologist
Richard Yanagihara, M.D. Medical Director
Mark A. Miller, M.D. Howard Hughes Fellow
Cooperating Units:
Julianne Imperato-McGinley and Ralph Peterson, Cornell University Medical College, New York,
New York; Charles Weitz, Temple University, Philadelphia, Pennsylvania; Chen-ting Chin, Beijing
University Medical School, Beijing, People's Republic of China
We have successfully established the first high-altitude research station in southeastern Qinghai
province of the People's Republic of China to study the basic biology and neurobiology of minority groups
(ethnic isolates) living above 3000 meters. Studies initiated among these sheep and yak-herding pastoral
populations exposed to hypoxic stress include pulmonary and blood physiology, genetic epidemiology and
the neurobiology of sleep, chronic mountain sickness and aging. Preliminary data indicate that Han
populations have greater difficulty than native-born ethnic minorities adapting to high altitude, even
after emigrating to such regions nearly two decades ago. During the ensuing years, epidemiologic, genetic,
virologic and human biologic studies are expected to yield important new insights into these unique
population groups.
During the past 25 years, we have studied a focus of male pseudohermaphroditism among small, remote,
inbred, forest-dwelling, hoe and digging-stick horticulturalists of the Simbari Anga linguistic group of the
Eastern Highlands of Papua New Guinea. Clinically, these male pseudohermaphrodites represent a
spectrum of congenital anatomic abnormalities including a foreskin forming a small fold above a
rudimentary clitoris-like penis and bilateral scrotal flaps resembling labia enclosing small testes. There is
a urogenital sinus containing a urethra and a blind vaginal pouch. Patients show no gynecomastia or
menses. At puberty, the clitoris-like penis and testes enlarge with concurrent extensive facial, pubic and
axillary hair growth and musculature development greater than in their normal male peers. Sera collected
17 - LCNSS
701 NS 01282-26 CNSS
from two of these young adult patients revealed elevated testosterone/ dihydrotestosterone ratios Both
had Weh urinary etiocholaolone/ androsterone, C19 and C21 5|3-5a metabolite ratios. The data mdicate a
5 a-reductase deficiency similar to patients studied in the Dominican Republic.
18 - LCNSS
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 00969-26 CNSS
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Chronic CNS Disease Studies: Slow, Latent and Temperate Virus Infection
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: D.C. Gajdusek, M.D.
Others: Clarence J. Gibbs, Jr., Ph.D.
David M. Asher, M.D.
Paul Brown, M.D.
Ralph M. Garruto, Ph.D.
Richard Yanagihara, M.D.
(continued see next page)
Chief LCNSS
Deputy Chief LCNSS
Research Medical Officer LCNSS
Medical Director LCNSS
Senior Research Biologist LCNSS
Medical Director LCNSS
COOPERATING UNITS (if any)
See Sub-Project Summaries
LAB/BRANCH
Laboratory of Central Nervous System Studies, Basic Neurosciences Program, Intramural Research
SECTION
INSTITUTE AND LOCATION
NINDS, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
12
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
I x | (a) Human subjects
x| (a1) Minors
x| (a2) Interviews
l"x~l (b) Human tissues ] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Studies focus on causes and pathogenesis of chronic degenerative CNS disorders with emphasis on MS;
Parkinson's , Pick's, Huntington's and Alzheimer's diseases; ALS/PD of Western Pacific; supranuclear
palsy; other presenile dementias; spinocerebellar ataxias; epilepsy; chronic encephalitis with focal
epilepsy; Viliuisk encephalopathy; muscular dystrophies; chronic schizophrenia; autism; SSPE; PML;
dialysis encephalopathy; goiterous cretinism; cysticercosis; and intracranial neoplasm.
We have defined the transmissible and nontransmissible dementias as cerebral amyloidoses caused by
post-translational modification of a specific host precursor protein to amyloid fibril deposits. We now
recognize the slow unconventional viruses causing kuru-CJD-scrapie as replicating polypeptides formed
de novo from a normal host precursor protein, specified on chromosome 20 in man and 2 in mice. The
molecular elucidation of the spontaneous configurational change to infectivity, basically a
crystallographic problem, is now becoming our major target. Molecular genetic analysis of familial CJD
already indicates several point mutations which enormously increase (x106 ) the probability of this
spontaneous de novo conversion to an infectious polypeptide. Microbiology must now contend with a
totally new paradigm for replicating, infectious, pathogenic agents in the nontransmissible brain
amylodoses. Our studies focus on the elucidation of the molecular configurational events conferring the
property of infectivity on a previously normal host precursor.
In normal aging, Alzheimer's disease (AD), and Down's syndrome a different host precursor protein
(specified on chromosome 21 in man, 16 in mice) is a cell excreted inhibitor of growth factors. Post-
translational degradation of this normal precursor forms the 42 amino acid amyloid polypeptide which
polymerizes to form the deposits of amyloid angiopathy, amyloid plaques and neurofibrillary tangles in
aging, AD and Down's. This occurs in all individuals who reach their 90s. Genetic, toxic, and infectious
factors may accelerate this aging brain amyloid deposition.
Conventional viruses causing slow, infectious, degenerative disease are intensely studied to elucidate the
neurotropism and mechanism of pathogenesis: retrovirus encephalomylopathies of HTLV-I and HIV (of
AIDS); herpesviruses (HSV, CMV, EB and virus varicella-zoster); papovavi ruses (JC); RSSE; measles; SSPE;
and many chronic virus infections of amyloid.
19- LCNSS
PHS 6040 (Rev 1 84)
Z01 NS 00969-26 CNSS
I. Molecular pathogenesis of the transmissible cerebral amyloidoses
Co-PI: Paul Brown, M.D. Medical Director
Clarence J. Gibbs, Jr., Ph.D. Research Microbiologist (Deputy Chief)
Jiri Safar, M.D. Visiting Associate
Mauro Ceroni, M.D. Visiting Associate
Pedro Piccardo, M.D. Visiting Associate
Don C. Guiroy, M.D. Visiting Associate
Pawel P. Liberski, M.D. Visiting Fellow
Our work indicates that the scrapie amyloid precursor protein is converted into an infectious form by
configurational changes of the normal precursor. In susceptible hosts, the scrapie monomer may act
much like the fibril amyloid-enhancing factors found in AA amyloidosis to autonucleate and
autopattern this conversion, resulting in its own polymerization or crystallization and precipitation as
insoluble arrays of amyloid fibrils. Using tissues stored from experimentally infected animals, we
have begun to determine the structure and sequence of the amyloid protein of different strains of virus
recovered from the same genetic host. In addition, we have determined the intracellular localization
of the amyloid precursor protein, and are now trying to define the pathway of scrapie amyloid
formation and to interrupt its formation in vivo.
II. Molecular generics of the PRIP gene in the subacute spongiform virus encephalophies
Co-PI: Paul Brown, M.D. Medical Director
Lev Goldfarb, M.D. Visiting Scientist
David M. Asher, M.D. Research Medical Officer
Pedro Piccardo, M.D. Visiting Associate
Cooperating Units:
Dmitry Goldgaber, State University of New York, Stonybrook, New York
We have demonstrated several mutations in the open-reading frame of the gene encoding the scrapie
precursor protein which seem to be linked to the human transmissible encephalopathies. An amino
acid-altering mutation in codon 102 was identified in three patients with GSS, adding a large and well
documented family of German origin to two previously reported unrelated American and English
families. Brain tissue from one of these patients has transmitted the disease to experimental animals.
This mutation was not found in 8 unaffected family members, 25 healthy control individuals, 3
transmitted cases of kuru, 17 transmitted cases of CJD (4 familial and 13 sporadic), and 11 patients with
other neurologic disorders.
A different double-allele mutation in codon 129 was found in 2 of 3 kuru patients, 3 of 3 patients with
familial CJD belonging to three related families, and 2 of 2 patients with iatrogenic CJD caused by
treatment with contaminated pituitary-derived human growth hormone. The nucleotide change in this
codon (ATG to GTG) resulted in an amino-acid subsitution of valine for methionine; it also abolished an
Nspl restriction site and created a new Maell site, which permited the use of a restriction-endonuclease
technique as a screening procedure for this mutation. Using direct sequencing and restriction-endonuclease
analysis in additional cases, we failed to find this double-allele mutation in patients with sporadic CJD
or in healthy controls, but an identical single-allele codon 129 mutation (heterozygosity) was found in 3 of
15 patients with sporadic CJD and in 3 of 24 healthy control individuals. Detection of a consistent double-
allele mutation in 7 studied patients with related disorders suggests that homozygosity for a PRIP-129
point mutation may serve as the genetic background for some cases of kuru and CJD. It is likely that other
amino acid-altering mutations in the PRIP gene will be found in patients with sporadic or familial CJD.
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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.
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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-
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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.
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Finally, attempts are being made to characterize the factor present in homogenates of immature rat
cerebellar cortex (prepared from 10-day old BD-IX rats when synaptogenesis is at its peak), which
reportedly produces lesions akin to the transmissible spongiform encephalopathies, such as the
mutlilamellated membranes seen in spider monkeys experimentally infected with kuru.
IX. Attempts to produce neuro-AIDS in experimental animals with human immunodeficiency viruses
PI: Clarence J. Gibbs, Jr., Ph.D. Research Microbiologist (Deputy Chief)
David M. Asher, M.D. Research Medical Officer
Bruce K. Johnson, Ph.D. Special Expert
Hiroko Minagawa, M.D. Visiting Fellow
Mark A. Beilke, M.D. Medical Staff Fellow
Gary Stone, M.S. Biologist
Maneth Gravell, Ph.D. Research Microbiologist
Cooperating Units:
Prem Sarin, NCI, LTCB, Bethesda; Jaap Goudsmit, University of Amsterdam, Amsterdam,
Netherlands
In an effort to develop an animal model of neuro-AIDS, we have inoculated multiple species of New
and Old world monkeys, chimpanzees, domestic horses, goats and small laboratory rodents with
autopsy tissues, whole blood or plasma from patients with AIDS or pre-AIDS, as well as with
supernatant fluids from cell cultures infected in vitro with different strains of HIV-1. Although
chimpanzees were readily susceptible to infection, and while some continue to remain viremic for more
than five years, none has developed clinical AIDS. By contrast, all other nonhuman primate species
and other experimental animals were rather resistant to infection, and to date, only one rhesus monkey,
one cynomolgus monkey and six horses have shown serologic evidence of subclinical infection.
X. Virological and molecular genetics studies on simian immunodeficiency viruses as a model for AIDS
PI: Maneth Gravell, Ph.D. Research Microbiologist
Clarence J. Gibbs, Jr., Ph.D. Research Microbiologist (Deputy Chief)
Mark A. Beilke, M.D. Medical Staff Fellow
Gary Stone, M.S. Biologist
Elaine Kay Jordan, D.V.M. Senior Staff Fellow
Marta Leon-Monzon, Ph.D. Visiting Scientist
Rebecca Hamilton Microbiologist
Cooperating Units:
P.R. Johnson, Georgetown University, Washington, D.C.
In an effort to develop an animal model of neuro-AIDS, we have inoculated multiple species of Old
World monkeys with simian retroviruses and both rhesus and pigtailed macaques with strains of
simian immunodeficiency virus (SIV) isolated from African green and sooty mangabey monkeys. Three
of six rhesus monkeys inoculated with an SrV isolate from a sooty mangabey monkey have developed
an AIDS-like disease with impaired motor function and orofacial dyskinesia. Enlarged lateral
ventricles and defects in the cerebral cortex were found by MRI brain scan in one of these monkeys one
week before death. Vascular gliosis and neuronal loss were evident, particularly in the brain stem, and
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virus was recovered from brain, spinal cord, CSF, peripheral nerve and muscle. Further studies are
underway to clarify the pathogenesis of AIDS encephalopathy and dementia.
XI. Prevention of AIDS: antigenic potency and immunogenicity of inactivated HIV vaccine and
synthetic HTV core polypeptide immunogen
PI: Clarence J. Gibbs, Jr., Ph.D. Research Microbiologist (Deputy Chief)
Maneth Gravell, Ph.D. Research Microbiologist
Bruce K. Johnson, Ph.D. Special Expert
Hiroko Minagawa, M.D. Visiting Fellow
Carlos A. Mora, M.D. Visiting Associate
Gary Stone, M.S. Biologist
Cooperating Units:
Jonas Salk, Salk Institute, La Jolla, California; Frederick Jensen, Immune Response Corporation,
La Jolla, California; Prem Sarin, NCI, LTCB, Bethesda; Allen Goldstein, George Washington
University, Washington, D.C.
Following repeated doses of an inactivated whole virus HIV vaccine devoid of env gp 120/160,
chimpanzees persistently infected with HIV developed non-anamnestic antibody responses and were
cleared of viremia for one year following virus challenge, beginning at 10 weeks following primary
vaccination, as evidenced by negative virus isolation attempts and inability to detect HIV DNA
sequences by PCR. Additional safety, toxicity and immunogenicity studies are underway using a
synthetic 30-amino acid peptide of a conserved pi 7 region of HIV-1. Studies are in progress to assess
the utility of each of these vaccines to prevent AIDS in humans already infected with HIV.
XII. Antigenic, virological and molecular biological characterization of HTLV-I strains circulating in
high incidence in the Caribbean basin, South America and Melanesia
Co-PI: Richard Yanagihara, M.D. Medical Director
Clarence J. Gibbs, Jr., Ph.D. Research Microbiologist (Deputy Chief)
Ralph M. Garruto, Ph.D. Senior Research Biologist
Carlos A. Mora, M.D. Visiting Associate
Vivek R. Nerurkar, Ph.D. Visiting Fellow
David M. Asher, M.D. Research Medical Officer
Marta Leon-Monzon, Ph.D. Visiting Scientist
Pawel P. Liberski, M.D. Visiting Fellow
Mark A. Miller, M.D. Howard Hughes Fellow
Cooperating Units:
Prem Sarin, NCI, LTCB, Bethesda; Michael P. Alpers, Institute of Medical Research, Goroka,
Papua New Guinea; Jaap Goudsmit, University of Amsterdam, Amsterdam, Netherlands; Steve
Alexander, Biotech Research Laboratories, Rockville, Maryland; Andrew Ajdukiewicz, Ministry of
Health and Medical Services, Central Hospital, Honiara, Solomon Islands.
Multiple HTLV-I isolates have been made from peripheral blood lymphocytes and CSF cells obtained
from Jamaican, Colombian and Chilean patients with tropical spastic paraparesis. Molecular genetic
analyses of some of these isolates indicate minor differences from strains of HTLV-I isolated from
patients with adult T-cell leukemia /lymphoma (ATLL). Further comparisons between TSP and ATLL
strains of HTLV-I now in progress should establish wheither distinct pathogenic markers exist.
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The high prevalences of HTLV-I infection, based largely on the results of screening tests such as enzyme
immunoassay, in remote population groups in Melanesia, have been contested because of the inability to
confirm seropositivity by Western analysis in many Melanesian sera, and the failure of such sera to
neutralize prototype strains of HTLV-I We have now isolated HTLV-I from T-cell lines derived from
peripheral blood mononuclear cells of Solomon Islanders and a New Guinean with confirmatory
Western immunoblots. By Western analysis, these isolates exhibit virus-specific bands at 15, 19, 24, 46,
53 and 61 kda, and HTLV-I sequences have been detected by PCR in DNA extracted from these cell
lines. Further molecular genetic characterization of these isolates should establish whether or not
they differ significantly from prototype strains of HTLV-I isolated from Jamaica, Colombia and Japan.
In addition, attempts are being made to determine if the high frequency of indeterminate HTLV-I
Western immunoblots among Melanesians is a direct result of the existence of closely related by distinct
retroviruses.
XIII. Experimental HTLV-I infection in nonhuman primates and rabbits: pathogenesis and virus
neurotropism
Co-PI: Clarence J. Gibbs, Jr., Ph.D. Research Microbiologist (Deputy Chief)
David M. Asher, M.D. Research Medical Officer
Hiroko Minagawa, M.D. Visiting Fellow
Bruce K. Johnson, Ph.D. Special Expert
Carlos A. Mora, M.D. Visiting Associate
Maneth Gravell, Ph.D. Research Microbiologist
Marta Leon-Monzon, Ph.D. Visiting Scientist
Pawel P. Liberski, M.D. Visiting Fellow
Gary Stone, M.S. Biologist
Cooperating Units:
Jaap Goudsmit, University of Amsterdam, Amsterdam, Netherlands
We have inoculated chimpanzees, African green monkeys and rabbits by various routes, including
intraspinally with different strains of HTLV-I, some of which were isolated from patients with
tropical spastic paraparesis (TSP). All animals have seroconverted and some remain viremic
Five chimpanzees have been studied, one naturally infected with an HTLV-I-related virus and four
experimentally inoculated with standard strains of human origin. Two chimpanzees were inoculated
with HTLV-I propagated in human cells: one was injected intravenously with complete HTLV-I-
infected human cells and the other with cell-free virus. Neither of those animals became persistently
infected with HTLV-I (repeatedly negative PCR for HTLV-I pol gene in DNA extracted from their
peripheral blood mononuclear cells). Although they mounted antibody responses to the virus, the fact
that their IgM responses were of short duration (<12 weeks) and limited spectrum (only against gag) in
comparison with other chimpanzees suggests that they were abortively infected.
Two chimpanzees were inoculated with HTLV-I propagated in chimpanzee cells: one with HTLV-I
grown in its own cultured peripheral blood mononuclear cells and the other with a transfusion of whole
blood from the first chimpanzee. Both chimpanzees had long lasting IgG and IgM antibody responses to
gag and env antigens, and both became persistently infected.
The pol genes of several HTLV-I isolates from the two experimentally infected chimpanzees were
compared with those from the naturally infected chimpanzee. No definite changes were found in the
nucleotide sequences of the area of pol gene tested over a 6-month period (one of 209 bases was
apparently altered in a single isolate from one animal, though that may have been a sequencing error)
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The same area of the pol gene of HTLV-I isolated from the naturally infected chimpanzee differed
from the others by 8 nucleotides (4%). None of the chimpanzees-naturally infected, abortively
infected or experimentally persistently infected with HTLV-I-had any signs of acute chronic illness.
The pol gene of HTLV-I in persistently infected chimpanzees is clearly not hypermutable.
Rabbits were successfully infected with two strains of HTLV-I, one isolated from a Colombian patient
with TSP. Rabbits inoculated intravenously or intracerebrally with HTLV-I-infected autologous
lymphocytes were viremic for more than 40 weeks, while rabbits inoculated intraperitoneally were
only intermittently viremic. All animals developed antibodies against various structural proteins of
HTLV-I. Further manipulations of this model may yield additional insights into the pathogenesis of
HTLV-I infection.
XIV. Detection of cryptic viral genomic sequences in tissues from patients with chronic neurological
diseases of unknown etiology
PI: David M. Asher, M.D. Research Medical Officer
Richard Yanagihara, M.D. Medical Director
Mark S. Godec, M.D. NRC Research Fellow
Bruce K. Johnson, Ph.D. Special Expert
Lev G. Goldfarb, M.D. Visiting Scientist
Zayd A. Eldadah, B.S. Biological Lab Aid
Kitty L. Pomeroy, B.S. Microbiologist
Michael P. Sulima Biological Lab Technician
Alfred Bacote Biological Lab Technician
Cooperating Units:
Steven Feinstone, CEBR, FAA, Rockville, Maryland; Peggy Swoveland, Univ. Maryland,
Baltimore, Maryland; Frederick Andermann, Montreal Neurological Institute, Montreal, Canada.
We have developed a PCR technique for detecting genes of several RNA and DNA viruses. Sensitivity
has been increased markedly using a second round of PCR employing oligioneucleotide primers
(interned)to the initial compared to classical virus isolation, the technique has to detect incomplete,
defective and degraded viruses in fixed and embedded tissues, as well as in frozen tissues after decades
of storage. The technique is highly specific and is capable of rapidly distinguishing genes of related
RNA viruses, such as enteroviruses. The technique is being applied to the amplification of viral RNA
and DNA in tissues of patients with chronic neurological disorders of unknown etiology, such as chronic
encephalitis, ALS, parkinsonism, and Viliusk encephalitis. Thus far, primer pairs have been
synthesized for multiple structural genes of several RNA viruses, including measles, mumps, rubella,
polio, coxsackie B, HTLV-I, and St. Louis encephalitis viruses, and DNA viruses, including Epstein-
Barr virus, herpes simplex virus types 1 and 2, cytomegalovirus, varicella-zoster virus and human
herpes virus type 6. Using this technique we failed to confirm reports of others who used an in situ
hybridization technique of dubious specificity and low sensitivity, claiming to have identified
genomes of cytomegalovirus and Epstein-Barr virus in brains of patients with chronic encephalitis.
Primer pairs for Treponema pallidum and Toxoplasma gondii are also used.
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XV. Search for an infectious cause of Alzheimer disease and multiple sclerosis
PI: David M. Asher, M.D. Research Medical Officer
Mark S. Godec, M.D. NRC Research Fellow
Cooperating Units:
Steven Feinstone, CEBR, FAA, Rockville, Maryland;Stanley Rapoport and Robert Friedland,
NIA, LN, Bethesda
Futher attempts are being made to recover infectious agents from patients with Alzheimer's disease
and MS. Buffy coat specimens from more than 100 unaffected family members of patients with familial
Alzheimer's disease have been inoculated into LVG hamsters to verify the claims, by Manuelidis and
his colleagues, of the successful transmission of a spongiform encephalopathy. In addition, CNS
tissues, intestinal mucosa and peripheral mononuclear cells from patients with MS are being examined
for viral genomic sequences by the PCR.
XVI. Pathogenesis of hantavirus infection in animals and cell culture
Co-PI: David M. Asher, M.D. Research Medical Officer
Richard Yanagihara, M.D Medical Director
Shuyuan Xiao, M.D. Visiting Fellow
Mark Godec, M.D. National Research Council Fellow
Zahd A. Eldadah, B.S. Biological Lab Aid
Xueyun Wu, M.P.H. Special Volunteer
Bruce K. Johnson, Ph.D. Special Expert
Kitty L. Pomeroy, B.S. Microbiologist
Axel V. Wolff, D.V.M. Facility Veterinarian
Cooperating Units:
David J. Silverman, University of Maryland, Baltimore, Maryland
We have developed and exploited several rodent and primate systems for the study of hantavirus
infection, including infant mice and weanling rats infected with Hantaan virus, bank voles
{Clethrionomys glareolus) infected with Puumala virus and cynomolgus monkeys infected with
Puumala and Prospect Hill viruses. We have extended these studies to include the use of the reverse-
tianscriptase directed-polymerase chain reaction (RT-PCR). Using primers specific for the
glycoprotein 2-endcoding region of hantavirus M segment, we demonstrated Hantaan virus sequences in
tissues of mice experimentally infected with Hantaan virus strain 76-118 and HV114, an isolate from
the urine of a Chinese patient with hemorrhagic fever with renal syndrome. We are now adopting
this method to detect hantavirus genomic sequences in clinical specimens and postmortem tissues of
patients with HFRS.
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Publications in Print:
1 . Bolis L, Gibbs CJ, Jr. Proceedings of an international roundtable on bovine spongiform
encephalopathy. Summary report and recommendations. J Am Vet Med Assoc 1990,196:1673.
2. Brown P. Central nervous system amyloidoses: a comparison of Alzheimer's disease and
Creutzfeldt-Jakob disease. Neurology 1989;39:1103-5.
3. Brown P. The phantasmagoic immunology of transmissible spongiform encephalopathy. In
Waksman BH, ed. Immunologic mechanisms in neurologic and psychiatric disease. New York:
Raven, 1990^05-13.
4. Brown P. Guidelines for high risk autopsy cases: special precautions for Creutzfeldt-Jakob
disease. In: Hutchins G, Kircher T, Peters HJ, eds. Autopsy Performance and Reporting,
Northfield, IL: College of American Pathologists, 1990;67-74.
5. Brown P. Transmissible spongiform encephalopathies in humans: kuru, Creutzfeldt-Jakob disease
and Gerstmann-Straussler-Scheinker disease. Can J Vet Res 1990;54:38-41.
6. Brown P, Jannotta F, Gibbs CJ Jr, Baron H, Guiroy DC, Gajdusek DC. Coexistence of Creutzfeldt-
Jakob disease and Alzheimer's disease in the same patient. Neurology 1990;40:226-8.
7. Brown P, Liberski PP, Wolff A, Gajdusek DC. Conservation of infectivity in purified fibrillary
extracts of scrapie-infected hamster brain after sequential enzymatic digestion or
polyacrylamide gel electrophoresis. Proc Natl Acad Sci USA 1990;87:7240-44.
8. Brown P, Wolff A, Gajdusek DC. A simple and effective method for inactivating virus
infectivity in formalin-fixed samples from patients with Creutzfeldt-Jakob disease. Neurology
1990;40:887-90.
9. Brown P, Wolff A, Liberski PP, Gajdusek DC. Resistance of scrapie infectivity to steam
autoclaving after formaldehyde fixation, and limited survival after ashing at 360°C: practical
and theoretical implications. J Infect Dis 1990;161:467-72.
10. Ceroni M, Piccardo P, Safar J, Gajdusek DC, Gibbs CJ Jr. Scrapie infectivity and prion protein are
distributed in the same pH range in agarose isoelectric focusing. Neurology 1990;40:508-13.
11. Fox KM, Garruto RM, Gajdusek DC, Plato CC. Dermatoglyphics of the isolated
Kapingamarangese of Micronesia. In: Durham NM, Plato CC, eds. Trends in dermatoglyphic
research. Dordrecht, Netherlands: Kluwer Academic Publishers,1990;278-287.
12. Fox KM, Plato CC, Garruto RM. Dermatoglyphics of the people of Micronesia: a review.
Proceedings of the Valsik Memoria Symposium, Bratislava, September 21-21, 1987. 1990; 325-38.
13. Gajdusek DC. Raymond Pearl Memorial Lecture, 1989. Cultural practices as determinants of
clinical pathology and epidemiology of veneral infections: implications of predictions about the
AIDS epidemic. Am J Hum Biol 1990;2:4347-51.
14. Gajdusek DC. Fantasy of a virus from the inorganic world: pathogenesis of cerebral amyloidoses
by polymer nucleating agents and/or "viruses". In Neth, M.J. , ed. Modern trends in human
leukemia VIII. Heidelberg: Springer Verlag, 1990; 481-99.
15. Gajdusek DC. Paradoxes of aspiration for and of children in primitive and isolated cultures.
Pediatr Res 1990;27:S59-61.
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16. Gajdusek DC. Subacute spongiform encephalopathies: Transmissible cerebral amyloidoses caused
by unconventional viruses. In: Knipe DM, Fields BN, eds. Virology, 2nd Edition. New York:
Raven, 1990;2289-324.
17. Gajdusek DC. Cycad toxicity not the cause of high incidence amyotrophic lateral
sclerosis/parkinsonism-dementia on Guam, Kii Peninsula of Japan or in West New Guinea. In
Hudson AJ, ed. Amyotrophic lateral sclerosis: concepts in pathogenesis and etiology, Toronto:
University of Toronto, 1990; 317-25.
18. Gajdusek DC, Gibbs CJ Jr Brain amyloidoses: precursor proteins and the amyloids of transmissible
and nontransmissible dementias: Scrapie-kuru-CJD viruses as infectious polypedpides or
amyloid-enhancing factors. In Goldstein AL, ed. Biomedical advances in aging, New York:
Plenum , 1990; 3-24.
19. Garruto RM. Cellular and molecular mechanisms of neuronal degeneration: amyotrophic lateral
sclerosis, parkinsonism-dementia and Alzheimer disease. Am J Hum Biol 1990;1:529-43.
20. Garruto RM. Review of Modern biological theories of aging. In: Warner HR, Butler RN, Sprott
RL, Schneider EL, eds. Ann Hum Biol 1990;1 6:285-6.
21. Garruto, RM. Introduction: biocultural Implications of AIDS and other retroviral infections. Am J
Hum Biol 1990; 2:, 343-5.
22. Garruto RM, Plato CC, Yanagihara R, Fox K, Dutt J, Gajdusek DC, Tobin J. Bone mass in
Guamanian patients with amyotrophic lateral sclerosis and Parkinsonism-dementia. Am J Phys
Anthropol 1989;80:107-13.
23. Garruto RM, Slover M, Yanagihara R, Mora CA, Alexander SS, Asher DM, Rodgers-Johnson P,
Gajdusek DC High prevalence of human T-lympho tropic virus type I infection in isolated
populations of the western Pacific region confirmed by Western immunoblot. Am J Hum Biol
1990;2:439-47.
24. Garruto RM, Yanagihara R, Gajdusek DC. Models of environmentally induced neurological
disease: epidemiology and etiology of amyotrophic lateral sclerosis and parkinsonism dementia
in the western Pacific. Environ Geochem Health 1990;12:137-51.
25. Garruto RM, Yanagihara R, Strong MJ. Insights from Pacific ALS: development of models of
chronic metal induced motor neuron degeneration in non-human primates. In: Norris FB, Rose FC,
eds. The etiology of amyotrophic lateral sclerosis: epidemiological and neurotoxicological
aspects. London: Smith-Gordon, 1990; 143-56.
26. Gibbs CJ Jr. AIDS: the biological, ethical and moral dilemmas of this twentiety century plague.
In: Walton CC, ed. Business ethics: other voices, other perspectives. New York: Plenum, 1990;
341-65.
27. Gibbs CJ Jr. Subcommittee on the production and standarization of reference reagents GM1 and
Anti-GMl antibodies. Ann Neurol 1990;27:no ppn.
28. Gibbs CJ Jr, Safar J, Ceroni M, DiMartino A, Clark WW, Hourrigan JL. Experimental transmission
of scrapie to cattle. Lancet 1990; 336, 551-4.
29. Godec MS, Asher DM, Swoveland FT, Eldadah ZA, Feinstone S, Goldfarb LG, Gibbs CJ Jr,
Gajdusek DC. Detection of measles virus genomic sequence in SSPE brain tissue by the polymerase
chain reaction. J Med Virol 1990; 30:237-44.
30-LCNSS
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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.
31-LCNSS
Z01 NS 01282-26 and Z01 NS 00969-26
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.
32-LCNSS
Z01 NS 01282-26 and Z01 NS 00969-26
60. Wills PR. Quantitative constraints on mechanisms of replication for information-carrying prions.
In: Iqbal, K., Wisniewski, H.M., Winblad, B., eds. Alzheimer's disease and related disorders.
New York: Alan R. Liss, 1989;669-77.
61. Wills PR. Induced frame-shifting mechanism of replication for an information-carrying scrapie
prion. Microb Pathogen 1989;6:235-49.
62. Wills PR, Hughes AJ. Stem loops in HIV and prion protein mRNAs. J AIDS 1990;3:95-7.
63. Wong T-W, Chan Y-C, Joo YG, Lee HW, Lee P-W, Yanagihara R. Hantavirus infection in humans
and commensal rodents in Singapore. Trans R Soc Trop Med Hyg 1989; 83:248-51.
64. Yanagihara R. Hantavirus infection in the United States: epizootiology and epidemiology. Rev
Infect Dis 1990;12:449-57.
65. Yanagihara R, Jenkins CL, Alexander SS, Mora CA, Garruto RM. Human T lymphotropic virus
type I infection in Papua New Guinea: High prevalence among the Hagahai confirmed by
Western analysis. J Infec Dis 1990;162:649-54.
66. Yanagihara R, Silverman DJ. Experimental infection of human vascular endothelial cells by
pathogenic and nonpathogenic hantaviruses. Arch Virol 1990;111:281-6.
33-LCNSS
701 NS 01282-26 and Z01 NS 00969-26
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
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ANNUAL REPORT
October 1, 1989 through September 30, 1990
Laboratory of Experimental Neuropathology
Basic Neurosciences Program, DIR, NINDS
Table of Contents
RESEARCH SUMMARY 1-5
PROJECT REPORTS
Herpesvirus Infections and Nervous System Diseases
Z01 NS 02549-09 LENP 6
Morphological Studies of Myelin Formation, Breakdown
and Regeneration
Z01 NS 01995-18 LENP 7
Biochemical and Immunologic Mechanisms in Virally-
Induced CNS Demyelination
Z01 NS 02550-09 LENP 8
Roles of Gangliosides in Neuronal and Myelin Function,
Cell Growth and Differentiation, and Neurotoxicity
Z01 NS 02699-05 LENP 9
Morphological Studies of Experimental and Human HIV
Infection
Z01 NS 02758-03 LENP 10
Lipid Peroxidation, Protein Oxidation and Demyelination
Z01 NS 02759-03 LENP 11
Mechanism of Latency and Pathogenesis of Herpes Simplex
Virus in the Nervous System
Z01 NS 02803-01 LENP 12
Nervous System Regeneration in a Herpesvirus Model
Z01 NS 02804-01 LENP 13
JC Human Polyomavirus Infection and Tumor Induction in the
Neonatal Brain
Z01 NS 02807-01 LENP 14
The Role of Protein Kinase C and raf Protein Kinase in
Cellular Functions
Z01 NS 02808-01 LENP 15
Glial Cells in Development and Viral Disease
Z01 NS 02809-01 LENP 16
i/LENP/DIR
ANNUAL REPORT
October 1, 1989 through September 30, 1990
Laboratory of Experimental Neuropathology, DIR
National Institute of Neurological Disorders and Stroke
Henry deF . Webster, Chief
The Laboratory of Experimental Neuropathology (LENP) includes the
Cellular Neuropathology Section (CN) and the Neurotoxicology Section (NT) .
The main goal of the Laboratory ' s research program is to investigate
cellular mechanisms of neurological diseases, especially those that are
directly related to progressive multifocal leukoencephalopathy (PML) , the
pathogenesis and latency of herpes simplex virus (HSV) infections,
multiple sclerosis (MS) , and age-related changes in peripheral nerves. Other
closely coordinated projects use biochemical and immunocytochemical
methods to explore gangliosides and protein phosphorylation . Some
neurotoxic actions also are being investigated.
The most important new finding in LENP1 s AIDS research was: Early
lesions in PML, an important complication of AIDS, were shown to consist
of glial hypertrophy in the absence of virus capsid production. In
established cases of PML, double- labeling showed that reactive astrocytes
resist productive JC virus (JCV) infection, suggesting a possible new
approach to treatment of this usually fatal disorder.
Other important discoveries were: 1 ) In the hamster model of JCV
infection, areas of gliosis were found in association with but independent of
tumor tissue, suggesting a second tissue response to JCV involving
hyperplasia without frank tumor induction. Further, we now have indica-
tions that endothelial cells are infected in the hamster model, and may play
a role in the pathogenesis of disease . Studies with hybrid JCV/SV40
regulatory regions in transgenic mice have shown that the regulatory
region, not the early protein (T-antigen) , determines the pattern of tumor
induction in this model . 2 ) In adult rat optic nerves, the a-subspecies of
protein kinase C existed in astrocytes in both native and partially degraded
forms . Some of the partially proteolyzed enzyme was shown to contain the
catalytic domain. By contrast, the 8- isozyme of protein kinase C was
always found as an intact macromolecule in the axons. These findings
suggest that the cellular mechanisms involved in the regulation of protein
kinase C activity in astrocytes and in axons are different. 3) An HSV-2
transcript which is expressed in neurons during latent infection has been
characterized; this transcript is similar but not identical to the latency-
associated transcript of HSV-1 . 4) Acute HSV-2 infection targets small
dorsal root ganglion neurons; selectivity is independent of virus strain and
only partly dependent on inoculation route. 5) Following corneal HSV
inoculation, the virus targets sensory sympathetic and parasympathetic
1/LENP/DIR
ganglia, and spreads to the central nervous system (CNS) by at least four
neural routes . 6 ) When nerve regeneration after crush injury was compared
in sciatic nerves of 6-month-old and 24-month-old mice, unmyelinated axon
regeneration was not significantly different. However, the number of myeli-
nated fibers in nerves of 24-month-old mice was only 38% of that seen in
the young adult ( 6-month-old) nerves. The myelinated fiber axons were
smaller and were surrounded by thinner myelin sheaths.
The following summary describes these discoveries and the most
significant new evidence obtained in FY 1990 LENP projects.
Human Polyomavirus JC in Animal Models and in Human Neurologic Disease
in AIDS and non-AIDS Patients.
The pathogenesis of JCV infection in the human brain has resisted
clarification ever since the recognition of PML as a fatal demyelinating
disease in 1 958 . In the absence of clear evidence, opinion has been about
equally divided between those who assume the disease is another oppor-
tunistic infection of patients with AIDS in which the virus invades tissue
where it is not normally resident, and those who assume that the virus is
reactivating frcm a latent state in the brain. Clearly, the latter alternative
holds more interest in terms of a possible role for JCV in other human
neurologic disease . The two areas of greatest interest in this regard are
the implications of JCV latency for brain tumor induction, particularly
tumors of childhood such as medulloblastomas, and the possible implications
of JCV latency for demyelinating diseases of unknown etiology, such as
MS . Since much evidence suggests that JCV is latent in the brain and can
reactivate following immunosuppression as occurs in AIDS, we have now
explored further the implications of latent JCV in the brain for demye-
linating diseases such as MS. For the first time, a comprehensive
hypothesis has been developed which has the potential to explain all facets
of the MS enigma. This hypothesis suggests numerous new avenues of
experimentation, some of which are currently being pursued. Our major
experimental model for JCV infection continues to be infection of the
neonatal hamster brain following intracerebral inoculation. Recent evidence
showing that JCV continues to express T-antigen in gliotic lesions of the
hamster brain independently of tumor induction is consistent with the
demonstration of early glial hypertrophy in incipient PML lesions, and
reemphasizes the relevance of the hamster model to several facets of the
host response to JCV in the human brain.
Herpesvirus Pathogenesis and Nervous System Disease
Mouse models of neurotropic herpesvirus infection provide important
opportunities to systematically examine disease mechanisms in the nervous
system. During FY 90, four areas of herpes simplex virus (HSV) patho-
genesis were investigated.
2/LENP/DIR
To determine which dorsal root ganglion (DRG) neuronal subpopulations
are targeted if inoculation route ( footpad and sciatic) and virus strain ( 1 86,
MS , HG52 ) are varied, mice were inoculated with HSV-2 strains. Peripheral
inoculation targets chiefly small neurons, while sciatic inoculation infects a
broader range of cell sizes . Altered neuronal peptide content was not
detected in immunohistochemical tests . HSV-2 strains differing in virulence
affect the numbers of DRG neurons that express viral antigen, but not the
specific subpopulation.
Three studies were performed in the mouse cornea model . In the
first, an HSV-2 transcript which is expressed in trigeminal ganglion neurons
during latent infection has been mapped and described. In addition to this
transcript , RNA transcription which could be detected and mapped by in
situ hybridization, occurred adjacent to and to the right of the map
position of HSV-2 LAT. The transcript is similar but not identical to that
found with HSV-1 infection.
In the second, HSV-1 spread from a peripheral (corneal) inoculation
site to tissues targeted by this injection was traced in the whole mouse
head . Results indicate that a much wider range of tissues are infected in
this model than was previously shown. Sensory, sympathetic and para-
sympathetic ganglia are infected, and observations suggest at least four
neural routes by which virus may enter the intracranial compartment.
In the third, studies to define characteristics of latent HSV-1 infection
in non-neuronal tissues have been undertaken.
Studies on nervous system disease caused by another neurotropic
herpesvirus, varicella-zoster virus (VZV), were initiated. In an autopsy
case , a meningoradiculopathy developed in the absence of detectable skin
lesions . Herpesvirus infection was identified at autopsy and using immuno-
histochemical and in situ hybridization methods, was shown to be due to
VZV infection . In a separate study, the literature on VZV-related arteritis
and angiitis of the CNS was reviewed.
Roles of Gangliosides and Protein Phosphorylation in CNS function and
Cellular Metabolism.
Gangliosides are sialic acid-containing glycosphingolipids ubiquitous in
eukaryotes . In mammals , these glycol ipids are most abundant in the CNS .
Studies in this laboratory have established that gangliosides can modulate
several protein phosphorylation systems in vitro. Yet, the mechanisms
involved in the perturbation or mobilization of cellular gangliosides for the
putative lipid second messenger function still remain unknown . To address
this problem, oligosaccharides were generated from different gangliosides
and their effects on protein phosphorylation were investigated . Preliminary
results indicated that the sialic acid-containing oligosaccharide portion of
G^ could enhance the autophosphorylation activity of a brain ganglioside-
stimulated protein kinase, albeit the effect is lower than those mediated by
ganglioside micelles. By contrast, the oligosaccharide derived
3/lenp/dir
from asialo-Gj^i is completely ineffective. These findings suggest that the
metabolic turnover of gangliosides, in analogy to the turnover of phosphati-
dylinositols and other phospholipids such as phosphatidylcholine, may play
an important role in regulating cellular signal transduction pathways .
The cellular biochemistry of ganglioside transport also was studied. In
skeletal and cardiac muscle, several ganglioside-binding proteins were
identified . Some of these proteins may be involved in the transfer of
gangliosides among different cellular compartments . Further investigation
of ganglioside-binding proteins may provide new insights into certain
ganglioside-related diseases . Gangliosides can bind myelin basic protein
(MBP) under in vitro conditions to form tight complexes that remain
undissociated in 8 M urea . Phosphorylation of MBP by protein kinase C
and by a ganglioside-stimulated protein kinase was altered by this protein-
lipid interaction . Electron microscopic (EM) experiments are in progress to
define the exact location of the myelin specific ganglioside GM1 in vivo and
to determine whether gangliosides and MBP could be colocalized in myelin.
Protein kinase C is a major Ca^+/phospholipid-dependent protein kinase
in the brain . The activity of this enzyme can be further regulated by
gangliosides . Using both immunocytochemical and immunological techniques,
we have shown that different isozymes of protein kinase C have different
subcellular distribution in the optic nerves of adult rats. The ma j or
isozyme is the type II (S) subspecies, and is found predominantly in the
axons as intact macromolecules . By contrast, the type III (a) isozyme is
localized almost exclusively in astrocytes. Some of type III isozymes were
in partially proteolyzed forms. Very little inmunostaining of the type I (y)
isozyme was observed in the optic nerves . Because different protein kinase
C isozymes have different responses to modulators such as arachidonic acid,
our results may provide a molecular basis for future studies on the
epigenetic and regulatory mechanisms of this protein phosphotransferase
system.
Nerve Regeneration During Aging
After inducing Wallerian degeneration by a crush injury, image analysis
and quantitative EM methods were used to compare nerve regeneration in
sciatic nerves of 6-month-old and 24-month-old mice. Two weeks after the
crush injury, there was no significant difference in the regeneration of
unmyelinated axons . In contrast, the number of regenerated myelinated
fibers in the aging nerves was only 38% of the number found in nerves of
the young adults , suggesting that regeneration of axons destined to become
myelinated might be impaired. In addition, the myelinated axons in the
aging nerves were smaller and had thinner myelin sheaths . Also, the aging
nerves had more single unmyelinated axons surrounded by one Schwann cell,
suggesting that Schwann cell ensheathment and segregation of single large
regenerating axons that precedes myelination also was slowed during aging.
Our discovery that in aging mice myelinated fiber regeneration is retarded,
4/LENP/DIR
is relevant to the management of peripheral nerve disease in older patients .
It may explain why older patients recover function more slowly in certain
types of peripheral neuropathy and after nerve injury.
5/LENP/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOINS 02549- 09 LENP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (SO characters or less. Title must fit on one line between the borders.)
Herpesvirus Infections and Nervous System Diseases
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: J. R . Martin, M.D. Medical Officer LENP, NINDS
Others: W.J. Mitchell, D.V.M., Ph.D. Sr. Staff Fellow LENP, NINDS
D.B.Henken,Ph.D. Staff Fellow LENP, NINDS
H.deF. Webster, M.D. Chief LENP, NINDS
COOPERATING UNITS (if any)
Department of Neurology, Colorado University, Denver, CO; Department of
Microbiology, USUHS, Bethesda, MD
LAB/BRANCH
Laboratory of Experimental Neuropathology
SECTION
Cellular Neuropathology Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
PROFESSIONAL: 1 1
OTHER: 0.6
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects [T] (b) Human tissues EH (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project seeks to identify nervous system diseases associated with the neurotropic
viruses herpes simplex types 1 and 2 (HSV-1, HSV-2) and varicella zoster virus (VZV), and
to examine mechanisms underlying production of neural lesions in experimental
models of infection. Problems of particular interest are: (i) the role of HSV and VZV
infection in the production of CNS and PNS disease, including acute encephalitis and
chronic demyelination, and (ii) mechanisms of neurotropic herpesviruses in CNS
arteritis and stroke-
During FY 1990, initial studies of human autopsy tissues to examine nervous system
disease caused by VZV showed infection of the CNS and PNS in an elderly patient with
neurological signs. Cutaneous manifestations of zoster were not evident, and VZV
infection was unsuspected. This case supports the hypothesis that VZV reactivations
may occur in the absence of peripheral cutaneous manifestations. In a separate study,
the literature on VZV-related arteritis and angiitis of the CNS was reviewed.
In experimental models, HSV-1 spread was traced from a peripheral (corneal)
inoculation site to other tissue targets in the whole mouse head. Virus spread in this
model, used for pathogenesis studies by many investigators, is more extensive than
previously recognized, including infection of an intracranial nerve and associated
artery. This provides the first direct evidence linking neural spread of a neurotropic
herpesvirus to arterial infection. In an associated study, replication of HSV-1 mutants
with genetic alterations in the joint region showed impaired replication together with
restricted spread in mouse tissues.
6/LENP/DIR
PHS 6040 (Rev 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBtR
ZOI NS0199S 18 LENP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (SO characters or less. Title mutt fit on one line between the borders.)
Morphological Studies of Myelin Formation, Breakdown and Regeneration
PRINCIPAL INVESTIGATOR (list other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI:
Others:
H.deF. Webster, M.D.
K..Tanaka,M.D.
M.G. Nunzi, M.D.
Chief
Visiting Fellow
Guest Researcher
LENP, NINDS
LENP, NINDS
LENP, NINDS
COOPERATING UNITS (if any)
None
LAB/BRANCH
Laboratory of Experimental Neuropathology
SECTION
Cellular Neuropathology Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
1.5
PROFESSIONAL:
0.4
OTHER:
1.9
CHECK APPROPRIATE BOX(ES)
I | (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues LLJ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The goal of this project is to use quantitative light (LM) and electron microscopy (EM)
along with other morphological and biochemical methods to study cellular mechanisms
of myelin formation, breakdown and regeneration. This year, to compare nerve
regeneration in young adult and aging mice, we transected sciatic nerves in 6- and 24-
month-old mice. After 2 weeks, the nerves were removed, transverse sections were cut
5 mm distal to the transection site, and regeneration of myelinated and unmyelinated
axons was studied quantitatively by LM and EM. The results showed that in sciatic
nerves of aging mice, regeneration of myelinated axons was retarded. In these nerves
there also were more Schwann cells that surrounded a single unmyelinated axon more
than one micron in diameter, suggesting that ensheathment of axons by Schwann cells,
a process that precedes the onset of myelin regeneration, also occurred more slowly
during aging. Mechanisms responsible for the delayed myelinated fiber regeneration
during aging are being explored in current experiments. Another important finding in
these experiments was that regeneration of unmyelinated axons in nerves from young
adult and aging mice did not differ significantly.
7/LENP/DIR
PHSHMdten. l««)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOI NS 02550-09 LENP
PERIOD COVERED
Octoberl, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or /ess. Title must fit on one line between the borders.)
Biolochemical and Immunologic Mechanisms in Virally-lnduced CNS Demyelination
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
PI:
Others:
G.L Stoner, Ph.D.
C.F. Ryschkewitsch.B.S.
H.deF. Webster, M.D.
H.G. Ressetar, Ph.D.
Chief, Neurotoxicology Section LENP, NINDS
Medical Technologist LENP, NINDS
Chief LENP, NINDS
Staff Fellow LENP, NINDS
COOPERATING UNITS (if any)
Dept. of Medical Microbiology, Univ. of Wise. Med. Sch. (D.L. Walker); Dept. of
Biochemistry, Penn. State Univ. (R.J. Frisque)
LAB/BRANCH
Laboratory of Experimental Neuropathology
SECTION
Neurotoxicology Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN-YEARS:
2.0
PROFESSIONAL:
1.0
OTHER:
1.0
CHECK APPROPRIATE BOX(ES)
J (a) Human subjects
] (a1) Minors
J (a2) Interviews
[X] (b) Human tissues Lj (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Evidence from several sources points toward the existence of latency by JC Virus (JCV)
in the CNS in a significant portion of the human population. This includes: (a) our own
studies of progressive multifocal leukoencephalopathy (PML) pathology, (b) The
appearance of PML in 2-5% of autopsied patients with acquired immunodeficiency
syndrome (AIDS), (c) Our evidence from the hamster model of JCV CNS infection, in
which JCV inoculated intracerebral^ in the neonatal hamster brain localizes in the
cerebellar granular layers, hippocampus and periventricular areas. This evidence
suggests a mechanism by which low levels of JCV entering the brain through the
circulation early in life might become focally distributed through replication as a
minichromosome in synchrony with host cell division, (d) Preliminary evidence from
studies of normal brain and brain with neurological disease other than multiple
sclerosis (MS) using the polymerase chain reaction (PCR) for amplification of JCV DNA
suggests that the virus might be present in a latent state in brains without overt
demyelinating disease. The weight of evidence in favor of latency by JCV in the brain
has led us to formulate a theory of pathogenetic mechanisms by which latent and
partially reactivating (limited to early region expression or other restricted expression
without independent DNA replication) might lead to immune-mediated demyelinating
disease, such as seen in MS. It is proposed that PML and MS may be the result of
differing host responses to the presence of virus focally distributed in the brain
following infection of glial cells or their precursors early in life. While JCV is a
candidate for involvement in both diseases, other DNA viruses with some specificity for
infection of and expression in glial cells should also be considered.
8/LENP/DIR
. .i
fn', 6040 (Hi-j 1 84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02699-05 LENP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ ECT (SO characters or less. Title must fit on one line between the borders)
Roles of Gangliosides in Neuronal and Myelin Function, Cell Growth and Differentiation, and Neurotoxicity
PRINCIPAL IN VE STIG A TOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: K.-F.J. Chan, Ph.D. Sr. Staff Fellow LENP, NINDS
Others: Y. Liu, M.D. Visiting Fellow LENP, NINDS
COOPERATING UNITS (itany)
None
LAB/BRANCH
Laboratory of Experimental Neuropathology
SECTION
Neurotoxicoloqy Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN-YEARS: --
PROFESSIONAL: 1 g
OTHER:
0.2
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects [T] (D) Human tissues ] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The main goal of this project is to establish a functional role for gangliosides as
multifunctional biomodulators. Studies in this laboratory revealed that gangliosides
have profound modulatory effects on protein phosphorylation and dephosphorylation
in the central nervous system and in several other tissues such as skeletal muscle. This
lipid second messenger-like action is mediated in part through the regulation of two
classes of protein kinases, namely, qanqlioside-dependent and qanqlioside-modulated
protein kinases. Preliminary studies revealed that certain sialic acid-containing
oligosaccharides derived from gangliosides could enhance the autophosphorylation
activity of a ganglioside-stimulated protein kinase. This finding suggests that
perturbation of cellular gangliosides may play a role in altering metabolic signal
transduction pathways
The mechanisms of intracellular trafficking of gangliosides also were investigated
We found that both skeletal and cardiac muscle contain unique qanqlioside-bindinq
proteins. Further characterization of these proteins may provide new information on
the biochemistry of cellular gangliosides and perhaps more insights in the pathogenesis
of ganglioside-related diseases.
9/LENP/DIR
PHSiMiVw. 1141
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOI NS 02758-03- LENP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less, rule must fit on one line between the borders.)
Morphological Studies of Experimental and Human HIV Infection
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) {Name, title, laboratory, and institute affiliation)
PI: H. deF. Webster, M.D. ~^ef LENP, NINDS
Others:
COOPERATING UNITS (,f an,)
None
LAB/BRANCH
Laboratory of Experimental Neuropathology
SECTION
Cellular Neuropathology Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN-YEARS: n~
PROFESSIONAL: Q Q
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects ] (b) Human tissues 0 (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Due to the departure of Drs. Lamperth (LENP, NINDS), Leonard (LMM, NIAID) and
Pezan (LMM, NIAID) this project was terminated.
Publications: None.
10/LENP/DIR
PHS 6040 (Rev 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBtR
ZOINS 02759 03 LENP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters ot less. Title must fit on one line between the borders)
Lipid Peroxidation, Protein Oxidation and Demyelination
PRINCIPAL INVESTIGATOR (list other professional personnel below the Principal In vestigator ) (Name, title, laboratory, and institute affiliation)
PI: Hubert Mickel, M.D. Guest Researcher LENP, NINDS
COOPERATING UNITS (,tany)
None
LAB/BRANCH
Laboratory of Experimental Neuropathology
SECTION
Cellular Neuropathology Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN YEARS.
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a)Human subjects ] (b) Human tissues EH (c) Neither
] (a1) Minors
I I (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project has been terminated.
Publications:
Mickel HS, Kempski O, Feuerstein G, Parisi JE, HdeF Webster. Prominent white matter
lesions develop in Mongolian gerbils treated with 100% normobaric oxygen after
global brain ischemia. Acta Neuropathol 1990;79:465-72.
11/LENP/DIR
PHSbWOIRt. 1 84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOINS 02803-01 LENP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Mechanism of Latency and Pathogenesis of Herpes Simplex Virus in the Nervous System
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: W.J. Mitchell, D.V.M., Ph.D. Sr. Staff Fellow LENP, NINDS
Others: J. R. Martin, M.D. Medical Officer LENP, NINDS
COOPERATING UNITS Of any)
None
LAB/BRANCH
Laboratory of Experimental Neuropathology
SECTION
Cellular Neuropathology Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
10
OTHER: q2
CHECK APPROPRIATE BOX(ES)
[_] (a) Human subjects J (b) Human tissues E (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project is aimed at understanding aspects of the pathogenesis of herpes simplex
virus (HSv) infection in the nervous system including the mechanism by which HSV
infections are regulated within neurons during acute, latent, and reactivated
infections. These studies which utilize a mouse model system and cultured cells should
allow a better understanding of the pathogenesis of herpesvirus-induced disease in
humans.
During FY 1990 an HSV-2 RNA transcript which is expressed in neurons during latent
infection has been characterized; this transcript is similar but not identical to the
latency associated transcript of HSV-1. Further studies are in progress to define the
differences between the latency associated transcripts of HSV-1 and HSV-2 Despite
numerous studies the role of these viral latency associated transcripts in regulation of
HSV infections is not clear Studies are in progress to define the characteristics of latent
HSV-1 infection in non-neuronal tissue. Experiments have been initiated to define a
model for investigating the role of nerve growth factor and other host gene products
in the regulation of viral gene expression during latent infections of neurons.
12/LENP/DIR
PHS 6040 (Rev 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOI NS 02804 01 LENP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ E CT (80 characters or less, title must fit on one line between the borders.)
Nervous System Regeneration in a Herpesvirus Model
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: D.B.Henken.Ph.D. Staff Fellow LENP, NINDS
Others: J. R. Martin, M.D. Medical Officer LENP, NINDS
COOPERATING UNITS vfmy)
None
LAB/BRANCH
Laboratory of Experimental Neuropathology
SECTION
Cellular Neuropathology Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN-YEARS: ?
PROFESSIONAL: 1 q
OTHER: q 2
CHECK APPROPRIATE BOX(ES)
|_J (a) Human subjects ] (b) Human tissues l~x~l (0 Neither
] (a1) Minors
] (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
One of the distinguishing features of alpha herpesviruses is their propensity to infect
and then establish latency in sensory ganglion cells. The initial portion of this project
aims at determining whether there are specific subpopulations of dorsal root ganglion
(DRG) neurons, as characterized by their morphology, connections and neuropeptide
content, that initially become infected following herpes simplex virus type-2 (HSV-2)
inoculation and later harbor latent virus. Major findings are: 1) Route of inoculation
plays an important role in determining which populations of DRG neurons are acutely
infected. Peripheral inoculation establishes HSV-2 infection in the small subpopulation,
while intraneural injection targets a slightly broader spectrum of cell sizes. 2)
Modification of host cell peptide production is not detectable with
immunocytochemical methods during the acute phase of infection. In situ
hybridization histochemistry for peptide mRNA would be a more sensitive indicator
and will be examined. 3) HSV-2 strains differing in virulence affect the numbers of DRG
cells that express viral antigen, but not the specific subpopulation. The effects of HSV-2
inoculation route and virus strain on the latent phase of DRG infection will next be
examined. These experiments lay the groundwork for the interpretation of
subseguent central and peripheral nervous system regeneration studies in a herpesvirus
model.
13/LENP/DIR
PHSMWOIRev 1 84|
DEPARTMENT OF HEALTH AND HUMAN SERVICES -PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOINS 02807-01 LENP
PERIOD COVERED
October!, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or leu Title must fit on one fine between the borders.)
JC Human Polyomavirus Infection and Tumor Induction in the Neonatal Brain
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI:
Others:
H.G. Ressetar, Ph.D.
G.L Stoner, Ph.D.
H.deF. Webster, M.D.
Staff Fellow
Section Chief
Chief
LENP, NINDS
LENP, NINDS
LENP, NINDS
COOPERATING UNITS (it any)
Dept. Medical Microbiology, University of Wisconsin Medical School (D.L. Walker); Lab.
of Molecular Oncology, Alton Ochsner Med. Foundation, New Orleans, LA (O. Prakash)
LAB/BRANCH
Laboratory of Experimental Neuropathology
SECTION
Neurotoxicoloqy Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN-YEARS:
1.0
PROFESSIONAL:
1.0
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
J (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues H (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The goal of this project is to examine the possible role of the human polyomavirus, JC
virus (JCV) in perinatal brain infection. Childhood acquisition of JCV is followed by
viral latency and reactivation of JCV in the kidney with shedding of virus occurring
during pregnancy and the postpartum period. When injected intracerebral^ into
newborn hamsters, JCV establishes a nonproductive brain infection with subsequent
tumor formation. The implications of this for human perinatal infection are being
explored through three main projects: a) Studies utilizing the hamster model have
revealed that JCV preferentially infects mitotic cells in the subventricular zone and sites
of postnatal granule neuron production in the developing brain. Further investigation
has suggested that JCV infection of blood vessel endothelial cells and local astrogliosis
may be involved in the mechanisms of tumor induction Tumors induced in 6 -month-
old hamsters exhibit variable expression of JCV early protein product, T-antigen,
emphasizing that the virus is not readily detected in the cells it transforms, b)
Preliminary studies examining human pediatric tumor tissue for the presence of JCV or
its T-antigen are being performed using immunostaining and PCR techniques, c) In
collaboration with Dr. Prakash, 4 transgenic mice have been produced that carry the
hybrid regulatory and coding early regions from JCV and SV40. Observed pathology
included an abdominal lymphoma and adrenal tumor in one mouse, a choroid plexus
papilloma in a second mouse and a brainstem tumor in a third animal. Splenic, thymic
and epidermal abnormalities were also noted.
14/LENP/DIR
PHS 6040 (Rev VS4)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOI NS 02808-01 LENP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less, rule must fit on one line between the borders.)
The Role of Protein Kinase C and raf Protein Kinase in Cellular Functions
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: S. Komoly, M.D.
Others: Z. Olah, Ph.D.
D.VAgoston.M.D.
E. Mezey, M.D.
K.-F. J. Chan, Ph.D.
Y. Liu, M.D.
H.deF. Webster, M.D.
Visiting Scientist
Visiting Fellow
Visiting Scientist
Visiting Scientist
Sr. Staff Fellow
Visiting Fellow
Chief "
LENP, NINDS
LCO, NCI
LCB, NIMH
LCB, NIMH
LENP, NINDS
LENP, NINDS
LENP, NINDS
COOPERATING UNITS (,fan,)
None
LAB/BRANCH
Laboratory of Experimental Neuropathology
SECTION
Neurotoxicoloqy Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN YEARS:
1.2
PROFESSIONAL:
1.2
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
I | (a) Human subjects
] (a1) Minors
J (a2) Interviews
Q (b) Human tissues [*J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The goal of this project is to study the role of protein kinases in cellular metabolism and
signaling using immunocytochemical methods along with biochemical techniques.
Current studies and major findings are: 1) We showed for the first time that protein
kinase C isozymes type II (beta) and type III (alpha) but not type I (gamma) can be found
in rat optic nerve. Type II (beta) is localized in axons, while type III (alpha) is expressed
exclusively by the astrocytes. Accordingly, in the retina type II (beta) isozyme was found
in the ganglion cells and nerve fiber layer, type III (alpha) in the Muller cells and
interneurons. Intracellular^ the protein kinase C-like immunoreactivity was associated
in addition with cell membranes with cytoskeletal structures such as glial filaments
(alpha-isozyme), neurotubules and neurofilaments (beta-isozyme). 2) Comparative
studies revealed that delipidation of the specimens abolishes the protein kinase C-like,
but not the raf protein kinase-like immunostaining in frozen and vibratome sections.
Quantitative image analysis of immunostained fibroblast cultures revealed cell density
dependent translocation of raf protein kinase from the cytoplasm into the nucleus.
Protein kinase C did not show this distribution. In contrast, phorbol ester treatment
caused nuclear translocation and down-regulation in a dose- and time-dependent
manner of protein kinase C while such an effect of phorbol ester was very limited on
raf protein kinase. 3) Translocation of raf protein kinase from cytoplasm into the
nucleus was also observed in hippocampal neurons in experimental cerebral ischemia.
4) In preliminary immunocytochemical experiments the localization of GM1
qanqlioside was examined with the use of cholera toxin B-subunit. In the brain the B-
subunit binding sites were most dense in myelinated pathways, while in tissue cultures
(fibroblast, neuroblastoma, embryonic spinal cord cell lines) the immunostaining
suggested highest GM1 concentration in neuronal cells. An increase in "B-subunit
binding capacity" of the cellular membranes was also noted with increasing cell
density.
15/LENP/DIR
PHS MHO (Rev 1 84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOI NS 02809-01 LENP
PERIOD COVERED
Octoberl, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or /ess. Title must fit on one line between the borders.)
Glial Cells in Development and Viral Disease
PRINCIPAL INVESTIGATOR (list other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: K.E. Astrom, M.D.
Others: G.L Stoner, Ph.D.
H.deF. Webster, M.D.
Visiting Scientist
Section Chief
Chief
LENP, NINDS
LENP, NINDS
LENP, NINDS
COOPERATING UNITS Ofany)
None
LAB/BRANCH
Laboratory of Experimental Neuropathology
SECTION
Neurotoxicoloqy Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN-YEARS:
1.0
PROFESSIONAL:
1.0
OTHER:
CHECK APPROPRIATE BOX(ES)
I | (a) Human subjects
] (a1) Minors
J (a2) Interviews
QT] (b) Human tissues LJ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The long-range goal of this project is to study the normal early development of glial cell
precursors in the telencephalon and the affect on of glial cells by virus, notably the JC
virus. Major findings and current studies are: (1) Methods for the study of cellular
shape, growth patterns and fine structure during early stages of development were
evaluated and modified to meet certain criteria. These methods can be used for
electron microscopic, immunocytochemical and in situ hybridization studies of early
glial and neuronal development. (2) The neopallial wall and area choroidea in 11-12-
day -old fetal rats form a continuous, nonstratified, cohesive monolayer of elongated,
radially oriented polarized cells. During the early development of telencephalon, the
epithelial character is maintained but modified locally to serve various functions. Of
particular interest is that the appearance of cells in area choroidea is similar to that of
polarized cells with high metabolic activity elsewhere in the body, e.g., the kidney
epithelium. This observation supports a notion that JC virus may enter CNS through the
apical surfaces of cells in the choroid area and plexus. (3) The study of two preclinical
cases of progressive multifocal leukoencephalopathy (PML) have given new
information on the early appearance and progress of PML-lesions in the brain. Cells
with capsid-antigen were never seen outside areas of myelin destruction and sections
stained for GFAP showed some areas which contained reactive astrocytes but lacked
destruction of myelin. The latter finding suggests that reaction of astrocytes as
manifestation of disease may exist independently of myelin destruction.
16/LENP/DIR
PHS 6040 (Rev. 184)
>
00
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03
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03
ANNUAL REPORT
OCTOBER 1, 1989 THROUGH SEPTEMBER 30, 1990
LABORATORY OF MOLECULAR BIOLOGY
TABLE OF CONTENTS
Page
RESEARCH SUMMARY 1
PROJECT REPORTS
Differentiation and Regulation of Gene
Expression in Astrocytes and Neurons
Z01 NS 02677-06 5
Neurotransmitter Gene Studies
Z01 NS 02800-02 6
Mechanisms of Neurotransmitter-
Receptor Interactions in Mammalian
Neurons
Z01 NS 02365-12 7
Biology of Mammalian Homeodomain
Proteins
Z01 NS 02698-05 8
Cloning and Functional Analysis of Genes
Active in Neurogenesis
Z01 NS 02820-01 9
Annual Report
October 1, 1989 Through September 30, 1990
Laboratory of Molecular Biology
Basic Neurosciences Program/DIR
National Institute of Neurological Disorders and Stroke
Franklin G. Hempel, Ph.D., Acting Chief
The Laboratory of Molecular Biology (LMB) is continuing to investigate the structure
and function of genes that code for enzymatic or structural proteins important in
neural cells. Enzymes that regulate neurotransmitter metabolism, receptors for
neurotransmitters, and filament proteins are included in these ongoing projects.
The Laboratory has concluded research studies on neural excitatory amino acid
receptors and the effects of substrate and ionic conditions on cell survival, while
beginning new work on neurogenesis and homeodomain proteins in Drosophila and
murine models.
1 . Differentiation and regulation of gene expression in neurons and astrocytes.
The synthesis of glutamine from glutamate is catalyzed by glutamine synthetase
(GS), and glutamine in turn is hydrolyzed to glutamate and NH4+ by glutaminase
(GA). This cycle and the synthesis of GABA from glutamate play a pivotal role in
neural function. The LMB has investigated the appearance of GS mRNA in rat brain
astrocytes during development from embyronicday 14 (E 14) through adult.
Northern blots, analyzed with full-length rat cDNA probes, show GS mRNA size
classes of 1.4 and 2.8 kb as early as day E14. Message levels reached near-adult by
postnatal day 1 5, when type II astrocytes differentiate. In primary cultures of rat
cortical astrocytes and C6 glioma cell lines, GS expression was elevated by
dexamethasone and forskolin, and induced in astrocytes by glutamate or by
coculturing with glutamatergic rat cerebellar granule cells. Neither human
neuroblastoma cells (SKN-SH-SY5Y) nor PC12 cells increased the GS mRNA pool.
To study the genomic regions that regulate GS expression, a 1 5 kb fragment of rat
genomic DNA was cloned from a Charon 4A genomic library. Southern blots of
restriction digests of the genomic clone, hybridized with a lab cDNA clone, iden-
tified regions coding for GS. Seven exons and a 2 kb intron in the 5' untranslated
region were found. To determine the start site for GS transcription, a synthetic
complimentary oligonucleotide was made for bases +95 to + 1 15 in the first exon.
The start site was identified 5 bases downstream from that of other species. A TATA
box was found 28 bp upstream from the start site; a GC-rich region was found at -50;
and at + 65 a CCAAT box was identified.
A rat brain cDNA library was constructed in the pCD2 vector and screened for GA,
using a 500 bp partial cDNA clone for GA. A 4 kb cDNA clone was isolated and its
identity con-firmed by sequence homology to the original GA sequence. This clone
contains a sequence extending from a poly A tail at the 3' terminus toward the 5'
end for 100 bp beyond the older 500 bp clone. The older GA cDNA clones all
extended into introns at their 5' termini as judged by a breakdown in the coding
sequence and a 3' splicing consensus sequence. The new clone contains divergence
atthe 5' end from that of the previous clones, and maintains the open-reading
frame, suggesting that it is derived from a processed message whose sequence
extends past this 5' intron. By using the additional 5' sequence past the intron,
oligonucleotides were designed and used in a RACE protocol to generate a series of
clone-containing sequences from the 5' end of the clone.
l -LMB/DIR
Astrocytes synthesize and secrete only the B2 chain of laminin, a potent neurite
growth-promoting factor consisting of three subunits: A chain, B1 chain, and B2
chain. Transfection of cDNA constructs containing gene promoter elements for B1
and B2 laminin into astrocytes and HepG2 cells (which produce all three subunits of
laminin) has shown that laminin subunit synthesis is controlled at the level of
transcription. By use of deletion mutants, the promoter element of the B2 laminin
gene was located within 200 bp of the gene.
Astrocytes also exhibit collagen type IV mRNA. Analysis of protein synthesis by
immunoblot and immunoprecipitation techniques, and double-labelling immuno-
fluorescence of cultured astrocytes with anti-GFAP (glial fibrillary acidic protein) all
show expression of collagen type IV.
GFAP, a major subunit of intermediate cellular fragments, found exclusively in
astrocytes and used to identify them, is expressed by the gfa gene. Transfection of
cultured cells with chloramphenicol acetyltransferase (CAT)-reporter gene constructs
is now being used to identify cis-acting sequences responsible for cell-specific
expression of the human gfa gene. A 2.2 kb 5' fragment has been found to drive
expression of the CAT gene in the human glioma cell line U251, but not in the
nonglial cell lines HepG2, HeLa, and BHK. Analysis of deletion mutants has localized
two regions within the segment which appear to interact synergistically to enhance
expression, either alone supporting little if any CAT expression. Footprint analysis
shows that both regions are strongly bound by proteins. The distal region is located
about 1 500 bp upstream of the RNA start site. Its displays two closely-spaced
footprints, each covering about 30 bp of DNA. The proximal region is located about
100 bp upstream of the RNA start site, and displays a single footprint of about 30 bp.
The promoter region of the human gfa gene was found to contain two elements
that can independently specify the correct transcription startpoint. One of the
elements is a TATA box found 25 bp upstream from the transcription startpoint,
while the other is located between 10 and 50 bp downstream from the transcription
initiation site. Surprisingly, most of the downstream element overlaps with the
protein encoding sequence. Binding of purified transcription factor IID (TFIID) to the
TATA box was affected by a downstream initiation element when human or yeast
TFIID is used.
To understand the genetic mechanisms controlling calcium gene expression, cDNA
and genomic clones for the calcium channel gene were obtained. By screening rat
brain cerebral cortex and hippocampus cDNA libraries with a set of oligonucleotide
probes based on nucleotide sequences of rabbit skeletal muscle dihydropyridine
(DHP) receptor a1-subunitcDNA, the LMB isolated and sequenced several cDNAs
corresponding to the mRNA encoding a1-subunit of L-type voltage-sensitive calcium
channel (VSCC). Nucleotide sequence analysis of the cDNAsshow at least four
different classes of cDNAS, indicating that rat brain expresses multiple forms of the
a1-subunit of VSCC, probably from separate calcium channel genes.
Expression in rat brain of mRNA corresponding to VSSC a1-subunit cDNAs was
examined by in situ hybridization histochemistry. While the mRNA for one of the
cDNA clones (BCCII) was widely distributed both in neurons and glial cells, the mRNA
for BCCI cDNA was found only in brain neurons, such as the olfactory bulb and
piriform cortex, hippocampal formation, suprachiasmatic nucleus and median
preoptic nuclei of the hypothalamus, the pituitary and pineal glands. Northern blot
analysis revealed that two poly(A) + RNA species of s8 kb and ^1 2 kb which
hybridized to BCCI cDNA markedly increased when intracellular levels of cAMP were
2-LMB/DIR
also increased in NG108-15 cells. The BCCI calcium channel gene is located on mouse
chromosome 14 and human chromosome 3.
2. Genes encoding neurotransmitter receptors.
The LMB has made considerable progress in characterizing the structure and genetic
expression of acetylcholine and dopamine receptors, and their localization in the
CNS. Each of the 5 muscarinic cholinergic subtypes has been expressed in CHO and
A9-L cells. In these cells, muscarinic receptors can be divided into two classes: ml,
m3, and m5 versus m2 and m4. The odd-numbered receptors increase cAMP levels
and phosphatidyl inositol metabolism, and also open potassium channels by
coupling to pertussis-insensitive G-proteins, while the even-numbered muscarinic
receptors decrease cAMP levels by coupling to pertussis toxin-sensitive G-proteins.
A region of strong homology exists within an 18-amino acid intracellular stretch of
the receptors that is predictive of their coupling selectivity of the odd- versus even-
numbered muscarinic receptors. In chimeric m2-m3 receptors, this small domain is
sufficient to reverse the coupling selectivity of m2 and m3 receptors with respect to
inositol phosphate metabolism as well as cAMP metabolism and coupling to G-
protems. The genetic identity of the individual G-proteins that mediate functional
responses of the muscarinic receptors was shown by coexpression of individual
receptor cDNAs with individual G-protein cDNAs. For m4 receptors, coexpression of
the receptor cDNA with the G-protein Go inhibits adenylate cyclase more than m4
receptors expressed alone.
From a previously published dopamine D2 receptor sequence, the LMB has prepared
oligodeoxynucleotide probes and applied in situ hybridization procedures to map
the distribution of this mRNA. It was shown that this mRNA encodes both pre- and
postsynaptic dopamine D2 receptors in three major ascending dopaminergic
pathways: the nigrostriatal, mesolimbicand mesocortical pathways. D2 mRNA is
also located on various cholinergic cell groups in the forebrain. Using the same
probes, the laboratory found that these mRNAs are present only in the inner retina
and not in the photoreceptors in rats. A similar distribution was seem in monkey
retina.
J. Boulter (Salk Institute) supplied the laboratory with cDNAs that encode for the
a2, a3, a4,and 82 subunits of the neuronal nicotinic receptors. These cDNAs were
subcloned into the expression vector pCD-PS, previously used to express the
muscarinic acetylcholine receptors and G-proteins in mammalian cells. If the a3 and
a4 cDNAs are coexpressed with the 82 cDNA/then membranes are obtained that
bind acetylcholine. Conversely, if the DNAs are expressed alone, there is no binding.
These data indicate that functional receptors of this type can be obtained using our
expression system. This work represents the first expression in mammalian cells of
neuronal nicotinic receptors.
From functional and pharmacologic data, it can be predicted that nicotinic acetyl-
choline and glutamate receptors may belong to a homologous family of genes. To
clone the glutamate receptors, oligodeoxynucleotide probes were assembled for a
region of the neuronal nicotinic receptors expected to be in the receptor channel
lining of the neuron. Using this probe, the same human retinal library was screened
as for the dopamine receptors. Full-length human a2, a 3, a 4, 82 and 84 clones,
and a human glutamate receptor have been sequenced and cloned.
3 -LMB/DIR
3. Neurotoxicity of excitatory amino acids.
The LMB has focused on the mechanisms of receptor-mediated EAA activity and the
transition of EAAs from neurotransmitters to neurotoxins. Such a transition has
been postulated as the possible cause of neuronal death in various neuropathology
conditions such as cerebral ischemia, Huntington's disease, and Alzheimer's disease.
Results show that glucose metabolism and ATP production are necessary to maintain
the voltage-dependent Mg2 + block of the NMDA receptor channel, and that relief of
the block renders the neurons vulnerable to the toxic effects of glutamate.
Glutamate istoxicto cerebellar granule cells in primary culture when Mg2+ is
removed from the incubation medium. With Mg2 + present but glucose removed
from the medium, glutamate is also toxic due to partial depolarization resulting
from energy depletion; the partial depolarization leads to relief of the Mg2+ block
which is known from electrophysiologic evidence to be voltage-dependent. When
the neurons are deprived of oxygen or when an inhibitor of oxidative phosphory-
lation is added, glutamate is toxic even in the presence of both Mg2 + and glucose,
again due to energy depletion and partial depolarization. In every case, the toxicity
of glutamate is blocked by CPP (( ± )-3-(2-carboxypiperazine-4-yl)-propyl-1-phos-
phonic acid) and D(-)-2-amino-5-phosphonovaleric acid (APV), the most potent and
selective competitive antagonists of the NMDA receptor. Adrenal corticosteroids
can potentiate the neurotoxicity of glutamate and other agonists at the NMDA
receptor. Pretreatment of neonatal rat granule cells with low levels of cortico-
sterone permits low concentrations of NMDA agonists to become toxic in the
presence of Mg2+ and glucose. Corticosterone appears to potentiate NMDA agonist
toxicity by relieving the NMDA channel Mg2+ block via compromised energy
availability.
4. Cloning and analysis of genes in neural development.
The LMB has generated 6 lines of healthy transgenic mice that harbor in their
genome between one and approximately 25 copies of a DNA fragment that contains
1.8 kb of the Mx1 promoter, a Hox 1.3 cDNA, and a 3' noncoding region derived
from the mouse beta-globin gene. Transgenic mRNA levels are below detectability
in most organs (i.e., below endogenous Hox 1 .3 mRNA). However, when interferon
is injected as a single dose into these transgenic mice, transgenic Hox 1 .3 mRNA and
protein rapidly accumulate to levels that far exceed those of endogenous Hox 1 .3
transcripts and protein. The induced protein accumulates in the nuclei of cells where
it can be labelled with a specific antibody. Western blot analysis shows that this
protein comes in multiple posttranslationally modified forms, much the same as the
endogenous Hox 1 .3 protein. Interferon injected into non-transgenic pregnant mice
that carry in utero transgenic embryos, will activate the transgene as early as day 11
of gestation, again to levels higherthan their endogenous Hox 1.3 expression. This
observation opens up the possibility of determining the exact time window in
development during which Hox 1 .3 expression may affect embryogenesis.
4 -LMB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02677-06 LMB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 character* or less. Title must fit on one line between the borders.)
Differentiation and Regulation of Gene Expression in Astrocytes and Neurons
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.:
Others:
E. Freese,* Ph.D., Chief, LMB, NINDS
M. Brenner, Ph.D., Special Expert, LMB
H. Chin, Ph.D., Senior Staff Fellow, LMB
Y. Nakatani, Ph.D., Visiting Associate, LMB
J. Mill, Ph.D., Senior Staff Fellow, LMB
J. Wujek, Ph.D., Senior Staff Fellow, LMB
R.King, Ph.D., IRTA, LMB
K. Mearow, Ph.D., Visiting Fellow, LMB
H. Purohit, Ph.D., Visiting Fellow, LMB
F. Besnard, Ph.D., Visiting Fellow, LMB
V. Kedar, Ph D., Visiting Associate, LMB
COOPERATING UNITS Of any)
Dr. Y. Yamada, LDBA, NIDR, NIH
Dr. R. Wenthold, LMO, NIDCD, NIH
Dr. M Nirenberg, LBG, NHLBI, NIH
LAB/BRANCH
Laboratory of Molecular Biology, BNP
SECTION
Developmental Biology Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
8.3
PROFESSIONAL:
76
OTHER:
0.7
CHECK APPROPRIATE BOX(ES)
J (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues 0 (0 Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The genes for a number of neurologically important proteins have been isolated and sequenced, and
their control mechanisms are being studied. In particular, we want to understand why certain genes are
expressed in neurons but not in astrocytes and vice versa. Laminin is a component of the extracellular
matrix of astrocytes and promotes neurite outgrowth. In basal lamina cells laminin normally consists of 3
chains (A, B1, B2). Using chain-specific antibodies and small cDNAs, we have shown that astrocytes make
only the B2 mRNA and protein of laminin. The promoter region responsible for this specificity has been
localized within 200 bp of the B2 gene. Glial fibrillary acidic protein (GFAP) is an intermediate filament
protein found only in mature astrocytes. It is transcribed by RNA polymerase II but, in contrast to other
genes, uses as promoter not only a TATA box 25 bp upstream of the start site but also a downstream
element 10 to 50 bp distant. Glutamine synthetase (GS) converts glutamate to glutamine which then
enters neurons and is converted to glutamate or GABA. The promoter for the GS gene contains a TATA
box, 28 bp upstream from the transcription start site. Sequences with homology to cAMP and
glucocorticoid response elements have also been found. Deletion mutants with the GS promoter and a
CAT reporter gene have been constructed and transfected into several cell lines in order to examine the
functional significance of the regulatory sequences. Genomic clones and cDNAs for the L-type calcium
channel gene have been obtained. At least 4 forms of the a1 subunit of the channel are expressed.
Hybridization experiments reveal high levels of neuron-specific calcium channel mRNA in the olfactory
bulb, hippocampal CA1 cells, dentate gyrus, suprachiasmatic nucleus, and the medial preoptic nucleus.
Other sites also expressed activity. Work is continuing toward isolating a human calcium gene.
*Dr. E. Freese died on March 30, 1990.
5-LMB/DIR
PHSMMOtRcv. I'M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02800-02 LMB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (SO characters or less. Title must fit on one line between the borders.)
Neurotransmitter Gene Studies
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: M.R. Brann, Ph.D. Senior Staff Fellow LMB, NINDS
Others: D. Weiner HHMI Fellow LMB, NINDS
J.Wess, PhD Visisting Associate LMB, NINDS
B. Novotny, M.S. Physiologist LMB, NINDS
A. Levey, M.D., Ph.D. Special Volunteer LMB, NINDS
D. Gdula, B.S. Biologist LMB, NINDS
S.H.Yu, M.D. Visiting Associate LMB, NINDS
COOPERATING UNITS of any)
S. Jones, J.L Barker, LNP, NINDS; W. Simonds, A. Spiegel, MPB, NIDDK; H. Arnheiter, LMG, NINDS; T.
Bonner, LCB, NIMH; J. Ellis, Univ. VT; S. Jones, N. Nash, T. Stormann, R-Gene, CRADA
LAB/BRANCH
Laboratory of Molecular Biology, BNP
SECTION
Developmental Biology Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
4.7
PROFESSIONAL: j 7
OTHER: 2 0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects [T] (b) Human tissues ] (c) Neither
] (a1) Minors
_J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Our work involves the study of genes encoding receptors that bind the neurotransmitters acetylcholine,
dopamine, and qlutamate. Previous work led to the molecular cloning, sequencing and expression of a
family of muscarinic acetylcholine receptor subtype genes. We have continued to characterize these
subtypes pharmacologically with, regard to agonists and antagonists, anatomic distribution, their
selectivity of coupling to G-proteins, second messengers and ion channels, and the development of
antibodies specific for each protein subtype. Using the recently reported sequence of a rat cDNA that
encodes a dopamine D2 receptor, we have mapped the distribution of the corresponding mRNA in rat
retina and brain, and have cloned, sequenced and expressed a cDNA that encodes the human homolog
of this receptor and likewise a human dopamine D1 receptor. This work has provided evidence that
dopamine receptors, like muscarinic receptors, may be derived from a family of genes. We are
attempting to clone the other members of the family, and to study the selectivity of coupling of the
cloned dopamine receptors to G-proteins, second messengers, and ion channels. Finally, sequences of
the rat cDNAs that encode neuronal nicotinic acetylcholine receptors have been reported, and we have
cloned some of the human homologs of these receptors. Because of a hypothetical evolutionary
similarity between glutamate and nicotinic acetylcholine receptors, we have been attempting to clone
the qlutamate receptors by homology cloning. As a result, we recently cloned, sequenced and expressed
the human homolog of the rat glutamate receptor.
6-LMB/DIR
PHS6M0(R»w. 1/MI
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02365-12 LMB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or loss. Title must fit on one line between the borders.)
Mechanisms of Neurotransmitter-Receptor Interactions in Mammalian Neurons
PRINCIPAL INVESTIGATOR (I 'it other professional per sonne/ below the Principal Investigator.) {Name, title, laboratory, and institute affiliation)
P. I.: R.C. Henneberry, Ph.D. Chief, Molecular Neurobiology Section LMB, NINDS
Others: P.G. Lysko, Ph.D. Guest Researcher
J A. Cox, Ph.D. Visiting Associate
M. Voigt, Ph.D. NRC Fellow
COOPERATING UNITS of any)
Enzyme Chemistry Section, Laboratory of Neurochemistry, BNP, DIR, NINDS
LAB/BRANCH
Laboratory of Molecular Biology
SECTION
Molecular Neurobiology Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL: . ■,
OTHER:
06
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [x~l (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The mechanisms by which neurotransmitters become neurotoxins and the role of these toxins in the
death of neurons characteristic of neurodegenerative disorders such as Alzheimer's disease,
Huntington's disease, Parkinson's disease, lathyrism, etc. have been the focus of our studies for the last
five years. We have shown that cerebellar neurons cultured from neonatal rats express several subtypes
of glutamate receptor, including the N-methyl-D-aspartate (NMDA) receptor. When this receptor is
occupied by an appropriate agonist, a receptor-gated channel opens, permitting sodium and calcium
influx. However, in the healthy brain this channel is normally blocked by magnesium in a voltage-
dependent manner, i.e., magnesium prevents ion influx through the channel at normal membrane
potential. Under physiological conditions, the NMDA channel may only permit ion flow in response to
high-frequency stimulation. We have shown that the magnesium block is relieved when neurons
partially depolarize in response to reduced energy levels in the neuron; decreases in adenine nucleotide
levels due to glucose starvation, oxygen deprivation, or metabolic poisons cause sufficient
depolarization to relieve the magnesium block of the channel. Thus, when neuronal energy levels are
compromised, endogenous agonists such as glutamate can persistently open the NMDA channel
resulting in excess ion influx; the increased energy demands by the pumps involved in maintaining ion
gradients cannot be met in the energy-poor neurons, and neuronal death ensues via a mechanism not
yet understood.
Our results provide experimental evidence for a mechanism which may trigger the transition of
endogenous glutamate from neurotransmitter to neurotoxin; this mechanism may have important
implications for a variety of neurodegenerative disorders.
Due to the retirement of the principal investigator from the USPHS this project was terminated 1
February 1990.
7-LMB/DIR
PHS 6040 (R»v. 1 M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02698-05 LMB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Biology of Mammalian Homeodomain Proteins*
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: Ward F. Odenwald, Ph.D. Staff Fellow LMB, NINDS
Others: Shang-Ding Zang, M.D. Visiting Fellow LMB, NINDS
COOPERATING UNITS (if any)
Heinz Arnheiter, Visiting Scientist, LVMP, NINDS
LAB/BRANCH
Laboratory of Molecular Biology, BNP
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: - ?[-
PROFESSIONAL: q jc
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects (b) Human tissues [x~| (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Most of the proteins whose dysfunction disturbs embryogenesis of the fly share a characteristic stretch of
60 amino acids called the homeodomain. Mammals likewise express homeodomain-containing proteins.
Based on their nuclear localization, their capacity to bind to specific DNA sequences, and their relation to
some well-characterized transcription factors, it is suggested that homeodomain proteins are involved in
regulating gene expression in mammals. In our effort to determine the function of mammalian
homeodomain proteins, we have cloned, sequenced, and studied the expression of one of the murine
Antennapedia class genes known as Hox 1.3.
To test in vivo biofunction, we have recently generated transgenic mice that contain either a single or
multiple (up to 25) copies of an inducible Hox 1.3 transgene. To control the levels of ectopic Hox 1.3
expression, we have used the inducible mouse Mx1 regulatory element as our transgene promoter which
allows the deliberate ubiquitous expression of Hox 1.3 in the presence of its inducers (interferon or
double-stranded RNA). Under physiologic conditions, that is, in the absence of induction, the mice
express their endogenous Hox 1.3 gene, and in those organs tested by Northern analysis, little or no
detectable transgene RNA was found. In the absence of transgene induction, these mice appear healthy.
After transgene induction with either interferon or double-stranded RNA, the mice start to express high
levels of transgenic RNA and protein in many different organs. In addition, intravenous injection of
interferon into non-transgenic females that carry in utero transgenic embryos results in the fetal
induction of our transgene. Our primary goal is to first identify if and when during embryonic or post-
natal development does ectopic expression of Hox 1.3 have an effect, and then to use this in vivo tool to
identify endogenous genes that are either up- or down-regulated by Hox 1.3.
*Formerly in LVMP Transferred on October 1, 1989.
PHS 6O40 (Rev. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02820-01 LMB
PERIOD COVERED
October 1 , 1 989 through September 30, 1 990
TITLE OF PROJECT (80 characters or lets. Title must fit on on© line between the borders.)
Cloning and Functional Analysis of Genes Active in Neurogenesis
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: WardF. Odenwald, Ph.D. Staff Fellow LMB, NINDS
Others: Dervla Mellerick-Dressler, Ph.D. Special Volunteer LMB, NINDS
COOPERATING UNITS Of any)
Judith Kassis, CBER, DBB
LAB/BRANCH
Laboratory of Molecular Biology, BNP
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: -
PROFESSIONAL: Q r
OTHER:
0.25
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects (b) Human tissues [x~| (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The mechanisms that underlie the orderly sequential changes in gene expression
during neurogenesis are unknown. Establishment of form and pattern within the
nervous system is dependent on cellular interactions that are initiated early in
development. These communications regulate cell proliferation, differentiation,
neuroblast migration, axonal growth and guidance, target recognition, and,
ultimately synapse formation. The molecular machinery that relays this positional
information is the foundation upon which neuronal diversity and function is based.
How the nervous system is "wired" during embryogenesis remains a fundamental issue
in molecular biology today.
We have initiated our search for novel genes involved in these processes by identifying
and characterizing genes involved in the neurogenesis of the fruit fly, Drosophila
melanogaster. The fly was selected because of its relatively simple nervous system, the
availability of developmental mutants, and the possibility of genetic analysis of
function. Thus far we have identified several candidate genes and are focusing our
efforts on one that is expressed in central nervous system neuroblasts during
development. This gene and the other candidates were identified by the enhancer
detection method of transposon tagging in transgenic flies. Our goal is to use the
knowledge gained from the molecular analysis of these genes, to build the appropriate
tools to search for mammalian genes that carry out related functions.
9-LMB/DIR
PHS6O40(R«v. 1/84)
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ANNUAL REPORT
OCTOBER 1, 1989 THROUGH SEPTEMBER 30, 1990
LABORATORY OF VIRAL AND MOLECULAR PATHOGENESIS
TABLE OF CONTENTS
Page
RESEARCH SUMMARY 1
PROJECT REPORTS
Biology of Myelin-Forming Cells In Vitro 5
and In Vivo Including Remyelination
Z01 NS 02034-18 LVMP
Regulation of Myelin Synthesis 6
Z01 NS 02528-09 LVMP
Biology of Mammalian Homeodomain Proteins 7
Z01 NS 02698-05 LVMP
Mechanisms of Viral Pathogenesis 8
Z01 NS 02742-04 LVMP
Neurotropism of Human Retroviruses 9
Z01 NS 02789-02 LVMP
Expression of Viral Proteins in Transgenic Mice 10
Z01 NS 02790-02 LVMP
Molecular Biology of Human Neurotropic Virus 11
Infections, HIV-1 and JCV
Z01 NS 01983-19 LVMP
Replication and Pathogenesis of Enveloped Viruses 12
Z01 NS 02791-02 LVMP
Neurologic and Systemic Manifestations of Retrovirus 13
and Autoimmune Mediated Diseases
Z01 NS 02756-03
Pseudotypic Defective Interfering HIV Particles as 14
an Antiviral Therapy for AIDS
Z01 NS 02818-01 LVMP
ANNUAL REPORT
October 1, 1989 through September 30, 1990
Laboratory of Viral and Molecular Pathogenesis
National Institute of Neurological Disorders and Stroke
Monique Dubois-Dalcq , M. D., Acting Chief
The Laboratory of Viral and Molecular Pathogenesis (LVMP) was established 2 years ago. It
investigates the viral and molecular bases of nervous system diseases using the most advanced
techniques of molecular and cell biology. The laboratory studies the cell and molecular biology
of neural cells in vitro and in vivo,, characterizes mutations in myelin genes and studies the
cells involved in myelin repair in mice and men. An important goal of LVMP is to elucidate
mechanisms of nerve cell dysfunction in human diseases caused by RNA and DNA viruses,
especially human retroviruses (HIV, HTLV-1) and other viruses associated with them, such as
JC papova virus and herpes virus. The mechanisms of demyelination in acquired
immunodeficiency syndrome, tropical spastic paraparesis, multiple sclerosis, and progressive
multifocal leukoencephalopathy are being studied. The laboratory attempts to elucidate how
human neurotropic viruses get to the brain and which viral and host factors determine the final
outcome of infection. Long-term goals are to find ways to stop virus entry in nerve cells, to
inhibit viral cytopathogenesis and persistence, and enhance nervous system repair. The new
laboratory gathers scientists with expertise in the cellular and molecular biology of glial cells,
and others with expertise in the molecular biology and pathogenesis of neurotropic viruses. It
has maintained the breadth of expertise in cellular and molecular biology which was
characteristic of the Laboratory of Molecular Genetics and there is an ongoing discussion of the
cellular and molecular aspects of biological phenomena between the different members of the
laboratory. In addition, the laboratory has an active transgenic mice facility which has started
several collaborations with other NINDS and NIH laboratories.
Advances made this year in the area of neural and molecular biology of glial cells and in the
transgenic mice strategy to analyze the role of homeobox containing proteins in mammalian
development and disease are described first. The Studies on viral pathogenesis [involving
viruses other than HIV] are then reviewed, followed by a summary of the laboratory's
approaches to understanding HIV neurotropism and to designing strategies to prevent virus
spreading.
MOLECULAR PATHOGENESIS
We have progressed in our studies of the oligodendrocyte-type 2 astrocyte(0-2A) lineage
during development and remyelination. (Dubois-Dalcq et al). First we showed that FGF can
modulate the PDGF-driven pathway of oligodendrocyte development in the rat by up-regulating
the PDGF receptor on 0-2A progenitor cells. This receptor is in fact highly expressed in rat
brain in the postnatal period preceding myelination. Second, we demonstrated in vitro the
directed migration of these progenitors to PDGF but not to FGF. Third, we showed that 0-2A
progenitor cells can be readily isolated from mice CNS tissue in remyelination and that all cells
of the 0-2A lineage can divide in response to the disease process. Moreover, we found evidence
that the adult human nervous system may harbor a "resting" oligodendrocyte precursor, an
observation which bears on the regeneration potential of the human CNS in demyelinating
diseases like Multiple Sclerosis.
1-LVMP/DIR
In our studies of transcriptional control of myelination (Hudson et al), we have delimited a
region of 310 bp in the PLP promoter which regulates the expression of this major myelin gene
in both rat and human glial cells. Four human DNA-binding proteins that recognize portions of
this regulatory region have been isolated from expression libraries. One of the DNA-binding
proteins which recognizes a sequence motif present in PLP as well as in other myelin genes,
could potentially form a leucine zipper structure. Our continued analysis of PLP mutations,
which was extended to identify a novel mutation in the dog (the shaking pup), has revealed that
this class of mutations disturbs oligodendrocyte development. In preparation for screening
individuals in families with the dysmyelinating disease, Pelizaeus-Merzbacher, we have
devised a strategy for rapidly automated sequencing of the human PLP gene. We have already
received over 30 inquiries and hope that our request for special NINDS support in this area
will soon be approved.
In our collaborative studies on the role of homeobox genes in mammalian development
(Arnheiter et al ), we have obtained 7 lines of transgenic mice expressing the Hox 1 .3 gene
under the interferon inducible Mx1 promoter. We demonstrated induction of mRNA and protein
after a single injection of interferon in adult mice, and recently, induction was obtained at 11
days of gestation. Thus, we are now in a position to determine the exact time during which
Hox 1.3 can affect embryogenesis and to identify, in this induction system, potential genes
which are regulated by Hox 1.3. In the course of our studies of transgenic animals we have
come across an insertional mutant. Mice homozygous for this insertion show hemivertebrae,
spina bifida occulta, fusions of vertebral bodies and short kinky tails. These features are
reminiscent of those seen in the natural mutation, undulated, alleles of which have alterations
in the coding region of the Pax1 gene. We are now beginning the molecular characterization of
the gene whose function is disturbed in the transgenic mice. Once identified, we will then
determine whether patients suffering from hereditary malformations of their vertebral column
also have mutations in the corresponding human gene.
VIRAL PATHOGENESIS
A. RNA and DNA neurotropic viruses other than HIV
1. Intracellular immunization in vivo (Arnheiter et al). One of the ultimate goals of
studying viral pathogenesis is to find ways to protect organisms against viral infections. Here
we report for the first time that through germ line transformation we have converted a mouse
from virus-susceptible to highly resistant to a virulent infection, influenza. To achieve
resistance, we generated transgenic mice that express a potent intracellular anti-influenza
protein, Mx1, under control of the virus/interferon-inducible Mx1 promoter. We have
obtained several lines of transgenic mice that differ in the level at which virus infection can
stimulate Mx1 synthesis. In high responder lines, high levels of Mx1 are induced upon
replication at the sites of initial viral infection, resulting in limited viral spread and survival
of the mouse. Low responders show a lower degree of resistance, but paradoxically only to low
dose infection; when infected at high virus doses, they are as resistant as high responders. This
is because high-dose infection induces higher amounts of Mx1 protein than low-dose infection,
thus apparently overcoming the generally lower response in these animals. This observation
demonstrates that influenza virus pathogenesis in mice results from a subtle balance between
the dose of the infecting virus and the levels of an intracellular antiviral host factor. The trick
of the system is that the virus itself induces Mx1 and thus controls the animal's fate. To test
2-LVMP/DIR
whether other Mx proteins act in a similar way, we have prepared transgenic mice that express
human Mx genes. These mice are currently being analyzed. It is anticipated that the Mx1
promoter will become very useful for a number of studies in viral pathogenesis but also for
many other questions of gene function in transgenic mice.
2. Expression of individual viral proteins in vitro and in transgenic mice. An
interesting question in viral pathogenesis is whether individual viral proteins expressed in the
absence of any other viral component can cause cytopathic changes or a modified immune
response. The model rhabdovirus VSV is useful for such studies. We have shown (Schubert et
al) that the VSV M protein is solely responsible for the cytopathic effect observed after viral
infection. The VSV G protein of the virus was introduced into the germ line of mice (Arnheiter et
al) and the immune response of these animals to that protein was studied. When challenged with
purified recombinant G protein, only IgM and not IgG antibodies able to neutralize wild type VSV
were elicited. In contrast, the animals respond with neutralizing antibodies to the challenge
with the wild type virus. Thus, immunotolerance breaks down and autoimmunity is established
in this case, suggesting that these mice could be used as models to study autoimmune processes
following a viral infection.
3. Studies on neurotropic viruses in vivo and in vitro: We have continued our studies
(Major et al ) on JC papovavirus, the agent of progressive multifocal leukoencephalopathy. We
have developed a method to identify viral genomic sequences in a few hours in brain biopsy
specimens. Studies on the JC large T protein demonstrate that the retinoblastoma protein binds
to that viral protein, suggesting a role for this interaction in viral-induced tumor formation in
rodents and monkeys. In our studies of another DNA neurotropic virus, we showed that
varicella-zoster virus can replicate in human fetal Schwann cells.
B. HIV infection: prevention and neurotropism
In our studies of engineering negative strand viruses using vaccinia virus vectors, we
expressed VSV genes N, NS and L in cells and, with the help of the T7 RNA polymerase, replicate
defective VSV genome (Schubert et al). Such studies pave the way for the making of target
recombinant negative strand virus. Viruses are often used as vehicles for other genes, provided
they are non-cytopathic for the cells they invade. Our goal is to target HIV-infected cells with
recombinant viruses able to interfere with HIV expression. Toward this objective we have
constructed chimeric envelope proteins (Schubert et al). The extracellular portion of the CD4
molecule has been fused to the VSV G transmembrane and intracellular regions using PCR
technology. Using the vaccinia virus vectors to express these genes, we now have evidence that
pseudotypic virus can be immunoprecipitated from the supernatant of infected cells with
anti-CD4 antibodies. Such a CD4-enveloped defective virus could specifically infect gp120
expressing HIV infected cells. Optimally such defective virus should be able to use HIV in the
infected cell as a helper virus. To this goal, we have now constructed a prototype pseudotypic
defective interfering HIV genome. This prototype contains both HIV LTRs but the GAG-POL-ENV
region has been replaced by a new gene coding for a CD4-ENV chimera protein. In other
constructs we have placed a ribozyme that can cleave the ENV gene in the ectodomain region in
order to render the HIV particles noninfectious.
In our studies of HIV neurotropism, we have infected and transfected human fetal astrocytes
with HIV1 (Major et al). After a transient period of viral production, such cells appear latently
infected since virus can be rescued by coculture with a CD4+ T lymphocyte cell line. This raises
3-LVMP/DIR
the question whether chronic infection of astrocytes and possibly other nerve cell types can
occur in HIV1 infection of the CNS of fetuses carried by sero-positive mothers.
HIV also frequently causes neurological dysfunction and is abundantly expressed in the central
nervous system (CNS) of adult AIDS patients with HIV encephalitis or myelopathy. The virus is
found mostly in cells of the monocyte-macrophage lineage within the CNS, but the possibility of
infection of other glial cells has been raised. Therefore, the effects of different HIV-1 and HIV-
2 strains were studied in primary cultures of adult human brain containing microglial cells^
the resident CNS macrophages, and astrocytes.(Dubois-Dalcq et al ). These cultures could be
productively infected with macrophage-adapted HIV-1 isolates but not with T lymphocyte-
adapted HIV-1 isolates or two HIV-2 isolates. As determined with a triple label procedure,
primary astrocytes did not express HIV gag antigens and remained normal throughout the three-
week course of infection. In contrast, virus replicated in neighboring microglial cells, often
leads to their cell fusion and death. The death of microglial cells which normally serve immune
functions in the CNS, may be a key factor in the pathogenesis of AIDS encephalitis and/or
myelopathy. We are actively investigating the ways that microglial cells infection can influence
the function and gene expression of other cell types such as astrocytes, oligodendrocytes and
neurons.
The next question we have addressed this year is: what is the receptor used by HIV for entry
into microglial cells? (Dubois-Dalcq et al ). CD4 molecules function as the HIV receptor in T
and B cells, monocytes and macrophages. Although the CD4 receptor is also expressed in brain,
recent in vitro studies suggest a CD4-independent pathway of HIV entry in glial cells
originating from tumors or fetal tissues. In our work, we have identified CD4 transcripts in
primary microglial cell cultures using the PCR technique. Moreover anti-CD4 monoclonal
antibodies directed to the HIV binding site prevent HIV-1 infection of microglial cells . Thus,
infection of brain-derived microglial cells is CD4-mediated, suggesting that therapies aimed at
blocking the CD4 receptor may be appropriate to inhibit entry and spreading of HIV1 to the
brain.
4-LVMP/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOI NS 02034-18 LVMP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Biology of Myelin-Forming Cells In Vitro and In Vivo Including Remyelination
PRINCIPAL INVESTIGATOR (list other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute afiihation)
P. I.: M. Dubois-Dalcq, M D. Acting Chief LVMP, NINDS
Others:
R. McKinnon, Ph D
R.Armstrong, PhD
H Dorn, B.S.
R. Rusten
Sr. Staff Fellow
IRTA
Biologist
Biol. Lab. Tech.
LVMP, NINDS
LVMP, NINDS
LVMP, NINDS
LVMP, NINDS
COORPERATING UNITS Of any)
K. V. Holmes, Dept. Pathology, USUHS, Dr S Aronson, LMG, NC I, and
Dr. K. Kufta, Neurosurgery Branch, NINDS
LAB/BRANCH
Laboratory of Viral and Molecular Pathogenesis
SECTION
Section on Neural and Molecular Biology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
3.5
PROFESSIONAL:
2.5
OTHER:
CHECK APPROPRIATE BOX(ES)
I | (a) Human subjects
] (a1) Minors
J (a2) Interviews
[ x | (b) Human tissues J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The myelin sheath is essential for normal conduction in major nerve tracks. Therefore neurological
dysfunction occurs in demyelinating diseases such as multiple sclerosis (MS) and viral encephalitis. We
study how myelin-forming cells can develop and regenerate both in vitro and in vivo.
Molecular analysis of oligodendrocyte type 2 astrocyte (0-2A) progenitor cells reveals that FGF
modulates the PDGF-driven pathway of oligodendrocyte development by up-regulating the PDGF
receptor on these cells. In a chemotaxis chamber assay PDGF, but not FGF, can trigger directed migration
which can occur in the absence of mitosis. 0-2A lineage cells were isolated and cultured from mice with
a demyelinating disease caused by a corona virus. Cultures from demyelinated tissue differed in several
ways from those of the controls: (1) the total number of 0-2A lineage cells was dramatically increased;
(2) the 0-2A cells consisted of a high proportion of 04 positive astrocytes and of cells with a mixed
oligodendrocyte-astrocyte lineage; (3) all 0-2A cells showed enhanced proliferation in response to an
episode of demyelination. In particular a large proportion of progenitor cells were incorporating DNA.
Our in vitro analysis of adult human brain cells also suggests the existence of a "resting"
oligodendrocyte precursor cell with an antigenic pheonotype similar to that seen in rodents Thus, our
interest lies in the characterization of the oligodendrocyte precursor cells present in the adult CNS and
their role in remyelination.
PHS 6040 (Rev 184)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02528-09 LVMP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Regulation of Myelin Synthesis
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: L. D. Hudson, Ph.D. Staff Scientist LVMP:NINDS
Others: J Kim, Ph.D. M.S. Fellow LVMP:NINDS
N. Nadon, Ph.D. Staff Fellow LVMP:NINDS
J. Berndt, B.S. Microbiologist LVMP:NINDS
COORPERATING UNITS Of any)
Dr. Heinz Arnheiter, Section of Viral Pathogenesis, LVMP, NINDS
LAB/BRANCH
Laboratory of Viral and Molecular Pathogenesis
SECTION
Neural and Molecular Biology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
3. bo
PROFESSIONAL: j SR
OTHER: 1 q
CHECK APPROPRIATE BOX(ES)
O (a) Human subjects 0 (b) Human tissues ] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The objective of this project is to define the regulatory signals that control myelination, the event where
oligodendrocytes and Schwann cells extend processes that enwrap and ensheath axons. Our search for
the molecular basis of transcriptional controls on myelination involves characterization of both the trans-
acting regulatory factors and their cognate cis-acting enhancer/repressor elements necessary for
expression of the prototype myelin gene, proteolipid protein (PLP). A combination of biochemical and
functional assays have revealed that the essential cis elements are clustered in a small region
encompassing the PLP promoter. Regulation of this region most likely requires an interplay of various
tissue-specific and ubiquitous binding proteins, some of which directly interact with the cis recognition
sequences and some which bind a protein-DNA complex. We have cloned four putative transactivator
factors of the former class and identified features of DNA-binding proteins in at least one partially
sequenced clone. The isolation of these clones permits a search for the growth factors and other
molecules that are critical to the initiation and maintenance of myelin gene transcription.
PLP is the most abundant constituent of central nervous system myelin and its loss has devastating effects
on myelinating cells. After defining mutations in the proteolipid protein (PLP) gene of a number of
animal and human dysmyelinatinq disorders, we have provided molecular evidence that these mutations
act, at least in part, to interrupt oligodendrocyte differentiation. Efforts to identify additional mutations
in PLP and in other myelin genes of patients affected with dysmyelinating diseases (such as Pelizaeus-
Merzbacher) are underway.
PHS 6040 (Rev. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02698-05 LVMP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 character* or less. Title muit fit on one line between the borders.)
Biology of Mammalian Homeodomain Proteins
PRINCIPAL INVESTIGATOR (L at other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
PI: Heinz Arnheiter, Ph.D. Visiting Scientist LVMP, NINDS
Others: Ronald J. DeSanto, B.S. Biologist LVMP, NINDS
COOPERATING UNITS (,fan„)
Ward. F.Odenwald, Ph.D. Staff Fellow LMB, NINDS
LAB/BRANCH
Laboratory of Viral and Molecular Pathogenesis
SECTION
Viral Pathogenesis Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland, 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
0.25
OTHER: q ^
CHECK APPROPRIATE BOX(ES)
□ (ajHuman subjects ] (b) Human tissues [x~l (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Homeodomain proteins are nuclear proteins that share a characteristic stretch of 60 amino acids, the
homeodomain. In the fly, members of this family of proteins are important factors for implementing the
correct developmental program of embryoqenesis. In vertebrates, they may have similar roles, but
actual evidence is scarce. It is thought that homeodomain proteins function by regulating the
expression of other genes, but we do not know which genes are being regulated.
To identify potential target genes regulated by a representative murine homeodomain protein, we have
introduced into the germ line of laboratory mice a piece of DNA that allows the deliberate ubiquitous
expression of Hox 1.3. We used as regulatory element the Mx1 promoter which is known to respond to
interferon and interferon-inducers such as double-stranded RNA. Under physiological conditions, that
is, in the absence of interferon, the transgenics express only their endogenous and not the transgenic
Hox 13 protein However, when stimulated with interferon or double-stranded RNA, they start to
express transgenic Hox 1 .3 RNA and protein at high levels in many different organs. These mice and their
descendants will be tested for up- or down-regulation of various endogenous genes following
experimental Hox 1.3 induction.
To identify potential genes regulated during embryonic development, we will test the effect of
interferon or interferon-inducers given during pregnancy. This approach may become extremely helpful
to identify the time window during which Hox 1.3 expression may effect organogenesis, but may be
limited by the efficiency with which Hox 1 .3 can be induced in embryos, and by the side effects interferon
treatment may have on embryos during development.
PHS6O40(Rev 1841
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOI NS 02742-04 LVMP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Mechanisms of Viral Pathogenesis
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI:
Others:
Heinz Arnheiter, M.D.
Ellen Meier, Ph.D.
Susan Skuntz, B.S.
Ronald J. DeSanto, B.S.
Visiting Scientist
Senior Staff Fellow
Biologist
Biologist
LVMP, NINDS
LVMP, NINDS
LVMP, NINDS
LVMP, NINDS
COOPERATING UNITS {if any)
LAB/BRANCH
Laboratory of Viral and Molecular Pathogenesis
SECTION
Viral Pathogenesis Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2.3
PROFESSIONAL:
1.5
OTHER:
0.8
CHECK APPROPRIATE BOX(ES)
[ | (a) Human subjects
] (a1) Minors
J (a2) Interviews
J (b) Human tissues | x | (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
In addition to generating an immune response with its specific antibodies and T cells, the vertebrate host
can control viral infections by induction of intracellular host factors that directly interfere with viral
replication. Our research focuses on a family of such intracellular proteins known as Mx proteins. One
member of this family, the Mx1 protein found in two strains of laboratory mice, is induced by interferon
and arrests growth of influenza viruses (but not other viruses) by interfering with an early step in viral
replication. We have introduced its corresponding cDNA into the germ line of Mx-negative, influenza-
susceptible mice, a procedure which results in protection of the mice against fatal influenza. This
procedure can be called " intracellular immunization" , because the mice become immune to infection,
and because the antiviral principle is acting inside cells, unlike antibodies and T cells which act on cells or
viruses from the outside.
Proteins with homology to Mx1 are expressed in organisms ranging from yeast to man, that is, they are
also found in organisms for which influenza is no threat. It is possible that Mx proteins of such species
have activities against other viruses. However, it is also possible that the main mission of Mx proteins is
to fulfill some basic cellular function, and antiviral action isjust a byproduct of this basic function.
To test the possibility that certain Mx proteins have activities against other viruses, we have analyzed the
Mx system of the rat. We found that a nuclear rat Mx protein has in fact activity not only against
influenza virus, but also against vesicular stomatitis virus (VSV), a negative-stranded RNA virus against
which mouse Mx1 has no activity. A cytoplasmic rat Mx protein has activity only against VSV. A
suggestion for a basic cellular function of Mx proteins comes from the observation that all published Mx
sequences contain in their more conserved ammo-terminal halves a consensus sequence characteristic of
GTP-binding proteins. Thus, Mx proteins may belong to a family of GTP-bindinq proteins.
PHS 6040 (Rev. 184
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUM8ER
ZOI NS 02789-02 LVMP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Neurotropism of Human Retroviruses
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
P.I M Dubois-Dalcq, M D Acting Chief, LVMP LVMP, NINDS
Others: B. Watkins, Ph.D. Visiting Fellow LVMP, NINDS
C. Jordan, Ph.D. Sr. Staff Fellow LVMP, NINDS
H. Dorn, B.S. Biologist LVMP, NINDS
W Kelly, B.S. Chemist LVMP, NINDS
COORPERATING UNITS (if any)
Dr. K. Kufta, Neurosurgery Branch, NINDS; Dr. B Potts, Repligen, Boston, Ma; and F. Michaels, Laboratory
Tumor Cell Biology, NCI.
LAB/BRANCH
Laboratory of Viral and Molecular Pathogenesis
SECTION
Section on Neural and Molecular Biology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL: j 3
OTHER:
1.2
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects [V] (b) Human tissues I I (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
In order to elucidate the pathogenesis of AIDS encephalitis and myelopathy, we have infected primary
cultures derived from cortex of patients with intractable epilepsy or malignant astrocytoma with
different isolates of HIV-1 and HIV-2. Viral infection was measured by reverse transcriptase (RT) activity
and HIV-1 core antigen (p24) in cell free supernatant. The three isolates which showed signs of
replication were derived from either macrophages (HIV-1 AD87(M)and HIV-1 BaL) or brain (HIV-1jr_fl).
whereas T-cell adapted and HIV-2 strains did not replicate. Peak levels of RT activity (1-2x105 cpm/ml)
and p24 antigen (50-100 ng/ml in productivity infected cultures were generally seen at 9-15 days post
infection The antigenic phenotype of infected cells was characterized by a triple label assay using
antibodies against HIV p24or pi 7 gag proteins, glial fibrillary acidic protein (GFAP) antibody to identify
astrocytes and Dil-labelled low density lipoprotein (Dil-LDL), which binds to microglia, a cell of the
macrophage lineage residing in the brain In infected cultures, Dil-LDL labelled microglia often stained
positively for gag but GFAP+ astrocytes did not. Viral production was further confirmed by electron
microscopy which showed budding of HIV particles from the plasma membrane of microglial cells. After
4-5 days post infection progressive syncitia formation began among infected microglia and after 3
weeks up to 25 times more Dil -LDL + cells had died as compared to controls. This progressive clustering
and fusion of microglial cells in vitro mimics the two pathological hallmarks of HIV infection in the
brain: microglial nodules and multi-nucleated giant cells. When cells were incubated prior to infection
with large amounts of anti-CD4 antibody which recognizes the gp1 20 binding site (25 ug/ml Leu 3a),
both viral replication and cytopathic effects were reduced to control levels. In addition, microglia
isolated from control cultures were found to express mRNA for CD4 by polymerase chain reaction (PCR).
This demonstrates that HIV can enter microglia via the CD4 receptor as it does in T-cells and monocytes .
We propose that the death of microglial cells which normally serve immune functions in the central
nervous system may be a key factor in the pathogenesis of AIDS encephalitis and/or myelopathy
f>HS6M0(fiev 1 S4|
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOI NS 02790-02 LVMP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Expression of Viral Proteins in Transgenic Mice
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: Heinz Arnheiter, Ph.D. Visiting Scientist LVMP, NINDS
Others: Susan Skuntz, B.S. Biologist LVMP, NINDS
Ronald J. De Santo, B.S. Biologist LVMP, NINDS
COOPERATING UNITS </f any)
Rolf M. Zinkernagel, Institute for Pathology, University of Zurich, Switzerland
Dr. Elisabeth Tournier-Lasserve, Institut Pasteur, Paris, France
LAB/BRANCH
Laboratory of Viral and Molecular Pathogenesis
SECTION
Viral Pathogenesis Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL: Q yc
OTHER: q 45
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues f~x~~l (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Individual viral proteins are expressed in transgenic mice in order to answer two fundamental questions
in viral pathogenesis: what are the mechanisms of induction of an immune response against a virus and
what effect does a particular viral protein have on the metabolism of a cell in vivo?
To address an example of the first question, induction of an immune response, the envelope
glycoprotein G of vesicular stomatitis virus, serotype Indiana, was expressed in transgenics. When these
mice are immunized with purified G protein, or when challenged with a recombinant vaccinia virus
expressing this protein, only IgM and not IgG antibodies able to neutralize wildtype Indiana VSV are
formed. However, the response with neutralizing antibodies is normal when the corresponding
wildtype Indiana VSV is used. Thus, tolerance breaks down, and autoimmunity is established, when
wildtype virus is used for immunization, and not when G protein is presented in other ways. These mice
may become a model for those autoimmune phenomena of humans for which viral infections may be the
triggering event.
To address an example of the second question, we have expressed the regulatory protein Tax of the
human retrovirus HTLV-1 known to be associated with certain forms of T-cell leukemias, and tropical
spastic paraparesis. To date we have found that low level Tax expression in transgenic mice may cause a
thymic hypoplasia but no gross pathology. Since in these mice the Tax protein is under the control of an
inducible promoter, we will be able in the future to study the effect of higher levels of expression.
However, in one transgenic line the Tax transgene is apparently inserted into a host gene whose product
is neccessary for normal embryonic development of the vertebra: mice of this line, when homozygous for
the transgene, show a severe malformation of the vertebra characterized by hemivertebras, spina bifida
occulta, fusions of vertebral bodies, and short kinky tails. The molecular characterization of the
corresponding gene that we have mapped to mouse chromosme 1 1 is under way.
10
PHS 6040 (Rev. 1/S4)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOI NSOWaJ 191VMH
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less, rule must fit on one line between the borders >
Molecular Biology of Human Virus Infections, HIV-1 and JCV
PRINCIPAL INVESTIGATOR (list other professional personne/ below trie Principal Investigator ) (Name, title, laboratory and institute affiluti^ I
P I
E.O Major, PhD Section Chief LVMP, NINDS
Others:
K. Amemiya, Ph.D. Special Expert LVMP, NINDS S. Finch, Ph D Microbiologist LVMP, NINDS
J. Assouline, Ph.D. Staff Fellow LVMP, NINDS A. Nath, M D Medical Staff LVMP, NINDS
B Curfman, B.S. Microbiologist LVMP, NINDS Fellow
L. Durham, M.S. Biologist LVMP, NINDS R. Traub, B S Microbiologist LVMP, NINDS
COOPERATING UNITS (if an,)
Microbiological Associates, Inc.Rockville, MD.
Veterans Administration Hospital, Washington, D.C.
LAB/BRANCH
Laboratory of Viral and Molecular Pathogenesis
SECTION
Section on Molecular Virology and Genetics
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: ?f.
PROFESSIONAL: 4 c
OTHER: 2 5
CHECK APPROPRIATE BOX(ES)
LJ (a) Human subjects [x~l (b) Human tissues ] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Progressive multifocal leucoencephalopathy (PML) and AIDS encephalopathy are viral
induced neurological diseases which occur almost exclusively in individuals whose
immune systems are impaired. We have investigated the molecular pathogenesis of
these diseases by developing laboratory assays that examine the human polyomavirus,
JCV, and the human immunodeficiency virus, HIV-1 the causes of PML and AIDS
encephalopathy, respectively. We have developed a new methodology that prepares
brain biopsy tissue for in situ DNA hybridization to detect viral genomes on the same
day of surgery. For example, in brain biopsy tissue from an AIDS patient with PML, JCV
DNA can be detected using a non-radioactive, nucleic acid probe. Data from this rapid
molecular diagnostic assay facilitates patient management decisions on a more timely
basis. We have also examined protein interactions between JCV and infected glial cells
The non-structural JCVT protein can bind the cellular retinoblastoma protein which
normally functions for growth regulation. Results from this study suggest that the
oncogenic properties of JCV may be related to viral proteins interfering with normal
cellular metabolism. Other human viruses have been studied for their association with
glial cells of the nervous system. Astroglial cells from human fetal brain respond very
rapidly to the introduction of the HIV-1 genome. AIDS virus proteins are synthesized
within hours of transfection. However, HIV-1 infection of astrocytes results in a
persistent infection without cytopathic effects. Varicella-zoster virus, the agent which
causes shingles, can productively infect Schwann cells, the myelinating cell of the
peripheral nervous system. VZV proteins are also produced very rapidly following
infection and are efficiently assembled into progeny virions
11
PHS 6040 (Rev I nil
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl MS 02791-02 LVMP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Replication and Pathogenesis of Enveloped Viruses
PRINCIPAL INVESTIGATOR (List other professional personnel below the Prin- ipa/ Investigator.) (Name, title, laboratory, and institute affiliation)
P.I. M.Schubert, Ph.D. Section Chief LVMP, NINDS
Others: D. Blondel, Ph.D.
G. Harmison, B.S.
B. Joshi,Ph.D.
Visiting Fellow
Chemist
Senior Staff Fellow
LVMP, NINDS
LVMP, NINDS
LVMP, NINDS
COORPERATING UNITS Of any)
Dr. Yong Kang, University of Ottawa, Ottawa, Canada.
LAB/BRANCH
Laboratory of Viral and Molecular Pathogenesis
SECTION
Viral Replication Section
INSTITUTE AND LOCATION
NINDS, NIH. Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.8
PROFESSIONAL:
1.5
OTHER:
0.3
CHECK APPROPRIATE BOX(ES)
] (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues [jT] (c Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project focuses on the molecular biology of enveloped viruses which include a broad group of
viruses from negative strand viruses (measles, rabies), to retroviruses ( AIDS virus), to DNA viruses (herpes
virus). The goal of this research is to study the basic molecular mechanisms of viral pathogenesis from
viral entry and assembly to viral gene expression and replication. The understanding of these
mechanisms will be necessary in a long term to design novel targeted gene delivery systems or gene
expression systems with possible applications in gene therapy and vaccine development. Our plan is to
develop defective virus particles with tropism for neuronal or nonneuronal cells. So far our studies have
centered around vesicular stomatitis virus (VSV) but the basic strategies can be applied to other
enveloped viruses.
Towards these goals we found that viral assembly and cytopathogenesis of VSV are most likely
independent events. We also found that the high affinity of the polymerase protein NS to the defective
interfering particle genome determines the level of autointerference. These are important observations
for the understanding of viral pathogenesis on one end of the spectrum and the establishment and
maintenance of persistent infections on the other end.
For the targeting of viruses to specific cells we were able to insert a chimeric HIV receptor CD4-VSV
envelope glycoprotein into a majority of VSV particles. These particles could be specifically
immunoprecipitated with antibodies to the CD4 protein. We were also able to insert normal human CD4
molecule into the VSV envelope. Experiments are in progress to test whether the efficiency of the
insertion as well as viral assembly depend on the presence of the cytoplasmic tail region of the VSV
envelope protein. We are in the process of developing a system which allows studing the molecular
mechanisms of viral assembly when the viral envelope protein is absent and/or replaced by a
recombinant chimeric envelope protein. Using vaccinia virus we were able to express the VSV N, NS and L
genes and replicate a defective virus genome in the absence of helper virus. This finding is particularly
promising in our efforts to generate the first recombinant rhabdovirus.
12
PHS 6040 (Rev. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
ZOl NS 02756-03 LVMP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one fine between the borders )
Neurologic and Systemic Manifestations of Retrovirus and Autoimmune Mediated Diseases
PRINCIPAL INVESTIGATOR ft ist other professional personnel below the Principal Investigator J (Name, title, laboratory, and institute affiliation)
P. I.: D M Klinman, M. D.Ph.D. Senior Staff Fellow LVMP, NINDS
COORPERATING UNITS Of any)
None
LAB/BRANCH
Laboratory of Viral and Molecular Pathogenesis
SECTION
Viral Pathogenesis Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues ] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project has been terminated.
13
(>HS 6040 (Rev V84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02818-01 LVMP
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Pseudotypic Defective Interfering HIV Particles as an Antiviral Therapy for AIDS
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
p.l.
M.Schubert Ph.D. Section Chief LVMP, NINDS
Others:
G. Harmisonll, B.S. Chemist LVMP, NINDS
B. Joshi, Ph.D. Sr. Staff Fellow LVMP, NINDS
D. Blondel.Ph.D Visiting Fellow LVMP, NINDS
K. Haglund Summer Student LVMP, NINDS
COOPERATING UNITS Ofany)
None
LAB/BRANCH
Laboratory of Viral and Molecular Pathogenesis
SECTION
Viral Replication Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
2.8
PROFESSIONAL:
2.5
OTHER:
0.3
CHECK APPROPRIATE BOX(ES)
J (a) Human subjects
] (a1) Minors
J (a2) Interviews
J (b) Human tissues I x I (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This newly initiated project has evolved from the young project on the Replication and Pathogenesis of
Enveloped Viruses (Z01 NS 02791-01 LVMP). The main topic covered by this project focuses on the
molecular virology of HIV, the AIDS virus, and on antiviral strategies designed to specifically interfere
with the replication of HIV. It is our hope this interference with virus replication may cause a significant
delay in the onset of AIDS or possibly prevent it.
We are currently developing what we call pseudotypic defective interfering particles of HIV. The goal of
this antivirual approach is to use these defective particles to target and specifically infect cells which are
already infected by HIV. It is our anticipation that this cell-targeting will allow deliverance of genes
and/or gene products specifically into HIV infected cells. The gene products are designed to interfere
with the replication of HIV itself. In addition, we anticipate that these defective interfering HIV particles
will not only down-regulate the number of HIV progeny but that they also will use HIV in an
opportunistic way as their helper virus for their own assembly. This will permit repetition of this cycle in
other HIV infected cells.
We have succeeded by using our gene fusion method to construct the first prototype pseudotypic
defective interfering HIV genome. The genome was assembled, cloned and sequenced and was found
suitable for further evaluation as a targeted antiviral delivery system. We have also constructed two
potential ribozymes which are designed to specifically cleave HIV genomic RNA, inactivate it and thereby
increase the inferfering potential of the defective virus. Functional tests are in progress.
14
PHS 6040 (Rev. I'M)
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ANNUAL REPORT
October 1, 1989 through September 30, 1990
Laboratory of Neural Control, Basic Neurosciences Program, Division of Intramural Research
National Institute of Neurological Disorders and Stroke
Table of Contents
RESEARCH SUMMARY j.g
PROJECT REPORTS
Motor Control Systems in the Spinal Cord 7
Z01 NS 01686-22 LNLC
Techniques for Making Connections with the Nervous and 8
Musculoskeletal Systems
Z01 NS 01687-22 LNLC
Cortical Mechanisms of Voluntary Motor Control 9
Z01 NS 01688-22 LNLC
Models of Neurophysiological Systems 10
Z01 NS 02079-17 LNLC
Intrinsic Properties of Motor Units 1 1
Z01 NS 02160-16 LNLC
Repair of Injured Nervous Tissue with Foreign Grafts 1 2
Z01 NS 02254-14 LNLC
Analysis of Network Function in the Developing Spinal Cord of the Chick Embryo 13
Z01 NS 02787-02 LNLC
Development of Primary Sensory Neurons 14
Z01 NS 02788-02 LNLC
i-LNLC/DIR
ANNUAL REPORT
October 1, 1989 through September 30, 1990
Laboratory of Neural Control, Basic Neurosciences Program, Division of Intramural Research
National Institute of Neurological Disorders and Stroke
Robert E. Burke, M.D , Chief
Introduction
Research work in the Laboratory of Neural Control (LNLC) is devoted to studies of the central and
peripheral neural mechanisms involved in the control of movement in mammals. Our work emphasizes
analysis of neural organizations at the level of the spinal cord and those regions of the brain stem and
cerebral cortex that project directly to the spinal cord in vertebrates, ranging from birds to primates.
Studies of spinal cord development mainly involve in vitro studies, ranging from tissue culture systems to
whole chick embryos, and include extra- and intracellular electrophysiological, as well as optical, recording
techniques Interactions among motoneurons, motor units, and elemental interneuronal circuits are
studied by single unit electrophysiological methods in adult cats. The organization of cortical motor
control systems is studied in primates, primarily using chronic recording techniques in awake, behaving
animals. Neuroanatomical, histochemical and immunocytochemical, and mathematical modeling
approaches are used as adjuncts to these basically physiological studies. The Laboratory also includes a
Section devoted to studies of nerve regeneration after injury, using light and electron microscopic
anatomical techniques. Finally, members of the Laboratory continue to be involved in studies of neural
prostheses that utilize state-of-the-art techniques to develop systems that can benefit neurologically-
handicapped individuals.
Present Organization
During FY 1990, the staff of the Laboratory of Neural Control (LNLC) included: thirteen professional
scientists (five permanent senior scientists, one special expert, and seven post-doctoral fellows). The
permanent staff also includes eight full-time permanent support personnel (a physiologist, two engineers,
one computer programmer, one biological technician, a histology technician, and one laboratory
secretary).
The FY 1990 research effort in LNLC can be described under six general headings:
1 . Electrophysiological and morphological analyses of the cellular physiology and neuronal circuitry
operating in the control of movement at the spinal cord level in anesthetized or unanesthetized animals
(primarily cats) following acute destruction of the supratentorial brain (decerebrate preparations).
2. Theoretical and computer modeling studies of cellular biophysical and morphological features of
individual neurons and of information processing in defined neural systems.
3. Studies of the discharge properties of individual neurons in the primate motor cortex and
supplementary motor area (SMA) during performance of voluntary movements.
4. Studies of the development of the vertebrate spinal cord, including maturation of cellular
electrophysiological and morphological characteristics, formation of specific synaptic connections, and
analysis of the maturation of intrinsic motor patterns in the chick embryo.
5. Studies of the mechanisms of injury repair in mammalian peripheral nerves following axotomy
and the role of the blood-nerve barrier in this process.
6. Activities concerned directly with the development of new methods for making contact with the
central nervous system. Many of the activities under this rubric serve to support ongoing research projects
within LNLC. During FY 1990, this project also included continued work on the development of a
functional prosthesis for stimulation of the human visual cortex.
1 -LNLGDIR
Project Summaries
Motor Control Systems and Intrinsic Properties of Motor Units in the Cat Spinal Cord: Control of
excitatory last-order intemeurons: Thissubproject utilizes cats, either anesthetized or after decerebration,
with destruction of thesupratentorial brain. This animal has been used as the ideal model system for work
on the anatomy and physiology of the spinal cord for over a century and there is thus a wealth of detailed
information about the cat spinal cord that serves as the basis for the design and interpretation of new
experiments. In addition, cats are relatively accessible, have the ideal body size for neurophysiological
experiments, and are well-adapted to the laboratory environment. The neural mechanisms that control
movement are inferred from data obtained in reduced, immobile preparations. When survival surgery is
required, as in studies of the morphology of motor nuclei using retrograde transport methods, surgery is
performed under anesthesia and aseptic conditions, and appropriate postoperative care is supplied. The
main goal of this project is to examine the organization of neuronal systems in the mammalian spinal cord
that are involved in the control of movement. Specific topics range from studies of cellular properties of
individual motoneurons and intemeurons in identified neuronal systems to examination of the
organization and function of specific neuronal circuits that are involved in movement control, including
the problem of central pattern generation at spinal segmental levels.
During FY 1990, we enlarged earlier results on the organization of excitatory intemeurons that
project directly to motoneurons in the cat spinal cord, about which relatively little detailed information is
currently available. We have emphasized studies of the short-latency excitatory pathways from distal areas
of hindlimb skin to particular groups of motoneurons, especially those that innervate the flexor digitorum
longus (FDL) muscle. We have shown that large, low-threshold afferents from the dorsal surface and tips
of the toes, traveling in the superficial peroneal nerve (SP), as well as those from the ventral surface
traveling in the medial plantar (PLNT) nerve, both produce excitatory postsynaptic potentials (EPSPs) with
minimum central latencies consistent with disynaptic connection to FDL motoneurons. This means that
that the fastest pathway from the respective afferents to FDL output cells consists of a single layer of
interposed intemeurons, which makes the analysis of the organization of the pathways by conventional
techniques possible.
We previously demonstrated that the initial excitatory components in SP PSPs in FDL motoneurons
are strongly facilitated during the early flexion phase of fictive locomotion (rhythmic patterned
motoneuron discharges in decerebrate, paralyzed animals that mimic those found in intact, freely walking
animals). This indicates that excitatory spinal intemeurons, including those in the disynaptic SP to FDL
pathway, receive convergent excitation from the spinal central pattern generator (CPG) for locomotion.
The same spatial facilitation technique was used to show that short-latency EPSPs produced in FDL
motoneurons by stimulation of cutaneous afferents in the PLNT nerve are also modulated by the
locomotor CPG, but in a pattern completely different from that observed with SP EPSPs. In contrast to SP
EPSPs, PLNT EPSPs are markedly reduced in amplitude during the entire flexion phase, with maximum
reduction during early flexion. In addition, PLNT EPSPs exhibit facilitation during the extension phase of
fictive stepping in occasional step cycles in which the FDL muscle shows a burst of activity during extension.
This facilitation is not observed in SP EPSPs. These results indicate that the last-order intemeurons that
convey excitation from these two skin nerves are completely separate and subject to independent central
control, despite the fact that the sensory territories of the two nerves are contiguous. This model system
demonstrates the existence of highly specific cutaneous reflex pathways that presumably subserve
particular functions in the intact animal. They also suggest that particular sets of excitatory intemeurons
that receive convergence of input from central neural systems as as from the periphery may serve multiple
roles in the generation of particular patterns of muscle output. The finesse exhibited by this system is quite
at variance with the common notion of cutaneous reflexes as global in character, as in the flexion
withdrawal reflex.
Studies of the morphology of motoneuron dendrites: In FY 1989, we began to examine whether it is
possible to define relatively simple mathematical rules that embody the complex architecture of
motoneuron dendrites. Initial results using data from a large sample of dendrites from adult triceps surae
2-LNLC/DIR
motoneurons indicate that the probability of branching and terminating are primarily functions of local
dendritic diameter, with a minor dependence on the length of dendritic branches. In addition, the rate of
dendritic taper was found also to depend on local dendritic diameter. During FY 1990, a full model system
was implemented in which relatively simple mathematical rules are used to govern the probabilities of
branching and termination in individual branches, and to constrain the diameters of daughter branches at
branching points The rules are the direct output from appropriate analyses of real dendrites and the
parameters are not varied beyond the limits observed in the data. The object has been to develop a modei
system that uses parameters directly obtained form actual data to reproduce structures with the
characteristics of actual dendrites. Thus far, the model produces simulated "dendrites" that mimic the
characteristics of real motoneuron dendrites in most, but not all, respects. Development of the model has
forced us to focus on very specific features of dendritic anatomy. The fact that all the rules depend
primarily on local dendritic diameter suggests that factors correlated with diameter, including features of
the local cytoskeleton, play critical roles in controlling dendritic morphology and maintenance.
We have continued this work, using morphological data sets developed within LNLC and additional
sets developed in the Department of Anatomy, Karolinska Institutet, Stockholm, Sweden. The Karolinska
data includes measurements from cat alpha-motoneurons from newborn and immature kittens, as well as
adult animals We anticipate that application of our current analysis methods and the implementation of
the mathematical simulation may reveal the factors that account for the morphological changes that
dendrites undergo during normal postnatal maturation.
We have continued a similar analysis of the dendrites of adult gamma motoneurons in order to test
our approach on a different species of neuron. It has been known for some time that gamma
motoneurons, although smaller in total membrane area than alpha cells, nevertheless have dendrites that
are equally long, albeit less highly branched. Our analysis to date suggests that the key parameters that
control dendrite branching and termination are relatively similar in alpha- and gamma-motoneurons, but
the rate of taper is markedly lower in gamma motoneurons. Studies are underway to pinpoint the factors
that can account for the large apparent differences in the morphologies of these two kinds of
motoneurons.
Models of Neurophvsioloqical Systems: Work on this project during FY 1990 has been closely
connected to the above attempt to simulate dendritic morphologies in computer simulations. In fact, two
parallel approaches have been used for the actual simulations - Monte Carlo and analytical simulations
Both have given equivalent results but each is used so as to check on the reliability of the other. Dr. Marks
has undertaken additional work to examine descriptive rules that embody the trajectories of individual
dendritic branches. These can be adequately simulated using a smoothed random walk. Motoneuron
branches have an average fractal dimension of about 1 .2. We are also exploring the possibility of
simulating fully branched dendritic trees in three dimensions, using constraints of the angles formed at
branching points. This direction may lead to clues as to how the 3-D space-filling characteristics of
neuronal dendrites can be quantitated in a meaningful way. This extremely difficult problem has no
current solution.
Network Function in Developing Spinal Cord of the Chick Embryo: This project began in FY 1989,
under the direction of Dr. Michael J. O'Donovan. The primary aim is to analyze the properties of spinal
cord neurons and networks during embryonic development in the chick spinal cord. This model system is
very favorable for experimental attack, since reduced preparations in vitro exhibit spontaneous motor
patterns that are qualitatively similar to those emitted by intact embryos
Real-time optical imaging of neuronal activity: A major focus of the effort during FY 1990 has been
the successful development of a real-time imaging system to detect changes in intracellular calcium
concentration during stimulated or spontaneous motor patterns in chick embryos, using the calcium
indicator, Fura-2AM and fluorescence microscopy with video image processing. Many spinal neurons,
including both motoneurons and interneurons, exhibit phasic changes in calcium fluorescence during
motor output bursts, enabling identification particularly of interneurons that may be involved in the
generation of such bursts Active interneurons are located mainly dorsal to the lateral motor column,
including cells in the dorsal horn. Some neurons exhibit oscillations in calcium signal in phase with
3-LNLQDIR
repetitive motor bursts while others show more tonic signal increases. The identity of the cells that give
•sitive signals is under study using a combination of optical imaging and conventional
electrophysiological recording techniques and by immunocytochemistry.
Immunocvtochemistry: We have found that cells loaded with Fura-2AM can be identified by photo-
oxidation and thus identified in subsequent histological preparations. Cells thus labeled can then be
studied using other reagents, such as antibodies to cell constituents specific for neurons and glial cells, as
well as putative neurotransmitters, calcium-binding proteins, etc. This approach has now been successfully
applied to chick embryo tissues and, in principle, offers the possibility of precisely identifying the cellular
elements that participate in generating motor patterns.
Lesion and stimulation studies of pattern generation: The results of the optical recordings have
been used to guide selective lesions to determine whether "active versus "inactive" areas are indeed
critical to pattern generation. We have found rostro-caudal differences in the ability to sustain patterned
output, with greater capacity in segments rostral to LS3 than caudal to it. Various components of the
normal motor patterns can be affected in isolation by restricted lesions within particular regions of low
thoracic and upper lumbar cord. In general, the greatest disruption of pattern generation occurred when
lesions were made in the ventro-lateral portion of the spinal cord, which is the region showing the largest
concentration of cellular activity in optical recordings.
The entire chick embryo nervous system can be sustained for several hours in vitro, enabling
experiments to determine the location and nature of descending influences on motor pattern formation.
Electrical stimulation of the brainstem in stage 37 embryos produces patterned motor output, as does
superf usion with NMDA. This preparation offers great promise in elucidating the mechanisms that
generate motor output during early development.
Patch clamp recording from embryonic neurons: We have used the whole cell patch clamp
technique to record membrane currents from very small neurons in both slice preparations and intact
spinal cords in vitro. Successful recordings have been made as early as 9 days of development, making it
possible to examine changes in specific membrane currents during stages of development during which
motor patterns mature. It has been possible to develop evidence that extensor motoneurons fire because
of excitatory synaptic input during patterned motor output, but flexor cells receive predominant inhibition
early in development, while excitatory conductances appear somewhat later. There are also indications
that other conductances, as yet unidentified, may also be involved in some cells. The accessibility of this
preparation and the ability to maintain it in vitro offers great promise for analysis of normal motor pattern
generation in a vertebrate nervous system.
Development of Primary Sensory Neurons: An additional project in the Section on Developmental
Neurobiology concerns an attempt to use a variety of cytochemical, immunocytochemical, optical, and
electrophysiological techniques to study the mechanisms that specify the identity of primary sensory
neurons and to direct their outgrowth to particular targets, both centrally and peripherally. One
subproject, done using bullfrog tadpoles in collaboration with investigators at the University of Pittsburgh,
has shown that re-routing of thoracic sensory afferents to innervate the forelimb causes the formation of
central connections appropriate for forelimb afferents. This suggests that the peripheral targets of sensory
neurons can influence their choice of targets within the spinal cord. However, there are complex
interactions between the level of entry of fibers into the spinal cord and the presence or absence of
adjacent sensory ganglia.
Experiments aimed at determining whether sensory neurons are also intrinsically specified have
used the finding that a subset of primary sensory neurons in chick embryos bind soybean agglutinin (SBA).
This binding can be demonstrated in some sensory neurons removed from embryos on the 5th day of
incubation, when few have innervated peripheral targets, and grown in tissue culture without target
tissues. Birthdate studies are underway to determine whether the labeled neurons were remnants of the
original tissue or were generated in culture.
4-LNLC/DIR
High resolution video microscopy is being used to examine the factors that control neurite
outgrowth from chick embryo primary sensory neurons grown in tissue culture. Certain substrates, such as
laminin, permit these cells to become bipolar, with one rapidly growing process and another growing
more slowly. These may be equivalent to the peripheral and central neurites, respectively, of cells in the
embryo. We have found that the presence of nearby neurons in culture promotes the formation of
neurites at the site of contact, suggesting that neuronal membranes may contain factors that promote
axon outgrowth. The predictability of this effect has enabled us to use time-lapse video microscopy to
follow the development of neurites and their relation to other cell constituents. Immunocytochemical
studies have shown that the neurite outgrowths contain microtubules and GAP43, a molecule known to be
enriched in growth cones.
These studies, which are still at an early stage of development, have considerable promise for
elucidating the interplay between intrinsic specification of primary sensory neurons and the influence of
the local and distant environment in governing their ultimate structure and function.
Cortical Mechanisms of Voluntary Motor Control: Work in this project is designed to increase our
understanding of the organization of neuronal systems in regions of the cerebral cortex, primarily the
sensorimotor cortex and supplementary motor area (SMA) in primates, that are associated with the control
of voluntary movement and which project directly to the spinal cord and brainstem. The major emphasis is
on control of voluntary arm, wrist, and finger movements. Most of this research is done using non human
primates (rhesus monkeys), which are intensively trained to perform specific tasks while recording from
individual cortical neurons mounted on chronically implanted chambers. Primates are used for this
research because they exhibit the same sort of fine control of hand and individual finger movement
present in humans and they can be trained to perform fine hand and finger movements to receive
desirable food rewards. Macaque monkeys readily adapt to the laboratory situation and have been used
extensively for earlier work on the hand-arm control by the motor cortex. The new data obtained can thus
be readily integrated with the large body of existing data on macaque neuroanatomy and motor cortex
physiology. LNLC has pioneered several new methods for ensuring the health of laboratory primates and
freedom from stress in the experimental situation.
During FY 1990, we concluded one phase of our studies of the influence of the SMA on movement
performance, which was reported in previous Annual Reports. We have made a major change in computer
equipment because of the increasing fragility and lack of vendor support for the original system. This
retooling has required a major investment of time and effort. In addition, we have begun a new set of
experiments to examine the activity patterns of motor cortex neurons during bimanual skilled tasks. This
has required training of several animals to perform skilled control of wrist flexion and extension with
either hand, or both hands together. When this training is complete, cortical recording session swill begin,
probably in the first quarter of FY 1991 .
Activity in this project has also been directed to the development of a safe and reliable visual
prosthesis for blind human subjects. This effort has extended over many years in LNLC and we have now
determined that the LNLC "hatpin" electrode design, developed for primate neurophysiology, is suitable
and safe for chronic implantation in human subjects. Chronic implantation of multiple hatpin electrodes in
a monkey for 1 05 days, with systematic stimulation protocols at intensities at and above those needed for
production of visual percepts ("phosphenes") have been shown to cause minimal tissue reaction and local
damage. Light and electron microscopic study of the tissue was done in LNLC (see report of Project Z01 NS
02254-14). The electrode system also proved to be mechanically sound. LNLC will continue to collaborate
with members of the Surgical Neurology Branch and the Neuroprosthesis Program, NINDS, in this project.
According to the current schedule, the first human volunteer will be implanted in FY 1991.
Mechanisms of Repair Following Injury to Peripheral Nerves: The major focus of work in this project
is to elucidate the mechanisms by which peripheral nerves are repaired following injury. We have
developed a model system in which peripheral nerves regenerate through a gap within silicone rubber
tubes. Motor axons successfully regenerate through such gaps but to do so they must traverse a tissue
system formed de novo within the silicone tube. The tube model thus represents an exaggeration in both
time and space of the situation at an end-to-end nerve anastomosis. Interactions between fibroblasts,
5-LNLC/DIR
blood vessels, Schwann cells, axons, and extracellular matrix in the tissue cable that forms across the gap
are being studied using light and electron microscopic techniques.
Schwann cells, which appear to migrate mainly from the distal nerve stump, are essential for axonai
regeneration within the cable, since substitution of tendons for nerve in the distal stump prevents
regeneration. The Schwann cells within the cable are not organized in columns, nor are they surrounded
by basement membrane, as they are in the distal stump of an end-to-end anastomosis. We are studying
whether axonai regeneration precedes or follows Schwann cell migration at the proximal stump, and wr.at
happens when the fibers meet the mass of Schwann cells moving into the cable from the distal stump.
Complex interactions occur within the regenerating cable with respect to the endoneurial blood-
nerve barrier (BNB) In an end-to-end anastomosis of a cut or crushed nerve, the initial breakdown of BNB is
rapidly restored but there is long-lasting permeability of BNB in the tissue cable within silicone tubes, even
when regeneration is well advanced. Horseradish peroxidase (HRP) is used as a tracer substance to study
the breakdown and repair of the BNB in cut or crushed nerves. Electron microscopy is being used to
examine the morphological correlates of BNB changes.
Techniques for Making Contact with the Nervous System: As in the past, this project includes all
LNLC activities related to the development, design, and fabrication of instrumentation, specialized
mechanical equipment, specialized electrodes, and transducer devices used to support the research work
of LNLC, as well as the development of computer software necessary to handle multiple simultaneous
streams of data from on-line experiments. The advent of optical recording methods and the combination
of these with electrophysiological recording and stimulation has required development of sophisticated,
special-purpose amplifiers, tissue chambers, microscope stands, and other ancillary devices The software
necessary to store, retrieve, and analyze thousands of video images represents a major effort under this
project.
Virtually all staff members of LNLC participate in one or more aspects of this project, both as an
adjunct to their own research and as a way to share the fruits of their efforts with other staff members and
projects. Many of the techniques and instruments developed in LNLC are new and without commercial
counterpart. In such cases, LNLC staff continue to provide assistance to other scientists at NIH and at other
institutions around the world who request information and advice about specific data acquisition and
processing problems. An example is the current collaborative effort to develop a workable visual
prosthesis. The centerpiece of this project is the hatpin electrode array for intracortical microstimulation,
which was developed and refined over many years by LNLC engineers to fit the requirements of
fundamental research projects. Without the hatpin electrode, there would be no visual prosthesis. In
order to be used in humans, we have had to refine the design of the cable used to connect the electrode
with the outside world. This required fabrication of a special wire, with multiple strands of stainless steel
for strength and a single strand of gold wire, to serve as the flexible link between the cable and the
electrode itself. We have also been engaged in evaluating the properties of the activated iridium surface
of the electrode tip after it is subjected to the rigorous sterilization procedures needed for human
implants.
6-LNLC/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 01686-22 LNLC
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (BO characters or less Title must fit on one line betvteen the borders.)
Motor Control Systems in the Spinal Cord
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (hame. title, laboratory, and institute affiliation)
PI: R.E.Burke, M.D. Chief LNLC, NINDS
Others: A.K. Moschovakis, M.D., Ph.D. Staff Fellow LNLC, NINDS
G.N. Sholomenko, Ph.D. Visiting Fellow LNLC, NINDS
COORPERATING UNITS (ifny)
LAB/BRANCH
Laboratory of Neural Control
SECTION
Section on Neural Mechanisms
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
2.25
PROFESSIONAL:
1.65
OTHER:
0.6
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues [T] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The project is designed to provide information about the structure and function of neuronal
mechanisms in the mammalian spinal cord which produce and control movement. These mechanisms
include reflex pathways that convey sensory information from primary afferents to alpha motoneurons,
interactions between different reflex pathways, modulation of information flow through reflex
pathways by supraspinal descending systems, and the production of autonomous rhythmic activity by
central pattern generators within the spinal cord. Electrophysiological, neuroanatomical, and computer
modeling approaches are used. Recent work has emphasized examination of the modulation of
transmission through excitatory cutaneous reflex pathways by the spinal central pattern generator for
locomotion in order to clarify the organization of spinal interneurons that control the basic patterning of
muscle activation during locomotion in the cat.
7-LNLODIR
pms |M0 (Raw. 1.&4I
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 01687-22 LNLC
PERIOD COVERED
Octoberl, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Techniques for Making Connections with the Nervous and Musculoskeletal Systems
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: M.J.Bak Electronics Engineer LNLC, NINDS
Others: RE. Burke, M.D. Chief LNLC, NINDS
G.M. Dold Engineering Technician LNLC, NINDS
M.J.O'Donovan, M.B.Ch.B. Visiting Scientist LNLC, NINDS
E.M.Schmidt, Ph.D. Biological Engineer LNLC, NINDS
W.J.Yee Biological Engineer LNLC, NINDS
COORPERATING UNITS (if any)
Fundamental Neurosciences Program, NINDS (FT. Hambrecht)
LAB/BRANCH
Laboratory of Neural Control
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
1.0
PROFESSIONAL:
0.20
OTHER:
0.8
CHECK APPROPRIATE BOX(ES)
] (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues 0 (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project is intended to develop techniques and instrumentation for the acquisition and processing
of neuroelectric signals from the central and peripheral nervous system in acute and chronic
neurophysiological preparations. Because of this laboratory's continuing interest in sensorimotor neural
activity during unrestrained movements, the project also includes development and fabrication of
chronically implantable mechanical transducers, catheters, and connectors. Due to the laboratory's new
interests in doing research on isolated preparations, such as the spinal cord of chick embryos, much
development work has been devoted to improving techiques associated with the recording and visual
observations associated with fluorescence changes of these preparations. Also included is the
development of computer programs of general utility for acquisition and analysis of neuroelectric and
mechanical records, as well as of neuroanatomical material.
8-LNLC/DIR
PHS6M0(Re» 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 IMS 01688-22 LNLC
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ E CT (80 characters or lesi. Title must fit on one line between the borders.)
Cortical Mechanisms of Voluntary Motor Control
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (nlame. title, laboratory, and institute affiliation)
Biological Engineer LNLC, NINDS
Electronics Engineer LNLC, NINDS
Engineering Technician LNLC, NINDS
Director, Neuroprosthesis Program FNP, NINDS
Neurosurgeon SNB, NINDS
Physiologist LNLC, NINDS
Pyschologist LNLC, NINDS
PI:
E.M.Schmidt, Ph.D.
Others:
M.J. Bak
G.M. Dold
FT. Hambrecht, M.D
C. Kufta, M.D.
J.S Mcintosh
M. Pomerantz
COORPERATING UNITS (If any)
Fundamental Neurosciences Program, NINDS (FT. Hambrecht); Neuroprosthesis Research Program,
NINDS; Surgical Neurology Branch (Dr. Kufta, Dr. Oldfield)
LAB/BRANCH
Laboratory of Neural Control
SECTION
Section on Neural Mechanisms
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
2.8
PROFESSIONAL:
08
OTHER:
20
CHECK APPROPRIATE BOX(ES)
I x | (a) Human subjects
] (a1) Minors
] (a2) Interviews
J (b) Human tissues J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project is designed to investigate the spatial distribution and functional properties of cortical
neuron 'colonies' in the primate motor cortex and supplementary motor area that project to the spinal
cord and are associated with individual muscles or closely related groups of muscles, as well as the activity
of neurons in such colonies during defined voluntary motor behaviors. Cortical cell discharge patterns
during normal movements are evaluated in terms of EMG patterns, and their responses to small loading
and unloading torque perturbations. Spinal cord location of motoneurons innervating selected forelimb
muscles and termination patterns in the spinal cord and brain stem of sensory receptors within these
muscles are studied using anterograde and retrograde tracing methods.
9-LNLGDIR
PHS 6040 (Rev 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02079-17 LNLC
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ ECT (80 characters or less. Title must fit on one line between the borders.)
Models of Neurophysiological Systems
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: W.B.Marks, Ph.D. Research Physiologist LNLC, NINDS
COORPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Neural Control
SECTION
Section on Neural Mechanisms
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
1.30
PROFESSIONAL:
0.8
OTHER:
0.5
CHECK APPROPRIATE BOX(ES)
Q (a) Human subjects ^] (b) Human tissues [T] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
As quantitative data become available for a particular form or function in the nervous system, it is
advisable to attempt to assimilate the information in a comprehensive model of the underlying
mechanisms and their interactions. This project consists in the development of such models and the
necessary analytical and mathematical techniques for their implementation and testing in several areas
of experimental investigation carried out by LNLC members and in other laboratories.
Drs. Marks, Burke, and Ulfhake, guided by the discovery described in last year's Annual Report, that
for motoneuronal dendrites, local dendritic diameter largely determines the rate of branching,
terminating, and tapering, showed that (a) these rules, augmented by the empirical distributions of
daughter branch diameter, satisfactorily predict the branch length distributions of dendritic trees of
alpha and gamma motoneurons to the gastrocnemius and soleus muscle, using the empirical branching
and tapering rates of these four neuron types; (b) the meanderinqs of the dendrites between branch
points can be described as random walk constrained to have a fractal dimension of about 1 .2; (c) the
membrane area beyond any branch obeys a simple law implied by the rules; and (d) to fit real neurons,
the rules must explicitly constrain the growth beyond any branch point not to exceed that allowed by the
diameters of the parent branches.
10-LNLC/DIR
PHS 6040 (Hew. I'M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02160-16 LNLC
°ERIOD COVERED
, October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders )
Intrinsic Properties of Motor Units
PRINCIPAL INVESTIGATOR (List other profession*! personnel below the Principal Investigator.) (Name, trtie. laboratory, and institute affiliation)
PI: R.E.Burke, M.D. Chief, LNLC LNLC, NINDS
Others: W.B. Marks, Ph.D. Research Physiologist LNLC, NINDS
A.K. Moschovakis. M.D. .Ph.D. Staff Fellow LNLC, NINDS
COORPERATING UNITS Of any)
(1) Mathematics Research Branch, NIDDK
LAB/BRANCH
Laboratory of Neural Control
SECTION
Section on Neural Mechanisms
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
1.25
PROFESSIONAL:
085
OTHER:
04
CHECK APPROPRIATE BOX(ES)
1 1 (a) Human subjects
] (a1) Minors
J (a2) Interviews
I | (b) Human tissues |jT] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project designed to provide information on the electrophysiological and morphological
properties of alpha motoneurons and of the muscle fibers (muscle units) innervated by them. The
properties of gamma motoneurons are also being investigated. Methods include electrophysiological
techniques of intra- and extracellular recording, mechanical recording of muscle unit properties,
neuroanatomical techniques of intracellular injection of horseradish peroxidase to label individual,
functionally identified motoneurons, retrograde transport of lectin-conjugated horseradish peroxidase
to label motor nuclei, and computer modeling studies to analyze the experimental data produced
Recent work has emphasized detailed analysis of the dendritic morphology of alpha- and gamma-
motoneurons, with development of a computer model to describe the structure of these dendrites using
a minimum number of parameters
11 -LNLC/DIR
PHS bMO IRev l/g4)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02254-14 LNLC
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT ($0 characters or less. Title must fix on one line between the borders.)
Repair of Injured Nervous Tissue with Foreign Grafts
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (htame, title, laboratory, and institute affiliation)
PI: A. A. Zalewski.M.D. Medical Officer LNLC, NINDS
Others: N. A. Azzam, Ph.D. Special Expert LNLC, NINDS
R. N.Azzam Histopathology Tech. LNLC, NINDS
J.D. Ziemnowicz NIH Special Volunteer LNLC, NINDS
COORPERATING UNITS (if any)
CNS Disorders Research, The Upjohn Co., Kalamazoo, Ml (L.R. Williams)
LAB/BRANCH
Laboratory of Neural Control
SECTION
Section on Neuronal Regeneration
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
3.8
PROFESSIONAL:
2.00
OTHER:
1.8
CHECK APPROPRIATE BOX(ES)
I | (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues QT] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Experiments are being performed in rats to determine how a new peripheral nerve segment
develops in a silicone tube. We believe the nerve formation in an artificial tube exaggerates the events at
a nerve anastomosis site in that they occur over a longer distance and time. Studies are in progress to
analyze the early cellular events that take place during the initial tissue cable formation and axonal
regeneration. We have prepared cables which, by light microscopy, have demonstrated that cells
migrate from both ends of nerve stumps inserted into opposite ends of a 14 mm long tube. Our focus of
interest was to find a time at which migrating cells had not yet met in the middle of the tube. We are
now performing an electron microscopic analysis of the growing tips of axons in relation to the non-
neuronal cells (e.g., Schwann cells) and matrix in the cable. We have continued our studies on the
development and regeneration of the permeability barriers of nerve. Previously, we found that whereas
the endoneurial blood-nerve barrier (BNB) of normal and in situ regenerated nerve is impermeable to the
vascular tracer horseradish peroxidase (HRP), the BNB of nerve segments in tubes is not. However, during
the early phases of nerve degeneration and regeneration in situ, there is a marked increase in BNB
permeability. Accordingly, we have begun to examine crushed and cut nerves in rats to elucidate the
cause and initiating event of the increased vascular permeability. We found by light microscopy that,
within 1-2 weeks, injured nerves are permeable to HRP. It is of interest that we did not see blood-borne
cells migrating through vascular walls, which might have accounted for the increased HRP permeability
An electron microscopic investigation is in progress to determine the route of HRP passage through the
endothelium of the endoneurial blood vessels. In a collaborative project, we have studied the effect of
chronic implantation and stimulation of microelectrodes on the visual cor\ex of a monkey. Neuronal loss
was minimal. The pia mater and astocytic foot processes reacted to ensheath the microelectrodes,
isolating them from the surrounding nervous tissue. The stimulated but not unstimulated electrodes
elicited a macrophage reaction that included some giant cells. We feel that the pathology observed is
not severe enough to contraindicate the use of our visual prosthesis in blind people.
12-LNLC/DIR
PHS 6040 (Re.. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02787-02 LNLC
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders J
Analysis of Network Function in the Developing Spinal Cord of the Chick Embryo.
PRINCIPAL IN VE STIG ATOR (list other professional personnel below in* Principal Investigator ) (Name, title, laboratory, and insniufe affiliation)
PI: M.J.O'Donovan, M.B.Ch.B. Visiting Scientist LNLC, NINDS
Other: M. Antal, M.D., Ph.D. Visiting Associate LNLC, NINDS
S. Ho, Ph.D.
W.B.Marks, Ph.D.
E. Sernagor, Ph.D.
G. Sholomenko, Ph.D.
Visiting Associate
Visiting Fellow
Research Physiologist
Visiting Fellow
Visiting Fellow
LNLC, NINDS
LNLC, NINDS
LNLC, NINDS
LNLC, NINDS
COORPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Neural Control
SECTION
Section on Developmental Neurobiology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
5.6
PROFESSIONAL:
34
OTHER:
22
CHECK APPROPRIATE BOX(ES)
J (a) Human subjects
] (al) Minors
J (a2) Interviews
] (b) Human tissues QT] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The project is concerned with analyzing the development and function of spinal networks in the
lumbosacral cord of the chick embryo. One focus of the study is on the synaptic organization of
motoneurons with particular emphasis on sensorimotor and recurrent pathways A second interest is in
analyzing the cellular and network mechanisms responsible for the genesis of spontaneous motor
activity. All experiments are performed on an isolated preparation of the spinal cord which is maintained
in vitro.
13-LNLC/DIR
PHS 6O40 (Slev. 1 tW)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02788-02 LNLC
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Development of Primary Sensory Neurons
PRINCIPAL INVESTIGATOR IList other professional personnel below the Principal Investigator.) (flame, title, laboratory, and institute affiliation)
PI: C.L.Smith, Ph.D. Senior Staff Fellow LNLC, NINDS
Other: E. Munro Biologist LNLC, NINDS
E. Frank, Ph.D. Univ. PA
COORPERATING UNITS (Ifany)
University of Pittsburgh
LAB/BRANCH
Laboratory of Neural Control
SECTION
Section on Developmental Neurobiology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
2.0
PROFESSIONAL:
1.00
OTHER:
1.0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [x~1 (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The general aim of this research is to provide information about the development mechanisms
responsible for the specificity of the synaptic connections formed by primary sensory neurons in the
spinal cord during embryonic development and to gain insight into factors that might promote
regeneration of functionally appropriate connections following injury. Developing sensory neurons
innervate peripheral targets before they form central connections and it has been suggested that the
central connections of sensory neurons actually are determined by their peripheral targets. This
hypothesis was tested by forcing sensory neurons in bullfrog tadpoles to innervate a foreign peripheral
target and then examining the central connections of these neurons by anatomical and
electrophysiological methods. The results support the suggestion that central connections of sensory
neurons are influenced by their targets but also suggest that they depend on the axial level at which the
central processes enter the spinal cord and on the presence or absense of ganglia in adjacent segments.
The formation of appropriate connections by sensory neurons requires that they develop terminal arbors
in specific areas of the spinal cord. In order to identify the environmental cues that guide sensory axons
and that induce them to form branches, we are using videomicroscopy to determine how the branching
patterns of neurons growth in vitro are influenced by contacts with other cells, purified substrate
molecules and growth factors. Our initial studies have focused on early stages in axon outgrowth and
the establishment of neuronal polarity.
14-LNLC/DIR
PHS6M0(Rev 1*4)
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00
CO
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30
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o
30
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03
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ANNUAL REPORT
October 1, 1989 through September 30, 1990
Laboratory of Neurobiology
Basic Neurosciences Program, DIR
National Institute of Neurological
Disorders and Stroke
Table of Contents
RESEARCH SUMMARY 1-9
PROJECT REPORTS
Permeability of Cellular Layers in the Vertebrate Nervous System
Z01 NS 01442-24 LN 10
Structural Basis of Synaptic Transmission and Development
Z01 NS 01881-20 LN 11
Structure of Neuronal Cytoplasm
Z01 NS 02551-09 12
Distribution of Mobile and Structural Components at Chemical Synapses
Z01 NS 02610-07 LN 13
Proteins Involved in Axonal Transport
Z01 NS 02700-05 LN 14
Membrane Structure of Astrocytes
Z01 NS 01805-22 LN 15
Regeneration Specificity in Transplanted Neural Tissue
Z01 NS 02086-17 LN 16
The Blood-Brain Barrier
Z01 NS 02144-16 LN 17
i-LN/DIR
ANNUAL REPORT
October 1, 1989 through September 30, 1990
Laboratory of Neurobiology, DIR
Basic Neurosciences Program
National Institute of Neurological Disorders and Stroke
Thomas S. Reese, M.D., Chief
The Laboratory of Neurobiology has two Sections, the Section on Structural
Cell Biology and the Section on Structural Plasticity. The Section on Structural Cell
Biology uses modern structural and biochemical techniques to investigate basic cell
biological problems germane to an understanding of the function of nerve cells; the
Section on Brain Structural Plasticity applies these and other appropriate
approaches to problems of both fundamental and clinical importance in the
mammalian central nervous system (CNS), emphasizing problems related to
regeneration and response to injury. Current emphasis of the Section on Structural
Cell Biology is on the mechanism of axoplasmic transport, axonal growth, and
synaptic function while the Section on Structural Plasticity is investigating factors
which promote establishment of blood-brain barrier function and neural
connections in neural tissues implanted in the brain.
It is the Section on Structural Cell Biology that discovered the molecular
basis of the directed organelle movements underlying fast axoplasmic transport.
The translocator protein for fast anterograde transport belongs to a new class of
motility proteins that we have named kinesin. Kinesin (with ATP) is able to mimic
the movements serving fast anterograde transport. Kinesin also occurs in many
non-neuronal cells, and appears to be a general and fundamental effector of cell
motility.
Movement induced by kinesin, however, is only unidirectional with respect
to microtubule polarity, which is away from the neuronal cell body; therefore,
kinesin must mediate anterograde but not retrograde axonal transport. There is a
separate retrograde translocator which we have now characterized as a species of
cytoplasmic dynein. The anterograde kinesin-induced movements and the
retrograde dynein-induced movements can be independently inhibited, suggesting
that they function as separate motors. Since organelles can bind both kinesin and
dynein, the next question is how kinesin or dynein activation is organized on the
organelle surface.
1-LN/DIR
This question can be approached using a new electron microscope that is now
available as a result of a major instrumentation development project. This
instrument is the field-emission scanning transmission electron microscope
(STEM) for high resolution quantitative mass mapping and elemental
microanalysis of rapidly frozen, isolated macromolecules (as well as tissue
cryosections). This microscope allows, for the first time, kinesin and other protein
motors to be visualized directly on the surfaces of microtubules and organelles. It is
equipped with a cryotransfer cold stage, a highly efficient parallel electron energy
loss spectrometer (PEELS), and an ultrathin-window energy-dispersive x-ray
spectrometer which permits obtaining low-dose (<103 electron/ nm2), dark-field
molecular mass maps with single electron sensitivity, as well as performing
sensitive elemental analysis of individual macromolecules. This STEM was used to
determine at 2 nm resolution the shapes and mass distributions of purified squid
brain kinesin molecules— both free and bound to microtubules. Individual kinesin
molecules appeared as asymmetrical dumbbell-shaped structures (47-51 nm long,
MW =374 ± 14 kD), with a large head, stalk, and smaller globular foot. This
structure is quantitatively consistent with models derived from biochemistry and
molecular biology. Maps of kinesin bound to purified, taxol-stabilized bovine
microtubules (MW=19.2 kD/A) by AMP-PNP revealed that both ends of kinesin
bind to microtubules, indicating that individual kinesin molecules provide the
bridges responsible for bundling microtubules in AMP-PNP. These results provide
the first direct evidence for cross-bridging of microtubules by single kinesins and
suggest that kinesins in cells might also translocate microtubules and therefore
have some role in both slow and fast axonal transport.
Metabolic uncouplers of various classes (e.g., DNP, CCCP, valinomycin)
uniformly block organelle movements along microtubules in vitro, but not
kinesin or dynein induced movements of latex beads. Since this block is not a direct
effect on the translocators, it appears that an ionic gradient across the organelle
membrane is responsible for programming an organelle to go in the anterograde or
retrograde direction. The nature of this ionic gradient is now being investigated.
The structure and dynamic properties of other biological motors are also being
studied for comparison with the axonal transport motors. The bacterial flagellar
motor in E coli has also been shown to depend on interactions of the flagellar
structure with membrane proteins although these motors are driven, rather than
controlled by, ionic gradients. This motor system, like the axonal motor system can
also switch direction of translocation. A new structural component of the flagellar
2-LN/DIR
motor was discovered which has led us to propose a novel structural model for this
motor. A molecular genetic analysis of the new component is underway, which
may lead to an understanding of its function, possibly including the directional
switching.
The function of the microtubule-based transport system in vivo is also under
further investigation. Since microtubules do not extend the full length of the axon,
reconstructions of serial section electron micrographs of vertebrate axons and
growth cones showed the distribution of organelles contacting individual
microtubules. Organelles contacting microtubules are, judging from previous work
on the squid axon, in transport. Each organelle in the vertebrate axon may contact
several microtubules, so it is the continuous microtubule bundles which constitute
the transport pathways down the axon. An analysis of microtubule distributions in
CNS cell bodies is underway. The number of organelles in transport along these
microtubules is being counted and it is apparent that there are different populations
of microtubules, some supporting organelles transport in the cell body and some
not.
A preparation of synapses on cell bodies in the mammalian brain that could
be studied with rapid freeze techniques was developed. The rostral anteroventral
cochlear nucleus (AVCN) of the chinchilla provides a preparation in which
neuronal cell bodies and synapses in the CNS can be examined after direct freezing
and freeze-substitution of rapidly excised brain stem slices. The four types of
synaptic terminals known to be in the AVCN were distinguished and correlated
with four types of terminals previously reported after chemical fixation. Since the
transmitters for each of the four types of terminals have been specified, the
transmitter type could be correlated with the detailed structure of the postsynaptic
density in each chemical type of synapse. Two types of filamentous components-
short vertical projections from the postsynaptic membrane and long filaments
protruding from these projections— comprised the basic structure of the postsynaptic
density; the sizes and distribution of these components differed specifically in each
type of terminal. Thus, the freeze-substitution images have provided new
information about structural differences between receptor arrays and associated
cytoskeletal components at different types of CNS synapses.
As part of a continuing interest in synaptic secretion by exocytosis, the
formation of membrane pores induced by electroporation was studied with rapid
freeze techniques. This approach provided detailed information on a millisecond
time scale regarding the opening of membrane pores of our initial test preparation,
3-LN/DIR
human erythrocytes. A surprising finding was that the pores continued to open
over the first tens of milliseconds before reaching a maximum size, suggesting that
membrane interactions with the cytoskeleton were important in pore formation.
Direct freezing, cryosectioning and quantitative x-ray microanalysis had
previously been used to characterize the calcium-sequestering activity of the
endoplasmic reticulum (ER) of dendritic spines and shafts of Purkinje cells, thereby
demonstrating a role for these organelles in intracellular calcium regulation.
Presently, the route of entry of extracellular calcium remains uncertain. However,
this question and others could be resolved by pharmacologic experiments which are
beyond the scope of the present brain slice preparation. Consequently, an effort has
been mounted to develop organotypic brain slice cultures that can be used for the
more complex manipulations of calcium that are required. Such cultures of mouse
hippocampus and cerebellum are now becoming available.
Parallel efforts are directed at developing fluorescent dye techniques to
investigate the distributions of internal calcium in living cells and cell fractions
where the time course of calcium movements can also be studied. The calcium
indicator dye fluo-3 has been loaded into the ER of a neurosecretion model, the sea
urchin egg cortex, allowing ER calcium uptake and release to be monitored directly.
An RNA staining dye, thiazole orange, stains ribosomes on the same cortical ER
and in cultured cells, thereby localizing the protein-synthesizing regions of
cytoplasm. These same regions are disorganized by microtubule depolymerization.
Confocal microscopy has been useful for studying the three-dimensional
organization of ER in living cells, and offers an opportunity to observe the response
of these organelles to morphogenetic and developmental changes in cell shape.
Recent results on actively myelinating oligodendrocytes in the CNS have
demonstrated that a specific isoform (the large, 72 kD form) of the myelin-associated
glycoprotein (MAG) is involved in receptor-mediated uptake and retrograde
transport from the periaxonal region of the oligodendrocyte to the cell body. Now,
new light and electron microscopic immunocytochemical studies on optic nerve
and isolated sciatic nerve have indicated that the distribution and polarity of
microtubule bundles in myelin-forming cells are consistent with organelle
transport on microtubules, and that depolymerization of microtubules reversibly
blocks the transport of myelin proteins from the Golgi apparatus. These results
suggest that in oligodendrocytes and Schwann cells, as in neurons, transport on
microtubules is the basis of directed vesicular transport.
4-LN/DIR
Light and electron immunocytochemical techniques have also proven useful
for studying the biogenesis of the excitation-contraction (E-C) coupling system in
skeletal muscle. In normal muscle, the ai and the 0:2 subunits of the
dihydropyridine receptor have been colocalized in the junctional membrane of the
T-tubule system, thus providing evidence for the close association of these
polypeptides in the E-C coupling complex. Furthermore, ankyrin (a protein
thought to link transmembrane proteins to the cytoskeleton) has been
demonstrated in the triad, suggesting a possible role for this protein in the
organization of this structure. The localization of dihydropyridine receptors in
normal muscle contrasts with the expression and association of these proteins in
dysgenic (mdg/mdg) muscle in vitro, where the virtual absence of the oq subunit is
associated with disorganization of the (X2 subunit. To study organelle biogenesis of
the T-tubule system, the fluorescent lipid probe [DiIC16,(3)] has been used, in
conjunction with antibodies against T-tubules and plasma membranes, for the
visualization of T-tubules in living and fixed cultured muscle cells. These studies
have shown that T-tubules and plasma membrane are formed independently and
that exchange of specific components between the two connected membrane
compartments is restricted.
Membrane renewal is an important feature of homeostasis in nondividing
cells such as neurons, and is especially prominent in the light-receptive membranes
of photoreceptors which is, therefore, a suitable model system for study of this
phenomenon. Turnover of the photoreceptive membranes was studied in Limulus
photoreceptors maintained in vitro. These experiments have shown that there are
two classes of ventral photoreceptors which differ in size, morphology, and
rhabdom renewal. Rapid, light-stimulated turnover of the photoreceptive
membranes was observed in the large photoreceptors, but not in small
photoreceptors. Experiments designed to examine the role of the cytoskeleton in
rhabdom turnover showed that light exposure reduced the numbers of phalloidin-
labeled actin filaments in the rhabdoms of large, but not small photoreceptors.
The Section on Brain Structural Plasticity has three primary objectives. The
first is to characterize the neuronal differentiation of PC12 pheochromocytoma cells
infected with ras oncogene in order to select the type of neuron, either adrenergic or
cholinergic, that the cells may be able most effectively to replace in damaged brain.
The second objective is to test the possibility that muscle isografts to the brain may
serve as reservoirs for circulating macrophages which may then be induced to enter
5-LN/DIR
the brain. The third is to explore the function of the pituicyte, the astroglial cell of
the pituitary gland's neural lobe.
PCI 2 cells, infected with retroviral vectors encoding for the K-ras oncogene,
undergo neuronal differentiation which, unlike that induced by nerve growth
factor (NGF), is irreversible. Current work is to see whether this profound
difference may be attributable to the pathway of differentiation. Does the path taken
by the ras oncogene differ from the one used by NGF? We have now found that
there are a number of similarities between the effects of ras and NGF. Both the ras
oncogene and NGF down-regulate EGF receptors, but ras is more effective; by the
third day of treatment an 80% loss of receptors occurs with ras as compared with a
56% loss with NGF. Both ras and NGF bring about the development of Na+
channels as measured by the binding of saxitoxin, a specific ligand for the Na+
channel. This binding in ras -infected cells was equal to or greater than that in
NGF-primed cells. Thus, ras-infected PC12 cells should be as electrically excitable as
the NGF-treated cells. The binding of tetanus toxin to neurons is considered to be a
developmental marker in the maturation of neurons in vivo and in vitro. Like the
saxitoxin binding sites, the tetanus binding sites are localized to the neurites and
growth cones. Both ras- and NGF-treated cells have a two- to three-fold increase in
tetanus binding sites after 6 days in vitro. The remarkable neuritic outgrowth
probably entails considerable reorganization of the cytoskeleton and might involve
a finely tuned interaction of proteases and protease inhibitors. It was thus decided
to examine possible changes brought about by either ras or NGF in the level of
calpain, a calcium-activated neutral thioprotease, that occurs in normal rat brain. It
is known that calpain activity decreases in neurite-forming PCI 2 cells given NGF.
We now find that a similar decrease is brought about by the ras oncogene.
Although the effects on neuronal differentiation are thus very similar in
PCI 2 cells given either NGF or ras, there are two differences. (1) K252a, an inhibitor
of protein kinase, blocks the formation of neurites in PC12 cells given NGF, but it
does not inhibit neuritic extension by PC 12 cells infected with ras. (2) The
glycoprotein, NILE, on the surface of neurons, is expressed by NGF-treated PC12
cells but not by ras-treated ones. Such dissimilarities suggest that the pathway
utilized by the regulatory G-protein, P21, that is encoded by ras diverges from the
path taken by NGF-treated PC12 cells. It is not known whether the two differing
responses described are involved in the irreversibility of neuronal differentiation,
which is a paramount requirement for transplanted cells.
6-LN/DIR
The next goal is to examine the potential of oncogene-differentiated PC 12
cells to form synaptic junctions after being transplanted to the brain. Such potential
is more readily assessed, at first, in vitro. Only one report has described synaptic
interaction between NGF-treated PC12 cells and two clonal cell lines originating
from muscle. We have begun to establish, by differential centrifugation, cultures of
myotubes from L-6 and L-8 myoblast lines, and have labeled NGF- and ras-treated
PCI 2 cells with "cell linker", a lipid soluble fluorescent dye with a label retention
half-time of about 12 days. The marked cells are then injected into a recipient. We
have also begun to examine the spatial relation of stained neurite terminals of
living PC12 cells and the myotube surface. Neurites, varicosities and growth cones,
as well as cell bodies, are stained in PCI 2 cells which subsequently appear to remain
viable in vitro. Myoneural contacts, in the form of clusters of acetylcholine (ACh)
receptors on the sarcolemma beneath the site of neuritic contact, are to be assessed
with a series of different fluorescent conjugates of a-bungarotoxin conjugates.
Concurrent work in progress is the stereotaxic injection of ibotenic acid into the
medial forebrain nucleus of adult rats, which destroys cholinergic neurons. The
objective is to see whether ras-differentiated PC 12 cells, grafted to the damaged area
emit neurites and release ACh in amounts sufficient to overcome the induced
deficiency in this neurotransmitter. We are also repeating the measurements of
radiolabeled choline uptake, ACh synthesis, and the K+-stimulated release of ACh
into the culture medium by naive PC12 cells and those treated with NGF and ras to
evaluate the feasibility of using the cells as neuronal replacements in situ.
Autografts of skeletal muscle, inserted into the fourth ventricle, are the site
of entry of blood-borne horseradish peroxidase. Can such grafts also act as
reservoirs of macrophages which may subsequently be induced to enter the
underlying brain? Autografts of superficial neck muscle, about 1x2 to 1x6 mm, are
placed in the IV ventricle of normal rats. After five weeks, peritoneal macrophages
are collected and labeled with the lipid soluble, fluorescent marker "cell linker".
The labeling of macrophages was confirmed by immunostaining the same cells
with MAC-I antibody or by histochemical methods for the detection of nonspecific
esterase. The labeled cells are then repelleted and infused as a bolus into an axillary
artery. From here, the cells are swept through a subclavian artery into a vertebral
artery and into the circulation to the medulla. This means of perfusing the
medullary vasculature in the living animal has not been tried until now. In these
normal rats, nonactivated, labeled macrophages remain within the vessels and
parenchyma of the muscle graft. No labeled macrophages are detected within these
7-LN/DIR
intact brains, even near the site of the muscle graft. In contrast, macrophages
activated by incubation with an endotoxin from E. coli, or in a phorbol ester,
accumulate in large numbers within the graft and a few enter the adjacent medulla.
The next step is to perfuse the cerebral ventricles with purified meningococcal
endotoxin during circulation of the marked, activated macrophages. The purpose is
to entice the macrophages to enter the brain substance. We shall also try to
differentiate between the sources of the intramedullary macrophages— the
accumulated macrophages from the graft or the labeled ones coming from the
capillary bed of the brain. Both graft and capillary bed may permit activated
macrophages to enter the surrounding brain substance in substantial numbers.
Repetition of the experiments in rats that do not bear grafts should enable the
distinction between entry directly from the intrinsic capillaries of the medulla and
entry from the transplant.
We are investigating two aspects of the pituicyte, the astroglial cell of the
pituitary gland's neural lobe: the mechanism and conditions under which pituicyte
cell processes retract and reexpand, and whether the pituicyte promotes the
ingrowth of regenerating neurosecretory axons (NSA). In normal, hydrated rats,
pituicyte cell processes embrace the terminals of NSA that lie next to fenestrated
capillaries within the neural lobe. In dehydrated rats, the processes retract, so that
the released secretory products are free to reach the capillary wall without having to
confront an intervening pituicyte process. What is the mechanism of this
reversible retraction and how is it triggered? We have confirmed that pituicytes,
like astrocytes of the CNS, undergo a pronounced changes in shape when placed in
culture medium devoid of serum. Such pituicytes change from an epithelioid,
scalloped contour to a highly stellate one. The responsive cells then form many
thin branches. We have begun to follow this change with video-enhanced
differential interference contrast microscopy and by time lapse photomicroscopy.
The alteration is reversed within 60 to 90 minutes after addition of serum to the
medium. A similar, though less pronounced branching is induced by dibutyryl-
cAMP when added to culture medium containing serum. We plan to follow the
changes in the distribution of actin with phalloidin staining and to see whether
actin is involved in the shape change by adding cytochalasin D to the medium.
Inasmuch as the shape of the pituicyte in vivo is stellate and the retraction would
involve the terminals of the processes, we decided to bring the cells to the stellate
form by removing serum and waiting for 1-2 hours for the shape change to stabilize.
It was then that we added various substances that might initiate this change in
8-LN/DIR
vivo. Such possible factors are vasopressin and dynorphin, (peptides that are
coreleased during neurosecretory activity) and oxytocin. As dynorphin receptors
have been found on the surface of pituicytes in vitro, this peptide seemed to be a
particularly good candidate. Other possible agents are the neurotransmitters or
their analogs that are involved in neural lobe innervation: isoproterenol,
norepinephrine, dopamine and serotonin. The peptides have not added any
appreciable change to the stellate shape imposed by serum withdrawal. The
neurotransmitters have yet to be tried. To compare the dramatic conversion of
epithelioid to stellate forms, with the change in shape and dimensions due to
shrinkage in hyperosmotic medium, we plan to add arabinose and monitor any
changes with time lapse phase microscopy. It is possible that the neurosecretory
peptides may activate Ca++ channels in pituicytes and we are to begin a collaboration
to explore that possibility. The second project is based on: (1) our previous
observations that NSA are attracted to and regenerate within neural lobe grafts,
presumably due to the presence of pituicytes; and, (2) the report that, in explants of
the rat's ventral hypothalamus from young fetuses, some magnocellular
neurosecretory cells survive but are only able to synthesize vasopressin and not
oxytocin. Do pituicytes have a trophic effect on neurosecretory cells in enhancing
regeneration of their axons and in the synthesis of all their peptides? In order to
test the notion of a trophic influence exerted by pituicytes, fetal hypothalamus is to
be grown on extracellular matrix only and on a bed of pituicytes that have grown
out from explants. The amount of neurite formation will be measured and the
induction of oxytocin synthesis identified immunohistochemically.
9-LN/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 -NS-01442-24LN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Permeability of Cellular Layers in the Vertebrate Nervous System
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: T.S Reese, M.D. Chief LN, NINDS
Others:
S B Andrews, Ph.D.
Research Chemist
LN, NINDS
COOPERATING UNITS frfany)
B. Kachar, LNO, NIDCD, NI11, Bethesda, MD 20892. R. C. Wagner, University of Delaware, Newark,
DE. N. Lane, University of Cambridge, Cambridge, England.
LAB/BRANCH
Laboratory of Neurobiology, BNP, DIR, NINDS
SECTION
Section on Structural Cell Cell Biology
INSTITUTE AND LOCATION
N 1 N PS, N 111 , Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.1
PROFESSIONAL:
0.1
OTHER:
CHECK APPROPRIATE BOX(ES)
] (a) Human subjects
] (al) Minors
J (a2) Interviews
] (b) Human tissues [>T] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
How ti^ht junctions might prevent small charged solutes from entering the brain
(across the blood-brain barrier) is made clear by our new model of tight junction
structure based on a lipidic backbone. Tight junctions in invertebrates also appear to
have lipid backbones though our most recent observations suggest that periodic
structures, presumably proteins are intercalated into these backbones. This new work
has been submitted for publication, and publications describing completed studies on
the three-dimensional organization of the vesicular system in capillaries are appearing
this year. Otherwise this project is in abeyance.
10-LN/DIR
PHS 6040 (Hev 1/841
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS-0 1881 20 1, N
PERIOD COVERED
October 1 , 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or loss Title must fit on one line between the border s)
Structural Basis of Synaptic Transmission and Development
PRINCIPAL INVESTIGATOR (list other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
PI: T S Reese, M.D. Chief LN, NINDS
Others: G. Benshalom, Ph.D. Visiting Scientist LN, NINDS
J. A. Drazba, PhD IRTA Fellow LN, NINDS
COOPERATING UNITS (if any)
Marine Biological Lab, Woods Hole, MA. H. Tatsuoka, Deptof Anatonmy, Chiba University, Chiba,
Japan I) Chang, Dept of Molecular Physiol and Biophys, Baylor College Medicine, Houston, TX
LAB/BRANCH
Laboratory of Neurobiology, BNP, DIR, NINDS
SECTION
Section on Structural Cell Cell Biology
INSTITUTE AND LOCATION
NINDS, NIH.Bethesda Maryland 20892
TOTAL MAN YEARS:
PROFESSIONAL: . o
OTHER: q 2
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [x~\ (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The rostral anteroventral cochlear nucleus (AVCN) of the chinchilla has provided a
preparation in which neuronal cell bodies and synapses in the CNS can be examined
after direct freezing and freeze-substitution of rapidly excised brain stem slices. The
four types of synaptic terminals known to be in the AVCN were distinguished and
correlated with four types of terminals previously reported after chemical fixation.
Since the transmitters for each of the four types of terminals have been specified, the
transmitter type could be correlated with the detailed structure of the postsynaptic
density in each chemical type of synapse. Two types of filamentous components
comprising the basic structure of the postsynpatic density differed specifically in each
type of terminal, thus providing new information about structural differences between
receptors arrays and associated cytoskeletal components at different types of central
nervous system synapses. As part of a continuing effort to understand the basis of
synaptic secretion by exocytosis, the formation of membrane pores induced by
electroporation was studied with rapid freeze techniques. This approach provided
detailed information on a millisecond time scale regarding the opening of membrane
pores of our initial test preparation, human erythrocytes. A surprising finding was that
the pores continued to open over the first tens of milliseconds before reaching a
maximum size, suggesting that membrane interactions with the cytoskeleton were an
important factor in pore formation. A new initiative uses confocal microscopy of
organotypic brain slice cultures to study dynamic changes in structure during normal
function and during development; reliable cultures and detailed imaging by confocal
(fluorescent) microscopy have now been achieved.
II I.N/DIR
PHS bU4U(Hev 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01-NS-02551-09LN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Structure of Neuronal Cytoplasm
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: T S. Reese, M.D. Chief LN, NINDS
Others: P. E. Gallant, Ph.D. Research Biologist LN, NINDS
J. E Moreira, Ph.D. Visiting Scientist LN, NINDS
KG. Herman, Ph.D. Senior Staff Fellow LN, NINDS
COOPERATING UNITS (,f any)
Marine Biological Lab, Woods Hole, MA. H. Tatsuoka, Department of Anatomy, Chiba University,
Chiba, Japan. T. Cheng, State University of New York Health Science Center, Brooklyn, NY.
LAB/BRANCH
Laboratory of Neurobiology, BNP, DIR, NINDS
SECTION
Section on Structural Cell Cell Biology
INSTITUTE AND LOCATION
NINDS, Nlll, Bethesda Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL: 2 3
OTHER:
CHECK APPROPRIATE BOX(ES)
Q (a) Human subjects Q (b) Human tissues |~x~] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project determines the structure of neuronal and glial cytoplasm, particularly as it pertains to
axoplasmic transport. A protein translocator, kinesin, is responsible for the anterograde organelle
movements along microtubules which are the basis of anterograde fast axonal transport . A high
molecular weight protein in squid axoplasm, which we have characterized as a cytoplasmic dynein,
transports exclusively in the retrograde direction. Since organelles can bind both kinesin and dynein, the
next question is how kinensin or dynein activation is programmed on the organelle surface. Metabolic
uncouplers of various classes uniformly block organelle movements along microtubules in vitro, but do
not block movements of latex beads induced by kinesin or dynein so it appears that an ionic gradient
across the organelle membrane is responsible for programming an organelle to go in the anterograde or
etrograde direction. The function of the transport system in vivo is also under investigation. Each
organelle contacts several microtubules in the axon, so it is the continuous microtubule bundles which
constitutes the transport pathways down the axon. Much of the pool of kinesin and dynein in vivo is in a
soluble form and new immunocytochemical methods are being developed to determine their
distributions in cytoplasm in relation to the transport pathways. An analysis of microtubule distributions
in central nervous system cell bodies is underway. The numbers of organelles contacting, and therefore
in transport, along these microtubules is being counted and it is apparent that there are different
populations of microtubules, some supporting organelle transport in the cell body and some not.
Turnover of the photoreceptive membranes, or rhabdom, was studied in Limulus photoreceptors
maintained in vitro in order to investigate interactions of these membranes with the cytoskeleton. The
distributions of actin and tubulin were examined by conventional and laser confocal fluorescence
microscopy. Light-induced reduction of actin labeling suggests a role for actin in turnover of the
microvilli, whereas microtubules may mediate the transport of shed microvilli.
12 LN/DIR
PHS 6040 (Rev- 1«M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS 02610 07 LN
PERIOD COVERED
October 1 , 1989 through September 30, 1990
TITLE OF PROJECT (do characters or lest. Title must fit on one line between the borders)
Distribution of Mobile and Structural Components at Chemical Synapses
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: S B Andrews, Ph.D. Research Chemist LN, NINDS
Others: TS Reese, M.D. Chief LN, NINDS
A Shainberg, Ph.D. Visiting Scientist LN, NINDS
B E Flucher, Ph.D. Visiting Associate LN, NINDS
M Terasaki,Ph.D. Senior Staff Fellow LN, NINDS
G Benshalom.Ph.D. Visiting Scientist LN, NINDS
M F O'Connell.B.S. Biologist LN, NINDS
COOPERATING UNITS (if any)
Marine Biol Lab, Woods Hole, MA. K.D. Leapman, BEIF, NCCR, NIH, Bethesda, Ml). 1). ML). Landis, Case-Western Reserve Univ
Cleveland, (>l I H.I). Trapp, Johns Hopkins Univ Sch of Med, Baltimore, Ml). Ml'. Daniels, l.BG, NHLBI, NIH, Belhesda, Ml)
LAB/BRANCH
Laboratory of Neurobiology, BNP, DIR, NINDS
SECTION
Section on Structural Cell Biology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda Maryland 20892
TOTAL MAN-YEARS: . .
4.4
PROFESSIONAL: ^ 4
OTHER:
10
CHECK APPROPRIATE BOX(ES)
LJ (a) Human subjects ] (b) Human tissues 0 (c) Neither
] (al) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project studies the organization of structural and diffusible components of nerve, muscle and glia,
particularly with respect to the function of specialized membrane regions such as synapses A new and
unique instrument - the low-temperature, high-resolution, field-emission scanning transmission
electron microscope (STEM) - has been developed and shown to reach new levels of sensitivity and
resolution for molecular weight mapping and chemical analysis of native proteins. This STEM has now
been used to determine the tertiary structure and molecular weight distribution of individual, native
molecules of kinesin, as described in Project Z01-NS-02700-05 LN. This instrument will also be essential
for extending analytical microscopy studies on calcium regulation in dendritic spines and shafts, which
have been advanced by the development of suitable organotypic cultures of hippocampal slices In
parallel, uptake and release of calcium from the endoplasmic reticulum (ER) of a model for calcium-
regulated neurosecretion, the sea urchin egg cortex, has been studied using calcium indicator dyes.
Light and electron microscopy immunocytochemistry has indicated that transport on microtubules is also
the basis of anterograde and retrograde vesicular transport of myelin-specific proteins in
oligodendrocytes Thus, the depolymerization of microtubule bundles, the distribution and polarity of
which support a role in organelle translocation, reversibly block transport of myelin proteins from the
Golgi apparatus Microtubule depolymerization also causes reorganization of organelles involved in
protein synthesis in cultured cells; in living cells, the three-dimensional organization of the ER in relation
to microtubules is being revealed by confocal microscopy Immunocychemistry has been used to
localize the al and u2 subunits of the dihydropyridine receptor to the t-tubule system of skeletal muscle,
thereby indicating the association of these polypeptides in the excitation-contraction (EC) coupling
complex This contrasts with the distribution of these receptors in dysgenic muscle (mdg/mdg) where the
absence ot theal subunit is associated with disorganization of the remaining a2 protein During
myogenesis in normal muscle, the development of the E/C coupling apparatus is characterized by the
formation ol t-tubule membranes prior to connection with the plasma membrane.
13-LN/DIR
PH4il)40|Rt</ 1114)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01-NS-027U0-05LN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Proteins Involved in Axonal Transport *
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: T S. Reese, M.D. Chief LN, NINDS
Others: S B Andrews, PhD.
S. Khan, Ph.D.
Research Chemist
Guest Researcher
LN, NINDS
LN, NINDS
COOPERATING UNITS <.f any)
Marine Biological Lab, Woods Hole, MA. R. I). Leapman, BE1P, NCCR, NIH, Bethesda, MD. B.J.
Schnapp, Dept of Physiology, Boston University, Boston, MA.
LAB/BRANCH
Laboratory of Neurobiology, BNP, DIR, NINDS
SECTION
Section on Structural Cell Cell Biology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
08
PROFESSIONAL:
0.6
OTHER:
0.2
CHECK APPROPRIATE BOX(ES)
J (a) Human subjects
] (al) Minors
J (a2) Interviews
] (b) Human tissues [xj (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The goal of this project is to understand how the motors which power fast axonal
transport promote movement. Metabolic uncouplers (eg., DNP, CCCP, valinomycin)
block organelle movements along microtubules in vitro, but do not block movements of
latex beads induced by kinesin or dynein. It thus appears that an ionic gradient across
the organelle membrane is responsible for programming an organelle to go in the
anterograde or retrograde direction. The next question is how kinesin or dynein is
organized on the organelle surface and microtubule substrate. A new scanning
transmission electron microscope (STEM), as described in Project # Z01-NS-02610-07-
LN, has allowed kinesin to be visualized directly on the surfaces of microtubules and
organelles. This STEM was used to determine at 2nm resolution the shapes and mass
distributions of purified squid brain kinesin molecules— both free and bound to
microtubules. Maps of kinesin bound to purified, taxol-stabilized bovine microtubules
provided the first direct evidence for cross-bridging of microtubules by single kinesins
which suggests that kinesins in cells might also translocate microtubules and therefore
have some role in slow as well as fast axonal transport. The dynamic properties of other
biological motors are being studied for comparison with the axonal transport motors; the
bacterial flagellar motor in E coli has also been shown to depend on interactions of the
flagellar structure with membrane proteins though these motors are driven by, rather
than controlled by, ionic gradients. This motor system, like the axonal motor system,
can also switch direction. A newly discovered component of the flagellar motor has led
us to propose a novel structure model. Molecular genetic analysis of the new structural
components may lead to an understanding of directional switching.
* Formerly: "The Mechanochemistry of Proteins Involved in Axonal Transport."
14-LN/DIK
PHS6O40(Rev 1 tW|
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 -NS-01 805-22 LN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 character* or lea. Title must fit on one line between the border*.)
Membrane Structure of Astrocytes
PRINCIPAL INVESTIGATOR (list other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: P Doe, M.D. Visiting Fellow LN, NINDS
Others: M. W. Brightman, Ph.D. Section Chief LN, NINDS
COOPERATING UNITS {if any)
LAB/BRANCH
Laboratory of Neurobiology, BNP, DIR, NINDS
SECTION
Section on Brain Structural Plasticity
INSTITUTE AND LOCATION
NINDS, Nlll.Bethesda Maryland 20892
TOTAL MAN-YEARS
PROFESSIONAL: .. ?
OTHER: Q4
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [x~\ (0 Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The pituicyte of the pituitary gland's neural lobe, resembles the more common
astrocyte of the CNS in being perivascular and in having intramembranous, orthogonal
aggregates of particles or assemblies. However, pituicytes subtend fenestrated vessels
permeable to peptides and have dynorphin receptors and cells processes that embrace
the neurosecretory terminals (NST) of the neural lobe. It is known that in a dehydrated
rat, these processes retract, exposing the NST directly to the fenestrated capillaries.
During rehydration, the processes reensheath the NST. We have examined explants to
determine the mechanism of reversible contraction and to detect whether Ca+ + is
involved. We have found that, like other astrocytes, pituicytes change shape from
epithelioid to stellate in serum-deprived culture medium. If the pituicytes are initially
kept in 5% fetal calf serum instead of the usual 10%, the shape change is completed
within one hour. By the use of video-enhanced differential interference contrast and
time-lapse microscopy, we have been able to follow the conversion of lamellipodia to
thin, varicose branches. A less pronounced change is caused by dibutyryl cAMP in the
presence of serum. If Ca+ + is involved in process retraction, we may then see whether
there are Ca f + channels sensistive to dynorphin and vasopressin, that are co-released
during neurosecretion, and to oxytocin and catecholamines by the use of calcium-
activated flurochromes (e.g., Fura-2) in a video-enhanced microscope system.
15 LN/DIR
PHS6040|Ke. 1 t>4|
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01-NS-02086 17 1.N
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT \8Q characten or toss. Title mutt fit on one line between the bordary)
Regeneration Specificity in Transplanted Neural Tissue
PRINCIPAL INVESTIGATOR (Lnt other professional personnel below the Prmapal inwesvgator J (Name, title, laboratory, and institute affiliation)
PI: DL Simpson, Ph.D. Special Expert LN, NINDS
Others: S Ishihara, M.D.
M W. Brightman, Ph D
Visiting Fellow
Section Chief
LN, NINDS
LN, NINDS
COOPERATING UNITS (ifiny)
G. Guroff, IliP.Clll), NIH, Bethesda, Ml)
LAB/BRANCH
Laboratory of Neurobiology, BNP, DIR, NINDS
SECTION
Section on Brain Structural Plasticity
INSTITUTE AND LOCATION
NINDS, N1H, Bethesda Maryland 20892
TOTAL MAN-YEARS:
1.5
PROFESSIONAL:
1.3
OTHER:
0.2
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues \x~] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Before PC 12 cells, permanently differentiated into neuron-like cells by ras oncogene,
are grafted into brain as replacements for missing neurons, it is necessary to further
define their neuronal phenotype. We had found tnatras-differentiated PC 12 cells in
vitro, are both catecholaminergic and cholinergic, but that the intracellular amounts of
dopamine and norepinephrine, are less than that in naive PC12 cells. The neuronal
phenotype induced by the ras oncogene appears to follow much of the differentiation
path taken by nerve growth factor (NGF). Both ras infection and NGF of PC12 cells
result in a down-regulation of receptors for epidermal growth factor, augmentation of
Na+ channels and tetanus binding sites, and a decrease in calpain activity. However,
the effects of the two agents diverged in two aspects. While NGF induced the production
of a surface glycoprotein, NILE, ras did not. The protein kinase inhibitor K252a, which
prevents neurite formation in NGF-treated PC12 cells has no such effect on ras-treated
cells. The pathway of differentiation in PC12 cells treated with ras thus diverges from
that taken by NGF. The regulatory G-protein, P21, encoded by the ras oncogene acts at
a different point from that of NGF.
As the Schwann cell (SC) is a neighbor of the PC12 cell progenitor, the chromaffin cell of
the adrenal gland, we have examined the interactions between the PC12 and SC in
vitro. SC, derived from sciatic nerve, forms clusters upon which naive PC12 cells
aggregate. This affinity for SC is expressed even earlier by ras -PC12 cells. There is no
affinity for fibroblasts in the same co-cultures, or for astrocytes or endothelial cells.
Neurite extension is supported by SC cells or their matrix and some SC ensheath PC12
neurites. In co-cultures, choline acetyltransferase increases while acetylcholinesterase
is largely unchanged.
16-LN/DIR
CriS b04lj [lUv I ts.ll
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01-NS-02144-16LN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (tit) characters or less. Title must fit on one line between the borders )
The Blood-Brain Barrier
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: S Ishihara, M.D.
Others D.l Simpson, PhD
M W Brightman, PhD
Visiting Fellow
Special Expert
Section Chief
LN, NINDS
LN, NINDS
LN, NINDS
COOPERATING UNITS Of 3ny)
M. Sawada, I, DMA, N1DR, Nlll, Belhesda, Ml)
LAB/BRANCH
Laboratory of Neurobiology, BNP, DIR, NINDS
SECTION
Section on Brain Structural Plasticity
INSTITUTE AND LOCATION
NINDS, Nlll, Belhesda Maryland 2U892
TOTAL MAN YEARS:
1.8
PROFESSIONAL:
1.4
OTHER:
04
CHECK APPROPRIATE BOX(ES)
j (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues QT] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Does a piece of skeletal muscle, transplanted to the brain's surface, provide an entry into
the brain for blood-borne macrophages? Pieces of neck muscle, 2-3 mm x 1 mm, are
grafted to the dorsal surface of the medulla in mature rats. Two to 12 weeks later, the
rats macrophages are withdrawn from the peritoneal cavity, pelleted and labeled with a
long-lasting fluorochrome "Cell linker," which is presumably a carbocyanin dye. The
macrophages are then infused as a single bolus into the rat's axillary artery. This
vessel, which has not been used heretofore as a means of perfusing the vertebral artery,
leads the cells into the brain stem circulation. A few labeled macrophages accumulate
in the graft and surrounding choroid plexus; very few enter the medulla under the
muscle graft. It is to be tested whether macrophages will enter the brain primarily from
the intrinsic capillaries of the CNS or from the graft, when endotoxin is perfused
through the cerebral ventricles prior to introducing the macrophages.
17 1,N/1)1R
PHS6M0(Rev 184)
Laboratory of Neurobiology
Publications
Brightman MW, Simpson DL, Tao-Cheng JH, Bressler J, Okuda O, Jhang L.
Some neuronal properties of PC 12 cells infected with kirsten-ras orcogene. J
Neurocytol 1990; 20, in press.
Brightman MW, Tao-Cheng JH. Mutually imposed structural changes in
plasma membranes of astroglia and brain endothelium. In: Levi G, ed.
Differentiation and function of glial cells. New York: AR Liss, 1990;107-15.
Chang DC, Reese TS. Changes in membrane structure induced by
electroporation as revealed by rapid-freezing electron microscopy. Biophys J
1990;58:1-12.
Flucher BE, Morton ME, Froehner SC, Daniels MP. Localization of the ai and
a2 subunits of the dihydropyridine receptor and ankyrin in skeletal muscle
triads. Neuron 1990; in press.
Herman KG. Two classes of Limulus ventral photoreceptors. J Comp Neurol
1990; in press.
Kerman KG. Light stimulated rhabdom turnover in Limulus ventral
photoreceptors maintained in vitro. J Comp Neurol 1990; in press.
Kadota Y, Pettigrew KD, Brightman MW. Regrowth of damaged
neurosecretory axons to fenestrated vessels of implanted peripheral tissues.
Synapse 1990;5:175-89.
Landis DMD, Reese TS. Substructure in the assemblies of intramembrane particles in
astrocytic membranes. J Neurocytol 1989;18(6):819-31.
Leapman RD, Andrews SB. High-resolution biological microanalysis in the field-
emission STEM. Proc Xllth Int Congress Electron Microsc 1990;154-55.
Leapman RD, Andrews SB. Analysis of directly frozen macromolecules and
tissues in the field-emission STEM. J Microsc 1990; in press.
Reese T. Molecular basis of axonal transport: Kinesin and other transport proteins. In:
Fidia Foundation Neuroscience Award Lectures 1989;3:99-119.
Schnapp BJ, Crise B, Sheetz MP, Reese TS, Kahn S. Interaction between nucleotide
bonding sites during kinesin-based microtubule movement. Proc Natl Acad Sci 1990;
in press.
Schnapp BJ, Reese TS. Dynein is the motor for retrograde axonal transport of
organelles. Proc Nad Acad Sci, USA 1989;86:1548-52.
Simpson D, Dickens G, Doll S, Koizumi S, Okuda O, Oshima I, Rudkin B,
Brightman MW, Guroff G. Differentiation of PC12 Cells with K-ras;
Comparison with nerve growth factor. J Neurosci Res 1990; in press.
Tao-Cheng JH, Nagy Z, Brightman, MW. Astrocytic orthogonal arrays of
intramembranous assemblies are modulated by brain endothelial cells in vitro. J
Neurocytol 1990;19:143-53.
Tatsuoka H, Reese TS. New structural features of synapses in the anteroventral
cochlear nucleus prepared by direct freezing and freeze-substitution. J Comp Neurol
1989;290:343-57.
Terasaki M. Recent progress on structural interactions of the endoplasmic
reticulum. Cell Motility and the Cytoskeleton 1990;15:71-5.
Trapp BD, Andrews SB, Cootauco C, Quarles RH. The myelin-associated
glycoprotein is enriched in multivesicular bodies and periaxonal membranes of
actively myelinating oligodendrocytes. J Cell Biol 1989;109:2417-26.
Wagner RC, Andrews SB. Cryofixation of vascular endothelium. J Electron
Microsc Tech 1990; in press.
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ANNUAL REPORT
October 1 , 1 989 through September 30, 1 990
Laboratory of Neurochemistry
National Institute of Neurological Disorders and Stroke
Table of Contents
RESEARCH SUMMARIES
Section on Cellular and Developmental Neurobiology l
3
4
Section on Enzyme Chemistry
Section on Molecular Neuroscience
PROJECT REPORTS
Enzymological Aspects of Neural Functions 6
Z01-NS-00813-29LNC
Peptides in the Adult and Developing Vertebrate Nervous systems 7
Z01-NS-02723-04LNC
Molecular Mechanisms in Neuronal Structure and Function 8
Z01-NS-02724-04LNC
Calcium Metabolism and Protein Phosphorylation in Neuronal Systems 9
Z01-NS-02725-04LNC
Molecular Biology of the Genes Encoding Prohormones for Bombesin-Like Peptides 10
Z01-NS-02753-02LNC
Molecular Cloning of the Bombesin Receptor H
Z01-NS-02774-02 LNC
Analyses of Peptide Receptors
Z01-NS-02757-03LNC
12
ANNUAL REPORT
October 1, 1989 through September 30, 1990
Laboratory of Neurochemistry, Division of Intramural Research
National Institute of Neurological Disorders and Stroke
Harold Gainer, Ph.D., Chief
The laboratory is concerned with the development and functional organization of
the nervous system, with a special focus on molecular mechanisms. The approach of
the laboratory is cellular and molecular biological in nature and utilizes a wide
variety of techniques and concepts from a number of disciplines, e.g., physiology,
biochemistry, biophysics, morphology, immunology, and molecular biology.
Specifically, we study neuropeptides and other neurotransmitters, neuropeptide
receptors, plasma membrane and intracellular membrane systems, cytoskeletal
proteins, and various enzymes (e.g., Na,K-ATPase, protein kinases, and proteases)
which are found in the nervous system and are essential to its development and
function.
The Laboratory of Neurochemistry is composed of three Sections: Cellular and
Developmental Neurobiology, Enzyme Chemistry, and Molecular Neuroscience.
I. Section on Cellular and Developmental Neurobiology
Studies on the role of neuropeptides in nervous system development and
function, and the molecular mechanisms underlying the unique morphologies of
neurons constitute the major activities in this Section. Our studies focus on selected
neuronal populations in the hypothalamus and peripheral nervous system which we
beiieve are models for the analysis of the regulation of peptideric phenotype and
neuronal morphology. These are the luteinizing hormone-releasing hormone
(LHRH), arginine vasopressin (AVP), and oxytocin (OT)-synthesizing neurons in the
Hypothalamus, and the peptidergic sensory neurons in dorsal root and trigeminal
ganglia. Some questions we are addressing, using these systems, are: 1) What are
the ontogenetic histories of these neuronal populations? How do their cell lineage
relationships and migratory patterns during development relate to their
differentiated phenotypes (e.g., specific peptide expression)? 2) What are the
mechanisms which underlie the establishment of the differentiated neuronal
phenotypes? These issues include considerations of the heterogeneity of
differentiated properties of the populations (subpopulations), their membrane,
receptor, and signal transduction systems, unique morphologies and relevant
intrinsic cytoskeletal proteins, and axonal outgrowth and nerve terminal
distributions. 3) What are the regulatory elements in the genes uniquely expressed
by these neurons during development and homeostatically after maturation? and 4)
To what extent do these neuronal populations exhibit "plasticity"?
Most peptidergic neurons in the central nervous system (e.g., OT and AVP
neurons) derive from precursor cells in the ventricular germinal zone. Recent studies
in our section, however, have demonstrated that LHRH neurons normally located in
the adult forebrain are in fact derived from progenitor cells outside the central
nervous system proper, i.e., in the olfactory placode, and subsequently migrate
along "tracks" in the nasal area towards the brain. These cells first accumulate at
the base of the telencephalon, after which they penetrate the brain and migrate
towards their final resting destinations in the forebrain. Our recent observations
1 -LNC/DIR
suggest that the LHRH neurons associate with an neural cell adhesion molecule (N-
CAM)-rich "track" during migration, but appear to avoid fibronectin-rich areas.
Transgenic mice containing a construct of a human LHRH promotor connected to the
SV-40 T-antigen, appear to produce tumor cells in the migratory pathway. When
these cells are cultured, they express LHRH in vitro. Although formation of the LHRH
system is a prenatal event, its function is primarily postnatal; i.e., occurring after
puberty. The analysis of postnatal development and function requires an in vitro
model system. Recently, organotypic slice explant cultures have been developed in
our Section which allow for the study of LHRH neurons (and other peptidergic
neurons, e.g., AVP, OT, corticotropin-releasing hormone (CRH), and thyrotropin-
releasing hormone (TRH)-containing neurons). Studies of the regulation of peptide
gene expression in these neurons under rigorously controlled environmental
circumstances are currently in progress, and we have found thus far that estrogen
inhibits LHRH gene expression in a subset of cells, whereas inhibition of spontaneous
electrical activity inhibits overall LHRH gene expression in vitro.
In previous studies we have found that various neuroendocrine cells (e.g., CRH,
AVP and OT cells) in the hypothalamus exhibit neuropeptide coexistence which can
have profound effects when the coexisting peptides (in contrast to each peptide
alone) are secreted near targets. A central issue of peptide coexistence is the
elucidation of the molecular mechanisms which underlie the various combinations
of peptide coexistence in individual neurons. Sensory ganglion neurons exhibit a
remarkable degree of peptide coexistence, and studies on the regulation of specific
peptide gene expression in these neurons in dissociated cell culture is a major activity
in this Section. In addition to containing neurons which secrete specific peptides, a
functional network must also contain peptide receptors. In our Section, the analysis
of peptide receptors has focused on two candidates: bombesin-like peptide family
(gastrin-releasing peptide and neuromedin B) receptor, and the vasopressin (V1)
receptor. Messenger RNAs from cultured cell lines containing these receptors have
been injected into Xenopus oocytes, which then express these receptors and are
assayed by pharmacological/electrophysiological methods. Efforts to clone these
receptors are in progress, using the Xenopus assay for identification of relevant
clones (see Section on Molecular Neuroscience summary for more details). Both
tumor cell lines and primary tissue cultured neurons which contain the above
receptors are studied by patch-clamp electrophysiological techniques. The central
issues here are the elucidation of signal transduction pathways interposed between
activation of the receptors and the mobilization of membrane effecters.
At present, the study of cytoskeletal proteins in relation to neuronal morphology
in our Section is restricted to the study of intermediate filament proteins. Studies on
the posttranslational regulation of neurofilament proteins have shown that these
proteins, which appear to stabilize axonal structures, are highly phosphorylated.
Using monoclonal antibodies and immunocytochemistry we have shown that the
higher molecular weight neurofilament proteins (NF-M and NF-H) are principally
phosphorylated in axons at later stages of development. We have also found that
the specific phosphorylated sites (epitopes) found in NF-H are not universally used by
all neurons. Analyses of phosphorylated epitopes in the rat spinal cord
demonstrates that myelinated axons of comparable diameter in the cuneate and
corticospinal tracts selectively phosphorylate different sites on NF-H. Probably the
most significant issue with respect to neurofilaments is what role these polymers and
their phosphorylation play in neuronal structure and function. The most popular
hypothesis at present is that these molecules stabilize axonal structure and are
involved in increasing axonal caliber. We have examined their function by injecting
antibodies to specific neurofilament subunits and forms into one blastomere of
2-LNC/DIR
Xenopus embryos at the two-cell stage. With subsequent development these
antibodies partition selectively into only those cells which derive from the injected
blastomere. In Xenopus, this leads to a bilateral difference in the antibody
distribution and also appears to modify axonal outgrowth and branching only in
those neurons which contain the antibodies, suggesting that neurofilaments may
play a role in axonal outgrowth and elongation. We have also found that
neurofilament phosphorylation appears to protect the neurofilaments from
degradation by calpain (a calcium-dependent protease presumed to act on NFs in
nerve terminals), and that both squid axoplasm and bovine spinal cord contain
unique protein kinases related to casein kinase I that can phosphorylate
neurofilament proteins. Experiments employing mammalian sensory ganglion
explants are currently underway to investigate the effects of specific protein kinase
activations on the phosphorylation of specific serine/threonine residues in NF-L, M,
and H and peripherin in cell bodies versus axons. The significance of site specific
phosphorylation on neurofilament assembly and function remains an enigma and a
major focus of this Section.
II. Section on Enzyme Chemistry
The major projects in the Section on Enzyme Chemistry are concerned with the
relationships between structure, function and the mechanism of the ATP-dependent
cation transport proteins. The ability of cells to use metabolic energy to create and
maintain gradients of Na + ,K + ,Ca2 + and protons depends directly on these
transport proteins. In the case of the sodium-potassium pump, some general
principles of the transport mechanism and considerable structural information are
now available, but relatively little is known about the relationships between
structure and function. Current projects in the Section on Enzyme Chemistry are
directed toward (1) resolving certain mechanistic questions, and (2) gaining
information about the relationships between structure and function.
We have been developing a series of antibodies against synthetic peptides that
correspond to strategically located segments of sodium pump proteins. We are
studying the isoforms of the sodium pump that are expressed in rat brain tissue. All
three isoforms are expressed in a single neuronal cell type, i.e., primary cultures of
the cerebellar granule cell. Studies of the selective regulation of these isoforms in
pituitary and pineal glands are in progress.
The sodium pump consists of equimolar a- and B-subunits. Little is known of the
function of the 8-subunit, which is heavily glycosylated. Although, as yet, there is
good evidence for only a single message for the principal B-subunit, the expressed
protein displays a marked degree of microheterogeneity. We have recently shown
that much of this, in Electrophorus electric organ, can be ascribed to the sialic acid
component. Current studies are directed at determining the sites of glycosylation in
the Electrophorus B-subunit. Because a second form of the B-subunit has been
identified in rat brain, we are planning to examine the physiological inter-
relationships among the isoforms of the a- and B-subunits.
We are also extending previous studies on the mechanism through which the
catalytic phosphorylation of the sodium pump is coupled to cation transport. This
involves a combination of steady-state kinetic studies of -ATPase and phosphatase
activities and radiometric studies of cation-binding.
3 - LNC/DIR
Several aspects of these studies are interrelated. Our recent kinetic studies of the
sodium pump from Electrophorus electric organ indicate that, even in a preparation
that appears to consist of a single isoform, the kinetics are complicated by oligomeric
interactions. This raises questions about cells that express more than one isoform.
Do isoforms interact within the same cell to form functional hybrids? Are the B-
subunits that combine with different isoforms identical? Are these variables
functionally significant? Some of the techniques to address such questions are now
at hand.
III. Section on Molecular Neuroscience
The goal of this Section is to use a molecular approach to explore the structure,
function, and regulation of neuropeptide hormone and neuropeptide receptor
genes in the mammalian nervous system. The current effort is focused on the
mammalian bombesin-like peptides (gastrin-releasing peptide (GRP) and
neuromedin B [NMB]) and their receptors. The bombesin family of peptides is
expressed in many brain nuclei, the dorsal root ganglia, and the posterior spinal
cord. They are potent neuropeptides, eliciting a variety of central homeostatic and
behavioral responses including poikilothermia, regulation of blood glucose levels,
anorexia, alterations in gastric acidity, and scratching behavior. These peptides have
also been detected in the intrinsic neurons of the gut, where they regulate smooth
muscle contraction, stimulate the release of gastrin from G cells of the antral gastric
mucosa, and function as secretogogues for a variety of gastroenteropancreatic
hormones. Bombesin-like peptides are mitogens for growth-arrested murine
embryonal Swiss 3T3 fibroblasts in culture, G cells in neonatal rat antral mucosa, and
autocrine growth factors for human small cell lung cancer, indicating that under
some circumstances these peptides can regulate cell division in addition to
transducing a secretory or neuromodulatory signal.
We have obtained cDNA and genomic clones for the prohormones encoding the
two known mammalian bombesin-like peptides, GRP and NM-B. The rat prepro GRP
gene is expressed in many brain nuclei, most prominently in the suprachiasmatic
nucleus of the hypothalamus. Two forms of the mRNA are found in brain: a more
abundant 1.1 kb form which initiates in both central and peripheral neurons, and a
less abundant 1.5 kb mRNA form, whose initiation sites are heterogeneous, located
several hundred bases upstream of the 1.1 kb initiation site, and is used only in spinal
cord and a subset of brain nuclei expressing prepro GRP mRNA. Studies performed
on cultured cells expressing the human prepro GRP gene indicate that gene
regulation occurs primarily at the level of transcription initiation, and involves
chromatin structural changes resulting in DNase hypersensitive sites. Transfection of
GRP promotor-luciferase reporter gene constructs into these cells have identified
two cis promotor elements of importance in regulating prepro GRP transcription.
These regions will be precisely mapped and characterized, with the central goal of
obtaining cDNA clones for any novel transcription factors which specifically bind
these cis acting elements. The molecular mechanisms responsible for cell-type
specific regulation of the prepro GRP gene constitute an area of intense interest that
we plan to pursue in detail in the future.
We have isolated and characterized cDNA and genomic clones for the rat prepro
NM-B gene. The gene has a three-exon structure analogous to that described
previously for the prepro GRP gene, consistent with the view that the two genes
diverged from a common ancestral precursor gene. Similarity between the two
genes is observed only over the carboxyl ten amino acids of the GRP and NM-B
4-LNC/DIR
peptides, which is the region of the peptide necessary and sufficient for high affinity
binding to bombesin receptors. The sequences of the two genes in the promoter
region show no regions of similarity, suggesting that the two genes are
independently regulated. Expression of the gene results in a 1.0 kb mRNA species
that is most abundantly expressed in brain and gut. The initiation site in the brain
appears to be heterogeneous, and not TATA-directed. In situ hybridization studies
localizing NM-B mRNA in the brain indicate that the distribution of cells and loci
expressing NM-B is quite distinct from those expressing GRP, and more limited. Both
genes contribute independently to the bombesin-like immunoreactivity described
previously in the brain, indicating that these two peptides modulate different
physiologic processes. Neuromedin B mRNA is expressed at high levels in trigeminal
and dorsal root ganglia neurons. In collaboration with Dr. Gainer, we are currently
exploring the possibility that primary cultures derived from these ganglia may
provide a population of neurons expressing the neuromedin B gene. These cells
would be appropriate hosts for promoter-reporter fusion gene transfection studies
done in collaboration with Dr. Gainer's lab to define transcription regulatory
elements in the neuromedin B promoter.
In collaboration with Dr. Richard Feldman at Triton Biosciences, and Drs. Kiyoshi
Kusano and Hagit Shapira of the Laboratory of Neurochemistry, we have isolated
cDNA clones encoding the GRP receptor expressed in Swiss 3T3 murine embryonal
fibroblasts. Amino acid sequences derived from an internal tryptic fragment of the
purified GRP receptor protein were used to design oligonucleotide probes, which in
turn were used to screen a subtracted cDNA library enriched for Swiss 3T3 GRP
receptor cDNA clones. Structural analysis of cDNA clones encoding the entire open-
reading frame show that the receptor is a member of the G-protein coupled receptor
superfamily with seven predicted transmembrane domains and numerous sites for
N-linked glycosylation. In vitro transcripts from cloned cDNA templates
enrompassing the predicted protein coding domain express functional GRP
receptors when injected into Xenopus oocytes.
Low-stringency screening of cDNA libraries derived from rat esophagus and
olfactory bulb using the Swiss 3T3 GRP-preferring receptor probe identified a clone
sharing 50% amino acid identity, which is an excellent candidate for an additional
bombesin receptor subtype (NMB-preferring) known to exist in these tissues.
Expression of this candidate receptor in Xenopus oocytes will confirm this result,
since there is an antagonist available which selectively blocks the GRP receptor from
Swiss 3T3 cells but does not block the NMB-preferring bombesin receptor found in
the esophagus. These clones will form the basis for site-specific mutagenesis
experiments probing the domains and residues involved in specific ligand binding
and coupling to G-proteins. Expression of cDNA clones in various model systems will
allow the production of large quantitites of this relatively rare protein, facilitating
the identification of subtype-specific antagonists which will prove useful in
dissecting the functions of the bombesin-like peptides and their receptors in the
nervous system. In addition, these antagonists may prove useful as therapeutic
agents in the subset of human small cell lung carcinomas which show bombesin-
dependent growth.
5 - LNC/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01-NS-00813 29LNC
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or /ess. Title must fit on one line between the borders.}
Enzymological Aspects of Neural Functions
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: Robert W. Albers, Ph.D. Chief, Enzyme Chemistry LNC, NINDS
Others: William T. Link, Ph.D. Senior Staff Fellow LNC, NINDS
CharleneP. Osborn, B.S. Biological Technician LNC, NINDS
Paul M. Rowe, Ph.D. Senior Staff Fellow LNC, NINDS
COORPERATING UNITS lit an,)
J. P. Froehlich, Ph.D., M.D., NIA, NIH, Baltimore, MD;
E. Bamberg, Max-Planck-lnstitut fur Biophysik, Frankfurt, FRG; D. Klein, Ph.D., NICHD, NIH
LAB/BRANCH
Neurochemistry, DIR, NINDS
SECTION
Enzyme Chemistry
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS: . n
4.0
PROFESSIONAL: o q
OTHER:
1.0
CHECK APPROPRIATE BOX(ES)
Q (a) Human subjects ^] (b) Human tissues [x~| (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project is comprised of research into the structure and functioning of ion transport systems. There
are currently 4 active subprojects: 1) Studies of the transient-state kinetics of phosphorylation and
dephosphorylation of the Na,K-ATPase catalytic site, utilizing rapid quenching techniques. 2) A study of
the regulation and expression of isoforms of the Na,K-ATPase utilizing site-directed antibodies raised
against synthetic peptides as identifying probes. 3) A study of the relation of the glycosylation state of
the B-subunit of the Na,K-ATPase to the expression and function of the sodium pump. 4) A study of the
interactions between the catalytic and the ionophoric domains of the sodium pump utilizing steady-
state kinetic and radiometric binding techniques.
6-LNGDIR
■HS6CM0(Row 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS 02723-04 LNC
PERIODCOVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT fAO characters or less Title must fit on one line between the borders.)
Peptides in the Adult and Developing Vertebrate Nervous Systems
PRINCIPAL INVESTIGATOR tList other profession*/ personnel below the Principal Investigator.) {Name, title, laboratory. *nd institute affiliation)
PI: Harold Gainer, Ph.D. Chief LNC, NINDS
Others: Carolyn Bondy, M.D. Senior Staff Fellow LNC, NINDS
MonaCastel.Ph D Visiting Scientist LNC, NINDS
Sharon Key, B.S. Biologist LNC, NINDS
Susan Wray, Ph.D. Senior Staff Fellow LNC, NINDS
COORPERATING UNITS (,fanf)
Dr. W.J. Schwartz, University of Massachussetts, School of Medicine; Dr. W.S. Young, NIMH;
Dr. S. ^adovick, NIDDK
LAB/BRANCH
Laboratory of Neurochemistry, DIR, NINDS
SECTION
Section on Cellular and Developmental Neurobiology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN-YEARS: . n
4.0
PROFESSIONAL: o Q
OTHER:
1.0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [~x~l (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do nor exceed the space provided.)
Studies of the extracellular matrix over the track upon which the LHRH cells migrate from the olfactory
placode to the CNS have shown that the appearance of N-CAM directly correlates with the appearance of
LHRH cells, whereas fibronectin does not. LHRH cells are present in tissue cultures deriveJ from
embryonic nasal regions of mice. A transgenic line was established using a human LHRH promoter joined
to the SV40 t-antiqen construct. These animals show abnormal reproductive function and on
examination very few LHRH immunopositive cells were detected in the forebrain. Transgenic mice
containing constructs which have both AVP and OT genes together exhibit cell-specific expression,
whereas transgenic mice with only one of these genes exhibit only ectopic expression. Studies of aldose
reductase mRNA in lens and kidney show that expression of this gene is inhibited by galactosemia and
hyperglycemia. Rat brain slice-explant cultures have been used to show that estrogen and inhibition of
spontaneous electrical activity by TTX leads to inhibition of LHRH gene expression. Exaggerated
coexistence of CGRP and substance P peptides in postnatal sensory neurons in vitro was found.
7-LNC/DIR
■"!'. blUOIK.v 1*4)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 -NS-02724-04LNC
PERIOD COVERED
Octoberl, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Molecular Mechanisms in Neuronal Structure and Function
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: Harold Gainer, Ph.D. Chief LNC, NINDS
Others: Margi Goldstein, Ph.D. Senior Staff Fellow LNC, NINDS
Philip Grant, Ph.D. Expert Consultant LNC, NINDS
Shirley House, B.S. Biologist LNC, NINDS
Ben Szaro, Ph.D. Senior Staff Fellow LNC, NINDS
COORPERATING UNITS of any)
L. Charnas, M.D., NICHD; V.M.Y. Lee, Ph.D., University of Pennsylvania
LAB/BRANCH
Laboratory of Neurochemistry, DIR, NINDS
SECTION
Section on Cellular and Developmental Neurobiology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL: j 5
OTHER:
1.0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [~x~~| (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Monoclonal antibodies directed against neurofilament (NF-M) proteins in Xenopus, have been injected
into individual blastomeres of Xenopus embryos at the two-cell stage. These antibodies remain
restricted in the intracellular space so that only cells derived from the injected blastomere contain the
antibody (i.e., about half the cells in the developing embryo contain antibody). The results are that the
axons derived from neurons containing these antibodies show a variety of dramatic deficiencies in
axonal outgrowth and elongation, representing the first demonstration in vivo that perturbation of a
neurofilament protein has structural consequences. Studies on rat central and peripheral nervous system
neurons in vivo and in vitro have shown that different post-translational modifications (i.e.,
phosphorylation of specific sites) of neurofilaments are characteristic of specific nerve pathways and
neuronal types, and not necessarily correlated with axonal caliber or their central or peripheral origins.
Dissociated sensory ganglion cells from postnatal rats survive in defined media in the absence of NGF,
but are deficient in general growth, neuropeptide and neurofilament expression and neuritic
outgrowth.
8-LNGDIR
•v-ioMOIRev. 1,84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01-NS-02725-04LNC
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 character s or Ion Title must tit on one line between the borders )
Calcium Metabolism and Protein Phosphorylation in Neuronal Systems
PRINCIPAL INVESTIGATOR (List other professional per sonnel 6e'ow the Principal Investigator ) (Name, title, laboratory, and institute affiliation}
PI: HarishC. Pant, Ph.D. Research Chemist LNC, NINDS
Others: Ayse Dosemeci, Ph.D. Visiting Fellow LNC, NINDS
Carl C. Floyd, PhD IRTA Fellow LNC, NINDS
Ben G.Szaro, Ph.D. Senior Staff Fellow LNC, NINDS
James Battey, M.D., Ph.D. Chief, Molecular Neurosciences Section LNC, NINDS
James Way, B.S. Biologist LNC, NINDS
Alexander Wheaton Biological Laboratory Technician LNC, NINDS
COOPERATING UNITS (if an,)
Dr. P.E. Gallant, LN, NINDS; Dr. Jan Metuzals, University of Toronto
LAB/BRANCH
Neurochemistry, DIR, NINDS
SECTION
Cellular and Developmental Neurobiology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS: . ..
4.3
PROFESSIONAL: , j
OTHER:
1.1
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LJ (a) Human subjects ] (b) Human tissues ["*! (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Studies have been continued to understand the regulation and role of neurofilament phosphorylation in
the nervous system. Axonal neurofilament-rich preparation from squid giant axon contains second
messenger-independent protein kinases that phosphorylate high molecular weight >400 and 220 kDa
squid neurofilament protein subunits, as well as exogenous substrates. Two major kinase activities were
separated and characterized in this preparation. One of these strongly phosphorylated kemptide and
was inhibited by the selective inhibitors of cAMP-dependent kinase, wiptide. The second kinase activity
effectively phosphorylated alpha-casein and was not inhibited by wiptide and heparin. The alpha-casein
phosphorylating activity was the principal activity responsible for neurofilament protein
phosphorylation and was not inhibited by various kinase inhibitors. A newly synthesized isoquinohne
derivative (CKI-7) that specifically inhibited purified casein kinase-l was the effective inhibitor of the
axonal neurofilament protein kinase. The physical, biochemical and pharmacological studies indicated
that the major kinase activity associated with axonal neurofilaments is like a casein kinase I. The
mammalian neurofilament associated kinases appear to be more complex. There was only a partial
inhibition of bovine neurofilament kinase activity by casein kinase I inhibitor and the tryptic peptide
maps of neurofilament protein subunit (NF-M) after its phosphorylation by bovine neurofilament kinase
showed some similarities when phosphorylated by purified casein kinase I or casein kinase II. The cAMP-
dependent kinase phosphorylated distinct peptides on NF-M. We have determined the sequence of a
squid neuronal intermediate filament protein, approximately 59 kDa, using a cDNA library made from
squid optic lobe. This protein is specific to neural tissue, and is present in the axoplasm of the giant axon.
Structurally, its rod domain possesses features common to mammalian Type IV (neurofilament) and Type
III (vimentin, GFAP) intermediate filament proteins as well as Lamms. By highlighting those sequences of
NF-proteins that are conserved between these two animals, which diverged around 570 million years
ago, the sequence of this mRNA will help elucidate functionally important domains in NF-proteins.
9-LNC/DIR
^HS bM |H«v 1 Ml
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01-NS-02753-02LNC
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (SO characters or less. Title must fit on one line between the borders.)
Molecular Biology of the Genes Encoding Prohormones for Bombesin-like Peptides
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: James Battey, M.D., Ph.D. Section Chief LNC, NINDS
Others: Zahra Fathi, Ph.D. IRTA Fellow LNC. NINDS
Etsuko Wada, M.D., Ph.D. Visiting Fellow LNC, NINDS
James Way, B.S. Biologist LNC, NINDS
COORPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Neurochemistry, DIR, NINDS
SECTION
Section on Molecular Neuroscience
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN-YEARS:
1.8
PROFESSIONAL:
1.3
OTHER:
0.5
CHECK APPROPRIATE BOX(ES)
I (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues 0 (0 Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The goal of this project is to understand aspects of the structure, function, and regulation of the genes
encoding the mammalian counterparts to bombesin-like neuropeptides. Two mammalian bombesin-like
peptides have been identified to date, gastrin-releasinq peptide (GRP) and neuromedin B (NM-B). Both
peptides are found in a subset of central and peripheral neurons, and share a structural motif at their
carboxyl-terminal domains, which is necessary and sufficient for binding to high-affinity cell surface
bombesin receptors. There are several subprojects being actively pursued: 1) cDNA and genomic clones
for rat and human prepro-GRP and prepro-NMB genes have been isolated and characterized to identify
potential regulatory motifs in the promoter regions. 2) In situ hybridization technigues are being
utilized to determine the localization of GRP and NMB mRNA at the cellular level in brain; 3) Promoter-
reporter gene constructs will be introduced into cultured cell hosts by transfection to map the elements
in the promoter region of the human GRP gene responsible for the cell-type specific regulation.
10-LNODIR
■'• "■> bWD IR.w 1.84J
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS02774 02 LNC
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or lesi. Title must fit on one line between the borders.)
Molecular Cloning of Bombesin Receptors
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Hame. title, laboratory, and institute affiliation)
PI: James Battey, M .D., Ph.D. Section Chief LNC, NINDS
Others: Martha Corjay, Ph.D. Staff Fellow LNC, NINDS
Kiyoshi Kusano, Ph D Visiting Scientist LNC, NINDS
HagitShapira, Ph.D. Visiting Fellow LNC, NINDS
Etsuko Wada, M.D., Ph.D. Visiting Fellow LNC, NINDS
James Way, B.S. Biologist LNC, NINDS
COORPERATING UNITS (if any)
Richard Feldman, Ph.D., Triton Biosciences, 1 501 Harbor Way Parkway, Alameda, CA 94501
LAB/BRANCH
Neurochemistry, DIR, NINDS
SECTION
Section on Molecular Neuroscience
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS: ^ n
PROFESSIONAL: j Q
OTHER: 1 q
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [x~l (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The goal of this project is to use molecular genetic techniques to study the structure and function of
mammalian bombesin receptors. We plan to accomplish this objective by first obtaining a full-length
cDNA clone for the murine bombesin receptor found on Swiss 3T3 fibroblasts in relatively high numbers.
The cDNA will be sequenced to determine the predicted amino acid sequence and general structural
features of the receptor. Specific antisera will be generated against synthetic peptides to allow further
characterization of the receptor protein. This cDNA clone can then be used in site-specific mutagenesis
studies where the effects of structural perturbations can be examined after expression of the mutant
receptors in Xenopus oocytes and DNA-mediated transfection into murine fibroblasts which do not
normally express the receptor Genomic clones will be isolated to examine the gene structure and
promoter region. The cDNA coding domain will be used as a low-stringency probe to screen cDNA
libraries from appropriate sources for other distinct but structurally related bombesin receptor cDNA
clones, including a distinct subtype found on esophageal smooth muscle and in the olfactory bulb of the
brain.
11 -LNC/DIR
'-'.bWD|>t>. 1 »4)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS-02757-03LNC
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Analyses of Peptide Receptors
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: Kiyoshi Kusano, Ph.D. Visiting Scientist LNC, NINDS
Others: James Battey, M.D., Ph.D. Chief Section on Molecular Neurosciences LNC, NINDS
Harold Gainer, Ph.D. Chief LNC, NINDS
HagitShapira, Ph.D. Visiting Fellow LNC, NINDS
COORPERATING UNITS Of any)
Dr. M.J. Brownstein and Dr. L. Mahan, LCB, NIMH; Dr. I. Tasaki, LCB, NIMH;
Dr. R. Wenthold, LND, NINDS
LAB/BRANCH
Laboratory of Neurochemistry, DIR, NINDS
SECTION
Section on Cellular and Developmental Neurobiology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS: c
1 .0
PROFESSIONAL: ., g
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues fx~| (c) Neither
] (a1) Minors
J (a2) Interviews
SU MMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This study on the cellular and molecular mechanisms of peptide receptor signal transduction processes
has used two approaches. The first approach is to analyze the electrophysiological responses to
application of neuropeptides in selected peptide receptor-bearing cells: Swiss 3T3 cells for bombesin
(BN) receptors and AR42J cells for cholecvstokinin (CCK) receptors. The second approach is to study the
peptide receptor signal transduction process in detail using the Xenopus oocyte surrogate system, after
injection of mRNAs extracted from the above cells. Whole cell voltage-damp and patch-clamp
techniques are applied to those cultured cells. The Swiss 3T3 cells do not have profound voltage sensitive
K-channels, but some cells possess a transient type of Ca-current (lca(t>) channels. Application of
bombesin to 3T3 cells induces a K-conductance increase by activating a Ca-mediated K-current (l«(Ca))
flow. AR42J cells possess voltage-sensitive Ik, l|\ia. 'ca(t) ancl 'ca(i). ancl Ca-sensitive lci(Ca)- Application of
CCK to AR42J cells induces a conductance increase to CI ions, whereas Na and K ions do not show
significant effects. In both cell lines, ligand binding with receptors triggers a rise in intracellular Ca ion
concentration by releasing from intracellular sources. The two-electrode voltage clamp technique has
been applied to assay the functionally transcribed CCK and bombesin receptors in the Xenopus oocytes,
after injection of their respective mRNAs. Size-fractionated mRNAs obtained by the sucrose gradient
technique are further assayed in order to clone and sequence these receptor cDNAs and genes.
Pharmacological analysis of BN receptors expressed by injecting mRNAs of various tissues indicates that
there are subtypes in brain and esophageal tissues.
12-LNC/DIR
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ANNUAL REPORT
October 1, 1989 through September 30, 1990
Laboratory of Neuropathology and Neuroanatomical Sciences
Basic Neurosciences Program, DIR
National Institute of Neurological Disorders and Stroke
Table of Contents
RESEARCH SUMMARY 1-9
PROJECT REPORTS
Blood-Brain Barrier: In Vitro Model for the Study of 10
Cerebrovascular Endothelial Permeability
Z01 NS 02324-14 LNNS
Cerebral Ischemia and Monoamines 11
Z01 NS 02357-13 LNNS
Evaluation of Electrical Impedance in Cerebral Ischemia 12
Z01 NS 02548-09 LNNS
Cerebial Ischemia and Edema: Tryptophan Uptake and 13
Metabolism
Z01 NS 02623-05 LNNS
Regulation of Carbohydrate Metabolism in 14
Cerebromicrovascular Cultures
Z01 NS 02689-06 LNNS
Study of Cerebral Electrical Activity Associated with 1 5
Ischemia
Z01 NS 02718-05 LNNS
Stress Protein Induction in Gerbil Brain After Ischemia 16
Z01 NS 02720-04 LNNS
The Measurement of Cerebral Blood Flow by Laser 1 7
Doppler Velocimetry
Z01 NS 02749-04 LNNS
Cultures of Mouse Capillary Endothelium: Establishment
Z01 NS 02751-04 LNNS 18
Postischemic Accumulation of Calcium in Brain Tissue 19
Z01 NS 02768-03 LNNS
Observations on Global Cerebral Ischemia in Rats 20
Z01 NS 02773-02 LNNS
Blood-Brain Barrier Changes Following Repeated 21
Ischemic Insults
Z01 NS 02775-02 LNNS
Mechanism of Production of Experimental Allergic 22
Encephalomyelitis
Z01 NS 02776-02 LNNS
Human Cerebromicrovascular Endothelium: 23
Studies in Vitro
Z01 NS 02777-02 LNNS
Cerebral Vascular Endothelial Cell-Specific 24
Monoclonal Antibodies
Z01 NS 02780-03 LNNS
Human Cerebromicrovascular Endothelial Culture: 25
Cholinergic and Histaminergic Receptors
Z01 NS 02795-02 LNNS
Transneuronal Effects of Cryogenic Brain Injury on 26
Calcium Uptake and Blood-Brain Barrier Changes
Z01 NS 02796-02 LNNS
Cultures of Human Cerebromicrovascular Endothelium: 27
Establishment, Growth and Characterization
Z01 NS 02797-02 LNNS
Ultrastructural Observations on Distribution of 28
Calcium in Cerebral Ischemia
Z01 NS 02798-02 LNNS
Interactions Between Cerebrovascular Endothelial 29
Cells and Immune Lymphocytes
Z01 NS 02801-02 LNNS
Dynamics of Postischemic Calcium Accumulation and
Protein Synthesis in Brain
Z01 NS 02821-01 LNNS 30
Glutamate Microdialysis During Repeated Ischemia
and Cold Lesions.
Z01 NS 02822-01 LNNS 31
Immune Mechanisms: Regulation of la Antigen Expression 32
Z01 NS 02802-03 LNNS
ANNUAL REPORT
October 1# 1989 through September 30, 1990
Laboratory of Neuropathology and Neuroanatomical Sciences
Basic Neurosciences Program, DIR
National Institute of Neurological Disorders and Stroke
Igor Klatzo, M.D. Chief
During the past year, the Laboratory of Neuropathology and Neuroanatomical
Sciences has continued its effort on elucidating the mechanisms operative in the
pathophysiology of ischemia and traumatic brain injury.
In the Section on Cerebrovascular Pathology, the main interest has been directed to
define and evaluate extent to which neuroexcitatory mechanisms were involved in the
final development of postischemic and posttraumatic lesions.
In 1975, our laboratory was first to describe a selective, delayed destruction of CA1
pyramidal neurons of the hippocampus following a short-lasting cerebral ischemia (Ito et
al. 1975). Later we were able to associate the development of this injury with a
protracted period of neuroexcitation of pyramidal CA1 neurons by a direct recording of
axonal discharges with microelectrodes (Suzuki et al. 1983). At the same time the
neuroexcitatory nature of CA1 lesions was further confirmed by Benveniste et al. (1984),
who demonstrated by microdialysis a marked extracellular release of glutamate and
aspartate during and after an ischemic insult. The demonstration of neuroexcitation as a
primary mechanism of ischemic injury has long been confined to the hippocampus. More
recently, however, it became increasingly apparent that excitatory damage may involve
different transmitter systems and may affect distant neuronal locations, according to
connecting circuitries. Thus, an excitatory effect of the dopaminergic system on ischemic
injury was demonstrated by Globus (1987) who reported a significant reduction of
striatal damage by lesioning the substantia nigra.
One of our three experimental models of cerebral ischemia in which we assume a
widespread involvement of excitatory mechanisms is repetitive ischemia in gerbils. Our
studies on the effect of three 2-minute occlusions at one-hour intervals revealed a
pattern of dense accumulations of 45Ca in various structures at different time sequences.
The earliest accumulations appeared in the ventral parts of the thalamus, particularly in
the nucleus reticularis thalami, then in the lateral portions of caudate, and the cerebral
cortex, and after 4 days in the medial geniculate bodies and substantia nigra. There was
an obvious correlation between caudate and substantia nigra, since the unilateral
preponderance of calcium uptake in the caudate corresponded to increased 45Ca on the
same side in the substantia nigra. The abnormal uptake of calcium correlated with the
appearance of histological neuronal changes which, however, were usually of a
moderate, chronictype, with exception of CA1 pyramidal neurons, which showed severe
destruction after 4 days.
The abnormal calcium uptake correlated also with changes in blood-brain bar Tier
(BBB) permeability to serum proteins, expressed by the noticeable presence of
extravasated albumin in the cytoplasm of ischemically affected neurons, which was
demonstrated by staining with conjugated anti-gerbil albumin serum. The abnormal
accumulation of calcium and intracytoplasmic albumin uptake were associated with
1 -LNNS/DIR
morphological picture of rather moderate neuronal injury and were absent in regions
showing severe, irreversible damage of the nerve cells.
The involvement of neuroexcitatory mechanisms in repetitive ischemia was
supported also by our observations using microdialysis to assess levels of glutamate
during and following repetitive ischemic insults. The release of extracellular glutamate
was evaluated by cortical microdialysis followed by an enzymatic cycling assay employing
glutamate dehydrogenase and glutamate B-oxaloacetate transaminase. Our assay
revealed in some animals, during and after repeated 5-minute occlusions, a progressive
increase in glutamate release after the second and third ischemic insult, often reaching
concentrations of glutamate in the dialysate comparable to those observed after a single
1 5-minute occlusion. Such progressively higher levels of glutamate may constitute the
main explanation for a cumulative effect of repeated insults, whereas this cumulative
effect appeared in our previous studies to be totally unrelated to the reduced cerebral
blood flow (hypoperfusion) which develops after recirculation, or to energy metabolism
disturbances during the immediate postischemic periods (Hossmann et al. 1990).
Studies on the the formation of secondary foci of injury following a cryogenic
cortical lesion in rat have further supported to the assumption of neuroexcitatory
mechanisms operative in the development of such lesions. The delayed appearance of
secondary lesions in the ipsilateral thalamus or bilaterally in CA1 sectors of the
hippocampus, when the cortical injury was extended to include the entorhinal cortex,
was characterized by the presence of 4$Ca accumulation, neuronal uptake of serum
albumin, and morphological changes indicating a moderate neuronal injury. To
establish whether these changes originate from neuroexcitation at the margin of the
cryogenic focus, the cold lesion was produced in proximity to the rat visual cortex from
which the evoked potentials were measured following stroboscopic light stimulation.
Our observations indicate a significant reduction in latency time which appeared
approximately 4 hours after induction of the cortical lesion. This reduction in the latency
period could be abolished by pretreatment with the noncompetitive glutamate
antagonist of MK-801. Further indication of glutamate involvement was obtained from
direct application of I mM glutamate solution on the exposed cortex, which reduced the
latency period similar to that following cold injury. The demonstrated reduction in the
latency time would be in line with facilitation of neuronal excitability. Another
observation, which would support an assumption that neuroexcitation may be primarily
responsible for the development of secondary lesions, was provided by comparative
evaluation of volumes of the areas of the cerebral cortex and the thalamus showing
dense 4$Ca accumulation. Such assessments, done by Dr. G. Mies, indicated that the
thalamic foci tended to be of uniform volume, whereas the cortical 45Ca areas varied
greatly in volume, without any correlation to the thalamic foci. This observation
indicates against a possibility that the thalamic lesions were merely a product of a
retrograde neuronal degeneration.
An assumption of neuroexcitatory involvement was further strengthened in our
studies on the pathophysiology of global ischemia produced by compression of the major
heart vessels. This model was characterized, as in repetitive ischemia, by 45Ca marking of
various anatomic locations affected by ischemia which appeared after different time
delays. The most striking was sharply outlined very dense marking of the nuclei
reticularis thalami as early as 6 hours after 10 minute global ischemia, thus appearing as
the first structure affected by ischemia. This may have etiologic significance, since the
ischemic affection of this nucleus which consists entirely of GABAergic neurons, may
remove inhibitory function on the otherthalamic nuclei and other structures,
contributing thus to development of ischemic lesions in "uninhibited" areas. After
24 hours accumulation of 45Ca calcium was noticeable in other regions such as cerebral
cortex, caudate, ventral thalamic nuclei, medial geniculate and substantia nigra. In the
hippocampus, CA1 neurons showed abnormal calcium uptake only after 48 hours. At the
2-LNNS/DIR
initial postischemic periods the abnormal calcium uptake was associated with neuronal
changes assessed with cresyl violet stain. After one week the abnormal calcium deposits
persisted in the CA1 sector of hippocampus and in the dorsolateral portions of the
caudate, which later revealed severe neuronal damage. In the other areas, animals
examined after 2 weeks and later showed fading or absent abnormal 45Ca radioactivity.
Otherwise those regions revealed an improved neuronal appearance, the nerve cells
being either well-preserved or showing a minimal injury. These observations provide
another indication that following ischemic injury although neurons, may show marked
histological changes, such as hyperchromasia, loss of Nissl bodies and cytoplasmic
vacuolization, nonetheless they preserve their capacity for recovery, this constitutes the
most important research challenge to find the means to facilitate such neuronal
recovery.
Concerning the abnormal deposition of 45Ca, it clearly requires participation of
living cells, since it does not occur in regions where neurons suffer irreversible injury,
with exception of CA1 sector of the hippocampus which may show persistently dense
45Ca radioactivity, even a few months after ischemic injury. In such cases, the increased
4$Ca content may be related to ultrastructurally observed dense calcium precipitates in
the surving pyramidal cells and interneurons as seen by EM with the oxalate-
pyroantimonate method.
Simultaneous demonstration of 4$Ca uptake and protein synthesis in the same
sections brought some insight into the problems of ability and extent of neuronal
recovery from ischemic insults. The separate demonstration of 4$Ca ar|d 3H-leucine
radioactivities was accomplished by exposure of the same sections to emulsions
selectively sensitive to these two markers. Observations with this procedure revealed
that the abnormal 45Ca uptake was frequently preceded by reduction of protein
synthesis in the ischemic regions, followed later by an inversely proportional increase in
calcium uptake and reduction in protein synthesis. With regard to the CA1 sector,
changes in protein synthesis were varied. An initial supression of synthesis during the
first recirculation days was superceded by an improvement evident at 7 days, which was
followed by a secondary reduction of protein synthesis several months after global
ischemia. Interestingly, in rats sacrificed after one year protein synthesis appeared to
show recovery; this was supported by histological sections showing a remarkable
number of well-preserved pyramidal neurons.
In summary, our studies on repetitive and global ischemia, as well as on cryogenic
cortical injury have further advanced the assumption that neuroexcitatory mechanisms
play a significant role in the pathophysiology of resulting injuries in these models. Our
current effort is directed primarily towards quantitative evaluation of changes in
excitatory transmitters during and after ischemic or traumatic insults. We intend to carry
out these evaluations as regionally as possible, in order to define the sequence of
excitatory changes with regard to neuronal structures, such as cortex, striatum,
hippocampus and thalamus. In addition to glutamate, we intend to evaluate, mostly by
immunohistochemical methods, changes involving other systems, such as the GABAergic
and dopaminergic ones. In view of the striking early changes involving the nuclei
reticularis thalami in global ischemia, we intend to elucidate more thoroughly the nature
of these changes and their possible effect on other interconnected structures. Finally,
with the information acquired we intend to test various compounds which, by
interferring with neuroexcitatory mechanisms, will be able to ameliorate the
development of secondary excitatory lesions. The studies of Dr. Nowak regarding
3 - LNNS/DIR
changes in gene expression after ischemia and other brain insults are increasingly
relevant to the framework outlined above, in that there is considerable evidence
suggesting that at least some aspects of the changes may result from activation of signal
transduction mechanisms coupled to neuronal activity. It is particularly striking that the
initial distribution of hsp 70 mRNA induction in gerbil brain after ischemia, identified by
in situ hybridization, is identical to that of the mRNA encoding the proto-oncogene c-fos.
It has been proposed that c-fos induction may provide an index for mapping functional
neuronal activity, and excitatory amino acids have been demonstrated to induce c-fos in
a number of experimental systems. While c-fos induction appears to occur in response to
relatively mild stimuli it would appearthat additional input is required to induce hsp 70,
making it a potential marker for pathologic activation. One goal of future studies is to
determine the precise signal transduction mechanisms responsible for hsp 70 induction.
The most striking feature of hsp70 mRNA induction after ischemia is its prolonged
expression in severely challenged neurons, such as those in the CA1 sector of the
hippocampus. While these cells fail to express immunoreactive hsp70 protein after a
5-min ischemic insult, they show a prolonged expression of hsp70 mRNA. Both of these
observations may be linked to the persistent reductions in protein synthesis in these cells,
with a superinduction of hsp70 mRNA in the absence of the functional protein. These
results suggest the using hsp70 mRNA expression as a marker for vulnerable neurons;
and this hypothesis is currently being tested in a variety of experimental injury models.
One interesting aspect of these studies is the entirely neuronal distribution of hsp70
induction after transient ishemia and its predominantly glial and vascular localization
after mild hyperthermic insults, with an increasing neuronal component under more
severe conditions. Focal injury models have been reported by others to induce
expression in diverse cell types. Future efforts will continue to expand the range of
probes used in these studies to include those expected to have complementary neuronal
and glial localizations, in order to evaluate the contributions of altered gene expression
in specific cell populations to the evolution of brain injury.
The continuous goals of the Section of Neurocytobiology have been to: (a) develop
and utilize model systems for the investigations of basic mechanisms operative on the
level of normal and pathologically altered blood-brain barrier (BBB), cerebral blood flow
(CBF) and hypertension; (b) to study metabolic processes occurring in ischemia and
ischemic edema, their prevention and therapy; and (c) to evaluate the influence of
genetic and immunologic factors on the generation of experimental autoimmune
diseases and ischemia involving the central nervous system.
During the last year, cultured endothelial cells (EC) derived from dissociated
microvesselsof human, mice and rat brains have been very useful.forthe continuous
studies of cerebromicrovascular function. Three different aspectsof cerebral EC
properties related to BBB function, regulation of CBF and hypertension have been
further investigated in the in vitro models using EC cultures: A) assess the susceptibility
of human cerebromicrovascular EC to oxygen-derived species (like H2O2) and compare it
with that of EC obtained from animal brains; B) characteriza-tion of dopaminergic
receptors and interaction with a-adrenergic and 5-HT receptors in human EC; and C)
interaction between cerebral EC and immune cells.
Formation of free radical species has been thought to affect the BBB permeability
and formation of brain edema. To determine whether exogenous H2P2 may alter may
alter permeability, we examined its effect on cultured endothelium derived from
cerebral microvessels from human and animals. Release of 5Kr from labeled
4-LNNS/DIR
endothelium exposed to these substances was used as a main marker for the assessing
of endothelial injury. In general, the 51 Cr-release from EC exposed to glucose/glucose
oxidase (representing a continous H2C>2-generated system was time- and dose-
dependent. Both human and murine EC exposed to 0.001 U/ml glucose oxidase in the
presence of glucose showed no loss of the isotopic marker, whereas the same
concentration of these substances caused 5iCr-releasefrom rat EC. atalase blocked the
release of 51 Cr-labeled EC exposed to all concentrations of glucose oxidase (0.1-0.01
U/ml) irrespective of EC origin. However, catalase inhibition was less effective in EC of
rats. SOD had no effect on the 51 Cr release from the EC. Addition of NaN3 or
aminotriazole (catalase blocker) increased (2-7 fold) the 51 Cr-release from all EC. A
higher dose of diethylmaleate (which depletes the reduced glutathione) was required to
affect the release of 51 Cr from human than from murine EC. The study represents the
first demonstration of human brain EC response to H2O2 insult. The results support the
existence of a balance between the endogenous anti-oxidant properties of cerebral EC
and exogenous oxidants. The disturbance of these parameters results in EC injury. Thus,
the altered EC function may contribute to the development and/or progression of brain
edema.
Dopamine (DA^ is a well known neurotransmitter and vasoactive substance. It may
affect the metabolism of the central nervous system (CNS) and reactivity of both central
and peripheral vessels. It has been suggested that DA may interact with specific
receptors in cerebrovascular smooth muscle to alter their diameter, thus modulating
blood flow and blood pressure. Since a marked DA induced enhancement of endothelial
AC activity was detected in cultured endothelium derived from human microvessels
(Spatz et al., 1989), we characterized the endothelial DA receptors and elucidated their
relationship to a-adrenergic-and 5-HT receptors. Cultured endothelium derived from
three fractions of human cerebral microvessels was used to characterize DA receptors
linked to adenylate cyclase activity. DA or Di-agonists [(SKF-38393, R( ± ) SKF-
82958(±)HBR)J stimulated endothelial cAMP formation in a dose-dependent manner.
The selective Di-antagonist [(SCH-23390, R( ± )] dose-dependently inhibited the DA or Di-
agonists stimulated production of cAMP. The selective 62-antagonist (sulpiride, S(-))
inconsistently augmented the DA stimulatory effect on cAMP formation in all three
endothelial fractions. The sensitivity of the endothelial adenylate cyclase was greater in
endothelium derived from large and small microvessels than from capillaries. Agonists of
ai-adrenergic receptors (phenylephrine, 6-fluoronorepinephrine) or 5-HT which
stimulated the production of cAMP by itself did not augment the DA-stimulated cAMP
formation. On the contrary, ai-adrenergic agonists or 5-HT incubated with DA reduced
the cAMP production, which could be partially blocked by the 5-HT2 or D2 antagonists,
respectively. These findings represent the first demonstration of Di (stimulatory) and D2
(inhibitory) receptors linked to the adenylate cyclase in microvascular endothelium
derived from human brain. The data also indicate that dopaminergic receptors can
interact with either ai-adrenergic or 5-HT receptors in endothelium on the AC level. The
observed presence of endothelial D1 and D2 receptors positively and negatively linked to
AC system strongly supports the contention of an existing central microvascular
dopaminergic mechanism which may be affected by dopaminergic agents and interact
with adrenergic and serotoninergic substances.
A continuous effort has been to examine: interactions between lymphocytes and EC
in order to establish possible mechanisms leading to alterations in BBB permeability and
the migration of peripheral blood immune cells across the BBB into the CNS; and the
role of class II MHC antigens in the pathogenesis of neuroimmunologic disorders.
The perivascular infiltration of peripheral blood lymphocytes and macrophages into
the CNS is a histopathologic feature of human demyelinating diseases such as
5-LNNS/DIR
multiple sclerosis and the animal model disease, experimental allergic encephalomyelitis
(EAE). To elucidate the mechanisms which could be involved in this event, we examined
the capacity of EC to function as targets for lysis by T cells capable of transferring EAE.
Lysis was proportional to E/T ratios, dependent upon the expression of la antigen and the
presence of specific antigen. Continuously cultured antigen-specific T-cell lines
ultimately lost the ability to lyse EC but continued to proliferate to MBP. These cell lines
also lost the ability to transfer EAE suggesting a relationship between lytic potential and
encephalitogenicity. Another aspect of these studies was concerned with the
observations that cerebral vascular EC inhibited antigen-induced proliferation by MBP-
specific lymphocytes. Attempts to characterize the inhibition indicated that IL-1 induced
synthesis of prostacyclin (PGI2) by EC. The amount of PGL2 synthesized in these
experiments was sufficient to account for the inhibitory response observed. These data
indicate that EC-lymphocyte interactions may result in effects on both EC (i.e., lysis) and
lymphocytes (i.e., anti-proliferative response). Such interactions may result in alterations
of the BBB permeability leading to vascular egression into the CNS, which are pathologic
hallmarks of neuroimmunologic disorders such as EAE and multiple sclerosis.
In EAE, la antigen expression by cerebral vascular EC (which comprise the BBB) is
believed to play an important role in migration of peripheral immune cells into the CNS.
In addition, la antigen expression by resident CNS cells (astrocytes, microglia, pericytes,
and smooth muscle cells) also plays important roles in the expression of pathogenesis.
The investigations focused on induction of la antigen on microvascular human
endothelium which has been successfully cultured in our laboratory. Cerebral cap;llary
EC isolated from human brain biopsy tissue demonstrated the absence of class II MHC
antigen expression on cultured EC. However, EC could be induced to express HLA-DR, -
DP and -DQ antigens by culture with the recombinant human IFN. The degree of HLA-DR
antigen expression, as determined by ELISA, immunofluorescence, FACS analysis and
Northern blot analysis, was proportional to both time in culture and concentration of
IFN-y (20-200 U/ml). This represents the first demonstration of class II MHC antigen
expression by cultured human cerebral vascular EC. Preliminary evidence indicated that
IL-1 treatment down-regulates IFN-induced expression of these antigens. These results
indicate that human cerebral vascular EC may participate in MHC class ll-restricted
interactions with peripheral immune cells. Such interactions may play a role in the
passage of immune cells across the BBB into the CNS which is a marker of certain CNS
autoimmune disorders.
The continuous studies of cerebral ischemia, its pathophysiology, prevention and
therapy have been focused on: (a) the relationship between ischemic changes in the
cerebral content of energy-related metabolites and biogenic amines in adult and young
gerbils; and (b) in vivo detection of regional ischemic release of monamines into
extracellularfluid. Changes in the brain content of biogenic amines are one of the main
factors implicated in the pathogenesis of ischemic CNS damage. We have recently
demonstrated a lack of correlation between the cortical ischemic changes of energy-
related metabolites and noradrenergic metabolites in adult orthe young gerbil brains.
This study was extended to the striatum (which is more vulnerable to ischemia than
cortex) to further elucidate the relationship between the ischemic cerebral monoamine
changes and the lesser susceptibility of younger than adult animals to ischemic insult.
Bilateral ischemia of 5 to 1 5 minutes duration with and without 1 hour reflow similarly
affected the glucose and energy-related metabolites in both groups of animals. A
reduction in striatal norepinephrine (NE) level was detected only in adults after each
period of ischemia. In adults, 1 5 minutes but not 5 minutes of ischemia reduced the
striated DA content which also decreased during recirculation. In young animals, the
same striatal DA changes were seen after 1 5 min ischemia with reflow. The delayed
alteration of monoamines in the striatum of the young animals observed in reflow but
6-LNNS/DIR
not during ischemia as seen in adults resembles the described "maturation"
phenomenon in the adult brain. The late manifestions of monoaminergic changes in
young as compared to adult brain structures most likely reflect a lesser extent of ischemic
injury in the young than adult animal. These observations suggest that the relative
resistance of young animals to ischemia may be related to the function of
neurotransmitters in the CNS.
To further elucidate monoamines participation in selective regional vulnerability to
ischemia, we determined and compared the cortical release into extracellular fluid (ECF)
of biogenic amines with those of striatum exposed to the same ischemic insult. Ischemia
induced a marked release of DA and 5-HT from the cortex and striatum. The levels of DA
released into ECF from the striatum was 4-2000 fold higher than in the cortex. Moreover,
the release of DA from both structures was 4,000-20,000-fold higher than that of 5-HT.
DA and 5-HT release from the cortex peaked at 15 minutes of ischemia. The levels of 5-
HT remained higher than that of DA during 1 5 minutes of reperf usion and normalized at
30 min of reflow. A similar pattern e* DA release was seen in thedialysate of the
striatum. However, the level of the released 5-HT during ischemia remained the same
after 1 5 minutes of reflow and returned to preischemic value at 30 minutes of
recirculation. DOPAC, HVA and 5-HIAA showed a similar pattern in the perfusate. These
metabolites decreased during ischemia and their accumulation above the preischemic
level was observed between 60-120 minutes after recirculation, 3-methyl tyrosine
accumulated in reflow. The findings support the idea that selective vulnerability to
ischemia may at least be in part related to ischemic disturbances of biogenic
neurotransmitters. The results of this investigation confirm data previously obtained by
indirect pharmacologic techniques concerning the behavior of monoamines in ischemia.
Thisdirect method allowing in vivo monitoring of the ischemic release of monoamines
will be very useful for elucidating the pathomechanisms and prevention of ischemic
sequelae.
Dr. McCarron continues to study the mechanism responsible for the production of
chronic relapsing EAE in F1 animalsand examines the role of parental immune response
antigens in the induction of relapses in animals with disease. A better understanding of
the regulation of immune response gene products expression in the F1 mice (i.e., .I-A,s, I-
Au and l-As/u hybrid molecules) is also a major objective of this project. MBP residues 90-
101 are encephalitogenic in SJL(H-2s), while residues 1-9 areencephalitogenic in PL mice
(H2u). Utilizing SJLxPL(F1) mice the relationship between encephalitogenicity and
genetic background were previously examined. It was found that the
encephalitogenicity of antigen was dependent upon l-A haplotype of the antigen
presenting cell. T-cellsfrom F1 mice immunized with MBP fragment 1-37 (containing 1-9
fragment) were positively selected on PL (l-Au) parental macrophages. These cells
transferred disease to 100% of the recipient naive F1 mice. Examination of spleen cell
populations during the course of disease in mice revealed the presence of T-cells
responsive to 1-37, as well as other MBPfractions (i.e. 89-169). In addition to l-A"
restricted responses, l-As restricted responses were also observed. These findings
demonstrated that T-cells with novel epitope specificities and class II MHC restriction
requirements were generated during the course of disease. Such cells may be involved in
the pathogenesis of chronic disease (i.e., initiation of relapses).
7-LNNS/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02324-1 3 LNNS
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJECT (SO characters or less. Trt/e must fit on one line between the borders.)
Blood-Brain Barrier: In Vitro Model for the Study of Cerebrovascular Endothelial Permeability
PRINCIPAL INVESTIGATOR (Ust other professional personnel below tne Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Pi: McCarron Ph.D., Senior Staff LNNS, NINDS
Other: M.Spatz M.D., Section Chief LNNS, NINDS
COOPERATING UNITS (if any)
Sumio Uematsu and M. Delong, John Hopkins Hospital, Baltimore, MD
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neurocytobioloqy
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: q n
PROFESSIONAL:
0.6
OTHER: Q 2
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues 0 (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Arachidonic acid release from tissue membranes and/or formation of free radical
species have been thought to affect the blood brain barrier permeability and formation
of brain edema. To determine whether exogenous arachidonic acid or H2O2 may alter
blood brain barrier permeability, we examined their effect on cultured endothelium
derived from cerebral microvessels of human and animals. Release of 5iCr from labeled
endothelium exposed to these substance was used as a main marker for the assessment
of endothelial injury. The results of these studies indicate that the endothelial cells are
susceptible to exogenous arachidonic acid or H2O2 insult irrespective of their origin.
However, human endothelial cells are less affected than animal endothelial cells by the
H202-generated system. The findings suggest that a disturbance of the existing
balance between the endogenous antioxidant properties of endothelial cells and
exogenous oxidant leads to EC injury.
10-LNNS/DIR
PHS 6040 (Rev. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBUC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
201 NS 02357-12 LNNS
PERIOD COVERED
i October 1, 1989 to September 30, 1990
TITLE OF PROJECT (tOdttntUn v Im. Tut* man M an m km Mmm «*• aoraMJ
Cerebral Ischemia and Monoamines
PRINOPAL INVESTIGATOR (an •ttarptffcuiaail jmommIMm u» ^vk*</ tmm§Hm ) (*•««. in* te*or«i<y>. •/>« mnnvM i»jiw)
PI: M.Spatz, M.D. Section Chief LNNS, NINDS
Others: C.J.Chang Visiting Fellow LNNS, NINDS
COOPERATING UNITS (Htn,)
Institute of Biochemistry, Faculty of Medicine, Belgrade, Yugoslavia, (Bogomir. Mrsulja)
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neurocytobioloqy
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (ajHuman subjects □ (b) Human tissues HH (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Uit flAncUrd unreduced type bo not txct*d the ipict proved .)
Changes in the brain content of biogenic amines constitute one of the main factors
implicated in the pathogenesis of ischemic central nervous system damage. We have
recently demonstrated a lack of correlation between the cortical ischemic changes of
energy related metabolites and noradrenergic metabolites in the adult or the young
gerbil brains. This study was extended to the striatum (which is more vulnerable to
ischemia than cortex) to further elucidate the relationship between the ischemic
cerebral monoamine changes and the lesser susceptibility of young than adult animals
to ischemic insult.
Bilateral ischemia of 5 to 15 minutes duration with and without 1 hour reflow similarly
affected the glucose and energy-related metabolites in both groups of animals. A
reduction in striatal norepinephrine (NE) level was only detected in adults after each
period of ischemia. In adults, 15 minutes but not 5 minutes of ischemia reduced the
striatal dopamine (DA) content which also decreased during recirculation. In young
animals, the same striatal DA changes were seen after 1 5 minutes ischemia with reflow.
The delayed alternation of monoamines in the striatum of the young animals observed
in reflow but not during ischemia as seen in adults resembles the described
'maturation" phenomenon in the adult brain. The late manifestations of
monoaminergic changes in young as compared to adult brain structures most likely
reflect a lesser intensity of ischemic injury in the young than adult animal. These
observations suggest that the relative resistance of young animals to ischemia may be
related to the function of neurotransmitters in the central nervous system.
ll-LNNS/DIR
•MSiMtflU*. I«4|
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02548-09 LNNS
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJ ECT (so dimeters or less. Title must fir on one line between the borders.)
Evaluation of Electrical Impedance in Cerebral Ischemia
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: H.G.Wagner, M.D. Scientist Emeritus LNNS, NINDS
Others:
S.Xu, M.D.
I. Klatzo, M.D.
Visiting Fellow
Chief, LNNS
LNNS, NINDS
LNNS, NINDS
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Cerebrovascular Pathology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.3
PROFESSIONAL:
0.3
OTHER:
CHECK APPROPRIATE BOX(ES)
I [ (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues QT] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Further study was made of cerebral electrical impedance (CEI) during the phase of
hypoperfusion. In the gerbil no substantial decrease in the CEI was found during this
phase after a single 5 minute bilateral carotid occlusion. This was in spite of noticeable
swelling of the brain and probable compression of the capillaries.
This project was terminated because of the retirement of the prinicipal investigator.
12-LNNS/DIR
WIS 6M0 (Dm. 1/M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02623-07
LNNS
PERIOD COVERED
i October 1 , 1 989 to September 30, 1 990
TITLE OF PROJ ECT (SO characters or less. Trite must fit on one lint bthntr. fh« borders.}
Cerebral Ischemia and Edema: Extracellular Biogenic Amines
PRINCIPAL IN VE STIGATOR (Uu other professional personnel below the frinapal Investigator.) (Name, title. laboratory, end institute affiliation)
Pi: C.J. Chang. M.D. Visiting Fellow LNNS, NINDS
Others: M. Spatz, M.D. Section Chief LNNS, NINDS
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neurocytobioloqy
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues l"x~l (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The cortical and '". iaiai .lease of biogenic amines induced by ischemia has been
evaluated in dialysates of extracellular fluid. Mongolian gerbils subjected to bilateral
common carotid artery occlusion for 1 5 minutes with a reflow up to 2 hours served as a
model for transient ischemia of brain. These studies are still incomplete, but the results
indicate that ischemia of short duration causes a marked release of dopamine and
serotnin (5-HT) from both structures. The level of the released amines was higher in
the striatum than in the cortex. Both metabolites of dopamine and 5-HT decreased
during ischemia and increased in reflow. The findings support the idea that the
selective vulnerability to ischemia may at least be in part related to ischemic
disturbances of biogenic neurotransmitters.
*Formerly Cerebral Ischemia and Edema: Tryptophan uptake and metabolism.
13-LNNS/DIR
PHS&IMOiRev. 1/M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02689-06
LNNS
PERIOD COVERED
Octoberl, 1989 to September 30, 1990
TITLE OF PROJ ECT (SO characters or less. Title must fit on one line between the borders.)
Reg u I ation of Carbohydrate Metabolism in Cerebromicrovascular Cultures
PRINCIPAL INVESTIGATOR (Ujt other professional personnel below the frindpal Investigator.) (Name, title, laboratory, and instrtute affiliation)
Pi: M.Spatz, M.D. Section Chief LNNS, NINDS
COOPERATING UNITS (if any)
David Lust, Case Western Reserve University, Cleveland, Ohio
B.B. Mrsulja, CVD Research Group, Institute of Biochemistry, Belgrade, Yugoslavia
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neurocytobioloqy
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues fxl (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Studies in vitro related to neurogenic regulation of cerebromicrovascular function
showed an involvement of B2-adrenergic system in controlling norepinephrine-
inducible glycogenosis in separately cultured cerebromicrovascular cellular elements.
The present investigation focused on the responsiveness of phosphorylase A and B to
adrenergic agonists and antagonists in order to elucidate the possible mechanisms
responsible for norepinephrine-inducible glycogenosis.
The study is still in progress and no sufficient data were generated for proper
evaluation.
14a - I.NNS/DIR
FHS6M0(Rev. 1/M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02718-05 LNNS
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJ ECT (so dtmmcttrs or hss. Tit/* must fit on on* Ina Worn tn* bordvi.)
Study of Cerebral Electrical Activity Associated with Ischemia and Brain Injury
PRINCIPAL INVESTIGATOR (Ust otr»r pmtosionil DWTOnntt b»low th* trtnaotl InnstiomtorJ (Htm*, trtl*. Itborttory, mnd mstrt </»• IffilHtion)
PI: H.G.Wagner, M.D. Scientist Emeritus LNNS, NINDS
Other: S.Xu.M.D. Visiting Fellow LNNS, NINDS
COOPERATING UNITS (rttny)
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neuropathology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: ft „
PROFESSIONAL: q q
OTHER: 0_0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [x~] (c) Neither
] (al) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Our initial efforts to detect and analyze single neurons in the cortex were
unsatisfactory for the purposes of analyzing the effects of ischemia and cold lesions to
the cortex. Improved success followed comparison of power spectra of occipital and
frontal EEG in the rat subjected to a parietal cold lesion. Normally, the dominant
frequency was about 5 Hz with higher frequency components. Rats subjected to a
parietal cold lesion showed immediate reduction in EEG amplitude and loss of high
frequency components. The dominant frequency in power spectrum analysis of the
EEG showed a shift to 1-3Hz and remained there for more than 1 week of animal
survival. The amplitude, however, recovered rapidly within about 20 minutes. Visually
evoked potentials were also examined using flash stimulation and extradurally placed
electrodes over visual area I. The cold lesions were applied as in the aforementioned
lesions.
A most striking result was a reduction of latency, starting at about 4 hours and
reaching a maximum at 24 hours to 3 days. On the assumption that this reflected
heightened excitability from excessive presence of glutamate, a non-competitive
antagonist of glutamate, MK-801 was given intraperitoneal^ (2 mg/kg) at 24 hours
after the cold lesion. The effect of this treatment was a prolongation of the latency
beginning within about 5 minutes of injection and lasted about 4 hours. This was
associated with a marked reduction of amplitude of the evoked response. This effect
of a direct application of glutamate (IM) resulted in reduction of latency, similar to the
cold lesion.. This provides further support to the hypothesis that glutamatergic
mechanisms were involved in the generation of the evoked response.
15-LNNS/DIR
mi M40 <K»v. 1/M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02720-04 LNNS
PERIOD COVERED
October 1 , 1 989 to September 30, 1 990
TITLE OF PROJ ECT (80 characters or less. T7f/e must fit on one tine between the borders.)
Stress Protein Induction in Gerbil Brain After Ischemia
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: T.S. Nowakjr. M.D. Special Expert LNNS, NINDS
Others: K. Kawai, M.D. Visiting Fellow LNNS.NINDS
N.Saito, M. D. Visiting Fellow LNNS.NINDS
G.Mies Visiting Associate LNNS.NINDS
COOPERATING UNITS (if any)
(j. F. Mill, LMB, NINDS. A.M. Marini, CNB. NINDS; S. Nadi, LNP, NINDS; P. Lindsberg. Dept. Neurol., USUHS; M. Blake and N.
Holbrook, LMG, NIA; M. Heyes, LCS, NIMH; M. Jacewia and W. Pulsinelli, Dept. Neurol, Cornell University School of Medicine.
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Cerebrovascular Pathology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: n?
PROFESSIONAL: q y
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues {x~\ (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
In situ hybridization studies of mRNA encoding the major 70 kDa heat shock/stress
protein, hsp70, have demonstrated its prolonged expression in vulnerable
lippocampal CA1 neurons after transient global ischemia. These cells also experience a
asting impairment of protein synthesis that may contribute to such a superinduction
phenomenon. The proto-oncoqene, c-fos shows only transiently increased expression
but with a distribution identical to that of hsp70 mRNA at early recirculation intervals.
These results suggest that related signal transduction mechanisms may be responsible
forthe induction of these RNAs, and support the utility of hsp 70 hybridization asa
possible indicator of some aspect of persistent neuronal activation. A threshold of 2
minutes ischemia has been established for induction of hsp70 in the gerbil.
A more complex pattern of induction is observed after hyperthermia, with a
predominantly glial and vascular distribution of immunoreactive protein but a
superimposed neuronal localization of hsp70 mRNA during increasingly severe
hyperthermia.
The major direction of future studies is to utilize changes in hsp70, c-fos and other
mRNAs and proteins as tools for identifying the precise contributions of individual cell
types to the evolution of pathophysiology following diverse brain insults, and to
elucidate the specific signal transduction mechanisms involved.
16-LNNS/DIR
PHS 6040 (Rev. 1(84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02749-04 LNNS
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJECT (80 chirectert or less, title must fit on one Itne between the borders.)
The Measurement of Cerebral Blood Flow by Laser Doppler Velocimetry
PRINCIPAL IN ViS7\GA70R (Ust other professiontl personnel below the rrinapet Investigator.) (Njrne. title, tiborttory, end institute tffilietion)
PI: H.G.Wagner, M.D. Scientist Emeritus LNNS, NINDS
Other: S. Xu, M.D. Visiting Fellow LNNS, NINDS
COOPERATING UNITS (rfeny)
Biomedical Engineering, Research Services, NIH, R.F. Bonner
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neurocytobioloqy
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: - q
PROFESSIONAL: q q
OTHER:
0,0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [x~l (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
A study has been made of cerebral blood flow (CBF) in gerbils by laser Doppler
velocimetry (LDV). The gerbils were subjected to repeated bilateral carotid occlusions
and studied for up to 3 hours. Continuous measurement of the CBF was made of both
parietal cortices simultaneously using 2 probes separately located. The purpose of
these experiments was to examine the agreement between the two sides. The essential
finding was that while the CBF dynamics were usually similar, they were not always the
same and the differences coula be substantial in time of recovery, magnitude of
recovery and in pattern of recovery.
This project is terminated because prinicipal investigator retired.
17-LNNS/DIR
P«i6O40(»e. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02751-04
LNNS
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJECT (60 characters or less. Title must fit on one line between the borders.}
Cultures of Mouse Capillary Endothelium: Establishment, Growth and
PRINCIPAL INVESTIGATOR (Ust other professional personnel below trie Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Pi: M.Spatz, M.D. Section Chief LNNS, NINDS
Others: R. M. McCarron, M.D. Senior Staff Fellow LNNS, NINDS
L.Wang, M.D. Guest Researcher LNNS, NINDS
COOPERATING UNITS (if any)
Dale E. McFarlin,CNP, NIB, NINDS
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neurocytobiology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: « -,
PROFESSIONAL:
0.2
OTHER:
0.5
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (fc>) Human tissues [T] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This study represents a continuous effort to develop and establish the most reliable and
reproducible method for culturing cerebromicrovascular endothelium derived from SJL
mice susceptible to experimental allergic encephalomyelitis.
The focus of the present investigation has been to analyze the nutritional requirements
needed forsustained long-term growth and propagation of the endothelium. High
concentration of endothelial cells grow equally well in medium containing 10-20%
fetal calf serum (FCS). They required endothelial cell growth factor (ECGF) for maximal
proliferation. Inclusion of heparin synergistically increased the proliferative response
of the cells. In the presence of high concentration of FCS (20%), heparin surpassed
ECGF in the ability to support increased proliferative response of the endothelium. A
more detailed analysis of various nutrients is still required for establishment and
characterization of long-term endothelial culture for expediting the investigation
concerned with interaction between capillary endothelium and immune cells.
Formerly: "Interaction Between Cerebral Capillary Endothelium and Immune Cells"
This investigation could not be continued in this fiscal year (1989-1990) due to fire
destruction of the 57.6 mouse breeding facilities.
18aLNNS/DIR
f>HS 6M0 (Rev. 1*4)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02768-03 LNNS
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROiiCT (»0 chtrtcttrs or less. Title musl tn on ont lint bttwvtn tht bordtn.)
Postischemic Accumulation of Calcium in Brain Tissue*
PRINCIPAL INVESTIGATOR (Usl othtt proftsuontl penonntl btlow tht Frinopal Innitigttor.) (Manx. Irt It. laboratory, tnd inttitutt ttfilittion)
PI:
T.S. Nowak, Jr. M.D.
Expert
LNNS.NINDS
Others:
K. Kawai.M.D.
Visiting Fellow
LNNS, NINDS
N.Saito, M.D.
Visiting Fellow
LNNS.NINDS
J. Lohr, M.D.
Lab. Technician
LNNS.NINDS
C. Ruetzler, M.D.
Biologist
LNNS.NINDS
1. Klatzo, M.D.
Chief
LNNS.NINDS
COOPERATING UNITS (rftny)
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section on Cerebrovascular Pathology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.0
PROFESSIONAL:
0.7
OTHER:
0.3
CHECK APPROPRIATE BOX(ES)
I 1 (a) Human subjects
] (a1) Minors
] (a2) Interviews
J (b) Human tissues [xT| (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Observations on the distribution and the time sequences of calcium accumulation
following single and repetitive ischemic insults using 45calcium autoradiography
revealed significant differences in the pattern of calcium uptake between single and
repeated occlusions. A striking feature of calcium accumulation in repeated ischemia
was intense uptake of Ca + + in thalamus, striatum and, later, in the medial geniculate
and substantia nigra. Also, a striking finding was an intense Ca + + uptake in CA1
when the pyramidal neurons were virtually destroyed. Our observations suggest that
abnormal accumulation of calcium may be due to other causes than a direct ischemic
injury and may not necessarily indicate irreversible neuronal damage.
*Formerly: "Post-lschemic Accumulation of Calcium in Brain Tissue and Histological
Ischemic Injury."
19- LNNS/DIR
PHS6O40(Rev. 1*a|
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02773-02 LNNS
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJECT (SOchjracferjortesi Title must fit on one line betvireen the borders.)
Observations on Global Cerebral Ischemia in Rats
PRINCIPAL INVESTIGATOR (List other professional personnel below the Frindpal Investigator.) (Name, title, laboratory, and institute affiliation)
PI:
Others:
K. Kawai, M.D.
N.Saito, M.D.
T.S. Nowak,Jr.,M.D.
G.Mies, M.D.
J. Lohr, M.D.
C. Ruetzler, M.D.
I. Klatzo, M.D.
Visiting Fellow
Visiting Fellow
Expert
Visiting Associate
Lab. Technician
Biologist
Chief
LNNS, NINDS
LNNS, NINDS
LNNS, NINDS
LNNS, NINDS
LNNS, NINDS
LNNS, NINDS
LNNS, NINDS
COOPERATING UNITS (rf any)
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section on Cerebrovascular Pathology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.3
PROFESSIONAL:
1.0
OTHER:
0.3
CHECK APPROPRIATE BOX(ES)
] (a) Human subjects
] (a1) Minors
] (a2) Interviews
] (b) Human tissues 0 (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Our studies on global cerebral ischemia in rats by compression of major cardiac vessels
revealed striking early changes in the nucleus reticularis thalami, which suggests a
possibility that loss of inhibitory function of this GABAergic nucleus may contribute to
aggravation of ischemic injury in the thalamus and in other structures. Morphological
observations in this study indicate that neurons in CA1 sector, cerebral cortex and
thalamus can survive very long and then recover.
20 - LNNS/DIR
PHS 60*0 (R*v. 1/M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02775-02 LNNS
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJECT (SO charecters or less. Trtte must fit on on* lint between the borders.)
Blood -Brain Barrier Changes Following Repeated Ischemic Insults
PRINCIPAL INVESTIGATOR (List other professional personnel Mow the frinapal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: T.S. Nowak, Jr. Expert LNNS, NINDS
Others: N. Saito Visiting Fellow LNNS, NINDS
C. Ruetzler Biologist LNNS, NINDS
I. Klatzo Chief LNNS, NINDS
COOPERATING UNITS (if an,)
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Cerebrovascular Pathology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: - ,
U.b
PROFESSIONAL: n g
OTHER:
0.3
CHECK APPROPRIATE BOX(ES)
LJ (a) Human subjects ] (b) Human tissues PH (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Rabbit antisera have been raised against purified gerbil albumin and used for the
immunocytochemical localization of the extravasated protein in brain following
repeated ischemic results. A striking accumulation of albumin immunoreactivity was
evident in thalamus as early as 6 hours after a series of repeated 5 minute carotid artery
occlusions while positive staining was first evident in this region at 24 hours following
repeated 2 minute occlusions. At longer recirculation intervals, thalamic staining was
intensified and theCAl region of hippocampus showed strong immunoreactivity which
accompanied the degeneration of neurons in this vulnerable area. In general, albumin
immunoreactivity was closely correlated with the distribution of enhanced 45calcium
45uptake as well as with the appearance of reactive glia, as evidenced by
immunocytochemical visualization of glial fibrillary acidic protein. The similarity of
intraneuronal uptake of albumin to the endocytotic uptake of a protein tracer,
observed in epileptic convulsions, suggests that neuroexcitatory mechanisms may be
involved in uptake of proteins from the extracellular space in both conditions.
21-LNNS/DIR
l>HS t(M0 (Rev 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02776-02
LNNS
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (SO characters or less. Title must fit on one line between the borders.)
Mechanism of Production of Experimental Allergic Encephalomyelitis
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: R.M. McCarron, M.D. Senior Staff Fellow LNNS, NINDS
Others: L.Wang, M.D. Guest Researcher LNNS, NINDS
COOPERATING UNITS (rtan,)
Dale McFarlin, Chief, NIB, NINDS
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neurocytobioloqy
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: n .
PROFESSIONAL:
0.4
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects | | (b) Human tissues [7] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
MBP residues 90-101 are encephalitogenic in SJL mice (H-2*), while residues 1-9 are
encephalitogenic in PL mice (H 2u). Utilizing SJLx PL(F1) mice the relationship between
encephalitogenicity and gentic background were previously examined. It was found
that the encephalitogenicity of antigen was dependent upon l-A haplotype of antigen
presenting cell. T-cells from F1 mice immunized with MBPfragment 1-37 (containing 1-
9 fragment) were positively selected on PL (l-Au) parental macrophages. These cells
transferred disease to 100% recepient naive F1 mice. Examination of spleen cell
populations during the course of disease in the mice revealed the presence of T-cells
responsive to 1-37, as well as other MBP factions (i.e. 89-169). In addition to l-Au-
restricted responses, l-As-restricted responses were also observed. These findings
demonstrated that T-cells with novel epitope specificities and class II MHC restriction
requirements were generated during the course of disease. Such cells may be involved
in the pathogenesis of chronic disease (i.e., initiation of relapses).
Significant progress on this project was not possible due to the inability to obtain
sufficient Fl mice. These mice are solely distubuted by Jackson Labs which suffered a
debilitating fire resulting in loss of many breeder pairs including the F1 mouse stock.
Attempts to breed cur own were successful, but resulted in only a limited number of
mice available for research. The supply at Jackson has been reestablished and we are
currently performing experiments with these mice.
22 - LNNS/DIR
PHS 6040 (Re». 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02777-02
LNNS
PERIOD COVERED
October!, 1989 to September 30, 1990
TITLE OF PROJECT (SO character* or less. Title must fit on on* line between «/>• border*.)
Human Cerebromicrovascular Endothelium: Studies in Vitro
PRINCIPAL INVESTIGATOR (Ijji other profession*! personnel below the rnnapal Investigetor.) (Heme, title, laboratory, end institute effilutron)
PI: F. Bacic, M.D. Visiting Fellow LNNS, NINDS
Others:
R.M. McCarron, M.D.
M.Spatz, M.D.
Senior Staff Fellow
Section Chief
LNNS, NINDS
LNNS, NINDS
COOPERATING UNITS (rf,n,)
The Johns Hopkins Hospital, Baltimore, MD (Donlin Long and Sumio Uematsu)
LAB7B RANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neurocytobioloqy
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.8
PROFESSIONAL:
0.5
OTHER:
0.3
CHECK APPROPRIATE BOX(ES)
I 1 (a) Human subjects
] (a1) Minors
J (a2) Interviews
LD
] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Cultured endothelium derived from three fractions of human cerebral microvessels
was used to characterize dopamine (DA) receptors linked to adenylate cyclase activity.
Dopamine or Di -agonists [SKF -38393, R( ± ), SKF -82958( ± ) HBR] stimulated
endothelial cAMP formation in dose - dependent manner. The selective Di -antagonist
[SCH -23390, R( ± )] dose -dependency inhibited the DA or Di -agonists stimulated
production of cAMP. The selective D2 -antagonist [sulpiride, S (-)] inconsistently
augmented the DA stimulatory effect on cAMP formation in all three endothelial
fractions. The sensitivity of endothelial adenylate cyclase was greater in endothelium
derived from large and small microvessels than from capillaries. Agonists of ai -
adrenergic receptors (phenylephrine, 6 -fluoronorepinephrine) or serotonin (5-HT)
which stimulated the production of cAMPby itself did not augment the DA-stimulated
cAMP formation. On the contrary, ai-adrenergic agonists or 5-HT incubated with DA
reduced the cAMP production which could be partially blocked by 5HT2- or D2-
antagonists, respectively.
These findings represent the first demonstration of Di -(stimulatory) and D2 -
(inhibitory) receptors linked to adenylate cyclase in microvascular endothelium derived
from human brain. The data also indicate that dopaminergic receptors can interact
with either ai-adrenergic or 5-HT receptors in endothelium on the adenylate cyclase
level.
23-LNNS/DIR
•HSMXXHRe.. I'M!
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02780-03
LNNS
PERIOD COVERED
October 1# 1989 through September 30, 1990
TITLE OF PROJECT (80 character* or less, mie must fit on one line between the borders.)
Cerebral Vascular Endothelial Cell-Specific Monoclonal Antibodies
PRINCIPAL IN VESTIGATOR(UitotrierproreM«>n*/ personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI:
Others:
R.M. McCarron, M.D.
M.Spatz, M.D.
J. Bembry, M.D.
N. Merkel, M.D.
Senior Staff Fellow
Section Chief
Biologist
Chemist
LNNS, NINDS
LNNS, NINDS
LNNS, NINDS
LNNS, NINDS
COOPERATING UNITS (rf any)
Dale McFarlin, Chief, NIB, NINDS
Laura Quigley, Lab Technician, NIB, NINDS
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neurocytobiology
INSTITUTE AND LOCATION
NINDS, N1H, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.4
PROFESSIONAL:
0.4
OTHER:
CHECK APPROPRIATE BOX(ES)
1 | (a) Human subjects
] (a1) Minors
J (a2) Interviews
□ (b)H
uman tissues
["*~l (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
In vitro studies on the role of the blood-brain barrier (BBB) in neuroimmunological
disorders involving migration of cells across the BBB have been hampered by the
inability to establish long-term cultures of cerebral vascular endothelial cells (EC), the
main component of the BBB. Using hybridoma technology, we have established a
panel of hybridomas (fusion of Lewis rat and mouse myeloma NS-1 cells) which secrete
antibodies which recognize EC. Characterization of these antibodies by
immunofluorescence microscopy, FACS analysis and ELISA techniques demonstrate
antibodies which react with only murine cerebral vascular EC, with EC isolated from
other than of the mice, or react with EC from other species. These antibodies were also
characterized according to their type (IgG vsIgM).
The results demonstrate the isolation of pure monoclonal antibodies to CNS-EC
which will be useful to further progress on establishment of long-term EC cultures and
other studies utilizing cerebral vascular EC.
24-LNNS/DIR
»HSM«0(R*v. 1/M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02795-02LNNS
PERIOD COVERED
October 1 , 1 989 to September 30, 1 990
TITLE OF PROJECT (BOcharaaers or less. Title must fit on one Sim bettveen the borders.;
Human Cerebromicrovascular Endothelial Culture: Cholinergic and Histaminergic Receptors
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal investigator.) (Name, title, laboratory, and institute affiliation)
PI: M.Spatz, M.D. Section Chief LNNS, NINDS
Others: F. Bacic, M.D. Visiting Fellow LNNS, NINDS
R.M. McCarron, M.D. Senior Staff Fellow LNNS, NINDS
COOPERATING UNITS (if an,)
The Johns Hopkins Hospital, Baltimore, MD, (Donlin Long and Sumio Uematsu)
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neurocytobioloqy
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: Ac
0.0
PROFESSIONAL: n j
OTHER: q 3
CHECK APPROPRIATE BOX(ES)
[7] (a)Human subjects [7] (b) Human tissues ] (c) Neither
J (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Acetylcholine and histamine are known as vasoactive substances which may be involved
in regulatinq cerebral blood flow (CBF) and/or blood-brain barrier (BBB) functions This
supposition has been supported by detection of cholinergic and histaminergic fibers in
the close vicinity of cerebral vessels. Moreover, receptors for both substances were
described in microvessels isolated from brain of animals but not from man. The aim of
this investigation was to examine the effect of acetylcholine, carbachol and histamine
on adenylate cyclase (AC) activity in the endothelium derived from three fractions of
human microvessels.
The response of adenylate cyclase to acetylcholine, carbachol and histamine was seen in
all examined homogenates of endothelium (4th-i 1th passage) derived from large
microvessels. Pirenzepine (Mi-muscarinic inhibitor) blocked the acetylcholine and
carbachol stimulated formation of cAMP while methoctramine (M2 muscarinic
inhibitor) had no effect on AC of endothelium derived from large microvessels.
Preliminary investigations demonstrated a dose-dependent cholinergic stimulation of
cAMP production in endothelium derived from capillaries and small microvessels. The
response of endothelial AC activity to histamine was inhibited (30-100%) by cimetidine
indicating the AC linkage to ^-receptors. These findings represent the first
demonstration of cholinergic and histaminergic receptors linked to the AC system in
cerebromicrovascular endothelium of human brain.
The loss of endothelial cultures due to toxic human serum prevented the completion of
this project.
25-LNNS/DIR
**ti WU (««v- 1/M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02796-02 LNNS
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJ ECT (80 characters ci lass. Title must fit an one line between the borders.)
Transneuronal Effects of Cryogenic Brain Injury on Calcium Uptake and Blood-Brain Barrier Changes
PRINCIPAL IN VE STIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
LNNS, NINDS
LNNS, NINDS
LNNS, NINDS
LNNS, NINDS
LNNS, NINDS
LNNS, NINDS
PI:
JN. Saito
Visiting Fellow
Others:
T.S. Nowak, Jr.
Expert
G. Mies
Visiting Associate
1, Lohr
Lab. Technician
C. Ruetzler
Biologist
I. Klatzo
Chief
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section on Cerebrovascular Pathology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.9
PROFESSIONAL:
0.6
OTHER:
0.3
CHECK APPROPRIATE BOX(ES)
] (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues 0 (0 Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Development of transneuronal, secondary, injuries developing in distant but
anatomically connected regions following primary, direct injury to the cerebral cortex
was shown to be associated with an abnormal uptake of calcium, and blood-brain
barrier (BBB) permeability changes in those areas. The delayed onset of these changes,
pattern of calcium uptake into dendritic structures, and intracytoplasmic uptake of
extravasated albumin suggest a neuroexcitatory nature of mechanisms involved. If
correct, studies will be developed to ascertain whether suppression or reduction of
neuroexcitation by some agents could substantially ameliorate or prevent development
of secondary transneuronal injuries. This could be of relevance to the treatment of
brain trauma patients.
26-LNNS/DIR
•-HS6M0 (Rev. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02797-02
LNNS
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJ ECT (80 dttntatrs or hss. rah must fit on on* lint t»tn>Mn th» borders.;
Cultures of Human Cerebromicrovascular Endothelium: Establishment, Growth and Characterization
PRINCIPAL IN VIST\GA70R (LHt other proftssnnil p*?onr*l b*k>w th* rnnaiHl lnv*itio*tor.) (Hrm. trtJe. laboratory, •nd irutrtufe fffitalion)
PI:
Others:
M.Spatz, M.D.
R.M. McCarron, M.D.
L Wang, M.D.
Section Chief
Senior Staff Fellow
Guest Researcher
LNNS, NINDS
LNNS, NINDS
LNNS, NINDS
COOPERATING UNITS (if my)
Drs. Doniin Long and Sumio Uematsu, The John Hopkins Hospital, Baltimore, MD
Dr. Ronald F. Dodson, University of Texas, Health Center, Tyler, TX
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neurocytobioloqy
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.9
PROFESSIONAL:
0.5
OTHER:
0.4
CHECK APPROPRIATE BOX(ES)
I | (a) Human subjects
] (a1) Minors
J (a2) Interviews
I x [ (b) Human tissues J (c) Neither
SUMMARY OF WORK ( Use standard unreduced type. Do not exceed the space provided.)
This study has been concerned with the development and establishment of endothelial
cell cultures derived from three microvascular fractions of human brain. The purity of
the endothelial cultures was found to be greater than 95% since most of the cells
expressed human Factor Vlll-related antigen and only an occasional cell stained for glial
fibrillary acidic protein. Both light and electronmicroscopy showed that the separately
cultured endothelium derived either from the capillaries (EC) or small [(<100ym) ES]
and large [ > 100ym) EL] arterioles and venules exhibited the same appearance and
?rowth pattern. The maximal proliferative response of endothelium was seen on the
irst day after exposure to fresh medium. A direct relationship was found between
various components of growth medium and individual proliferative response of
endothelium dependent on its origin indicating different growth requirements. The
characterization of the cultured endothelium is still incomplete and in progress.
Nevertheless the successfully developed technique for cultivating pure
cerebromicrovascular endothelium derived from human brain (dependent on the
availability of brain tissue) provides a model in vitro for investigation of endothelial
properties related to the normal or altered function of blood-brain barrier and cerebral
blood flow in man.
The loss of endothelial cultures due to toxic human serum prevented the completion of
this project.
27-LNNS/DIR
•MS tOa(MB.» V»J|
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02798-02 LNNS
PERIOD COVERED
October 1 , 1 989 to September 30, 1 990
TITLE OF PROJECT (SO character* or less. Title must fit on one line between the borders.)
Ultrastructural Observations on Distribution of Calcium in Cerebral Ischemia
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: I. Klatzo, M.D. Chief LNNS, NINDS
Others: J. Lohr, M. Lab. Technician LNNS, NINDS
C. Ruetzler, M.D. Biologist LNNS, NINDS
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Cerebrovascular Pathology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.0
PROFESSIONAL:
0.2
OTHER:
0.8
CHECK APPROPRIATE BOX(ES)
I | (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues [x~] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Using the pyroantimonate method for precipitating calcium to electron-dense calcium
pyroantimonate, the ultrastructural distribution of calcium was evaluated following
single and repeated ischemic insults, produced by bilateral occlusions of common
carotid artery in gerbils. Our observations revealed an excessive accumulation of
calcium deposits in the mitochondria of pyramidal cell dendrites preceding their
destruction. Following the disappearance of pyramidal cells, which becomes evident
approximately 3 days after ischemic insults, a striking accumulation of electron-dense
calcium deposits was observed in the dendrites of the interneurons, whereas
mitochondria of these dendrites were free of calcium. Occasionally the reverse was
true and calcium deposits were conspicuous in the center of mitochondria with well-
preserved cristae, whereas there was no calcium in the cytosol of the dendrites. The
pattern of calcium distribution in the dendrites appears similar to that observed
following convulsive seizures and indicates a possibility of neuroexcitatory mechanisms
playing an important role in accumulation of calcium in injured by ischemia neurons.
Otherwise, the intraneuronal presence of calcium itself does not seem to be invariably
associated with a neuronal death.
28-LNNS/DIR
PHS MW0 (Rev. 1/84]
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02801-02
LNNS
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 character! or less. Trtle must fit on one line tennn the borden.)
Interactions Between Cerebrovascular Endothelial Cells and Immune Lymphocytes
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator .) (Name, title, laboratory. and institute aft station)
P.I. R. M. McCarron, M.D. Senior Staff Fellow LNNS, NINDS
Other: M.Spatz, M.D. Section Chief LNNS, NINDS
COOPERATING UNITS (rfany)
Dale McFarlin, Chief, NIB, NINDS
Michael Racke, Senior Staff Fellow, NIB, NINDS
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neurocytobioloqy
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: q -,
PROFESSIONAL:
0.7
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [T] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The studies were designed to examine interactions between lymphocytes which
induce the model autoimmune disease experimental allergic encephalomyelitis (EAE)
and cerebral vascular endothelial (EC) which comprise the blood-brain barrier (BBB).
The first part of these experiments examined the capacity of EC to function as
targets for lysis by T-cells capable of transferring EAE. Lysis was proportional to E/T
ratios, dependent upon the expression of la antigen and the presence of specific
antigen. Continuously cultured antigen-specific T-cell lines ultimately lost the ability to
lyse EC but continued to proliferate to MBP. These cell lines also lost the ability to
transfer EAE suggesting a relationship between lytic potential and encephalitogenicity.
The second part of these experiments concerned observations that cerebral vascular
EC inhibited antigen-induced proliferation by MBP-specific lymphocytes. Attempts to
characterize the inhibition indicated that IL-I induced synthesis of prostacyclin (PGI2) by
EC. The amount of PGI2 synthesized in these experiments was sufficient to account for
the inhibitory response observed.
These data indicate that EC-lymphocyte interactions may result in effects on both EC
(i.e., lysis) and lymphocytes (i.e., antiproliferative response). Such interactions may
result in alterations of BBB permeability leading to vascular egression into the CNS,
which are pathologic hallmarks of neuroimmunological disorders such as EAE and
multiple sclerosis.
29-LNNS/DIR
»HS6O60(l>*» 1*4)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02821-01
LNNS
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Dynamics of Postischemic Calcium Accumulation and Protein Synthesis in Brain Tissue
PRINCIPAL INVESTIGATOR (list other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I. G. Mies, M.D. Visiting Associate LNNS, NINDS
Other: T.S. Nowak, Jr., M.D.
K. Kawai, M.D.
N.Saito, M.D.
I.KIatzo, M.D.
Expert
Visiting Fellow
Visiting Fellow
Chief
LNNS,NINDS
LNNS.NINDS
LNNS/NINDS
LNNS/NINDS
COOPERATING UNITS (if an,)
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Cerebrovascular Pathology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.8
PROFESSIONAL:
0.8
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
I | (a) Human subjects
] (a1) Minors
J (a2) Interviews
J (b) Human tissues I x | (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
After 10 minute global of ischemia in rat brain, a marked increase in abnormal
calcium uptake and suppression of protein synthesis in nuclei reticularis of thalamus
was observed as early as 6 hours postischemia, followed by a significant increase in
abnormal accumulation of calcium at moderately reduced rates of protein synthesis in
the ventral thalamic nucleus and inferior colliculus which lasted up to 7 days
postischemia. In hippocampal CA1 sector, protein synthesis of CA1 neurons was
severely suppressed until 4 days postischemia, but recovered significantly to subnormal
levels of protein synthesis after 7 days postischemia. Metabolic amelioration of
hippocampal CA1 neurons correlated with their improvement of morphological
appearance. Our findings suggest 1) that early postischemic calcium accumulation
occurring predominantly in GABAergic brain areas appears to reflect the consequences
of inhibition failure of GABAergic interneurons which may result in a subsequent
'disinhibition' of interconnected projection areas and the development of remote
neuron injury; and 2) that following a phase of chronic neuronal injury, hippocampal
CA1 neurons are able to recover from an ischemic insult.
30-LNNS/DIR
MfSWXO (Rev. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02822-01 LNNS
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJ ECT (BO ctortcfn or Itss. 7/tte must ftlOfiOMlint betwn (h« borders.)
Giutamate Microdialysis During Repeated Ischemia and Cold Lesions
PRINCIPAL INVESTIGATOR (Ust other professional ptrsonnel below the frinaptl Investioetor.) (Name, title, laboratory, end institute affiliation)
PI: I. Klatzo, M.D. Chief, LNNS, NINDS
N.Saito, M.D. Visiting Fellow LNNS.NINDS
K. Kawai.M.D. Visiting Fellow LNNS.NINDS
T.S. NowakJr., M.D. Specital Expert LNNS.NINDS
COOPERATING UNITS (if en,)
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Cerebrovascular Pathology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.6
PROFESSIONAL:
0.6
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
J (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues [T] (0 Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Previous studies have demonstrated a cumulative effect of repeated ischemic insults in
thegerbil. The present studies have been designed to address the potential role of the
amino acid, giutamate, in this phenomenon, since giutamate is known to be released
during ischemia and has been suggested to contribute to excitotoxic damage following
ischemia and other insults. Extracellular giutamate was monitored in several brain
regions by in vivo cerebral microdialysis. Current results demonstrate that a burst of
giutamate release occurs during each ischemic insult, but that this appears to be
cleared more rapidly than the larger increase seen after single longer occlusions.
Further studies will attempt to determine whether the timing of giutamate release
plays a critical role in the increased pathogy seen after repeated occlusions.
31 -LNNS/DIR
PHSWMDIBtv 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02802-03
LNNS
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (so characters or less. Title must fit on one line between the borders.)
Immune Mechanisms: Regulation of la Antigen Expression
PRINCIPAL INVESTIGATOR (LJst other profession*! personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: R. M. McCarron, M.D.
Others: M.Spatz, M.D.
L Wang, M.D.
Senior Staff Fellow
Section Chief
Guest Researcher
LNNS, NINDS
LNNS, NINDS
LNNS, NINDS
COOPERATING UNITS (if any)
Masami Tanaka, Brain Research Institute, Niagata University, Niagata City, Japan
Elliot Cowan, Special Expert, NIB, NINDS
LAB/BRANCH
Laboratory of Neuropathology and Neuroanatomical Sciences
SECTION
Section of Neurocytobioloqy
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.4
PROFESSIONAL:
1.4
OTHER:
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects
] (a1) Minors
J (a2) Interviews
|x| (b) Human tissues _J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The role of class II MHC antigen expression in neuroimmunological disorders is being
studied using the model autoimmune disease experimental allergic encephalomyelitis
(EAE) in SJL mice. Studies are focused on the expression of la antigen by SJL cerebral
vascular endothelial cells (EC) which comprise the blood-brain barrier (BBB). Previous
experiments characterized expression of la antigen by EC and functional significance
(i.e. , antigen-presentation). The potential role of la-positive EC in the extravasation of
peripheral blood lymphocytes (across the BBB into the CNS) is examined by analyzing
regulation of EC la antigen expression in vitro.
Observations include inhibition of IFN-induced la antigen-expression by cytokines IL-
1 and TNF. Similar experiments utilizing human cerebrovascular EC indicate that IFN
induced the expression of HLA-DR, -DP, and- DQ antigens. Preliminary experiments
indicate that cytokines may regulate expression of human class II MHC antigens as was
observed in mice.
The mechanism of IFN-induction of la antigen expression was studied by examining
effect of agents which alter secondary messangers (i.e., cAMP and protein kinases),
which are involved in IFN-induced up-regulation of la antigen. Results demonstrate
that agonists and antagonists of adrenergic receptors modulate induction of la
expression. The mechanism involves interaction between the two signal transduction
pathway leading to activation of protein kinase a (and cAMP formation) and protein
kinase C.
32 - LNNS/DIR
»HSSO40<R«v. 1*4)
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ANNUAL REPORT
October 1, 1989 through September 30, 1990
Laboratory of Neurophysiology
Basic Neurosciences Program, DIR
National Institute of Neurological
Disorders and Stroke
Research Summary II-rV
Project Reports
Physiological Properties Developing on CNS Cells 1
Z01NS 02019-18 LNP
Cell Biological Studies of Developing CNS Cells 2
Z01NS 02330-13 LNP
Synaptic Contacts of Retinal Neurons 3
Z01NS 01659-22 LNP
Structure and Function in Retinal Neurons 4
Z01NS 02631-07 LNP
Anatomical Studies of Neurons, Neurotransmitters and 5
Neurotransmitter Receptors
Z01NS 02705-05 LNP
Image Processing and Analysis of Cellular Structures 6
Z01 NS 02767-03 LNP
I-LNP, BNP, Dm
Annual Report
October 1, 1989 through September 30, 1990
Laboratory of Neurophysiology, BNP, DIR
National Institute of Neurological Disorders and Stroke
Jeffery L. Barker, M.D., Chief
The Laboratory of Neurophysiology's research program has been divided
primarily between application of well-established techniques in electrophysiology to
generate new insight into intercellular communication and signal transduction
mechanisms and the refinement of relatively novel strategies in physiological
recording techniques to expedite the rate at which such insights can be obtained.
Most of the research endeavors are related to the physiology of intercellular
communication and signal transduction mechanisms expressed by vertebrate,
Central Nervous System (CNS) cells or cells that are primary targets of CNS signals.
The broad and long-term goal of this program is to systematically elucidate
details regarding the development, differentiation and cellular distribution of
specific circuits in vertebrate nervous systems. All CNS cells and their peripheral
targets exhibit receptors, ion channels and signal transduction mechanisms that
mediate synaptic and extrasynaptic types of intercellular communication. How,
when and where specific excitable membrane properties develop in vertebrate
nervous systems is the focus of much of our research activities. Multidisciplinary
study of the differentiation of transmitters, receptors, ion channels and signal
transduction mechanisms during development should reveal fundamental insights
into the complex process of distributing such mechanisms among cells and their roles
both in differentiating and in differentiated circuits. In time, we aim to provide a
useful reference for studying specific pathophysiologies of the vertebrate CNS that
involve dysfunctions of specific circuits and signals.
All the projects are at cellular or molecular levels of study. Experimental
substrates and lines of investigation include: 2) monolayers of primary neuronal and
glial cells cultured from embryonic and early postnatal mammalian and avian CNS;
2) monolayers of cultured fibroblasts transfected with genes encoding specific
transmitter receptors; 3) acutely isolated central elements from specific regions of
the embryonic and postnatal CNS; 4) retinal eye cup and intact retina; 5)
quantitative electrophysiological analysis of cellular excitability expressed in short
(hours) or long-term (days- weeks) cultured embryonic CNS neurons, as well as
tumoral and primary pituitary cells; 6) quantitative electrophysiological analysis of
cellular excitability resident in retinal circuits; 7) flow cytometric analysis of
physiological properties exhibited in populations of acutely suspended central
elements; 8) light- and electron-microscopic resolution of cellular form and
subcellular structure in normally developed mammalian CNS tissues including
retina and spinal cord; 9) cytochemical and biochemical characterization of specific
transmitter phenotype expressions in vivo and in monolayer culture; and
10) quantitative optical recordings of physiological signals emitted by embryonic
neurons or associated with the morphology of differentiating glia and neurons.
Conceptually, these diverse strategies and lines harmonize to allow quantitative
resolution of receptor functions, ion channel expression and signal transduction
E-LNP, BNP, DIR
mechanisms. The strength of the Laboratory's research lies in the spectrum of
contemporary and innovative strategies at single-cell, circuit and population levels,
and the opportunity for multidisciplinary and collaborative study of basic problems
into the physiology of intercellular communication emerging in the embryonic CNS.
Several accomplishments this year have long-lasting implications with regard to
the role of y-aminobutyric acid (GABA) and its receptors in the differentiation of
embryonic neurons and circuits, which has become a major focus of LNP research
activity.
Dr. Marc Walton, in collaboration with Dr. Anne Schaffner, has applied voltage-
sensitive dye to cultured embryonic rat spinal cord cells, and probed the emergence of
functional Na + channels and amino acid receptors, which may be among the first to
develop in postmitotic elements. Their results demonstrate that depolarizing
GABAa receptors are widespread and co-expressed with functional Na+ channels
and kainic acid receptors during the early postmitotic period. This multicellular
recording strategy provides an especially insightful perspective for examining
excitable membrane properties of intact cells differentiating for hours to weeks in
monolayer culture.
The early embryonic appearance of functional GABAa receptors has prompted
parallel study of the synthesis and release of GABA during embryogenesis.
Dr. Suzan Nadi has used biochemical methods to study GABA synthesis and release
during rat and chick CNS development. She has found that polyamines precede
glutamate as a source of GABA during embryogenesis. Preliminary data also
indicate that GABA can be released from embryonic cortical neurons cultured by
Ms. Smallwood.
Ms. Toby Behar and Dr. Schaffner have used immunological techniques to
demonstrate that during the course of rat spinal cord development many neurons (up
to 40% ) contain GABA along with proteins related to the enzyme that decarboxylates
glutamate to GABA (GAD). Immunoblot analysis of spinal cord proteins probed with
anti-GAD antibodies reveals that low molecular weight GAD-related proteins
precede the developmental appearance both of high molecular weight forms and of
GABA. Ms. Behar and Dr. Walton have found that depolarizing GABAa receptors
precede the appearance of GABA immunoreactivity, and that virtually all GABA +
neurons express functional GABAa receptors. Together these results suggest that
GABAa receptors may play differentiating roles in the emergence of functional, high
molecular weight GAD proteins and the synthesis of GABA in spinal cord neurons.
Dr. Anita Prasad has cultured embryonic chick spinal cord neurons, which, like
embryonic rat spinal cord neurons also express depolarizing GABAa receptors, in the
presence of the GABAa antagonists bicuculline and picrotoxin, and discovered that
pharmacological block of GABAa receptor function prevents cell adherence and
neurite outgrowth. These toxic actions are neither shared by the Na+ channel
antagonist tetrodotoxin nor by the kainic acid receptor antagonist CNQX, and can be
reversed by co-application of muscimol, a GABAa agonist. Thus, GABAa receptors
may be critical to morphogenesis.
m-LNP, BNP, DIR
Postnatal GABAa receptors consist of various permutations and combinations of
subunits. What is the molecular composition of depolarizing GABAa receptors?
Dr. Michael Poulter, in collaboration with members of the Laboratory of Cell Biology,
NIMH, has used in situ hybridization techniques to begin to elucidate the
developmental appearance of GABAa receptor mRNA. His preliminary results
indicate that {5-subunit mRNA is more abundant in the embryonic rat CNS than a-
subunit mRNA.
The presence of embryonic GABAa receptors has led to an electrophysiological
analysis of GABAa receptor functions at membrane and cytoplasmic levels.
Drs. Alexander Valeyev and Ricardo Cruciani have used whole-cell recording
techniques to study GABAa receptor functions on cells cultured from embryonic
hippocampal tissue by Ms. Smallwood. Their preliminary results indicate that the
GABAa receptors couple to a CI- ion-selective conductance whose kinetics are
complex. Using cell-attached recording techniques, Dr. Jan Suszkiw has observed
that the response to GABA in otherwise intact cells is functionally excitatory,
triggering action potentials in early postnatal septal neurons cultured by
Dr. Schaffner. Suszkiw's results are consistent with the depolarizing effects of
GABAa receptor activation indirectly recorded by Dr. Walton in cultured spinal
neurons and by others in this laboratory (Drs. Nadi, Wu Ma, and Keiichi Torimitsu
all working with Ms. Smith) using flow physiological recording techniques applied to
embryonic and early postnatal neurons dissociated from spinal and supraspinal
regions of the rat and chick central nervous systems. Finally, Dr. Torimitsu has
recorded Ca2 + -indicator dye signals in response to GABAa receptor stimulation
using cells suspended from the embryonic chick telencephalon in a fluorescence
spectrophotometer. GABAa receptors depolarize responsive embryonic elements
leading to Na+ influx as well as Ca2+ increases.
From all these results it is clear that functional GABAa receptors emerge soon
after terminal cell division in the early period of postmitotic differentiation
throughout rat and chick CNS, well in advance of functional synaptic circuits.
Continued systematic and multidisciplinary analysis of GABA should reveal what
roles GABA and its receptors play in the differentiation of specific cellular
phenotypes, including GABAergic neurons and their synapses.
D7-LNP, BNP, DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS 02019-18 LNP
PERIOD COVERED
October 1 , 1989 through September 30, 1990
TITLE OF PROJ ECT (SO chincters or less Till* must fit on one line between the borders.)
Physiological properties developing on CNS cells*
PRINCIPAL INVESTIGATOR (List other profession*! personnel betom the frinaptl Investigator ) (Mime, title, laboratory, and institute affiliation)
PI: J.L. Barker, Chief, LNP, BNP, DIR, NINDS. Others (LNP, BNP, DIR, NINDS): A.E. Schaffner,
Biologist; M.K. Walton, Senior StaffFellow; A. Y. Valeyev, Visiting Scientist; R. Cruciani, Visiting
Associate; MO Poulter, Visiting Fellow; K.S. Madden, NRC Fellow; J. Suszkiw, 1PA; K. Torimitsu,
Special Volunteer; R.E. Study, Special Volunteer; B. Dufy, Special Volunteer; N. Hardegen, Electronics
Technician; L. Mahan (LCB, NIMH).
COOPERATING UNITS litany)
G.D. Lange (RSB, NINDS); J.M.H. ffrench-Mullen (1CI Pharmaceuticals, Wilmington, DE)
LAB/BRANCH
Laboratory of Neurophysiology, BNP, DIR, NINDS
SECTION
Office of the Chief
INSTITUTE AND LOCATION
NINDS, N1H, Bethesda, Maryland 20892
TOTAL MAN YEARS: „ .
8.5
PROFESSIONAL:. - g
OTHER: j q
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [T\ (c) Neither
] (a1) Minors
] (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Electrophysiological and optical recording techniques are used primarily to elucidate
the development, differentiation and cellular distribution of physiologically important
properties expressed in vitro by vertebrate CNS neurons or by fibroblasts transfected
with transmitter receptor genes. Electrical studies involve direct, high-fidelity
amplification of ion fluxes generated in single cells or in synaptically coupled pairs of
cells maintained in monoloayer culture. Optical recordings include simultaneous
indirect measurements of membrane potential or of intracellular ion concentration in 5-
50 cells in culture. Principal findings this year include: 1) depolarizing GABAa
receptors are co-expressed with functional voltage-dependent Na+ channels and
depolarizing kainic acid receptors in the earliest period of postmitotic differentiation of
the rat spinal cord; 2) GABAa receptors couple to CI- ion selective conductances in
hippocampal neurons during the embryonic period; 3) GABAa receptors activated on
physiologically intact early postnatal septal neurons are functionally excitatory,
triggering action potentials; 4) dose-response curves indicate that submicromolar
agonist concentrations are effective and that a Hill coefficient close to 1 describes the
dose-response; 5) pharmacological effects of anesthetic and naturally occurring steroids
at GABAa receptors can be detected before birth on embryonic hippocampal neurons; 6)
fluctuation analysis of GABAa receptor-coupled CI- conductances shows complex, two-
component kinetics of ion channel behavior both in embryonic hippocampal neurons and
in fibroblasts expressing specific GABAa receptor subunit combinations; 7) septal
neurons express a full complement of electrical and chemical excitability during the
first week postnatal; 8) TRH activates K + -ion selective conductances in tumoral
pituitary cells via 1,4,5-inositol trisphosphate- and diacylglycerol-induced mobilization
of Ca^+ from intracellular stores; 9) fluctuation analyses of endogenous voltage
trajectories in cultured embryonic chick spinal cord cells at the resting membrane
potential reveals complex behavior suggestive of a deterministic system.
l-T.NP R\TP, DTR
PHSMMOfRtv 1 M
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS 02330-13 LNP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters orless. Title must fit on one lute between the borders.)
Cell Biological Studies of Developing CNS Cells
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: J.L. Barker, Chief, LNP, BNP, DIR, NINDS. Others (LNP, BNP, DIR, NINDS): S. Nadi, Special
Expert; A.E. Schaffner, Biologist; K. Thor, Senior Staff Fellow; W. Ma, Senior Staff Fellow; M. Fiszman,
Visiting Fellow; A. Prasad, Staff Fellow; MO. Poulter, Visiting Fellow; K. Torimitsu, Special Volunteer;
T.N. Behar, Microbiologist; S.V. Smith, Biologist, N. Hardegen, Electronics Technician.; V. Smallwood,
Bio. Lab. Technician; J. Cuebas, Lab. Technician.
COOPERATING UNITS (it any)
G.D. Lange (RSB, NINDS): S. Lolait (LCB, NIMH)
LAB/BRANCH
Laboratory of Neurophysiology
SECTION
Office of the Chief
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues PH (c) Neither
] (a1) Minors
] (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Flow cytometry and immunochemical methods are applied to embryonic rat spinal
and supraspinal regions to study the development, differentiation and cellular
distribution of transmitters and their corresponding receptor-coupled alterations in
membrane excitability. At present this multidisciplinary investigation is focused on
elucidation of a developmental calendar outlining the expression of specific transmitters
and their functional receptors with a primary focus on the development of the GABA
phenotype and a secondary focus on the glutamate phenotype. Principal observations
this year include: 1) immunological detection of GAD-related proteins during embryonic
development of the rat spinal cord with low molecular weight proteins apparent in many
neurons before the advent of GABA, the product of GAD enzyme activity; 2) progressive
appearance of GABA + fibers, then GABA+ neurons during embryogenesis along rostro-
caudal and ventrodorsal axes in the spinal cord coincident with the appearance of high
molecular weight GAD proteins; 3) postnatal emergence of a second set of GAD-related
proteins with a subcellular distribution and colchicine sensitivity characteristic of
vesicular transport in neurons also immunoreactive for embryonic GAD proteins; 4)
biochemical detection of GABA transiently derived from ornithine and increasingly
synthesized by GAD in embryonic rat cortex; 5) sizable secretion of GABA in vitro under
resting conditions from embryonic cortical neurons; 6) early embryonic appearance of
functional voltage-gated Na+ channels and depolarizing GABAa progesterone
metabolite receptors in rat cortex, cerebellum and thalamus; 7) in situ hybridization
evidence of GABAa receptor subunit (5 receptor message preceding a subunit message
throughout the embryonic rat CNS; 8) emergence of depolarizing GABAa receptors along
a rostrocaudal gradient throughout the early embryonic chick CNS; 9) critical
requirement of GABAa receptors for chick spinal cord neuron adherence in vitro and
process formation; 10) arrival of spinothalamic tract projections in the thalamus about
the time of birth in the rat.
2-LNP,BNP,DIR
?HS£IW<">"
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 01659-22 LNP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (SO chanctari ot luii. Tnl9 murt frt on on* hna kMMM tha burden .)
Synaptic Contacts of Retinal Neurons
PRINCIPAL \NVISTIGA70R (Lm olhar enfatsmnal partonnal baltn* tht Principal ln¥*s1iQa<oc.) (Him*, tnla. laboratory, and mstituta affiliation)
PI: A. Lasansky, Unit Chief, LNP, BNP, DIR, NINDS
COOPERATING UNITS (if any)
LAB/BRANCH
Laboratory of Neurophysiology, BNP, DIR, NINDS
SECTION
Unit on Cell Biology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN YEARS:
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues fxl (c) Neither
] (a1) Minors
] (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The current-voltage relationship of salamander depolarizing bipolar cells displays a
strong outward rectification. As a result of this membrane nonlinearity, increases in
the intensity of bright lights cause relatively smaller amplitude increases in the voltage
than in the current responses and the latter have a proportionally smaller after effect.
The light-evoked responses appear to consist of at least two components: a chloride-
dependent on-off increase in membrane conductance and a faster depolarizing input
that is lost through diffussional exchange between the cytosol and the content of patch
pipettes. Because of its on-off pattern, the chloride-dependent component is thought to
reflect input from amacrine or interplexiform cells.
3-LNP, BNP, DIR
PHSMMOIitev lltl
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01-NS-02631-07
LNP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or lass. Title must fit on one line between the borders..)
Structure and Function in Retinal Neurons
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: Ralph Nelson, Unit Chief, LNP, NINDS
Others: Michael Freed, StaffFellow, LNP, NINDS; Renate Pflug, Instructor, U. of Vienna; Helga Kolb,
Professor, U. of Utah; Steven Baer, Asst. Prof, U. of Arizona, Tempe
COOPERATING UNITS (if my)
Physiology, University of Vienna, Austria (Renate Pflug); Physiology, University of Utah School of
Medicine, Salt Lake City (Helga Kolb); Mathematics, Arizona State University, Tempe and
Mathematical Research Branch, NIDDK (Steven Baer)
LAB/BRANCH
Laboratory of Neurophysiology, DIR, NINDS
SECTION
Neural Circuitry Unit
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL: 2 Q
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a)Human subjects □ (b) Human tissues [71 (c) Neither
J (a1) Minors
] (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
. Neural organization and neural interactions in mammalian retinas are investigated
using intracellular recording and staining techniques, electron-microscopic (EM) and
pharmacological approaches. In the inner plexiform layer of cat retina amacrine cells
with depolarizing responses to both onset ant offset of photic stimuli (ON-OFF cells) are
filled with horseradish peroxidase and identified as wide-field, monostratified types
(A19). In addition to a medium-field dendritic arbor surrounding the soma, A19
amacrine cells have multiple axon-like processes extending from dendritic tips.
Impulses of variable height accompany depolarizations, and also occur spontaneously.
Current clamp reveals that depolarizations result from a conductance increase. In the
EM A19 cells synapse on alpha ganglion cells (GC's), and may energize their nonlinear
subunits.
OFF-beta cat retinal GC's reveal about equal numbers of amacrine and bipolar cell
inputs whereas OFF-alpha GC's receive mainly amacrine input, as seen in serial EM
reconstructions. OFF-alpha cells receive input from only one type of bipolar cell
whereas OFF-beta cells receive input from two types. OFF-depolarizations arise from a
conductance increase. Investigations of ON-alpha GC's reveal about 2000 chemical
synapses distributed in dome-like fashion across the dendritic field. This dome-shaped
synaptic weighting, as convolved with dendritic, and electrotonic factors, contributes to
the Gaussian profile of alpha-GC receptive-field sensitivity.
In the outer plexiform layers of cat and rabbit retinas, the dopaminergic agonist
apomorphine depolarizes horizontal cells and both suppresses and delays cone-flicker
signals. The effect is mimicked by SKF38393 and may be Dl-like. The potentiating
effects on cone signals of rod-desensitizing backgrounds is modelled by a feedback
circuit in which dark-adapted, depolarized horizontal cells, release a substance that
antagonizes cone feed-forward synaptic transmission.
4-LNP, BNP, DIR
WtMOIBt, IM,
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02705-05 LNP
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT ffiO characters or Ian. Title rnuif fit on one line b«l»««n rh« borOe'i )
Anatomical Studies of Neurons, Neurotransmitters and Neurotransmitter Receptors
PRINCIPAL INVESTIGATOR (Usx other prorfuskmai personnel below the Principal tnvestiaetor.) (Name, title, laboratory, and institute affiliation)
A. P. Mariani, Expert, LNP, BNP, D1R, NINDS
COOPERATING UNITS Of my)
Ohio State University College of Medicine (N.H. Neff and M. Hadjiconstantinou)
LAB/BRANCH
Laboratory of Neurophysiology
SECTION
Office of the Chief
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a)Human subjects □ (b) Human tissues [x~1 (c) Neither
J (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Amacrine cells of the retina are central nervous system interneurons. Knowledge of
their morphology, connections and neurotransmitters could aid in the understanding of
the local-circuit neurons in the brain. Amacrine cells of the rhesus monkey, Macaca
mulatto., were studied in 38 retinas Golgi-impregnated as whole, flat preparations. By
using criteria of dendritic morphology, span of arborization, and level of arborization in
the inner plexiform layer, 26 types of amacrine cell ranging in size of dendritic span
from 30 mm to 2 mm were identified. This study provides evidence for an unprecedented
number of amacrine cell types in the primate retina. The similar morphologies of
different types of amacrine cell types within a group suggests other common features
within these groups such as neurotransmitter phenotype. Two types of amacrine cell
(type 1CA and type 2CA) immunoreactive for tyrosine hydroxylase (TH), the rate-
limiting enzyme in the catecholamine (CA) synthetic pathway, are present in the retina
of the rhesus monkey, Macaca mulatta. Although type 2CA cells are more numerous
than type 1CA amacrines, type 2 cells contain 3.5 times less TH than the type 1 cells.
Electron microscopy of retinal tissue immunoreactive for TH by the peroxidase-
antiperoxidase (PAP) method revealed synaptic input from amacrine cells at
conventional synapses, and bipolar cells at ribbon synapses onto the type 2CA amacrine
cells. Curiously, although the synaptic input is comparatively easily found, the output
synapses, or synapses of the type 2CA amacrine cells onto other neuronal elements, are
rarely found. Some synapses of the type 2CA cells onto non-immunoreactive amacrine
cells have been identified however. This unusual pattern of synaptic organization, with
many identifiable input synapses but few morphologically characterizable output
synapses, suggests a paracine role for nonsynaptic release of dopamine by the type 2CA
amacrine cells in the primate retina.
5-LNP, BNP, DIR
PHS WMOIBev 1 gdl
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02767-03 LNP
PERIOD COVERED
October 1, 1990 through September 30, 1991
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Image Processing And Analysis of Cellular Structures
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: T.G. Smith, Jr., Unit Chief, LNP, BNP, DIR, NINDS.
Others (LNP, BNP, DIR, NINDS): Kathleen Madden, Guest Researcher; Anita Prasad, Staff Fellow;
T.N. Behar, Technician
COOPERATING UNITS lifmy)
G.D. Lange, (IACS, NINDS), W.B. Marks (LNLC, NINDS), E.A. Neale (LDB, NICHD); W.H. Sheriff, Jr.
(1ACS, NINDS)
LAB/BRANCH
Laboratory of Neurophysiology
SECTION
Unit on Sensory Physiology
INSTITUTE AND LOCATION
NINDS, N1H, Bethesda, Maryland 20892
rOTAL MAN-YEARS:
PROFESSIONAL: 2 fj
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [x~| (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
We have continued to employ the concepts of Mandelbrot's fractal geometry to the
quantitative studies of central nervous system neurons, Glia and other cell types grown
in tissue culture or from whole animals. We do this by employing image processing
techniques to measure the fractal dimension (FD), which is a quantative measure of the
complexity of the structure under investigation. In particular, the FD relates to the
degree ofpranching (e.g. of dendrites), the ruggedness of borders, and the degree of
space-lilling of theobject of interest.
We have undertaken, in separate studies, how the fractal dimension changes during
the differentiation and growth of glial and neuronal cells in tissue culture. We have
found that optic nerve-derived oligodendrocytes differentiate faster and to a greater
extent than do nerve-derived astrocytes. A subsequent study has found that nerve-
derived glia also differentiate taster and to a greater extent than do brain-derived glia.
interestingly, the rates of differentiation as measured by the FD can be described by a
single time constant. The work on cultured spinal neurons is not yet complete, but it
appears that they differentiate in a similarly simple lashion. We nave proposed the FD
is a useful, quantitative measure of morphological differentiation.
We have begun separate studies 1) of the development of the internal structures of
cultured chick spinal cord neurons with fluorescence microscopy, 2) of the phase-plane
plots of the electrical activity of spinal cord activity by measuring their FD's, and
6) Courier methods of analysing cellular structure.
6-LNP, BNP, DIR
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ANNUAL REPORT
October 1, 1989 through September 30, 1990
Laboratory of Molecular and Cellular Neurobiology
Basic Neurosciences Program, DIR
Table of Contents
Page #'s
RESEARCH SUMMARY 2-13
PROJECT REPORTS
Investigation of the Structure and Function of 14
Gonadotropins and their Receptors
Z01 NS 02784-02 LMCN
Regulation of Hormone-Responsive Adenylate Cyclase 1 5
Z01 NS 02366-12 LMCN
Biosynthesis and Function of Glycosphingolipids and 16
Othtr Glycoconjugates
Z01 NS 01309-25 LMCN
Information Processing in Simple Nervous Systems 17
Z01 NS 02151-16 LMCN
Antibodies to Glycoconjugates in Neurological Diseases 18
Z01 NS 02786-02 LMCN
Glycoproteins of Myelin in Development and Disease 19
Z01 NS 01808-21 LMCN
Molecular and Immunological Aspects of Myelin Abnormalities 20
in Neuro-AIDS
Z01 NS 02805-01 LMCN
Molecular Biology of Neurotransmitter Receptors 21
Z01 NS 02710-05 LMCN
Megabase DNA Sequencing 22
Z01 NS 02754-03 LMCN
Human Brain cDNA Project 23
Z01 NS 02806-01 LMCN
1 LMCN/DIR
ANNUAL REPORT
October 1, 1989 through September 30, 1990
Laboratory of Molecular and Cellular Neurobiology
National Institute of Neurological Disorders and Stroke
Peter H. Fishman, Ph.D. , Chief (Rotating)
The Laboratory of Molecular and Cellular Neurobiology consists of four independent
Sections, each of which is engaged in a comprehensive research program in the
neurosciences. Thus, there is overlap in areas of interest, especially in biochemistry,
cell biology and molecular biology. Our recently renovated library and conference
room provides a focal point for staff interactions and the development of
collaborative endeavors. The future status of the Laboratory, however, remains
unclear. It is envisioned that at least two of the Sections will return to the main
campus upon completion of the Child Health and Neurosciences Building (Building
49) whereas the other Sections may remain off campus.
Membrane Biochemistry Section
The Membrane Biochemistry Section actively investigates the structure, biosynthesis
and regulation of cell membrane components involved in various recognition
phenomena and incellular signaling. These include complex glycoconjugates such as
gangliosides and the receptor-coupled adenylate cyclase and phospholipase C
systems which mediate the cellular responses to various hormones,
neurotransmitters, and growth factors as well as pathological toxins and viruses.
Most of our studies involve using cultured cell lines which express these components
and respond to physiological and environmental signals.
1. Role of Ganqlioside Lipid Moiety in Action of Cholera Toxin.
In an extensive series of experiments, we had previously established that ganglioside
GM1 is the only endogenous cell surface receptor for cholera toxin, the active agent
in the disease cholera. The toxin intoxicates target cells by ADP-ribosylation of the
stimulatory G protein, Gs, of adenylate cyclase which thus results in a persistent
activation of adenylate cyclase. We recently demonstrated that Givn-type
"ganglioproteins" were unable to serve as functional receptors for cholera toxin
even though they were able to bind the toxin. In order to further explore the role of
the ceramide lipid moiety of Gmi, we synthesized a series of neoglycolipids by
attaching the oligosaccharide of GM1 to cholesterol, aliphatic amines and
phospholipids. We incubated the derivatives with rat C6 glioma cells which are
deficient in GM1, bind only traces of cholera toxin and are poorly responsive to the
toxin. C6 cells exposed to the various derivatives exhibited an increase in both
iodinated cholera toxin binding and toxin activation of adenylate cyclase. There
were differences among the neoglycolipids in terms of the amounts of cyclic AMP
formed per amount of toxin bound to the treated cells. Those derived from long-
chain aliphatic amines and cholesterol were more efficient as toxin receptors than
2 LMCN/DIR
GM1 whereas those derived from phospholipids were less efficient . When the
distance between the oligosaccharide and the phospholipid was increased by adding
spacers, the efficiency decreased even more. Thus, the lipid moiety of GM 1 is
important for the action of cholera toxin, and these neoglycolipids may be useful as
probes of GM 1 bioactivity in other systems such as the neuritogenic and
neuronotrophic effects of the ganglioside.
2. Receptors for Neurotransmitters and Neuropeptides
We are continuing to mak progress on the identification, characterization and
regulation of neurotransmitter and neuropeptide. These include ^-adrenergic, Di
dopaminergic, and neuropeptide Y receptors. Large advances have been made by us
and others to understand the mechanisms of desensitization and down-regulation
of 0-adrenergic receptors. Although there are two major subtypes of P-adrenergic
receptors, most of the progress has been directed toward the {^-subtype as it is the
subtype found on all the established ^-adrenergic responsive cell lines to date. We
have been able to demonstrate that human neurotumor SK-N-MC cells express only
01-adrenergic receptors. Agonist stimulation of cyclic AMP accumulation was
inhibited more potently by {$r than by [32-selective antagonists as was binding of
labeled antagonists to both intact cells and cell membranes. Northern blot analysis
of mRNA from SK-N-MC cells using cDNA probes for {5i- and {32-adrenergic receptors
revealed only the presence of {ii-adrenergic receptor mRNA. When SK-N-MC cells
were exposed to isoproterenol, there was no loss of maximum responsiveness to the
agonist upon rechallenge during the first hour. After longer exposure,
desensitization slowly occurred and the Pi-adrenergic receptors slowly down-
regulated to 50% of control levels by 24 hours. We explored this unusual resistance
to regulation by preparing membranes from control and agonist-treated cells and
assaying them for adenylate cyclase activity. There was no loss of maximum
stimulation of adenylate cyclase by isoproterenol in membranes from cells exposed
to the agonist for 1 hour. There was, however, a 3-fold shift to the right in the dose
response. As receptor phosphorylation has been implicated in desensitization, we
determined which, if any, protein kinases might be involved. Cells were rendered
permeable and loaded with either an inhibitor of cyclic AMP-dependent protein
kinase A or heparin, an inhibitor of the p-adrenergic receptor-specific kinase.
Inhibiting protein kinase A but not the receptor-specific kinase blocked the shift in
the agonist dose response. Furthermore, membranes incubated with ATP and the
purified catalytic subunit of protein kinase A exhibited a similar shift. The shift,
however, was not observed in cells treated with dibutyryl cyclic AMP. Thus, in intact
cells, this unusual desensitization requires both agonist-occupied receptors and
activation of protein kinase A. We are now generating antipeptide antibodies
against specific sequences in the B-adrenergic receptor and will use these to
determine the state and site of phosphorylation of the receptor in control and
desensitized cells. A key issue to explore is whether the receptor is being
phosphorylated by the receptor-specific kinase and if not, why not. We have
preliminary evidence that the dopamine Di receptor-coupled adenylate cyclase
undergoes a normal desensitization when SK-N-MC cells are exposed to dopamine.
As the current dogma predicts that the receptor-specific kinase will phosphorylate
and initiate desensitization of Di receptors, it would appear that this kinase is
present in SK-N-MC cells.
Using our recently developed procedures for solubilizing and purifying the Di
receptor from rat striatum, we have scaled up the procedure and obtained sufficient
receptor protein for partial amino acid sequencing and the raising of antibodies,
both of which will be used to screen a rat striatal cDNA library by standard
3 LMCN/DIR
techniques. With the availability of suitable molecular probes for the Di receptor,
we will be able to investigate both human and animal models for defects in the
receptor gene. One potential possibility is the spontaneous hypertensive rat model.
Kidney proximal convoluted tubules contain a dopamine-sensitive adenylate cyclase
and it is believed that dopamine is involved in the regulation of sodium transport.
We found that in tubules from spontaneous hypertensive rats, adenylate cyclase
activity was not stimulated by dopamine Di agonists but exhibited a normal
response to guanine nucleotides, forskolin and parathyroid hormone. Maximum
binding activity and affinity for a Di selective antagonist was the same in both
control and hypertensive tissues as was the apparent molecular weight of the Di
receptor as determined by photoaffinity labeling and separation by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Di agonists, however, were able to
compete more effectively for antagonist binding in control than in hypertensive
specimens. We believe that these results are consistent with a defect in the Di
receptors in proximal kidney tubules of hypertensive rats and this defect impairs
receptor coupling to G proteins.
Neuropeptide Y (NPY) is the most abundant neuropeptide in the mammalian
nervous system. It is widespread, found in both central and peripheral neurons,
often colocalized with catecholamines, and has several physiological effects,
including appetite stimulation and vasoconstriction. Little is known about its mode
of action or its receptors. We have previously established that human neurotumor
cells express functional NPY receptors and thus are useful to investigate various
aspects of NPY bioactivity. In this regard, we found that an NPY analog in which
amino acid residues 7-17 had been replaced with an amino-octanoicacid moiety and
further stabilized with an intramolecular disulfide bridge, [D-Cys7-Aoc817-
Cys20]pNPY, both competed for [125I]NPY binding to SK-N-MC cells and inhibited
isoproterenol-stimulated cyclic AMP accumulation in these cells, but with slightly
lower affinity and potency than native pNPY. Thus, the central a-helix of the
molecule does not appear to be required for either binding or biological activity but
may be important for stabilizing the N-and C-terminal regions of the peptide.
Recently, NPY receptors have been classified into Yi and Y2 subtypes which can be
distinguished both physiologically and pharmacologically. Y1 receptors bind only
the full-length NPY and are associated with vasoconstriction; Y2 receptors will also
bind the C-terminal fragment of NPY (residues 1 3-36) and mediate the inhibition of
norepinephrine release from presynaptic neurons. Whereas SK-N-MC cells and its
subclone MC-IXC cells have only Y1 receptors, another human neurotumor cell line,
SMS-MSN, contains only Y2 receptors. We have used these two cell lines to
investigate the mechanism(s) of signal transduction mediated by these two NPY
receptor subtypes. High affinity binding of NPY to membranes from both cell lines
was sensitive to pertussis toxin, indicating that the receptors are linked to G proteins.
The toxin catalyzed the ADP-ribosylation of a 41,000 dalton protein in both cell lines.
NPY inhibited effector-stimulated cyclic AMP production in MC-IXC cells but not in
SMS-MSN cells. In contrast, fluoroaluminate, a general activator of Gj in intact cells,
inhibited cyclic AMP production in both cell lines. Western blot analysis of G
proteins with specific antibodies showed that MC-IXC cells contained Gs and Gj
whereas SMS-MSN cells contained Gs, Gj and G0- Whereas Gj is involved in coupling
receptors to the inhibition of adenylate cyclase, G0 has been implicated in coupling
receptors to the modulation of ion channels. Our results suggest that Y1 and Y2
receptors mediate cellular signaling through different G proteins. We are now
exploring which ion channels may be coupling to the latter receptor subtype.
Initially, we will investigate Ca2+ and K+ channels which have been implicated for
other inhibitory neurotransmitters.
4 LMCN/DIR
3. Differential Activation of G Proteins in Cells and Membranes
It is well-established that many receptors for hormones and neurotransmitters are
coupled to their effector systems through guanine nucleotide binding proteins (G
proteins). Thus, stimulatory receptors activate adenylate cyclase through Gs and
inhibitory receptors inhibit adenylate cyclase through Gj. These G proteins are
heterotrimers with common By and distinct a subunits. It is believed that hormone-
bound receptors activate their respective G proteins by promoting the exchange of
GDP with GTP which results in dissociation of the heterotrimers and release of the a
subunits containing bound GTP. In time, the bound GTP is hydrolyzed to GDP by the
intrinsic GTPase activity of the a subunit which then reassociates with the By
subunits to complete the cycle. Hydrolysis-resistant analogues of GTP and
f luoroaluminate also can persistantly activate G proteins as the GTPase "turnoff " is
no longer operating. We have shown previously that fluoroaluminate is a potent
inhibitor of hormone-stimulated adenylate cyclase in intact cells but by itself
activated adenylate cyclase in permeable cells and membranes. Our results indicated
that the interactions or relationships between the G proteins and the catalytic
subunit of adenylate cyclase are altered when the cell membrane is perturbed.
We now have applied another probe to explore these interactions. The diterpene
forskolin, is a potent activator of the catalytic subunit of adenylate cyclase but its
effect is potentiated by the presence of Gs. We observed that different cells varied in
their responses to forskolin. Some cell lines accumulated large amounts of cyclic
AMP whereas as others accumulated very little when exposed to it. We found that
even in the "unresponsive" cells, forskolin potentiated the stimulation of cyclic AMP
production by hormones and neurotransmitters. Thus, the drug was not being
excluded by "unresponsive" cells. Forskolin, however, was able to activate
adenylate cyclase in membranes prepared from both "responsive" and
"unresponsive" cells. Again, it appears that the relationships between G proteins
and the catalyst are altered upon cell disruption. Although most studies have used
preparations of cell membranes or purified components (receptors, G proteins,
catalyst) reconstituted into lipid vesicles, it is clear that a better understanding of the
biological effects of hormones and neurotransmitters as well as drugs such as
forskolin will require the use of methods to elucidate the interactions of these
components in intact cells.
Neural Systems Section
The Neural Systems Section takes a multidisciplinary approach to the question of
how information is stored during associative learning and how it is made available
for later recall. Biophysical and molecular mechanisms of associative learning are
being analyzed in parallel for a mollusc (the sea snail Hermissenda crassicornis), the
rabbit, and, most recently, the rat. Parallel analyses offer the important opportunity
for uncovering general cellular principles of learning and memory - principles which
have been conserved over the course of evolution and which therefore could have
relevance for human cognition. Parallel analyses also permit exploitation of critical
experimental advantages unique to diverse species. For Hermissenda we have
demonstrated the first causal relationship of biophysical and molecular
transformations within individual neurons to Pavlovian conditioning of a living
animal. Casual relationships of cellular physiology and associative learning have not
yet been approximated for any vertebrate preparation. Nevertheless, we have
found evidence of biophysical transformations which are common to both mollusc
and mammal. An identified group of neurons, the CA1 cells (rather than individual
5 LMCN/DIR
identified neurons) was shown to have a distribution of conditioning-specific
modification of K + channels within hippocampal slices removed from rabbits on
days after they had been conditioned. Such slices provide vastly greater amounts of
tissue (than does the snail) for biochemical studies. Indeed, we have already
observed conditioning-specific translocation of protein kinase C (PKC) not only in the
hippocampus CA1 neurons, but also in a restricted region of the cerebellar cortex
called "H6". PKC regulation of identical K+ channels occurs in both Hermissenda
and hippocampal neurons. In Hermissenda this regulation is being investigated with
isolated membrane patches whose intracellular surfaces are accessible to precise
ionic and biochemical manipulations. The distribution of PKC-mediated neural
changes with associative learning in large neuronal arrays is currently being
analyzed autoradiographically. Within the past year, image analyses have revealed
changes within neuronal populations of conditioned but not control brain areas.
Also recently, PKC regulation has been linked to conditioning-specific modification
of Hermissenda mRNA metabolism. This molecular storage step occurs within a
specific temporal window during the retention period of the associative memory.
Convergence of CS and UCS pathways activated during associative conditioning of
the nudfibranch mollusc Hermissenda involves inhibition of type B photoreceptors by
hair cells caudally located in statocysts. Recently we demonstrated that this GABA-
mediated inhibition is entirely transformed into excitation when stimulation of the
visual pathway is precisely timed in relation to stimulation of the vestibular pathway.
The pairing-specificity of this vestibular-visual synapse has important implications
not only acquisition for associative memory in the snail Hermissenda but for
mammalian systems as well. Furthermore, theoretical constructs formulated on the
basis of this unique synaptic function have been extremely successful in improving
the design of artificial learning networks. Conditioning-specific changes of
particular proteins have been related to mRNA changes during the last year. The
functional roles of these proteins as well as their identitiesare now being explored.
One, for example, has been linked to GTP-binding protein signal transduction.
Another may have more importance for cell structure. These learning-induced
changes of protein availability may represent an important step for consolidating,
i.e., making more permanent the physiologic memory trace which could have
expression in conditioning-specific structural changes of Hermissenda neurons.
These latter changes, as studies with the formation of Hermissenda associations,
involve an apparent reorganization of the cell's terminal branches on which synaptic
interactions occur.
The experimental psychology program of the Section uses associative learning
paradigms to produce persistent behavioral changes in the nudibranch mollusc
Hermissenda crassicomis as well as vertebrate such as rabbits and rats. Quantitative
assessments are made of the animals' responses to the conditioned and
unconditioned stimuli before and after classical conditioning paradigms. These
assessments include precise dissection of generalized behavioral transformations
into modification of individual muscular components of the behaviors. A full range
of psychological manipulations has been used to clearly establish the sensitivity of
the learning behavior to the exact temporal relationship of the stimuli which are
associated during learning acquisition. Also of interest to psychologists is the close
linkage of the learning behaviortothe specific stimuli associated and discriminative
functions involving those stimuli not associated. Recently this aspect of the program
has been extended to include more cognitively oriented learning tasks involving
spatial mapping of an organism's environment.
The Section's neurophysiology program is concerned first with the definition of
those neural systems relevant to the learning capability. Multiple intracellular
6 LMCN/DIR
recordings from pre- and postsynaptic neurons have been employed within the
visual, vestibular and chemosensory pathways of Hermissenda to establish a working
knowledge of the critical neural systems and to describe how information flows in a
stepwise fashion beginning with sensory cells at the output. A similar approach is
being taken with the rabbit hippocampus, and more recently the cerebellum, and
critical afferent and efferent pathways within these structures. Neurophysiological
correlates are then obtained for conditioned (as well as a variety of control) animals.
These neurophysiological correlates are recorded in intact animals, isolated nervous
systems, and isolated neuronal membranes. Based on such correlates,
electrophysiological sequences are constructed to trace the transformation of the
information in electrical terms of the neural system.
The Section's biophysics program measures persistent modification of specific ionic
channels during and following the learning. In the past, a two microelectrode
voltage clamp was employed to characterize genetically specified membrane
currents within identified neurons which were demonstrated to play a causal role in
the acquisition and retention of associative learning. More recently the patch-clamp
technique has been used in both the cell-attached and "inside-out" configurations
to determine which subcellular biochemical processes (e.g., Ca2 + -dependent
phosphorylation) are critical for regulating those ionic channels which change
during learning. All these biophysical approaches have also been applied to
demonstrate unequivocally that it is persistent modification of specific ionic
channels which encode a learned association for later recall.
The biochemistry research of the Section seeks to uncover the molecular basis for the
persistent ionic channel modifications shown to underlie associative learning (both
in Hermissenda and the rabbit). A variety of biochemical and molecular biological
methods are being used for this purpose. Microgel analysis of phosphorylation of
individual neuronal proteins, for example, has revealed that Ca2 + -dependent
phosphorylation of a specific low-molecular weight protein changes within certain
neurons of conditioned animals but not those exposed to control paradigms.
Exposure of neurons to prolonged depolarization, which simulated the integrated
visual-vestibular network effects on identified neurons during conditioning, is also
followed by long-lasting phosphorylation differences for particular low-molecular
weight proteins. More recently, learning-induced differences in the amount and/or
synthesis of these and other proteins have been detected. Recently, it has been
possible to isolate specific proteins and inject them into individual neurons, thereby
reproducing the actual learning effects on membrane channels. Furthermore, a
number of intracellular manipulations have supported the hypothesis that learning-
induced modification of ionic channels involves Ca2 + -calmodulin-dependent and
Ca2 + -lipid-dependent phosphorylation. Such manipulations include iontophoretic
injection of Ca2 + -calmodulin-dependent protein kinase, inositol trisphosphate or
PKC, or preincubation with PKC activators such as phorbol esters or OAG. Modern
molecular biological techniques are enabling the Section to use monoclonal
antibodies to enzymes implicated in the learning process. Other antibodies may
also be helpful to reconstruct of the biochemical and associated biophysical
sequences which make biological records of memory possible. Recombinant
techniques may be particularly helpful in identifying specific proteins whose
synthesis is modified as a result of the recently observed conditioning-specific
increase in mRNA turnover.
The cellular anatomy aspects of the Section's programs contributes in several ways to
the various levels of inquiry into the learning process already mentioned.
Ultrastructural measurements of the cells and their synaptic interaction have
7 LMCN/DIR
provided further definition of the relevant neural systems. Activity-dependent
uptake of radioactive labels within these systems has been monitored by
autoradiographic methods. Morphometric techniques together with serial
sectioning and computerized reconstruction, have recently uncovered structural
manifestations of the biophysical and biochemical changes already shown for
neurons within conditioned but not control animals. Differential absorption
spectrophotometry allows intracellular localization of fluctuations of cytosolic Ca2 + -
dependent modulation of the channelsduring learning.
Finally, the developmental biologists within the Section have established laboratory
strains of Hermissenda. Such strains permit assessment of how genetic and
environmental factors may interact to determine individual differences in the ability
of the animals to undergo associative learning. Furthermore, critical biochemical
steps have now been identified which may serve potent cell-transforming functions
in learning, developmental, and oncogenic contexts.
Perhaps most important in all these efforts is the accumulated evidence that a
remarkable similarity exists between means of encoding learned associations in the
snail, rabbit, and now the rat. The same learning-induced reduction of well-
characterized K + currents has been found to provide such encoding in Hermissenda
as it does within identified neurons of rabbit hippocampal slices. Similar regulation
of these channels appears to occur atthe molecular level for both the mollusc,
rabbit, and rat. Such parallel mechanisms may ultimately provide the basis for
clinical intervention and thereby the amelioration of pathologic symptoms.
Section on Myelin and Brain Development
Research in the Section on Myelin and Brain Development is divided into three
related projects. The first project, entitled "Glycoproteins of Myelin in Development
and Disease," has been existed for many years and emphasizes the myelin-associated
glycoprotein (MAG). However, other myelin proteins and lipids are also studied with
the ultimate objective of understanding molecular mechanisms of myelin formation
and breakdown. The second project, entitled "Antibodies to Glycoconjugates in
Neurological Diseases" grew out of the first as a consequence of the discovery that
monoclonal IgM antibodies to MAG in patients with neuropathy cross reacted with
other glycoproteins and glycolipids. This research emphasizes the occurrence and
pathogenic significance of antibodies to MAG and to sphingoglycolipids in patients
with neurological diseases, especially peripheral neuropathies. Very recently, when
it became apparent that a significant aspect of the neuropathology in acquired
immunodeficiency syndrome (AIDS) involves white matter, a third area of research
was undertaken to elucidate molecular mechanisms involved in the neurological
aspects of this disease. This new project is entitled "Molecular and Immunological
Aspects of Myelin Abnormalities in Neuro-AIDS".
1. Structure and function of the myelin-associated glycoprotein (MAG). The
localization of MAG in periaxonal Schwann cell and oligodendroglial membranes of
myelin sheaths, its membership in the immunoglobulin (Ig) superfamily together
with other neural adhesion molecules and experiments directly demonstrating its
capacity to promote cell-cell interactions all suggest that this glycoprotein functions
in the interaction of myelin-forming cells with the axolemma. MAG is also present in
Schmidt-Lanterman incisures, lateral loops and the outer mesaxons of peripheral
nervous system (PNS) myelin sheaths where it appears to play a role in maintaining
the spacing of adjacent Schwann cell membranes in these structures where
8 LMCN/DIR
extracellular membrane surfaces are separated by 12-14 nm and there is always
cytoplasm at the inner membrane surface. In addition, during active myelination,
oligodendrocytes contain numerous multivesicular bodies (M VBs) that are enriched
in MAG. MVBs are known to be endocytosed structures in other cells, and this raises
that possibility that these MAG- enriched bodies are carrying a signal from the
periaxonal regions back to the oligodendrocyte cell bodies.
The myelin-associated glycoprotein has a single membrane spanning domain
separating its C-terminal tail from a heavily glycosylated N-terminus that is
composedof 5 domains related in amino acid sequence to each other and to
members of the immunoglobulin superfamily such as neural cell adhesion molecule
(N-CAM). These extracellular immunoglobulin-like domains must mediate the
function of MAG in glia-axon interactions, possibly by specifically interacting with
another member of the superfamily on the axolemma. Homophilic MAG binding
may be involved in the interactions of adjacent Schwann cell membranes in incisures,
lateral loops, and mesaxons. An important factor that has become apparent in the
past few years is that the functional binding properties possessed by the native MAG
molecule may be lost when the glycoprotein is purified by conventional methods
involving strong surface active agents such as lithium diiodosalicylate. Therefore,
experiments are underway to isolate native MAG for functional studies by using
milder detergents.
MAG has 8 extracellular sites for N-linked glycosylation, and detailed investigation
of the oligosaccharides are underway utilizing the Dionex BioLC Carbohydrate
System with an amperometric detector that is capable of quantifying low picomole
amounts of carbohydrate. Adult rat brain MAG contain a heterogeneus mixture of
complex bi-, tri- and tetraantennary oligosaccharides, many of which are highly
negatively charged due to sialic acid moieties and sulfate groups. Differences in
oligosaccharide structure may modulate the functioning of MAG in membrane-
membrane interactions. MAG is abnormally glycosylated in the dysmyelinating
quaking mutant, and this may lead to some of the abnormalities of myelin structure
in this mutant.
MAG occurs in two developmental^ regulated isoforms differing in the lengths of
their C-terminal domains and arising by alternative splicing of the primary mRNA
transcript. The larger isoform (L-MAG) is most prominent at the time of active
myelination in rodent brain, whereas nearly all the MAG in rodent peripheral nerve
is the shorter isoform (S-MAG) at all ages. Both CNS and PNS MAG are
phosphorylated on their cytoplasmic domains, but the sites of phosphorylation are
different. Oligodendrocytes phosphorylate MAG on serine, threonine and tyrosine
residues in L-MAG, whereas Schwann cells phosphorylate MAG primarily on serine
residues of S-MAG. This may be relevant to cellular differences in the process of
myelination in the CNS and PNS, respectively. Regulation of expression of the two
forms of MAG as well as phosphorylation of the cytoplasmic domains may modulate
interactions with cytoskeletal components. Overall, the MAG molecule seems well-
suited to mediate interactions between intracellular cytoskeletal elements and
adjacent extracellular membrane surfaces. In this manner, MAG could play a key
role in transmitting the chemomechanical forces involved in generating spiraled
myelin sheaths.
An investigation of MAG in cultured oligodendrocytes and Schwann cells is under
way with the ultimate objective of determining factors that control its expression
and probing its function as an adhesion molecule. Cultured oligodendrocytes
constitutively express MAG and other myelin proteins. Recent experiments show
9 LMCN/DIR
that cultured oligodendrocytes isolated from myelinated bovine brain appear to
express more MAG than those enriched from primary cultures of embryonic rat
brain, and therefore appear to provide the preferable system for studies on the cell
biology of MAG. Cultured Schwann cells do not constitutively express MAG, but can
be induced to express a small amount by elevating cAMP levels with forskolin.
Recently, an immortalized line of Schwann cells (S-1 6) has been generated by
multiple passaging that expresses a remarkably high level of MAG, similar to that in
adult sciatic nerve. These cells appear to resemble Schwann cells early in the process
of myelination, since they are also galactocerebroside positive, but express little or
none of the major myelin proteins, PO glycoprotein or myelin basic protein. The
MAG expressed in the S-1 6 cells resembles that in vivo since it is primarily the shorter
isoform, is glycosylated to about the same extent and is both sulfated and
phosphorylated . Most of the MAG expressed by the cells is on the surface, and it is
expected that these immortalized Schwann cells will be useful for studying the cell
biology of MAG and its function in cell-cell interactions.
2 MAG in multiple sclerosis (MS) Previous studies have demonstrated a greater loss
of MAG than other myelin proteins at the periphery of MS plaques, suggesting an
important role for MAG in the molecular pathology of this disease. Currently, our
favored hypothesis to explain the selective loss of MAG involves its high
susceptibility to a myelin-associated, calcium -activated, neutral protease. This
enzyme converts the glycoprotein to a partially soluble, lower Mr weight derivative,
dMAG, that is missing tne C -terminus. Experiments are planned to further define
the reason for the elevated activity of this enzyme in tissue from MS patients.
3. Antibodies to qlycolipids in peripheral neuropathies This area of investigation
began with the demonstration of monoclonal anti-MAG antibodies in patients with
polyneuropathies occuring in association with IgM gammopathy. It was
subsequently demonstrated that these anti-MAG antibodies cross-reacted with the
newly discovered sphingoglycolipid, sulfate-3-glucuronyl paragloboside (SGPG).
Further studies showed the monoclonal IgM antibodies in other neuropathy patients
did not react with MAG and SGPG, but that they did react with various ganglioside
antigens. Patients with motor neuropathies and monoclonal antibodies to GM 1
ganglioside have been of particular interest in recent years. Overall, about 80% of
patients with neuropathy occurring in association with IgM paraproteinemia have a
monoclonal antibody that reacts with SGPG or a ganglioside antigen, suggesting
that glycolipids may be important target antigens in this type of neuropathy. An
extension of the research on monoclonal gammopathies has been the
demonstration that some patients with neuropathy in the absence of a monoclonal
antibody have polyclonal antiganglioside antibodies. For example, 10 to 20% of
patients with Guillain-Barre syndrome have high levels of anti-ganglioside
antibodies that may play a pathogenic role. Also, high levels of polyclonal
antibodies reacting with GM1 ganglioside in some patients with primarily motor
neuropathies have been of special interest.
Recent research has revealed differences in the fine specificities of both the anti-
MAG antibodies in the paraproteinemic neuropathies and the anti-GM1 antibodies
in association with motor neuropathies. For example, experiments with chemically
modified derivatives of SGPG revealed that some of the MAG/SGPG antibodies
absolutely require the sulfate for reactivity, others require the carboxyl group of
glucuronic acid, whereas still others will react as long as one or the other of the two
negatively charged groups is present. The fine specificities of anti-GMI antibodies in
patients with motor neuropathy also vary as revealed by differing cross-reactivity
with other gangliosides. However, since GM1 is the only common antigen in all the
10 LMCN/DIR
patients examined by us, the results suggest that it could be the pathogenically most
significant target antigen in motor nerves. Nevertheless, differences in fine
specificity and the spectrum of cross-reacting neural antigens could affect the clinical
presentation in these patients. It should be emphasized that our research and that
of others indicates that serum antibodies occurring in association with neuropathy
should be analyzed both qualitatively by TLC-overlay and quantitatively by ELISA,
since their pathogenic significance appears to depend both on their specificity and
titer. Although anti-MAG/SGPG antibodies have been demonstrated to be capable
of causing neurological disease, an important priority for the future is to evaluate
the capacity of anti-ganglioside antibodies to induce neural damage or interfere
with normal function in appropriate animal models or tissue culture systems.
4. Biochemical neuropathology in acquired immunodeficiency syndrome (AIDS).
Relevant areas of our research described in the preceding section are being
extended in an attempt to determine the molecular basis for some of the
abnormalities of myelin occurring in neuro-AIDS including myelin pallor, vacuolar
myelopathy and multifocal demyelination in the CNS as well asdemyelinating
peripheral neuropathy. This research has just begun and results are very preliminary.
Specific aspects of the research include the following: 1) Postmortem CNS tissue
from AIDS patients is being analyzed for quantitative and qualitative alterations of
myelin proteins. 2) Since the myelin-associated glycoprotein (MAG) and the CD4
receptor for HIV are both in the immunoglobulin superfamily and have a modest,
but statistically significant, sequence homology, possible interactions between MAG
and GP1 20 are being tested. 3) Sera from AIDS patients with neuropathy are being
tested for antibodies to spingoglycolipids as have been observed in other types of
neuropathy.
Receptor Biochemistry and Molecular Biology Section
The Section of Receptor Biochemistry and Molecular Biology conducts research on
?iene super families of neurotransmitter receptors, including receptor structure,
unction and evolution. The Section has major programs underway on genomic
structure and evolution and on the the genetic basis of neurological diseases. New
personnel have been recruited to the Section in a number of important areas. Dr.
Mark Dubnick, a molecular biologist and computer programmer, joined the
laboratory in January 1990 as a Staff Fellow. He has already made significant
contributions along with Senior Staff Fellow Dr. Anthony Kerlavage, Chief, Unit on
Molecular Structure and Evolution, and John Powell of DCRT, in the area of data
handling and analysis. They have established a computer network consisting of six
IBM, fifteen Macintosh, two Sun 4 and two Silicon Graphics workstations in the
laboratory and networked these machines to the NIH Convex, Cray and other
computers essential for the analysis of large genomic sequences; and have
developed software to facilitate data analysis. Dr. Mark Adams joined the
laboratory from the University of Michigan where he recently completed his Ph.D. in
biochemistry. Dr. Adams is the lead scientist on a major new project in the
laboratory to isolate and sequence all the genes expressed in the human brain (see
below). Working with Dr. Adams is another recruit to the laboratory, Dr. Bjorn Olde,
a recent graduate of University of Stockholm. Dr. Antonia Martin was recruited
from Praxis Biologies to head up a new unit established on gene therapy. Dr. Martin
has lead a team at NIH, in collaboration with Dr. Francis Collins of the University of
Michigan, to sequence several exons of the neurofibromatosis gene from
chromosome 17, and is working on the genomic structure of the alpha-3 subunit of
the GABA receptor mapped to Xq28 and the mytonic dystrophy region of
chromosome 19 (see below).
11 LMCN/DIR
Dr. W. Richard McCombie, Chief, Unit on Neurological Disease Gene Structure and
Function, is the lead scientist on sequencing three cosmid clones (40kb each) isolated
from the region on chromosome 4 where the Huntington's disease gene has been
mapped. The cosmid clones were provided as part of a collaboration with the
Huntington's disease collaborative group. Dr. McCombie has also established a
collaboration with Lev Goldfarb in LCNSS to sequence DNA from patients with
various spongiform encephalopathies, in search of point mutations associated with
these disorders. This project involves sequencing multiple clones from each of 43
patients, or over 600,000 base pairs of raw sequence data.
Jeannine Gocayne, microbiologist, has integrated the new robotics added to the
laboratory. These completely automate DNA sequencing reactions. This work is part
of a CRADA with Applied Biosystems, Inc.
The laboratory has completed over 2 million bases of raw DNA sequence and over
300 kb of finished sequence this year, including the following projects. Over 15 kb
from the disease gene for neurofibromatosis I (NF1) have been sequenced,
identifying several new exons. Determining the intron-exon boundaries will allow
us to develop polymerase chain reaction (PCR) probes to sequence exons from NF1
patients in search of point mutations associated with disease symptoms. Three
cosmids from the Huntington's region of chromosome 4 have been sequenced.
These cosmids total over 1 20 kb of finished sequence and comprise one of the two
largest segments of human chromosomal DNA sequenced to date. The sequence
from this region is currently being analyzed. The second largest region which has
been sequenced by the laboratory in collaboration with the DOE and Applied
Biosystems, is a three cosmid contig of over 100 kbfrom chromosome 19. This region
is where a number of DNA repair enzymes and the gene for mytonic dystrophy have
been tentatively mapped. Lambda and cosmid clones have been obtained from the
human Xq28 region which contains several loci of neurological significance
including the alpha-3 subunit of the GABA receptor.
Progress on the project to characterize the gene and protein structure and evolution
of neurotransmitter receptors belonging to two major multigene families continues.
The first gene family contains adrenergic, muscarinic, opsin, serotonin and
octopamine receptors; the nicotinic cholinergic, GABA/benzodiazepine, NMDA, and
glycine receptors. To data we have cloned and sequenced a significant number of
receptor genes from both multigene families. These include several alpha and beta-
adrenergic receptors from human and rat cDNA and genomic libraries; muscarinic
cholinergic receptors from human, rat, and Drosoph'na genomic libraries;
octopamine receptors from Drosophila; human alpha and beta subunitsof the
GABA/benzodiazepine receptor; and nicotinic receptors from locust and C. elegans.
For example, the human GABA beta-1 subunit has been cloned, sequenced and
localized to chromosome 4. This gene is over 65 kb and comprises 9 exons. The
exons and splice sites are highly conserved in other GABA receptor subunits from
humans and lower species. Permanent cell lines expressing the unique
neurotransmitter receptor proteins provide key new information concerning the
mechanism of receptor activation by neurotransmitters. Extensive site directed
mutagenesis studying on adrenergic and muscarinic cholinergic receptors have
identified a number of conserved amino acid residues essential for ligand binding
and receptor activation.
Another major new project within the laboratory has the goal of isolating,
sequencing and characterizing all genes expressed in the human brain. As many as
12 LMCN/DIR
half of the over 50,000 human genes are believed to be expressed in the brain.
While sequencing the human genome is expected to take over 1 5-20 years,
sequencing a large number of cDNA clones can readily provide coding sequence data
on genes expressed in tissues. We are building a large library of clones and
sequences of human brain cDNAs. Sequence data will be obtained on 1000 human
brain clones in the first 3-4 months of this project. Over half the genes isolated are
new ones, not previously identified or cloned. An additional percentage are new
human genes, having been previously isolated from other species. Computer
analysis of the protein sequences at primary and secondary structural levels is
assisting in clone identification. DNAand predicted protein sequences will be
examined for the presence of conserved primary and secondary structure motifs and
relationships to previously sequenced genes. Further characterization of potentially
interesting clones will include chromosome localization, examination of tissue
distribution of expression, and evolutionary conservation. The availability of a
broad-based library of cDNA sequences will also facilitate identification of coding
regions in genomic sequences as well as provide a starting point for individual
cloning projects. A variety of approaches are being used to select brain-specific
clones and to eliminate highly represented sequences.
The Section of Receptor Biochemistry and Molecular Biology also runs the NINDS
DNA facility. This facility synthesizes oligonucleotides for institute laboratories and
branches and performs DNA sequencing. During FY 1990 the DNA synthesis facility
under the direction of Mike FitzGerald will have synthesized over 900
oligonucleotides for eight institute laboratories and branches. The DNA facility has
also sequenced over 50,000 bases for other laboratories.
13 LMCN/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02784-02LMCN
PERIOD COVERED
October 1, 1990 through September 31, 1990
TITLE OF PROJECT (aOchtrtcUrtorlta. Tnlt mull tn on sot lint Minan Hit boiOtrt )
investigation of the Structure and Function of Gonadotropins and their Receptors
PRINCIPAL INVESTIGATOR (tol olhw ontmtueotl pmHonntl btlew Uw fruiaptl kiwiiiuttoi ) (Htm; till*. Itbotttary, tnd instituf tttilmion)
PI: R. V. Rebois, Ph.D. Head, Unit on Receptor Structure and Function LMCN, NINDS
OTHERS: S. Okuya, M.D.. Ph.D. Visiting Associate LMCN, NINDS
V. J. B. Reddy, Ph.D. Visiting Fellow LMCN, NINDS
COOPERATING UNITS (itt*,)
LAB/BRANCH
Laboratory of Molecular and Cellular Neurobiology, BNP
SECTION
Membrane Biochemistry Section
INSTITUTE AND LOCATION
NINDS, NIH, Park Building, Room 408, Bethesda, Maryland 20892
TOTAL MAN-VEARS:
2.5
PROFESSIONAL:
25
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects
] (a1) Minors
J (a2) Interviews
GO (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use sundsrd unreduced type. Do not exceed the space provided.)
The carbohydrate moieties of glycopeptide hormone human chorionic gonadotropin (hCG) have sialic
acid (NeuAc) residues which are required for full biological activity. hCG stimulates adenylate cyclase in
murine Leydig tumor MLTC-1 cells, and we used this system as a model to study the role of NeuAc in the
biological activity of hCG. Removing NeuAc produced asialo-hCG which had only 50% of the biological
activity of hCG. The NeuAct was replaced with a modified form of NeuAc or with the carbohydrate
moiety of the ganglioside Gmi which also contains NeuAc. Both derivatives had the same activity as the
asialo-hCG indicating the importance of both position and structure of NeuAc for biological activity. 2.
The drug forskolin activates adenylate cyclase, but we found that different cells varied in their response
to the drug as measured by cyclic AMP accumulation. Cyclic AMP accumulation in cells with a strong
response to forskolin was blocked by activating the inhibitory G protein (G,), with fluoroaluminate.
Forskolin potentiated cyclic AMP accumulation stimulated by hormones that activate the stimulatory G
protein (Gj), even in cells the responded poorly to forskolin alone. Activating G, with fluoroaluminate
prevented the hormone response, as well as its potentiation by forskolin. Forskolin stimulated adenylate
cyclase activity in membranes, even if the membranes were prepared from cells that responded poorly to
the drug. Activating G, with GTP analogues inhibited forskolin-stimulated adenylate cyclase activity in
membranes. The results indicate that interactions between Gs G„ and the catalytic subunit of adenylate
cyclase affect the ability of forskolin to stimulate cyclic AMP accumulation. The observation that some
cells responded well to forskolin and other responded poorly suggests that these interactions are not the
same in all cell types. Furthermore, differences in the response of cells and their membranes to forskolin
suggest that changes in these interactions occur during the preparation of membranes.
14a-LMCN/DIR
'HStMSIfto I'M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02366-12LMCN
PERIOD COVERED
October 1 , 1 989 through September 31,1 990
TITLE OF PROJECT fwchmctwtartau hti» i»«»i tit an on» iu>* Mm** iht txxant.)
Regulation of Hormone-Responsive Adenylate Cyclase
PRINCIPAL \NV ISHG AT OR tun olhw *nt*ui»*tl ftunnml *»lem th» tiituittl IniHHigttoi.) (Mam*, into. Itkoftat/. tn4 inmtufttilHtion)
PI:
P. H. Fishman, Ph.D.
Chief, Membrane Biochemistry Section
LMCN, NINDS
OTHERS:
A. Sidhu.Ph.D.
Biochemist (IPA)
LMCN, NINDS
E.A.Gordon, Ph.D.
IRTA Fellow
LMCN, NINDS
X.-M. Zhou, M.D.. Ph.D.
Visiting Fellow
LMCN, NINDS
M. Lee
Special Volunteer
LMCN, NINDS
T.L. Miller
Biologist
LMCN, NINDS
COOPERATING UNITS fi'myj
LAB/BRANCH
Laboratory of Molecular and Cellular Neurobiology
SECTION
Membrane Biochemistry Section
INSTITUTE AND LOCATION
NINDS, NIH, Park Building, Room 411, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
4.3
PROFESSIONAL:
3.3
OTHER:
1.0
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects
] (a1) Minors
J (a2) Interviews
f"x~1 (b) Human tissues ] (c) Neither
SUMMARY OF WORK (Ut* lUniUrd unnduttf typ«. Do not M(Nd !/)• ijmc* provldtd.)
Neuropeptide Y (NPY) binds to Y ^ -subtype receptors on MC-IXC cells and to Y2 receptors on SMS-MSN,
two human neuroblastoma cell lines. NPY inhibits adenylate cyclase in MC-IXC but not SMS-MSN cells.
NPY binding in both cell lines is inhibited by pertussis toxin, which ADP-ribosylates 41 kDalton G proteins
in both. Western blotting with specific antibodies to Ga subunits showed that MC-IXC has Gia, the
inhibitory G protein of adenylate cyclase whereas SMS-MSN has both Gio and Goa, which has been
implicated in activation of ion channels. Thus, Yt receptors appear to mediate inhibition of adenylate
cyclase and Y:, activation of ion channels. In contrast to other 0-adrenergic responsive cell lines which
have B?-adrenerqic receptors (02AR), SK-N-MC have BjAR and undergo an unusual desensitization when
exposed to agonists. There is no loss of SiAR or maximum agonist-stimulated adenylate cyclase activity
but a shift in the dose response which can be mimicked by incubating membranes with ATP and the
catalytic subunit of cyclic AMP-dependent protein kinase (PKA). Desensitization is blocked in permeable
cells by a specific inhibitor of PKA but not an inhibitor of BAR kinase. A large-scale purification of the
dopamine Pi receptor from rat striatum has been carried out and the purified receptor is being used to
obtain peptide sequences, antibodies, and eventually the receptor gene. The latter will be useful for
identifying defects in the receptor that occur in disease states. In kidney, Oy receptors coupled to
adenylate cyclase have been implicated in regulation of sodium transport Dopamine fails to stimulate
adenylate cyclase in proximal convoluted ('joules from spontaneous hypertensive rats although the
enzyme responds normally to other effectors. As the binding activity and apparent molecular weight of
the kidney receptors from the hypertensive rats are normal, it appears that the D i receptors are defective
in their ability to couple to the stimulatory G protein of adenylate cyclase.
15a-LMCN/DIR
f Hi tiwu man I Ml
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS01309-25LMCN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (aochtrtatn 01 iiu- Till* null lit on on* lm* Mnui th* bo/0*i>.)
Biosynthesis and Function of Glycosphingolipids and Other Glycoconjugates
PRINCIPAL INVESTIGATOR dm oth*r pronuUonsI p*nenn*l a*low th* ftlnxla*! kiy*UhJ»toi.) {Ntm*. ml*. Itoo/ttory. tndlnsMul* ttftlntlon)
PI: P.H. Fishman, Ph.D.
OTHERS: T. Pacuszka, Ph.D.
D.R. Critchley, Ph.D.
R.M. Bradley
Chief, Membrane Biochemistry Section LMCN, NINDS
Visiting Scientist LMCN, NINDS
Visiting Scientist LMCN, NINDS
Chemist LMCN, NINDS
COOPERATING UNITS (it*n,)
LAB/BRANCH
Laboratory of Molecular and Cellular Neurobiology
SECTION
Membrane Biochemistry Section
INSTITUTE AND LOCATION
NINDS, NIH, Park Building, Room 41 1, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.9
PROFESSIONAL:
1.3
OTHER:
0.6
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects
(al) Minors
(a2) Interviews
I I (b) Human tissues [T] (c) Neither
SUMMARY 05 WORK (Us* sundsrd unreduced typ*. Do not txifd th* ipse* provided.)
Ganqlioside Gmi is the cell surface receptor for cholera toxin. The oligosaccharide chain of Gmi is
recognized by the B or binding subunits of the toxin whereas the A subunit of the toxin activates
adenylate cyclase by ADP-ribosylation of the stimulatory G protein of the cyclase system. Less is known
about the role(s) that the lipid moiety of Gmi plays in toxin action. We synthesized derivatives of Gmi in
which the oligosaccharide was coupled to different lipids such as phospholipids, cholesterol and aliphatic
amines. We tested the derivatives on rat glioma C6 cells which are deficient in Gmi . bind only traces of
the toxin and are poorly responsive to it. C6 cells incorporated the neoglycolipids into the plasma
membranes as measured by the increase in cholera toxin binding. However, neoglycolipids consisting of
GMi-oligosacchande attached to short chain aliphatic amines were not taken up by the cells; at least a
dodecylamine was required. The neoglycolipids also conferred enhanced cholera toxin responsiveness
ci the cells as measured by increased stimulation of adenylate cyclase. There were differences among
the neoglycolipids in terms of the amounts of cyclic AMP formed per amount of toxin bound to the
treated cells. Those derived from long-chain aliphatic amines and cholesterol were more efficient than
Gm i as toxi n receptors whereas those derived from phosphol i pids were less efficient that Gmi- When the
distance between the oligosaccharide and the phospholipid was increased by adding spacers, the
efficiency as receptors decreased. Thus, the lipid moiety of Gmi is important for the action of cholera
toxin and these neoglycolipids may be useful as probes of Gmi bioactivity in other systems such as the
neuritoqenic and neuronotrophic effects of the ganglioside.
16a-LMCN/DIR
PHi MM0 <«•« 1<M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
HKOJtU NUMbtK
Z01 NS02151-16
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT fMdtWMMnarJtu T"l» mvU tit on on* bna b*tm—n iht boio*ri.)
Memory Storage in Neural Networks
PRINCIPAL IN VE STlGATOR (tin olli*i otantmontl a*iMnn*l »*tom I'm hinuotl Inmitiotioi) <*u<m. Ulh. Utbtunoif. »no" MKilul* tffilislion)
PI: D L Alkon Medical Officer LMCN, NINDS
Others: LMCN, NINDS: C Collin, Vis. Fellow; P Huddie, Vis. Fellow; C Bramham, Vis. Fellow; R
Etcheberrigaray, Vis. Fellow; JV Sanchez-Andres, Vis. Fellow; E Yavin, Vis. Scientist; D Lester,
Vis. Assoc; E Maduh, Vis. Fellow; L Matzel, IRTA Fellow; B Schreurs, Staff Fellow; I
Lederhendler, Staff Fellow; T Nelson, Staff Fellow; J Olds, Staff Fellow; D McPhie, Biologist; J
Blaszcyk, Sp. Volunteer; AM Craig, Spec. Volunteer; S Moshiach; Spec. Volunteer; L Keith,
Spec. Volunteer; S Hyman, Guest Researcher; E Yamoah, Guest Researcher.
COOPERATING NITS<ir«w
Marine Biological Laboratory, Woods Hole, MA 02S43 (A. Kuzirian); California Institute of Technology (C.
Chen); Medical Research Council, Canada (AM. Craig)
LAB/BRANCH
Laboratory of Molecular and Cellular Neurobiology, BNP, DIR, NINDS
SECTION
Neural Systems Section
INSTITUTE AND LOCATION
Park Building, Room 431 and Building 9, Room 1W125, NINDS, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
10.0
PROFESSIONAL:
9.0
OTHER:
1.0
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects
] (a1) Minors
J (a2) Interviews
I I (b) Human tissues [Tj (c) Neither
SUMMARY OF WORK {Use standard Unreduced type. Do not tk-ctd iht tpact provided.)
The principal objective of the program is to define molecular and biophysical mechanisms of learning
and memory. Emphasis is placed on learning and memory which can be related to human cognition.
Ultimate goals of such research are to arrive at clinically meaningful interventions and to design and
construct artificial intelligence which has advanced learning and memory capabilities. With human
cognitive function as the principle frame of reference, the research focuses on associative processes (such
as Pavlovian conditioning) rather than non-associative behavioral modifications (such as sensory
adaptation, habituation, arousal, and sensitization). The biological basis of learning and memory is of
interest at several levels of complexity: behavioral phenomena, neuronal systems, neuronal
membranes and molecular transformations. To literally reconstruct the physiology involved (and to
model it in artificial contexts) it is necessary to use both "simple system" preparations such as the
nudi branch mollusc Hermissenda crasiicomis as well as "complex system" preparations such as rabbits
and rats. The molluscan work thus far has yielded the first unequivocal biological record of an
associative memory. This record consists of persistent transformations of specific ionic channels. Because
these records have been found within the membranes of identified single neurons it has proven possible
to define biochemical pathways which regulate such long-term membrane modifications as well as to
analyze how this biological memory record is expressed by the integrative functions of an entire
neuronal system. The work on the vertebrate brain offers two essential opportunities. First, the
generality of mechanisms determined for much less evolved species can be tested. Remarkably, the same
ionic channel transformations have been shown in our program to record associative memory in the
rabbit as were found in Htrmiutnd*. Rabbit and now rat neural systems have also provided sufficient
quantities of tissue so that conditioning-specific alterations of critical enzymatic (e.g., protein kinase C)
pathways which control membrane excitability have recently been demonstrated.
17a-LMCN/DIR
WMMIM> 1 Ml
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02786-02LMCN
PERIOD COVERED
Octoberl, 1989 through September 30, 1990
TITLE OF PROJ ECT (ao chtracfrt or Mm. fill* must hi on on* lint Mi»no iht bortory)
Antibodies to Glycoconjugates in Neurological Diseases
PRINCIPAL IN VESTI6ATOR(UitofnwpranuiioiutpwsoflfM/s*k>i*l/w*rlMvfltanni9ilwJ (Mtrn*. Ml*. Memory, tndmttituftfiltttion)
PI: Richard H.Quarles, Ph.D. Section Chief LMCN.NINDS
Others: Genevieve Daune, Ph.D. Visiting Fellow LMCN, NINDS
Antonio Noronha, Ph.D. Staff Fellow LMCN.NINDS
Carl Lauter Chemist LMCN, NINDS
Jeffrey Hammer Biologist LMCN.NINDS
COOPERATING UNITS Oltny)
Medical Neurology Branch, NINDS; Dept. Neurology, Johns Hopkins Univ., Baltimore, MD;
The Kennedy Institute, Baltimore, Maryland
LAB/BRANCH
Laboratory of Molecular and Cellular Neurobiology, BNP
SECTION
Section on Myelin and Brain Development
INSTITUTE AND LOCATION
Park Building, Rm. 425, NINDS, NIH Bethesda, MD 20205
TOTAL MAN-YEARS: , c
1 .0
PROFESSIONAL: 1 ,
OTHER:
0.3
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects [T] (b) Human tissues ] (c) Neither
] (a1) Minors
_J (a2) Interviews
SUMMARY OF WORK (Use st»nd»rd unreduced type. Do not exceed the sptce provided.)
This area of investigation began with the demonstration of monoclonal anti-MAG antibodies in patients
with mixed motor-sensory polyneuropathies occuring in association with IqM paraproteinemia. It was
subsequently demonstrated that these anti-MAG antibodies were all directed toward carbohydrate
epitopes in MAG and cross-reacted with 19 to 28 kD glycoproteins of PNS myelin and a sphinqoqlycolipid.
sulfate-3-qlucuronvl paraqloboside (SGPG). Although all these human anti-MAG antibodies have similar
specificities since they react with the same family of glycoconjugates in nerve, experiments with
chemically modified derivatives of SGPG have revealed differences in fine specificities. Some of the
human antibodies and the related HNK-1 antibody absolutely require the sulfate for reactivity, others
require the free carboxyl group of glucuronic acid, whereas still others will react if either one or the
other of the two negatively charged groups is present. The fine specificity of the anti-MAG antibodies
may affect the clinical presentation in these patients. Our research in recent years has emphasized
antibodies to GM1 qanqlioside in patients with motor nerve disorders. Four patients with multifocal
motor neuropathy were found to have high titers of polyclonal antibodies reacting with GM1
qanqlioside. The fine specificities of these antibodies also differed in the individual patients as revealed
by identification of different cross reacting gangliosides. Since GM 1 was the only common antigen in
these patients, GM1 may be the pathogenically significant target antigen in motor nerves. Although
these findings reveal a strong correlation between the occurrence of antibodies to GM 1 and one type of
motor nerve disorder, we did not find high levels of antibodies to GM1 in motor neuron disease or
amyotrophic lateral sclerosis. Preliminary results have revealed moderately elevated antibodies to
gangliosides in some cases of adrenoleukodvstrophy which may play a role in variable secondary
autoimmune aspects of this genetic disease caused by a defect in the peroxisomal degradation of
saturated, very long chain fatty acids. Finally, it should be emphasized that our work and that of others
indicates that serum antibodies in patients with neurological diseases should be analyzed both
qualitatively by TLC overlay and quantitatively by ELISA. since the pathogenic significance of antibodies
to glycolipids appears to depend both on their specificity and titer.
18a-LMCN/DIR
f>HS 6040 (»•«. I/M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECTNUMBER
Z01NS01808-21 LMCN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT faoctaracun or itti Tula muif ni on <ww lint tuMtn i/m botdtnj
Glycoproteins of Myelin in Development and Disease
PRINCIPAL INVESTIGATOR (List o<nu orotmuiontl ptnonnil btlow tho trinaptt In— ttiottor ) (Htm*, utk. Itbotnory. tnd mmtutt ttfilittion)
PI: Richard H.Quarles, Ph.D. Section Chief LMCN, NINDS
Others: Antonio Noronha, Sr. Staff Fellow, LMCN, NINDS; Kenichi Toda, Vis. Fellow, LMCN, NINDS
Johanna Moller, Sr. Staff Fellow, LMCN, NINDS; Carl Lauter, Chemist, LMCN, NINDS
Sung Hye Yim, Special Expert, LMCN, NINDS; Jeffrey Hammer, Biologist, LMCN, NINDS
Judy Small, Sr. Staff Fellow, LMCN, NINDS; Ritu Khanna, Biologist, LMCN, NINDS
Shuichiro Goda, Vis. Fellow, LMCN, NINDS
Zbigniew Bartoszewicz, Vis. Fellow, LMCN, NINDS
COOPERATING UNITS lifm/i
Dept. Neurology, Johns Hopkins Univ., Balto., MD; Dept. Ped., Washington Univ., St. Louis, MO; Lab.
Exp. Neuropath., NINDS; Developmental and Metabolic Neurology Br., NINDS; Lab. Clinical Sci., NIMH
LAB/BRANCH
Laboratory of Molecular and Cellular Neurobiology
SECTION
Section on Myelin and Brain Development
INSTITUTE AND LOCATION
Park Building, Rm.425, NINDS, NIH I, Bethesda, MD. 20892
TOTAL MAN-YEARS: ca
PROFESSIONAL: e 2
OTHER:
1.7
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects [T] (h) Human tissues □ (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use sundard unreduced type. Do not exceed the iptce provided.)
The myelin-associated glycoprotein (MAG) is localized in the periaxonal membranes of PNS and CNS
myelin sheaths where it appears to be involved in glia-axon interactions. MAG is a member of the
immunoglobulin gene superfamily along with other neural adhesion proteins, and alternative splicing of
its mRNA generates two developmental! v regulated isoforms with differing C-terminal tails. The longer
isoform (L-MAG) is the principal form synthesized during active myelination early in CNS development,
whereas the shorter isoform (S-MAG) is the principal form synthesized in the PNS at all ages. MAG is
phosphorylated both in the CNS and PNS, but the sites of phosphorylation by oligodendrocytes and
Schwann cells differ. The extracellular domains of the two forms of MAG are identical with 5
immunoglobulin-like domains and 8 potential sites for N-linked glycosylation. The carbohydrate consists
of a mixture of neutral and negatively charged, complex oligosaccharides whose structures are now
under detailed investigation. MAG appears to be abnormally glycosylated in dvsmyelinating guaking
mice. The expression of MAG in cultured oligodendrocytes and Schwann cells is being studied with the
ultimate objectives of identifying factors that control its synthesis and probing its function in cell-cell
interactions. Cultured oligodendrocytes from adult bovine brain constitutively express a substantial
amount of MAG on their surface. Although cultured Schwann cells do not normally express MAG in the
absence of neurons, an immortalized Schwann cell line generated in our laboratory expresses a
remarkably high level of MAG but little or none of the major myelin proteins. Furthermore, the post-
translational glycosylation. sulfation and phosphorylation of MAG occuring in these immortalized cells is
similar to that /nv/vo, and this continuous cell line expressing a lot of MAG on its surface should be useful
for investigating the cell biology and function of this glycoprotein. MAG and other myelin proteins were
studied during the demyelination and remyelination occurring following cuprizone treatment of mice.
Finally, a novel autoantibody was identified in an apparently normal rabbit that specifically reacts with
the shorter isoform of 2'.3'-cvclic nucleotide 3'-phosphodiesterase (CNP).
19a-LMCN/DIR
'HiUWJIHi. 114)
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02805-01LMCN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 chmrtavt or 1***, ntl* mutt fit on on* lint botw—n tnt batdmrt.)
Molecular and immunological aspects of myelin abnormalities in neuro-AIDS
PRINCIPAL INVESTIGATOR tUttothoiont*umt»lo*rtonnoloolowth*hlndp*llnnsttg*tor,) (ktmt. tit*. l»borttory, tnd Inn/tut* ttillittton)
PI: Richard H. Quarles. Ph.D.
Others: Johanna Moller, M.D.
Antonio Noronha, Ph.D.
Mary McLenigan
Jeffrey Hammer
Ritu Khanna
Section Chief
Sr. Staff Fellow
Sr. Staff Fellow
Chemist
Biologist
Biologist
LMCN, NINDS
LMCN, NINDS
LMCN, NINDS
LMCN, NINDS
LMCN, NINDS
LMCN, NINDS
COOPERATING UNITS otm,)
Dept. Neurology, Johns Hopkins University, Baltimore, Maryland; Medical Neurology Branch, NINDS
LAB/BRANCH
Laboratory of Molecular and Cellular Neurobiology, BNP
SECTION
Section on Myelin and Brain Development
INSTITUTE AND LOCATION
Park Building, Rm. 425, NINDS, NIH Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.7
PROFESSIONAL:
0.7
OTHER:
1.0
CHECK APPROPRIATE BOX(ES)
[ I (a) Human subjects
] (a1) Minors
J (a2) Interviews
|~x | (b) Human tissues ^] (c) Neither
SUMMARY OF WORK (Use standard unreduced typo. Do not exceed the space provided.)
This is a new project undertaken to elucidate biochemical and immunological aspects of myelin disorders
associated with neuro-AIDS including myelin pallor, vacuolar myelopathy and multifocal demyelination
in the CNS as well asdemyelinatinq peripheral neuropathy. Specific areas to be investigated fall into
three catagories: 1) Postmortem CNS tissue from AIDS patients will be analyzed for quantitative and
qualitative alterations of myelin proteins for comparison to results previously obtained in other
demyelinating diseases. Adjacent sections of tissue will be examined histologically and
immunocytochemically to evaluate the pathological changes for correlation with the biochemical data.
Preliminary biochemical results have already demonstrated a loss of myelin proteins in some areas of
AIDS CNS; 2) Since the myelin associated glycoprotein (MAG) and the CD4 receptor for HIV a re both in
the immunoglobulin superfamily and have a modest, but statistically significant, sequence homology,
possible interactions between MAG and GP120will tested. The capacity of GP120 to interfere with the
normal role of MAG in olia-axon interactions will be studied as will the possible role of MAG as a neural
cell receptor for HIV. The effects of GP120 on cultured oligodendrocytes will also be examined. 3) Since
high levels of antibodies to qangliosides and other qlvcoconjuqates were previously detected in patients
with some types of neuropathy (including a few that were HIV-positive), a larger number of sera from
AIDS patients with neuropathy will be examined systematically for antibodies to qlycoconiuoates.
20a-LMCN/DIR
'HSKMOIRk 1/W)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS02710-05LMCN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (w<Mnmnwl<ii Mrt/nurt hi on ont lint b»t~9*n ihtbordtn.)
Molecular Biology of Neurotransmitter Receptors
PRINCIPAL INVESTIGATOR (l«f olntr enHiuontl pwwoMl b*low tl» frinaptl m ^^tV)ilot ) (htmt. IfUft Ijfco/jeory. «ntfin«nut« i«.iui.on)
PI: J.C. Venter, Ph.D., Chief, RBMB, LMCN, NINDS. OTHERS (LMCN, NINDS): E. Kirkness, Ph.D., Fogarty
Fellow; T. Onai, M.D., Fogarty Fellow; S. Arakawa, M.D., Special Volunteer; J. Flemming, B.S., Special
Volunteer; J. Kusiak, Ph.D., Staff Scientist; M. Fitzgerald, B.A., Biologist; R. Bevan, Ph.D., Special
Volunteer; T. Saverese, Ph.D., Special Volunteer; J. -P. Giacobino, Ph.D., Special Volunteer; M. Adams,
Ph.D., IRTA; A. R. Kerlavage, Ph.D., Sr. Staff Fellow; W.R. McCombie, Ph.D., Sr. Staff Fellow; J. Gocayne,
M.S., Microbiologist; A. Martin, Ph.D., Sr. Staff Fellow
COOPERATING UNITS <>'»»>;
Section of Neurobiology, LPPS, NIAAA (C. M. Fraser, Ph.D. & M. Buck, Ph.D., IRTA Fellow);
Department of Biochemical Pharmacology, SUNY at Buffalo (L.M. Hall, Ph.D., Professor)
LAB/BRANCH
Laboratory of Molecular and Cellular Neurobiology, BNP, DIR, NINDS
SECTION
Section of Receptor Biochemistry and Molecular Biology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: , .
PROFESSIONAL: 5 g
OTHER:
0.2
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects ] (b) Human tissues [T] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project is to characterize the gene and protein structure and evolution of neurotransmitter
receptors belonging to two major multigene families. The first gene family comprises adrenergic.
muscarinic, opsin, serotonin and octopamine receptors and the second comprises nicotinic cholinergic.
GABA/benzodiazepine NMDA. and glycine receptors. The specific aims are to clone and seouence the
genes for receptors in these two multigene families; to obtain high density receptor expression and to
use the expressed proteins to determine the complete receptor structure in part by computer enhanced
molecular modeling; to determine the evolution of the neurotransmitter receptor gene families; and to
search for and characterize receptor gene polymorphisms. To date, we have cloned and sequenced a
significant number of receptor genes from both multigene families. These include several alpha' and
beta-adrenergic receptors from human and rat cDNA and genomic libraries; muscarinic cholinergic
receptor from human, rat and Drosophila genomic libraries. Octopamine receptors for Drosophila,
human alpha and beta subunits of the GABA/benzodiazepine receptor; and nicotinic receptors from
locusts and C. e/eqans. For example the human GABA beta 1 subunit has been cloned and sequenced
and localized to chromosome 4. This gene is over 65kb and is composed of nine exons. Theexonsand
splice sites are highly conserved in other GABA receptor subunits from humans and lower species.
Permanent cell lines expressing the unique neurotransmitter receptor proteins are providing key new
information concerning the mechanism of receptors activation by neurotransmitters.
21a-LMCN/DIR
► Hi WKOIHt. I'M I
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
201 NS 02754-03 LMCN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (BO cn*r*c1*r< or /•>>. nt/» must fit on on* lint btftmn <rt* bordtn )
Megabase DNA Sequencing
PRINCIPAL INVESTIGATOR (Um otbtr pnttiuonil prtonnti octet* lh» hmaptl lnmtig»tor.) (M*m*. mi*, ijooMiory. tnd msiitui* Mttihttion)
PI: J.C. Venter, Ph.D., Chief, RBMB, LMCN, NINDS . OTHERS (LMCN, NINDS): W.R. McCombie,
Ph.D., Sr. Staff Fellow; A. Martin, Ph.D., Sr. Staff Fellow; A.R. Kerlavage, Ph.D., Sr. Staff Fellow; M.
Dubnick, Ph.D., Staff Fellow; M. Adams, Ph.D., IRTA; J. Gocayne, M.S., Microbiologist; J. Kelley, M.S.,
Microbiologist; S. Trapp, B.S., Microbiologist; M. FitzGerald, B.A., Biologist; E. Kirkness, Ph.D., Fogarty
Fellow; J. Kusiak, Ph.D., Staff Scientist; J. Xin, Ph.D., Fogarty Fellow
COOPERATING UNITS (lfiV)
DCRT (M. Hunkapiller); University of Michigan (F. Collins, M.D.); DOE (A. Corrano, DOE); L. Goldfarb,
LCNSS;
LAB/BRANCH
Laboratory of Molecular and Cellular Neurobiology, BNP
SECTION
Section of Receptor Biochemistry and Molecular Biology
INSTITUTE AND LOCATION
NINDS, NIH, Park Building, Room 405, Bethesda, MP 20892
TOTAL MAN-YEARS: „-
PROFESSIONAL: e 1
OTHER:
2.9
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues HH (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project has as its goal the acquisition and analysis of the sequence of genomic DNA that is associated
with receptor multigene families and neurological disorders and to understand genomic structure at the
sequence level. To accomplish this, sequencing strategies and technologies are being developed that
will allow the sequencing of megabase regions of DNA. Our use of four Applied Biosystems automated
DNAseguencers allows the generation of up to 48 kilobases of raw sequence per day. A computer
network consisting of Macintosh, Sun 4, and Silicon Graphics computers in the laboratory connecting to
the NIH Convex and Cray computers is assisting the analysis of large genomic sequences. The use of Tag
polymerase and PCR cycle-sequencing reactions has greatly enhanced the quantity and the quality of the
sequence data generated. New automation has been implemented in the laboratory by the addition of
new robotics which completely automate DNA sequencing reactions. These techniques have been
applied to sequencing clones associated with several important neurological disease gene regions of
human chromosomes. The laboratory has completed over 2 million bases of DNA sequence this year,
including the following projects. Over 1 5kb from the disease gene for neurofibromatosis [ has been
sequenced, identifying several new exons. Having the intron-exon boundaries has allowed us to develop
PCR probes to sequence exons from NF1 patients in search of point mutations associated with disease
symptoms. A three cosmid contig from the Huntington's region of chromosome 4 has been sequenced.
This region of approximately 1 0Okb is one of the two largest segments of human chromsomal DNA
sequenced to date and is currently being analyzed. The second largest region is a 1 0Okb region of
chromosome 19 which has been sequenced. This region is where a number of DNA repair enzymes and
the gene for mytonic dystrophy have been mapped. Cosmid clones have been obtained from the human
Xq28 region which contains several loci of neurological significance including the alpha3 subunit of the
GABA receptor. These clones are currently being processed for sequencing. We have begun sequencing
clones obtained from patients with spongiform encephalopathies in collaboration with Lev Goldfarb.
This project will involve sequencing about 240 clones from 40 patients. This will require obtaining about
600,000 bases of raw sequence data.
22a-LMCN/DIR
► HS MM0(«*v. 1*4)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS02806-01LMCN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT f*0cn«r*ct*r<o/ Mis. Titltmult Monona lint MMM th* oorote'l)
Human Brain cDNA Project
PRINCIPAL INVESTIGATOR (Urt out*/ prvttiuon*! p»nonn»l potow fn* *rinao<( Inriiitttoi.) (Manx, inn, laboratory, •no' mnituf tttiiuuon)
PI: J.C. Venter, Ph.D., Chief, RBMB, LMCN, NINDS; OTHERS (LMCN, NINDS): M. Adams, Ph.D., IRTA;
B. Olde, Fogarty Fellow; W.R. McCombie, Ph.D., Sr. Staff Fellow; A.R. Kerlavage, Ph.D., Sr. Staff Fellow; J.
Gocayne, M.S., Microbiologist; J. Kelley, M.S., Microbiologist; S. Trapp, B.S., Microbiologist; M.
FitzGerald, B.A., Biologist
COOPERATING UNITS V*»n,)
A. Corrano, Lawrence Livermore (DOE); G. Evans, Salk Institute;
LAB/BRANCH
Laboratory of Molecular and Cellular Neurobiology, BNP, DIR, NINDS
SECTION
Receptor Biochemistry and Molecular Biology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: . 5
PROFESSIONAL:
1.1
OTHER:
0.4
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues |T] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The goal of this project is to isolate, sequence, and characterize the genes expressed in human brain. As
many as half of the more than 50,000 human genes are believed to be expressed in the brain. While
sequencing the human genome is expected to take over 1 5-20 years, sequencing a large number of cDNA
clones can readily provide coding sequence data on genes expressed in tissues. We are building a large
library of clones and sequences of human brain cDNA clones. One thousand human brain sequences
have been completed in the first 3-4 months of this project. Over half of the genes isolated are totally
new genes, not previously identified or cloned. An additional percentage are new human genes, having
been previously isolated from other species. Computer analysis of the protein sequences at the primary
and secondary structural level is assisting clone identification. DNAand predicted protein sequences will
be examined for the presence of conserved primary structure motifs and relationships to previously
sequenced genes. Further characterization of potentially interesting clones will include chromosome
localization, examination of tissue distribution of expression, and evolutionary conservation. The
availability of a broad-based library of cDNA sequences will also facilitate identification of coding
regions in genomic sequences as well as provide a starting point for individual cloning projects. A variety
of approaches are being used to select brain-specific clones and to eliminate highly represented
sequences.
23a-LMCN/DIR
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ANNUAL REPORT
October 1, 1989 through September 30, 1990
Biometry and Field Studies Branch
Division of Intramural Research
Clinical Neurosciences Program
National Institute of Neurological Disorders and Stroke
Table of Contents
RESEARCH SUMMARY 1 - 7
CONTRACT NARRATIVES:
Statistical and Collaborative Biomedical Research 8
Data Management Support
PROJECT REPORTS
Statistical Collaboration and Consultation 9
Z01 NS 02652-06 BFSB
Research in Statistics 10
Z01 NS 02490-10 BFSB
Predictive Value of the EEG in Febrile 11
Seizures
ZOl NS 02483-10 BFSB
Stroke Data Bank 12
ZOl NS 02598-08 BFSB
Traumatic Coma Data Bank 13
ZOl NS 02516-09 BFSB
Factors Predictive of Reading and Writing 14
Skills in the Congenitally Deaf
ZOl NS 02594-08 BFSB
Headache in Pregnant Women 15
ZOl NS 02505-10 BFSB
Statistical Coordinating Center for Collaborative 16
Clinical Studies
ZOl NS 02810-01 BFSB
Publications 17 - 19
i - BFSB/DIR
ANNUAL REPORT
October 1, 1989 through September 30, 1990
Biometry and Field Studies Branch
Division of Intramural Research
Clinical Neurosciences Program
National Institute of Neurological Disorders and Stroke
Jonas H. Ellenberg, Ph.D., Chief
The Biometry and Field Studies Branch (BFSB) supports a program in
biostatistics to advance the mission of NINDS in the areas of neurologic
disorders. The Branch participates in a wide range of intramural and extramural
collaborative projects, including large- and small-scale observational studies,
clinical trials and laboratory studies. These collaborative studies have been
conducted both through direct staff research and research and development
contracts. In addition to collaborative work, the Branch has an important
research component in statistical methodology.
The Branch is composed of an Office of the Chief and three Sections. Dr James . .
Dambrosia is Deputy Chief of the Branch. In that capacity he assists the Branch
Chief in policy decisions and assumes part of the administrative responsibilities
of running the Branch. The Mathematical Statistics Section, headed by Dr. James
Dambrosia is the main statistical consulting unit for other branches and
laboratories in the Division of Intramural Research, the Extramural Research
Divisions as well as neuroscience units outside of NINDS. This Section is also
responsible for research in statistical methodology. The Computer Applications
Section is currently occupied primarily with the activities of the Stroke and
Traumatic Coma Data Banks. Dr. Mary Foulkes is Acting Chief of this Section, and
Project Director for both of the Data Banks. With the completion of the enormous
data management and data collection activities and of the Data Banks , the Section
is focusing on analysis of the data from these projects as well as the development
of statistical collaborative initiatives for clinical studies in other areas such
as the clinical trial on treatment of AIDS Dementia Complex. A change in the name
of the Section to the Collaborative Studies Section has been proposed to reflect
the current focus of the Section's activities and a permanent Section Chief will
be appointed. The Data Processing Section provides computer programming, systems
analysis, and data management support to the Branch. The Branch Chief is the
Acting Chief of this Section.
Over the past two years, the Branch has aggressively recruited six recent Ph.D.
statisticians for positions through the Staff Fellow Program, and a Senior
Statistician. As a result of these efforts, we have been able to attract only two
candidates, Dr. Paul Albert of Johns Hopkins University and Dr. Lisa McShane of
Cornell University, to accept our Staff Fellow positions. With the departure of
Dr. Young Jack Lee to take a position as the Biometry Branch Chief in NICHD and
the departure of Dr. Sherrie Emoto on extended maternity leave, the Branch is
BFSB/DIR
currently recruiting for three positions: a Visiting Scientist and two Staff
Fellows. Our recruitment efforts to fill vacant positions for entry level
biostatisticians have been hampered by increased competition for well -trained
Ph.D. level statisticians and a major differential in Staff Fellow salaries as
compared to salaries offered by academic institutions and industry.
Several of our previous projects were long-term and labor intensive, requiring a
large amount of effort for routine activities such as data entry, data editing and
day-to-day monitoring of protocol compliance. These activities, although
essential, consume an excessive amount of staff time, with the drawback that
little time is left for statistical methodological research and expansion of our
collaborative efforts into new areas. BFSB collaboration on new large-scale
projects with a substantial data management component will depend, in large part,
on whether routine data management operations of such studies can be contracted
out under our supervision. To support ongoing projects and other future
collaborative studies, an R&D contract, "Statistical and Collaborative Biomedical
Research Data Management Support" (NOl-NS-9-2325) , was awarded to Information
Management Services, Inc. on December 31, 1988. This R&D contract, with an
initial funding period of three years, will provide expertise in statistical
programming, data entry, data monitoring, and systems analysis, in partial support
of collaborative projects and statistical methodological research.
I. STATISTICAL COLLABORATION AND CONSULTATION
Our current program of collaborative research has developed primarily in response
to requests for collaboration from intramural and extramural scientists at NINDS
and from researchers outside of NIH. Typically, BFSB assumes responsibility for
the statistical design, data management, statistical analysis, and interpretative
aspects of the projects, with the subject matter specialists providing the project
initiatives, subject matter expertise, and overall leadership. The Branch selects
projects on the basis of scientific merit, a high probability of successful
completion, and potential for scientific contributions consistent with the goals
of the DIR.
In collaboration with the Division of Convulsive, Developmental and Neuromuscular
Disorders, (DCDND) , BFSB was the statistical coordinating center for the clinical
trial of behavioral and cognitive side effects of phenobarbital used for the
prevention of febrile seizure recurrence. This trial required extensive
monitoring of patient accrual, extensive data quality control and several interim
data analyses for the trial's Safety and Data Monitoring Committee. Patient
accrual was completed in December 1985 with 217 children with febrile seizures
randomized to treatment and 150 seizure free controls recruited for the study.
The two and one-half year follow-up of the last patients was completed in July
1988. The primary results of this clinical trial have been published (NEJM 1990;
332: (6) 364-9) and the results of other analyses such as the effect of
phenobarbital on sleep of children with febrile seizures, and the prediction of
recurrence of febrile seizures, have been submitted for publication.
A second collaborative effort with the DCDND and the Neuroepidemiology Branch is a
population-based study of the prognostic value of the EEG for subsequent seizure
activity in children who experienced a febrile seizure. The cooperating medical
2 - BFSB/DIR
center is the Pediatric Clinic in Skopje, Yugoslavia. The recruitment of new
cases ended in December 1984, and follow-up (including repeat EEGs and neurologic
and physical examinations) continued through December of 1989. The study includes
400 children with a normal or non-specific abnormal EEG following a first febrile
seizure, as well as about 300 children with a specific abnormal EEG following a
seizure. The major outcomes of the study are recurrent febrile and afebrile
seizures and their relationship to the initial EEG, subsequent EEG changes, and
the influence of other medical and demographic factors. Univariate statistical
analysis of the data for the baseline visit examined a large number of potential
factors predictive of abnormal specific EEG classification. For example, number
of previous febrile seizures was associated with an increased rate of EEG
abnormality: 18% in children with no previous seizures and 63% in those with four
or more prior attacks. When these factors were considered jointly in a logistic
regression model the significant prognostic factors for abnormal specific EEG
were: age at initial EEG; number of prior febrile seizures; focal febrile
seizures; and motor activity abnormalities. Data editing and further analyses are
in progress .
The BFSB continues to collaborate with many Branches and Laboratories in the
Division of Intramural Research (DIR) . The feasibility of a randomized controlled
trial of treatment with anti-convulsant medication following a first convulsion in
subjects presenting for care to the Beijing Tiantan Hospital is being evaluated.
This collaborative study involving BFSB, the Neuroepidemiology Branch, and the
Tiantan Hospital in the Peoples Republic of China will address the issue of
whether early treatment after a first seizure can reduce the likelihood of
developing chronic epilepsy. A similar protocol is being considered for
implementation with a consortium of hospitals in Israel. BFSB would collaborate
as the statistical coordinating center for these projects.
BFSB is collaborating with the Medical Neurology Branch on two clinical trials of
felbamate: one trial is evaluating the effect of felbamate in controlling seizures
in adults with intractable partial epilepsy; the other is assessing efficacy in
children with Lennox -Gas taut syndrome.
Other collaborative studies in DIR include: development of optimal sampling
procedures for estimation of the size of a population of neuron cells (CN) ;
evaluation of the effect of the levorotatory forms of S-Hydroxytryptophan in the
amelioration of gait and limb ataxia (MN) ; mapping of the cerebral cortex using
EMG amplitude and latency responses to electro-magnetic stimuli to the scalp (MN) :
preliminary evaluation of environmental and occupational risk factors for multiple
system atrophy (CN) ; clinical trial of high dose prednisone in the treatment of
post-polio muscular atrophy (MN) ; examination of psychopathology of epileptics
(MN, NIMH); analysis of time to stroke using time dependent covariates in a
proportional hazards regression model (NE) ; a case-control study of the potential
association of serologically confirmed infection during pregnancy with morbidity
in the child; analysis and comparison of the amplitude of blink responses evoked
by mechanical or electrical stimuli for normal controls and spasmodic dysphonic
patients (MN) ; examination of catecholamine, neuropeptide and amino acid levels in
epilepsy patients at baseline and ictal periods (MN) ; survey of attitude and
potential behavior of patients with von Recklinghausen's neurofibromatosis with
respect to genetic screening (NE) ; clustering of occurrences of somatic and
affective symptoms in epilepsy patients(MN) ; nature of parkinsonism-dementia
3 - BFSB/DIR
complex on Guam (NE) ; prevalence of neurological diseases in the Navajo tribe
(MN) ; hyperarousal in chronic insomnia patients (MN) ; study of epilepsy
progression to generalized tonic-clonic seizures (MN) ; an examination of seizure
frequency in patients with intractable complex partial seizures (MN) ; the effect
of dexamethasone suppression tests in medicated patients with poorly controlled
partial seizures (MN) ; clinical evaluation of Ceredase^ glucocerebrosidase
infusion for treatment of Gaucher 's disease (DMN) ; and determination of the effect
of time from last seizure and seizure type on the dynamics of inter- ictal
metabolic change (MN) .
BFSB will act as the statistical coordinating center for a collaborative study
with MNB and the National Naval Medical Center on a clinical trial involving a
dose comparison of 3'-Azido-2', 3' -Dideoxy thymidine (zidovudine) in the treatment
of Human Immunodeficiency Virus Type 1 infection of the nervous system. This
double-blind clinical trial is designed to compare the efficacy of two different
daily doses of zidovudine in the treatment of mild/moderate/severe AIDS Dementia
Complex (ADC) and will involve the treatment and one year follow-up of
approximately 124 ADC patients. This study is now in the planning and approval
stage.
Collaborative research with biomedical research units not in NINDS includes: the
comparison of expert systems, AI , and statistical classification for
differentiation of cerebral infarction and intracerebral hemorrhage; development
of laboratory quality control procedures for the measurement of selenium;
evaluation of the effects of weather and ambient light on mood in patients with
seasonal affective disorders; area surveys for epidemiologic studies of neurologic
disorders in Latin America, India and Italy; study on the incidence of primary
intracranial neoplasms in Israel; and publication of a monograph on the
epidemiology of neurologic disorders.
A major commitment has been made for collaboration with the Parkinson's
Epidemiology Research Committee (PERC) , a group comprised of individuals from the
fields of movement disorders (neurology), biometry, epidemiology, occupational
health, chemistry, toxicology and neuropathology. PERC is charged with the
development of research protocols for identifying industrial, agricultural and
naturally occurring environmental chemicals and compounds that might play a role
in Parkinson's disease (PD) , and with the assessment of past and current
epidemiologic efforts. PERC has evolved beyond its original "think tank" mission
to become a working group, and the members took full advantage of the
multidisciplinary nature of the committee. PERC has taken on the feasibility
evaluation or initiation of several specific projects, such as (1) Studying cases
of Parkinson's disease that occur in both husband and wife (conjugal Parkinson's
disease); (2) Establishing a central registry to study Parkinson's disease
clusters; (3) Using the resources of the Mayo Clinic for studies of etiology and
progression of Parkinson's disease; (4) Identifying chemicals in natural products
that may cause Parkinson's disease; (5) Determining if occupational exposure to
MPTP-like chemicals increases the risk of Parkinson's disease; (6) Examining
available data bases to determine their utility in studying either the etiology of
Parkinson's disease or the progression of Parkinson's disease; and (7) Summarizing
what is known about the etiology of Parkinson's disease in a comprehensive,
critical overview of the literature.
BFSB/DIR
BFSB has taken the primary role for projects (6) and (7). To date, the vital
registration system, the provider based surveys and the population based surveys
of the National Center for Health Statistics (NCHS) have been examined in project
(6) with regard to ascertainment bias of PD cases, correctness of diagnosis,
completeness of records, recall bias, etc. From among the myriad of NCHS data
bases, the National Death Record System and the National Health and Nutrition
Examination Survey have been identified for possible studies of the association of
PD with occupation and diet respectively. Other ongoing assessments of available
data bases include the Twins Registry of the Medical Follow-up Agency (MFA) of the
Institute of Medicine (16,000 white male twin pairs, of service age during World
War II) and the MFA roster of Parkinson's disease patients.
We have now compiled approximately 700 papers related primarily to the etiology of
PD and the critical review and annotation for project (7) is in progress. The
published studies have not, in general, been definitive due to such problems as:
difficulty of diagnosis; modest prevalence; and retrospective and environmental
histories requiring long-term memory recapture in PD patients or their surrogates.
We are proceeding with the evaluation and annotation of the previous research,
based on stated hypotheses, appropriateness of study design, and inference. An
annotated bibliography along with the assessment of the knowledge base for
etiology of PD will be published.
II. CLINICAL DATA BANKS
BFSB continues its responsibility for the management and operation of the Stroke
and Traumatic Coma Data Banks. Each data bank was a collaborative effort between
BFSB, which was the statistical coordinating center, and four hospital centers.
The data banks were involved in the collection of prospective, observational,
clinical and laboratory data at the multiple clinical centers using a common set
of data forms. These data banks have provided a resource for addressing research
questions on the characteristics, clinical course, and outcome of hospitalized
stroke and traumatic head injury patients.
For the duration of both data bank projects, resources will be allocated to ensure
the continued maintenance, updating and easy access by the principal investigators
and BFSB to complete and to provide accurate databases. BFSB will continue in its
primary scientific leadership role in the collaborative analysis efforts of these
projects, providing both statistical collaboration and oversight of the
preparation of the scientific reports.
Stroke Data Bank
Data collection for the main phase of the Stroke Data Bank began in FY 1983.
By the end of new patient accrual in June 1986, 1,805 patients had been entered.
All acute care data were entered by the centers, edited and corrected by BFSB and
the centers, and a final acute care data file was created in September 1986.
Primary analysis of acute care data began at that time. Collection of follow-up
data continued until March 1987, and the final edited data base was completed in
May 1987. A priority for analysis and publication has been established to focus
on the areas of primary interest. A policy for publication and a Publication
Committee were established to review and critique all potential publications.
5 - BFSB/DIR
Examples of research studies being addressed include: the investigation of racial
and sexual differences in stroke type, site and vascular territory; the
examination of stroke severity to determine whether motor weakness and/or sensory
loss can be predicted by the location or size of the CT scan abnormality in
infarcts; evaluation of trends in institutionalization of post-stroke survivors
has shown that race, sex and marital status are predictive of institutional-
ization; and a homunculus profile analysis is in progress which will demonstrate
the association, or lack thereof, between lesion location and corresponding motor
deficits, using digital mapping of CT scan data on lesion location.
Traumatic Coma Data Bank
Data collection for the main phase Traumatic Coma Data Bank (TCDB) began in
FY 1984. By the end of September 1987, 1,030 severely head- injured patients were
enrolled in this project. Patient follow-up ended in February 1988, and data
editing was completed in July 1988.
The first major analyses began after the completion of data collection and
editing. Reports submitted for publication include: outcome following severe head
injury; the influence of age on outcome; verbal learning deficits following severe
head injury; initial CT scan findings in patients with severe head injury; the
impact of intracranial pressure instability and hypotension on outcome; and
several methodologic reports on TCDB diagnostic classifications, intracranial
pressure monitoring methods, and longitudinal neurobehavioral assessments
following severe head injury. Additional publications are also in progress, and
these include the relationship between intracranial pressure and Glasgow Coma
Outcome Score; post-injury recovery of memory and attention; time related
prognostic factors for outcome in severe head injury; recovery from a vegetative
state; obstreperousness and depression; and the role of secondary injury in
determining outcome.
III. METHODOLOGICAL RESEARCH IN STATISTICS
BFSB statisticians continue to develop new statistical methodology and derive
innovative modifications of statistical techniques to meet the needs of the
Institute for the design of experiments and field studies, analysis of data, and
statistical modeling of biological processes and phenomena. Most of the
statistical problems addressed arise from collaborative studies with the Division
of Intramural Research and neuroscience units outside of NINDS . In general, there
are two objectives associated with these various statistical activities of BFSB.
The primary objective is the development and improvement of statistical
methodology to meet the needs of the Institute. The secondary objective is to
make contributions to the development of statistical methodology which may be more
generally useful in neurologic and other medical research.
A partial listing of areas in which BFSB staff is developing new statistical
applications to neurologic problems includes: statistical analysis of shapes with
spatial dependencies; growth functions for responses with contributions from two
compartments; sampling strategies for rare neurologic disorders; determination of
the order of categories in attribute data; methods of inference on frequency of
events in follow-up data; analysis of longitudinal data with missing observations;
6 - BFSB/DIR
statistical designs for two-state episodic diseases using follow-up data; and
statistical design and analysis of randomized clinical trials in neurology.
Theoretical statistical work has included: an empirical Bayes approach for
examining multiple time series; the effect of informative and/or prognostic
censoring on survival analysis; comparison of common parametric and nonparametric
methods for survival analysis in the presence of mismodeling; proportional hazard
model estimators for transitions in discrete state semi-Markov processes;
derivation of consistent, efficient estimators for the bivariate Weibull
distribution; mixture models for time series count data; regression models for
interval censored time-to-event data; optimal checking methods for laboratory
quality control; and a new Weibull survival function for dependent censoring.
IV. BRANCH RECOGNITION AND OTHER PROFESSIONAL ACTIVITIES
Several of our staff have been active on important national and inter-
national review committees, and have participated in major meetings or received
other peer recognition this fiscal year. Dr. Ellenberg continues as an officer of
the International Biometric Society (immediate past President). He is also a
member of the Parkinsonism Epidemiology Research Committee; the NINDS , Monitoring
Committee for the Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism
Clinical Trial ( DAT ATOP ) ; the NINDS, DIR Clinical Review Board; and the NIH, Ad
Hoc Epidemiologist and Statistician Review Panel. Dr. Dambrosia is the Biometrics
Society (Eastern and Western North American Region) representative to the AAAS
Medical Science Section (N) and Chair of the Regional Advisory Board of the
Eastern North American Region of the Biometrics Society. Dr. Anderson serves on
the American Statistical Association's Committee on Committees and will be an
invited lecturer in Bressanone at the First International Workshop on Statistics
in Epidemiologic and Pharmacologic Research speaking on case finding strategies
for neurologic disorders. Dr. Foulkes is a member of the Monitoring Committee for
the VA Cooperative Study of Carbamazepine versus Valproic Acid for Treatment of
Partial Seizures, and organized and chaired a workshop at the annual meeting of
the Society for Clinical Trials entitled "When a Coordinating Center Assumes an
Ongoing Trial." Drs . Ellenberg, Anderson and Dambrosia were invited chapter
contributors for medical monographs.
In summary, BFSB is involved in a strong program of collaborative research. Our
collaboration extends throughout the Institute on projects with both Intramural
and Extramural scientists, and also involves collaboration with scientists outside
of NINDS. The scope of our research activity ranges from small, one-on-one
collaboration with intramural scientists, to the conduct of large-scale,
multicenter clinical studies. BFSB also makes an important and continuing
contribution to statistical methodology applicable to neurological research.
BFSB/DIR
CONTRACT NARRATIVE
Biometry and Field Studies Branch, CNP, DIR, NINDS
Fiscal Year 1990
Information Management Services. Inc.. Rockville. Maryland
CNOl-NS-9-2325')
Title: Statistical and Collaborative Biomedical Research
Data Management Support
Date Contract Initiated: December 30, 1988
Contractor's Project Director: William Lake, Jr.
Current Annual Level FY 90: $69,500
Objectives : To provide statistical programming and data management support
for both collaborative research projects and the development of statistical
methodology.
Major Findings: This contract provides statistical programming and data
management support for data entry, editing, quality control and report
generation for all BFSB collaborative projects. Software for new statistical
methods as well as all data management support is developed, tested and
implemented by the Contractor on the NIH computer system.
Significance to the NINDS Program and Biomedical Research: The statistical
staff of BFSB engages in collaborative biomedical research and conducts
statistical research evolving generally from problems encountered in these
collaborative studies. The Contract provides timely and efficient systems
development, data management (including data entry), data processing and
programming for both ongoing and future collaborative studies. Statistical
programming, an essential element of biostatistical research, is provided
under the direction of the Branch. New statistical methods developed by BFSB
are coded, evaluated and then made compatible with existing interactive
statistical software packages by the Contractor.
Proposed Course of the Project: The Contract began on December 30, 1988 and
continues through December 29, 1991.
Publications : None
8 - BFSB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02652-06 BFSB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must tit on one line between the borders )
Statistical Collaboration and Consultation
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
PI: James M. Dambrosia, Ph.D.
Co-PI : Jonas H. Ellenberg, Ph.D.
Others: Paul S. Albert, Ph.D.
Dallas Anderson, Ph.D.
Sherrie E. Emoto, Ph.D.
Mary A. Foulkes , Ph.D.
Lisa McShane, Ph.D.
Chief, Mathematical
Statistics Section
Chief
Mathematical Statistician
Mathematical Statistician
Mathematical Statistician
Mathematical Statistician
Mathematical Statistician
BFSB,
DIR,
NINDS
BFSB,
DIR,
NINDS
BFSB,
DIR,
NINDS
BFSB,
DIR,
NINDS
BFSB,
DIR,
NINDS
BFSB,
DIR,
NINDS
BFSB,
DIR,
NINDS
COOPERATING UNITS (if any)
Bombay Hospital, India (Dr. N. Bharucha; Dr. Z. Fu, Dr. Z. Zhang, D. S. Li (PRC)
Y. Leibowitz (Israel); Dr. J. de Pedro Cuesta (Sweden), National Institute of
Mental Health (Dr. Norman Rosenthal)
LAB/BRANCH
Biometry and Field Studies Branch
SECTION
Mathematical Statistics Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
6.2
PROFESSIONAL
4.2
OTHER
2.0
CHECK APPROPRIATE BOX(ES)
B (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
This project encompasses a wide scope of statistical collaboration and
consultation with Laboratories and Branches within the Division of Intramural
Research (DIR), and with other units outside of NIH. Particular consideration i^
given to statistical planning and design of experiments, statistical analysis of
data, and statistical inference. Our collaboration has involved seven
Laboratories/Branches, and the scope of the studies has ranged from the coordin-
ation and statistical management of clinical trials to consultation on the
appropriateness of the statistical analysis used for small laboratory experi-
ments. Examples of studies with DIR include: randomized clinical trials of
felbamate for the treatment of intractable complex partial seizures (MN) ;
development of time series models for the effect of weather and ambient light on
mood in patients with seasonal affective disorder (NIMH) ; measurement of the
effect of time from last seizure and seizure type on the dynamics of inter- ictal
metabolic change (MN) ; clinical trial of predisone for the treatment of post
polio muscle atrophy (MN) ; optimal sampling procedures to estimate the size of a
population of neuron cells (CN) ; identification of risk factors for febrile
seizures with a population based case-control study of six cities in China (NE) ;
statistical analysis of shape and spatial relationships of maps of the cerebral
cortex based on EMG responses to electomagnetic stimulation of the scalp (MN) ;
clinical course and outcome of patients with Gaucher's disease (DMN) ; clinical
evaluation of Ceredasera glucocerebrosidase in Gaucher's diseases (DMN);
prevalence of neurological diseases in the Navajo tribe (MN) ; use of quasi-
likelihood models to demonstrate that seizure frequencies are not random in time
(MN) ; study of epilepsy progression to general tonic-clonic seizures (MN) ; and
the use of hyperarousal scores for diagnosis of chronic insomnia (MN) .
9 - BFSB/DIR
PHS 6040 (Rev 1/84)
CPO SI 4-S16
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02490-10 BFSB
PERIOD COVERED
October 1, 1989 through Sppt-pmhpr ^0, 1990
TITLE OF PROJECT (80 characters or less Title must fit on one line between the borders.)
Research in Stafisfir';
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
PI: James M. Dambrosia, Ph.D.
Co -PI : Jonas H. Ellenberg, Ph.D.
Others: Paul S. Albert, Ph.D.
Dallas W. Anderson, Ph.D.
Sherrie E. Emoto, Ph.D.
Mary A. Foulkes , Ph.D.
Lisa M. McShane. , Ph D
Chief, Mathematical
Statistics Section
Chief
Mathematical Statistician
Mathematical Statistician
Mathematical Statistician
Mathematical Statistician
Mat-hpmafiral Stati sM rian
BFSB, DIR, NINDS
BFSB, DIR, NINDS
BFSB, DIR, NINDS
BFSB, DIR, NINDS
BFSB, DIR, NINDS
BFSB, DIR, NINDS
BFSB, nTR, NTNDS
COOPERATING UNITS (if any)
LAB/BRANCH
Biometry and Field Studies Branch
SECTION
Mathematical Statistics Section
INSTITUTE AND LOCATION
NINDS. NIH, Bethesda,
Maryland 7089?
TOTAL MAN-YEARS:
2.5
PROFESSIONAL:
2.0
OTHER.
0 5
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues H (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project addresses statistical problems generated from collaboration with
scientists in other program areas and general statistical problems of current
interest. This project is a continuing activity of the Section on Mathematical
Statistics. Papers have been submitted, are in review or were published in FY
1990 on the following statistical subjects: estimation of the joint survival
and censoring distribution in the presence of dependent censoring; statistical
planning, design and analysis of randomized clinical trials in neurology;
development of a Weibull model for survival data with dependent censoring; an
empirical Bayes procedure for examining the relationships between multiple time
series; establishing statistical quality control methods for biomedical
laboratories; design of panel studies under alternating Poisson process
assumptions. Other work in progress includes: selection criteria for use of
the Kaplan-Meier or parametric MLE for survival analysis; influence of missing
data in randomized clinical trials; methods to improve coverage in surveys;
analysis of time-to-event data with non-regular censoring; estimation of
time-to-event with interval data in the presence of left and right censoring;
- site selection for epidemiological surveys; adjustments for covariates in the
analysis of categorical data; two-state models for analyzing time series count
data; analysis of response surface data with both spatial and temporal
components; development of sampling strategies for count data in the presence
of multiple types of clustering; estimation of hazard functions with time-
dependent covariates in the presence of competing risks.
10 - BFSB/DIR
PHS 6040 (Rev. 1/84)
GPO 614-818
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02483-10 BFSB
PERIOD COVERED
October 1, 19ft9 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must lit on one line between the borders )
Pre.rii rHve Value of the EEG in Febrile Seizures
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
PI: Sherrie E. Emoto, Ph.D
Others: Jonas H. Ellenberg, Ph.D.
Deborah G. Hirtz, M.D.
Karin B. Nelson, M.D.
Jack Panossian
Mathematical Statistician BFSB, DIR, NINDS
Chief
Pediatric Neurologist
Medical Officer
Programmer
BFSB, DIR, NINDS
DNB, DCDND, NINDS
NEB, DIR, NINDS
BFSB, DIR, NINDS
COOPERATING UNITS (It any)
Cerebral Palsy and Other Motor Disorders Section, DNB, DCDND, NINDS;
Pediatric Clinic, University of Skopje, Yugoslavia (Nikola Sofijanov)
LAB/BRANCH
Biometry and Field Studies Branch
SECTION
Mathematical Statistics SecfcJ
1 on
INSTITUTE AND LOCATION
NINDS. NTH, Bethesria,
Maryland 7089?
ry
5FE
TOTAL MAN-YEARS
0.35
PROFESSIONAL
0 IS
OTHER
0.20
CHECK APPROPRIATE BOX(ES)
LS (a) Human subjects
S (a1) Minors
□ (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
This population based study will evaluate the significance of the EEG as a
predictor for recurrence of seizures in those children who have had a simple
febrile convulsion. Outcomes reported are febrile seizure recurrence and
afebrile seizure occurrence. The evolution of the EEG pattern will be described,
and patterns will be correlated with the clinical outcome. The clinical study is
being carried out in Skopje, Yugoslavia, at the Pediatric Clinic of the
University of Skopje.
The study began in FY 1982 and final follow-up visits will be completed in
FY 1991. Patient accrual was completed in December, 1984, by which time
approximately 400 patients with a febrile seizure, no prior complex or multiple
seizures and with a normal or nonspecific abnormal EEG, were registered into the
study and began the study protocol and follow-up. An additional 300 patients
with a specific abnormal EEG were entered for baseline information and follow-
up. Data editing and file creation are continuing. After an on-site review
visit, it was determined that additional efforts by the clinical center were
needed to collect data from those patients lacking a return visit and those who
did not have return visits after 24 months following study entry. This will
facilitate study of long-term recurrence of febrile seizures, change in EEG, and
the predictive qualities of EEG for febrile seizure occurrence. The extended
follow-up effort is now in progress. Statistical analysis of baseline EEG and
its association with characteristics of the child and family and the clinical
characteristics of the seizure has been completed and a manuscript has been
submitted for publication. The master file, including follow-up data at all
visits for all patients, should be completed in FY 1991, at which time further
analyses will be undertaken.
11 - BFSB/DIR
PHS 6040 (Rev. 1/84)
GPO 91 4-916
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02598-08 BFSB
PERIOD COVERED
October 1. 1989
through September ^n, iQQn
TITLE OF PROJECT (80 characters or less Title must HI on one line between the borders.)
Stroke Data Bank
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: Mary A. Foulkes, Ph.D.
Others: James M. Dambrosia, Ph.D.
Margaret Meadows
Rebecca Rohde
Jack Panossian
Alan Polls
Mathematical Statistician
Chief, Mathematical
Statistics Section
Statistician Asst.
Statistician
Programmer
Comp. Sys . Analysis
BFSB, DIR, NINDS
BFSB,
DIR,
NINDS
BFSB,
DIR,
NINDS
BFSB,
DIR,
NINDS
BFSB,
DIR,
NINDS
BFSB,
DIR,
NINDS
COOPERATING UNITS (If any)
Departments of Neurology: Boston University Medical Center, Michael Reese
Hospital, Neurological Institute - Columbia University, and University
of Maryland .
LAB/BRANCH
Biometry and Field Studies Branch
SECTION
Computer Applications Section
INSTITUTE AND LOCATION
NINDS, NIH, Be thesda. Maryland 20892
TOTAL MAN-YEARS:
2.4
PROFESSIONAL:
1.6
OTHER
0-8
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The Stroke Data Bank is a prospective observational study which collected data
on hospitalized newly diagnosed stroke patients, at four clinical centers. The
collaborating clinical centers were responsible for the collection of acute care
and longitudinal follow-up information using common definitions and procedures,
under contracts NO1-NS-2-2302 , 2398-9, N01-NS-5-2384. The general objective for
the project was to provide a comprehensive body of data for clinical research on
the factors influencing survival . morbidity and quality of life following onset
of a stroke. The BFSB served as the statistical coordinating center for the
project. The data collection phase was completed in FY 1988 including follow-
up, with a final cohort of 1805 patients. Now the BFSB is responsible for
statistical collaboration with the clinical investigators for the analysis of
the research questions. An observational study reporting the occurrence within
the Stroke Data Bank cohort of dementia both at first exam post- stroke onset and
incident dementia over the first year of follow-up summarizes the factors
associated with dementia within this cohort. An homunculus profile analysis is
in progress which will indicate the association, or lack thereof, between lesion
location and corresponding motor deficit. Institutionalization after hospital
discharge is the focus of an ongoing investigation which indicates that race,
sex and marital status may be associated with institutionalization among stroke
survivors. An investigation of the factors predictive of recurrence within two
years post-stroke indicated that prior stroke, diabetes mellitus, blood pressure
on admission, and an identifiable cause of infarct were predictive of recurrent
infarction. Two analyses of lacunar infarctions contrast these with other
infarction types and with lacunar infarctions from the Schlaganfall - Datenbank
from Austria.
12 - BFSB/DIR
PHS 6040 (Rev. 1/84)
GPO » 14-81 6
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02516-09 BFSB
PERIOD COVERED
October 1, 1 989 through SpptPmhpr 30, 1990
TITLE OF PROJECT (80 characters or less Title must tit on one line between the borders )
Traumatic C.nma Data Rank
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator j (Name, title, laboratory, and institute affiliation)
PI:
Others:
Mary A. Foulkes, Ph.D. Mathematical Statistician BFSB, DIR, NINDS
Margaret Meadows
Rebecca Rohde
Jack Panossian
Alan Polis
Statistician Asst.
Statistician
Programmer
Comp. Sys . Analysis
BFSB, DIR, NINDS
BFSB, DIR, NINDS
BFSB, DIR, NINDS
BFSB, DIR, NINDS
COOPERATING UNITS (if any)
Departments of Neurosurgery: Medical College of Virginia, University of
California - San Diego, University of Texas - Galveston, University of Virginia
LAB/BRANCH
Biometry and Field Studies Rranr.h
SECTION
Computer Applications; SprM'nn
INSTITUTE AND LOCATION
NINDS. NIH. Bethesda.
TOTAL MAN-YEARS
2.2
Maryland 2089?
PROFESSIONAL
1 U
OTHER
0 8
CHECK APPROPRIATE BOX(ES)
E (a) Human subjects
H (a1) Minors
SI (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The Traumatic Coma Data Bank is a prospective observational study which
collected data on severely head injured patients at four clinical centers.
The collaborating centers were responsible for the collection of acute care
and longitudinal follow-up information using common definitions and procedures,
under contracts N01-NS- 3-2339-42 . The research objectives for the project were
formulated by a Steering Committee composed of the principal investigators from
the clinical centers, other outside experts, and BFSB staff, with the concur-
rence of the BFSB Advisory Committee. The research objectives were the basis
for determining the specific data to be collected, the format of the data
collection forms and the data collection procedures. The general objective for
the project was to provide a comprehensive body of data for clinical research on
the factors influencing survival . morbidity and quality of life following a
severe head injury. The BFSB was the statistical coordinating center for the
project. Accrual of 1030 patients was completed in September 1987, and patient
follow-up was completed in January 1988. Reports are being prepared for
publication on such topics as the relationship between intracranial pressure and
Glasgow Coma Score, post- injury recovery of memory and attention, time related
prognostic factors for outcome, recovery from a vegetative state,
obstreperousness and depression, and the role of secondary injury in determining
outcome .
13 - BFSB/DIR
PHS 6040 (Rev 1/84)
GPO 814-Sia
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02594-08 BFSB
PERIOD COVERED
October 1, 1989 through Sepi-emher /*", 19QQ
TITLE OF PROJECT (80 characters or less Title must tit on one line between the borders.)
— Factors Predictive nf Reading and Writing Skills in the Congeni tally Deaf*
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: Paul S. Albert, Ph.D.
Others: Christy Ludlow, Ph.D.
Judith Cooper, Ph.D.
Staff Fellow
Speech Pathologist
Speech Pathologist
BFSB, DIR, NINDS
DCSD, NIDCD
DCSD, NIDCD
COOPERATING UNITS (it any)
Central Institute for the Deaf, St. Louis, MO (Ann Geers) ;
Gallaudet College, Washington, D.C. (Donald Moores)
LAB/BRANCH
Biometry and Field Studies Rranrh
SECTION
Mathematical Statistics Section
INSTITUTE AND LOCATION
NINDS. NIHT Bethesda,
Maryland ?089?
TOTAL MAN-YEARS:
0.05
PROFESSIONAL
n ns
OTHER:
0.00
CHECK APPROPRIATE BOX(ES)
SI (a) Human subjects
S (a1) Minors
D9 (a2) Interviews
D (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project consists of the statistical and data editing and file creation
aspects of a NIDCD project. Collaboration includes design of data collection
and monitoring procedures, and statistical analysis of study data.
The study examined factors that may be associated with development of reading and
writing skills in the congenitally deaf. Study subjects comprised three groups
of deaf 16- to 17 -year-olds , with 65 subjects in each group. Each group included
only subjects who received their preschool language training through one of three
approaches: aural-oral, total communication, and American Sign Language. Data
were collected on the audiologic, familial, and educational background of the
subjects, and on their present language skills. Data will be examined for their
association with present reading and writing skills of the subjects. Familial
and educational data for the main phase have been received and entered onto the
NIH computer system. Substantial amounts of missing data dictated the need for
new efforts to obtain the information required for the study.
*[This project is the BFSB/NINDS support of the NIDCD contract study NIH-NINDS-
84-19. The project officer is Dr. Judith Cooper, NIDCD. This project has been
subsumed under Statistical Collabor?tion and Consultation (Z01 NS 02652-06).]
14 - BFSB/DIR
PHS 6040 (Rev. 1/84)
GPO 914-918
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02505-10 BFSB
PERIOD COVERED
October 1. 1989 through SeptPrnhpr 3D, 1 990
TITLE OF PROJECT (80 characters or less Title must tit on one line between the borders )
Headache in Pregnant Womp.n
PRINCIPAL INVESTIGATOR (List other protessionai personnel below the Principal Investigator ) (Name, title, laboratory, and institute attiliation)
PI: Ta-Chuan Chen, Ph.D.
Other: Jonas H. Ellenberg, Ph.D.
Mathematical Statistician
Chief
BFSB, DIR, NINDS
BFSB, DIR, NINDS
COOPERATING UNITS (it any)
Boston Children's Hospital (Dr. Alan Leviton)
LAB/BRANCH
Biometry and Field Studies Branch
SECTION
Office of the Chief
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda.
Maryland 2089?
TOTAL MAN-YEARS
0.35
PROFESSIONAL
0-30
OTHER
0.05
CHECK APPROPRIATE BOX(ES)
E (a) Human subjects
H (a1) Minors
□ (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
This project investigates the relationship between migraine headache and
other diseases based on the data collected from the large group of gravidae
in the Collaborative Perinatal Project. The report of the investigation of the
influence of smoking on associations between migraine and other diseases such as
heart and thrombotic diseases and some respiratory and allergic diseases was
published in the Archives of Neurology, in FY '1988.
We are currently examining the possible association of maternal migraine in
pregnant women with the health status of their children. Subgroups of women
characterized by the absence and presence of migraine and other recurrent
headaches prior to or during pregnancy, were identified. Children of mothers
with a history of migraine appear to have higher incidence of some infectious and
allergic diseases than children born to mothers in the non-migraine group.
Statistical investigation of the latter results has revealed an association of
occurrence of bronchial asthma in children born to mothers with migraine, even
after partitioning out the potential influence of maternal asthma and allergies.
The risk of occurrence of bronchial asthma in children born to mothers with a
history of migraine was estimated to be 1.8 times larger than in the children
whose mothers did not have migraine headaches. A manuscript has been accepted
for publication in the Archives of Neurology.
Further studies examined the frequency and pattern of headache attacks during
pregnancies of the women with a history of migraine to test the hypothesis of
improvement of headache during pregnancy, and the possible association of
pregnancy complications such as uterine bleeding with migraine and headache
medication.
This project will be subsumed under Statistical Collaboration and Consultation
(Z01 NS 02652-06) .
15 - BFSB/DIR
PHS 6040 (Rev 1/84)
GPO SI 4-9 1 f
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02810-01 BFSB
PERIOD COVERED
Or.rnhp.r 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Sfafi Sti cal Cnnrriinat-ing Center for Col 1 ahnraf.i vs Clinical Studies
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: Mary A. Foulkes.
Others : Marinos Dalakas .
Jordan Graf man,
George Grimes
Rebecca Rohde
Jack Panossian
Alan Polis
Ph.D. Mathematical Statistician
M.D. Chief
Ph.D. Chief
Pharmacist
Statistician
Programmer
Comp. Sys . Analyst
BFSB, DIR, NINDS
NDS, MNB, DIR, NINDS
CNS, MNB, DIR, NINDS
CC, PHAR, NIH
BFSB, DIR, NINDS
BFSB, DIR, NINDS
BFSB, DIR, NINDS
COOPERATING UNITS (if any)
Depts . Of Neurology and Psychiatry: National Naval Medical Center, Bethesda,
and Oak Knoll Naval Medical Center, Oakland
LAB/BRANCH
Biometry and Field Studies Branch
SECTION
Computer Applications Section
INSTITUTE AND LOCATION
NINDS. NIH, Bethesda, Maryland 2QM1
TOTAL MAN-YEARS:
UL
PROFESSIONAL:
1 .6
OTHER:
0-4
CHECK APPROPRIATE BOX(ES)
S (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project encompasses all statistical coordinating center responsibilities for
collaborative clinical studies undertaken by this Section. Currently the major
collaborative clinical study is a double-blind clinical trial, designed to
compare the efficacy of two different daily doses of zidovudine in the treatment
of mild/moderate/severe AIDS Dementia Complex (ADC) . It is a collaborative trial
between the National Institute of Neurological Disorders and Stroke (NINDS), the
National Naval Medical Center (NNMC) , and Naval Hospital, Oakland. The major
objective of the trial is to compare the effects of two different daily doses of
zidovudine on the rate of progression of HIV-1 infection of the brain. The trial
is designed to determine if zidovudine at a reduced dose offers a therapeutically
effective alternative to zidovudine at 1250 mg daily dose in the management of
patients with mild/moderate/severe ADC. One hundred twenty- four patients will be
enrolled, 62 in each of two treatment arms. All patients will either be Depart-
ment of Defense (DOD) health care beneficiaries who meet established criteria for
ADC stage 1 (mild) , stage 2 (moderate) , or stage 3 (severe) and who are currently
being treated/monitored at Naval hospitals in Bethesda or Oakland, or non-DOD
health care beneficiaries with stage 1,2, or 3 ADC who reside in the Washington-
Baltimore area and are under the care of local private practitioners. All
patients will be followed for one year from the time of enrollment with periodic
studies to monitor their response to the dose assigned. These studies will
include routine clinical evaluations to detect adverse response to the drug as
well as specialized clinical, neuropsychological, imaging, neurochemical,
neurovirologic , and immunologic studies to measure the efficacy of the drug
regimens employed in this trial.
16 - BFSB/DIR
PHS 6040 (Rev. 1/84)
GPO 91 4-818
BIBLIOGRAPHY
ZOl NS 02652-06 BFSB
Gilboe DD, Kintner DB, Emoto SE, Fitzpatrick JH. Brain intracellular pH during
graded hypoxia and subsequent reoxygenation. Am J Physiol; (in press).
Gilboe DD, Fitzpatrick JH, Kintner D, Emoto SE, Bazar NG, Braquet P.
Biochemical changes in normoxic and post ischemic brain tissue following
treatment with BN52021. In: Braquet P, ed. Ginkgolides - chemistry, biology,
pharmacology and clinical perspectives, vol 2. Barcelona: JS Prous Science;
(in press) .
Eldridge R, Denkla MB, Bien E, Myers S, Kaiser MI, Pikus A, Schlesinger SL,
Parry DM, Dambrosia JM, Zasloff MA, Mulvihill JJ . von Recklinghousen
neurofibromatosis: neurological and cognitive assessment. Am J Dis Child
1989; 143:933-7.
Von Lubitz DKEJ , Dambrosia JM, Redmond DJ . Protective effect of cyclohexyl
adenosine in the treatment of cerebral ischemia in gerbils. Neuroscience
1989; 30:451-62.
Nelson KB, Ellenberg JH. Prenatal and perinatal antecedents of febrile
seizures. Ann Neurol 1990; 27:127-31.
Zhang Z-X, Anderson DW, Lavine L, Mantel N. Patterns of acquiring
parkinsonism- dementia complex on Guam: 1944 through 1985. Arch Neurol; (in
press) .
Zang Z-X, Anderson DW, Mantel N. Geographic patterns of parkinsonism- dementia
complex on Guam: 1956 through 1985. Arch Neurol; (in press).
Zhao F, Emoto SE, Lavine L, Nelson KB, Wang C, Li S, Cheng X, Bolis CL,
Schoenberg BS . Risk factors for febrile seizures in the People's Republic of
China. Epilepsia; (in press).
McShane LM, Clark LC , Combs GF, Turnball BW. Reporting the accuracy of
biochemical measurements for epidemiologic and nutrition studies. Am J Clin
Nutr ; (in press) .
Kessler M J , London WT, Madden DC, Dambrosia JM, Hillard JK, Soike KF, Rawlings
RG . Serological survey for viral diseases in the Cayo Santiago rhesus Macaque
population course. Puerto Rico Health Sci J 1989;8:95-7.
Farwell JR, Lee YJ , Hirtz DG, Sulzbacher SI, Ellenberg JH, Nelson KB.
Phenobarbital for febrile seizures: effects on intelligence and on seizure
recurrence. NEJM 1990; 322: (6) 364-9.
17 - BFSB/DIR
Z01 NS 02490-10 BFSB
Emoto SE, Matthews PC. A Weibull model for dependent censoring. Ann Stat;
(in press) .
Albert PS. A two-state mixture model for a time series of epileptic seizure
counts. Biometrics; (in press).
Lee Y J . Nonparametric statistical methods for Salmonella/Ames mutagenesis
assay. Proceedings of the First International Conference on Statistical
Computing 1990; (in press).
Dambrosia JM. Statistical and epidemiological considerations for clinical
trials in neurology. In: Porter R J , Schoenberg BS , eds . Controlled clinical
trials in neurology. Boston: Kluwer Academic, 1990; 31-57.
Foulkes MA. Design issues in chemosensory trials. Arch Otolaryngol Head Neck
Surg 1990; 116:65-68.
Ellenberg JH. Clinical trials. In: Theodore W, ed. Clinical Neuro-
pharmacohology. Philadelphia: WB Saunders; 1990; vol.8, (1) 15-30.
Anderson DW, ed. Neuroepidemiology . A tribute to Bruce Schoenberg. Boca Raton:
CRC Press; (in press).
Gourie-Devi M, Anderson DW, Rao VN. Neuroepidemiologic survey in a developing
country: some questions and answers. In: Anderson DW, ed.
Neuroepidemiology. A tribute to Bruce Schoenberg. Boca Raton: CRC Press,
1990; (in press).
Ellenberg JH. Biostatistical collaboration in medical research (with
discussants). Biometrics 1990; 46, 1-32.
Dambrosia JM. Comments on some personal remarks on clinical trials. Brazilian
J Probability Stat 1990; (in press).
Albert PS, Brown CH. The design of a panel study under an alternating Poisson
process. Biometrics; (in press).
Z01 NS 02598-08 BFSB
Tatemichi TK, Foulkes MA, Mohr JP, Hewitt JR, Hier DB, Price TR, Wolf PA.
Dementia in stroke survivors in the Stroke Data Bank cohort. Stroke 1990;
21:858-66.
Kittner SJ , Sharkness CM, Price TR, Plotnick GD, Dambrosia JM, Wolf PA, Mohr
JP, Hier DB, Kase CS , Tuhrim S. Infarcts with a cardiac source of embolism in
the NINDS Stroke Data Bank: historical features. Neurology 1990; 40:281-4.
18 - BFSB/DIR
Project No. Z01 NS 02505-10 BFSB
Significance to NINDS Program and Biomedical Research: Headache is an area of
interest to the NINDS Program. It has been reported in the published medical
literature that there are notable indications of the association of migraine with
many other diseases such as hypertensive disorders, functional heart diseases,
cerebral vascular accident, hayfever etc. However, the results of these studies
have not been consistent because (1) the clinical groups of migraine patients
under study might often have been biased by their mechanism of selection or (2)
the reports were produced based on insufficient medical observations. Further
studies with sufficient data and unbiased- sampling groups of subjects, therefore,
are still needed.
Proposed Coures: This project will be subsumed under Statistical Collaboration
and Consultation (Z01 NS 02652-06).
Publications : Chen TC, Leviton A. Asthma and exzema in children born to women
with migraine. Arch Neurol; (in press).
19 - BFSB/DIR
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ANNUAL REPORT
October 1, 1989 through September 30. 1990
Developmental and Metabolic Neurology Branch
Clinical Neurosciences Program, DIR
National Institute of Neurological Disorders and Stroke
TABLE OF CONTENTS
RESEARCH SUMMARY 1
CONTRACTS 5
PROJECT REPORTS
Metabolism of Complex Lipids of Nervous Tissue
Z01 NS 0081 5-30 DMN 10
Synthesis of Compounds Analogous to Glycolipids
Z01 NS02162-16DMN 11
Development of Analytical Methods for the Use of
Reseaix i Sphingolipidoses
Z01 NS02163-16DMN 12
Gaucher's Disease: Biochemical and Clinical Studies
Z01 NS 02453-10 DMN 13
Molecular and Genetic Studies of Niemann-Pick Disease
Z01 NS 02657-06 DMN 14
Clinical Studies of Neurogenetic Diseases
Z01 NS 02664-06 DMN 15
Retroviral Mediated Transfer of Human Globin Genes
Z01 NS 02730-04 DMN 16
Gene Therapy of Inherited Enzyme Deficiencies
Z01 NS 02731-04 DMN 17
Exploration of Strategies for the Treatment of AIDS
Z01 NS 02769-03 DMN 18
Strategies for the Treatment of Autoimmune Neuropathies
Z01 NS 2770-03 DMN 19
Modification of Growth Factor Genes by Genes Targeting
Z01 NS 02771-02 DMN 20
Preparation of Transgenic Murine Analogs of Human
Metabolic Storage Disorders
Z01 NS 02782-02 DMN 21
Generation of Mice with Sickle Cell Anemia
Z01 NS 02785-02 DMN ll
Synthesis of Inhibitors of N-Myristoyltransferase
Z01 NS 02816-01 DMN zs
ii
ANNUAL REPORT
OCTOBER 1, 1989 THROUGH SEPTEMBER 30, 1990
DEVELOPMENTAL AND METABOLIC NEUROLOGY BRANCH, DIR
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
ROSCOE O. BRADY, M.D., CHIEF
The principal activities of the Branch include the following areas of investigation :
1. Basic studies of lipid and mucopolysaccharide synthesis, catabolism, and
enzymatic abnormalities in hereditary human disorders. 2. Investigations of the
molecular basis of metabolic storage disorders. 3. Pathogenic and therapeutic
investigations in animal models of disorders of metabolism. 4. Development of
transgenic analogs of human disorders. 5. Clinical investigations of lysosomal
storage disorders and neurogenetic diseases. 6. Development of therapy for
patients with heritable diseases. 7. Development of gene replacement technology.
I. BASIC INVESTIGATIONS IN HEREDITARY METABOLIC DISORDERS
A. Gaucher's Disease
Fundamental studies are in progress to examine the effects of lipid activators
and small molecular weight protein "cohydrolases" on sphingolipid degrading
enzymes. It is anticipated that appropriate combinations of these substances will
improve the efficiency of exogenous enzymes administered to patients with lipid
storage disorders. Maximally effective mixtures of these agents have been
identified. Their application to the treatment of Gaucher's disease is being
examined in animal and tissue culture modelsof thisdisorder.
B. TypeC Niemann-Pick Disease
We have extended our investigation of the effect of dimethylsulfoxide (DMSO)
on the intracellular accumulation of unesterified cholesterol in cultured skin
fibroblasts obtained from patients withType C Niemann-Pick disease. This
information is being used in our phase I clinical trial of DMSO in patients with this
disorder.
C. Fabry's Disease
A comprehensive investigation is underway concerning the targeting of
exogenous ceramidetrihexosidase to cells in which ceramidetrihexoside accumulates
in patients with Fabry's disease. This strategy has been employed with extraordinary
success in enzyme replacement in Gaucher's disease. When the pertinent tissue
lectins have been conclusively identified, a clinical trial with carbohydrate-modified
ceramidetrihexosidase will be undertaken in Fabry's disease.
1 DMN/DIR
II. INVESTIGATIONS OF THE MOLECULAR BASIS OF METABOLIC STORAGE
DISORDERS
A. Type C Niemann-Pick Disease
Recent studies have provided exceptionally promising leads concerning the
molecular defect in Type C Niemann-Pick disease. A protein polymorphism has been
discovered in subcellular particles in an Hispanic pedigree with severely affected
individuals with this disorder. Investigations are underway to determine whether
this molecular variant is involved in, or closely linked to Type C Niemann-Pick disease.
III. PATHOGENIC ANDTHERAPEUTIC INVESTIGATIONS IN ANIMAL MODELS
A. Murine Analog of Type C Niemann-Pick Disease
The treatment of patients with Type C Niemann-Pick disease is currently based on
the concept that cholesterol is the principal offending metabolite in this disorder.
Investigations with a spontaneously occurring murine analogue of the human
disease revealed that modifying the quantity of dietary cholesterol can affect the life
span of affected mice. Restricting cholesterol led to increased longevity whereas
cholesterol supplements hastened morbidity and mortality. These studies provided
the basis for our attempts to alter the cholesterol burden in patients with Type C
Niemann-Pick disease.
IV. DEVELOPMENT OFTRANSGENIC ANIMAL MODELS OF HUMAN DISORDERS
A. Gaucher's Disease
Progress has been made in developing a transgenic murine model of Gaucher's
disease using genomic clones of the mouse glucocerebrosidase isolated by DMNB
scientists. Retroviral constructs have been made with specific mutations in the gene
in orderto produce a transgenic mouse analogue of Gaucher's disease.
B. Sickle Cell Anemia
Transgenic mice have been generated that contain the abnormal Antilles beta-
sickle globin gene. The human alpha-globin gene isalso inserted in orderto
generate mice that produce high levels of beta-sickle Antilles and human alpha-
globins. Transgenic mice have been produced in which human globin chains are
produced at a level between 1 5 and 20 percent of mouse globin. These transgenic
mice will be mated with alpha-thalassemic mice. In addition, founder mice will be
mated to obtain mice homozygous for the sickling trait. Investigations with these
mice will greatly accelerate studies of therapeutic agents to correct sickling.
C. Transgenic Mice Containing a Defective Interleukin 3 (IL-3) Gene
A principal objective of this investigation is to determine the consequences of
altering the functional state of genes that control cell growth and maturation. This
goal will be examined through studies that include the introduction of the IL-3 gene
2 DMN/D1R
into an IL-3-dependent cell line. Other attempts will be made to develop transgenic
mice with a defective IL-3 gene. If such animals can be produced, studies will be
undertaken to correct the induced growth and maturation problems through
homologous recombination with a normally functioning IL-3 gene.
V. CLINICAL INVESTIGATIONS OF METABOLIC DISORDERS AND NEUROGENETIC
DISEASES
A. Familial Diurnally Variant Dystonia
Genetic linkage studies and an examination of the mode of inheritance of this
condition that is associated with low cerebrospinal fluid biopterin levels have been
carried out. Beneficial effects of supplemental biopterin have been documented in
one of these patients.
B. Fabry's Disease
Symptomatic therapies have been devised to correct the autonomic nervous
system dysfunction that occurs in patients with this disorder. Specific treatment
regimens are being developed that relieve the painful acroparesthesias without
exacerbating signs and symptoms of autonomic dysfunction. Additional studies are
directed toward correcting gastric hypomotility in Fabry patients.
C. Lennox-Gastaut Syndrome
Potential metabolic alterations in patients with this syndrome are under
investigation. Two patients with this disorder that exhibit non-ketotic
hyperglycinemia have been identified. Such studies may lead to the development of
specific remedial therapy in patients with this clinical presentation.
D. Type C Niemann-Pick Disease
The incidence of seizures and the occurrence of cataplexy have been determined
in patients with this disorder. A neurological staging system to evaluate disease
progression is under development. The effect of modifying dietary cholesterol and
drugs that alter cholesterol production and its intracellular disposition are under
investigation in Type C Niemann-Pick patients.
VI. THERAPY FOR HEREDITARY METABOLIC DISORDERS
A. Gaucher's Disease
1 . We completed a pharmacodynamic investigation of the effect of varying
quantities of intravenously injected glucocerebrosidase targeted to macrophage
storage cells in patients with Gaucher's disease. Reduction of hepatic
glucocerebroside and ultrastructural improvement of the liver was observed
following infusion of this enzyme. The threshold quantity of enzyme at which
these changes occur was determined. This study provided the guideline for the
selection of the therapeutic dose of mannose-terminated glucocerebrosidase in
the clinical efficacy trial summarized in the following paragraph.
3 DMN/DIR
2. A phase ll/lll enzyme replacement trial was carried out in which the efficacy of
enzyme replacement was evaluated in 1 2 patients with Gaucher's disease.
Dramatically beneficial responses followed biweekly infusions of 60 I.U./kg of
mannose-terminated human placental glucocerebrosidase. In all patients, there
was striking improvement of the anemia and thrombocytopenia that are
characteristic of Gaucher's disease. In addition, there was a surprisingly large
reduction in the size of the spleen in the recipients. Further, a significant
decrease of hepatomegaly was also observed in all of the patients. Evidence of
skeletal improvement was seen in four patients. Major skeletal improvement was
documented in another patient with Gaucher's disease treated with
macrophage-targeted glucocerebrosidase over a four-year period. These salutary
responses indicate that enzyme replacement is effective and that it is the therapy
of choice for Gaucher's disease at this time. These benefits augur well for the
successful enzyme replacement therapy in additional human metabolic disorders.
B. Type C Niemann-Pick Disease
A phase-l clinical protocol to examine the therapeutic potential of orally
administered dimethylsulfoxide (DMSO) and other agents known to modify organ
and tissue cholesterol in patients with Type C Niemann-Pick disease is nearing
completion. The results obtained in this investigation will be used to develop a
protocol to examine their therapeutic efficacy in Type C Niemann-Pick disease.
VII. DEVELOPMENT OF GENE REPLACEMENT TECHNOLOGY
A. Gaucher's Disease
High-titer recombinant retroviruses have been constructed that contain the
human glucocerebrosidase gene. The gene has been transferred to human
hematopoietic cell lines and progenitor cells. Glucocerebrosidase activity has been
increased to normal levels following gene transfer into hematopoietic progenitor
cells obtained from patients with Gaucher's disease. These results provide a sound
experimental basis for further exploration of gene replacement for the treatment of
Gaucher's disease.
B. Human qlobin disorders
Investigations with retroviral vectors containing the human beta-globin gene
resulted in regulated expression of human globin mRNA and protein in murine
erythroleukemia cells. A novel high-titer retrovirus vector was produced that
effectively transfers the human beta-globin gene into CFU-S spleen colonies that
express the human beta-gene. In addition, human beta-globin protein has been
produced in long-term reconstituted mice. Bone marrow from a primary murine
recipient that contained the human beta-globin gene for more than a year was
transplanted into a secondary recipient. The latter mouse expressed the human
beta-globin gene in erythroid cells more than three months after transplantation.
This study proves that the initial gene transfer was targeted to repopulating bone
marrow stem cells and provides the basis for further exploration of gene transfer
technology to correct human globin disorders such as beta-thalassemia.
4 DMN/DIR
CONTRACT NARRATIVE
DEVELOPMENTAL AND METABOLIC NEUROLOGY BRANCH
DIVISION OF INTRAMURAL RESEARCH, NINDS
OCTOBER 1, 1989 THROUGH SEPTEMBER 30, 1990
Contractor: GENZYME CORPORATION, BOSTON, MA. (N01-NS-9-2358)
Title: Preparation of Ceramidetrihexosidase from Human Placental Tissue
Contractor's Project Director: F. Scott Furbish
Current Annual Level of Support: $100,000
Objectives: To isolate human placental ceramidetrihexosidase in sufficient purity
and quantity for use in enzyme replacement trials in patients with Fabry's disease.
Major Findings: A procedure has been developed for the large-scale purification of
human placental ceramidetrihexosidase in sufficient purity and specific catalytic
activity so that it can be safely administered to patients with Fabry's disease. Its
carbohydrate portion has been analyzed. We are developing methods to target the
enzyme to cells and tissues where ceramidetrihexoside is stored.
Significance to Biomedical Research and to the Program of the Institute:
A principal mission of the Institute is to develop effective therapy for human
diseases. If salutary clinical results can be obtained, an extraordinary milestone will
have been accomplished regarding this type of human genetic disease.
Proposed Course of the Contract: We have made significant progress in our effort to
increase the delivery of this enzyme to specific cells in which ceramidetrihexoside
accumulates. When this preclinical investigation is completed, we shall resume
enzyme replacement trials in patients with Fabry's disease. We shall examine the
effectiveness of the enzyme with regard to clearance of accumulated
ceramidetrihexoside in the liver, kidney and blood, and monitor clinical responses in
thistherapeutictrial.
5 DMN/DIR
CONTRACT NARRATIVE
DEVELOPMENTAL AND METABOLIC NEUROLOGY BRANCH
DIVISION OF INTRAMURAL RESEARCH, NINDS
OCTOBER 1,1989 THROUGH SEPTEMBER 30, 1990
Contractor: GENZYME CORPORATION, BOSTON, MA. (N01-NS-9-2360)
Title: Preparation of Sphingomyelinase from Human Placental Tissue
Contractor's Project Director: F. Scott Furbish
Current Annual Level of Support: $100,000
Objectives: To isolate human placental sphingomyelinase in sufficient purity and
quantity for use in enzyme replacement trials in patients with Niemann-Pick disease.
Major Findings: A procedure is being developed for the large-scale isolation of
human placental sphingomyelinase. The final purification steps will be performed at
NIH. The highly enriched enzyme will be administered to patients with Type B
Niemann-Pick disease and its effect on liver and blood sphingomyelin levels will be
determined.
Significance to Biomedical Research and to the Program of the Institute:
A principal mission of the Institute is to develop effective therapy for human
diseases. If salutary biochemical results can be obtained, a clinical efficacy trial will
be conducted to determine the response of patients with Type B Niemann-Pick
disease to enzyme replacement.
Proposed Course of the Contract: We have carried out experiments to isolate human
placental sphingomyelinase in a high degree of purity. Large quantities of a
partially purified preparation are required in order to obtain sufficient enzyme for
an investigation of its effect on stored sphingomyelin in Niemann-Pick patients.
When a sufficient quantity of pure sphingomyelinase is available, we shall determine
if it causes a reduction of accumulated lipid in the liver and blood of patients with
this disorder.
6 DMN/DIR
CONTRACT NARRATIVE
DEVELOPMENTAL AND METABOLIC NEUROLOGY BRANCH
DIVISION OF INTRAMURAL RESEARCH, NINDS
OCTOBER 1, 1989 THROUGH SEPTEMBER 30, 1990
Contractor: GENZYME CORPORATION, BOSTON, MA. (N01-NS-9-2365)
Title: Preparation of Glucocerebrosidase from Human Placental Tissue
Contractor's Project Director: F. Scott Furbish
Current Annual Level of Support: $300,000
Objectives: To isolate human placental glucocerebrosidase in sufficient purity and
quantity for use in enzyme replacement trials in patients with Gaucher's disease.
Major Findings: A procedure has been developed for the large-scale purification of
human placental glucocerebrosidase targeted to cells in which glucocerebroside
accumulated in patients with Gaucher's disease. The intravenous infusion of this
enzyme causes a decrease in the quantity of glucocerebroside stored in the liver and
in association with erythrocytes in the blood. Long-term administration of the
enzyme has caused significant hematologic improvement, reduction of
hepatosplenomegaly, and skeletal benefit in 13 patients with Gaucher's disease.
Significance to Biomedical Research and to the Program of the Institute:
A principal mission of the Institute is to develop effective therapy to treat human
diseases. The results indicated in the preceding paragraph indicates that we have
achieved this goal in Gaucher's disease, the most prevalent human metabolic storage
disorder.
Proposed Course of the Contract: We shall determine the minimal amount of
exogenous glucocerebrosidase required to stabilize patients in whom the beneficial
responses indicated above have occurred. We shall also determine the minimal
amount of enzyme required to bring about these salutary changes. In addition, we
shall examine enzyme replacement therapy in patients with Type 3 Gaucher's
disease, the chronic neuropathic form of this disorder.
7 DMN/DIR
CONTRACT NARRATIVE
DEVELOPMENTAL AND METABOLIC NEUROLOGY BRANCH
DIVISION OF INTRAMURAL RESEARCH, NINDS
MARCH 1, 1990 THROUGH SEPTEMBER 30, 1990
Contractor: LIPITEK, INC. (NO1-NS-0-2386)
Title: Production of Radiolabeled Sphingolipids
Contractor's Project Director: Alexander L. Weis, Ph.D.
Current Annual Level of Support: $85,200
Objectives: To prepare glucocerebroside, sphingomyelin, and ceramidetrihexoside
labeled with 14C in critical portions of the molecule for diagnostic tests for Gaucher's
disease, Niemann-Pick disease, and Fabry's disease.
Maior Findings: Synthetic chemical procedures have been developed to incorporate
radioactive carbon-14 jn specific portions of sphingolipid molecules. These
compounds are used to diagnose patients with the sphingolipid storage disorders
listed above, to identify heterozygous carriers of these conditions, to diagnose these
disorders prenatally, and to monitor enzyme isolation procedures for
glucocerebrosidase, sphingomyelinase, and ceramidetrihexosidase.
Significance to Biomedical Research and to the Program of the Institute:
The ability to diagnose patients, identify heterozygotes, and to monitor pregnancies
at risk for sphingolipid storage disorders represents major contributions to the
control of the incidence of these diseases. These procedures are in wide use at the
present time.
Proposed Course of the Contract: The contract has recently been negotiated.
Contractor's performance will be monitored for compliance under a fixed-price
agreement concerning the reguisite deliverables.
8 DMN/DIR
CONTRACT NARRATIVE
DEVELOPMENTAL AND METABOLIC NEUROLOGY BRANCH
DIVISION OF INTRAMURAL RESEARCH, NINDS
JULY 16, 1990 THROUGH SEPTEMBER 30, 1990
Contractor: UNIVERSITY OF NEW MEXICO (NO1-NS-0-2394)
Title: Preparation of Homogeneous Human Liver Sterol Carrier Protein-2
(SCP-2) And Production of High-Titer Antibody TO SCP-2
Contractor's Project Director: Terence J. Scallen, M.D., PH.D.
Current Annual Level of Support: $74,577
Objectives: To isolate homogeneous SCP-2 from human livertissue and prepare
anti-SCP-2 monospecific polyclonal antibody for investigations in Type C Niemann-
Pick disease.
Major Findings: An isoform of SCP-2 appears to be lacking in tissue specimens
obtained from patients with Type C Niemann-Pick disease. We need to determine
whether this finding is consistent in all of the patients we are monitoring, and
whether exogenous SCP-2 can correct the impairment of cholesterol homeostasis in
cultured skin fibroblasts and other specimens derived from patients with this
disorder. If these results are positive, we will need to determine the amino acid
seguence of SCP-2 and to clone the gene for this protein in order to evaluate the
molecular investigations in Type C Niemann-Pick disease.
Significance to Biomedical Research and to the Program of the Institute:
The ability to identify biochemical and molecular abnormalities in patients with
previously unsolved metabolic disorders is a major goal of the Institute. Acguisition
of this information is likely to be required to develop effective therapy for such
conditions.
Proposed Course of the Contract: The contract has just been awarded and SCP-2 and
its antibody will be used in metabolic and molecular investigations in Type C
Niemann-Pick disease.
9 DMN/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS00815-30DMN
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJ ECT (80 characters or less. Title must fit on one line between the borders.)
Metabolism of Complex Lipids of Nervous Tissue
PRINCIPAL INVE STIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: R.O. Brady, M.D.
Others: P.G. Pentchev, Ph.D.
R.R. O'Neill, Ph.D.
J.M. Quirk, M.S.
C. Roff, Ph.D.
E.Goldin, Ph.D.
M. Comly, B.S.
A. Coonev. B.S.
Chief
DMN
NINDS
Section Chief
DMN
NINDS
Senior Staff Fellow
DMN
NINDS
Biochemist
DMN
NINDS
Special Expert
DMN
NINDS
Visiting Fellow
DMN
NINDS
Biologist
DMN
NINDS
Bioloaist
DMN
NINDS
COORPE RATING UNITS Of any)
Laboratory of Cellular and Developmental Biology, NIDDKD, Laboratory of Biochemistry, Faculty of
Medicine, Lyon-Sud, France
LAB/BRANCH
Developmental and Metabolic Neurology Branch
SECTION
Molecular and Cellular Pathophysiology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
6.0
OTHER:
2.0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects QT] (b) Human tissues ] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Summary: The metabolic defect in patients with Types C and D Niemann-Pick disease has been shown to
be due to abnormal intracellular cholesterol homeostasis. The molecular lesion in these disorders results
in: (1) failure to down-regulate LDL receptors on cell membranes; (2) lack of down-regulation of
HMGCoA reductase, a key enzyme in cholesterol biosynthesis; and (3) inability to up-regulate acyl
cholesterol acyl CoA transferase, the enzyme that catalyzes the esterification of intracellular cholesterol.
Tests have been developed and introduced into medical practice for the diagnosis of Types C and D
Niemann-Pick disease and the identification of heterozyqotes, and the prenatal diagnosis of these
conditions. Current emphasis is on the development of effective therapy for patients with this disorder
and the elucidation of the molecular basis of this novel metabolic disorder.
10 DMN/DIR
PHSMMOIRev 1 84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02162-16DMN
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJECT (80 characters or 'ess. Title must fit on one line between the border*.)
Synthesis of Compounds Analogous to Glycolipids
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator J (Name, title, laboratory, and institute affiliation)
PI: S P. Miller, Ph.D.
Others: S. A. French, B.S.
Senior Staff Fellow
Chemist
DMN NINDS
DMN NINDS
COORPERATING UNITS Of an, )
LAB/BRANCH
Developmental and Metabolic Neurology
SECTION
Neurochemical Methology Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAI MAN-YEARS:
08
PROFESSIONAL:
0.5
OTHER:
0.3
CHECK APPROPRIATE BOX(ES)
| J (a) Human subjects
] (a1) Minors
J (a2) Interviews
[~x | (b) Human tissues J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Summary: The object of this work is to prepare artificial substrates of sphinqolipid hydrolases that can
detect enzyme activity within intact cells Chromogenic substrates synthesized earlier in this section have
proven highly useful for enzyme measurements in vitro. However, the requirements of in vivo
measurement of enzyme activity necessitate the preparation of fluoroqenic glycolipids. The substrates
required for such an approach should be nonfluorescent prior to enzymatic cleavage of the glycoside
bond. The released product should then be fluorescent at lysosomal pH. A group of strongly fluorescent
phenols, obtained by 0-4 monoalkylation of 2,3-dicyanohydroquinone (DCH) were synthesized for this
project. These were converted into gjucosides and qalactosides which were then tested as enzyme
substrates. Most interesting was the beta-qalactoside of a lysosomotropic derivative of DCH. The
membrane-permeant tetraacetate of this substrate was hydrolysed within intact human fibroblasts.
Moreover, beta-galactosidase deficient fibroblasts derived from patients with Gmi gangliosidosis
hydrolysed this compound at 6-17% of the rate seen with normal cells. Attempts to apply these
techniques to the beta-glucocerebrosidase which is deficient in Gaucher's disease are in progress.
Ultimately, these probes will be used to measure the efficiency of gene-transfer experiments with
lysosomal hydrolase genes. In this setting, jn vivo fluorogenic substrates have the potential to greatly
increase the sensitivity for detection of gene expression in transformed cells. In conjunction with
fluorescence activated cell sorting, fluorogenic probes should also expand the possibilities for study of
the developmental biology of these recombinant cells.
11 DMN/DIR
PHSSMOIRe- 184)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02163-16DMN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Development of Analytical Methods for the Use of Research of Sphingolipidoses
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: S. P. Miller, Ph.D. Senior Staff Fellow DMN NINDS
COORPERATING UNITS (if my)
LAB/BRANCH
Developmental and Metabolic Neurology
SECTION
Neurochemical Methodology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD. 20892
TOTAL MAN-YEARS:
0.2
PROFESSIONAL:
0.2
OTHER:
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects
] (a1) Minors
J (a2) Interviews
[x~l (b) Human tissues ] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
New analytical techniques were developed and used in enzymatic research and in clinical investigations
of lipidoses. Quantitation of all major phospholipid and neutral lipid species was performed on hepatic
biopsy specimens from Type C Niemann-Pick patients. Analytical techniques were applid to the study of
glycerolphosphorylcholine requlation in kidney cells. Structural studies of polyglycosylated saponins
were carried out. Techniques were developed for the quantitation and study of new fluorescent enzyme
substrates.
12 DMN/DIR
PHS 6040 (Rev. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02453-10DMN
PERIOD COVERED
Octoberl, 1989 to September 30, 1990
TITLE OF PROJECT (80 characters or less. Titlemust fit on one line between the borders.)
Gaucher's Disease: Biochemical and Clinical Studies
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
Chief
Section Chief
Special Volunteer
Biologist
Special Volunteer
Special Volunteer
Special Volunteer
PI:
R 0 Brady, M D
OTHERS:
N Barton, M.D.,Ph D
G. Murray, PhD
G. Zirzow, B.S.
S. Reisz, B.S.
K. Howard, B.S.
F.S.Jin, M.D.
DMN
NINDS
DMN
NINDS
DMN
NINDS
DMN
NINDS
DMN
NINDS
DMN
NINDS
DMN
NINDS
COORPERATING UNITS (.f any)
Massachusetts Gen. Hospital, Dept. of Orthopedic Surgery, Boston, MA: (H Mankin, D Rosenthal,
S. Doppelt); Children's Hospital, Washington, D. C. (P. Guzetta)
LAB/BRANCH
Developmental and Metabolic Neurology
SECTION
Clinical Investigations & Therapeutics Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
5.5
PROFESSIONAL:
35
OTHER:
2.0
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects
] (a1) Minors
J (a2) Interviews
|~x | (b) Human tissues ] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Summary: Successful therapy for Gaucher's disease depends upon a comprehensive clinical and basic
scientific knowledge of the disorder. Patients have been extensively studied and complications
identified Research on qlucocerebrosidase addresses the biochemistry, cell biology, and molecular
genetics of the enzyme. A pharmacodynamic assessment of mannose-terminated human placental
glucocerebrosidase has been completed. Extraordinarily gratifying results have been obtained with
enzyme replacement therapy in patients with Gaucher's disease.
13 DMN/DIR
PHS 6040 (Rev. 1/841
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02657-06DMN
PERIOD COVERED
October 1 , 1 989 to September 30, I990
TITLE OF PROJECT [80 characters or less. Title must fit on one line between the borders.)
Molecuar and Genetic Studies of Niemann-Pick Disease
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: N. Barton, M.D., Ph.D. Section Chief DMN NINDS
OTHER: K. Oliver, B.S. Biologist DMN NINDS
COORPERATING UNITS (•! any)
LAB/BRANCH
Developmental and Metabolic Neurology
SECTION
Clinical Investigations and Therapeutics
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD.
20892
TOTAL MAN-YEARS:
0.9
PROFESSIONAL:
0.3
OTHER:
0.6
CHECK APPROPRIATE BOX(ES)
I x | (a) Human subjects
| x 1 (a1) Minors
J (a2) Interviews
I x | (b) Human tissues J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Niemann-Pick disease is a proqressly debilitating, neurogenetic disorder which is characterized
biochemically by the accumulation of sphingomyelin in several tissues and organs in conjunction with
deficiency of the lysosomal hydrolase, sphingomyelinase. Detailed description of various phenotypes in
terms of cellular pathochemistry and molecular genetics has not been accomplished to date. A major
obstacle in this area has been the absence of reproducible techniques for the isolation of homogeneous
preparations of sphingomyelinase. Employing novel detergent and chromography systems, we have
purified sphingomyelinase to homogeneity. The purified enzyme migrates with an apparent molecular
weight of 67,000 daltons is SDS-polyacrylamide gels under both reducing and nonreducing conditions.
Kinetic analyses and determinations of the primary protein structure and carbohydrate composition are
in progress. Polyclonal antibodies have been raised to the purified enzyme that will assist in cloning the
c-ene for sphingomyelinase. Characterization of the phenotypes of Niemann-Pick disease in terms of
protein polymorphisms and specific mutations at the DNA level will be undertaken.
14 DMN/DIR
PHS6(M0(Kev 1/M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02664-06DMN
PERIOD COVERED
October!. 1989 to September 30. 1990
TITLE OF PROJECT (80 characters or less Title must fit on one line between the borders }
Clinical Studies of Neurogenetic Diseases
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal In vestigator.) (Name, title, laboratory, and institute affiliation)
PI: N. Barton, M.D., Ph.D. Section Chief DMN NINDS
OTHERS: R. Brady, M.D. Chief DMN NINDS
J. Fink, M.D. Sen. Staff Fellow DMN NINDS
K. Yu, M.D. Visiting Associate DMN NINDS
R. Grewal, M.D. Visiting Associate DMN NINDS
COORPERATING UNITS OUn,)
Armed Forces Institute of Pathology, ( K. Ishak)
and S Doppelt)
Massachusetts General Hospital, (H. Mankm and
LAB/BRANCH
Developmental and Metabolic Neurology Branch
SECTION
Clinical Investigations
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD. 20892
TOTAL MAN-YEARS
7.0
PROFESSIONAL:
65
OTHER
05
CHECK APPROPRIATE BOX(ES)
1 x | (a) Human subjects
| x | (a1) Minors
J (a2) Interviews
[x~| (b) Human tissues ] (c) Neither
Summary OF WORK (use standard unreduced type. Do not exceed the space provided.)
A number of novel neurogenetic diseases have been identified by studies carried out under this project.
Genetic linkage studies are underway in familial diurnally variant dystonia associated with low CSF
biopterin levels and a beneficial effect of supplemental biopterin has been documented in one of these
patients. Corrective therapy has been developed for autonomic nervous system dysfunction associated
with Fabry's disease. Potential metabolic alterations in the Lennox-Gastaut syndrome have been
examined. The incidence of seizures and cataplexy has been determined in patients with Type C
Niemann-Pick disease. The effect of modifying dietary cholesterol and the responses of patients to
drugs that alter cholesterol synthesis are under investigation in this disorder.
15 DMN/D1R
miHMII(«t» I'M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02730-04DMN
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Retroviral Mediated Transfer of Human Globin Genes
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: S. Karlsson, M.D.,Ph.D
OTHERS: D. Bodine, Ph.D.
L. Perry, B.S.
A. Nienhuis, M.D.
Acting Chief, M&MG
Sen. Staff Fellow
Biologist
Chief
DMN NINDS
CHB NHLBI
DMN NINDS
CHB NHLBI
COORPERATING UNITS i,h-.,i
National Heart, Lung and Blood Institute
LAB/BRANCH
Developmental & Metabolic Neurology
SECTION
Molecular & Medical Genetics Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD.
20892
TOTAL MAN-YEARS:
2.3
PROFESSIONAL:
1.5
OTHER:
0.8
CHECK APPROPRIATE BOX(ES)
J (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues [T] (c) Neither
Summary OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Retroviral vectors can be used to transfer genes effectively into cell lines and primary cells. Less is known,
however, whether these vectors can be used to transfer genomic genes into tissue culture cells and
animals to yield tissue- specific expression of the transferred gene. We have now shown, that retroviral
vectors carrying the human beta-globin gene can lead to regulated expression of human globin RNA and
protein in murine erythroleukemia cells. In addition, we have recently designed a new high titer
ecotropic retroviral vector that has been used to transfer the human beta globin gene into CFU-S murine
spleen colonies. The human beta- globin gene is expressed in a vast majority of the CFU-S colonies
indicating preferential integration at transcriptionally active sites, the expression level of the transferred
human beta globin gene was found to be 1-5% of the mouse beta globin- genes.
Infected bone marrow was transplanted into genetically anemic WAA/v mice resulting in production of
h;jman beta globin chains in circulating red cells of long term reconstituted mice. The human beta-
globin gene is detected in all hematopoietic lineages of these mice and it is expressed in a tissue-specific
manner. Bone marrow from a primary recipient that had contained the beta- globin gene for a whole
year was transplanted into a secondary recipient. The secondary recipient contains and expresses the
human beta-globin gene in erythroid cells more than three months after transplantation proving that
the initial gene transfer was targeted to repopulating bone marrow stem cells. Furthermore it has been
demonstrated, that the growth factors IL-3 and IL-6 preserve stem cell function in culture and enhance
retroviral-mediated gene transfer into murine hematopoietic stem cells.
16 DMN/DiR
PHSbMO(Rev. VIM)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02731-04DMN
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJECT (SO characters or less Title must fit on one fine between rne borden.)
Gene Therapy of Inherited Enzyme Deficiencies
PRINCIPAL INVESTIGATOR (i/srofne/,pror'ess/ona/pe/'sonne/6e/otvfhePnnc/pa//nvesf/gafor J (Name. title, laboratory, and institute affiliation)
PI:
S. Karlsson, M.D.
,Ph.D.
Acting Chief, M&MG
OTHERS:
J. Fink, M.D.
Senior Staff Fellow
L. Perry, B.S.
Biologist
P. Correll.B.S.
Special Volunteer
Y Kew, B.S.
Biologist
R. Brady, M.D.
Chief
DMN
NINDS
DMN
NINDS
DMN
NINDS
DMN
NINDS
DMN
NINDS
DMN
NINDS
COORPERATING UNITS (.( *nY>
LAB/BRANCH
Developmental and Metabolic Neurology
SECTION
Molecular and Medical Genetics
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland
20892
TOTAL MAN-YEARS:
9.0
PROFESSIONAL:
4.5
OTHER:
4.5
CHECK APPROPRIATE BOX(ES)
I « ] (a) Human subjects
] (a1) Minors
J (a2) Interviews
I | (b) Human tissues ] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Gaucher's disease is an inherited disorder caused by a mutation of the gene for the enzyme
qlucocerebrosidase. The normal gene for this enzyme has been cloned by several laboratories. We have
constructed high-titer, helper-free recombinant retroviruses containing this gene We have shown that
infection of cell lines from normal individuals and patients with Gaucher's disease with this retroviral
vector results in increased glucocerebrosidase activity. The glucocerebrosidase gene has been transferred
efficiently into progenitor cells and repopulating stem cell of mouse bone marrow and is expressed at the
RNA and protein level in the progeny of CFU-S multipotential progenitor cells following gene transfer.
The gene has also been transferred efficiently into murine hematopoietic stem cells that can be used to
repopulate secondary transplant recipients. The vector genome can be detected in all hematopoietic
lineages and produces human glucocerebrosidase RNA in all hematopoietic tissues tested. The human
glucocerebrosidase gene has been introduced into human hematopoietic progenitor cells with a high-
degree of efficiency (at least 35%). Vector transduced hematopoietic progenitors from Gaucher's
patients produce progeny cells with glucocerebrosidase enzyme values similar to those of normal
individuals.
17 DMN/DIR
PHUMIKIItv 1,114)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02769-03DMN
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between trie borders.)
Exploration of Strategies for the Treatment of AIDS
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Pi: R. O. Brady, M.D.
OTHERS: R. R. O'Neill, Ph.D.
/ the Principal ,
Chief
Staff Fellow
DMN
DMN
NINDS
NINDS
COORPERATING UNITS (if any)
LAB/BRANCH
Developmental and Metabolic Neurology
SECTION
Enzymology and Genetics
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD. 20892
TOTAL MAN-YEARS:
1.5
PROFESSIONAL:
0.5
OTHER:
CHECK APPROPRIATE BOX(ES)
] (a) Human subjects
] (a1) Minors
] (a2) Interviews
J (b) Human tissues [ x | (c) Neither
SUMMARY OF WORK (use standard unreduced type. Do not exceed the space provided.)
Two novel approaches for the treatment of AIDS will be examined. The first encompasses the
development of anti-retroviral agents that traverse the blood-brain barrier. The second utilizes
molecular biological approaches to inhibit virus replication.
18 DMN/DIR
PHSf>(MO(Kev 1.84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01-NS2770-03DMN
PERIOD COVERED
Octoberl, 1989 to September 30, 1990
TITLE OF PROJECT (80 characters or lea. title must fit on one line between the border* I
Strategies for the Treatment of Autoimmune Neuropathies
PRINCIPAL INVESTIGATOR R/tt other professional petvonne/ 6- 'iwrhe Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: R O Brady, M.D
OTHERS: R H. Quarles, Ph D
M.C. Dalakas, PhD
N. W. Barton, M. D.,
A. L. Weis, PhD
Ph D
Chief
Chief
Unit Chief
Section Chief
DMN
NINDS
LMCN
NINDS
MN
NINDS
DMN
NINDS
Eastman Kodak Co
COORPERATING UNITS (,fany)
Laboratory of Molecular and Cellular Neurobiology
Eastman Kodak Company Resarch Laboratories, Rochester, NY.
LAB/BRANCH
Developmental and Metabolic Neurology Branch
SECTION
Enzymology and Genetics
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland
20892
TOTAL MAN-YEARS:
PROFESSIONAL:
1.5
OTHER:
CHECK APPROPRIATE BOX(ES)
I x I (a) Human subjects
] (a1) Minors
] (a2) Interviews
~] (b) Human tissues J (c) Neither
SUMMARY OF WORK (use standard unreduced type. Do not exceed the space provided.)
A number of patients with peripheral neuropathy associated with benign monoclonal qammopathy have
been shown to have circulating antibodies to specific glycoconjugates The principle immunoreactive
epitopes in a number of these individuals have been identified It is proposed that immunoaffinity
columns be prepared with appropriate ligands for selective apheresis trials to remove the reactive
immunoglobulin from the patients's serum and to assess the effect tof this procedure on the clinical
course of the patients.
19 DMN/DiR
PHSbMOIltev 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02771-02DMN
PERIOD COVERED
October 1 , 1 989 to September 30, 1 990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Modification of Growth Factor Genes by Gene Targeting
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: A. Kulkarni.Ph.D.
OTHERS: S. Karlsson, M.D., Ph.D.
L. Perry, B. S.
L. Cairns, Ph.D.
Sen. Staff Fellow
DMN
NINDS
Acting Section Chief
DMN
NINDS
Biologist
DMN
NINDS
Visiting Fellow
DMN
NINDS
COORPERATING UNITS (if any)
LAB/BRANCH
Developmental and Metabolic Neurology Branch
SECTION
Molecular and Medical Genetics
INSTITUTE AND LOCATION
NINDS, NiH.Bethesda, MD. 20892
TOTAL MAN-YEARS:
2.5
PROFESSIONAL:
2.0
OTHER:
0.5
CHECK APPROPRIATE BOX(ES)
] (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues QT] (c) Neither
SUMMARY OF WORK (Use standard unreduced type- Do not exceed the space provided.)
Gene targeting by homologous recombination can be used to activate or inactivate cellular genes in
eukaryotic cells. The objective of this project is to alter the functional status of genes that control growth
and maturation of specific tissues and study the biological consequences of these molecularly defined
alterations. The interleukin 3 gene has been chosen for this study as it is a well- characterized gene
whose biological funtions are well known. Activation of the interleukin 3 (IL-3) gene will be attempted
in a cell line (32-D) which is IL-3 dependent for growth. Successful activation will therefore lead to a
selective phenotype which will minimize screening and iidentification of the cells where the gene has
been targeted to the correct location. Similarly, the biological importance of the IL-3 gene to its
will be studied in the context of a whole organism by targeting a defective IL-3 gene to its cognate
counterpart in embryonic stem cells by homologous recombination. These cells will subsequently be
used to generate transgenic mice containing a defective IL-3 gene. Using the same approach, attempts
are being made to target the transforming growth factor beta gene in embryonic stem cells in order to
better define the biological role of this growth factor gene.
20 DMN/D1R
PHS 6040 (Rev. 1/114)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02782-02DMN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (SO characters or less. Title must fit on one line between the borders.)
Preparation of Transgenic Murine Analogs of Human Metabolic Storage Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: R.R. O'Neill, Ph.D. Senior Staff Fellow DMN NINDS
Others: S Karlsson, M.D., Ph.D. Acting Section Chief DMN NINDS
E.Carstea, Ph.D. Staff Fellow DMN NINDS
L. Cairns, PhD. Visiting Fellow DMN NINDS
A. Kulkarni.Ph.D. Senior Staff Fellow DMN NINDS
R. Brady, M.D. Chief DMN NINDS
COORPERATING UNITS f/f any;
LAB/BRANCH
Developmental and Metabolic Neurology
SECTION
Enzymology and Genetics
INSTITUTE AND LOCATION
NINDS, NIH.Bethesda, MD. 20892
TOTAL M",f "EARS:
3.0
PROFESSIONAL:
3.0
OTHER:
CHECK APPROPRIATE BOX(ES)
I [ (a) Human subjects
] (a1) Minors
J (a2) Interviews
J (b) Human tissues I x | (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Summary: Molecular genetic principles will be used to produce murine analogs of inherited human
diseases for which spontaneous mutants are not available. Development of a transgenic mouse model of
Gaucher's disease is a high priority aspect of this project. Murine genomic and cDNA clones were
obtained, characterized, and mapped to mouse chromosome 3 and introduced into murine embryonic
stem cells grown in cell culture. Methods have been devised using the PCR and Southern blot techniques
to identify which few clones of the 108 progenitor cells that were electroporated are the cells carrying an
inactivated glucocerebrosidase gene.
21 DMN/DIR
PHS 6M0 (Rev 1«4|
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02785-02DMN
PERIOD COVERED
October 1, 1989 to September 30, 1990
TITLE OF PROJ ECT (SO characters or less. Title must fit on one line between the borders.)
Generation of Mice with Sickle Cell Anemia
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: S. Karlsson, M.D.Ph.D.
OTHERS: B. Dropulic, Ph.D.
L. Perry, B.S.
A. Schecter, M.D.
C. Noguchi.Ph.D.
F. Shafer.Ph.D
Acting Chief, M&MG
DMN
NINDS
Visiting Fellow
DMN
NINDS
Biologist
DMN
NINDS
Chief,
LCB
NINDS
Sen. Scientist
LCB
NINDS
Staff Fellow
LCB
NINDS
COORPERATING UNITS Ofsny)
Laboratory of Chemical Biology, NIAID
Dept. of Biology, Univ. of S. Carolina, INSERM, CreteM, France
LAB/BRANCH
Developmental and Metabolic Neurology Branch
SECTION
Molecular and Medical Genetics
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD. 20892
TOTAL MAN-YEARS:
3.7
PROFESSIONAL:
2.5
OTHER:
1.2
CHECK APPROPRIATE BOX(ES)
] (a) Human subjects
] (a1) Minors
] (a2) Interviews
J (b) Human tissues [ * | (c) Neither
SUMMARY OF WORK (use standard unreduced type. Do not exceed the space provided)
Transgenic mouse technology can be utilized to produce animals expressing a foreign gene to high levels
of its RNA and protein. The objective of this project is to generate mice that contain an abnormal beta-
globin gene (beta- sickle Antilles). This gene has two mutations and generates sickle cell anemia in a
heterozygous individual. High level expression of the gene is obtained by inserting the dominant control
region from the human beta globin locus in cis to the Antilles gene. This dominant control region allows
high level globin expression to occur. The human alpha- globin gene is also inserted in cis in order to
generate mice that can produce high levels of beta sickle Antilles and human alpha globins. Three
independent transgenic mouse lines that express both human globin genes in a coordinated fashion have
been generated. The production levels of the human globin chains in the founder mice is 15-20%
compared to the mouse globin levels. The founder mice exhibit sickling erythrocytes in vitro but not m
vivo. To generate in vivo sickling, the levels of human globins will be increased by breeding the animals
to homozygosity and by mating them with thalassemic mice.
I'd DMN/DIR
PHSbMOfllev l'Bfl)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01NS02816-01DMN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must fit on one line between the borders.)
Synthesis of Inhibitors of N-Myristoyltransferase
PRINCIPAL \NV£S7\GATOR (List other professional personnel belo* the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
PI: S.P.Miller, Ph.D. Senior Staff Fellow DMN NINDS
Others: S.A. French, BS Chemist DMN NINDS
J. M. Quirk, M S. Biochemist DMN NINDS
R.R. O'Neill, Ph.D. Senior Staff Fellow DMN NINDS
R.O. Brady, M.D. Chief DMN NINDS
COORPERATING UNITS dtany)
LAB/BRANCH
Developmental and Metabolic Neurology
SECTION
Neurochemical Methodology
INSTITUTE AND LOCATION
NINDS, NIH.Bethesda, MD 20892
TOTAL MAN-YEARS:
0.8
PROFESSIONAL:
0.4
OTHER:
CHECK APPROPRIATE BOX(ES)
] (a) Human subjects
] (a1) Minors
J (a2) Interviews
[x~1 (b) Human tissues ] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Summary: This research project was begun this year with the object of preparing new inhibitors of the
enzyme myristoyl-CoA: protein N-myristoyltransferase (NMT). This enzyme catalyzes the covalent
modification of specific proteins by acylation of the N-terminal amino group with myristoyl-CoA.
Biomedically important proteins which are myristoylated include oncoproteins such as p60srcand the gag
polyproteins of a number of retroviruses including HIV. Interference with myristoylation has been shown
to greatly reduce the transforming potential of p60src without affecting its tyrosine kinase activity.
Blocking of the myristoylation of the HIV gag protein by site-directed mutagenesis (N terminal glycine
alanine) prevents viral particle release. Thus, potent chemical inhibitors of NMT should be useful for
studies of the biochemical function(s) of protein myristoylation, and could potentially have therapeutic
significance A group of target structures have been identified as potential transition state or combined
product inhibitors of NMT. Both irreversible and competitive inhibitors are sought. Synthesis,
purification and characterization has been completed for 12 new compounds. These are being tested for
inhibition of NMT both in vitro and with cells in tissue culture
23 DMN/DIR
PHSMMOIRav l «4)
>
w
VI
X
m
30
30
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5
H
00
Annual Report
October 1, 1989 through September 31, '990
Experimental Therapeutics Branch
Clinical Neurosciences Program, DIR
National Institute of Neurological Disorders and Stroke
Table of Contents
RESEARCH SUMMARY
page
Pharmacology and Cellular Biology of 17
Peptidergic Neurons
Z01 NS 02578-08 ET
Biochemical and Pharmacological Studies 18
of Dopamine Receptors
Z01 NS 02263- 14 ET
Pharmacology and Physiology of the 19
Substantia Nigra and Basal Ganglia
Z01 NS 02139-16 ET
Pharmacology, Biochemistry and Physiology 20
of Central Neurotransmitters
Z01 NS 02265-14 ET
ANNUAL REPORT
October 1, 1989 through September 31, 1990
Experimental Therapeutics Branch
Clinical Neurosciences Program, DIR
National Institute of Neurological Disorders and Stroke
Thomas N. Chase, M.D., Chief
The goal of the Experimental Therapeutics Branch is to develop improved
pharmacotherapies for neurologic disease. To this end the Branch operates a
vertically integrated program of research extending from basic neurobiology
to clinical trials. The focus of these investigative efforts remains on
neurodegenerative disorders which impair motor and cognitive function.
The Branch is organized into four highly interactive operating components:
1) The Molecular Pharmacology Unit, directed by Dr. David Sibley, conducts
molecular and biochemical investigations to characterize central transmitter
receptors and information transduction processes; 2) The Genetic Pharmacology
Unit, since July of 1990 led by Dr. M Maral Mouradian, seeks at the molecular
level to develop pharmaceutical approaches to the selective regulation of
gene expression within the mammalian central nervous system (CNS); 3) The
Neurophysiologic Pharmacology Section, operating under the leadership of Dr.
Judith Walters at the neuronal network level, studies basal ganglia function
especially in relation to dopamine receptor mechanisms and the effect of
drugs that influence motor behavior; and 4) The Clinical Pharmacology Section
under Dr. Thomas Chase works with neurologic patients and in animal models to
elucidate pathophysiologic mechanisms and evaluate novel pharmaceutical
interventions.
Genetic Pharmacology Unit
Molecular Regulation of Transmitter and Receptor Genes
The goal of the Genetic Pharmacology Unit is the study of molecular
regulation of neurotransmitter and transmitter receptor genes, and through
this understanding, develop novel strategies in the design of improved
therapeutic agents for neurologic disorders. The major thrust of the Unit
over the past six years has been examination of the regulation of
neuropeptide gene expression using the pro-opiomelanocortin (POMC) gene as a
model system. In FY90, the Unit initiated a program to study the genetic
regulation of the D1 and D2 dopamine receptor genes of relevance to basal
ganglia diseases and their treatment.
In FY88, experiments were begun concerning the regulatory elements in
the 51 upstream region of the mouse POMC gene. The promoter region of this
gene was subcloned into pGEM and used in exonuclease protection assays to
determine protein binding sequences that are involved in modulating
transcription. Initial experiments conducted in FY88-89 have identified
several protein binding sites in the -1000 to +1 bp region of the mouse POMC
promoter. One of these sites located between -119 and -106 base pairs (bp)
upstream from the transcription start site, was found to be homologous to the
consensus sequence of AP-2 (a DNA-binding protein thought to convey second
1 - ET/IR
messenger signals to transcriptional machinery). In FY89, this putative AP-2
site was characterized in detail using gel retardation experiments. Results
indicated that this site binds to nuclear factor(s) present in AtT-20 cells
in a specific manner, completely abolished with excess DNA of the authentic
AP-2. In addition, this putative AP-2 site appears physiologically relevant,
with its ability to bind to nuclear factor(s) dependent on phosphorylation.
In FY90, another strong exonuclease stop site located between -137 and
-131 bp from the transcription initiation site of the POMC gene was
characterized further because of its sequence homology to AP-1 binding site
and the previously shown inducibility of POMC transcription in AtT-20 cells
with phorbol esters. Gel shift studies using a double-stranded
oligonucleotide containing three copies of this sequence and AtT-20 cell
nuclear extract revealed that a heat labile factor(s) exhibited specific
binding to this sequence. Although this DNA sequence had about 70% homology
to the consensus sequence for AP-1, its interaction to the nuclear factor(s)
was unaffected by an oligonucleotide containing authentic AP-1 sequence.
Furthermore, using specific antibodies against each peptide component of AP-
1, c-fos and c-Jun, no significant changes were noted. Current studies
address the role of phorbol ester treatment and phosphorylation in the
interaction of nuclear factor(s) with this new protein binding sequence.
Also in FY90, studies of AtT-20 cell signal transduction machinery
involved in POMC gene expression were continued. Based on findings from this
laboratory in FY88-89 about the role of protein kinase C (PKC) in
corticotroph releasing hormone (CRH)-elicited 8-endorphin secretion, signal
transduction pathways in relation with other secretagogues were studied. The
cytokine, interleukin-1 (IL-1), which has been shown to stimulate 8-endorphin
release from AtT-20 cells and to potentiate the effects of other agents such
as CRH, forskolin and TPA, were studied in detail. Unlike CRH, IL-1-induced
B-endorphin release was found to be independent of PKC. Prolonged treatment
with TPA, which was shown to desensitize an 87-KDa protein (a substrate for
PKC), had no effect on IL-1-induced phosphorylation of 20-, 60- and 87-KDa
proteins, indicating that the phosphorylation of these proteins does not
involve PKC. Treatment of AtT-20 cells with IL-1 markedly phosphorylated 19-,
20- and 60-KDa proteins within minutes, presumably by early activation of
protein kinases. Since IL-1 does not generate cAMP in AtT-20 cells, cAMP-
dependent protein kinase is not involved either. The presence of IL-1 was
required only initially for a short time to induce late secretion of 8-
endorphin in AtT-20 cells suggesting that once IL-1 generates an early
signal, its presence is no longer necessary for the subsequent secretion of
8-endorphin. Based on recent observations that c-fos and c-jun have an
important physiologic role in IL-1-induced 8-endorphin secretion, the
possible involvement of these proto-oncogenes in relation to CRH and other
secretagogues are currently being investigated using antisense
oligonucleotides.
In FY90, studies aimed at investigating the molecular regulation of
dopamine receptor genes were begun. For the D2 receptor, the cDNA of which
has been recently cloned by several groups, efforts are currently underway to
clone the gene, including the 5' upstream elements. Because the D2 receptor
gene has long introns, and the available cDMA clones are not full-length, two
strategies are adopted to obtain genomic clones having the most 5' sequences.
One strategy uses a 5' stretch cDNA library subjected to polymerase chain
2 - ET/IR
reaction (PCR), while the second uses a full-length cDNA clone ootained from
an initial screen of a rat striatal cDNA library with oligonucleotide probes
homologous to the published D2 sequence. We have completely sequenced the 5'
region of this cDNA clone which is upstream from all published sequences thus
far. In addition, this cDNA clone was subjected to PCR analysis to obtain a
DNA copy of this 51 region. Northern blot analysis using rat striatal
poly(A)+ mRNA has indicated that this product hybridizes with a message of
the same size as the known rat D2 mRNA. Subsequently, this PCR product was
used to screen a rat genomic library, yielding several candidate clones that
are currently being characterized. For the D1 dopamine receptor which appears
to be intronless, the recently cloned cDNA for the rat D1 receptor (by the
Molecular Pharmacology Unit of the Experimental Therapeutics Branch) was used
as a probe to screen a human genomic library; this screen has yielded 13
positive clones that are now being characterized using Southern blots,
restriction analysis and DNA sequencing.
Molecular Pharmacology Unit
The Molecular Pharmacology Unit continued investigating the biochemical,
molecular, and pharmacologic properties of dopamine receptors in FY90. The
long term goal of the Unit is to characterize neurotransmitter receptor-
mediated information transduction, and its regulation, across neuronal
membranes. Dopamine receptors are used as representative model systems for
the large class of neurotransmitter receptors which are linked to their
biochemical effectors via guanine nucleotide-binding regulatory (G) proteins.
In order to characterize the D1 and D2 receptors at biochemical and molecular
levels and study their regulation, there are two major interrelated lines of
research which are ongoing: 1) investigation of the cell biology, function
and regulation of the receptors at the protein level; and 2) the molecular
cloning of the receptor genes and investigation of gene structure and
regulation in normal and pathophysiological states.
1. Cell Biology and Regulation of Dopamine Receptors.
Our efforts in FY90 have been to capitalize on our discovery of various
mammalian cell lines that express either D1 or D2 receptors and conduct
studies of dopamine receptor regulatory mechanisms. For D1 receptors we have
been utilizing the NS20Y murine neuroblastoma cell line which expresses high
levels of D1 receptors functionally coupled to the Gs protein and the
adenylate cyclase. Experimentation in FY90 has indicated that acute exposure
of these cells to dopamine results in a profound desensitization of the D1
receptor-stimulated adenylate cyclase activity as well as a loss in D1
receptor radioligand binding activity. This agonist-induced desensitization
response appears to be homologous in nature as only the dopamine-mediated
response is attenuated. This process also appears to be multimechanistic
involving receptor uncoupling, internalization and degradation. We have also
found in FY90 that the D1 receptors in these cells can undergo a cAMP-
mediated heterologous form of desensitization where the response to multiple
hormones as well as nonhormonal stimulators is attenuated. We are currently
investigating the underlying biochemical and molecular mechanisms associated
with these desensitization processes and are also investigating the effects
of chronic (days) agonist or antagonist exposure as well as evaluating the
withdrawal periods to detect a potential supersensitization response. In
addition, we are examining the effects of treating the cells with other
3 - ET/IR
agents such as steroid hormones to see if they may also modulate receptor
activity.
For D2 receptors, we have been characterizing the Y-79 human
retinoblastoma cell line which expresses significant quantities of functional
D2 dopamine receptors after attachment and differentiation with sodium
butyrate. In FY90 we have found that pretreatment of the cells with dopamine
results in a diminution in the subsequent ability of dopamine to inhibit
adenylate cyclase activity. This effect is time-dependent, reaching maximal
desensitization after ~24 hr. Preincubation of the cells with dopamine also
promoted a time-dependent increase (~3 fold) in the Kd for [3H]methylspiperone
with no change in its Bmax- *n contrast, after dopamine preincubation, the
KD for p25i]sulpride is unchanged while its Bmax is reduced by -50? upon
maximum desensitization. These results suggest that agonist preincubation
results in a functional uncoupling of the D2 dopamine receptor as well as a
loss in its ligand binding activity. We have thus established in FY90 that
the Y-79 cell line serves as an ideal model system to study agonist
regulation of D2 dopamine receptors and are currently investigating the
underlying biochemical and molecular mechanisms associated with this
process(es) .
In FY90, we completed development of an irreversible D2 receptor
antagonist. Previously, we synthesized N-(p-isothiocyanatophenethyl)
spiperone (NIPS) a derivative of the high affinity D2 selective antagonist,
spiperone. The isothiocyanate moiety in this ligand renders it a potential
affinity probe of the D2 receptor. This property was evaluated in FY90. In
vitro evidence was obtained using rat striatal membranes for an irreversible
inhibition of D2 receptors. Experiments in vivo indicated that NIPS can
almost completely inactivate D2 receptors without affecting D1 dopamine
receptors in the striatum or alphal, alpha2, or muscarinic cholinergic
receptors in the frontal cortex or 5-HTia receptors in the hippocampus.
These results suggest that NIPS is a highly selective irreversible
inactivator of D2 dopaminergic receptors and may prove useful in in vitro and
in vivo functional studies of this receptor subtype.
In FY90 we continued our synthesis program for novel fluorescently-
labeled ligands with high affinity and specificity for D1 and D2 dopamine
receptors. The interaction of these fluorescent ligands with dopamine
receptors was evaluated by examining their ability to compete for the binding
of the D1 and D2 selective radiolabeled antagonists. In addition, we
evaluated their affinities for 5HTia, 5HTic, and 5HT2 serotonin receptors.
In order to further evaluate the utility of these fluorescent probes for the
cellular visualization of dopamine receptors, we have collaborated with Dr.
Majorie Ariano who has successfully demonstrated receptor-specific regional
and cellular fluorescent labeling of D1 and D2 receptors in slices of rat
forebrain, pituitary, retina, and superior cervical ganglion. The regional
localization of the dopamine receptors reflect previous work ascertained
using in vitro receptor autoradiography. We are currently using these
probes in simultaneous labeling experiments to ascertain whether or not D1
and D2 receptors can be shown to colocalize in the same neurons within the
striatum and other brain regions.
In FY90 we initiated a project to develop antibodies to the D2 dopamine
receptor by raising antibodies to synthetic peptides derived from the cloned
4 - ET/IR
cDNA sequence of this receptor (see below). This approach of preparing
antibodies against synthetic peptides has proven most successful in preparing
antibodies against native proteins of which the sequence is known but not
enough of the protein purified to use directly as an immunogen.
Immunohistochemical and immunocytochemical localization of the D2 receptor in
rat brain slices was performed using the antisera raised against the NH2
terminal 18 residues. Within the striatum, about 50% of the medium-sized
neurons were labeled as well as an occasional large cholinergic interneuron.
Electron microscopic examination of the striatal neuropil demonstrated that
numerous reactive synaptic structures were present of both symmetrical and
asymmetrical classifications. The data show that the D2 dopamine receptors
can be localized in the projection neurons and also in the two
morphologically defined aspiny interneuron populations of the striatum.
2. Molecular Cloning and Expression of Dopamine Receptors.
In FY90 we continued investigations related to our discovery of RNA
splice variants of the D2 dopamine receptor. These RNA splice variants
result in long (D2L) and short (D2S) receptor isoforms. It should be noted
that this finding represents the first known example of alternative mRNA
splicing giving rise to two different neurotransmitter receptor isoforms. To
investigate the functional properties of the short (D2S) and long (D2L)
isoforms of the D2 receptor, we have stably expressed both rat cDNAs in CHO
cells in FY90. Following transfection, several clonal cell lines were
isolated which exhibited D2 receptor binding with Bmax values ranging from
0.5 to 5 pmol/mg protein as determined by [3H]methylspiperone binding. Both
receptor isoforms revealed a Kd for [3H]methylspiperone of -60 pM.
Competition assays for [3H]methylspiperone binding by dopamine antagonists
to both D2L and D2S exhibited identical affinities and rank orders of
potency. Inhibition of [3H]methylspiperone binding by dopamine revealed the
presence of high and low affinity agonist binding states with both receptor
forms exhibiting similar values for the affinities (KH and KL) and
proportions of the agonist binding states. In addition, both receptor
isoforms show a complete loss of high affinity agonist binding with the
addition of guanine nucleotides. Both of the D2 receptor isoforms expressed
in CHO cells exhibit dopamine- induced inhibition of forskolin-stimulated cAMP
accumulation by -60% with EC50 values of -100 nM. In each case, dopamine
inhibition of cAMP accumulation is prevented by pretreatment of the cells
with pertussis toxin. Finally, for both receptor isoforms, pretreatment of
the cells with 100 uM dopamine for 4 to 24 hours results in desensitization
of the ability of dopamine to inhibit cAMP accumulation. These data suggest
that the D2L and D2S receptors are functionally similar in their ligand
binding properties and linkage to adenylate cyclase inhibition.
In FY90, we have collaborated with Dr. Chris Felder in the Laboratory of
Cell Biology, NIMH to examine what role the D2 receptor might play in the
production of the second messenger, arachidonic acid. In transfected CHO
cells expressing the rat D2L receptor, the calcium ionophore, A23 1 87 ,
stimulated the release of arachidonic acid (AA) and this was potentiated by
dopamine in a dose-dependent fashion. Dopamine alone, however, had no effect
on basal AA release. Quinpirole, a D2-selective agonist, augmented A23187-
stimulated AA release, and sulpiride, a D2-selective antagonist blocked this
augmentation. Forskolin, prostaglandin E2, dibutyryl cAMP, 8-(4-
chlorophenylthio) cAMP and pertussis toxin all had no appreciable effect on
5 - ET/IR
both A23l87-stimulated AA release and on the dopamine enhancement.
Inhibition of PKC using long-term phorbol ester desensitization and
pharmacologic inhibitors diminished the dopamine potentiation of AA release.
Preliminary evidence with CHO cells expressing the D2S receptor indicates
that this form of the receptor also augments AA release. These results
suggest that both forms of the D2 receptor may be increasing the release of
AA by a mechanism involving PKC but adenylate cyclase independent.
In FY90 we continued our investigation of a novel D1 receptor subtype
using functional expression assays in Xenopus oocytes that have been injected
with rat striatal poly(A)+ RNA (mRNA). We have found that dopamine is able
to induce Ca2+ mobilization as detected by stimulation of 45ca2+ efflux in
striatal mRNA-inJected oocytes. This response was mimicked by SKF-38393 and
blocked by the D1-selective antagonist, SCH-23390. No efflux was observed
with quinpirole and the dopamine response was not blocked with domperidone, a
D2-selective antagonist. Assay of size-fractionated striatal mRNA using the
oocyte/45Ca2+ efflux assay revealed a single peak of dopamine-stimulated
activity corresponding to an mRNA size of 2.5-3.0 kb. Oocytes which were
injected with the peak mRNA fractions then prelabeled with myo[3H]inositol ,
additionally showed a 4-fold increase in IP3 production in response to
dopamine. In contrast, oocytes injected with the peak mRNA showed no
elevation of cAMP levels in response to dopamine application. Moreover,
exogenously applied dibutyryl cAMP did not elicit the electrophysiological or
the 45ca2+ efflux response. These data thus indicate the existence of a D1
dopamine receptor subtype which is coupled to phosphatidyl inositol (PI)
turnover and Ca2+ mobilization in addition to a D1 receptor subtype which
activates adenylate cyclase.
In FY90 we have begun to clone this novel D1 receptor subtype coupled to
PI turnover. We have constructed a cDNA library, using the size-fractionated
rat striatal mRNA discussed above, in the lambda SWAJ-2 vector. This vector
contains RNA polymerase sites flanking the polylinker and thus the cDNA
insert region. It is thus possible to transcribe RNA from the cDNA off the
vector and inject it into oocytes for translation and expression of the
protein. The cloning strategy consists of dividing the cDNA library into a
number of pools consisting of 10,000-100,000 clones each, running mRNA off
each pool individually and injecting it into oocytes and assaying for a
positive 45ca2+ efflux response. Once a positive pool is identified, it is
subdivided into daughter pools and the process is repeated until a pure clone
is obtained. This approach has been validated by the fact that it has been
recently used to clone the serotonin 5HTic and the substance K receptors,
both of which are coupled to PI turnover and Ca2+ mobilization.
In FY90 we have used NS20Y cells, which express D1 dopamine receptors
linked to adenylate cyclase activation, to investigate the molecular biology
of this receptor subtype. In order to clone this D1 receptor, the polymerase
chain reaction (PCR) method was used to selectively amplify a cDNA sequence
from NS20Y cell mRNA. Poly (A)+ RNA was used to synthesize cDNA by reverse
transcription followed by PCR amplification with sets of highly degenerate
primers derived from the transmembrane sequences of the previously cloned
adrenergic, D2 dopaminergic, and serotonin receptors. This amplification
produced a novel cDNA fragment which exhibits considerable sequence homology
to previously cloned G protein-coupled receptors. In order to characterize
this cDNA further, a full-length clone was isolated from a rat striatal
ET/IR
library using the cDNA fragment as a probe. Sequence analysis of this cDNA
clone indicated that it is indeed a member of the G protein-coupled receptor
family and exhibits greatest homology wLth the previously cloned
catecholamine receptors. Northern blot analysis in various neural tissues
revealed a transcript size of ~U kb which was predominantly located in the
striatum with lesser amounts in the cortex and retina. In contrast, no mRNA
was detected in the cerebellum, hippocampus, olfactory bulb, mesencephalon or
pituitary. In situ hybridization analysis also revealed a high abundance of
mRNA in the striatum as well as in the olfactory tubercle. To establish the
identity of this cDNA, we performed transient expression experiments in COS-7
cells. [3HJSCH-23390, a D1 selective radioligand, exhibited specific,
saturable binding only in cells which were transfected with this cDNA.
Competition binding analysis with a variety of dopaminergic ligands
demonstrated a D1 dopaminergic pharmacology. In addition, dopamine as well
as other D1 -selective agonists stimulated cAMP accumulation in transfected
COS-7 cells. We have concluded that we have cloned a cDNA encoding the D1
dopamine receptor linked to the activation of adenylate cyclase activity.
Importantly, we have found that when RNA is transcribed from this D1 receptor
cDNA clone and injected into Xenopus oocytes, dopamine will stimulate cAMP
accumulation but is incapable of producing a Ca2+ mobilization response.
Consequently, we propose that the D1 receptor subtypes linked to the
activation of adenylate cyclase and phospholipase C be designated D1A and
D1B, respectively.
Neurophvsiological Pharmacology Section
Focus in the Neurophysiological Pharmacology Section is on the
physiology of the basal ganglia and the potential for correcting basal
ganglia dysfunction with drugs. We are concerned with the organization of
the various components of the basal ganglia with one another and with the
roles of the individual nuclei in modulating basal ganglia function. We are
especially interested in the role of the nigrostriatal dopamine system in
regulating basal ganglia output. Dysfunction of these neuronal systems has
been implicated in the etiology of many neurologic diseases, including
Parkinson's disease, tardive dyskinesia, Huntington's chorea, and torsion
dystonia.
1) D1 and D2 dopamine receptors in the basal ganglia.
In the past year, the section has continued to study the roles of the
different dopamine receptor subtypes in the substantia nigra and basal
ganglia and the consequences of selectively stimulating various combinations
of these receptor subtypes. We have previously found that it is necessary
for postsynaptic Dl and D2 receptors to be simultaneously stimulated for the
induction of processes once thought to be independently mediated by the D2
receptor. This observation led to a fundamentally new and exciting perception
about the relative roles of the D1 and D2 receptors located in the basal
ganglia and has had important implications for the use of dopamine agonists
and antagonists in the treatment of neurologic disease which are currently
being explored by ETB clinicians.
A current area of focus is the mechanism underlying the synergistic
interactions of the D1 and D2 receptors. A key question is whether the D1 and
D2 receptors are segregated from one another with respect to their
7 - ET/IR
Localization on the different types of striatal neurons, or whether they
coexist on the same cells. Our electrophysiologic data has indicated that D1
receptors exert a greater effect on the str iatonigral pathway than do D2
receptors, while both appear to regulate the str iatopallidal neurons. Very
recent mRNA localization studies carried out by others in the ETB support our
conclusions. Evidence for some degree of segregation of the receptor subtypes
provides an exciting challenge for the neurophysiologist because it implies
that multisynaptic processes involving multiple neurotransmitters and their
receptors mediate the complex synergistic interactions between the dopamine
receptor subtypes.
During the past year we have made a very significant addition to our
technical capabilities by establishing two intracellular recording setups in
the Section. This has allowed us to begin to investigate how D1 and D2
receptor stimulation affects the physiology of striatal neurons and alters
local synaptic transmission in this nucleus. Striatal neurons in our slice
preparation exhibit a membrane potential and resistance at rest which is
similar to that reported by other investigators. Baseline striatal
recordings generally reveal no fluctuations in membrane potential or
spontaneous action potentials while accumbal and septal neurons are
spontaneously active. We have studied the apparently lower excitability of
striatal neurons by applying cathodal pulses to elicit neuronal activity.
Two types of response have been observed. One group of striatal neurons
responds with repetitive spiking activity. The evoked spikes exhibit a
characteristic waveform beginning with slow depolarization followed by a fast
spike and then repolarization below baseline. A second group of striatal
neurons do not exhibit spikes with steps of constant depolarizing current.
However, electrically-evoked EPSPs may elicit a fast action potential in
these cells without the slow convex ramp of depolarization which is
characteristic of cathodally-evoked spikes in the former group of neurons.
Steady-state current-voltage data obtained using slowly depolarizing ramps of
current has also provided evidence of different membrane properties in
responsive and silent groups of striatal neurons. These differences raise
interesting questions as to whether groups of striatal neurons possess
different ionic channels (intrinsic membrane properties) and what the
functional significance of such differences may be with regard to striatal
organization. Different levels of membrane excitability, for instance, may
be required in projection neurons subserving striatonigral and
striatopallidal pathways. The application of practical anatomical and
electrophysiological techniques to identify neurons in our slice preparation
is an issue currently being explored.
We have also used focal electrical stimulation in the slice studies to
induce neurotransmitter release from local nerve terminals. We have found
this stimulation evokes synaptic potentials in the recorded neurons as well
as slower changes in current voltage relationships. The EPSP elicited in
striatal neurons by focal stimulation is composed of at least three
potentials with different latencies. We are investigating the possibility
that these potentials are mediated by separate transmitters. The potentials
may be mediated directly by an excitatory amino acid or indirectly by amino
acid driven release of another excitatory transmitter in our slice
preparation. The fastest potential appears to involve a non-NMDA receptor.
The slower potentials can be blocked by an NMDA receptor antagonist.
8 - ET/IR
Following focal electrical stimulation of slices, we have also observed
changes in current-voltage relationships suggesting increased membrane
resistance near rest. Current-voltage changes are also mduced by
superfusion of dopamine agonists, but these appear to be different from those
produced by electrical stimulation of the slice, suggesting endogenous
dopamine is not mediating the latter effects. When dopamine agonists are
superfused, however, the evoked EPSPs are depressed with a slow time course
similar to that associated with changes in the I-V relationship. D1 and D2
agonists could be acting pre- and postsynaptically to produce this depression
and may have mechanistically different actions on each of the EPSPs we have
recorded in striatal neurons. From these first results, dopamine appears to
be a modulator of voltage-dependent conductances and may regulate repetitive
firing in striatal neurons. While dopamine does not appear to be "the
modulatory factor" liberated by low frequency electrical stimulation of our
slice preparation, we believe that dopamine may act together with other
neuromodulator candidates (e.g. enkephalin, substance P, dynorphin,
somatostatin) as possible comediators of this action. Pharmacologic
characterization of this dopaminergic action and the affected membrane
conductances using selective agonists and antagonists is needed, however, to
establish the specificity of this action and significance in neuronal
function. Future studies will extend these investigations to determine
whether concurrent D1 and D2 stimulation is necessary for full expression of
dopaminergic effects and will attempt to address the mechanism of this action
at a cellular level.
2) D2 Autoreceptor/D2 Postsynaptic Receptor Studies
It has long been thought that one site where D2 dopamine receptors are
located in the substantia nigra and basal ganglia is on the dopamine cells
themselves. The existence of these receptors, termed autoreceptors, has been
supported by a large body of experimental evidence and now appears confirmed
by localization of mRNA for the receptor protein. The autoreceptors on the
dopamine cell body induce inhibition of dopamine cell firing and those on the
dopamine terminals induce a decrease in dopamine synthesis and release. The
possibility that these D2 autoreceptors might be functionally or
fundamentally different from those located on the postsynaptic neurons has
been one area of active interest in both preclinical and clinical areas in
the Branch.
The recent discovery that the dopamine autoreceptor is genetically
identical to the D2 receptor located on the neurons postsynaptic to the
dopamine cells rules out fundamental receptor differences between the
autoreceptor and postsynaptic D2 receptors; attention has now turned to
understanding the mechanisms which underlie the apparent functional
differences between the D2 receptors at the two sites. Our demonstration of
the existence of a greater receptor reserve at dopamine autoreceptor sites
relative to postsynaptic D2 dopamine receptor sites has provided an
explanation for the properties of the so called "autoreceptor selective"
agonists. Differences in spare receptor number at the different sites can
allow a partial agonist to act like a fully efficacious agonist at the D2
autoreceptors while providing a "clamp" at postsynaptic D2 receptors. By
occupying but only weakly stimulating at postsynaptic sites where there are
fewer spare receptors, a partial agonist can provide some low level chronic
stimulation while blocking detrimental fluctuations or excessive stimulation
9 - ET/IR
from endogenous neurotransmitter. This could have advantages over trying to
maintain therapeutically appropriate levels of receptor stimulation or
blockade by careful adjustment of blood levels of more efficacious agonists
or antagonists. We have recently evaluated N-0923 and N-0924 mth respect to
these properties. The former is a highly potent and apparently efficacious
dopamine D2 agonist, but the latter is less potent and also less efficacious,
acting as a partial agonist. The Pharmacology Section is investigating the
therapeutic properties of these drugs in Parkinson patients.
3) Consequences of Dopamine Cell Degeneration in the Basal Ganglia
A third focus of investigation this year has been the neurophysiological
consequence of dopamine cell degeneration. The effects of stimulating the
individual dopamine receptor subtypes in the basal ganglia are altered by
chronic dopamine denervation. Moreover, we have shown that lesion of the
dopaminergic nigrostriatal pathway differentially affects dopamine receptor
mediated effects on the striatopallidal versus the striatonigral pathways.
Consequences of intermittent administration and chronic infusion of 1-DOPA
have been investigated in these animals in conjunction with studies on the
behavioral and biochemical effects of these treatments currently being
conducted in the Clinical Pharmacology Section. These studies have shown that
large variations in blood levodopa levels leads to desensitization of D1 and
GABA receptor mediated mechanisms of basal ganglia output through the
substantia nigra pars reticulata. We have also been investigating the changes
in dopamine receptor function following chronic reserpine treatment; a
strategy which is providing new insight into the compensatory responses of
systems postsynaptic to the dopamine cells following dopamine depletion.
Systemic administration of a D1 agonist causes changes in activity at sites
downstream to the striatum which are different from those seen in control but
in the opposite direction from those seen in the more chronically lesioned
rats. Most recently we find similar changes in rats with relatively short-
term lesions of the striatonigral pathway. This evidence for a biphasic
response pattern in dopamine agonist effects after dopamine depletion
indicates a greater range and complexity in the compensatory responses to
dopamine loss than previously appreciated. Mechanisms will be explored in
the coming year. Intracellular recording studies may provide significant
insight into these phenomena. The findings are immediately relevant to
strategies for treatment of parkinsonism and tardive dyskinesia.
4) Excitatory Amino Acids and the Pedunculopontine Tegmental Nucleus in
Regulation of Basal Ganglia Function
Another series of questions involves the relationships of the various
components of the basal ganglia to one another. These "systems"-related
issues have been receiving clinical attention as new strategies are
considered for compensating dopamine cell loss by addressing "downstream"
change induced by the lesion. More selective glutamate antagonists have
become available leading to concern about how excitatory amino acids affect
movement and regulate globus pallidus and dopamine cell activity, especially
via the subthalamic nucleus. In addition to intracellular recording
investigations into glutamate' s role in striatal function, we have initiated
studies using ketamine- induced anesthesia and other NMDA receptor antagonists
to examine the role of NMDA receptors in drug and stimulation induced
responses in various basal ganglia and substantia nigra nuclei.
10 - ET/IR
The role of cholinergic and excitatory amino acid outputs from the
pedunculopontine tegmental nucleus in regulating basal ganglia function is
another newly appreciated and potentially significant issue. The
pedunculopontine tegmental nucleus is a relatively unexplored region only
recently emerging as an area important for basal ganglia function.
Neuroanatomic tracing studies have identified extensive connections between
the pedunculopontine tegmental nucleus and the basal ganglia and substantia
nigra. Implication of the pedunculopontine tegmental nucleus in certain
neurologic disorders is derived from demonstration of pedunculopontine
tegmental nucleus degeneration in progressive supranuclear palsy, Parkinson's
disease, and Alzheimer's disease. A further link between the
pedunculopontine tegmental nucleus and changes associated with Parkinson's
disease has been demonstrated in 2-deoxyglucose studies in MPTP-treated
monkeys by Perino and coworkers. We have found that the pedunculopontine
tegmental nucleus appears to exert a very significant tonic influence on
substantia nigra dopamine cell activity. No other afferent of the region has
been shown to have such an effect. Indications for a functional link between
the pedunculopontine tegmental nucleus and the substantia nigra dopamine
cells have raised questions about whether pedunculopontine tegmental nucleus
(PPN) degeneration and dopamine cell loss in Parkinson's disease are
independent phenomena or somehow causally related. We have successfully
recruited a new Staff Fellow with an extensive research background in
pedunculopontine tegmental nucleus neurophysiology and are initiating new
studies in this area. Preliminary evidence suggests that both mono- and
polysynaptic pathways may be involved in mediating the influence of the
pedunculopontine tegmental nucleus on the dopamine cells. A combination of
systemic and microiontophoretic studies are being employed to address the
nature of the neurotransmitter(s) and receptor subtypes involved in
pedunculopontine tegmental nucleus projections to midbrain dopamine neurons
to further elucidate the role this nucleus plays in regulation of dopamine
cell function.
Clinical Pharmacology Section
Research conducted by the Clinical Pharmacology Section links basic
neuroscience investigations carried out by other Branch components with the
neurologically-disordered patient. Clinical and preclinical studies mainly
apply transmitter pharmacologic approaches to the development of improved
symptomatic therapies. In addition, pathogenetic mechanisms are studied that
might provide a basis for pharmaceutical interventions that attempt to modify
the basic disease process. The Section's investigative efforts remain
focused on Parkinson's disease and related extrapyramidal disorders and to a
lesser extent on Alzheimer's disease and related degenerative dementias.
Extrapyramidal Disorders
Studies of the pathogenesis and treatment of the peak dose dyskinesias
and fluctuations of the wearing-off and on-off types that ultimately disable
most levodopa-treated Parkinsonian patients continue as a major target of
Section research. Our earlier clinical pharmacologic studies suggested that
wearing-off phenomena may be attributable to a progressive shortening of the
antiparkinsonian action of levodopa due to nigrostriatal dopamine system
degeneration. On-off fluctuations and the abnormal involuntary movements
11 - ET/IR
occurring at maximal drug levels, on the other hand, appear to reflect the
participation of pathogenetic mechanisms in addition to dopamine cell
degeneration. Previous Section studies indicated that the levodopa dose-
antiparkinsonian response relation becomes progressively steeper as both the
duration of symptoms and the duration of levodopa exposure increase. As this
dose-response curve becomes nearly vertical, small variations in dopamine
levels at postsynaptic receptor sites may become sufficient to abruptly
switch patients between the on and off states. Furthermore, our finding of a
progressive narrowing of the therapeutic window for levodopa such that the
threshold dose for dyskinesia induction ultimately approximates the threshold
dose for parkinsonism reduction could explain the increasing frequency of
peak dose dyskinesias that complicate levodopa therapy in advanced
Parkinson's disease. Indirect clinical evidence suggests that these changes
may reflect secondary alterations at the postsynaptic level arising as a
consequence of chronic intermittent stimulation. Work during the past year
has sought to both extend and explicate these observations.
Consistent with the view that wearing-off fluctuations reflect the loss
of natural storage sites within dopamine terminals, we were the first to
report these phenomena promptly disappear when optimal dose levels of
levodopa are administered by continuous intravenous infusion. Results from
studies of continuous parenterally administered levodopa conducted during the
past 3 years in more than 100 Parkinsonian patients have recently been
analyzed. The variability in plasma Dopa levels attending standard oral
dosing schedules could immediately be reduced by more than 80% by converting
to intravenous administration. Although wearing off fluctuations disappeared
during the first day of levodopa infusion, fluctuation of the on-off type
only gradually improved over the ensuing days and weeks. Thus, when patients
were examined at the end of their first day of continuous infusion, those
Judged clinically to exhibit only wearing off phenomena on oral levodopa had
a 52% decrease in motor response variability, while those considered to also
manifest on-off fluctuations had only a 12% decrease. Most patients judged to
have either wearing-off or on-off phenomena actually have a combination of
both responses, although in widely differing ratios. The extent to which
their motor fluctuations diminish with conversion to infusion therapy
depended on the degree to which on-off phenomena contribute to their overall
response instability. These observations support the view that since wearing
off fluctuations clear promptly upon stabilization of circulating levodopa
levels, they are most likely due to the loss of natural storage sites for
dopamine as a consequence of the progressive loss of dopaminergic neurons.
With advancing disease, a diminishing proportion of intrasynaptic dopamine
originates in residual dopaminergic terminals that are able to store and
release the transmitter amine under appropriate neuronal control, and more
derives from nondopaminergic cells lacking these capacities. Under such
circumstances, swings in dopamine precursor levels that attend oral dosing
produce concomitant swings in dopamine receptor stimulation.
Recent findings have also clarified the pathogenesis of the putative
postsynaptic changes underlying on-off fluctuations and peak dose
dyskinesias. In response to 10 days of around-the-clock intravenous levodopa
infusions, motor fluctuations gradually diminished. Clinical improvement was
associated with a shift of the levodopa dose-antiparkinsonian response curve
to the right and the therapeutic index improved substantially as patients
evidenced a marked increase in their antiparkinsonian response at a dose that
12 - ET/IR
produced no significant change in the severity of their dyskinetic response.
These results indicate that alterations in central dopaminergic mechanisms
contributing to motor complications in advanced Parkinson's disease can be
modified by procedures that provide continuous dopamine replacement and
further that on-off phenomena relate to postsynaptic changes, possibly
occurring as a consequence of chronic intermittent stimulation. Conceivably,
interactions between mechanisms mediated by the Dl and D2 subclasses of
dopamine receptors or alterations in other striatal transmitter systems may
explain both the improvement in clinical fluctuations and the restoration in
neuropharmacology indices with continuous levodopa therapy. The
stabilization of motor fluctuations over several days of continuous levodopa
administration reassures that virtually all patients who have already
developed motor complications will respond to steady-state infusions of
levodopa of sufficient duration. Hence, we continue to develop practical
means for providing stable level dopaminergic replacement for ambulatory
Parkinsonian patients.
Investigations conducted in the experimental animal appear to have
provided additional insight into the pathogenesis of the postsynaptic changes
postulated to underlie on-off fluctuations and peak dose dyskinesias. Our
earlier studies indicated that twice daily injections of levodopa for 30 days
enhanced the rotational response to the mixed dopamine agonist, apomorphine,
in rats with unilateral 6-hydroxydopamine-induced lesions of the
nigrostriatal dopaminergic pathway; no such effect was obtained when the same
daily dose of levodopa was infused continuously around-the-clock. Now we have
found that the chronic administration of levodopa by continuous infusion
enhanced the rotational response to the D2 dopamine receptor agonist
quinpirole, but had no effect on rotation induced by the Dl agonist SKF
38393. The rotational responses to the selective dopamine agonists differed
dramatically in rats given levodopa by intermittent injection, a treatment
schedule resembling that ordinarily used in Parkinsonian patients;
intermittently treated rats evidenced a substantially increased response to
quinpirole, a greatly diminished response to SKF 38393, and a modestly
enhanced response to apomorphine. These findings suggest that the
intermittence of central dopamine receptor stimulation may be an important
factor in determining the subsequent responses of the dopamine system.
Specifically, our data appear to indicate that intermittent treatment with
the dopamine precursor increases the sensitivity of D2 dopamine receptor
mechanisms and decreases the sensitivity of mechanisms associated with D1
receptors. The dissociation between the effects of both continuous and
intermittent levodopa on Dl and D2 agonist-induced rotation indicates that Dl
and D2 dopamine receptor-mediated mechanisms respond differently to chronic
levodopa treatment. We hypothesize that chronic intermittent stimulation of
the normally tonically active dopamine system may produce alterations at the
level of the postsynaptic receptor or in more distal neural elements that
give rise to on-off fluctuations and peak dose dyskinesias.
Additional insight into these matters were derived from studies of the
effects of striatal lesions on both turning behavior and globus pallidus
single unit responses to the administration of a dopamine agonist. Pallidal
neurons in normal rats are excited by the systemic administration of
postsynaptically active doses of apomorphine. The role of the striatum in
mediating this phenomenon was examined by investigating the effects of
apomorphine on neuronal activity in the globus pallidus and on turning
13 - ET/IR
behavior in rats with unilateral quinolinic acid lesions of the striatum. The
lesion, which reduced striatal choline acetyltransferase activity by over 65%
and GABA content by more than 60%, markedly attenuated the effect of
systemically administered apomorphine on pallidal neuron activity. In
lesioned animals, both the degree of attenuation of the pallidal
electrophysiologic response and the neurotoxin-induced decreases in striatal
choline acetyltransferase activity and GABA content correlated with the
degree of apomorphine- induced turning. These findings indicate that the
stimulatory effect of apomorphine on pallidal neuron firing in normal rats is
probably mediated indirectly via striatopallidal neurons. Because a striatal
lesion attenuated the response of pallidal neurons to apomorphine, the
present data suggest that at least one of the two dopamine receptor subtypes
involved in mediating these phenomena are localized on, or acting through,
the striatal efferent pathway.
The contribution of D1 and D2 receptor-mediated mechanisms to the
pathogenesis of disorders other than those primarily involving extrapyramidal
motor function have received increased investigative attention during the
past year. In one study, we sought to evaluate the effects of selective D1
and D2 receptor stimulation and/or blockade on the propagation and motor
expression of pilocarpine-induced seizures. At moderate doses, pilocarpine
produced limbic stereotypies but no convulsions. Following pretreatment with
the D1 agonist, SKF 38393, but not its (S)- enantiomer, the same dose of
pilocarpine-induced convulsive activity as revealed by behavioral,
electroencephalographic, and histologic alterations. These features were
identical to those produced by a higher, convulsant dose of pilocarpine. On
the other hand, pretreatment with the D2 agonist, LY 171555, failed to induce
seizures. Furthermore, the D1 receptor antagonist SCH 23390, prevented
convulsive activity induced by either SKF 38393 plus pilocarpine or by higher
dose pilocarpine given alone. However, neither dopamine agonists nor
antagonists altered the limbic stereotypies induced by pilocarpine,
apparently indicating dopamine system involvement primarily in the mechanisms
of seizure generalization. These results suggest the participation of D1
dopamine receptors in the propagation of convulsive activity, possibly via D1
receptors on terminals of striatonigral GABAergic projections.
Increasing evidence suggests that striatal dopaminoceptive cells may be
affected in Parkinsonian patients both by the basic disease process as well
as by chronic dopaminomimetic treatment. As one early result of our efforts
to focus both clinical and preclinical studies on peptides found in GABA
containing striatal efferents we have recently reported on measurements of a
proenkephalin derived peptide, met5-enkephalin-arg-gly-leu in the lumbar
spinal fluid of patients with Huntington's disease and progressive
supranuclear palsy (PSP). Decreases averaging 37? in Huntington patients and
47% in PSP patients were found in comparison to healthy, age-matched control
subjects. No significant correlation could be established between CSF levels
of the proenkephalin precursor derivative and the age or symptom duration of
patients in either group. The reduction found in spinal fluid from
Huntington's disease patients likely reflects the loss of proenkephalin-
containing neurons. In PSP, on the other hand, deficits in enkephalinergic
neurons are not reported and the observed spinal fluid abnormality may
reflect a functional decline in the activity of this system secondary to the
striatal cholinergic deficit. These studies are now being extended to
patients with Parkinson's disease both in the untreated and levodopa treated
14 - ET/IR
states and could have important implications for the design and monitoring of
future therapeutic interventions.
Cognitive disorders
Widely disparate rates of symptom progression have long been recognized
in patients with Alzheimer's disease. We ranked U6 patients who met NINCDS-
ADRDA and DMS-III-R criteria for Alzheimer's disease and who had received a
complete neuropsychologic evaluation by an index of progression rate. The
index for 13 of the patients exceeded the median value for the entire group,
and these individuals were considered rapid progressors; remaining patients
were classified as slow progressors. Comparison of the rapid and slow
progressing groups revealed no significant differences in age at symptom
onset, sex, education or global intellectual function. There were, however,
characteristic differences in neuropsychologic profile: those with rapidly
progressing symptoms had significantly lower scores on the Mental Control
subtest of the Wechsler Memory Scale, on the Initiation and Perseveration as
well as Conceptualization subtests of the Mattis Dementia Scale and the
Vocabulary subtest of the WAIS-R. Interestingly, all these tests, with the
exception of Vocabulary, which may capture a more general level of
intellectual functioning, are considered primarily as a measure of frontal
lobe function. Performance on neuropsychologic tests of memory as well as of
parietotemporal lobe function were indistinguishable between the two groups.
These results suggest that the diagnostic rubric of Alzheimer's disease may
subsume two distinct subgroups: one characterized by relatively slow
clinical progression and neuropsychologic evidence of mainly parietal lobe
dysfunction, the other with faster progression of dementia symptoms
associated with functional abnormalities in both parietal and frontal lobes.
The pathogenesis of Alzheimer's disease remains unclear, although there
is some evidence to suggest that excitatory amino acids might contribute to
the pathogenetic cascade culminating in selective neurodegeneration. Among
the various excitotoxins studied, quinolinic acid is the only endogenous
compound known to be present together with its metabolic machinery in human
brain. We analyzed postmortem brain tissues and spinal fluid samples for
quinolinic acid in Alzheimer patients and in age-matched controls. In the
control group, frontal cortex and caudate nucleus had higher concentrations
of quinolinic acid than the other regions studied. No significant differences
were found between Alzheimer brains and controls in any region analyzed.
Studies in lumbar spinal fluid showed no gradient for quinolinic acid along
the neuraxis, a trend for increasing levels with normal aging, and no
difference between Alzheimer patients and age-matched control subjects. Since
quinolinic acid does not appear to cross the blood-brain barrier, levels in
brain and spinal fluid most likely reflect local production. The lack of
increased central quinolinic acid levels does not necessarily negate the
possibility of this or other excitotoxins contributing to cell death: levels
in the lumbar sac may not reflect changes in regional brain levels; moreover,
a similarity in levels hardly excludes the possibility of altered metabolic
turnover of quinolinic acid.
The cognitive impairment occurring in patients with progressive
supranuclear palsy (PSP) has been the subject of two recent neuropsychologic
studies. In one, which sought to evaluate verbal memory, significant
abnormalities in learning, consolidation, and retrieval were found in
15 - ET/IR
comparison with age-, sex- and education-matched controls. Information
scanning, which requires the use of short-term memory processes, remained
intact. The duration of symptoms and degree of motor dysfunction correlated
with intrusions during learning. Mo relation between cerebral dopamine
metabolism, as assessed by spinal fluid levels of dopamine and of its major
metabolite, and memory dysfunction could be established. The observed memory
deficits may at least be partially explained by an inability to initiate and
maintain a systematic and strategic search of stored information. This
inability could be related to a disturbance in the organizational and
temporal aspects of encoding and retrieval subserved by the frontal lobe.
Clearly, memory dysfunction contributes significantly to disability in this
disorder and appears to be operationally independent of, yet correlated with,
motor dysfunction.
To test the hypothesis that PSP patients may be impaired on tasks used
to assess "executive and attentional" processes presumably subserved by the
prefrontal cortex, a second neuropsychologic investigation evaluated patients
with this disorder during baseline, placebo, and cholinesterase inhibitor
treatment periods. The results indicate that patients with PSP are
particularly impaired in tasks requiring sequential movements, conceptual
shifting, monitoring the frequency with which stimuli are presented, and the
rapid retrieval of verbal knowledge. These deficits could not be accounted
for by slowed information processing or by problems with representational
knowledge. Conceivably, "weak activation" of frontal lobe representational
knowledge characterized by an observed attentional deficit results in the
neuropsychological impairments noted in these patients. The oral
administration of physostigmine failed to facilitate executive or attentional
performance as evaluated by our tasks. Our results indicate that above and
beyond a general slowing of cognitive processes, PSP patients have difficulty
performing some tasks traditionally associated with the prefrontal lobe. The
contribution of basal ganglia dysfunction to this impairment remains to be
elucidated.
16 - ET/IR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02578-08 ET
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (tOcnaraclarlOr >«lf. f'tlamutl ttt on ona tin* oatwaan Pfea boroart.)
Pharmacology and Cellular Biology of Peptidergic Neurons
PRINCIPAL INVESTIGATOR dill olnar protatvonat oartofinal batom tha Prm<iaat Invasttgator.) (Nama. titla. laboratory, ana .nutluta attthaiionl
PI: M. Maral Mouradian, M.D. Head, Genetic Pharmacology ETB/NINDS
Others: Takashi Minowa, Ph.D. Visiting Fellow
ETB/NINDS
COOPERATING UNITS fVmyj
Laboratory of Cellular Biology, NIMH; Department of Physiology, Uniformed Services University of the
Health Sciences
LAB/BRANCH
Experimental Therapeutics Branch
SECTION
Genetic Pharmacology Unit
INSTITUTE AND LOCATION
NINDS. NIH, Bethesda. Maryland 20892
TOTAL MAN-YEARS:
2.3
PROFESSIONAL:
1.3
OTHER:
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects
(an) Minors
(a2) Interviews
I I (b) Human tissues I I (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
In FY90, the Genetic Pharmacology Unit concentrated on the molecular regulation of neurotransmitter
and transmitter receptor genes. Two model systems were studied:
1) Regulation of POMC gene transcription
Studies done in FY90 were based on earlier findings in this laboratory about several exonuclease stop
sites in the 1Kb 5' flanking region of the mouse POMC gene suggesting the presence of a number of
potential sites for DNA-protem interaction. Two of these have been characterized in detail. One located
between -119 and -106 bp upstream from transcription start site is homologous to the transcription
factor AP-2 thought to be involved in signal transduction, and the other located between -137 and -131
has 70% homology to, but is distinct from, AP-1 .
2) Regulation of striatal dopamine receptor genes
This project was initiated in FY90. For the D-2 receptor, a full length cDNA clone was characterized
and the 5' region sequenced Screening a rat genomic library has yielded several candidate clones that
are currently being characterized. Similarly for the D-1 receptor, screening a human genomic library with
the recently cloned rat D-1 receptor cDNA that is coupled to adenylate cyclase has yielded 13 candidate
clones. Current studies focus on characterizing the promoter region of these genes and study their
transcriptional regulation.
17-ET/IR
PHStO«0(R«». I Ml
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOOCOVEREO
October I, 1989 through September 30, 1990
PROJECT NUMBER
Z01 NS 02263-14 ET
TITLE OF PROJECT (90 ihtraamrt or fit. r;rf«musf tit on or>9 imm b*t**—n thmborOmrt.)
Biochemical and Pharmacological Studies of Dopamine Receptors
PRINCIPAL INVESTIGATOR (tin othmr profmutonsi ptrionnrnt miow thm Printipsi tnvtt>gstor.) (N»mm. titlm. laboratory, andmstitutm atiiiiationf
PI: David R. Sibley, PhD Head, Molecular Pharmacology Unit ETB/NINDS
Others: Frederick J. Monsma, Jr., Ph.D. IRTA Fellow ETB/NINDS
LorisD. McVittie, Ph.D. IRTA Fellow ETB/NINDS
Anne C. Barton, Ph.D. IRTA Fellow ETB/NINDS
Lauren E. Black, Ph.D. IRTA Fellow ETB/NINDS
Mario Rinaudo, M.D. Visiting Fellow ETB/NINDS
Elizabeth L. Webster, Ph.D. Staff Fellow ETB/NINDS
COOPERATING UNITS (lt»n,)
Lab Cell Biology, NIMH; Genetic Pharmacol Unit, ETB/NINDS; Dept Anatomy & Neurobiology, U.
Vermont; Ctr for Molecular & Behavioral Neurosci, Rutgers U; Molecular Probes, Inc.; Nova Pharm Corp.
LAB/BRANCH
Experimental Therapeutics Branch
SECTION
Molecular Pharmacology Unit
INSTITUTE AND LOCATION
NINDS. NIH, Bethesda.MD 20892
TOTAL MAN-YEARS:
PROFESSIONAL: 5 75
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a)Human subjects □ (b) Human tissues HH (c) Neither
(a 1) Minors
| ] (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The long-term goal of this project is the biochemical and molecular characterization of dopaminergic
receptor mediated information transduction, and its regulation, across neuronal membranes. Two major
interrelated areas of research on D1 and D2 dopamine receptors are currently under investigation:
1. Cell Biology and Regulation of Dopamine Receptors. The functional and regulatory properties of D1
and D2 dopamine receptors on various neuroblastoma and retinoblastoma cell lines were characterized.
The D1 receptors were shown to undergo homologous desensitization in response to agonist stimulation
while a heterologous desensitization was observed in response to cAMP. D2 receptors were also shown
to undergo desensitization and down-regulation in response to agonist treatment. A variety of
fluorescent, affinity, and antibody probes for dopamine receptors were developed. Utilizing these
probes, it was shown that D1 receptors were localized to most of the cells in the striatum as well as
numerous fibers while the D2 receptors appeared to be confined to a smaller population of neurons.
2. Molecular Cloning of Dopamine Receptors. Investigations concerning the RNA splice variants of the
D-2 dopamine receptor were continued in FY 90. A series of stably transfected cell lines were constructed
which express either the short (D-2S) or long (D-2L) isoforms of the D2 receptor. Both isoforms were
shown to exhibit identical pharmacologic and functional properties with respect to adenylyl cyclase
inhibition. Both isoforms were also found to augment arachidonic acid release. A cDNA encoding the
D1 dopamine receptor linked to the stimulation of adenylyl cyclase was cloned and expressed. A novel
subtype of the D-1 receptor was discovered by expressing rat striatal mRNA in Xenopus oocytes. This
new D-1 receptor subtype is linked to the stimulation of phosphatidylinositol turnover and calcium
mobilization.
18-ET-IR
PH5 6040 («». I. Ml
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS02139-16ET
PERIOO COVERED
October 1, 1989 through September 31, 1990
TITLE OF PROJECT (lt<iu'i(t«i«i)ii TW* »»uii W on on* ihw mi«hi rn« oo'wi )
Pharmacology and Physiology of the Substantia Nigra and Basal Ganglia
PRINCIPAL INVESTIGATOR a'nor/l«rsroh>iiion<'Mrionn«lo«la»fn««rlncis«/lnv«IIi9<Cor.> (Ate/n*. till: ImbOflQty. tntt HllflfllM tMillBlton)
PI: Judith R.Walters Chief, Neurophysiological Pharmacology Section ETB/NINDS
Others: Debra Bergstrom
Michael Twery
Kai-Xing Huang
MarkKelland
Lisa Thompson
Pharmacologist
Senior Staff Fellow
Special Volunteer
Senior Staff Fellow
Staff Fellow
ETB/NINDS
ETB/NINDS
ETB/NINDS
ETB/NINDS
ETB/NINDS
COOPERATING UNITS (iftnr)
Clinical Pharmacology Section, Experimental Therapeutics Branch
LAB/BRANCH
Experimental Therapeutics Branch, CNP
SECTION
Neurophysiological Pharmacology Section
INSTITUTE AND LOCATION
NINDS.NIH.Bethesda^MD 20892
TOTAL MAN-YEARS:
4.3
PROFESSIONAL:
3.3
OTHER:
1.0
CHECK APPROPRIATE BOX(ES)
I I (a) Human subjects
(a 1) Minors
I ! (a2) Interviews
] (b) Human tissues [x] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
1) Roles of D1 and D2 Dopamine Receptors in Basal Ganglia. Mechanisms underlying the synergistic
interactions of D1 and D2 receptors are being studied in striatal slices via intracellular recording
techniques, an approach new to the Section this year. Cathodal pulses induce at least two types of
response in the striatal neurons; relevance to striatal mechanisms will be studied. Focal stimulation
elicits EPSP's composed of at least three potentials; the fastest appears to involve a non-NMDA receptor,
the slower potentials involve NMDA receptors. Focal electrical stimulation also appears to release a
modulatory factor which induces changes in current-voltage relationships. First results show that
dopamine is not this modulatory factor but dopamine does appear to be a modulator of the voltage-
dependent conductances and may regulate repetitive firing in striatal neurons.
2) D2 Autoreceptor / D2 Postsynaptic Receptor Studies. N-0923 and N-0924 are two stereoisomers of
current clinical interest; we find N-0923 is a potent and efficacious D2 agonist and N-0924 acts as a partial
D2 agonist. Differences in spare receptor number at pre- and post-synaptic D2 receptor sites account for
why a partial dopamine agonist can be fully efficacious at D2 autoreceptors while providing a "clamp" at
postsynaptic D2 receptors, stimulating weakly while blocking further stimulation. In fact, maintaining
therapeutic levels of postsynaptic dopamine receptor stimulation or blockade may be more effectively
done with partial dopamine agonists than by adjustment of blood levels of more efficacious agents.
3) Conseguences of Dopamine Cell Degeneration in the Basal Ganglia. Changes in dopamine receptor
function following chronic reserpine treatment are different from those seen in control but in the
opposite direction from those seen in long term 6-hydroxydopamine lesioned rats. Similar changes are
being found in rats with relatively short-term lesions of the striatonigral pathway. This evidence for a
biphasic pattern in dopamine receptor-mediated effects after dopamine depletion indicates a greater
range and complexity in the compensatory responses to dopamine loss than previously appreciated.
4) Role of the Pedunculopontine Tegmental Nucleus (PPN). We have found the PPN exerts effects on
substantia nigra dopamine cell activity which appear mediated by both indirect and direct mechanisms.
This raises questions about the role of PPN degeneration shown occurring in certain neurological
disorders including progressive supranuclear palsy, Parkinson's disease and Alzheimer's disease.
19-ET/IR
PHS 6O40 !■.•». I Ml
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02265-14 ET
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (SOchatmcffS or f«I. T<ttm mutt t>t on on* hnm brntwrnn thm bor<*rt.)
Pharmacology, Biochemistry and Physiology of Central Neurotransmitters
PRINCIPAL INVESTIGATOR (Lift othtr protosuontt pmrsonnml b*io*t th9 FrtMpal inwstigstof.) (Nam*, tittm. laboratory, and mstttutm stiillation)
PI: Thomas N. Chase, M.D. Chief ETB/NINDS
Others: Fabio Baronti, Visiting Fellow; Jerome Blin, Visiting Associate; Thomas Davis, Special
Volunteer; Thomas M. Engber, Senior Staff Fellow; Marianne Giuffra, Clinical Associate;
Ulrike Mann, Visiting Fellow; Concepcion Marin, Visiting Fellow; M. Maral Mouradian, Senior
Staff Fellow; John H. Ownby, Clinical Associate; Zvi Susel, Visiting Fellow, ETB/NINDS
COOPERATING UNITS ut.ny)
NIMH Clinical Brain Disorders Branch, NIDR Neurobiology and Anesthesiology Branch, NIMH Laboratory
of Clinical Science
LAB/BRANCH
Experimental Therapeutics Branch
SECTION
Clinical Pharmacology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda. MP 20892
TOTAL MAN-YEARS:
PROFESSIONAL: 1Q
OTHER:
CHECK APPROPRIATE BOX(ES)
[xj (aHHuman subjects |~x~l (b) Human tissues I I (c) Neither
|X (a1) Minors
I x I (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The goal of this project is to develop improved pharmacotherapies for selected neurodegenerative
disorders, especially Parkinson's disease and Alzheimer's disease.
In Parkinson's disease, on-off fluctuations declined by nearly 50% during a 10-day around-the-clock
infusion of optimal dose levodopa. Clinical improvement was associated with a marked shift of the
levodopa dose-antiparkinsonian response curve towards desensitization, a substantial improvement in
the therapeutic index for the dopamine precursor, and a significant prolongation in its duration of
antiparkinsonian action. On-off phenomena, which ultimately disable most parkinsonian patients, now
appear to reflect relatively plastic postsynaptic changes, occurring as a consequence of nonphysiological
intermittent stimulation. Findings in an animal model of parkinsonism indicate that Dl and D2 dopamine
receptor-mediated mechanisms respond differently to chronic levodopa treatment, and suggest that
intermittence of central dopamine receptor stimulation, which increases sensitivity of D2 dopamine
receptor mechanisms and decreases sensitivity of mechanisms associated with the D1 receptor, may be an
important factor in determining subsequent responses of the dopamine system.
In Alzheimer's disease, recent results indicate that the diagnostic rubric may subsume two distinct
subgroups: one characterized by relatively slow clinical progression and neuropsychological evidence of
mainly parietal lobe dysfunction, the other with faster progression of dementia symptoms associated
with functional abnormalities in both parietal and frontal lobes, a distinction having obvious
implications relative to the pathogenesis and treatment of this disorder. Neuropsychologic evaluations
of patients with progressive supranuclear palsy (PSP) found significant abnormalities in learning,
consolidation, and retrieval in comparison with matched controls. PSP patients were also observed to be
particularly impaired in tasks requiring sequential movements, conceptual shifting, and the rapid
retrieval of verbal knowledge. Orally administered physostigmine failed to improve performance on any
of the measures tested.
20-ET/IR
PHS 4040 («•». IWI
Annual Bibliography
Experimental Therapeutics Branch, DIR, NINDS
Ariano MA, Monsma FJ, Jr, Barton AC, Kang HC, Haugland RP, Sibley DR. Direct
visualization and cellular localization of D1 and D2 dopamine receptors in
rat forebrain using novel fluorescent ligands. Proc Natl Acad Sci USA
1989;86:8570-4.
Barone P, Palma V, Parashos SA, Marin C, Chase TN, Carapanella G. D-1 dopamine
agonist administration reduces the threshold for convulsions produced by
pilocarpine. Boll Soc Ital Biol Sper 1 989; 65: 337-4 1 .
Barone P, Parashos SA, Palma V, Marin C, Campanella G, Chase TN. Dopamine D-1
receptor modulation of pilocaropine-induced convulsions. Neuroscience
1990;34:209-17.
Bishop JF, Rinaudo MS, Ritter JK, Chang ACY, Conant K, Gehlert DR. A putative
AP-2 binding site in the 5U flanking region of the mouse POMC gene. FEBS Lett
1990;264:125-9.
Braun A, Mouradlan MM, Mohr E, Fabbrini G, Chase TN. Selective D-1 dopamine
receptor agonist effects in hyperkinetic extrapyramidal disorders. J Neurol
Neurosurg Psychiat 1989;52:631-5.
Carlson, J.H., Bergstrom, D.A., Demo, S.D. and Walters, J.R.: Nigrostriatal
lesion alters neurophysiological responses to selective and nonselective D-1
and D-2 dopamine agonists in globus pallidus. Synapse 1990;5:83-93.
Chase TN, Baronti F, Fabbrini G, Heuser IJ, Juncos JL, Mouradian MM.
Rationale for continuous dopaminomimetic therapy of Parkinson's disease.
Neurology 1989;39(Supl 2) :7- 10.
Engber TM, Susel Z, Juncos JL, Chase TN. Continuous and intermittent levodopa
differentially affect rotation induced by D-1 and D-2 dopamine agonists. Eur
J Pharmacol 1989;168:291-8.
Fagarasan M0, Bishop JF, Rinaudo MS, Axelrod J. Interleukin 1 induces early
protein phosphorylation and requires only a short exposure for late induced
secretion of b-endorphin in a mouse pituitary cell line. Proc Natl Acad Sci
USA 1990;87:2555-9.
Grafman J, Litvan I, Gomez C, Chase TN. Frontal lobe function in progressive
supranuclear palsy. Arch Neurol 1990;47:553-8.
Juncos JL, Mouradian MM, Fabbrini G, Chase TN. Levodopa infusion therapy. In:
Koller WC, Paulson GW, eds. Therapy of Parkinson's disease. New York: M.
Dekker, 1990; 185-203.
Litvan I, Gomez C, Atack JR, Gillespie M, Kask AM, Mouradian MM, Chase TN.
Physostigmine treatment of progressive supranuclear palsy. Ann Neurol
1989;26:404-7.
Litvan I, Grafman J, Gomez C, Chase TN. Memory impairment in patients with
progressive supranuclear palsy. Arch Neurol 1989;46:765-7.
Mahan LC, Monsma FJ, Jr, Burch R, Sibley DR. Expression of rat mRNA coding
for a D1 dopamine receptor coupled to Ca2+ mobilization in Xenopus oocytes,
Proc Natl Acad Sci USA 1990;87:2196-200.
Mann UM, Mohr E, Chase TN. Rapidly progressive Alzheimer's disease. Lancet
1989;2:799.
Monsma FJ, Jr, Barton AC, Sibley DR. Expression of functional D2 dopamine
receptors following differentiation of human retinoblastoma cells. J
Neurochem 1989;52:1641-4.
Monsma FJ, Jr, McVlttie LD, Gerfen CR, Mahan LC, Sibley DR. Multiple D2
dopamine receptors produced by alternative RNA splicing. Nature 1989;342:926-
9.
Monsma FJ, Jr, Sibley DR. Identification and characterization of D1 and D2
dopamine receptor subtypes expressed in cultured neuroblastoma and
retinoblastoma cell lines. Brain Res 1989;492:314-24.
Mouradian MM, Chase TN. Clinical perspectives in neuropeptides in the central
nervous system. Curr Opin Neurol Neurosurg 1989;2:517-9.
Mouradian MM, Chase TN. Parkinson's Disease: therapeutic aspects. Current
Opin Neurol Neurosurg 1989;2:309-13.
Mouradian MM, Heuser IJE, Baronti F, Chase TN. Modification of central
dopaminergic mechanisms by continuous levodopa therapy of advanced
Parkinson's disease. Ann Neurol 1990;27:18-23.
Mouradian MM, Heyes MP, Pan J-B, Heuser IJE, Markey SP, Chase TN. No changes
in central quinolinic acid levels in Alzheimer's disease. Neurosci Lett
1989;105:233-8.
Pan HS, Engber TM, Chase TN, Walters JR. The effects of striatal lesion on
turning behavior and globus pallidus single unit response to dopamine agonist
administration. Life Sci 1989;46:73-80.
Vyas S, Bishop JF, Gehlert DR, Patel J. Effects of protein kinase C (PKC)
down regulation on secretory events and POMC gene expression in anterior
pituitary tumor (AtT-20) cells. J Neurochem 1990;54:248-55.
Weinberger DR, Mann U, Gibson RE, Coppola R, Jones DW, Braun AR, Berman KF,
Suderland T, Reba RC, Chase TN. Cerebral muscarinic receptors in primary
dementia as evaluated by SPECT with iodine-123 labeled QNB. Adv Neurol
1990;51:147-9.
Experimental Therapeutics Branch
"Thomas N. Chase, M.D., Chief
Senior Staff
•Judith R. Walters, Ph.D.
Fellows
Michael Twery, Ph.D.
Mark Kelland, Ph.D.
Lisa Thompson, Ph.D.
Thomas M. Engber, Ph.D.
Marianne Giuffra, M.D.
M. Maral Mouradian, M.D.
John H. Ownby, M.D.
Frederick J. Monsma, Jr., Ph.D.
Loris D. McVittie, Ph.D.
Anne C. Barton, Ph.D.
Lauren E. Black, Ph.D.
Elizabeth L. Webster, Ph.D.
David R. Sibley, Ph.D.
Visiting Program
Fabio Baronti, M.D.
Jerome Blin, M.D.
Concepcion Marin, M.D.
Zvi Susel, M.D.
Ulrike Mann, M.D.
Takashi Minowa, Ph.D.
Mario Rinaudo, M.D.
Special Volunteers
Kai-Xing Huang, Ph.D.
Rafael G. Blesa, M.D.
Thomas L. Davis, M.D.
>
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ANNUAL REPORT
October 1, 1989 through September 30, 1990
Medical Neurology Branch
Clinical Neurosciences Program, DIR
National Institute of Neurological Disorders and Stroke
Table of Contents
RESEARCH SUMMARY 1-36
PROJECT REPORTS
Clinical Pharmacology of Antiepileptic Drugs 37
Z01 NS 02318-13 MNB
Diagnostic and Therapeutic Reevaluation of Patients With Intractable Epilepsy 38
Z01 NS 02236-1 5 MNB
Physiological Analysis of Involuntary Movements 39
Z01 .V 02667-06 MNB
Trial of Isoniazid for Action Tremor 40
Z01 NS 02668-06 MNB
Physiological Analysis of Voluntary Movement 41
Z01 NS 02669-06 MNB
Utility and Physiology of Botulinum Toxin for Involuntary Movement Disorders 42
Z01 NS 02711-05 MNB
Non-invasive Stimulation of Human Central Nervous System 43
Z01 NS 0271 2-05 MNB
Hemispheric Development and Specialization of the Intellectual Functions 44
Z01 NS 01658-23 MNB
Behavioral Modulation by the Limbic System in Man 45
Z01 NS 01424-24 MNB
EEG Learning Correlates Using Scalp and Intracranial Depth Electrodes 46
Z01 NS 01245-25 MNB
Cognitive and Emotional Profile of Neuropsychiatric Disorders 47
Z01 NS 00200-36 MNB
Pharmacological Studies of Ion Channels in Cultured Cells 48
Z01 NS 02732-04 MNB
i-MNB/DIR
Excitability Properties of Enzymatically Dissociated CNS Neurons 49
Z01 NS 02733-04 MNB
Development of Uncompetitive NMDA Antagonists as Anticonvulsants 50
Z01 NS 02772-03 MNB
Neuropsychological Investigations of Human Cognition and Mood State 51
Z01 NS 02792-02 MNB
Cognitive Neuroscience 52
Z01 NS 02793-02 MNB
Event-Related Potential Studies of Normal and Abnormal Cognitive Processing 53
Z01 NS 02794-02 MNB
Combined Clinical, Viral and Immunological Studies of Neuromuscular Diseases 54
Z01 NS 02038-18 MNB
Studies in Neuromuscular and CNS Diseases and Their Experimental Models 55
Z01 NS 02531-09 MNB
Radiographic, Radioisotopic, CT, and MR Angiography of the Spinal Cord 56
Z01 NS 01 195-26 MNB
Nuclear Magnetic Resonance and Computed Tomography (Transmission) 57
Z01 NS 02073-17 MNB
Positron Emission Tomography 58
Z01 NS 0231 5-13 MNB
ii-MNB/DIR
ANNUAL REPORT
October 1, 1989 through September 30, 1990
Medical Neurology Branch
Clinical Neurosciences Program, DIR
National Institute of Neurological Disorders and Stroke
Mark Hallett, M.D., Chief
The Medical Neurology Branch conducts research on human epilepsy, including new
approaches to diagnosis and treatment; investigates basic questions related to
normal and abnormal neuronal excitability; performs studies on human motor
control; investigates cognitive and emotional processes in man; conducts research
on memory, perception, language, and problem solving in neurological patients;
investigates immunological and virological aspects of neuromuscular disorders; and
investigates the central nervous system (CNS) using various research imaging
techniques.
The Branch is divided into seven sections in addition to the Office of the Chief. On
June 8, 1990, the Neuroimaging Section of the Clinical Neurosciences Program was
transferred to the Medical Neurology Branch. Giovanni Di Chiro, M.D., serves as
Chief, Neuroimaging Section. The Section investigates the CNS in health and
diseases using as research tools X-rays, radionuclides, and magnetic resonance
imaging. The Section also studies brain tumors, movement disorders, intracerebral
bleedings, iron distribution in the aging and pathological brain, cerebrospinal fluid
circulation, vascular malformations andtumors of the spinal cord, pituitary gland
adenomas, and certain aspects of cerebral ischemia. In addition, the Neuromuscular
Diseases Unit and the Cognitive Neuroscience Unit became sections on June 8, 1990.
William H.Theodore, M.D., Deputy Chief, MNB, also serves as Chief of the Clinical
Epilepsy Section. The Clinical Epilepsy Section includes the Unit on Cerebral Blood
Flow and Metabolism headed by Dr. Theodore, and the Unit on Neurophysiology
headed bySusumu Sato, M.D. Mark Hallett, M.D., is Chief of the Human Motor
Control Section. The Human Motor Control Section includes the Cortical Physiology
Unit headed by Dr. Leonardo G. Cohen. Paul Fedio, Jr., Ph.D., is Chief of the Clinical
Neuropsychology Section. Marinos Dalakas, M.D., is Chief of the Neuromuscular
Diseases Section. Jordan Grafman, Ph.D., is Chief of the Cognitive Neuroscience
Section. In 1990, Michael Rogawski, M.D., Ph.D., was named as Chief, Neuronal
Excitability Section.
CLINICAL EPILEPSY SECTION
The Clinical Epilepsy Section is undertaking a series of studies in patients with severe
uncontrolled seizures using new techniques in order to improve clinical control, as
well as to elucidate the pathophysiology of epilepsy. The studies being carried out
at this time focus on two main groups of patients: adults or children with complex
partial seizures; and children with atonic / myoclonic seizures (the Lennox-Gastaut
syndrome). After seizure frequency and type are characterized by intensive
monitoring techniques, the patients are placed in an appropriate research protocol.
Before patients are offered surgical therapy or trial of an experimental antiepileptic
drug, the therapeutic regimen is adjusted to obtain optimal seizure control using
conventional agents. Several investigations are performed in collaboration with the
1 -MNB/DIR
Clinical Neuropsychology Section, the EEG Laboratory, the Neuronal Excitability
Section, and the Surgical Neurology Branch.
Neuroimaging techniques may be used to obtain both physiologic and anatomic
data and assist in the clinical evaluation of patients with severe seizures. Positron
emission tomography (PET) is a technique using the intravenous injection of a
radioactive isotope to determine regional rates of cerebral metabolism, blood flow,
or tracer distribution. Magnetic resonance imaging (MRI) is being used to help
elucidate the anatomical substrates of altered physiologic patterns revealed by PET.
Initial attempts have been made to use nuclear magnetic resonance (NMR)
spectroscopy to study biochemical parameters of human epileptic tissue in vivo.
Single photon emission computed tomography (SPECT) is being used as an
additional tool to study cerebral blood flow. Clinical, electroencephalographic
(EEG), and neuropsychological data, as well as ultrastructural and biochemical
investigations of epileptic tissue removed at surgery, are correlated with function
and structural results of imaging studies.
One of the major interests of the Clinical Epilepsy Section has been the use of
neuroimaging techniques to study neuropharmacology. In clinical pharmacology
research, it has always been much easier to derive pharmacokinetic than
pharmacodynamic data. We have been taking advantage of the ability of PET to
provide quantitative information to study the effect of drugs on cerebral blood flow
and metabolism, as well as the distribution of putative neuroreceptor liqands in vivo.
Recent and ongoing studies:
Estimation of cerebral glucose metabolism using 18-flouro 2-deoxyglucose PET
showed that patients with uncontrolled complex partial seizures have regions of
focal hypometabolism which correspond to electrographic localization of epileptic
foci. In patients with structural lesions on computed tomography (CT) or MRI,
hypometabolism was often much more extensive than suggested by the lesion itself.
In the Lennox-Gastaut Syndrome, a severe childhood epileptic encephalopathy, we
found diffuse neocortical hypometabolism with relative preservation of function in
the basal ganglia and diencephalon, even when CT and MRI are normal. Further
studies are in progress to study the relation between metabolic dysfunction, age of
seizure onset, severity and duration of seizures. The results were in marked contrast
to those obtained in patients with absence seizures, who had normal glucose
metabolism. Distinct metabolic pathophysiological processes were present in
patients with distinct electroclinical epileptic syndromes. We have used 150 water to
study cerebral blood flow in patients with uncontrolled partial seizures. With this
tracer it is possible to compare ictal and interictal states, as well as to compare blood
flow with FDG scans performed in the same patients. Preliminary results indicate
that there may be mismatches between blood flow and metabolism in epileptic
tissue, and that glucose metabolism is reduced to a greater degree than blood flow.
This may be an important marker for altered physiology in epileptic foci. Further
studies are in progress, using improved PET techniques. Moreover, focal increases in
blood flow have been found in patients who have had secondary generalized
seizures during I5fj PET scans. These studies may help to provide a means of
localizing seizure onset.
In patients with complex partial seizures, a speech discrimination task was used to
"activate" cerebral glucose metabolism, and investigate the possibility of functional
reorganization due to unilateral temporal lobe damage. Patients with complex
partial seizures and a unilateral EEG focus were compared to normal controls.
2-MNB/DIR
Preliminary results suggested that metabolic activation during speech performance
may have a broader distribution than suspected on the basis of purely anatomic
studies, and that hypometabolism in patients with left temporal foci became
relatively more severe during the task. Using ^O water, we have investigated the
effect OT verbal learning and recall on cerebral blood flow. Further studies of word
and object recognition, and verbal and nonverbal memory are planned.
We studied the effect of antiepileptic drugs on cerebral glucose metabolism.
Phenobarbital depressed global LCMRglu by 30-40% while carbamazepine and
phenytoin lowered glucose utilization by only 10-15%. These results are consistent
with a possible difference between the mechanisms of action of the compounds.
Phenobarbital interacts with the GABA-benzodiazepine receptor complex while
phenytoin and carbamazepine appear to affect currents in active sodium channels.
Moreover, other studies have shown that GABA agonists reduce glucose metabolism
in experimental animals. Thus, the PET results may be of particular relevance to
investigations into the mechanism of action of antiepileptic drugs. Moreover, they
may provide evidence for explaining the difference in neuropsychological toxicity,
whicn is much greater for phenobarbital than for phenytoin or carbamazebine.
Subsequently, valproic acid was found to reduce LCMRglc by 20-25%, suggesting
that it may have a GABAerqic effect at therapeutic doses in vivo.
Two approaches have been used to evaluate the opiate receptor sytem, which has
been implicated in the pathophysiology of epilepsy. [18F] cyclofoxy, a naltrexone
analogue, was used to image Mu and Kappa receptors. We did not find any
alterations in activity in the region of the epileptic focus. We administered 1 mg/kg
naloxone in order to see whether blocking opiate receptors would alter blood flow
in epileptic foci. Although a transient decrease in blood flow occurred, there
appeared to be no specific effect. These studies suggest that opiate receptors may
not play a dramatic role in the pathophysiology of human epilepsy.
Recent investigations in the clinical pharmacology of antiepileptic drugs include
studies of carbamazepine metabolism and phenytoin protein binding. We have
been evaluating the effect of drug withdrawal on seizure frequency, in order to
assess the presence or absence of transient exacerbations which could be
distinguished from a simple loss of drug effect. This was clearly present in the case of
phenobarbital, but absent for phenytoin. Data on carbamazepine are being
collected. These data are important for clinical practice. A physician wishing to
withdraw a drug known to cause a transient exacerbation during taper may be more
likely to perservere when seizures increase.
The Clinical Epilepsy Section has completed a trial of a promising antiepileptic drug
which was identified through the NIH Antiepileptic Drug Development program.
The drug, known asfelbamate, is2-phenyl-1, 3-propanediol dicarbamate. In pre-
clinical testing, the drug is effective in animal models which correlate with partial
seizures in man and is quite nontoxic in animals. Evaluation of the protective indices
indicate that felbamate has a significant and adequate margin of safety. A
randomized placebo-controlleddouble-blind study was performed. The purpose of
this study was to obtain definitive information regarding the efficacy and safety of
felbamate in patients with uncontrolled partial seizures who are receiving
concomitant carbamazepine therapy. The study used a unique three-period cross-
over design in order to eliminate carry-over effects from drug to placebo periods.
The results suggested that felbamate can reduce the frequency of partial seizures. A
trial of felbamate monotherapy for complex partial seizures, and of felbamate in
addition to valproic acid in the Lennox-Gastaut syndrome is being planned.
3-MNB/DIR
Sphenoidal, and in some cases, subdural electrodes are used in the evaluation of
potential surgical candidates, coupled with long-term video-EEG recording
techniques. These techniques allow the acquisition of EEG data not available via
surface recordings. These data are correlated with PET and MRI to obtain the best
possible presurgical localization of epileptic foci. A prospective evaluation of the
relative value of invasive (subdural) and noninvasive methods of presurgical
evaluation is in progress. Twenty-six patients with medically refractory complex
partial seizures had temporal lobectomy after evaluation which included prolonged
scalp EEG recordings, PET, MRI, and X-ray computed tomography. PET showed a
region of focal interictal temporal hypometabolism corresponding to electrographic
localization of seizure onset in twenty-one patients. Five patients had a region of
increased MR signal intensity on the spin echo image in the region of the EEG focus;
two, an abnormality ipsilateral to but distinct from the EEG focus; and one had
bilateral findings. CTscan was abnormal in three cases; two had tumors. Three
patients had low-grade tumors (one with a normal PET scan). PET can detect
metabolic dysfunction associated with mild pathologic changes in epileptic foci, but
increased signal intensity on MRI does not necessarily correlate with the degree of
pathologic abnormality. Tumors may be less likely when both CT and MRI are
normal.
During surgery, direct electrocorticographic recordings are made before and after
tissue is resected, in order to guide the surgical approach. Tissue from both
epileptogenic and nonepileptogenic regions is obtained for biochemical study.
Preliminary results indicate that glycine, glutamate, and aspartate were significantly
higher in spiking than nonspiking regions, while GABA, taurine, and alanine were
unchanged. These data suggest that alterations in excitatory rather than inhibitory
amino acids may occur in epileptic foci.
Magnetoencephalography (MEG) is a new approach to the problem of localizing
abnormal cerebral potentials which may represent an epileptic focus. Initial studies
suggest that MEG may provide more precise three-dimensional information than
EEG, allowing detection and localization of epileptic foci in the depths of the brain,
without the need for invasive procedures.
HUMAN MOTOR CONTROL SECTION
In the past year, Dr. Leonardo G. Cohen received Intent for Tenure and was
appointed Head of the Cortical Physiology Unit in the Section. Research was pursued
in a variety of areas related to human movement and its disorders.
VOLUNTARY LIMB MOVEMENTS.
Further data analysis has been done on data collected in previous years in relation to
(a) the silent period in the antagonist muscle prior to a ballistic movement in normal
subjects; (b) adaptation to movement with mirror-vision in patients with Parkinson's
disease (PD); and (c) reaction time studies in patients with cerebellar disease and PD.
Major effects have been directed to the role of the cerebellum in coordination and
motor skill learning.
(1) The contribution of the cerebellum to coordination: There exist long-standing
questions about the precise role of the cerebellum in coordination. Much
theoretical and experimental work has emphasized the action of the cerebellum in
4-MNB/DIR
calculating the appropriate distribution of neural activity to various muscle groups
involved in a movement. More recently, work by Keele and others has focused on
the possibility that the cerebellum's fundamental computation is that of clocking
neural motor control signals. To evaluate the validity of these and other viewpoints,
the detailed kinematics of upper extremity movements in two dimensions in human
cerebellar patients and in normal controls are being examined. In addition,
electromyography (EMG) signals are obtained from involved muscle groups. These
data are then used in conjunction with a biomechanical model of the upper
extremity to infer the joint torques which are developed during the movement. A
comparison is then made between the force patterns in normals and cerebellar
patients with regard to magnitude, distribution and timing. This is essentially an
extension to two-joint movements of the work on EMG patterns in single-joint
movements performed by our group earlier. The goal is to identify, if possible, the
fundamental motor control derangement in cerebellar patients which leads to ataxic
movements. Studies performed this year comparing hand trajectories executed
under either fast or accurate movement constraints have revealed intriquing
differences between patients with cerebellar disease and controls. Under the "move
accurately" constraint, the cerebellar patient's trajectories had normal paths and
final position accuracy. In contrast, in the "move fast" condition, the cerebellar
patients exhibited deficits in final position control and significant deviations from
straight-line trajectories, consistent with the notion that functions subserved by the
cerebellum are critical to the portion of the motor execution process involving
compensation for limb dynamic changes during high-speed movements.
A detailed biomechanical analysis of cerebellar movements is being undertaken . To
date, an extensive computer program has been developed to first filter and then
analyze trajectories individually and as a group with regard to several parameters
including degree of deviation from linearity, maximum velocity, movement time,
linear and angular acceleration, and jerk. A submodule then simulates the
movement using a two-link rotary-joint biomechanical arm model and thereby
predicts the torque and torque-change at each joint. The output is presented
graphically and can be exported in data files for further analysis. Preliminary
evaluation of data from several cerebellar patients reveals that the patients make
systematic errors when attempting to rapidly produce a straight line. These errors
appear to differ primarily in magnitude, rather than in character, from the trajectory
errors of normal volunteers. Work is now being done to quantify the degree of
kinematic abnormality and to attempt to relate this, if possible, to a consistent
underlying abnormality in the dynamics and EMG findings.
(2) The contribution of the cerebellum to learning: It has long been postulated that
the cerebellum and its input and output pathways play a significant role in long- and
short-term changes in motor behavior and quality of motor performance in response
to an alteration of either the sensory inputs or task demands. Studies in the past
mainly focusing on modifications ot the vestibulo-ocular reflex (VOR) have
demonstrated in various species that the integrity of cerebello-olivary pathways is
required in order to be able to modify the gain of pre-existing reflex pathways in
response to an alteration of the normal input-output pattern. Based on these
findings, it has been proposed that the modification of Purkinje cell firing pattern
caused by an alteration of climbing fiber input might represent one part of the
neuronal substrate of learning ana memory in humans.
To evaluate the role of the cerebellum in the acquisition of motor skills in humans,
we have initiated a series of experiments that study the time course and mechanisms
of skill aquisition in a paradigm involving complex two-dimensional multijoint arm
5-MNB/DIR
movements. Subjects are instructed to track a visually-guided path on a digitizing
tablet while attempting to increase speed and accuracy of their movements.
Initially, evaluation of kinematic movement parameters (trajectory variability,
tangential velocity and acceleration profiles) will be emphasized. Operating
characteristics in trie speed-accuracy domain shall be assessed before and after
various practice trials. Shifts in the speed-accuracy operating characteristic will be
used to quantify improvements of motor performance. Short-term skill acquisition,
i.e., skill improvement within practice sessions and long-term retention over periods
of several days, will be compared in normal subjects and in patients with cerebellar
deficits. In a second stage, the dynamic aspects (torque and torque-change) of the
employed optimization strategies during practice of multijoint arm movements as a
model of motor learning shall De investigated.
PET STUDIES.
Using [18F]-2-deoxyglucose for PET scans, we have investigated regional cerebral
metabolism of glucose with voluntary movement and with sensory activation. We
have failed to detect any increase in regional cerebral cortical metabolism with
simple hand movements. Using H2O15 as a marker for cerebral blood flow, we have
also looked for changes in the sensorimotor cortex with simple hand movements and
with sensory stimulation. We have found an increase in the cerebral blood flow in
the sensorimotor cortex contralateral to voluntary wrist movement. Also, there was
increased blood flow in the supplementary motor cortex and in the ipsilateral
sensorimotor cortex.
With this as a background, we have gone on to study changes in regional cerebral
blood flow (rCBF) during various sensorimotor tasks. The studies this year have
included: (1) comparisons of regional activation in self-paced movements and
passive vibration of the same body part; (2) comparisons of self-paced and visually-
triggered hand movements; (3) comparisons of activation associated with eye
movements of different kinds ( saccades, pursuit, and fixation ); and (4) rCBF changes
associated with acquisition of a manual motor skill. Approximately six to ten normal
volunteers have been studied in each of these experiments.
A major problem in the interpretation of our data has been the neuroanatomical
localization of the rCBF changes observed in particular behavioral states. Existing
techniques that utilize either a neuroanatomical brain atlas or generic stereotaxic
coordinates are subject to large errors in spatial localization due to the large
numbers of degrees of freedom involved in the process of determining a conformal
mapping between two imaging modalities. For this reason we have mounted a
major effort, in collaboration with Dr. Pellizari of the University of Chicago, to
develop a computational technique that allows rapid and accurate spatial
registration of pairs of three-dimensional data structures. Using this approach, it is
possible to mark regions of interest (ROI) in data obtained with one imaging
modality and then, through the mapping matrix, retrieve values from the
corresponding locations in a dataset obtained with a different imaging modality.
This allows determination of ROI's using a subject's MRI scan and then retrieval of
the corresponding changes in rCBF from the corresponding PET study.
Preliminary analysis of the self-paced wrist movements has provided information
relevant to the understanding of functional organization of cortical motor areas.
The largest increases in rCBF were observed in cortical motor areas, including: the
contralateral sensorimotor cortex (mean -42%, S.D. - 13%), the ipsilateral
sensorimotor cortex (mean - 19%, S.D. - 8%), and the medial frontal cortex (mean -
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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
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component on the affected side was the subcortical N18, which had a widespread
scalp distribution similar to controls. In the future, we plan to evaluate long-latency
SEP components and patients with more focal lesions.
(6) Congenital mirror movements: Short latency SEPs to right median nerve
stimulation were recorded in two patients. The scalp distribution of the postcentral
N20 and precentral P22 was similar to that observed in normal volunteers (100)
indicating that bilateral representation observed in motor systems is not present in
somatosensory systems.
(7) Amputees: Two patients with unilateral upper limb amputation were studied.
The goal was to determine side to side differences in the scalp distribution and
amplitudes of SEPs to stimulation of symmetric body parts immediately proximal to
the stump. In these two cases, we have found no differences.
(8) Spinal cord injury: We have finished collecting data from ten patients with
thoracic lesions leading to paraplegia. Results have not yet been analyzed.
MOVEMENT RELATED POTENTIALS.
We have used the standard, widely-spaced electrode montage in topographic
electroencephalography (EEG) mapping to study the scalp distribution of the
terminal phase of the Bereitschaftspotential using a collection window of 499 ms.
After establishing normative data on the subpotentials preceding the voluntary
index finger movements in a normal population, we have been able to do detailed
analysis on the generators of the different subpotentials using a tightly spaced
electrode montage over the sensorimotor area of the scalp. Four distinct negative
events were identified: the peak of the negative slope (NS1), the initial slope of
motor potential (isMP), the parietal peak of motor potential (ppMP), and the frontal
peak of motor potential (fpMP). For self-paced movements, NS' and isMP occurred
before the onset of electromyographic (EMG) activity, and ppMP and fpMP occurred
afterthe onset of EMG activity. NS' had a wide distribution, covering the parietal
region with slight contralateral predominance. The isMP mapped focally over the
contralateral hand motor area on the scalp. The location of ppMP was similar to that
of isMP. The fpMP was localized anterior and medial to motor cortex with a
contralateral preponderance and possible location over the supplementary motor
area (SMA). The isMP appears to reflect activation of the cortical cells in the hand
area of motor cortex for the execution of voluntary movement, and the fpMP
appears to reflect proprioceptive feedback from the periphery.
We studied the scalp-recorded, movement-related cortical potentials occurring
immediately before and after the onset of movement in five patients with
asymmetric Parkinson's disease. The topographic distribution of the isMP was
diffuse for voluntary finger movements of the more affected hand but normal for
movements of the less affected hand. The fpMP was located more posterior in
patients than in normal subjects. The peak amplitudes of the potentials were
normal in all patients. The topographic abnormalities might reflect inadequate
excitatory activity from the basal ganglia to the primary motor cortex and the SMA.
We studied six patients with cerebellar degenerative disease. All patients had an
abnormal topographic pattern of potentials compared with normal subjects. The
amplitudes and latencies of the potentials were normal in all patients except two, in
whom NS' was absent. The isMP was diffuse and bilateral in the patients. The
topography of the fpMP was more posterior in the patients than in normal subjects.
8-MNB/DIR
These patterns indicate a derangement of sensorimotor cortex activity in voluntary
movement as a consequence of cerebellar dysfunction.
Eight patients with dystonia were evaluated with the particular goal of measuring
the FPmp which might reflect SMA function. Preliminary evaluation shows that the
scalp distribution and the magnitude of this component was similar in both groups.
BALANCE AND GAIT.
The focus of studies in human balance has been the maintenance of upright posture.
Detailed quantification of postural sway has been applied to normal subjects at
various ages. Our studies snow that acceleration of individual body segments
characterizes normal upright stance; older normal subjects maintain a more rigid
posture, utilizing large and inconsistent adjustments to maintain stability. This may
relate to the instability of stance in the elderly. Additionally, we have begun an
evaluation of EMG phase relationships to descriptors of sway. Gastrocnemius, for
example, appears to act in a feedforward mode as a brake to forward sway.
We have completed studies of standing balance in patients with PD and patients on
gentamycin who might be developing vestibular deficits. The data have not yet
been analyzed in detail, but it appears that postural studies might be a sensitive
method for detecting vestibular toxicity.
Our previous studies have demonstrated that normal subjects make postural
adjustments in advance of voluntary arm movements. This has been studied in detail
in self paced and reaction time movements. Postural adjustments precede arm
movement more in the self paced situation (when there is more time). However, we
have not been able to detect any increased instability in the reaction time
circumstance. The linkage of postural adjustments to limb movements is not fixed,
but varies with the circumstance.
We have begun an analysis of gait in patients with autism and cerebellar ataxia.
Cerebellar gait is characterizedby marked variability compared to normal and
disorganization of interlimb coordination. Patients with autism are said to have
cerebellar lesions, but our analysis does not show any cerebellar features in their
gait.
REFLEXES.
In past years the laboratory has done extensive studies with reciprocal inhibition in
patients with dystonia ana PD. We conclude that there is an important deficit in
reciprocal inhibition in these disorders. There are a number of phases in the
reciprocal inhibition curves and the physiology of each is not clear. During further
studies of this project, it became clear that the method can be improved and better
standardized. Therefore, a study of different parameters that influence the
reciprocal inhibition of the H-reflex in wrist flexors was undertaken. The most
important parameter forthe inhibition in all three periods (with peaks at or around
0 ms, 10 ms, and 75 ms of delay between the shock to the radial and median nerves)
is the intensity of the radial nerve shock. The same parameter also strongly
influences the relative excitation of the H-reflex around 30 ms of delay between the
shock to the radial and median nerves. The duration of the stimulus delivered to the
radial nerve is comparatively less important; however, there is a trend for more
inhibition when the duration of the stimulus to the radial nerve is 0.5 ms instead of
longer or shorter durations. Moreover, preliminary data suggest that another
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-
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motoneuron pool after amputation suggests that reorganization occurs at a
suprasegmental level.
(b) Spinal cord injury: We studied seven patients with severe thoracic T9-T1 2
spinal cord injury occurring between two and five years before testing. EMG
recordings were made from three levels (T6to T12) on the right external oblique
abdominal muscle and from one level on the left. Magnetic stimulation evoked
larger MEPs with shorter latencies from a larger number of scalp positions in muscles
immediately proximal to the level of a spinal cord injury than in corresponding
muscles in controls. These phenomena were present at rest but not during voluntary
activation of the target muscles. Our findings suggest an enhanced excitability of
motor representation areas targeting muscles proximal to the level of a spinal
transection. These findings are compatible with the notion of a limited flexible
relationship between primary motor cortex and its target muscles following
alterations of normal input-output patterns.
Patients were asked to report sensations felt after each stimulus. Magnetic
stimulation evoked tingling and numbness sensations lasting for up to ten seconds
referred to different parts of legs and toes in three patients. Sensations were felt
more distally the closer the site of stimulation was to the midline. These results
indicate that at least portions of the cortical representation areas for body parts
deafferented by a complete spinal cord injury can remain related to those body parts
for up to several years following the lesion.
(c) Congenital mirror movements: We studied two patients with congenital
mirror movements by means of various neurophysiological and metabolic
techniques, including mapping of MEPs to transcranial electric and magnetic
stimulation, premovement and SEPs, kinematics of voluntary movements, muscle
reflexes, and PET. Abnormalities in maps of motor and premovement potentials and
in PET scans were consistent with a bilateral representation of hand muscles in the
motor cortex, the existence of physiologically active connections capable of
conducting fast efferent volleys from the motor cortex to ipsilateral muscles, the
presence of mirror EMG activity in either hand with intended voluntary movement
of the other hand, the absence of mirrored EMG responses from wrist flexors and
extensors to mechanical perturbation of the contralateral wrist, and normal scalp
distribution of SEPs to right and left median nerve stimulation. Our findings are
consistent with aberrant organization of motor representation areas and
corticospinal pathways with ipsilateral as well as contralateral control of voluntary
movement.
(d) Hemispherectomy: We have so far studied two patients with
hemispherectomy performed for treatment of unresponsive seizure disorder.
Stimulation of the remaining hemisphere evoked bilateral muscle responses in
proximal and distal muscles at similar latencies indicating a bilateral representation
of arm muscles and the existence of physiologically active ipsilateral pathways.
(e) Stroke: At this time we are studying changes in sensorimotor maps in patients
with stroke.
(4) Timing, locus, topographical extension: So far we have observed reorganization
in motor pathways as early as four to six months following amputations and spinal
cord injuries leading to quadriplegia. These changes are not present in every patient
after such a short time. Part of our future efforts will be dedicated to determining
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the factors that influence the development of reorganization and its timing
characteristics.
(5) Epilepsy: In this area, we have studied three patients with intractable epilepsy
who had a grid of subdural electrodes positioned over sensorimotor areas for
evaluation of surgical intervention. The purpose of this study was to determine: (a)
focality of noninvasive magnetic stimulation in activating hand motor
representation areas; (b) development of seizures or afterdischarges in epileptic
patients after magnetic stimulation (60). Magnetic stimulation with an 8- shaped
coil was accurate in localizing the peak of the hand motor representation area for
abductor pollicis brevis within 1 cm in the coronal axis in the three patients.
Magnetic stimulation was unable to elicit afterdischarges or other EEG changes in
any of the patients with subdural electrodes.
(6) Test of language laterality: A goal of our work is the development of a test of
language laterality using magnetictranscranial stimulation which is less invasive and
egually accurate to sodium amytal testing (Wada test). Pilot studies have been
carried out on one subject. Naming was maximally disturbed by stimulation over the
right or left superior marginal gyrus (SMG) with a latency of magnetic stimulation of
200 ms after the target. Rhyming, a phonological processing task, was interfered
with to a greater degree by stimulation to the left SMG than the right SMG.
(7) Evaluation of motor and sensory physiology: In a reaction time situation, it is
possible to delay the expected voluntary response by a magnetic stimulus delivered
over sensorimotor cortex given just before the movement. Using a relatively weak
magnetic stimulus that does not produce a motor response (MEP) when the body
part is at rest, but will produce a response when the body part is activated, it is
possible to divide the reaction time period into two phases. In the first phase there is
no MEP and the motor cortex remains "unexcitable. ' In the second phase there is a
gradual increase in MEP amplitude even though the voluntary EMG activity has not
yet appeared. This "excitable" phase demonstrates an activation of motor cortex
before the motor command isdelivered. Applying the lasttechnigue to the analysis
of prolonged reaction time (akinesia) in patients with PD, it can be demonstrated
that the excitable phase is prolonged. This demonstrates a mechanism for difficulty
in generating a motor command in these patients.
We studied the effects of focal transcranial magnetic stimulation on ipsilateral
muscles in six normal volunteers. An 8-shaped coil was used to deliver stimuli to
different scalp positions over one hemisphere. EMG recordings were made from
muscles of both upper extremities, at rest and at several degrees of voluntary
ipsilateral muscle activation. No ipsilateral responses were observed with the arm at
rest. During muscle activation, stimuli evoked a transient attenuation of ongoing
voluntary EMG activity (ipsilateral silent period, ISP). ISPs were highly reproducible
and could be evoked in scalp locations which activated corresponding contralateral
muscles. The ISP in abductor pollicis brevis had an onset latency of 35 + 4 msec (1 2
msec longerthan the onset of the contralateral excitatory response), and a duration
of 46 + 15 msec. At 50% muscle activation, the peak attenuation was 71 + 18%. In
wrist flexors, biceps and deltoid, ISPs tended to be less prominent. On some
occasions, excitatory responses preceded the ISPs. This study demonstrates the
existence of a powerful inhibitory influence of sensorimotor cortex on ipsilateral
upper extremity muscles and on distal muscles in particular.
Particular attention is being paid now to the evaluation of interhemispheric
right/left differences in motor representation areas targeting different arm and leg
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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.
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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
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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
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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.
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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
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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.
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In an attempt to improve upon some of the methodological flaws of past research,
we presented a large number of words to subjects at sub- and suprathreshold levels.
The significance of affective vs. cognitive processing of emotional words was also
explored by requiring subjects to read each word and/or identify emotional valences,
and to perform implicit memory tasks. Data analysis derived from normal subjects
confirmed the superiority of perceiving material delivered to the central field such
that reaction times were fastest, responses were most accurate, and response bias
was minimal. Materials projected at the longer durations yielded faster responses
during the label than label/read condition and faster and more accurate responses to
right visual field (RVF) compared with left visual field (LVF) words. Reading accuracy
was superior for stimuli projected to the right, compared with the left, visual field.
Responses were faster to emotional (positive and negative) than neutral stimuli, and
emotional stimuli were read more accurately than neutral stimuli. Stimuli appearing
in the LVF generally elicited a greater response bias than those appearing in the RVF.
At subthreshold levels, neutral stimuli were reported most accurately, owing in part
to a response bias to classify material conservatively as neutral. Subjects accuracy to
label words projected to the lateral fields at subliminal speeds did not significantly
exceed chance level, and at the briefest delivery rate, no accurate reading responses
were recorded from the lateral fields. Reaction times at 10 msec were fastest to label
words displayed in the LVF, followed by RVF and then the central visual field (CVF).
Parenthetically, at suprathreshold levels the usual pattern was for fastest responses
to occur from CVF, RVF, and LVF, which corresponded to accuracy levels.
Increased accuracies and longer reaction times occurred at suprathreshold levels
where the reading condition led to greater accuracy of labeling negative stimuli in
the RVF. Lateral field projection elicited different biases; there was a greater
negative than positive emotional bias for stimuli in the RVF and a LVF bias toward
positive rather than negative emotions. Also, more negative than positive or neutral
words were correctly read in the RVF, while more positive than negative or neutral
words were read in the LVF. Accuracy of labeling scores corrected for guessing
showed the reverse pattern under the label condition : the RVF perceived positive
and the LVF, negative, stimuli more accurately.
Analysis of word stems completed after the 10 msec level showed no field or
emotion advantage. At 120 msec, the expected superiority of central field
perception was followed by right and then left field accuracy. More word stems
were completed following read/label than just label command. In the reading
condition, more positive word stems were completed from the LVF and negative
words from the RVF, although overall completion rate was minimal.
That positive words were labeled faster than negative, which were labeled faster
than neutral, may reflect a strategy whereby subjects tended initially to evaluate
emotional coloration of stimuli. The neutral bias at subthreshold levels, in turn,
suggests that subjects were cautious to classify positive or negative stimuli when
insufficient material was presented from which to make a confident judgment. The
reversal of field pattern (LVF < RVF < CVF) for reaction time at the briefest
presentation suggests that syncretic cognition within the right hemisphere takes
precedence at this brief exposure.
A three-way interaction involving gender, condition, and visual fields emerged such
that the labeling accuracy scores for males were highest in the RVF under the
reading condition and in the LVF under the labeling condition. Females, however,
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scored most highly with the labeling instructions in both visual fields. Such findings
are suggestive of a more strict lateralization of verbal skills in males and a more
diffuse and integrated verbal organization in females.
The present findings are at variance with tachistoscopic studies reporting a right
hemisphere (left visual field) advantage in perceiving emotions. One explanation
may be that the majority of former studies used "facial expressions", advantaging
the native processes of the right brain. In the same vein, by using verbal stimuli, trie
present study tended to favor left brain functions, reflected by the overall right
visual field (left brain) superiority. This functional advantage, however, does not
account for the finding that subjects dissociated positive and negative emotions by
hemisphere and type of cognitive processing required. Results of the implicit stem
completion task also support this interpretation which assigns negative emotions to
the left, and positive to the right, hemisphere. These data suggest that positive and
negative mood reactions following right and left brain injury, respectively, do not
reflect contralateral disinhibition, but rather, each hemisphere's basic emotional and
cognitive style. Under more highly verbal conditions, the left hemisphere is biased
toward negative, and the right, toward positive, emotions. This protocol will be
extended to study how accurately patients with right or left TLE perceive
emotionally tinted material projected into lateral visual fields.
Studies of lateral brain differences in emotionality have utilized EEG while patients
view and rate pleasant, neutral, and horrific materials. This preliminary project uses
both standard scalp electrodes and recordings from chronic indwelling flap
electrodes in TLE patients. The paradigm demands either cognitive judgments about
emotionally evocative material or "feelings" and attempts to examine the role of
the left and right temporal lobe in this differential processing. Finally, EEG indexing
(power spectrum) of attentional and linguistic processes will be derived from EEG
recordings taken during the Wada procedure in which sodium amytal is injected into
each hemisphere. The analysis may provide anatomical references to patients who
allegedly display bilateral language, amnesia, and euphoria/dysphoria.
These procedures have been recently supplemented by electrophysiological studies
in collaboration with Dr. Barry Smith (University of Maryland). In one study, normal
subjects were selected for "hemisphericity dominance"; that is, reliance on a primary
cognitive strategy that emphasizes analytical (left) or syncretic (right) processing.
The subjects were required to view a series of photographs of individuals portraying
different emotions, and to perform either a cognitive or affective chore; spectral
analysis of alpha band activity was performed off-line from leads positioned over the
left and right temporal and parietal regions.
The preliminary results indicate greater diminished alpha with cognitive vs.
emotional processing. Moreover, left hemisphericity subjects showed increased
activity from left recording sites, especially during cognitive processing. The
converse was seen with right hemisphericity subjects who showed increased activity
from right temporoparietal leads, especially while performing an emotional
processing task.
NEURONAL EXCITABILITY SECTION
During the past year, the Neuronal Excitability Section under the direction of
Dr. Rogawski continued pharmacological studies of voltage-dependent K* channels
and N-methyl-D-aspartate (NMDA)-gated cation channels in cultured mammalian
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CNS neurons using whole cell voltage-clamp and single channel recording
techniques. The aim of these studies was to explore new strategies for the rational
development of antiepileptic drugs based upon their interaction with ion channel
systems that are critical to the regulation of CNS neuronal excitability and
epileptogenesis. Work was focused in three areas: (1) drugs that block the
ionophoreof the NMDA receptor-channel complex, (2) K+ channel activator drugs,
and (3) cloned K+ channel genes expressed in fibroblasts. In addition, a theoretical
analysis of T-type voltage-dependent Ca2 + channels and their role in the generation
of the low threshold spike (LTS) was completed in collaboration with researchers in
the Mathematical Biology Branch, NIDDK. Finally, Drs. Rogawski and Yamaguchi in
conjunction with the Laboratory of Medicinal Chemistry, NIDDK, continued the
evaluation of a series of novel NMDA antagonists for their activity as
anticonvulsants.
Cellular Electropharmacoloqy of Uncompetitive NMDA Antagonists
Excitatory neurotransmission mediated by NMDA receptors plays a critical role in
epileptogenesis. Blockers of the NMDA receptor-associated ion channel, such as the
dissociative anesthetics phencyclidine (PCP) and MK-801, are powerful
anticonvulsants. However, side effects, including ataxia and cognitive disturbances,
limit the practical usefulness of these drugs in the treatment of seizure disorders.
Researchers in the Section have identifiedseveral dissociative anesthetic analogs
that are effective anticonvulsants in animal seizure models but which fail to cause
motor toxicity at anticonvulsant doses, suggesting that they may provide clues to the
development of NMDA antagonists that are clinically useful to treat seizure
disorders. During the present reporting period, Drs. Jones and Rogawski
investigated the interaction of two of the compounds, PCA (an analog of PCP) and
ADCI (an analog of MK-801) with the NMDA receptor channel complex using cellular
electrophysiological techniques. They found that both drugs are able to block
NMDA responses and that they do so in an uncompetitive (use- and voltage-
dependent) fashion. However, a kinetic analysis revealed that the novel analogs
effect their block much more rapidly than do the parent compounds. It has
therefore been proposed that fast NMDA receptor blockade, at least in part,
accounts for the more favorable therapeutic indices of the novel compounds.
Synthesis and Evaluation of Novel Uncompetitive NMDA Antagonists
During the past year, the collaborative program with the Laboratory of Medicinal
Chemistry, NIDDK, for the synthesis and evaluation of novel anticonvulsants
targeted at the NMDA receptor-channel complex was continued. Compounds
provided were screened in several animal seizure models and in a motor toxicity test.
A study was completed of 38 PCA analogs. Many of these analogs protected against
maximal electroshock seizures in mice and several showed a substantial
improvement in therapeutic index for seizure protection compared with the parent
compound. Of particular interest were 3-fluro-PCA and 1-phenylcyclopentylamine
which had exceptionally high therapeutic indices when administered orally. For
example, the therapeutic index of 3-fluro-PCA in the rat was > 62. In addition,
studies were continued on the MK-801/carbamazepine analog 5-aminocarbonyl-5H-
dibenzo[a,d]cyclohepten-5,10-imine (ADCI). ADCI was found to have a broad
spectrum of anticonvulsant activity in various animal seizure models. In addition to
high potency in the maximal electroshock test, ADCI was effective against
pentylenetetrazol seizures and at relatively low doses also against NMDA induced
seizures. This latter result suggested that ADCI is an NMDA antagonist and
prompted the confirmatory electrophysiological studies discussed above. ADCI has
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the most favorable parenteral therapeutic index of any NMDA antagonist described
to date (but is also active orally), and is currently being considered for clinical trials.
K * Channel Activator Drugs
Voltage-dependent K* channels regulate neuronal excitability by acting to
repolarize the neuronal membrane. Recently, several different antihypertensive
drugs have been shown to stimulate the opening of K* channels in muscle cells.
Drs. Politi and Rogawski have completed studies showing that one of these drugs,
cromakalim, promotes the opening of ATP-sensitive K + channels in cultured
hippocampal neurons. These channels, which have not previously been identified in
neuronal cells, could play a role in protecting against brain ischemia, including that
which may occur during prolonged seizures. Moreover, there is evidence that
cromakalim is an effective anticonvulsant in several animal seizure models when
injected intracerebroventricularly. Thus, K+ channel activator drugs could be of use
in the treatment of seizure disorders (and possibly also in protecting against seizure-
induced brain damage) if blood-brain barrier permeable analogs can be developed.
Cloned K* Channel Genes Expressed in Fibroblasts
In view of the recently discovered molecular hetergeneity of voltage-dependent ion
channel proteins, it has become apparent that pharmacological studies of ion
channels will be much more useful if attention is focused on molecularly defined
populations of channels. Cells bearing homogeneous populations of channels can
be obtained by expressing cloned channel genes in cells that lack voltage-dependent
ion channels, such as fibroblasts. The Neuronal Excitability Section has undertaken a
long-range program to explore the physiological and pharmacological properties of
molecularly defined channel proteins with the aim of discovering agents that
selectively affect the activity of specific molecular forms of the channels. During the
present reporting period, studies were completed on one isoform of a delayed
rectifier type K+ channel expressed in a fibroblast cell line. The results indicate that
the channel has a specific pharmacological sensitivity that is different from that of
other delayed rectifier type channels, and support the concept that specific
molecular forms of the channel can be selectively targeted with drugs.
Theoretical Model of the Low Threshold Spike in Thalamic Neurons
Based upon data obtained from electrophysiological studies, a theoretical model of
the low threshold spike (LTS) in thalamic neurons was formulated in collaboration
with Drs. Xiao-Jing Wang and John Rinzel of the Mathematical Research Branch,
NIDDK. The LTS isa Ca2 + -dependent potential mediated primarily by T-type (low
voltage activated) Ca2 + channels that is responsible for the transition between tonic
and burst firing in thalamic neurons and may play a critical role in the generation of
absence (petit mal) seizures. T-type Ca2 + channels may also be an important target
for anti-absence drugs (such as ethosuximide and dimethadione). This study
demonstrated that the shape of the LTS can be accounted for almost entirely by the
intrinsic properties of T-type Ca2 + channels, and provides support for the role of the
LTS in rodent absence seizure models.
COGNITIVE NEUROSCIENCE SECTION
The primary objectives of the Cognitive Neuroscience Section are to identify and
model the components of information processing, the cognitive computations that
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underlie each component, and the categories and architecture of knowledge
representation systems. Furthermore, we make an effort to map cognitive processes
onto human brain physiology, structures, and systems.
Investigators in the Cognitive Neuroscience Section are currently studying a wide
range of cognitive processes including problem solving and reasoning; memory and
knowledge representation; number processing and calculation; reading, writing,
and naming; visual perception; object recognition; and the relationship of mood
state and emotions to stored knowledge. Although many of these studies utilize
young and old normal subjects, the majority of our studies are conducted with
central nervous system (CNS)-impaired patients. CNS-impaired patients are studied
because their cognitive deficit pattern often implies dissociations between types of
information processing components, cognitive computational properties, or
knowledge domains. Thus, not only can such patients teach us about the structure
of cognition on the basis of their dissociations (and associations too), but the nature
and direction of the dissociations may lead to inferences regarding the contribution
of anatomical and neurotransmitter systems to cognitive processing. Both single-
case within-subject and group study designs are utilized.
The methodological appropriateness of studying the components of information
processing is supported by several lines of research. For example, the study of
memory in multiple sclerosis patients has narrowed their memory processing deficits
to two components: the articulatory rehearsal loop in working memory which
temporarily stores information in a buffer when it cannot be processed on-line, and
a post-representation retrieval pathway. Dr. Ray Johnson, Jr. has demonstrated that
event-related potentials (ERP) provide a physiological index of the temporal course
of information processing with specific wave-forms and their topography associated
with particular information processing components. His recent work has
demonstrated that ERPs can reflect both the information load, and the time course,
of rehearsal processes in working memory. Additional work in his Cognitive
Psychophysiology Unit by Dr. Marten Scheffers is examining the role of visual
attention in controlled and automatic processing. Error analysis in CNS-impaired
patients has provided clues as to the nature of the computational processes. For
example, Dr. Rhonda Friedman is currently analyzing different forms of acquired
reading, writing, and naming disorders using error analyses to identify both the
disordered component (e.g., semantic lexicon) as well as the probable impaired
computational property (e.g., activation of associational links between stored lexical
items). Dr. Angela Sirigu is studying the effects of damage to an early visual
information extraction system that aids in object recognition. Ms. Marian Stewart is
examining the effects of visual spatial contrast sensitivity function on later visual
information processing components (i.e., form and object discrimination and
recognition). If patients have a problem in visual-spatial contrast sensitivity, Stewart
will attempt to identify which computational property (i.e., specific contrast
sensitivity channel) is responsible. Finally, Drs. Rhonda Friedman and Jeffrey Hadley
using priming tasks, and Dr. Jordan Grafman, using tasks requiring retrieval of items
from different domains of knowledge, are mapping out the structure and categories
of knowledge representation.
As a result of these studies, we have been able to tentatively assign cognitive
components and knowledge representation systems to brain locations. For example,
structural analysis of visual stimuli takes place in the posterior cortex in regions
distinct from where meaningful analysis of stimuli takes place. In addition, the more
complex the nature of stimulus representation ( e.g., schemas), the more anterior in
the brain is its representation. Representational systems are organized both serially
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and in parallel, can be activated in parallel, are partially information redundant, and
while informationally hierarchical, can be activated selectively via attentional
mechanisms. Basal ganglia structures appear to aid in the execution of
representations (e.g., via motor procedures or cognitive planning). The involvement
of many different neural structures and neurotransmitter systems in cognition are
currently being studied. While we are concerned with specifying the cognitive
components of specific neural systems and structures, we also expect in the next few
years to describe generic principles of cognitive processing and representational
knowledge and to map these broad principles to brain chemo- and neuroanatomy.
As alluded to above, specific projects carried out in the Cognitive Neuroscience
Section have supported the conceptual distinction between cognitive components,
computational properties, and knowledge domains that are at the heart of the
models of cognition developed by Section members.
NEUROMUSCULAR DISEASES SECTION
The Neuromuscular Diseases Section conducts clinical studies and laboratory
investigations to determine etiology (infection/immunity and/or genetics) of
patients with neuromuscular disorders and explore new therapeutic modalities.
Current studies include: (1) motor neuron disease syndromes such as amyotrophic
lateral sclerosis (ALS) and post-polio syndrome; (2) Demyelinatinq polyneuropathies;
(3) neuromuscular diseases associated^ with HIV infection; (4) Duchenne's muscular
dystrophy; (5) experimental models of retroviruses-induced polymyositis; (6) studies
on muscle regeneration; (7) studies involving the interactions of the lymphoid
system with the central or peripheral nervous system; (8) studies involving the
infection of muscle or Schwann cells in tissue culture with various viruses, especially
HIV and enteroviruses; and (9) clinical experimental therapeutic studies in patients
with post-polio syndrome, polymyositis, and HIV-related neurological diseases.
We have defined the clinical symptomatology of the post-polio syndrome and
provided evidence that the site of pathologic involvement is in the distal nerve
terminals. We have found changes in all the muscles regardless of the presence of
new symptoms suggestive of an ongoing neuronal dysfunction which appears to
continue slowly since the original polio attack. Abnormal immunoregulatory
mechanisms may also play a role in the manifestation of these patients' symptoms
based on the presence of inflammation in the histological specimens of muscle
biopsies and spinal cords and abnormal lymphocyte subsets in the circulation.
We have studied the metabolic activity of the cortex in ALS patients and correlated
the glucose utilization of the cortical neurons with the histopathological findings of
the same brains at autopsy. We found that ALS is a complex disorder affecting
multiple cortical regions tnat extend beyond the resolution of routine
histopathology.
In an effort to determine if in Duchenne's muscular dystrophy the weakness is due to
absent dystrophin or to loss of muscle fibers, we measured the force generated by
skinned muscle fibers and correlated it with the composition of muscle proteins in
the same fibers separated electrophoretically. We found that in Duchenne's
dystrophy the remaining muscle fibers generate normal force in spite of the absence
of dystrophin.
We have defined the spectrum of neuromuscular diseases associated with HIV
infection and investigated the immunopathogenesis of these disorders in an effort
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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
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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
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in pathological conditions (experimental parkinsonism). Iron distribution is verified
by Perls' stain of brain specimens.
Successful demonstration by MRI of selective basal ganglia damage following
intracarotid injection of MPTP.
The design and fabrication (under contract) of a variable field T1-T2 analyzer
that will enable rapid measurement of relaxation times in tissue samples at fields
from 0.05 to 1 .5 Tesla is well underway.
Probably our most important NMR contribution this year is represented by the
successful initiation of localized proton spectroscopy (1 H-MRS) at 1 .5 Tesla in human
patients. For the moment, we have concentrated our attention on brain tumors
(some 50 patients studied already). Our emphasis has been on correlations between
MRS, results, particularly lactate recognition, FDG-PET findings, and clinical
evaluations. Cats and other animals were studied at 2.0 T and 4.7 T. Prior to 1989,
31 P and 1H MRS data were obtained with surface coil localization. This technique
samples mostly cortical, but poorly defined tissue volumes, and requires surgical
retraction of overlying scalp. Methods of achieving reliable water suppression using
these techinques have been evaluated.
Post-ischemic metabolism in a cat model of transient cerebral ischemia was
studied to determine whether MRS-detectable abnormalities correlated with long-
term functional deficiencies. Despite prominent alterations of 31 P and 1 H spectra
(i.e., lactate production, energy failure and acidification) during 10 minute global
ischemia, the spectra renormalized within an hour of reestablishing blood flow.
Repetition of transient ischemia produced only minor changes in the metabolic
recovery rate. MR spectra appeared highly sensitive to flow, and high time
resolution monitoring is likely to be useful for evaluating the completeness of
ischemia. Localized spectroscopic measurements in the MCAO model revealed
lactate elevations that were stable for many hours.
(3) Positron Emission Tomography.
A follow-up FDG-PET study has been completed of a series of patients initially
harboring low grade gliomas which later changed grade. The FDG-PET method has
been found optimal for monitoring the grade change (malignant degeneration).
Confirmation has been obtained that the FDG-PET method is the optimal procedure
for differentiating tumor recurrence from postradiation and/or post chemotherapy
cerebral necrosis.
The FDG-PET method of tumor evaluation has been extended from the gliomas
to other intracranial tumors, particularly meningiomas. We have clear indications
that FDG-PET is an excellent method to predict ' post-removal" recurrence of
meningiomas. We have completed a study of pituitary microadenomas using the
FDG-PET method. This technique has proven to be a valuable adjunct to the other
imaging methods (CT, MRI).
We have continued our PET research on the dopaminergicsystem using FDG
and 6-[18F]fluoro-L-dopa (6FD) in both animals (primate hemiparkinson level) and
human hemiparkinsonian patients. Our results follow:
(a) Primate Hemiparkinson Model: Unilateral intracarotid administration of
MPTP results in a striking decrease of the 6FD-derived activity retention in the
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exposed putamen, whereby 60-1 50 minutes after 6FD administration there is
approximately a 67% decrease. The decrease in the caudate is much less. A
differential involvement of these structures is also found in idiopathic PD. MRI with
Gd-DTPA revealed a striking increase of signal intensity on T2-weighted sequences
and decreased intensity on T1 -weighted sequences beginning 3-6 days after
exposure. This change most likely reflects cytotoxic (rather than vasogenic) edema
associated with dopaminergic neuronal injury. The MRI findings persist for 2-3
weeks. The absence of any basal ganglia MRI abnormalities in the chronic animals is
similarto findings in idiopathic PD. In the MPTP-exposed putamen, rCBF was
decreased up to 25% while exposed caudate rCBF was decreased by less than 10%.
These findings indicate thatdopaminergicdenervation produces quantifiable
regional physiologic changes.
(b) Cerebral Metabolism in Human Hemiparkinson's Disease: In the
hemiparkinsonian patients, no statistically significant metabolic abnormalities were
encountered either in cortical structures, or in the caudate, thalamus and
cerebellum. However, hypermetabolism was present in the lenticular nuclei
contralateral to the most affected body side. Lenticular metabolic asymmetry
correlated both with degree of clinical akinesia and a scale of overall disease severity
(modified Columbia University Rating Scale).
(c) Cerebral Metabolism after Intravenous L-Dopa Infusion in Human PD: The
L-dopa infusion increased glucose metabolism globally, except in the basal ganglia
where the relative glucose utilization was actually decreased. The L-dopa infusion
significantly improved the patients' PD signs.
(d) Caudate Grafts in Primates: MRI with Gd-DTPA revealed that the blood-
brain barrier disruption (BBBD) was only present in the first three months after
surgery. Hence, BBBD is an unlikely explanation forthe long-term improvement
seen in some of the subjects. Among the surgically grafted primates, the time course
and extent of clinical improvement was much greater for the fetal mesencephalic
grafts as compared to the other groups. 6FD-derived activity was elevated only in
areas containing viable fetal mesencephalic graft. 6FD-PET revealed a diffuse
elevation of regional activity at or near the surgical target area in primates which
displayed some clinical improvement. This type of change was seen in the control
grafted subjects and in those receiving cavitation alone. Histology revealed the
presence of tyrosine hydroxylaseimmun-oreactive fibers coursing toward the graft
bed. It is proposed that these (sprouting) fibers are being detected by 6FD-PET and
are possibly responsible for the clinical improvement.
(e) Adrenal Medullary Autografts in Human PD: In the four grafted subjects,
the post-operative clinical improvement was judged to be mild. Enhancement with
Gd-DTPA, consistent with BBBD was seen in at least two of the operated subjects.
However, there was no detectable difference on PET scanning between patients
who received grafts and those who did not.
(f) Peripheral Metabolism of 6FD and PET Image Quality: The main metabolite
of 6FD, 3-methoxy-6-[18F]fluorotyrosine, crosses the blood brain barrier and
contributes to the background signal. In preliminary work, we have been studying
the effect of carbidopa and OR-462, inhibitors of the enzymes dopa decarboxylase
and catechol-O-methyl-transferase, respectively. The best improvement in image
quality is obtained using both drugs simultaneously.
35-MNB/DIR
Finally we have carried out the critical experiments necessary to proceed with the
investigation by PET of the NMDA receptors. An analog of PCP bearing the fluorine
atom, FTCP, has been recently synthesized (Kieswetter et al., 1989). PCP and its
analogs bind with high affinity to an allosteric site of the NMDA receptors only when
glutamate is present to open the cationic channels. In other words, the PCP-like
ligands are tools to investigate functionally active NMDA receptors, and also to
measure indirectly glutamate concentrations in different brain areas.
We have at present shown that FTCP retains the "in vitro" binding characteristics
(affinity, specificity) of the related compounds PCP, TCP, and MK801 . Moreover,
3H-FTCP binding is subjected to regulation by glutamate and glycine. We have also
shown brain uptake and "in vivo" pharmacological actions after intravenous
injection of the drug in mice, rats, and one monkey.
36-MNB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02318-13 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (ao characters or less. Title must fit on one line between the borders.)
Clinical Pharmacology of Antiepileptic Drugs
PRINCIPAL IN VE STIGATOR (List other professional personnel below (he Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: William H.Theodore, M.D. Chief, CES MNB NINDS
Others: Susumu Sato, M.D. Chief, EEG LAB OCD NINDS
Paul Fedio, Ph.D. Chief, CNS MNB NINDS
Frank Nice, M.S., M.P.A. Pharmacist MNB NINDS
COOPERATING UNITS (if any)
Office of The Clinical Director, NINDS
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Clinical Epilepsy Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
1.2
PROFESSIONAL:
1.2
OTHER:
CHECK APPROPRIATE BOX(ES)
| x | (a) Human subjects
|~x~| (a1) Minors
J (a2) Interviews
] (b) Human tissues L] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Current studies include a double-blind randomized placebo add-on controlled trial of felbamate. The
unique three period cross-over design allows unbiased estimates of drug effects even in the presence of a
carry-over effect from one period to the next
The first study has been completed, and the results suggest the drug may be effective against partial
seizures. A study of felbamate monotherapy is planned, as well as an add-on to valproic acid in the
Lennox-Gastaut syndrome. We are studying carbamazepine withdrawal, to establish the presence or
absence of transient seizure exacerbation. We have evaluated the effect of valproic acid on cerebral
glucose metabolism, using positron emission tomography (PET). This drug is of particular interest due to
its possible inhibition of GABA degradative enzymes. We compared its effect to that of phenytoin,
carbamazepine, and phenobarbital. Valproic acid reduced cerebral metabolism by 20%, suggesting an
interaction with the GABA-benzodiazepine receptor complex. PET is also being used to study the opiate
receptor system in patients with epilepsy. 18 F-cyclofoxy binding was assessed in patients with complex
partial seizures, and naloxone administered to study its effect on blood flow and metabolism.
37-MNB/DIR
PHS 6040 (Rev MA)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02236-15 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ ECT (SO characters or less. Title must fit on one line between the borders.)
Diagnostic and Therapeutic Reevaluation of Patients With Intractable Epilepsy
PRINCIPAL IN VE STIG ATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: William Theodore, M.D. Chief, CES MNB NINDS
Others: Susumu Sato, M.D. Chief, EEG Lab OCD NINDS
Paul Fedio, PhD Chief, CNS MNB NINDS
COORPERATING UNITS (if any)
Office of The Clinical Director, NINDS
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Clinical Epilepsy Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
PROFESSIONAL: 1 q
OTHER:
CHECK APPROPRIATE BOX(ES)
[xj (a) Human subjects [7~| (b) Human tissues I I (c) Neither
| x [ (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The Clinical Epilepsy Section has been developing and testing new techniques to improve seizure control,
medication tolerance, and rehabilitation in patients with severe epilepsy. Patients with uncontrolled
seizures are admitted for a complete evaluation, including simultaneous video and telemetered
electroencephaloqraphic (EEG) recording of seizures, daily determinations of antiepileptic drug serum
concentrations, positron emission tomography (PET), magnetic resonance imaging (MRI). and
maqnetoencephaloqraphy (MEG). A specific seizure diagnosis is established allowing each patient to be
assigned to an appropriate research protocol and therapy. PET in patients with localized brain lesions
has demonstrated focal hypometabolic cerebral areas corresponding to the interictal seizure EEG focus.
In some patients, PET has been able to detect a focus when other methods have failed. Studies of
patients during partial seizures have shown a change from hypo- to hypermetabolism at the site of the
focus. In the Lennox-Gastaut syndrome, PET has revealed the existence of two separate metabolic
patterns despite clinical seizure similarity. PET studies allow more definitive identification of the
epileptic lesion and suggest new avenues of investigation into the basic mechanisms of the epilepsies.
MRI may show small structural lesions underlying PET hypometabolism even when computed
tomography (CT) is normal. Further studies will elucidate the relation of metabolic and pathologic
changes. MEG may have the potential to accurately localize the subsurface origin of spikes. EEG
provides little information on the spatial distribution of epileptiform discharges in cortical depths; MEG
may be superior. Comparison of invasive localization of epileptic foci using subdural electrodes and non-
invasive evaluation is being performed. After surgery, patients are followed with serial clinical,
neuropsychological, and electroencephalographs evaluation.
38-MNB/DIR
PHS 6040 (Rev. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02667-06 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ ECT (80 characters or less. Title must fit on one line between the borders.)
Physiological Analysis of Involuntary Movements
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: Mark Hallett, M.D.
Others: Leo Cohen, M.D
Peter Fuhr, M.D.
HelgeTopka, M.D.
Joseph Matsumoto, M.D.
Clinical Director
OCD
ODIR
DIR
NINDS
Chief
HMCS
MNB
DIR
NINDS
Visiting Scientist
HMCS
MNB
DIR
NINDS
Visiting Fellow
HMCS
MNB
DIR
NINDS
Special Volunteer
HMCS
MNB
DIR
NINDS
Special Volunteer
HMCS
MNB
DIR
NINDS
COOPERATING UNITS (if any)
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Human Motor Control Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.5
PROFESSIONAL:
1.0
OTHER:
0.5
CHECK APPROPRIATE BOX(ES)
I x | (a) Human subjects
] (a1) Minors
J (a2) Interviews
J (b) Human tissues J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Involuntary movements have often been difficult to classify clinically. Clinical and physiological analysis
of a continuing series of patients has led to new classifications and pathophysiological insights.
Dystonias and Parkinson's disease (PD) have been the focus of our recent work.
The laboratory has done extensive studies with reciprocal inhibition studies. The conclusion of these
studies is that there is an important deficit in reciprocal inhibition in dystonia and PD. There are a
number of phases in the reciprocal inhibition curves and the physiology of each is not clear. During
further studies of this project, it became clear that the method can be improved and better standardized.
Therefore, a study of different parameters that influence the reciprocal inhibition of the H-reflex in wrist
flexors was done.
Cutaneous reflexes were measured in patients with PD. The reflex is composed of successive excitatory
and inhibitory events. While the latencies of the different reflex components and the amplitudes of the
excitatory peaks were not different from normal, the first inhibitory peak, occurring at a mean latency of
51 ms, was less pronounced in patients . The result is compatible with the loss of a spinal inhibitory
mechanism elicited by cutaneous afferents and can be a partial explanation for increased tone in PD.
We developed a technique to evoke perioral reflexes by stimulating branches of the trigeminal nerve
either electrically or indirectly via stimulation of cutaneous receptors by delivering well-defined taps to
the skin in the vicinity of the lips, mimicking the clinical test. We have established normative data and
studied patients suffering from orofacial dyskinesias and spasmodic dysphonia. In both groups of
patients, thresholds for eliciting R2 components of facial reflexes were lower and recruitment curves
were steeper as compared to normals.
We have studied several patients in a family with hyperekplexia.
in these patients is an exaggerated startle reflex.
39-MNB/DIR
Our results showed that the movement
PHS 6040 (Rev 184)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02668-06 MNB
PERIOD COVERED
October 1 , 1 989 through September 30, 1 990*
TITLE OF PROJ ECT (80 characters or less. Title must fit on one line between the borders.)
Trial of Isoniazid for Action Tremor
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: MarkHallett, M.D. Clinical Director OCD ODIR DIR NINDS
Chief HMCS MNB DIR NINDS
COOPERATING UNITS (if any)
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Human Motor Control Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS: -
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects ] (b) Human tissues ] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
*This project was terminated 9/89.
40-MNB/DIR
PHSfcMOOttv. 1/M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02669-06 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or toss. Title must fit on on* line between the borders.)
Physiological Analysis of Voluntary Movement
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.:
Others:
Mark Hallett, M.D.
Clinical Director
OCD
ODIR
DIR
NINDS
Chief
HMCS
MNB
DIR
NINDS
Leo Cohen, M.D.
Visiting Associate
HMCS
MNB
DIR
NINDS
Victoria Panzer, PhD
Staff Fellow
HMCS
MNB
DIR
NINDS
Rocco Agostino, M.D.
Visiting Fellow
HMCS
MNB
DIR
NINDS
InaTarkka, Ph.D.
Volunteer
HMCS
MNB
DIR
NINDS
COOPERATING UNITS at <«,)
Department of Rehabilitation Medicine, Clinical Center
Department of Nuclear Medicine, Clinical Center
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Human Motor Control Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
5.5
PROFESSIONAL:
4.0
OTHER:
1.5
CHECK APPROPRIATE BOX(ES)
I x [ (a) Human subjects
] (a1) Minors
J (a2) Interviews
J (b) Human tissues J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Studies of voluntary movement focused on the role of the cerebellum. One issue was the contribution of
the cerebellum to coordination. The results seem to indicate that the cerebellum is critical to the portion
of the motor execution process involving compensation for limb dynamic changes during high-speed
movements. A second issue is the role of the cerebellum in motor learning. Tasks are being developed to
assess motor skill learning. A study was done of the role of the silent period in the antagonist muscle
prior to a ballistic movement.
Using 0-15 labelled water as a marker for cerebral blood flow in positron emission tomography ( PET)
studies , we have been working on methods for improved anatomical correlation of regions of metabolic
change by superimposing the PET image onto an MRI image. Studies include analysis of the role of the
cerebellum in motor learning and the supplementary motor area in self-paced movement.
In evoked potential studies, we have been assessing changes in topography of sensory potentials with
lesions of the central and peripheral nervous system looking for evidence of plasticity. Studies of the N30
component of the median nerve sensory evoked potential was found to be enhanced in patients with
dystonia.
In studies of movement related potentials, we have studied patients with Parkinson's disease, cerebellar
degenerations and dystonia. Abnormalities in the first two groups suggest disturbances in the cortical
control of movement in these patients.
Studies in the Biomechanics Laboratory of the Department of Rehabilitation Medicine have focused on
the control of balance and gait. In a study of balance in aging, we found that older normal subjects
maintain a more rigid posture, utilizing large and inconsistent adjustments to maintain stability. This
may relate to the instability of stance in the elderly.
41 -MNB/ DIR
PHS 6040 (Rev 1 84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 0271 1-05 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ ECT (80 characters or less. Title must fit on one line between the borders.)
Utility and Physiology of Botulinum Toxin for Involuntary Movement Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: Mark Hallett, M.D.
Others: Leo Cohen, M.D.
Reginald Cole, M.D.
Clinical Director
OCD
ODIR
DIR
NINDS
Chief
HMCS
MNB
DIR
NINDS
Visiting Scientist
HMCS
MNB
DIR
NINDS
Clinical Associate
HMCS
MNB
DIR
NINDS
COOPERATING UNITS (if any)
Speech Pathology Unit, NIDCD
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Human Motor Control Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.0
PROFESSIONAL:
0.5
OTHER:
0.5
CHECK APPROPRIATE BOX(ES)
I x | (a) Human subjects
] (a1) Minors
J (a2) Interviews
J (b) Human tissues J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Botulinum toxin injected in small doses directly into muscle binds to the neuromuscular junction and
inactivates it for approximately three months.
Studies of utility of botulinum toxin are been carried out in writer's cramp (and its varients such as
pianist's cramp) in open label and double-blind trials. Treatment appears effective.
We have begun a trial of botulinum toxin in spasmodic torticollis with the purpose of analyzing the
dysphagia that some of these patients have following injection.
42-MNB/DIR
PHS 6040 (Rev 1/841
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02712-05 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ ECT (80 characters or lass. Title must fit on one line betvreen the borders.)
Non-invasive Stimulation of Human Central Nervous System
PRINCIPAL IN VESTIGATORUist other professional personnel belom the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: MarkHallett, M.D.
Others: Leo Cohen, M.D.
Peter Fuhr, M.D.
Rocco Agostino, M.D.
HelgeTopka, M.D.
Eric Wassermann, M.D.
Clinical Director
Chief
Visiting Associate
Visiting Fellow
Visiting Fellow
Special Volunteer
Clinical Associate
OCD
ODIR
DIR
NINDS
HMCS
MNB
DIR
NINDS
HMCS
MNB
DIR
NINDS
HMCS
MNB
DIR
NINDS
HMCS
MNB
DIR
NINDS
HMCS
MNB
DIR
NINDS
OCD
ODIR
DIR
NINDS
COOPERATING UNITS (if any)
Speech and Voice Pathology Unit, IRP, NIDCD
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Human Motor Control Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
4.0
PROFESSIONAL:
3.5
OTHER:
0.5
CHECK APPROPRIATE BOX(ES)
I x | (a) Human subjects
] (a1) Minors
] (a2) Interviews
3 (b) Human tissues Q (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Recently techniques have become available for the non-invasive stimulation of the human cortex and
deep proximal peripheral nerves. Stimulation can be with a high voltage, extremely brief electrical pulse
or with magnetic stimulation. One purpose is to use these methods for noninvasive localization of
different parts of the human cortex including motor cortex, sensory cortex and language cortex.
Another purpose is to study cortical physiology in different disease states.
We have made a number of advances in understanding the technical aspects of magnetic stimulation,
trying to define the optimal method to map different body part representations in motor cortex. In
detailed mapping studies, distal muscles were significantly more excitable than proximal muscles and
motor representations targeting proximal arm muscles were significantly more excitable in the right
than in the left hemisphere. Plastic reorganization of the brain has been demonstrated in a number of
circumstances. In amputees, muscles ipsilateral to the stump could be activated from a larger area than
those contralateral to the stump. In patients with spinal cord injury, magnetic stimulation evoked larger
motor evoked potentials with shorter latencies in muscles immediately proximal to the level of a spinal
cord injury than in corresponding muscles in controls. In patients with hemispherectomy, stimulation of
the remaining hemisphere evoked bilateral muscle responses in proximal and distal muscles at similar
latencies indicating a bilateral representation of arm muscles and the existence of physiologically active
ipsilateral pathways. Detailed mapping studies have also been done with paresthesias produced by
magnetic stimulation.
Akinesia in Parkinson's disease was studied with motor evoked potentials elicited prior to a reaction time
movement. Results showed that it took longer than normal for patients to bri ng the cortex to a
sufficient level of excitation to produce a voluntary movement.
43-MNB/DIR
PHS 6040 (Rev l 84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 01658-23 MNB
PERIOD COVERED
Ort-nher 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Ht-misphpr^ DggeJ opmenr. and Specialization of the Intellectual Functions
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI:
P.
Fedio, Ph.D
E.
Mohr, Ph.D.
Others :
T.
Blaxton, Ph
D.
S.
Bookhe imer ,
Ph.D
L.
Ryan , M . A .
Chief
Psychologist
Senior Staff Fellow
IRTA Fellow
Psychologist
CNS, MNB, NINDS
Ottawa, Canada
CNS, MNB, NINDS
CNS, MNB, NINDS
CNS, MNB, NINDS
COOPERATING UNITS (if any)
LAB/BRANCH
Medical Neurology, CNP. DIR
SECTION
Clinical Neuropsychology
INSTITUTE AND LOCATION
NTNDS, NTH, Bethesda, MP 20892
TOTAL MAN-YEARS:
1 o
PROFESSIONAL:
0.5
OTHER:
-Q.J.
CHECK APPROPRIATE BOX(ES)
S (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The effects of chronic progressive neurological disorders in adults and children
were evaluated by a broad range of neuropsychological tests evaluating
bra in - behavior relations. A neuropsychological profile was plotted for patients
with Alzheimer's (AD). Huntington's (HP), or Parkinson's (PD) disorder. The
evaluations extended into memory . learning and perception, applying standard and
experimental tasks to identify functional changes accompanying aging processes.
The results implicated dopamine deficiencies and frontal pathophysiology in PD,
most notably, losses in executive capabilities and visuospatial and generic memory
functions. With HD patients, perceptuomotor capacity and the abilility to
manipulate spatial information were affected whereas spatial discrimination was
relatively intact. With a dichotic task, AD patients did poorer and were unable
to selectively attend to serial information. The behavioral data extend
neuropathologic impressions of degeneration of the frontal striatal system in HD
and temporoparietal . cortical Involvement in AD.
44-MNB/DIR
PHS 6040 (Rev. 1/84)
OPO 814-918
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 01424-24 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (BO characters or less Title must fit on one line between the borders )
Behavioral Modulation by the Limbic System in Man
PRINCIPAL INVESTIGATOR (List other protessional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
Chief
Senior Staff Fellow
IRTA Fellow
Psychologist
Medical Officer
Medical Officer
Medical Officer
Psychologist Catholic University
PI:
P.
Fedio, Ph.D
Others :
T.
Blaxton, Ph
D.
S.
Bookheimer ,
Ph.D
L.
Ryan, M.A.
C.
Kufta, M.D.
S.
Sato, M.D.
W.
Theodore, M
D.
CNS,
MNB,
NINDS
CNS,
MNB,
NINDS
CNS,
MNB,
NINDS
CNS,
MNB,
NINDS
SNB,
NINDS
CES,
MNB,
NINDS
CES,
MNB,
NINDS
A. August, M.A.
COOPERATING UNITS (it any)
Surgical Neurology Branch, DIR, NINDS
Department of Medical Psychology, John Hopkins University, Baltimore, MD
Department of Psychology, Catholic University, Washington, D.C.
LAB/BRANCH
Medical Neurology, CNP, DIR
SECTION
Clinical Neuropsychology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS
1.5
PROFESSIONAL
1.0
OTHER
0.5
CHECK APPROPRIATE BOX(ES)
D3 (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided)
Emotional and cognitive characteristics were studied in epileptic patients before
and following unilateral left or right temporal lobe resection, and during brain
stimulation and intracarotid amvtal -injection (Wada ) . Physiological events (skin
conductance; and EEG measures were also monitored during select test performance.
The research examined the role of the temporal lobe in establishing limbic sensory
associations as a basis for cognition and emotion.
In affective spheres, dysphoria followed pharmacological deactivation of the left
hemisphere whereas euphoria accompanied amytal injection into the right internal
carotid. The transient mood state was more common for patients with right temporal
lesions, with late age at onset of seizure disorder. With neuropsychometric proce-
dures, the patients differed along an introver s ion- extrover s ion continuum, the left
being more inclined to rate themselves as avoidant, depressed and overly anxious.
In contrast, the right temporal patients presented themselves in a more favorable
light, but the test profile showed histrionic and aggressive features. Patients
presenting an aura of fear are more likely to exhibit maladaptive behaviors. These
data suggest that unilateral temporal lobe injury disrupts the normal linkage of
cognitive-affective associations mediated by frontal -limbic interaction.
Following unilateral temporal lobectomy, physiological hypoarousal was more common
with left, and hyperarousal, with right, resections. Changes in activation level
may account for the clinical signs of dysphoria and euphoria with left and right
brain lesions, respectively.
45-MNB/DIR
PHS 6040 (Rev. 1/84)
GPO 914-018
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 01245-25 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (BO characters or less. Title must lit on one line between the borders.)
EEG Learning Correlates Using Scalp and Intracranial Depth Electrodes
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI:
P.
Fedio, Ph.D.
Others :
S.
Sato, M.D.
M.
Balish, M.D.
B.
Smith, M.D.
C.
Kufta, M.D.
Chief
Medical Officer
Medical Officer
Psychologist
Medical Officer
CNS, MNB NINDS
CES, MNB, NINDS
CES, MNB, NINDS
University of MD
SNB, NINDS
COOPERATING UNITS (it any)
Surgical Neurology Branch, DIR, NINDS
Department of Psychology, University of MD.
College Park, MD
LAB7BRANCH
Medical Neurology, CNP, DIR
SECTION
Clinical Neuropsychology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS
1.0
PROFESSIONAL:
0.5
OTHER:
0.5
CHECK APPROPRIATE BOX(ES)
(S (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided.)
Perception and judgment with cognitive and emotional tasks were monitored by
electroencephalographic (EEG) activity recorded from left and right brain regions,
of patients following unilateral temporal lobectomy. EEG disturbances in
bra in -behavior relations in neuropsychiatric patients were also evaluated,
relating left and right brain dysfunctioning to maladaptive ideative and emotional
reactions, respectively.
With temporal lobectomy patients, preliminary results indicate that electrographic
shifts in frequency-amplitude differed in that right temporal patients showed
greater responsivity to pleasant and horrific materials, and less so while
applying cognitive stategies to deal with imaginary emotionally charged
situations. The converse was true with left temporal patients who generated
greater activity while intellectually resolving emotional tasks. These data
underscore the dual cognitive and emotional roles of the limbic system in
modulating human behavior.
Left brain stimulation (indwelling flap electrodes) of posterior sites, produced
storage and retrieval memory errors with anterior and posterior, temporal sites,
respectively. Stimulation of frontal cortex produced defects suggestive of mechan-
isms that collate immediate and long-term plans. There was a dissociation between
aphasia and amne s ia with inferior, posterior temporal stimulation, emphasizing the
importance of this region to retrieval from episodic memory registers. With right
brain stimulation, paralinguistic disturbances were produced in prosody, and there
were errors in interpreting ambiguous statements, and in pattern discrimination
and recognition.
46-MNB/DIR
spo »i 4-an
PHS 6040 (Rev. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 00200-36 MNB
PERIOD COVERED
October 1. 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must fit on one line between the borders )
Cognitive and Emotional Profile of Neuropsychlatrlc Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
PI: P. Fedlo, Ph.D.
T. Blaxton, Ph.D.
S. Bookheimer, Ph.D.
Others: L. Ryan, M.A.
D. Ronsaville, Ph.D.
T. Chase, M.D.
E. Mohr, Ph.D.
A. August, M.A
Chief
Senior Staff Fellow
IRTA Fellow
Psychologist
Psychologist
Neurologist
Psychologist
Psychologist
CNS, MNB, NINDS
CNS, MNB, NINDS
CNS, MNB, NINDS
CNS, MNB, NINDS
CNS, MNB, NINDS
Chief, ETB, NINDS
Ottawa, Canada
Catholic University
COOPERATING UNITS (if any)
Experimental Therapeutics Branch, DIR, NINDS
LAB/BRANCH
Medical Neurology, CNP, DIR
SECTION
Clinical Neuropsychology
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN- YEARS
2.0
PROFESSIONAL
1.0
OTHER:
1.0
CHECK APPROPRIATE BOX(ES)
B (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues D (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided.)
Experiments were initiated to identify the neuroanatomical basis underlying
different types of memory and perceptual dysfunctions exhibited by patients with
neurological disorders. One theoretical line focused on using implicit and
explicit memory tasks within the framework of data- versus conceptua 1 1 y - dr iven ;
other investigations examined the role of the temporal lobe in perceiving
information containing high and low spatial frequencies.
Normals and right brain-damaged patients did better when memory processing engaged
at study (data-or conceptually-driven) matched that required at test. Patients
with left hemisphere lesions showed normal performance levels on data-driven tests
but were impaired on both implicit and explicit conceptually driven tests.
Although controls and left lesion patients performed normally, patients with right
hemisphere lesions showed deficits with data-driven, nonverbal materials.
When sets of perceptual stimuli were identical, high spatial frequency information
was preferred, but when stimuli differed, low frequency information was perceived
better. This pattern did not hold for left brain damage subjects who were
impaired in strategic processing. Also, patients performed worse than controls in
processing upright faces but actually performed better with the inverted faces.
Perceptual asymmetries are less pronounced between the hemispheres than processing
asymmetries, and the mode of processing interacts with perceptual characteristics
of incoming stimuli to bias perception toward high or low spatial frequencies.
47-MNB/DIR
PHS 6040 (Rev. 1/84)
GPO 914-819
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02732-04 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ ECT (SO chtrtctan or toss. Tltfe must lit on ona Una barman !/>« borders)
Pharmacological Studies of Ion Channels in Cultured Cells
PRINCIPAL INVESTIGATOR (Uft olhar prohtiStonml pononntl balow tht rrinaotl Inwstigttor.) (Utmo. t/lta. iMborttory. tnd institute tffilittion)
PI: Michael A. Rogawski, M.D., Ph.D. Chief, NEXS, MNB, NINDS
Others: Dora M.T. Politi, M.D.
Norman Hershkowitz, M.D.
Taco R. Werkman, Ph.D.
Susan M. Jones, Ph.D.
Karen Wayns
Visiting Fellow, NEXS, MNB, NINDS
NRC Fellow, NEXS, MNB, NINDS
Visiting Fellow, MNB, NINDS
NRC Fellow, NEXS, MNB, NINDS
Bio. Lab. Tech., NEXS, MNB, NINDS
COOPERATING UNITS Vltny)
Bruce Smith, Ph.D., Research Services Branch, NIMH; Haruhiro Higashida, M.D., Kanazawa University,
Japan; J.M.H. ffrench-Mullen, Ph.D., ICI Americas, Wilmington, DE.
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Neuronal Excitability Section
INSTITUTE AND LOCATION
NINDS, Bethesda, MD 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
1 | (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues [x] (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Drug interactions with voltage-dependent K+ channels and N-methyl-D-aspartate (NMDA) receptor
coupled-cation channels were studied in cultured hippocampal neurons and in fibroblasts transfected
with K + channel genes using whole-cell voltage-clamp and single channel recording techniques. The
aim of this work was to explore new strategies for the rational development of antiepileptic drugs based
upon their interaction with neuronal ion channel systems. Work was focused in four areas: (1) kinetic
analysis of NMDA antagonism by novel dissociative-anesthetic-like anticonvulsant drugs; (2) tetrahydro-
aminoacridine (THA) block of NMDA-activated cation channels; (3) mechanism of action of K> channel
activator drugs; and (4) drug effects on K + channel currents carried by molecularly defined K + channel
proteins in fibroblast cells transfected with K + channel genes. The novel anticonvulsants phenylcvclo-
hexylamine (PCA) and 5-aminocarbonvl-5H-dibenzo[a,dkvclohepten-5,10-imine (ADCI) are structurally
related to the dissociative anesthetics phencyclidine and MK-801 . However, unlike their parents which
cause motor toxicity at low doses, PCA and ADCI protect against seizures in animal models at doses that
fail to cause motor impairment. We have determined that the more favorable toxicity profile of PCA and
ADCI may relate to their ability to block NMDA responses more rapidly than do PCP and MK-801 . Tetra-
hydroaminoacridine (THA), a centrally active cholinesterase inhibitor that may provide symptomatic
benefit in Alzheimer's disease, was found to produce a voltage-dependent block of NMDA responses in
cultured hippocampal neurons and also to reduce the frequency and duration of NMDA evoked single
channel currents in outside-out membrane patches. The antihypertensive cromakalim has been prev-
iously found to activate a K + current in cultured hippocampal neurons. We have demonstrated that
metabolic inhibitors can activate a similar K + current. We have also obtained evidence for the existence
of ATP-sensitive K + channels in hippocampal neurons, and our data indicate that cromakalim and met-
abolic inhibitors can activate these channels. These channels may play a role in protecting central neur-
ons from brain ischemia. Drugs like cromakalim that activate K + channels in CNS neurons have potential
as anticonvulsants, and perhaps also in the treatment of brain ischemia. The effects of various K +
channel blockers and activators are being studied on K + channels expressed in fibroblast cells transfect-
ed with K + channel genes. We observed that the NGK1 K+ channel is insensitive to tetraethylammon-
ium. but is effectively blocked by 4-aminopyridine, PCP, and histrionicotoxin. an alkaloid from frog skin.
48-MfiE7DIR
PHSSOMOUv. 1/M)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02733-04 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or lau. Tula must fit on ono Una bodveon the borders.)
Excitability Properties of Enzymatically Dissociated CNS Neurons
PRINCIPAL INVESTIGATOR (List othar professional personnel Mm (ho Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: Michael A. Rogawski, M.D., Ph.D. Chief, NEXS, MNB, NINDS
Others: Xiao-Jing Wang, Ph.D. Visiting Fellow, Mathematical Research Branch, NIDDK
John Rinzel, M.D. Chief, Mathematical Research Branch, NIDDK
COOPERATING UNITS (if an,)
Mathematical Research Branch, NIDDK
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Neuronal Excitability Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN-YEARS:
0.5
PROFESSIONAL:
0.5
OTHER:
CHECK APPROPRIATE BOX(ES)
] (a) Human subjects
] (a1) Minors
] (a2) Interviews
] (b) Human tissues (jT] W Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Electrophysiological techniques were used to characterize the ionic channels of neurons isolated from
slices of the adult guinea pig thalamus. Thalamic neurons undergo a shift from tonic to phasic (burst)
firing upon hyperpolarization. This state transition results from deinactivation of a regenerative
depolarizing event referred to as the low-threshold spike (LTS). We have previously shown that LTS-like
events can be evoked in isolated thalamic (dorsal lateral geniculate) neurons and that these potentials
are mediated by a low-threshold, rapidly inactivating (T-type) calcium current. Hodgkin-Huxley
modeling of the T-type calcium current indicated that the shape of the LTS can be accounted for almost
entirely by the intrinsic properties of T-type calcium channels. Our model is novel in that we used two
inactivation gates to account for the slow recovery from inactivation of the T-type calcium current. Burst
firing in thalamic neurons mediated by the LTS is believed to be critical to the generation of absence
seizures since drugs which specifically block the LTS (T-type calcium channels) prevent absence seizures.
Our theoretical model of the LTS is compatible with this idea.
49-MNB/DIR
PHSWHMRev 1441
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02772-03 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (SO characters or less, rule must fit on one line between the borders.)
Development of Uncompetitive NMDA Antagonists as Anticonvulsants *
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI:
Michael A. Rogawski, M.D., Ph.D. Chief, NEXS, MNB, NINDS
Others:
Shun-lchi Yamaguchi, Ph.D.
Andrew Thurkoff, Ph.D.
Brian de Costa, Ph.D.
KennerC. Rice, Ph.D.
Arthur E. Jacobson, Ph.D.
Psychologist, NEXS, MNB, NINDS
NRSA Fellow, Laboratory of Medicinal Chemistry, NIDDK
Fogarty Fellow, Laboratory of Medicinal Chemistry, NIDDK
Chief, Laboratory of Medicinal Chemistry, NIDDK
Senior Research Scientist, Laboratory of Medicinal
Chemistry, NIDDK
COOPERATING UNITS (if any)
Laboratory of Medicinal Chemistry, NIDDK
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Neuronal Excitability Section
INSTITUTE AND LOCATION
NINDS, Bethesda, MD 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
J (a) Human subjects
] (a1) Minors
J (a2) Interviews
] (b) Human tissues \j~\ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The dissociative anesthetics PCP and MK-801 are powerful anticonvulsants in a wide variety of animal
seizure models. However, undesirable side effects that occur in the same dosage range as seizure
protection limit their practical usefulness in the treatment of seizure disorders. Despite their
unfavorable therapeutic indices, PCP and MK-801 can be considered prototypes upon which to base the
design of less toxic and potentially more clinically useful antiepileptic drugs. We have examined the
anticonvulsant activity of more than 40 PCP analogs in an attempt to obtain compounds with enhanced
anticonvulsant activity relative to their neurotoxic side effects. Drugs were screened for anticonvulsant
activity in mice with the maximal electroshock (MES) test and by administration of the chemoconvulsants
pentylenetetrazol (PTZ) and N-methyl-D-asparate (NMDA). PCP had approximately equal potency in the
MES test and in a motor toxicity test so that its "therapeutic index" (Tl; ratio of dose causing toxicity in
50% of animals to dose causing seizure protection in 50% of animals) was about 1. Certain of the
analogs had Tl values higher than that of PCP. The most favorable of these were derivatives of 1-
phenylcyclohexylamine modified by (1) certain stereochemically orientated cyclohexane ring methyl
substituents, (2) ortho substituents on the phenyl ring, and (3) contraction of the cyclohexane ring.
Studies were also conducted on compounds related to the dibenzocyclohepteneimine MK-801. Of
particular interest is 5-aminocarbonyl-5H-dibenzo[a,d]cyclohepten-5,10-imine(ADG) which is
structurally related to the commonly used antiepileptic carbamazepine. ADCI was a potent
anticonvulsant in the MES test (ED50, 8.9 mg/kg) and showed a six-fold higher Tl than PCP. Unlike
carbamazepine, ADCI was able to protect against PTZ (ED50, 37 mg/kg) and NMDA-induced seizures
(ED50, 15 mg/kg). The ability of ADCI to block NMDA receptor-mediated responses was confirmed in
cellular electrophysiological studies (see project Z01 NS 02732-04 MNB). We conclude that ADCI may
offer advantages over carbamazepine in the treatment of resistant seizure disorders because of its ability
to block NMDA receptor-mediated responses.
* (Formerly, "Development of Phencyclidine Analogs as Anticonvulsants")
50-MNB/DIR
PHS 6040 (R«v 1 84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02792-02 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or /ess. Title must fit on one Una between the borders.)
Neuropsychological Investigations of Human Cognition and Mood State
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: Jordan Grafman, Ph.D. Chief CNS MNB NINDS
Others
Ray Johnson, Jr., Ph.D.
Special Expert
CNS
MNB
NINDS
Rhonda Friedman, Ph.D.
Special Expert
CNS
MNB
NINDS
MarkHallett, M.D.
Chief
MNB
NINDS
COOPERATING UNITS (if any)
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Cognitive Neuroscience Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN-YEARS:
0.4
PROFESSIONAL:
0.2
OTHER:
0.2
CHECK APPROPRIATE BOX(ES)
I x | (a) Human subjects
] (a1) Minors
J (a2) Interviews
J (b) Human tissues J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Current studies in the Cognitive Neuroscience Section focus on amnesia, thinking, neurolinquistics,
event-related evoked potentials, social cognition, and visual processing. Both single-case and group
design studies are used. Normal controls, inpatients and outpatients are evaluated. Memory is
studied in experiments focusing on implicit and explicit retrieval, priming, autobiographical recall,
discourse processing, naming and word retrieval, and categorization tasks. Reasoning and problem-
solving are studied in experiments focusing on planning, syllogisms, analogical thinking, and schema
organization. Dyslexia, dvsgraphia, and dysnomia, are studied in experiments focusing on single
word reading and writing, lexical decision, associative and semantic priming, and similar tasks.
Event-related evoked potentials are measured for latency, amplitude, and distribution and used as a
physiological indice of information-processing stages, working memory, visual attention, and
automatic processing. Emotions, impression and preference formation, and social judgment are
studied in experiments focusing on judgment of interpersonal behavior, word association, and mood
state. Finally, visual information processing is studied beginning with experiments examining spatial
frequency contrast-sensitivity, object recognition, and visual categorization. Although developing
theoretically valid and testable models of cognitive processing is the primary aim of the Section,
there is also a strong effort to relate the profile of cognitive deficits in patients to lesion location in
order to topographically map the components of cognitive processing to brain regions and systems.
Pharmacologic challenge and infusion studies are planned to evaluate the dissociability of
hypothesized components of memory processing. PET scan studies are planned to examine whether
familiar and unfamiliar faces are processed in a different brain location from real and non-objects
and forms.
51-MNB/DIR
PHS 6040 (Rev V84|
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02793-02 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ ECT (80 characters or less. Title must fit on one line between the borders.)
Cognitive Neuroscience***
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: Jordan Grafman, Ph.D.
Others: Ray Johnson, Jr., Ph.D.
Rhonda Friedman, Ph.D
Jeffrey Hadley, Ph.D.
Angela Sirigu, Ph.D.
Marten Scheffers, Ph.D.
Chief
Special Expert
Special Expert
IRTA
Visiting Fellow
Visiting Fellow
CNS
MNB
NINDS
CNS
MNB
NINDS
CNS
MNB
NINDS
CNS
MNB
NINDS
CNS
MNB
NINDS
CNS
MNB
NINDS
COOPERATING UNITS (if any)
Walter Reed Army Medical Center, Wash, DC; National Naval Medical Center, Bethesda, MD; Centre Paul
Broca, Paris, France; Hopital Salpetriere, Paris, France; Hospital Clinicas, Montevideo, Uruguay; **
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Cognitive Neuroscience Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
0.4
PROFESSIONAL:
0.2
OTHER:
0.2
CHECK APPROPRIATE BOX(ES)
I x | (a) Human subjects
] (a1) Minors
J (a2) Interviews
J (b) Human tissues J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Memory and cognition are studied in experiments focusing on representational knowledge, working
memory, priming, procedural learning, number processing and calculation, autobiographical memory,
visual attention, naming, and categorization. Normal subjects and patients with progressive dementia,
focal lesions, and psychiatric disorders are studied. New studies focusing on the composition of mental
structures in the frontal lobes have just begun.
*Continued:
A. Salazar, M.D.
S.Rao, Ph.D.
F. Boiler, Ph.D.
Y.Agid.M.D.
J.Sergent, Ph.D.
C. Chouza, M.D.
D. Hermann, Ph.D.
J. Hallenbeck, M.D.
A. Newell, Ph.D.
R. Desimone, Ph.D.
C. Chaviox, Ph.D.
J. Duncan, Ph.D.
I. Biederman, Ph.D.
J. Rapoport, M.D.
S. Swedo, M.D.
E.Zaidel.Ph.D.
Department of Neurology, Walter Reed Army Med. Ctr.
Dept. of Neurology, Medical College of Wisconsin
INSERM U. 324 Centre Paul Broca, Paris, France
INSERM U. 289 Hopital Salpetriere, Paris, France
Department of Neurology, Montreal Neurological Institute
Neurology Institute, Hospital Clinicas, Montevideo, Uruguay
Department of Labor, Washington, DC
Department of Neurology, National Naval Medical Center
Computer Science Dept., Carnegie-Mellon Univ., Pittsburgh, PA
National Institute of Mental Health
INSERM Unit 320, Caen, France
Applied Psychology Unit, Cambridge, England
Dept. of Psychology, USC, Los Angeles, CA
National Institute of Mental Health
National Institute of Mental Health
Dept. of Psychology, UCLA, Los Angeles, CA
** Medical College of Wisconsin, Milwaukee, Wisconsin; Montreal Neurological Institute, Montreal,
Canada; National Institute of Mental Health, NIH.
** (Formerly, "Amnesia and Cognitive Neuroscience")
52-MNB/DIR
PHS 6040 (Rev 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02794-02 MNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ ECT (80 characters or less. Title must fit on one line between the borders.)
Event-Related Potential Studies of Normal and Abnormal Cognitive Processing
PRINCIPAL INVESTIGATOR (list other professional personnel belojv the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: Ray Johnson, Jr., Ph.D. Special Expert CNU MNB DIR NINDS
Others: Marten Scheffers, Ph.D.
Jordan Grafman, Ph.D.
Daniel Ruchkin, Ph.D.
Wolfgang Miltner, Ph.D.
Visiting Fellow CNU MNB DIR NINDS
Chief CNU MNB DIR NINDS
Elec. Engineer U. of Maryland School of Medicine
Psychologist U. of Tuebingen, West Germany
COOPERATING UNITS (if an,)
University of Maryland School of Medicine, University of Tuebingen, West Germany
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Cognitive Neuroscience Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN-YEARS:
20
PROFESSIONAL:
2.0
OTHER:
CHECK APPROPRIATE BOX(ES)
I x | (a) Human subjects
] (a1) Minors
] (a2) Interviews
I I (b) Human tissues J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Event-related brain potentials (ERP) were used to study cognitive processes such as short- and long-term
memory, spatial attention and visual search, mental rotation, mental arithmetic, and language compre-
hension. ERP studies of normal subjects were intended to reveal the brain mechanisms underlying cogni-
tion. Studies of patients with neuropsychiatry disorders were intended to provide information on the
physiological sources of these cognitive processes while allowing us to characterize better the patients'
information processing deficits. Data analysis continues on studies of temporal lobectomy patients.
Turner's patients, and the maturation of cognitive processes. ERP studies of dementia are continuing for
patients with Alzheimer's disease while data collection has been completed for patients with HIV disease
and progressive supranuclear palsy. The results from the HIV and PSP studies indicate that, in the earliest
stages of subcortical disease, processing at the cortical level is more affected than processing at the sub-
cortical level (i.e., resembling a cortical dementia). This pattern reverses as the subcortical disease pro-
gresses. Studies of the mechanisms underlying and affecting attentional processes continue. Data col-
lection was completed recently in two studies, one on how normal controls visually search a spatial array
for items previously stored in short-term memory and one on the effects of fatigue on attention in pa-
tients with chronic fatigue syndrome and normal controls. Studies with Daniel Ruchkin have been aimed
at understanding a newly emerging class of ERP components known as slow waves. Completed studies
have investigated the processes of mental arithmetic, mental rotation, and short-term memory. Data
collection is nearly complete in an experiment on the differences between rehearsal processes for verbal
and spatial material. Studies with Wolfgang Miltner have been aimed at providing additional data on
the neural sources of ERP components. Completed studies have provided a comparison of the ERP com-
ponents elicited by auditory and somatosensory stimuli using large electrode montages. A new experi-
ment was recently finished so that the neural source localization program, BESA, can be applied to the
data. Patient and control data have been used to validate the predictions of Johnson's model of the vari-
ables controlling P300 amplitude. These data revealed that, contrary to the widely accepted notion, the
P300 is a modality-dependent component whose amplitude represents the sum of a number of distinct
neural generators which we are attempting to characterize and localize.
53-MNB/DIR
PHS 6040 (Rev 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02038-18 MNB
PERIOD COVERED
October 1,1989 through September 30,1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Combined Clinical, Viral and Immunological Studies of Neuromuscular Diseases
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: M.C. Dalakas, M.D.
Other: M. Hallett, M.D.
B. Sonies, Ph.D.
R. Quarles, Ph.D.
P. Plotz, M.D.
G. DiChiro, M.D.
R. Podolsky, Ph.D.
W.A. Gahl.M.D.
Medical Officer
Clinical Director
Speech Pathologist
Biochemist
Rheumatologist
Neuroradiologist
Biochemist
Medical Officer
MNB, DIR, NINDS
OCD, DIR, NINDS
CC, DIR, NINDS
DMN, DIR, NINDS
ARB, DIR, NIMSD
OD, DIR, NINDS
ARB, DIR, NIMSD
HGB, DIR,NICHD
COORPERATING UNITS (if any)
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Neuromuscular Diseases
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.5
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
I x I (a) Human subjects
| ] (a1) Minors
J (a2) Interviews
J (b) Human tissues j (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Clinical and laboratory studies are conducted to determine etiology (infection, immunity and/or
genetics) of chronic diseases of the neuromuscular system. Current studies include polymyositis/derm-
atomyositis. post-polio syndrome, amyotrophic lateral sclerosis (ALS), demyelinatinq polyneuropathies.
neuromuscular diseases associated with HIV infection, and Duchenne's muscular dystrophy. The post-
polio syndrome, a neuromuscular disease that occurs in patients who have had poliomyelitis at an early
age, has been clinically defined. A longitudinal study is now in progress to define the rate of progression
by quantifying the muscle strength with a series of dynamometric measurements. The pathogenesis of
this syndrome has been also investigated with a series of electrophysiological, virological, immunological
and histological studies. Its relevance to other motor neuron diseases is now being examined and
possible persistence of mutated poliomyelitis virus is sought using tissue cultures, PCR, and in situ
hybridization. Because abnormal immunoregulation was found in some patients with post-polio
syndrome, a double-blind placebo-controlled trial using prednisone has begun. The spectrum of
neuromuscular disorders associated with H]V infection has been studied and the role of the virus as the
cause of neuropathies or myopathies in HIV-positive patients was investigated with a variety of
immunocytochemical studies and in situ hybridization. The neuromuscular complications of antiretro-
viral drugs AZT and DDC were studied and factors that could limit their neurotoxicity were identified.
Patients with polymyositis are studied and the muscle changes before and after a randomized double-
blind controlled study with intravenous methotrexate, azathioprine or plasmapheresis/lymphocyto-
pheresis are investigated. The force of individual (skinned) muscle fibers from the muscle biopsies of
patients with Duchenne's muscular dystrophy was measured and correlated with the biochemical
composition of the contractile muscle proteins. We have found that in spite of the absence of
dystrophin, muscle fibers in Duchenne's dystrophy generate normal force. The metabolic activity of the
cortex in ALS patients was studied using the PET scan and 18FDG and the metabolic abnormalities were
correlated with the morphological changes in the neurons on brain tissues obtained at autopsy. A
variety of neuromuscular diseases associated with nephropathic cvstinosis and renal Fanconi syndrome
were studied morphologically and biochemically. A myopathy with lipid or cystine storage in the muscle
was found prompting a therapeutic trial with carnitine or cysteamine.
54-MNB/DIR
PHS 6040 (Rev. 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02531-09 MNB
PERIOD COVERED
October I, 1 989 through September 30, 1 990
TITLE OF PROltCJ (SO characters or less. Title must fit on one line between the borders.)
Studies in Neuromuscular and CNS Diseases and Their Experimental Models
PRINCIPAL INVESTIGATOR (List other professional personnel belo» the Principal Investigator.) (Name, tit/e. laboratory, and institute affiliation)
PI: M.C. Dalakas, M.D.
OTHERS: R. Quarles, Ph.D.
G.H. Pezeshkpour, M.D.
M.Mutchnich, M.D.
I. Ilia, M.D, Ph.D.
M. Monzon, Ph.D.
L. Lamperth, M.D.
Medical Officer
Biochemist
Neuropathologist
Gastroenterologist
Neurologist
Special Expert
Visiting Associate
MNB, DIR, NINDS
DMN, DIR, NINDS
AFIP, Washington, D.C.
Detroit, Ml
MNB, DIR, NINDS
MNB, DIR, NINDS
MNB, DIR, NINDS
COORPERATING UNITS (if any)
Department of Gastroenterology, Wayne State University, Detroit, Ml
AFIP, Washington D.C.
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Neuromuscular Diseases
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.5
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
I X | (a) Human subjects
] (a1) Minors
J (a2) Interviews
[xl (b) Human tissues O (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Immunocytochemical studies were conducted using specific antibodies to thymic peptides to investigate
the interaction of the immune system with the central and peripheral nervous systems. Thymosin beta 4,
an immunomodulating thymic polypeptide, was found to be a common antigen shared by macrophages,
dendritic lymphoid cells and the myelin producing cells in the CNS (oligodendrocytes) and the PNS
(Schwann cells). Prothymosin, a nuclear protein, and thymosin-a1, were found present in astrocytes of
normal human brain and could play a role in cell proliferation and gliosis. The IgM of certain patients
with paraproteinemic polyneuropathies has been identified as a specific antibody to acidic qlycolipids;
intraneural injection of IgM in the sciatic nerve of the cat induced demyelination suggesting a direct role
in the pathogenesis of the neuropathy. The nature of amyloid protein in patients with "sporadic"
amyloid polyneuropathy was identified using specific antibodies to amyloid proteins; point mutations
and direct sequencing of prealbumin genes, the precursor protein, were studied in the amyloid tissue
using the polymerase chain reaction. The mechanism of inflammatory myopathy in monkeys with
immunodeficiency (Simian AIDS) cause by SRV-1 andSIV-1 retroviruses, was studied. Antibodies to SRV-1
immunoreacted with inflammatory cells invading muscle fibers; SRV-1 was capable of infecting
myoblasts in tissue culture without exerting a cytopathic effect in the muscle. The role of SIV-1 is
similarly studied. The effect of aging on the neuromuscular system of monkeys from age 5 to 25 is being
studied with a detailed morphological and morphometrical analysis of their muscle and nerve biopsies.
The mechanism of muscle regeneration is studied examining markers on satellite cells including the role
of adhesion on molecules such as laminin, N-CAM,and ICAM. The monoclonal antibody Leu-19(NKH)
that identifies natural killer cells was found to share common antigenic determinants with the satellite
muscle cells. NKH also stains regenerating muscle fibers and could play a role in muscle regeneration.
The induction of abnormal mitochondria in the muscle by several nucleoside analogues is investigated in
the muscle fibers in tissue culture.
55-MNB/DIR
PHS 6040 (Rev 184)
PROJECT NUMBER
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT z01 NS 01195-26 MNb-
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must tit on one line Between me Borders I
Radiographic, Radioisotopic, CT, and MR Angiography of the Spinal Cord
PRINCIPAL INVESTIGATOR (List other orotessional personnel Below the Principal Investigator.) (Name, title. laBoratory. ana institute affiliation)
Giovanni Di Chiro, M.D., Chief, NIS, MNB, CNP , DIR, NINDS Others:
NIS, NINDS
DRD, CC
SN, NINDS
NMD, CC
Neuroradiology, GTU
Neurosurgery, Childrens
L.
J.
M. Levy, M.D. , Ph
L. Doppman, M.D.
D.
Guest
Chief
Researcher
E.
H. Oldfield, M.D.
Chief
R.
D. Neumann, M.D.
Chief
D.
Schellinger, M.D.
Chief
D.
C. McCullough
Chief
COOPERATING UNITS (it any)
Diagnostic Radiology and Nuclear Medicine Departments,
Clinical Center, NIH; Surgical Neurology Branch, NINDS; Medical Examiner's
Office, Department of Public Health Philadelphia, PA.
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Neuroimaging
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN-YEARS:
0.3
PROFESSIONAL:
0.3
OTHER:
CHECK APPROPRIATE BOX(ES)
H (a) Human subjects □ (b) Human tissues □ (c) Neither
Q (a1) Minors
□ (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Selective arteriography (radiographic) of the spinal cord is a
diagnostic technique which has proven to be informative in cases of
arteriovenous malformation (AVM), tumor, obstructive vascular disease,
trauma, and postradiation damage of the spinal cord.
Radioisotope angiography of the spinal cord offers some advantages as a
screening method, and in certain types of intraspinal pathology may £ive
information not available by any other diagnostic test.
Experience with the techniques of dynamic computed tomography, digital
subtraction angiography (DSA), positron emission tomography (PET) using
lbp_2-deoxyglucose and nuclear magnetic resonance imaging (MRI) of the
spine (without and with a contrast agent such as Gd-DTPA) indicates that
these methods may be useful screening and follow-up procedures in the
evaluation of certain vascular lesions and tumors of the spinal cord.
* Formerly in OCD ; transrerred to MNB on 6/8/90.
56-MNB/DIR
PHS 6040 (Rev. 1/84) OPO >I44II
DEPARTMENT OF HEALTH AND HUMAN SERVICES ■ PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02073-17 MNB*
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must tit on one line Between the borders )
Nuclear Magnetic Resonance and Computed Tomography (Transmission)
PRINCIPAL INVESTIGATOR (List other prolessional personnel belo* the Principal Investigator I (Name title, laboratory, and institute affiliation)
Giovanni Di Chiro, M.D., Chief, NIS, MNB, CNP, DIR, NINDS Others (NIS) : J. R.
Alger, Ph.D., Special Expert; R. A. Brooks, Ph.D., Staff Physicist; L. M. Levy,
M.D., Ph.D., Guest Researcher; A. Bizzi, M.D., Visiting Fellow; A. Brunetti, M.D. ,
Visiting Fellow; T. Francavilla, M.D., Staff Fellow; D. LeBihan, M.D. , Ph.D.,
R. Turner, Ph.D.; C. Moonen, Ph.D.; B. DeSouza, M.D., Staff Fellow; R. S.
Miletich, M.D., Ph.D., Senior Staff Fellow; M. J. Fulham, M.D., Visiting
Associate, N. Patronas, M.D., DRD , CC; J. Frank M.D., DRD, CC; J. L. Doppman, M.D.
Chief, DRD, CC; Joanna M. Hill, M.D., Senior Staff Fellow, NSB, NIMH
cooperating units (,t any, Diagnostic Radiology, Clinical Center, NIH; "In Vivo NMR
Research Center, BEIB, NIH; Division of Neuropathology of Case Western Reserve
University Medical School, Cleveland.
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
section
Neuroimaging
INSTITUTE ANO LOCATION
NINDS, NIH, Bethesda, MD
20892
TOTAL MAN-YEARS
2.0
PROFESSIONAL
2.0
OTHER
CHECK APPROPRIATE BOX(ES)
Cid (a) Human subjects
Q (a1) Minors
D (a2) Interviews
D (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
NMR: Our NMR imaging research is developing along eleven main lines: 1)
study of patients with spinal cord pathology, particularly arteriovenous
malformations (AVM); 2) recognition of artifactual linear regions of
abnormal signal intensity along the length of the spinal cord; 3)
assessment of pulsatile CSF flow using longitudinal imaging; 4) studies
of the "mobile" (normal) and "fixed" (pathologic) spinal cord; 5) con-
tinuing the in vivo and i_n vitro investigation of extravasated
intracranial blood; 6) analysis of the signal intensity from critical
areas (basal ganglia) in patients affected by a variety of movement
disorders; 7) trying to learn more about the NMR signal of various
abnormal tissues; 8) comparing clinical MRI imaging results with those
of CT and particularly PET; 9) Analysis of iron accumulation in the
basal ganglia of normal primates of various ages as well as in
parkinsonian (MPTP) animals; 10) MRI of MPTP induced lesions in the
basal ganglia of primates; 11) animal CNS imaging and spectroscopy with
experimental, small bore devices (2.0 and 4.7 Tesla). Finally, we have
initiated A) investigation of NMR spectroscopy (proton) in patients with
brain tumors; B) cine-MRI applications Lo the assessment of patients
with syringomyelia and other cord cavities; C) MR angiography (flow
based) in cord AVMs ; and D) comparison of diffusion (Intra-Voxel
Incoherent Motion Analysis) with PET in brain tumors.
CT: Ongoing research studies of tumoral, degenerative, demyelinating and
atrophic processes of the brain, plus hydrocephalus, brain edema, post-
radiation necrosis and epilepsy.
*Formerly in OCD; transferred to MNB on 6/8/90.
57-MNB/DIR
I
PHS 6040 (Rev 1/84)
SCO 114-tll
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02315-13 MNB*
PERIOD COVERED
October 1, 1Q8Q through September 30, 1 QQO
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.)
Positron Emission Tomography
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Giovanni Di Chiro, M.D., Chief, NIS, MNB, CNP, DIR, NINDS. OTHERS:
R. A. Brooks, Ph.D.
R. S. Miletich, M.D.,
M. J. Fulham, M.D.
C. F. Ferrarese. M.D.
J. W. Melisi, M.D.
M. 0. Duhaney, M.D.
A. Brunetti , M.D.
Staff Physicist
Ph.D. Sr. Staff fellow
Visiting Associate
Visiting Associate
Special Volunteer
Irta Fellow
Visiting Fellow
NIS, NINDS
NIS, NINDS
NIS, NINDS
NIS, NINDS
NIS, NINDS
NIS, NINDS
NTS, NTNuS**
COOPERATING UNITS (it any)
NM, CC; MN, NINDS, SN, NINDS; DIR, NINDS; BEIB, DRS; DRD, CC; LCM, NIMH;
or.n, NTNDS, r.NR, ntnds
LAB/BRANCH
Medical Neurology Branch, CNP, DIR
SECTION
Neuroimaging
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, MD 20892
TOTAL MAN- YEARS:
2.0
PROFESSIONAL:
2.0
OTHER:
CHECK APPROPRIATE BOX(ES)
0 (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Positron emission tomography (PET) allows us to obtain anatomical data
(e.g., axial transverse, coronal or sagittal images of the brain) as
well as dynamic functional data (such as regional cerebral glucose
consumption rate, using 1 ^F-2-deoxyglucose (FDG). Besides FDG, other
radiopharmaceuticals (tagged with 1°F, 15q, 11c, 13n) can be used-with
PET to study the blood-brain barrier (BBB), oxygen metabolism, protein
synthesis, and neuroreceptors. The unique property of PET is that it
provides physiologic and pathophysiologic information not available with
any other imaging procedure.
**Continued:
T. Francavilla
G. Comi
B. DeSouza
I. J. Kopin
E. H. Oldfield
C. V. Kufta, M.D.
M. Hallett, M.D.
R. J. Polinsky, M.D.
L. Sokoloff, M.D.
Staff Fellow
Visiting Fellow
Staff Fellow
Director
Chief
Staff Physician
Clinical Director
Chief, CNS
Chief
NIS, NINDS
NIS, NINDS
NIS, NINDS
DIR, NINDS
SN, NINDS
SN, NINDS
DIR, NINDS
CN, NINDS
LCM, NIMH
*Formerly in OCD; transferred to MNB on 6/8/90
58-MNB/DIR
PHS 6040 (Rev. 1/84)
FPO fit 4-018
>
00
C
30
O
m
O
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03
30
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03
ANNUAL REPORT
October 1, 1989 through September 30, 1990
Neuroepidemioloqy Branch
Clinical Neurosciences Program, DIR
National Institute of Neurological Disorders and Stroke
Table of Contents
RESEARCH SUMMARY 1-9
PROJECT REPORTS
Clinical, Genetic, Pathophysiologic Study of 10
Hereditary Movement Disorders
Z01 NS 01924-20 NEB
Clinical, Genetic, Pathophysiologic Study of 11
Hereditary Nervous System Tumors
Z01 NS 01927-20 NEB
denetic Epidemiology Studies in MS and Other 12
Multifactorial Neurologic Disorders
Z01 NS 02167-16 NEB
Epidemiology of Dementia 13
Z01 NS 02240-14 NEB
Pediatric Neuroepidemiology 14
Z01 NS 02243-14 NEB
Mortality from Neurologic Disorders: National 15
and International Comparisons
Z01 NS 02297-14 NEB
Reviews of Epidemiologic Aspects of Neurologic 16
Disease
Z01 NS 02299-14 NEB
Clinical Course and Medical Care for Neurologic 17
Disorders
Z01 NS 02300-14 NEB
Collaborative Studies of Less Common or Less 18
Debilitating Neurologic Disorders
Z01 NS 02301-14 NEB
Educational Resources in Neurological Epidemiology 19
Z01 NS 02307-14 NEB
i NFB/DIR
Table of Contents (cont'd)
Racial and Geographic Differences in Occurrence 20
of Neurologic Disease
Z01 NS 02370-12 NEB
Development of Data Resources for Neuroepidemiology 21
Z01 NS 02423-11 NEB
National History of ALS-PD in Guam 22
Z01 NS 02570-08 NEB
Epilepsy Neuroepidemiology 23
Z01 NS 02715-05 NEB
Phenobarbital Clinical Trial in Children with 24
Febrile Seizures
Z01 NS 02746-04 NEB
Dental Markers of Maldevelopment 25
Z01 NS 02747-04 NEB
Dermatoglyphic Markers of Maldevelopment 2 6
Z01 NS 02748-04 NEB
California Cerebral Palsy Registry 27
Z01 NS 02819-01 NEB
ii NEB/DIR
Annual Report
October 1, 1989 through September 30, 1990
Neuroepidemiology Branch
Division of Intramural Research
Clinical Neurosciences Program
National Institute of Neurological Disorders and Stroke
Jonas H. Ellenberg, Ph.D., Acting Chief
Gustavo C. Roman, M.D. Chief Designate
The Neuroepidemiology Branch (NEB) is responsible for the development and
implementation of epidemiologic and genetic programs to investigate the cause,
prevention, and treatment of neurologic disorders in human populations. Emphasis
has been placed on major neurologic diseases in which the diagnoses can be
clinically verified to the satisfaction of skilled neurologists.
On July 14, 1987, Bruce S. Schoenberg, M.D., Dr.P.H., Chief of the
Neuroepidemiology Branch since its inception, passed away at a very early age.
Dr. Gustavo C. Roman, Professor of Neurology and Acting Chairman of the
Departs it of Medical and Surgical Neurology at Texas Tech University School of
Medicine in Lubbock, Texas, was appointed as Chief Designate of the NEB as of
January, 1990. Dr. Roman, a long time collaborator of Dr. Schoenberg, will be
joining the NIH on a full-time basis as of Sept. 1, 1990.
Neuroepidemiologic research studies require collaboration of many individuals.
However, since there is a severe shortage of available manpower in
neuroepidemiology, the Branch has developed an information program for current
and future collaborative investigators. A series of six videotapes on aspects
of neuroepidemiologic methods produced by the Branch is available. A monograph
entitled Neurological Epidemiology: Principles and Clinical Applications was
published in 1978. A new monograph which will cover current methodology and
disease specific overviews of etiology, incidence, prevalence and intervention
studies is in press.
Another important problem for the neuroepidemiologist is the enormous cost of
maintaining neurologic surveillance on a large number of patients. Therefore,
we have attempted to utilize existing registries of neurologic diseases. We
collaborate, for example, with the Mayo Clinic in Rochester and utilize their
records-linkage system to study neurologic diseases in the population of
Rochester, MN; the California Birth Defects Monitoring Program registry; the
Israeli National Disease Registry; and the Israeli Cancer Registry.
Epilepsy
Epilepsy is a major cause of morbidity and mortality on a world-wide basis. A
considerable research effort has been devoted by personnel of the Branch to
studying this disease. A protocol is in place for a clinical study of the
Lennox-Gastaut syndrome (LGS) , a severe childhood epileptic encephalopathy with
significant morbidity and mortality, characterized by uncontrolled seizures,
1 - NFB/DIR
mental retardation, and possible mental deterioration. Evaluations include sleep
studies, auditory evoked potentials, EEGs and videotape-EEG monitoring,
psychologic testing, and a battery of immunologic tests. Neuroimaging studies
obtained under an ongoing protocol will be evaluated with the clinical data.
The project is being undertaken in collaboration with the Clinical Epilepsy
Section, DIR, NINDS.
In collaboration with the University of Skopje in Yugoslavia, we are examining
the utility of the electroencephalogram as a predictor of recurrence of febrile
seizures in a defined population in the region of Macedonia; 676 children have
been examined clinically and by EEC An initial manuscript has been submitted
concerning clinical and EEG findings at study entry.
The international literature indicates that many persons who experience a first
convulsion do not have a recurrence in the subsequent two to five years, while
many others develop chronic epilepsy. It is unknown whether treatment early
after an initial seizure can reduce the likelihood of developing chronic
epilepsy. NEB is collaborating with the Biometry and Field Studies Branch in
examining the feasibility of randomized controlled trials of treatment with
anticonvulsant medication following a first convulsion in subjects ages seven
to 60 years who present for care to the Beijing Tiantan Hospital, a municipal
hospital affiliated with the Beijing Neurosurgical Institute and to hospitals
in the Western Galilee Region of Israel. The initial efforts are focusing on
testing the feasibility of recruitment and standardization of procedures and
piloting of protocols. The number of patients meeting study criteria, the ease
of identification of eligibility, the proportion of patients to be excluded for
each indication, the establishment and testing of rules for initiation of
treatment in the placebo arm (e.g., would patients continue to accept placebo
after a first recurrence), the willingness of patients to participate and return
for scheduled visits, the time from seizure onset to study entry, will all be
evaluated. Pharmacy procedures in obtaining placebo, and establishing and
maintaining the blind will also be tested. Procedures for the informed consent
will be developed by staff.
Alzheimer's disease
Another neurologic disease of increasing importance in most of the developed
countries, including the U.S., is Alzheimer's disease. This is another area of
major research interest to Branch personnel. Uniform diagnostic criteria are
applied to all studies of Alzheimer's disease being conducted in the Branch.
The clinical diagnosis is based on the criteria proposed by the NINDS-
Alzheimer's Disease and Related Disorders Association, Inc. work group. Where
possible, pathological verification of the diagnosis is obtained.
Alzheimer's disease/senile dementia, despite its high apparent clinical frequency
among the elderly, has not been well studied in a U.S. population. Descriptive
studies are yielding etiologic hypotheses which can be further investigated using
the case-control approach, and will provide the opportunity to investigate
dementia occurring with other neurologic disorders, such as Parkinson's disease.
These studies should also provide evidence whether Alzheimer's disease occurring
in the very elderly and that in the presenium represent the same disease process.
Utilizing a common protocol will permit international comparisons.
In addition, three case-control studies to determine risk factors for Alzheimer's
2 ,- NEB/DIR
disease have been completed or are in progress. The first uses cases and
controls selected from the Rochester, MN population. Past medical records have
been utilized to obtain information concerning possible associations between
Alzheimer's disease and other medical conditions or surgical procedures. This
study has the advantage that recall bias cannot affect the results of the study
since data are being abstracted from medical records. Information on the
occurrence of head trauma with loss of consciousness (LOC) was abstracted from
the medical records of 274 cases and controls. A statistically significant
difference in LOC between the two groups was not detected.
A second case-control study of factors associated with Alzheimer's disease in
collaboration with the Burke Rehabilitation Center, White Plains, New York, has
been completed and analysis is in progress. A third case-control study of
possible environmental etiology is underway at Mt. Sinai (New York) Department
of Community Medicine and Neurology. The study, using interviews for deriving
past environmental exposures, has enrolled approximately 65 patients and an
equal number of hospital based controls. Data collection was completed by the
end of FY 1989.
Vascular Dementia
Dementia due to cerebrovascular disease (also known as multi-inf arct dementia)
is the second-most common cause of dementia in the elderly. However, diagnostic
criteria for this condition are deficient, and this has hampered the development
of comparable international data. New concepts in the pathogenesis of vascular
dementia include the possible role of white matter hypoperfusion as playing a
major role in this condition. The term Binswanger's disease has been suggested
as a better option for this form of dementia. Dr. Roman has championed this idea
in several reviews (JAMA 248:1782-1788, 1987) and international meetings
("Cerebral Ischemia and Dementia" International Workshop, June 25-28, 1990 Prien
Am Chiemsee/Bavaria, Germany, and "Study Group on Neuroepidemiology" AIREN/NINDS-
NIH/WHO Geneva, Switzerland, July 9-11, 1990).
A number of studies on the neuroepidemiology of vascular dementia are in the
planning stage and a workshop for the definition of diagnostic criteria of
vascular dementia has been scheduled.
Analysis of death certificate data
Although death certificate data are limited by possible misdiagnosis, incomplete
case ascertainment, errors in coding, etc., detailed mortality information on
some neurologic diseases for the entire U.S. is not available. Analysis of
mortality data can be particularly useful for some neurologic diseases because,
indirectly, these may indirectly contribute to death. Since there are no uniform
criteria for what constitutes the underlying cause of death in patients, it is
important to examine all deaths in which a disease is listed as an underlying,
immediate, associated, or a contributory cause of death to get more complete
information about the relationship between the disease and death. Association
of diseases occurring at the time of death was studied for all deaths occurring
in the U.S. for many neurologic diseases. Diseases occurring together may
provide important information in the search for their etiology. Such detailed
analysis of mortality data have been done for Huntington's disease, Alzheimer's
disease and related diagnoses, Friedreich's disease, motor neuron disease,
Parkinson's disease, etc., for 1971-78. The overall patterns which have emerged
3 - NEB/DIR
have been useful in evaluating trends over time and in formulating etiologic
hypotheses .
All death certificates for the entire U.S. for the years 1971, and 1973 through
1978 were searched for the diagnosis of Huntington's disease. Age-, race-, and
sex-specific mortality rates for deaths due to and deaths with Huntington's
disease were calculated. Time trends in the age-adjusted mortality rates between
1971 and 1978 were also calculated. To determine which conditions may be
associated with reduced survival in patients with Huntington's disease, all death
certificates in the United States for 1978 on which Huntington's disease was
mentioned have been studied. Each case was compared with two control deaths
which were matched for age, race, sex, county and year of death. In the case-
control study, pneumonia, choking, nutritional deficiencies, and chronic skin
ulcers were increased in cases relative to controls. The overall mortality rate
was 2.27 per million population per year.
All cases of Friedreich's disease (FD) diagnosed between 1945 through 1984 among
residents of a defined area of northwestern Italy were ascertained (N=58). These
patients were followed to death or to December 31, 1984 (whichever came first)
to determine the patterns of survival. The 10-, 20-, and 30-year survival rates
were respectively 96%, 80%, and 61%, suggesting a better prognosis than
previously reported. Survival of FD patients was poorer than expected from the
general population. Survival for males was poorer than females even after
adjustment for expected survival. Age of onset was not a significant prognostic
factor. Survival for patients diagnosed in 1960 or later was better than for
those diagnosed before 1960; however, the difference was not statistically
significant.
A study of reported international average annual age-adjusted mortality rates
for primary tumors of the nervous system was undertaken for the periods 1951-58
and 1967-73. International comparisons of average annual age-adjusted mortality
rates for primary tumors of the nervous system showed marked geographical
variation for both study periods. For the majority of countries, the mortality
rates increased by at least 40% in the intervening 15-year period, while in 20%
of the countries the rates increased by over 100%. The percentage increases
varied from 12.2 to 345.5. The improvement in the diagnosis of these tumors,
particularly among elderly individuals (who have the highest age-specific
incidence rates for these neoplasms), presumably accounts for most of this
change.
Potential risk factors for various types of stroke were studied using a case-
control study design. All the U.S. death certificates in 1978 which had the
registered underlying cause of death as subarachnoid hemorrhage (SAH), cerebral
hemorrhage (CH) , or cerebral infarction (CI) were identified. In a case-control
Btudy for potential risk factors for various types of stroke, the data provided
new information, such as the occurrence of peripheral vascular disease in
association with SAH, the risk of CH in epileptic and cirrhotic patients, and
the association of benign neoplasms of the nervous system, motor neuron disease,
and 'paralysis agitans' with CI.
Prevalence surveys
There is continuing controversy regarding racial differences in the occurrence
of major neurologic diseases. Thus, a study was conducted in Copiah County,
4 - NER/DIR
Mississippi to specifically address this question. A strategy was developed
which eliminated the requirement that persons must have entered the health care
system for detection of disease. The basis of the investigation was a
door-to-door survey which utilized a detailed questionnaire inquiring not only
about diagnoses, but also about signs and symptoms suggestive of neurologic
dysfunction. Over 99% of the households agreed to the interview. Those
household members suspected of having one of the disorders of interest were then
asked to have a neurologic examination conducted by a senior, board-certified
neurologist. This two-stage procedure has proven most effective, and the
strategy for this type of survey is being utilized in other areas of the world.
Prevalence and incidence data from different parts of the world are often not
directly comparable because of the use of different definitions of disease, and
results affected by the availability and accessibility to experts in neurology.
A uniform protocol developed by the Branch in collaboration with the World Health
Organization has helped diminish these differences. The protocol which is being
used to estimate the prevalence of major neurologic disorders in many parts of
the world, will enable international comparisons.
The prevalence of a wide spectrum of neurologic disorders using the World Health
Organization protocol has been reported, based on the Copiah County door-to-door
model. Research protocols for these neurologic studies have been completed in
India and Spain. The prevalence study of the Parsi Community of Bombay, India
provided point prevalence estimates of 328.3/100,000 for Parkinson's disease and
1591.7/100,000 for essential tremor. In the pilot prevalence study in an urban
area of Madrid, Spain, migraine and epilepsy were the most frequent disorders
with point prevalence ratios of 163.3/1000 and 9.3/1000, respectively.
A large prevalence survey for Amyotrophic Lateral Sclerosis - Parkinsonism
Dementia Complex of Guam (ALS-PDC) was undertaken in three rural villages in
southern Guam. The survey, utilizing a door-to-door approach, screened all
individuals 40 years and over for ALS-PDC. Only rare cases of ALS were
encountered, although PDC was found to be still common in the population. Data
editing is in progress and comprehensive analysis and publication of results
will follow completion of the final data file.
Neurogenetics
The Clinical Neurogenetics Unit of the Neuroepidemiology Branch seeks the cause,
cure and prevention of specific neurologenetic diseases. For those diseases of
apparent single gene origin etiologic efforts focus on mapping and then isolating
the mutant gene. For those disorders of more complex origins, a balanced search
is made for both genetic and environmental factors. Specific activities and
responsibilities of the unit include clinical care, clinical and laboratory
research, teaching, and liaison with several patient organizations.
Over 1000 families, some consisting of over 500 individuals, have been studied
in the course of research on single neurogenetic disorders such as the dystonias,
the neurofibromatoses, the hereditary leukodystrophies and the hereditary
ataxias .
More than 200 twin pairs and their families have been evaluated in the course
of study of complex neurological disorders such as Alzheimer's disease,
Parkinson's disease, multiple sclerosis, and post polio syndrome.
5 - NEB/DIR
Clinical care is delivered through the NIH Interinstitutes Genetics Clinic.
Presently, we and a team of 8 specialists participate in the screening,
diagnosis, evaluation and management of persons at high risk for selected
neurologic disorders, particularly neurofibromatosis 1 (NF1) which formerly was
known as von Recklinghausen's disease, and NF2 which was formerly known as
bilateral acoustic neurofibromatosis. Earlier, we pointed out that the
neurofibromatoses together constitute the most common of all neurogenetic
conditions. NF2 has been shown to be due to a mutation of a single gene in the
region of chromosome 22 in one family. Subsequently, the gene for NF1 was
localized to chromosome 17 suggesting there are two distinct forms of NF.
Recently the NF1 gene and its product have been identified.
Since NF2 is often associated with multiple brain and spinal cord tumors in
addition to acoustic neuromas, with over 10 meningiomas, neurofibromas and
astrocytomas in some cases, management of an individual case requires careful
coordination. However, we now consider NF2 a preventable cause of deafness,
facial disfigurement and even death, with optimum management.
An approach has evolved involving two other neurosurgical centers, and of over
50 cases followed during the past three years only one individual has died (a
man in his 30 's whose first tumor was noted at age 18) and only two other have
become completely deaf (both females with multiple brain tumors and deteriorating
neurological status). We are gratified with this record for a condition that
generally has been associated with severe morbidity and premature death.
Clinical research has consisted of: description of the nosology and natural
history and inheritance pattern of a number of neurogenetic diseases based on
family study; genetic epidemiologic studies seeking either factors influencing
the severity of the disorder often using sibs or co-twins as one of the control
groups; formal descriptive neuroepidemiologic studies of selected, defined
populations; and evaluation of treatment programs using our extensive patient
series.
Laboratory studies have been carried out with collaborators in a number of fields
including: brain imaging with CT scan, MRI or PET; cell biology looking for
factors influencing growth of NF2 tumor cells; molecular genetics for gene
mapping or gene isolation and characterization; and neuropathology for detailed
anatomic and neurochemical analysis of brain tissue.
Teaching responsibilities begin with students and fellows in the NIH
Interinstitutes Genetics Program and the clinical staff at NIH and other area
hospitals, and extends to professional and lay groups interested in the combined
genetic-epidemiologic approach we employ to understand specific neurologic
disease.
An especially active and successful area of responsibility for the Neurogenetics
Unit involves liaison with a number of voluntary lay organizations. Over the
years we have assisted in the formation of eight local or national foundations,
including the National Neurofibromatosis Foundation and the Washington, D.C.
chapter; the Dystonia Medical Research Foundation and the Washington, D.C.
chapter; the National Jewish Genetic Disease Foundation, the Washington, D.C.
chapter of the National Ataxia Foundation, Neurofibromatosis, Inc. and its local
chapter; and most recently iNFormer 2 which is a network for NF2 families. We
6 - NEB/DIR
have tried to help these organizations with advice. They in turn have enabled
us to perform three recent clinical studies by enlisting the cooperation of their
membership.
Sinole gene disorders; Recent work of the Neurogenetics Unit include the
following:
NF2 - The natural history of NF2 appears different in males with the gene
than it does in females. Affected men tend to have fewer tumors, require less
surgical intervention and, when they pass on the gene, have it expressed later
in their children than their affected sisters do. A pathognomonic sign has been
described by our team. It is the presence at a young age of one or more discreet
opacities in the posterior portion of the lens of the eye which can appear before
any other signs or symptoms. With hand-held ophthalmoscope it is seen as a black
fleck and is known locally as the 'Kaiser-Kupfer Cinder'. A review has been
prepared on the practical management of NF2, which makes the following points:
delineates 5 groups at high risk for NF2; emphasizes that no one should be
operated on for a presumed solitary, sporadic acoustic neuroma unless this
syndrome with bilateral acoustic neuromas is first ruled out; and that watchful
waiting rather than aggressive surgical intervention is the proper course in the
majority of cases. A consensus conference dealing with these issues is planned
for June of 1991.
NF1 - A study has been completed of an appraisal of attitudes of over
300 affected individuals and their relatives toward NF1 itself and toward
predictive testing for NF1.
Hereditary Leukodystrophy - We have described an adult form which has been
often misdiagnosed as chronic progressive multiple sclerosis. Four families in
the U.S. have now been ascertained. A high concentration of a previously
undescribed familial leukodystrophy of early onset has been identified by
colleagues in Saudi Arabia.
Complex, or multifactorial, neurologic disorders
Alzheimer's disease - demonstration in the first and only nationwide twin
study that most monozygotic twins are discordant and remain so for over 12 years,
indicating that environmental factors are important in many cases.
Multiple sclerosis - Noted the similarity between MS and paralytic polio
concordance rates in monozygotic and dizygotic twins, indicating that
environmental and genetic factors both must play a role in each condition.
Parkinson's disease - Demonstrated in the first nationwide twin study in
the U.S. low concordance rates, a finding since confirmed in English and Finnish
studies. In the U.S. study there were far more full sibs and parents affected
than co-twins, suggesting a familial tendency for Parkinson's and a possible
'protective effect' on the co-twin. A characteristic life-long personality
difference suggested an early environmental event 'set' a twin's risk. These
observations plus mouse data indicating genetic influence on dopamine neuron
number led us to offer the 'Initial Neuron Number', or INN, theory as one of the
processes contributing to Parkinson's disease. If true it would mean that there
is a humoral factor capable of reducing one's risk or of treating one's symptoms.
7 - NEB/DIR
Pediatric neuroepidemioloqy
In the area of pediatric neuroepidemiology, the efficacy and side effects of
phenobarbital in young children with febrile seizures, randomized to
phenobarbital or placebo is being studied. A number of endpoints have been or
will be assessed: IQ, seizure recurrence, sleep pattern, temperament scale, and
a number of behavioral and cognitive measures. The design of this study permits
comparison of measures of tested intelligence and of behavior in children with
febrile seizures who were treated with phenobarbital, in those randomized to
placebo, and in a group of seizure-free control children. It was presumed that
the group randomized to placebo would experience more recurrences of seizures,
while the group assigned to phenobarbital might be subject to unfavorable side
effects of medication. This comparison allows assessment of benefit and risk
of treatment in a common childhood neurologic problem. Children in this study
were also tested six months after completion of receipt of phenobarbital, so that
the reversibility of any observed effects could be judged. All subjects have
been recruited, follow-up testing has been completed. The initial manuscript
describing the primary results has been published and two others submitted. Work
has begun on other manuscripts from this data.
Studies of dental and dermatoglyphic markers of maldevelopment have been
initiated in cooperation with the National Institute of Dental Research and the
Institute of Aging. These studies of enamel defects and characteristics of
dermatoglyphics and palmar creases in children with cerebral palsy or mental
retardation will attempt to recognize the occurrence and timing of events adverse
to neurologic development.
NEB staff were involved in the analysis of a randomized controlled clinical trial
undertaken in the NICHD to evaluate the safety and efficacy of phototherapy in
the prevention/reduction of hyperbilirubinemia in the neonate. An earlier study
indicated the efficacy of the treatment in preventing hyperbilirubinemia. This
phase investigated the frequency of adverse outcomes, most of them neurological,
in this sample at follow-up examination at six years of age. Infants randomized
to treatment with phototherapy experienced lower ranges of bilirubin as neonates
but did not experience increased frequency of adverse neurologic outcomes. One
manuscript has been published, one is in press, and another is in preparation.
NEB staff in collaboration with the California Birth Defects Monitoring Program,
and the Health Officers Association of California, is establishing a
population-based registry of children with cerebral palsy (CP) in five counties
of the San Francisco Bay Area born between 1983-1985. Children are ascertained
through client records at California Children Services and the Department of
Development Services and their Regional Centers for the Developmentally Disabled.
CP cases meeting the registry criteria will receive a clinical examination to
establish a research diagnosis, and data will be obtained on associated features.
Obstetric and pediatric records will be reviewed by the staff. Information will
be used to establish trends in incidence, to examine for time-space clustering,
and to formulate hypotheses for testing suspected etiologic factors, medical and
environmental, in case- control studies. Ecological studies will also be
conducted comparing rates of CP among births in Santa Clara County census tracts
which contain EPA superfund waste disposal sites, to rates in the county as a
whole and to rates in the remainder of the Bay area; the purpose of this aspect
of the project is to examine the possibility that toxic exposures may contribute
8 - NEB/DIR
to some forms of neurologic morbidity as well as to other birth defects.
Examinations for case classification have been completed and birth record
abstraction is underway.
Rare disorders of the nervous system
Emphasis is placed on research on the major diseases of the nervous system, and
the Branch also investigates other diseases which may be less frequent or less
debilitating, but important in terms of pathogenesis or clues to disease
etiology.
In collaboration with other governmental agencies (Centers for Disease Control),
other governments (Colombia, Republic of Seychelles) , international organizations
(World Health Organization), and universities (Mayo Medical School), methods have
been developed to investigate lees common neurologic disorders. Several such
studies have been completed as: a prevalence and case-control study of
progressive supranuclear palsy (PSP); and a door-to-door survey of 21 provinces
of the People's Republic of China to determine the prevalence of migraine.
The prevalence and natural history survey of PSP in two counties in New Jersey
provided a prevalence ratio of 1.39/100,000. The median intervals to onset of
requiring assistance was 3.1 years; of visual symptoms, 3.9 years; dysarthria,
3.4 years; dysphasia, 4.4 years; requiring wheel chair, 8.2 years; and death
9.7 years. In the case-control study of PSP with two matched controls, as
adults, PSP cases lived in areas with low population significantly more
frequently than controls. The study identified no other factors associated with
PSP including a history of stroke, hypertension, or smoking. The prevalence
survey of migraine in the People's Republic of China yielded a point prevalence
ratio of 690/100,000. The prevalence ratio of migraine for females was higher
than that for males.
HTLV-I Infections of the Nervous System
The first human retrovirus, HTLV-I, has been confirmed as the causal agent of
tropical spastic paraparesis (TSP) and HTLV-I-associated myelopathy (HAM), as
well as some forms of polymyositis. Neuroepidemiologic studies have been carried
out in Tumaco( Colombia) and the Seychelles Islands.
The NEB will begin, in collaboration with the Pan American Health Organization,
a Registry of HTLV-I Infections of the Nervous System, in order to obtain data
on the magnitude of this problem in the Americas.
9 - NEB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 01924-20 NEB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must fit on one line between the borders.)
Clinical, Genetic, Pathophysiologic Study of Hereditary Movement Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: Roswell Eldridge, M.D. Medical Geneticist NEB, DIR, NINDS
Others: Robert Cohen, M.D.
Elizabeth Mathew, M.D.
Chief
Neurologist
LBI, IRP, NIMH
LBI, IRP, NIMH
COOPERATING UNITS (il any)
CNB, DIR, NINDS; LCS, DCBR, NIMH; Dept. of Neurology, University of Mississippi
School of Medicine
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
0.35
PROFESSIONAL:
0.3
OTHER
0.05
CHECK APPROPRIATE BOX(ES)
[X (a) Human subjects
. (al) Minors
QLj (a2) Interviews
LS (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided )
In this project, we seek to 1) clarify and expand the nosology of the
hereditary movement disorders; 2) contribute to the understanding of the
underlying biochemical basis; 3) determine the most effective treatment and
predictive testing for each disorder; and 4) suggest guidelines for counseling
individuals at risk. General syndromes under study include the dystonias, tic
disorders, the ataxias and myoclonus. Approaches include standard epidemiologic
and clinical genetic studies, and collaborative efforts in evaluating the role
of neurotransmitters such as dopamine, and PET Studies.
We are performing PET studies of at risk members of two large kindreds with
hereditary ataxia. Preliminary results suggest: a more generalized involvement
than previously recognized; presymptomatic changes by PET; and evidence for
genetic heterogeneity.
10 - NEB/DIR
PHS 6040 (Rev 1/84)
SPO S14*91«
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 01927-20 NEB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must tit on one line between the borders j
Clinical, Genetic, Pathophysiologic Study of Hereditary Nervous System Tumors
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, ana institute affiliation i
P.I.t Roswell Eldridge, M.D Medical Geneticist NEB, DIR, NINDS
Others:
Bracie Watson, M.S.
Murial Kaiser-Kupfer,
Anita Pikus
Wesley McBride, M.D.
Dilys Parry, Ph.D.
Geneticist
M.D. Chief
Audiologist
Molecular Geneticist
Acting Chief
Judith Schaeffer, Ph.D.JD Vestibulographer
NEB, DIR, NINDS
OGB, IP, NEI
CA, IP, NIDCD
LBC, IP, NCI
CEB, IP, NCI
CA, IP, NIDCD
cooperating units (if any) OP, CC : SN , DIR, NINDS; Division of Medical Genetics, Dept. of
Pediatrics, Children's Hospital National Medical Center; Dept. of Neurosurgery,
Massachusetts General Hospital, Boston, MA; Uniformed Services University of
Health Sciences
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
3.5
PROFESSIONAL
3.0
OTHER
0.5
CHECK APPROPRIATE BOX(ES)
E (a) Human subjects
Lx (a1) Minors
LE (a2) Interviews
B (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
In this project we seek to define and classify hereditary tumors of the nervous
system such as occur in the neurofibromatoses ; to add to the clinical
description and natural history of these diseases; to suggest methods for early
diagnosis; to evaluate present modes of treatment: and to develop methods for
preclinical detection and screening.
Our studies have led to the recognition of a preventable cause of deafness,
visual loss or even death: neurofibromatosis 2 or bilateral acoustic
neurofibromatosis . The genes for two distinct forms of neurofibromatosis have
now been mapped to specific chromosomes: 1) the classical form as described by
von Recklinghausen (neurofibromatosis 1) recently isolated on #17, and 2) a form
in which bilateral acoustic neuromas are the hallmark (neurofibromatosis 2) may
be on #22. Efforts in the latter have been directed at improving and
simplifying screening of high-risk individuals, confirming diagnosis,
establishing criteria for intervention, and isolating and characterizing the
gene. A consensus conference dealing with these issues is planned for June,
1991.
Our first major study involving neurofibromatosis 1, a multi-disciplinary
project, demonstrated mild but consistent impairment of neurologic and cognitive
status in these patients compared to their unaffected 6ibs. A second study
assessing the burden of NFI and attitudes towards predictive testing has been
completed. There is great interest in such testing but most would not terminate
a positive pregnancy.
11 - NEB/DIR
PHS 6040 (Rev 1/84)
spo i i«-» ■■
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02167-16 NEB
P6c€olDerEF¥,D 1989 through September 30, 1990
TITLE OF PROJECT, (80 characters or tess_ Title, must fit on age line between Jhe bordersj. . , , . , ., , . _ . ,
Genetic Epidemiology Studies in MS ana other Multifactorial Neurologic Disorders
>£INCIPAL INVF^TIGAT^^
ridge
Others: Henry F. McFarland, M.D.
Halter A. Rocca, M.D.
Gustavo Roman, M.D.
Marinos Dalakas, M.D.
Linda Nee, MSW
title, laboratoi
Assistant Chief
Epidemiologist
Chief
Neurologist
Research Associate
NI, DIR, NINDS
NEB, DIR, NINDS
NEB, DIR, NINDS
N, DIR, NINDS
CNB, DIR, NINDS
cWPllR,AT!filiBJ,NI'Ii§f,antiNB, NINDS; M; National Rehabilitation Hospital, D.C. Italian
Multicenter Study on Dementia, S.M.I.D. Centers, Florence, Italy; Departments
Pediatrics and Neurology, King Faisal Specialty Hospital, Riyadh, Saudi Arabia
l^euroepidemiology Branch
SECTION
l'MsE,AW,CABmhe6da, Maryland 20892
TOTAL MAN-YEARS-
PROFESSIONAL
0.3
OTHER
CHECK APPROPRIATE BOX(ES)
LXJ (a) Human subjects
E (a1) Minors
E (a2) Interviews
DO (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided I
In this project we are coupling genetic and environmental studies in selected
families, twin pairs £ populations with disorders such as multiple sclerosis,
Parkinson's disease, and Alzheimer's disease, in an effort to distinguish
specific contributing factors.
Multi-disciplinary twin studies of Parkinson's disease, multiple sclerosis and
Alzheimer's disease indicate each is complex and involves an interaction of
I environmental and genetic factors.
1 A study similar in design involving twins with documented polio over 30 years
I ago is designed to look at factors contributing to 'post polio' syndrome.
: An autosomal dominant, hereditary leukoencephalopathy simulating MS with onset
! at about age 35 is under study in a kindred with over 20 affected. Derangement
of the autonomic nervous system is often seen early in the course and when
recognized clinically, serves to distinguish this single gene disorder from
I multiple sclerosis.
i
i
In Saudi Arabia a multidisciplinary study of an extended kindred with a novel
form of leukodystrophy is planned. Epidemiologists and geneticists working
together will assess the relative burden of environmental and genetic factors in
thiB inbred population.
12 - NEB/DIR
PHS 6040 (Rev 1/84)
GPO fit 4-G1 e
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02240-14 NEB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must tit on one line between the borders )
Epidemiology of Dementia
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
Acting P.I.J Jonas H. Ellenberg, Ph.D. Acting Chief NEB, DIR, NINDS
Gustavo C. Roman, M.D. Chief Designate NEB, DIR, NINDS
(1/1/90-9/30/90)
COOPERATING UNITS (it any)
Vijay Chandra, M.D., Ph.D. New Dehli, India; Francis M. Baker, M.D., University
of Texas Health Science Center
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
0.05
PROFESSIONAL
0.05
OTHER
0.0
CHECK APPROPRIATE BOX(ES)
B (a) Human subjects
C (a1) Minors
SJ (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
Analytic studies to determine risk factors for Alzheimer's disease (AD) are
planned or being conducted. A large case-control study of head trauma with loss
of consciousness as a risk factor for AD has been completed in Rochester, MN.
There was no detected increase in risk of AD following head trauma. A
case-control study of Alzheimer's disease is in progress in White Plains, New
York. An attempt has been made to evaluate and improve instruments used in
case-control studies of dementia.
13 - NEB/DIR
PHS 6040 (Rev 1/84)
SPO 81 4-*l»
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02243-14 NEB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must fit on one line between the borders.)
Pediatric Neuroepidemiology
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
P.I.: Karin B. Nelson, M.D. Medical Officer NUB, DIR, NINDS
Others: Jonas H. Ellenberg, Ph.D.
Sherrie Emoto, Ph.D.
Acting Chief
Staff Fellow
NEB, DIR, NINDS
BFSB, DIR, NINDS
COOPERATING UNITS (if any)
Peter Scheidt, M.D. Medical Officer, NICHD
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland
20892
TOTAL MAN-YEARS
0.4
PROFESSIONAL.
0.3
OTHER
0.1
CHECK APPROPRIATE BOX(ES)
Lx (a) Human subjects
LX (a1) Minors
Lj (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
Several studies in pediatric neuroepidemiology are ongoing: 1) analysis of
case-control data for putative predictors of febrile seizures, based on a data
set from six cities from the People's Republic of China has been completed; 2)
The prenatal and perinatal antecedents of febrile seizures were examined in
NINDS data; 3) With Yugoslav colleagues, we are examining the utility of the
electroencephalogram as a predictor of recurrence of febrile seizures in a
defined population in Yugoslavia; 4) NINDS participants were involved in the
analysis of a randomized controlled clinical trial undertaken in the NICHD to
evaluate the safety and efficacy of phototherapy in the prevention/reduction of
hyperbilirubinemia in the neonate. We investigated the frequency of adverse
neurologic outcomes at six years of age.
14 - NEB/DIR
PHS 6040 (Rev 1/84)
cpo si4-«ia
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02297-14 NEB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must tit on one line between the borders )
Mortality from Neurologic Disorders: National and International Comparisons
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
Acting P.I.: Jonas H. Ellenberg, Ph.D. Acting Chief NEB, DIR, NINDS
Gustavo C. Roman, M.D. Chief Designate NEB, DIR, NINDS
(1/1/90-9/30/90)
COOPERATING UNITS (it any)
VJ Chandra M.D., India; W.A. Rocca M.D.
Firenze, Italy
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland
20892
TOTAL MAN-YEARS
0.05
PROFESSIONAL
0.05
OTHER
0.0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects
D (a1) Minors
□ (a2) Interviews
□ (b) Human tissues L5 (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
Although death certificate data are limited by possible misdiagnosis, incomplete
case ascertainment, errors in coding, etc., detailed mortality information on
some neurologic diseases for the entire U.S. is not available. Analysis of
mortality data can be particularly useful for some neurologic diseases because
these may contribute to death indirectly. Since there are no uniform criteria
for what constitutes the underlying cause of death in patients, it is important
to examine all deaths in which a disease is listed as an underlying, immediate,
associated, or contributory cause of death to get more complete information
about the relationship between the disease and death. Association of diseases
occurring at the time of death are also being studied for all deaths occurring
in the U.S. for many neurologic diseases. Diseases occurring together may
provide important information in the search for etiology of diseases.
Detailed analyses of mortality data have been accomplished for Huntington ' s
disease (comorbidity at time of death), Friedreich's disease (survival
I analysis), international comparisons of average annual age-adjusted mortality
rates for primary tumors of the nervous system, and stroke (case-control study
j of risk factors).
15 - NER/DIR
PHS 6040 (Rev 1/84)
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02299-14 NEB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must tit on one line between the borders.)
Reviews of Epidemiologic Aspects of Neurologic Disease
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Acting P.I.: Jonas H. Ellenberg, Ph.D. Acting Chief NEB, DIR, NINDS
Gustavo C. Roman, M.D. Chief Designate NEB, DIR, NINDS
(1/1/90-9/30/90)
COOPERATING UNITS (if any)
N. Bharucha, M.D., Bombay, India; William Koller, M.D., Ph.D., University of
Kansas; Ian Irwin, Ph.D., Institute for Medical Research, San Jose
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
0.05
PROFESSIONAL:
0.05
I OTHER
0.0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues GO (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
Development of new neurologic studies requires thorough historic and
methodologic reviews of prior investigations. These yield important unexplored
etiologic clues that may be investigated using current technology. Major
emphasis has been given to: cerebrovascular disease; otitis media; inherited
ataxias; Huntington's disease; febrile seizures; Tourette's syndrome; peripheral
neuropathy; neurologic disease in the elderly; controlled therapeutic trials of
motor neuron disease; epilepsy; descriptive, analytic, and experimental methods
in neuroepidemiology; procedures for neuroepidemiologic investigations in
developing countries; and epidemiologic studies of Alzheimer's disease;
myasthenia gravis; cerebral malaria; and autism.
16 - NEB/DIR
PHS 6040 (Rev 1/84)
6PO 914416
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02300-14 NEB
PERIOD COVERED
October 1, 1989 through September 30,
1990
TITLE OF PROJECT (80 characters or less Title must lit on one line between the boroers )
Clinical Course and Medical Care for Neurologic Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory ana institute affiliation)
Acting P.I.: Jonas H. Ellenberg, Ph.D. Acting Chief NEB, DIR, NINDS
Gustavo C. Roman, M.D. Chief Designate NEB, DIR, NINDS
(1/1/90-9/30/90)
COOPERATING UNITS (it any)
Shi-Chuo Li, M.D.
Beijing Neurosurgical Institute, PRC
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
0.0
PROFESSIONAL
0.0
OTHER
0.0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues LS (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
The study uses a review and abstraction of data from records for a selected
group of neurological disorders. It obtains the items of data necessary to
determine onset of the disorder, comorbid conditions, duration, date and cause
of death, or current status. These data will be used to construct modified life
tables to estimate the expectation of life after diagnosis, the survival curve,
and morbidity and severity estimates. It will also include analysis of type and
duration of medical care received by patients with neurologic disorders derived
from a well-defined population.
17 - NEB/DIR
PHS 6040 (Rev 1/84)
SPO 8 1 4-9 1 »
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02301-14 NEB
PERIOD COVERED
October 1, 1989 through September 30,
1990
TITLE OF PROJECT (80 characters or less Title must lit on one line between the borders.)
Collaborative Studies of Less Common or Less Debilitating Neurologic Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute atliliation)
Acting P.I.: Jonas H. Ellenberg, Ph.D. Acting Chief NEB, DIR, NINDS
Gustavo C. Roman, M.D. Chief Designate NEB, DIR, NINDS
(1/1/90-9/30/90)
COOPERATING UNITS (it any)
B. Chen, M.D., Beijing, China; G. Roman, M.D., Lubbock, Texas; L. Golbe, M.D.,
New Brunswick, New Jersey; C. Tanner, M.D., Chicago, 111
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
0.05
PROFESSIONAL:
0.05
OTHER
0.0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues ffl (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided)
A number of collaborative efforts involve the investigation of the
characteristics of unusual or less debilitating (e.g., headache) neurologic
disease phenomena. Unusual associations or space/time clusters of neurologic
disorders may provide leads to etiology or therapy. These may be tested through
more formal approaches.
Surveys of prevalence and natural history and a case control study of
progressive supranuclear palsy (PSP) have been completed in two counties in New
Jersey. The point prevalence was 1.39/100,000. The median intervals to onset
of requiring assistance was 3.1 years and to death was 9.7 years. The case
control study did not implicate a history of stroke, hypertension or smoking as
risk factors for PSP. A prevalence survey of migraine in the People's Republic
of China yielded a point prevalence ratio of 690/100,000, and indicated that
the prevalence ratio for females was higher than that for males.
18 - NEB/DIR
PHS 6040 (Rev 1/84)
GPO 9\4-»IB
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02307-14 NEB
PERIOD COVERED
October 1, 1989 through September 30,
1990
TITLE OF PROJECT (80 characters or less Title must tit on one line between the borders. )
Educational Resources in Neurological Epidemiology
PRINCIPAL INVESTIGATOR (List other protessional personnel below the Principal Investigator I (Name, title, laboratory, and institute artiHation)
Acting P.I.: Jonas H. Ellenberg, Ph.D. Acting Chief NEB, DIR, NINDS
Gustavo C. Roman, M.D. Chief Designate NEB, DIR, NINDS
(1/1/90-9/30/90)
Other:
Dallas W.Anderson, Ph.D. Mathematical Statistician BFSB, DIR, NINDS
COOPERATING UNITS (it any)
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
0.05
PROFESSIONAL
0.05
OTHER
0.0
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues LH (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
Because there is severe shortage of available manpower in neuroepidemiology, the
Branch has developed an active teaching program for current and future
collaborative investigators. A series of six video tapes produced by the Branch
are distributed on a loan basis without charge. A monograph, entitled
NEUROLOGICAL EPIDEMIOLOGY: PRINCIPLES AND CLINICAL APPLICATIONS was published
in 1978. A new monograph which will cover current methodology and disease
specific overviews of etiology, incidence, prevalence and intervention studies
has been completed.
A set of video tapes have been produced for training interviewers in the
methodology of interviewing for case-control studies. This has been done in
both Italian and English.
19 - NEB/DIR
PHS 6040 (Rev 1/84)
spo 814-aia
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02370-12 NEB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Racial and Geographic Differences in Occurrence of Neurologic Disease
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute attiliation)
Acting P.I.: Jonas H. Ellenberg, Ph.D. Acting Chief NEB, DIR, NINDS
Gustavo C. Roman, M.D. Chief Designate NEB, DIR, NINDS
(1/1/90-9/30/90)
Other:
Dallas Anderson, Ph.D. Mathematical Statistician BFSB,DIR, NINDS
COOPERATING UNITS (if any)
OBFS, OD, NINDS: A. Haerer, M.D., Univ. of Mississippi; U.S. Bureau of the
Census; N. Bharucha, M.D. (India)
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
0.05
PROFESSIONAL
0.05
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
E (a) Human subjects
13 (al) Minors
SI (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided.)
The purpose of these studies is to accurately document possible racial,
environmental and geographic differentials in the prevalence of major neurologic
disorders by surveying an entire geographically defined population. The
disorders investigated include cerebral palsy, dementia, psychomotor delay,
epilepsy, Parkinson's disease, essential tremor, multiple sclerosis and
cerebrovascular disease.
Research protocols for these neurologic studies have been completed in the
United States (Copiah County), India and Spain. The prevalence study of the
Parsi Community of Bombay, India provided point prevalence estimates of
328.3/100,000 for Parkinson's disease and 1591.7/100,000 for essential tremor.
In the pilot prevalence study in an urban area of Madrid, Spain, migraine and
epilepsy were the most frequent disorders with point prevalence ratios of
163.3/1000 and 9.3/1000, respectively.
20 - NEB/DIR
PHS 6040 (Rev 1/84)
SPO 914-916
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02423-11 NEB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must tit on one tine between the borders )
Development of Data Resources for Neuroepidemiology
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) {Name, title, laboratory, and institute atliliation)
Acting 9.1. i Lawrence Lavine, D.O., M.P.H. Medical Officer NEB, DIR, NINDS
COOPERATING UNITS lit anyl
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
0.00
i PROFESSIONAL
0.00
OTHER
0.0
CHECK APPROPRIATE BOX(ES)
E (a) Human subjects
□ (a1) Minors
G (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
To develop 1) a registry of hospitalized patients with neurologic disease in a
geographically well-defined population; 2) resources for case-control studies of
neurologic diseases using uniform methods of data collection; and 3) a registry
of neurologic disease in the well-defined population of the United States
military.
This project is completed,
other project areas.
Future projects in this area will be subsumed under
21 - NEB/DIR
PHS 6040 (Rev 1/84)
SPO II 44 II
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02570-08 NEB
PERIOD COVERED
October 1, 1989 through September 30,
1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Natural History of ALS-PD in Guam
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: Lawrence Lavine, D.O., M.P.H. Medical Officer NEB, DIR, NINDS
Other: Zhen-xin Zhang, M.D. Guest Researcher NEB, DIR, NINDS
Dalas W. Anderson, Ph.D. Mathematical Statistician BFSB,DIR, NINDS
Karin B. Nelson, M.D. Pediatric Neurologist NEB, DIR, NINDS
cooperating units (it any) Leonard Kurland, M.D. , Mayo Clinic, Rochester, MN; John
Steele, M.D., Veterans Administration, Guam; Xhen-xin Zhang, M.D., People's
Republic of China; Donald Calne, M.D., Univ. of British Columbia, Vancouver, BC;
Peter Spencer, Ph.D., Albert Einstein College of Medicine, NY.
uvb/branch
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
1.0
PROFESSIONAL:
0.9
OTHER:
0.1
CHECK APPROPRIATE BOX(ES)
IE (a) Human subjects
□ (a1) Minors
LH (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided.)
As a continuation of previous projects on clinical, pathological, and
epidemiologic surveillance of Guamanian amyotrophic lateral sclerosis (ALS) and
Parkinsonism-dementia (PD) in the Marianas Islands, identified survivors,
including suspects, in the NINDS Registry as of January 1, 1983, were followed
at intervals of six months for detailed clinical descriptions of patterns of
progression, by a qualified neurologist. The patients still alive have entered
a chronic state and final exams were completed in the fall of 1988. As these
patients expire, attempts will be made to obtain the appropriate pathologic
material for examination by various neuropathologists around the world. All
attempts to examine this material should be made because of the uniqueness of
these patients.
The prevalence survey of three southern Guamanian villages to determine the
current prevalence of ALS/PDC was completed in 1988. As this is a transitional
society, dietarily and culturally since WW-II, a simultaneous survey for other
major neurologic diseases and major diseases of aging populations was also
performed. A chart review study using the NINDS Guam registry for ALS/PDC for
the period 1944-85 was performed. Downward trends in age-adjusted incidence
rates and upward trends in age of onset were seen for both sexes. Since 1980,
new cases occurred only among persons over 50 years of age, whereas younger age
at onset had been noted in the past.
The NINDS no longer is supporting this program. It is now being operated under
a grant from NIA, with Dr. Leonard T. Kurland as principal investigator. This
project is completed.
22 - NEB/DIR
PHS 6040 (Rev 1/84)
GPOII 4-0 is
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02715-05 NEB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must lit on one line between the borders .)
Epilepsy Neuroepidemiology
PRINCIPAL INVESTIGATOR (List other protessional personnel below the Principal Investigator ) (Name, title, laboratory ana institute attiiiation/
P.I.: Karin B. Nelson, M.D. Medical Officer NEB, DIR, NINDS
Others: Jonas H. Ellenberg, Ph.D.
William Theodore, M.D.
Chief
Medical Officer
BFSB, DIR, NINDS
MNB, DIR, NINDS
COOPERATING UNITS (it any)
Shi-chuo Li, M.D., Beijing Neurosurgical Institute, PRC; Judith Manelis, M.D.,
Western Galilee Regional Hospital, Israel
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland
20892
TOTAL MAN-YEARS
.35
PROFESSIONAL
0.3
OTHER
0.5
CHECK APPROPRIATE BOX(ES)
Lx! (a) Human subjects
H (a1) Minors
S (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
Several studies on convulsive disorders are being planned and tested for
feasibility, or in progress. A protocol is in effect for a clinical study of
the Lennox-Gastaut syndrome (LGS), a severe childhood epileptic encephalopathy
with significant morbidity, characterized by uncontrolled seizures, mental
retardation, and possible mental deterioration, to define the pathophysiology
and anatomic locus of disturbance in LGS. We are evaluating the feasibility of
performing randomized and placebo controlled clinical trials of treatment after
an initial convulsion in subjects presenting for care to the Beijing Tiantan
Hospital and to a consortium of hospitals in Jerusalem. With Yugoslav
colleagues, we are examining the utility of the electroencephalogram as a
predictor of recurrence of febrile seizures in a defined population in
Yugoslavia.
23 - NEB/DIR
PHS 6040 (Rev 1/84)
SPO SI 4411
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02746-04 NEB
PERIOD COVERED
October 1, 1989 through September 30,
1990
TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.)
Phenobarbital Clinical Trial in Children with Febrile Seizures*
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
P.I.: Karin B. Nelson, M.D. Medical Officer NEB, DIR, NINDS
Others: Deborah Hirtz, M.D.
Young Jack Lee, Ph.D.
Jonas H. Ellenberg, Ph.D.
Pediatric Neurologist DNB, DCDND, NINDS
Mathematical Statistician NEB, DIR, NINDS
Chief BFSB, DIR, NINDS
COOPERATING UNITS (it any)
Jacqueline Farewell,
M.D., Dept. of Neurosurgery, Univ. of Washington, Seattle
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
0.1
PROFESSIONAL
0.1
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
[X (a) Human subjects
S (a1) Minors
□ (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
The objectives of the study are: To assess the effects on tests of intelligence
and behavior of phenobarbital , a commonly prescribed anticonvulsant in children.
The design of this study permited comparison of measures of tested intelligence
and of behavior in children with febrile seizures who had been treated with
phenobarbital, and in a group of seizure-free control children. A comparison of
the groups allowed assessment of benefit and risk of treatment for a common
childhood neurologic problem.
*[This study supports the DNB/DCDND/NINDS contract study entitled: "Behavioral
and cognitive side effects of phenobarbital used for prevention of febrile
seizure recurrence." The project officer is Dr. Deborah G. Hirtz, DNB, DCDND,
NINDS, and the contractor of the study is the University of Washington. ]
24 - NEB/DIR
PHS 6040 (Rev 1/84)
GPO 914-918
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02747-04 NEB
PERIOD COVERED
October 1, 1989 through September 30,
1990
TITLE OF PROJECT (BO characters or less Title must lit on one line between the borders )
Dental Markers of Maldevelopment
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
P.I.: Karin B. Nelson, M.D. Medical Officer NEB, DIR, NINDS
COOPERATING UNITS (if any)
Mohandas Bhat, D.D.S.
D.P.H., EODPP, NIDR
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland
20892
TOTAL MAN-YEARS
0.1
PROFESSIONAL
0.1
OTHER
0.0
CHECK APPROPRIATE BOX(ES)
LH (a) Human subjects
13 (a1) Minors
□ (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
This is an exploratory effort to examine, in a group of children with chronic
motor disability of early onset and nonprogressive course (cerebral palsy, CP),
potential markers of maldevelopment. It will focus on the frequency and nature
of dental abnormalities in affected children, in comparison with the frequency
of similar characteristics among healthy children.
The objectives are to examine whether dental abnormalities, especially enamel
defects, can serve as markers of maldevelopment, and whether such findings can
provide information concerning timing of adverse events or exposures.
The significance of the research is that enamel hypoplasias and other dental
anomalies can offer clues as to the timing of insults or exposures that occur
from the fourth month of gestation to the age of about 12 months of extrauterine
life. Correlation of dental with clinical data may offer a means to explore the
timing of departure from the normal course of development in a group of children
with chronic motor disability of early onset and nonprogressive course (cerebral
palsy, or mental retardation) .
This study of local populations is in abeyance while investigators concentrate
on the dental findings in the California CP registry study (q.v.)
25 - NEB/DIR
PHS 6040 (Rev 1/84)
CPO IU4II
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTtCE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02748-04 NEB
PERIOD COVERED
October 1, 1989 through September 30,
1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders )
Dermatoglyphic Markers of Maldevelopment
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
P.I.: Karin B. Nelson, M.D. Medical Officer NEB, DIR, NINDS
COOPERATING UNITS (if any)
Chris Plato, Ph.D., GRC, NIA; Mrs. Cathy Fox, University of Maryland
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland
20892
TOTAL MAN-YEARS
0.1
PROFESSIONAL
0.1
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
S (a) Human subjects
Lxl (a1) Minors
n (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY CF WORK (Use standard unreduced type Do no! exceed the space provided.)
Fingerprints are in place by nine to 1€ weeks of intrauterine life, dermal
creases by 19 weeks. The objective of this pilot study is to examine whether
dermatoglyphic abnormalities and dermal creases can serve as markers of
maldevelopment, and whether such findings can provide information concerning
timing of adverse events or exposures in children with abnormal neurologic
development. This is an exploratory effort to examine, in a group of persons
with chronic motor disability of early onset and nonprogressive course (cerebral
palsy, CP),or with mental retardation, potential markers of maldevelopment. It
will focus on the frequency and nature of dermatoglyphic abnormalities in
affected individuals, in comparison with the frequency of similar
characteristics among healthy persons.
The significance of the research is that a time course of establishment of
dermatoglyphics and palmar creases has been developed. The hypothesis has been
offered, and some data accrued to indicate, that dermal findings may offer clues
as to the timing of insults or exposures during early prenatal life, providing a
means to explore the timing of departure from the normal course of development
in a group of neurologically handicapped children.
This project is completed. Future projects in this area will be subsumed under
other project areas.
26 - NEB/ DIP.
PHS 6040 (Rev. 1/84)
opo ei 4-bib
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02819-01 NEB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must tit on one line between the borders )
California Cerebral Palsy Registry
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory and institute affiliation)
P.I.J Karin B. Nelson, M.D. Medical Officer NEB, IRP,NINCDS
COOPERATING UNITS (H any)
Judith Grether, Ph.D., Susan Cummins, M.D., Birth Defects Monitoring Group,
Environmental Epidemiology and Toxicology Branch, Department of Health Services,
California; Health Officers Association of California.
LAB/BRANCH
Neuroepidemiology Branch
SECTION
INSTITUTE AND LOCATION
NINCDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
0.
PROFESSIONAL
0.1
OTHER
0.0
CHECK APPROPRIATE BOX(ES)
B (a) Human subjects
Lxi (a1) Minors
S (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided )
In cooperation with the California Birth Defects Monitoring Program of the
California Department of Health and the March of Dimes, NEB is participating in
the establishment and utilization of a population-based registry of children
with cerebral palsy in four counties of the San Francisco Bay Area for births in
1983-1985. Cases were ascertained through client records of the California
Childrens' Services and the Regional Centers for the Developmentally Disabled.
Cases meeting CP registry criteria have received a standardized examination by a
single study pediatrician, including videotaping of all children, photographs of
teeth and hands and systematic scrutiny and measurement for dysmorphologic
features.
All children meeting registry criteria have been examined. In this birth cohort
of approximately 165,000 children, 230-290 cases have been identified, excluding
those with abnormalities apparently related to events after the first month of
life. Analyses using the examinations, birth certificates, initial interview,
and linkage with birth defects registry information are imminent.
The information will permit descriptive studies of prevalence and distribution
of cerebral palsy, evaluation of markers of maldevelopment , and exploration of
risk factors, medical and environmental. Ecologic studies will compare rates of
CP among births in Santa Clara County census tracts containing EPA superfund
waste disposal sites, to rates in the county as a whole and to rates in the
remainder of the Bay area. Trends in incidence and time-space clustering will
be sought and hypotheses formulated for testing in case-control studies.
27 - NEB/DIR
PHS 6040 (Rev 1/84)
spo si 4-»H
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ANNUAL REPORT
October 1, 1989 through September 30, 1990
Clinical Neurosciences Program, DIR
National Institute of Neurological Disorders and Stroke
Neuro immunology Branch
Table of Contents
RESEARCH SUMMARY 1-6
PROJECT REPORTS
Immunological Studies in Patients with Multiple
Sclerosis and Other CNS Diseases
Z01 NS 02202-15 NI 7
Membrane Molecules Involved in Immune Reactivity
Z01 NS 02203-15 NI 8
Immunologic Mechanisms Operative in Experimental
Autoimmune Diseases of the Nervous System
Z01 NS 02204-15 NI 9
Interactions Between the Human Immune System and
Antigens in the Nervous System
Z01 NS 02205-15 NI 10
Molecular Mechanisms of Lymphoid Cell-Cell Interactions
Z01 NS 02603-07 NI 11
Immunologic, Virologic, and Molecular Aspects of
Retroviruses Associated with Chronic Neurologic
Diseases
Z01 NS 02817-01 NI 12
NIB/DIR
Annual Report
October 1, 1989 to September 30, 1990
Clinical Neurosciences Program, DIR
National Institutes of Neurological and
Communicative Disorders and Stroke
Dale E. McFarlin, Chief
Research in the Neuro immunology Branch (NIB) includes a number of
interrelated fundamental, immunological studies and clinical investigations of
disorders affecting the nervous system. This is conducted in four
administrative groups, the Office of the Chief, the Neurological Disease
Section (NDS), the Molecular Immunology Section (MIS), and the Cellular
Immunology Section (CIS). During FY 1990, considerable emphasis was placed on
the genetic influence on immune reactivity and the application of molecular
biological strategies to both fundamental and clinical investigations.
The clinical research remained focused on multiple sclerosis (MS). Recent
findings from the NIB, as well as other institutions, indicate that magnetic
resonance imaging (MRI) is valuable for investigating the natural history,
pathogenic mechanisms, and the possible effect of treatment in MS.
Consequently, the use of MRI in the investigation of MS has been a major
clinical activity in the NIB, over the past year. Results from our studies,
as well as other institutions, suggest that an early event in the development
of MS lesions is breakdown in the blood-brain barrier (BBB). This can be
identified by the demonstration of enhancing lesions, after the administration
of gadolinium. In order to determine the frequency, magnitude, and natural
history of gadolinium-enhancing lesions, a new protocol was conducted in FY
1990. In this study a small group of patients with mild (Kurtzke disability
status scale of 3.5 or less) relapsing-remitting MS were evaluated at monthly
intervals by complete neurological assessment and MRI scanning, including the
administration of gadolinium. During the course of this protocol, 60 clinical
evaluations were conducted and 3 exacerbations were documented. MRI
evaluations, during the same period, showed the appearance of 91 new
gadolinium-enhancing lesions. This indicated that a marked amount of
clinically undetectable disease activity was occurring. The design of this
protocol enabled evaluation of the natural history of gadolinium-enhancing
lesions. Sixty-three lesions persisted for less than four weeks, twenty-four
for less than eight weeks, and only four beyond eight weeks. The gadolinium
enhancing lesions frequently evolved into nonenhancing lesions. During the
course of the study, it appeared that the amount of pathology detectable by
MRI gradually increased. Attempts to quantitate these changes by computer
technology are in progress. These MRI findings have major implications for
the investigation of a pathological process in MS and to the evaluation of
experimental therapeutic agents in the treatment of the disease. Future
studies of experimental therapeutic agents in the NIB will use MRI technology
for assessing efficacy.
The MRI findings reinforce the importance of investigation of the BBB.
Brain capillary endothelial cells (EC) that have tight junctions are a major
component of the BBB. For the past five years, imnuinological functions of EC
have been studied in the NDS in collaboration with Drs . Richard McCarron and
1 - NIB/DIR
Maria Spatz in the LNP. Isolated EC lack class II major histocompatibility
complex (MHC) molecules, but their expression can be induced by the
administration of gamma-interf eron. EC that express class II HLA molecules
are capable of presenting antigen to sensitized T-lymphocytes in vitro. This
finding led to the hypothesis that antigen recognition occurs at the luminal
surface of EC. A new finding is that EC treated with gamma- interferon and
pulsed with myelin basic protein (MBP) can be lysed by MBP-specific CD4+
cytotoxic T lymphocytes (CTL). The occurrence of this reactivity in vivo
would provide an immunological mechanism for a partial breakdown of the BBB.
Recent experiments have shown that other cytokines such as tumor necrosis
factors (TNF) and interleukin 1 (IL1) down-regulate the induction of class II
molecules on the surface of EC.
Both clinical and fundamental studies in the NIB continued to examine
genetic components possibly related to the pathogenesis of MS. A major
conclusion from our studies of genetic factors in MS is that multiple genes
are related to susceptibility. An immunopatho logical process has long been
postulated to be operative in MS, and products of genes on three different
chromosomes are involved in the recognition of antigen by immune T cells. The
T-cell receptor (TCR) for antigen consists of an alpha- and beta-chains
encoded by chromosomes 14 and 7, respectively. These receptors recognize
antigen presented by HLA molecules encoded on chromosome 6. For some time, MS
in some Caucasian populations has been known to be linked to class II HLA
molecules, namely, HLA-DR2. Two years ago, association between germline genes
encoding the beta-chain of the T-cell receptor was identified by the MIS in
collaborative studies with Dr. Leroy Hood at California Institute of
Technology. Subsequently, similar conclusions were reached by investigators
at Harvard University and a team of investigators at Stanford University have
described linkage between MS and a gene that encodes a variable gene segment
of the TCR alpha-chain. Studies in the MIS compared the TCR germline
repertoire of 40 chronic MS patients with normals. In order to characterize
this association in greater detail, germline TCR alpha- and beta-chain gene
haplotypes, as well as HLA molecules, are being assessed in families with
multiple affected members in a continuation of the collaboration with Dr.
Leroy Hood. In order to identify the affected individuals in each family as
precisely as possible, each of the family members is evaluated both clinically
and by MR I.
Extensive investigations of various aspects of HLA structure function
relationships and regulation of HLA expression are being conducted. Studies
of the association between MS and class II HLA molecules have included a
comparison of the sequences of these genes in MS and normal individuals.
Although susceptibility and resistance to diabetes has been correlated to a
single base change in the coding region of the HLA-DQ beta-chain, similar
sequence differences that are related to susceptibility have not been
identified in MS. Total genomic sequences for HLA-DQ beta genes from a
homozygous MS patient and a normal homozygous cell line showed no sequence
differences. The findings in a single patient were extended and focus was
placed on exon 2 of the alpha and beta gene which code for the antigen binding
regions. Genes for DQ-alpha, DQ-beta, and DR-beta of five MS patients have
been sequenced and no differences from the reported sequences in normal
individuals were detected. From this series of investigations, it has been
concluded that there is no coding difference in class II HLA genes that is
2 - NIB/DIR
related to susceptibility or resistance of MS. Therefore, the above-mentioned
association of susceptibility with class II genes probably is due to genetic
effects that do not involve specific antigen binding by class II MHC
molecules. Current efforts are designed to analyze the segregation of
noncoding regions of class II-associated polymorphic markers with various HLA
haplotypes in a series of 15 multiplex families.
A major difference between the nervous system and other tissues is that
MHC molecules are expressed in lower amounts or are undetectable. Major
emphasis is being placed on the fundamental mechanisms responsible for reduced
expression. Three experimental systems are being studied: 1) the regulation
of class I MHC molecules is being assessed in explanted murine fetal CNS cells
in collaboration with Dr. Keiko Ozato, in the Laboratory of Developmental and
Molecular Immunity, NICHHD; 2) the expression and regulation of both class I
and class II molecules on adult human glial cells (AHGC) is being studied in
vitro, in collaboration with Dr. Conrad V. Kufta, SNB; and 3) the molecular
regulation of class II molecules is being studied in cell lines derived from
human gliomas and medulloblastomas which retain the properties of neurons in
collaboration with Dr. Darell D. Bigner at Duke University.
A major conclusion from the studies of fetal CNS cells is that class I MHC
molecules are regulated differently in astrocytes, oligodendrocytes, and
neurons. An implication of this finding is that each of these cell types
varies in the capacity to function as targets for CD8+ cytotoxic T
lymphocytes that recognize antigen in association with class I MHC molecules.
Cultured neonatal astrocytes spontaneously express class I molecules in tissue
culture and have been used to investigate the molecular mechanisms responsible
for regulation of these molecules. In non-CNS cells, the transcription of
class I genes is regulated, in part, by a series of enhancer regions including
the class I regulatory element (CRE) and the interferon consensus sequence
(ICS) that are 5' to the promoter. Studies using cultures of neonatal
astrocytes transfected with constructs consisting of portions of the 5'
enhancers coupled to a reporter gene, chloramphenicol acetyl transferase,
indicate that the induction of class I MHC molecules is mediated through the
ICS. In contrast to all other cells so far examined, the CRE in astrocytes
has minimal enhancer activity and may even contain negative regulatory
elements. Nuclear binding proteins which react with the 5' enhancer motifs
are being characterized using gel mobility shift assays. Non-CNS tissues
express high levels of proteins that bind to the CRE region I and the ICS.
These proteins are undetectable in extracts of normal brain, but are
spontaneously expressed in cultured astrocytes at a time that coincides with
the production of class I molecules.
Procedures for the maintenance of AHGC in tissue culture have been
developed. Treatment of AHGC with gamma-interf eron leads to the induction of
class I and class II molecules and enables these cells to function as
immunological targets for CD4+ and CD8+ CTL. Because the T cell response
of MS patients to MBP is almost entirely HLA class II-restricted (discussed
below), focus is being placed on the molecular events responsible for the
regulation of these molecules. These experiments have been initiated with
established human glial cell lines and will be extended to AHGC when
sufficient numbers of these cells become available. The observations, to
date, indicate that class II molecules are up-regulated by gamma-interf eron,
3 - NIB/DIR
but other cytokines, particularly IL1, down-regulate the expression of these
molecules. As with class I molecules, the regulation of class II molecules is
controlled by 5' enhancer sequences. The molecular sites of action for the
various cytokines is being mapped with a group of molecular constructs in
collaboration with Dr. Jeremy Boss, at Emory University. The preliminary
findings indicate that one particular regulatory segment, the "Z box", has a
major influence on the regulation of these molecules by gamma-interf eron. The
negative influence of IL1 also maps to the same 5' region. This suggests that
different nuclear binding proteins are responsible for opposite effects of
these cytokines on class II regulation.
In an elegant series of investigations, the MIS has obtained new insight
into the molecular basis of antigen presentation by class I molecules. In
these experiments, a functional peptide competition assay was used to assess
the presentation of the influenza matrix (Ml) protein 55-73 by HLA-2.1
molecules to CD8+ specific CTL. Initial findings showed that approximately
25 percent of a panel of unrelated peptides bound to HLA-A2.1 and blocked
presentation of the Ml peptide to CTL. Subsequent studies examined the
capacity of these peptides to bind to another class I molecule, HLA-B37.
Although hi percent of the same panel of unrelated peptides bound and
inhibited antigen presentation by HLA-B37, none of these HLA-B37 binding
peptides were in the group initially shown to bind to HLA-2.1 molecule. These
observations demonstrate that individual HLA class I molecules can bind
distinct subsets of peptides.
The MIS previously demonstrated that the fungal antibiotic Brefeldin A
(BFA) abolishes the capacity of a cell to present endogenously synthesized
influenza viral peptides to class I-restricted CTL. This indicated that in
virus-infected cells, viral peptides bind to the class I molecules in the
endoplasmic reticulum and are then transferred to the cell surface where
recognition by CD8+ CTL occurs. During FY 1990, these studies were extended
to class II molecules using the influenza matrix protein Ml. Two distinct
pathways involved in the presentation of this antigen were identified. One is
an exogenous, chloroquine-sensitive, BFA-insensitive pathway and the second is
an endogenous BFA-sensitive pathway. The conclusion from these studies is
that endogenous antigenic peptides can associate with both class I and class
II molecules in the endoplasmic reticulum and are subsequently transferred to
the cell surface for recognition by TCR. These fundamental observations on
antigen presentation and recognition have major implications for the
assessment of immune function in MS and HTLV-I-associated myelopathy/tropical
spastic paraplegia (HAM/TSP).
The CIS previously demonstrated that patients with MS have impaired
capacity to generate CTL against measles virus. The measles virus-specific
CTL can be identified in individuals long after infection and are CD'++, HLA
class II-restricted. Because impaired generation of measles virus-specific
CTL is the only known antigen-specific defect in MS, the CIS has examined the
significance of this finding. In order to understand the generation of
measles-specific T cell response, adults with acute measles infection have
been studied. The results of these studies indicate that the infected
individuals who are apparent vaccine failures are fully capable of responding
to measles virus after natural infection. Thus, their failure to respond to
the virus after vaccination was not related to a genetically determined
k - NIB/DIR
impairment in immunity. Immediately following acute infection, these
individuals develop CD8+ class I-restricted CTL; subsequently, this cellular
response disappears and is replaced by circulating precursors of CD4+ class
II-restricted CTL. The findings are important in understanding mechanisms of
vaccine failure and indicate that the CD4+ class II-restricted CTL response
which is defective in MS probably contributes to the development of long-term
immunity against the virus.
MS has long been believed to have an immunopathological component, but the
antigen(s) responsible for the immunopathological process has not been
identified. A leading candidate is MBP, and during FY 1990, the CIS placed
major emphasis on characterizing the human cellular reaction to this
molecule. T cell lines selected by repeated antigenic stimulation with the
whole MBP molecule have been used to identify the major antigenic epitopes.
Two major immunodominant regions have been detected. These include an epitope
in residues 87-106 in the center of the molecule and a second epitope residing
in residues 154-172 in the C-terminal portion. Cell lines specific for these
epitopes have been derived from patients with MS and normal individuals.
Related experiments are examining the HLA molecules that present these
epitopes and the TCR molecules involved in recognition. The findings show
that at least three different HLA-DR types, each of which is over-represented
in certain MS populations, can present the epitope contained in residues
87-106 to T cells. The data obtained, to date, indicate that multiple TCRs
are used. The implication of these observations is that in humans a variety
of antigen presentation molecules and TCRs are used to react with MBP. This
is in contrast to findings in inbred rodents that develop experimental
allergit encephalomyelitis (EAE). In both rats and mice, TCR usage has
recently been shown to be restricted and has provided the rationale for
experimental treatment of EAE by the administration of monoclonal antibodies
to the TCR or vaccination with peptides corresponding to the sequence of the
TCR used by encephalitogenic T cells. The findings by the CIS indicate that
if a cellular immune response contributes to the pathogenesis of MS,
experimental treatment of this disease would require a more customized
strategy in individual patients.
Recent experiments in the Autoimmune Unit of the NDS are relevant to the
treatment of human immunological disorders. This group has obtained data
which indicate that during chronic-relapsing EAE, the specificity of T cell
reactivity shifts. In these studies, encephalitogenic T cells against one
epitope of MBP were selected in vitro by repeated antigenic stimulation with
synthetic peptides of the encephalitogenic epitope. Transfer of these cells
into normal mice produced relapsing EAE, however, T cells that recognize other
encephalitogenic epitopes appeared during the chronic phase of the disease.
This suggests that inhibition of immune reactivity to a single epitope may not
be effective for treatment of the chronic disease. An alternative would be to
use an approach that down-regulates immune reactivity without dramatically
producing immunosuppression. In other experiments, this group has demonstrated
that treatment with the cytokine transforming growth factor beta can both
prevent EAE and when given to mice, after an initial episode, reduces the
severity of chronic-relapsing disease.
During FY 1990, considerable research has been conducted on human
retroviruses and a new project (Z01 NS 02817-01) on the investigation of the
5 - NIB/DIR
immune reactivity to these viruses has been initiated. In previous studies of
HAM/TSP, patients with this disorder were found to have activated lymphocytes
in the blood that undergo spontaneous proliferation in vitro. During FY 1990,
studies of normal carriers of HTLV-I performed in collaboration with Dr.
William A. Blattner, Chief of the Viral Epidemiology Section, EEB, NCI have
shown that these individuals also have circulating lymphocytes that undergo
spontaneous proliferation in tissue culture and express surface molecules
including interleukin 2 receptor consistent with lymphocyte activation. A new
study has been initiated in collaboration with Dr. Blattner 's group to examine
lymphocyte function in individuals persistently infected with HTLV-II. Over
the past year, it has become apparent that this virus is prevalent in users of
intravenous drugs. The findings indicate that blood lymphocytes from carriers
of this virus spontaneously also proliferate in vitro. The molecular
mechanisms for this have not been identified, but the HTLV-I 5' tax protein is
believed to be responsible and is involved in the trans-activation of a number
of host genes.
Immunological studies of HTLV-I have produced a number of new findings. T
cell lines derived from patients with HAM/TSP by repetitive stimulation with
autologous infected lymphocytes react with the envelope protein in association
with class II HLA molecules. The epitope recognized by CD4+ CTL has been
characterized in collaboration with Dr. Thomas Palker at Duke University and
is identical to an immunodominant region of the envelope protein that is the
major site for antibody recognition. During the immune response to most
viruses, T cells and B cells respond to different epitopes. Consequently, the
demonstration that antibody and T cells react to the same region may be a
unique aspect of immune reactivity to human retroviruses because similar
observations have been made in HIV infection.
A second group of experiments conducted in collaboration with Drs. Scott
Koenig and Anthony Fauci in the Laboratory of Immunoregulation, NIAID, have
identified the presence of circulating CD8+ class I-restricted CTL in
patients with HAM/TSP. The presence of these CTL in circulating blood
indicate that these cells must persist in high concentrations in this
disease. Of particular interest is that circulating CTL have not been found
in the blood of normal carriers of HTLV-I or patients with ATL associated with
HTLV-I. The peptide specificity of the class I restricted CD8+ CTL has been
investigated in transfection experiments using vaccinia constructs coupled to
genes for individuals HTLV-I peptides. A second striking finding is that
circulating CTL are largely directed to the tax protein specified by 3'
regulatory regions of the virus. This series of observations has led to the
hypothesis that HAM/TSP has an immunopathological component consisting of CTL
reactivity to regulatory proteins encoded by the virus.
NIB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02202-15 NI
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT '80 characters or less Title must tit on one line between the borders.)
Immunological Studies in Patients with Multiple Sclerosis and Other CNS Diseases
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, ana institute affiliation)
PI: Dale E. McFarlin, M.D.
Others:
Henry F. McFarland, M.D.
Ajay Gupta, M.D.
David Mattson, M.D., Ph.D.
Jonathan Harris, M.D.
Joseph A. Frank, M.D.
Elliot Cowan, Ph.D.
Chief
Deputy Chief
Visiting Associate
Medical Staff Fellow
Clinical Associate
Radiologist
Special Expert
NI
DIR
NINDS
NI
DIR
NINDS
NI
DIR
NINDS
NI
DIR
NINDS
NI
DIR
NINDS
DR
CC
NI
DIR
NINDS
COOPERATING UNITS (if any)
Leroy Hood, M.D., Chairman, Dept. of Biology, Cal. Institute of Technology; Steven
Beall, M.D., Cal. Institute of Technology; Nicholas Patronas, M.D., Dept.
of Diagnostic Radiology, Georgetown University.
LAB/BRANCH
Neuroimmunology, CNP
SECTION
Office of the Chief
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
12.0
PROFESSIONAL:
8.0
OTHER
4.0
CHECK APPROPRIATE BOX(ES)
53 (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
E (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project is designed to evaluate multiple factors related, either singly or
in combination, to the pathogenesis of neurological diseases. Focus is currently
placed on multiple sclerosis (MS). Magnetic resonance imaging (MRI ) is important
in the assessment of MS and the recent detection of gadolinium-enhancing lesions
by this technology provides evidence that an early event in the pathogenesis is
breakdown of the blood-brain barrier. To determine the frequency and natural
history of such lesions, a group of patients with mild, early MS are being studied
monthly. MRI studies are being conducted in familial leukodystrophy.
Particular attention is being given to genetic and immunological factors which
contribute to pathogenesis of MS. Genes encoding for HLA molecules and the T-cell
receptors are being evaluated in patients with sporadic disease and families witn
multiple affected members.
Trials of experimental therapeutic agents for MS are conducted. The findings from
MRI investigations with gadolinium enhancement are being used to develop
strategies for the future evaluation of new treatments. A recent double-blind
study of cyclosporine A showed that patients treated with this agent progress less
rapidly than patients who received placebo; however, renal toxicity was a
significant side effect. The relationship between cyclosporine A treatment and
nephrotoxicity is currently being assessed in an open-label study.
The chronic myelopathy, HTLV-I-associated myelopathy/t ropical spastic paraplegia
( HAM/ T SP ) is being investigated. Immunological, clinical, and MRI findings in
HAM/TSP are being compared to normal carrier of HTLV-I.
7 - NIB/DIR
PHS 6040 (Rev. 1/84)
GPO 91 4-9K
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02203-15 NI
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.)
Membrane Molecules Involved in Immune Reactivity
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
PI: Dale E. McFarlin, M.D. Chief NI DIR NINDS
COOPERATING UNITS (it any)
LAB/BRANCH
Neuroimmunology, CNP
SECTION
Office of the Chief
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0
PROFESSIONAL:
0
OTHER:
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
□ (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project was terminated 10/89.
8 - NIB/DIR
PHS 6040 (Rev. 1/84)
GPO Bl 4-918
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02204-15 NI
PERIOD COVERED
lOctober 1, 1989 through September 30, 1990
j title of project ao characters or lass Tula must in on on» iir* Datwten tnt borders i Immunologic Mechanisms
Operative in Experimental Autoimmune Diseases of the Nervous System
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Same Me. laboratory, ana institute affiliation)
PI: Dale £. McFarlin, M.D.
I Others:
Michael R. Racke, M.D.
Robert Fallis, M.D.
Paul rtassa, Ph.D.
Mary E. Smith, M.D.
Chief
Special Volunteer
Senior Staff Fellow
Senior Staff Fellow
Clinical Associate
NI
DIR
NINDS
NI
DIR
NINDS
NI
DIR
NINDS
NI
DIR
NINDS
NI
DIR
NINDS
8eTrEfcT'^UR'aVnen,WPh.D., Professor, Albert Einstein U.; Maria Spatz, M.D. , Section
Chief, LNNS, DIR, NINDS; Richard McCarron, Ph.D., Senior Staff Fellow, LNNS, DIR,
NINDS; Keiko Ozato, Ph.D., Section Chief, LDMI, DIR, NICHHD
| LAB/BRANCH
iNeuroimmunology, CNP
SECTION
Neurological Diseases Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS
3.5
PROFESSIONAL
2.0
OTHER:
1.5
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects
D (a1) Minors
n (a2) Interviews
D (b) Human tissues LXJ (c) Neither
SUMMARY OF WORK (Use stanaera unreduced type Do not exceed the space provided)
Experimental allergic encephalomyelitis (EAE), a model of autoimmune disease
is being used to study immune reactivity and inflammation in the central
nervous system (CNS). Current research is focused on a model produced in
syngeneic animals by the transfer of lymphocytes sensitized against myelin
basic protein. Under optimal conditions, neurological dysfunction occurs;
this is characterized pathologically by inflammation and primary
demyelinat ion. Many mice recover and develop chronic-relapsing disease. The
immunological mechanisms responsible for both the initial and the chronic
disease are being investigated. An early event is the migration of immune
cells across the blood-brain barrier into the CNS. Initially, there is
interaction with the capillary endothelial cells, but astrocytes and microglia
are in close proximity. Antigen presentation by microglia, brain capillary
endothelial , and astrocytes are being compared. The regulation and expression
of MHC molecules in these cells is also being evaluated. The studies are
Deing conducted with cultures of murine fetal CNS cells, adult human glia
cells, and human cell lines derived from glioblastomas. The expression and
regulation of MHC molecules by neurons is also being studied in fetal murine
cultures and human medul loblastoma cell lines. The effect of various
cytokines on chronic-relapsing EA£ is being studied in order to gain insight
to the pathogenic mechanisms involved and to provide rationale for
experimental treatment of human demyelinat ing disorders.
9 - NIB/DIR
PHS 6040 (Rev t(84i
SPO >I44H
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02205-15 NI
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.)
Interactions Between the Human Immune System and Antigens in the Nervous System *
PRINCIPAL INVESTIGATOR (List other protessional personnel below the Principal Investigator.) (Name, title, laboratory.
PI: Henry F. McFarland, M.D.
Others:
Dale E. McFarlin, M.D.
William E. Biddison, Ph.D.
Suhayl Dhib-Jalbut, M.D.
Shari de Silva, M.D.
Rhonda Voskuhl, M.D.
Roland Martin, M.D.
Section Chief
Chief
Section Chief
Senior Staff Fellow
Clinical Associate
Clinical Associate
Special Volunteer
NI
and institute affiliation)
DIR NINDS
NI
DIR
NINDS
NI
DIR
NINDS
NI
DIR
NINDS
NI
DIR
NINDS
NI
DIR
NINDS
NI
DIR
NINDS
COOPERATING UNITS (if any) . _ _ . n -cc
John R. Richert, M.D. , Assist. Prof., Georgetown U. ; Diane Griff
Prof., Dept. Neurology, Johns Hopkins U. ; Richard Johnson, M.D
Neurology, Johns Hopkins U. ; Eric Long, Ph.D. Visiting Scientist
in, M.D., Ph.D. ,
Chairman, Dept.
BRB, DIR, NIAID
LAB/BRANCH
Neuro immunology, CNP
SECTION
Cellular Immunology Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
4.5
PROFESSIONAL:
4.0
OTHER:
0.5
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
HI (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The purpose of this project is to examine the manner in which immunological
mechanisms may contribute to diseases of the central nervous system (CNS) in
humans. These studies included examination of the cellular immune response to
viruses which can commonly effect the CNS and which may be related to the
induction of autoimmune processes of the nervous system. In addition, these
studies have examined the immune response, and in particular, the cellular
immune response to antigens of the nervous system such as myelin basic protein
and proteolipid protein. The emphasis in these studies has been on
identifying differences which may occur in patients with diseases of the CNS
such as multiple sclerosis as compared to healthy individuals. Particular
attention has been paid to the influence of genetic makeup on both the
induction and effector phases of the immune response to viruses as well as
antigens of the CNS. Focus has been placed on identification of cytotoxic T
cells specific for both viruses or antigens of the nervous system. In
addition, the capacity of cells of the CNS to process and present antigens to
immune T cells has been examined in order to help understand how immunological
mechanisms are induced within the nervous system.
* (Formerly, "Interaction Between Viruses and the Host Immune System")
10 - NIB/DIR
PHS 6040 (Rev 1/84)
GPO 914-918
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02603-07 NI
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must tit on one line between the borders )
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, ana institute affiliation)
PI: William E. Biddison, Ph.D.
Others:
Beatriz Carreno, Ph.D.
Ursula Utz, Ph.D.
Lael A. Stone, M.D.
Section Chief
Visiting Fellow
Special Volunteer
Special Volunteer
NI DIR
NINDS
NI
DIR
NINDS
NI
DIR
NINDS
NI
DIR
NINDS
COOPERATING UNITS (it any) , „ , „,_ „,.__ „ . , , _
John E. Coligan, Ph.D., Chief, Biological Resources Branch, DIR, NIAID; Richard D.
Klausner, M.D., Chief, Cell Biology and Metabolism Branch, NICHD; Leroy Hood, M.D.,
Ph.D., Professor, Department of Biology, California Institute of Technology
LAB/BRANCH
Neuro immunology, CNP
SECTION
Molecular Immunology Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
6.0
PROFESSIONAL:
3.0
OTHER:
3.0
CHECK APPROPRIATE BOX(ES)
EX (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
£3 (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided )
The general objective of this project is to define the mechanisms by which
human lymphoid cells interact with antigen-presenting cells in order to
produce and regulace immune responses. Over the past year, there have been
three major efforts underway that are targeted on this objective: 1)
dissection of tne molecular basis tor T cell recognition of antigens presented
by riLA class I molecules; 2) determination of endogenous versus exogenous
antigen presentation pathways for class I and II HLA molecules; and 3)
analysis of the potential roles of human T cell receptor (TCR) alpha and beta
cnain germline genes and HLA genes in susceptibility to multiple sclerosis
(MS). The principle findings are as follows: 1) two different class I
molecules, HLA-A2 . 1 and HLA-B37, bind largely nonover lapping sets of peptides;
2) the kinetics of peptide binding to RL.A-A2 and the conformation of the
peptide-A2 complex can be determined Dy amino acid side chains on the floor of
the peptide binding groove; 3) class II HLA molecules can use the endogenous
pathway of antigen presentation; and 4) in certain families that have multiple
members with MS, TCR and/or HLA genes can be shown to be associated with
susceptibility to MS.
11 - NIB/DIR
PHS 6040 (Rev 1/84)
GPO 914-018
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02817-01 NI
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders- ) Immunologic, Virologic, and
Molecular Aspects of Retroviruses Associated with Chronic Neurologic Diseases
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation)
PI: Steven Jacobson, Ph.D.
Others:
Dale £. McFarlin, M.D.
Masataka Nishimura, M.D.
Scott Koenig, M.D.
Suhayl Dhib-Jalbut, M.D.
Thomas Palker, Ph.D.
Cedric S. Raine, Ph.D.
Senior Staff Fellow
NI
DIR
NINDS
Chief
NI
DIR
NINDS
Visiting Fellow
NI
DIR
NINDS
Senior Staff Fellow
I
DIR
NIAID
Senior Staff Fellow
NI
DIR
NINDS
Assoc. Professor
Duke
University
Professor
Albert Einstein U.
COOPERATING UNITS (if any)
Dr. William Blattner, Chief, VES, NCI; Dr. Ashley Haase, Chairman, Dept.
of
Microbiology, University of Minnesota; Dr. Thomas Waldmann, Chief, MET Branch,
NCI, Dr. Anthony Fauci, Director, NIAID
LAB/BRANCH
Neuroimmunology, CNP
SECTION
Neurological Disease Section
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
5.0
PROFESSIONAL:
2.5
OTHER:
2.5
CHECK APPROPRIATE BOX(ES)
E (a) Human subjects
□ (a1) Minors
□ (a2) Interviews
Q (b) Human tissues □ (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The objectives of this project are to assess the involvement of human
retroviruses in chronic-progressive rayeloneuropathy termed HTLV-I-related
myelopathy /tropical spastic paraparesis (HAM/ISP) and multiple sclerosis.
These investigations have two major components: 1) the molecular/viro logical
identification of the virus(es) that are associated with the pathogenesis of
these disorders, and 2) the immunological response of the host to the
identified agent(s). Parallel studies are being conducted in normal carriers
of HTLV-I.
Human retroviruses are isolated from cells in the peripheral blood and
cerebrospinal fluid. The polymerase chain reaction is used to detect and
characterize viral genes in lymphocytes and in the central nervous system.
These viral genomes are being cloned and sequenced. Tissue sections from
spinal cord and brain from individuals with neurological diseases also are
analyzed for the presence of retroviral DNA by in situ hybridization. Immune
reactivity to these agents are characterized by demonstrating virus-specific
cytotoxic T cell (CTL) responses in these patients, as well as
lymphoproliferative responses. CTL are generated from long-term cultures,
addition, the presence of CTL in the peripheral blood of individuals with
active infection is being examined. The responding cells and the antigenic
specificity of the response are being characterized.
In
12 - NIB/DIR
PHS 6040 (Rev. 1/84)
GPO 9 1 4-9) S
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DO
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30
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30
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ANNUAL REPORT
October 1, 1989 through September 30, 1990
Surgical Neurology Branch - Clinical Neurosciences Program, DIR
National Institute of Neurological Disorders and Stroke
Edward H Oldfield, M.D., Chief
Table of Contents
Summary of Studies 1
I. Clinical Neurosurgery Section 6
II. Biochemistry Section 14
III. Central Nervous System Implantation Unit 18
IV. Tumor Biology Unit 21
V. Molecular Biology Unit 22
VI. Brain Imaging Unit 25
PROJECT REPORTS
Studies of Human Pituitary Tumors
201 NS02454-010SN-PI: Oldfield EH 27
Pharmacokinetics of Direct Brain Infusion
Z01 NS 02813-01 SN-PI: Bobo RH 28
Pentobarbital Effects on Damage of the Primate Brain by Fractionated Whole Brain Radiation
Z01 NS 02812-01 SN-PI: Bobo RH 29
Phase II Clinical Trials of Suramin and Hydrocortisone in the Therapy of Current Malignant Gliomas
201 NS 0281 1-01 -SN-PI: Bobo RH 30
Protection of the Brain Against Injury by Ionizing Radiation with Pentobarbital
Z01 NS02697-06SN-PI: Olson JJ 31
Studies on the Heterogeneity of Drug Delivery During Intracarotid Chemotherapy
Z01 NS 02728-04 SN-PI: Saris S 32
Adoptive Immunotherapy of Brain Tumors
Z01 NS 02778-03 SN-PI: Saris S 33
Monoclonal Antibodies Linked to Ricin for Use in Human Bone Marrow Transplantation
Z01 NS02673-06SN-PI: Youle RJ 34
Antibody-Toxin Conjugates for the Treatment of Human Brain Tumors
Z01 NS 02823-01 SN-PI: Youle RJ 35
Monoclonal Antibody-Toxin Conjugates for Tumor Therapy in vivo
Z01 NS 02674-06 SN-PI: Youle RJ 36
Tissue Implantation in Parkinsonian Models
Z01 NS 02781-03 SN-PI: Plunkett RJ 37
Adrenal Medullary Autografts in Parkinsonian Patients
Z01 NS 02729-04 SN-PI: Plunkett RJ 38
Vascular Permeability Factor Produced by Human Glioma Cells
Z01 NS 02708-05 SN-PI: Merrill M 39
The Role of Insulin and Insulin-like Growth Factors in Glioma Cells
Z01 NS 02707-05 SN-PI: Merrill M 40
Genetic Abnormalities in Primary Glial Tumors
Z01 NS 02814-01 SN-PI: AM 1 41
Molecular Genetics of Pituitary Conticotroph Adenomas
Z01 NS 02815-01 SN-PI: Alii 42
Metabolic Mapping of the Brain During Rewarding Brain Stimulation
Z01 NS 02760-03 SN - PI: Porrino L 43
Quantitative Autoradiographic Determination of Dopamine Receptor Distribution in Primates
Z01 NS 02762-03 SN-PI: Porrino L 44
Metabolic Mapping of Animal Models of Parkinsonism
Z01 NS 02761-03 SN-PI: Porrino L 45
Clinical and Laboratory Investigation of Central Nervous System Vascular Disorders
Z01 NS 02739-04 SN-PI: Oldfield EH 46
i SNB/DIR
October 1, 1989 through September 30, 1990
Surgical Neurology Branch, Division of Intramural Research
Clinical Neurosciences Program
National Institute of Neurological Disorders and Stroke
Edward H. Oldfield, M.D. - Chief
SUMMARY OF STUDIES
The Surgical Neurology Branch (SNB) principal activities are conducted in the
following: I) Clinical Neurosurgery Section; II) Biochemistry Section; III) Central
Nervous System Implantation Unit; IV) Tumor Biology Unit; V) Molecular Biology
Unit; VI) Brain Imaging Unit.
The Surgical Neurology Branch has as its major research functions: (1) the study
of the biology and therapeutic approaches to malignant and benign tumors of the
brain and pituitary gland; (2) investigation of the capability of implanted tissues into
the brain of animals and humans to survive and integrate anatomically or
biochemically with the host brain to influence brain function; (3) the investigation
of certain vascular disorders of the Central Nervous System (CNS); and (4) the
investigation and surgical treatment of patients with epilepsy. Its clinical function is
to provide the neurosurgical services to its own research protocol patients and to
patients seen in consultation in the NIH, Clinical Center. The SNB is presently located
in Buildings 9 and 10. Its staff includes 14 clinical neurosurgeons at various levels of
training and experience, as well as 2 senior scientist, 3 junior scientists and a support
staff of scientific, technical and administrative individuals.
In addition to its primary functions of clinical and basic research, the SNB
provides young neurosurgeons with experience in clinical and laboratory
investigation in a combined clinical and neuroscience environment. Of those who
have participated in the SNB program as Medical Staff Fellows and Senior Staff
Fellows, many have entered academic positions in neurosurgery.
The following significant observations have been made during the past 24
months. In the area of brain tumors we have made several observations. Using
Positron Emission Tomography (PET) scanning and positron-labeled fluoro-2-deoxy-
glucose, we previously demonstrated that thiopental selectively suppresses brain
metabolism while leaving tumor metabolism unaffected. Similar, but less
suppression was demonstrated with sedative doses of pentobarbital. Means to
exploit this capacity to selectively influence brain metabolism without effecting
tumor metabolism for therapy were then investigated.
Early and delayed cerebral radiation toxicity in rats was prevented by
pretreatment with pentobarbital. Survival of animals receiving whole brain
irradiation was increased from 0-20% to 45-70% by sedation with pentobarbital just
before irradiation exposure. The mechanism of this protection was investigated and
shown to be linked to interaction of barbiturates with the GABA receptor. Other
agents which functionally bind the GABA receptor were shown to be equally
effective. Since tumors lack functional GABA receptors, and tumor metabolism had
been shown not to be influenced by barbiturates, animals with implanted tumors
were investigated. No reduction of tumor sensitivity was demonstrated. Hence,
pentobarbital suppressed metabolism of normal brain and protected against
1 SNB/DIR
radiation toxicity, but tumors had no metabolic suppression and were not protected
against irradiation injury.
Further progress was made in the development of immunotoxins for treatment
of CNS tumors. The major goal of this project is to develop immunotoxins which will
selectively kill tumor cells in vivo. An antitransferrin receptor antibody-toxic peptide
conjugate was created against the transferrin receptor which specifically kills tumor
cells in vitro and also when injected into the cerebrospinal fluid (CSF). A cloned
diphtheria toxin was created in which the C-terminal 1 5 kD region was deleted. This
eliminated much of the nonspecific binding of the diphtheria toxin, but retained the
entry function of the toxin into the cytosol of cells and retained toxicity. Recent
results indicate that point mutations can increase selective cytotoxicity of antibody-
toxin conjugates up to 10,000-fold. Animal studies indicated a 5-log kill of tumor
cells inoculated into the peritoneal cavity in vivo. Utility of the immunotoxins in
animal models of leptomeningeal tumor was demonstrated. Treatment of tumor in
a guinea pig model of leptomeningeal neoplasia demonstrated a 2- to 3-log, and in
some animals a 5-log kill of tumor cells in the CNS with this conjugate. Clinical trials
in patients with carcinomatous meningitis began this year with a monoclonal
antibody against the human transferrin receptor linked to recombinant ricin A
chain. Five patients have been treated in a phase I trial without toxicity.
Several investigations of adoptive immunotherapy for treatment of malignant
brain tumors were completed. Techniques were perfected to successfully implant
reproducible lymphokine activated killer (LAK) cells into the brain of rodents and
primates. Clinical investigations of adoptive immunotherapy using interleukin-2
(IL2)and LAK against cerebral gliomas indicated that repeated injections of IL2 and
LAK into tumor cavities or directly into brain tumors produced increased cerebral
edema and transient or permanent new neurologic deficits in all patients.
Investigation of intravenous IL2 therapy in patients with glioblastomas
demonstrated increased edema from the tumor-bearing region. The results of these
studies indicate that adoptive immunotherapy using IL2 and LAK cells produces
significant toxicity and rarely elicits a measurable tumor response. In separate
clinical studies, IL2 was (1) demonstrated to penetrate the blood-CSF barrier in
patients without CNS tumor involvement in sufficient concentration to maintain
activity of LAK cells in the CSF, and (2) using magnetic resonance imaging (MRI)
scanning, IL2 was shown to increase the water content of the normal brain,
suggesting alteration of the normal blood-brain-barrier by IL2. Animal experiments
indicated that the cerebral edema results from IL2-induced increased vascular
permeability in the vessels of the tumor and the surrounding brain. It was shown in
an animal model of cerebral metastases that, although lung metastases responded
to I L2/LAK treatment, brain deposits of the same tumor in the same animal had no
response to treatment, indicating that the blood-brain-tumor barrier restricts
effectiveness of immunotherapy of this type. The requirement of continued
exposure of LAK cells to IL2 for maintenance of optimal cytotoxicity was
demonstrated in an in vitro analysis of LAK cytotoxicity against glioma cells.
Intraarterial infusion of IL2 was shown to increase delivery of IL2 to brain by 20-fold
compared to intravenous infusion.
In addition, the importance of aminoacid 309 of the B chain of diphtheria toxin
for translocation of the toxin across cell membranes was demonstrated; studies were
initiated to examine the pharmacokinetics of immunotoxins after direct infusion
into the brain and tumors; and ways to block the immune response to immunotoxins
were found. That drug streaming, which may cause toxicity and limit efficacy after
intraarterial infusion of chemotherapy, can be eliminated in humans by phased
2SNB/DIR
diastolic delivery was demonstrated in humans using PET scanning and positron-
labeled water to demonstrate the distribution and to quantify the delivery of
infusate to the brain after intracarotid delivery. Patients treated with intracarotid
cisplatin using a prototype pump which delivers drug into the internal carotid artery
only during a brief period of diastole, so that drug streaming is eliminated, had no
retinal or neural toxicity related to drug streaming. However, a trial of intraarterial
high dose BCNU via phased diastolic delivery in patients with malignant glioma
demonstrated retinal and brain toxicity despite the elimination of drug streaming.
A protein that is secreted by malignant brain tumors and increases vascular
permeability to macromolecules was isolated from the incubation media from
malignant brain tumors. We have demonstrated that this material is a protein of
approximately 45,000 daltons molecular weight and that its secretion is eliminated
by incubation of the tumor cells with dexamethasone. Further experiments
indicated that it binds a heparin-like moiety on the cell surface of endothelial cells
and that influx of Ca2 + into the endothelial cell occurs in response to this
interaction. These observations explain the origin of brain edema in patients with
brain tumors and may permit development of therapy against tumor-associated
cerebral edema, which now requires high dose glucocorticoid therapy and is
associated with significant complications. Recent advances have shown that this
protein is the same molecule as pituitary-derived vascular endothelial growth factor,
a potent mitogen selective for endothelial cells. These observations indicate that
vascular permeability factor (VPF) secreted by malignant brain tumors may be
responsible for brain edema and tumor angiogenesis.
Insulin and the insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) were
investigated for a role in the growth of malignant gliomas. High levels of IGF-1
receptors were demonstrated on several gliomas and shown to be functional as
determined by autophosphorylation of the receptor and DNA synthesis.
Additionally, high levels of high affinity IGFI-binding proteins were shown to be
produced by human glioma cells in vitro and in vivo. These results suggest a role of
IGF-1 and its receptor in the regulation of glioma growth. The clinical, genetic and
radiographic features of patients with symptomatic von Hippel-Lindau disease (VHL)
were investigated. In collaboration with Drs. Bert Zbar and Marston Linehan of NCI
and Dr. Michelle Filling-Katz of NINDS, we investigated the genetics of 9 tumors
from 5 patients with VHL disease and hemangioblastomas of the brain and spinal
cord. The tumors were shown to have absence of the wild type (normal) allele and
were shown to arise by a mechanism analogous to that of familial retinoblastomas,
i.e., by homozygous absence of a tumor-suppressing gene. In addition, the same
genetic abnormality was shown in sporadic hemangioblastomas of the CNS. MRI
scanning was used to demonstrate the incidence of syrhingomyelia and the extent of
the syrhingomyelia cavity in patients with hemangioblastomas of the spinal cord.
MRI with Gadolinium Ga-EDTA contrast enhancement was used to investigate the
brain and spinal cord in VHL disease and shown to the more sensitive than MRI
without Gd-EDTA and CT scanning, and to have similar sensitivity as arteriography,
for tumor detection. The syrhingomyelia that is associated with most
hemangioblastomas of the spinal cord, and which is responsible forthe neurologic
deficit in many patients, was shown to resolve with treatment directed only at tumor
removal and not to require separate consideration for treatment. A previously
unrecognized distribution of hemangioblastomas of the CNS to preferentially
involve the cervical segments of spinal cord was demonstrated.
Further experience with selective pituitary venous sampling for Cushing's
syndrome indicated that selective bilateral simultaneous inferior petrosal sinus
3SNB/DIR
sampling successfully (100% accuracy) distinguishes patients with Cushing's disease
from those with ectopic ACTH syndrome. This technique permits tumor localization
within the pituitary gland in patients with Cushing's disease with about 75%
accuracy. It was also demonstrated that repeat surgery could be used to successfully
treat (70% cure rate) patients with persistent or recurrent Cushing's disease after
prior pituitary surgery or pituitary irradiation. Predictive features of successful
outcome and guidelines for intraoperative management were established by a study
of 48 patients with repeat surgery. Gadolinium-EDTA enhancement with MRI
scanning was shown to be superior to current imaging techniques for demonstration
of the site of pituitary microadenomas in Cushing's disease pre-operatively. The
critical aspect of timing the image procurement within 5-10 minutes after the
injection of Gd-EDTA was demonstrated. However, in a study of 65 patients with
surgically proven Cushing's disease, MRI pituitary scanning was normal in 45%. In
addition, 100 normal subjects were scanned to estimate the incidence of pituitary
adenomas in the normal population and 1 5% were found to have pituitary tumors.
Encouraging progress was made in the area of implantation of tissues from the
CNS into the host brain in primates. Initially, a model of parkinsonism which affects
only one-half of the brain was developed by infusing MPTP into the internal carotid
artery. Using this model of hemiparkinsonism and the previously available model of
MPTP-induced full parkinsonism in the primate, we investigated functional recovery
and graft survival after implanting dopaminergic tissues into the caudate nucleus of
lesioned animals. Both adrenal and fetal substantia nigra allografts to induced
partial functional recovery in the host animals. However, the recovery was much
more pronounced using fetal substantia nigra for implantation. Immunohistological
assessment demonstrated functional survival of the fetal grafts and prominent
sprouting of dopaminergic fibers from resident fibers in the host brain surrounding
the area of implantation. By screening monkey and human fetal tissues for evidence
of neurite-promoting activity, using the chick dorsal root ganglion in vitro assay, the
fetal amnion was identified as having potent activity. Primates implanted with fetal
amnion were then shown to induce sprouting of dopaminergic fibers and functional
recovery in the hemiparkinsonian primate model that was comparable to that which
occurred after fetal mesencephalic implant. These results indicate that sprouting of
the intrinsic dopaminergic pathways of the host brain is induced by either surgical
cavitation or fetal implants. If the basic mechanisms responsible for this induced
sprouting can be identified and used for other neuronal systems, practical
application of CNS implants for Parkinson's disease and other CNS disorders may be
possible. The sprouting in response to cavitation and implantation which appeared
to be related to functional recovery of the animals appeared to derive from the
ventral tegmental tract, a dopaminergic neural tract which is separate from the
nigrostriatal tract and which is not affected by MPTP to the same extent as the
substantia nigra. To study this phenomenon in greater detail, and without requiring
of large numbers of subhuman primates, a rat model of parkinsonism was developed
with preservation of the ventral tegmental tract. In addition, accumulation of a
material which induces neural sprouting in an in vitro assay was found in the cavities
of monkeys and humans after surgical cavitation. Finally, a technique was
developed to assess the biochemistry of the basal ganglia of implanted primates in
vivo by implanting fine dialysis fibers into the basal ganglia. This technique should
not only be rewarding for investigating of implantation to tissues to reverse
parkinsonism, but should have wide application clinically.
Finally, the investigation of the mechanism and physiologic role of
programmed cell death, which occurs in both the CNS and the immune system
during normal development, demonstrated that thymocytes can be induced to die
4SNB/DIR
by the same signal that stimulates mature T-cells to proliferate. RNA and protein
synthesis inhibitors which block new gene expression interrupted the programmed
cell death. This is consistent with a new model of immune system self-tolerance that
states that potentially autoreactive cells are induced to die by crosslinking the T-cell
receptor early in development, the same signal that induces mature T-cells to react
against foreign antigens. Studies are being extended into the nervous system by
examining the mechanism of glutamate-induced neuron death in vitro and in vivo.
Immunotoxin-induced selective Purkinje cell death in an animal model has been
observed. This model is being used to examine the possible mechanism(s) of Purkinje
cell death in a variety of neurologic disorders.
A Molecular Biology Unit was established to study genetic abnormalities
associated with various nervous system disorders, especially brain and pituitary
tumors. The clonality of ACTH-secreting pituitary tumors was examined using the X-
chromosomal inactivation technique. Nine of 27 tumors proved to be evaluable with
this technique. The results demonstrated that these adenomas may be monoclonal
(6 patients) or polyclonal (3 patients, one of whom had Nelson's syndrome), and
indicate two mechanisms of oncogenesis are possible. The Brain Imaging Unit was
developed in the Surgical Neurology Branch this year to integrate quantitative
autoradiographic studies with our studies of parkinsonism neural implantation,
physiology of brain tumors, and certain aspects of pharmacology associated with
these problems. Using [3H]naloxone an increased density of opiate receptor sites in
MPTP-induced hemiparkinsonism monkeys was observed in the striatum of the
MPTP-treated side. The distribution of which was similar to the distribution of
dopaminergic Di receptor binding sites. This suggests potential interactions of
dopamine and opiate peptides in the striatum. Using the 2-[Ci4]deoxyglucose
method and selective lesioning of the substantia nigra behavioral effects were
correlated with precise loci of damage along the medial-lateral axis of the substantia
nigra.
5SNB/DIR
I. CLINICAL NEUROSURGERY SECTION
Edward H. Oldfield, M.D., Chief
The clinical activities of the Surgical Neurology Branch are primarily directed at
investigating the biologic behavior and mechanisms of pathophysiology of
malignant primary brain tumors, pituitary tumors, certain vascular disorders of the
CNS, surgical management of epilepsy refractive to medical therapy, and
investigation of tissue implantation in the CNS to reverse neurological disorders.
A. Brain Tumors
1. Barbiturate Sedation in the Management of Brain Tumors
Study of the radioprotective effect of alternative barbiturates to
pentobarbital. pPentobarbital has been shown to have significant radioprotective
effect on the brains of rats who undergo high-dose, single-fraction, whole-brain X-
irradiation. As pentobarbital is a radioprotectant, further study of alternative
barbiturates was carried out to evaluate their potential radioprotective effect. This
was performed in the standard rat model, utilizing Fisher 344 rats receiving 70 Gy of
whole-brain X-irradiation in a single fraction. Neither phenobarbital nor
methohexital delivered any radioprotection as measured in this model. Thiopental
enhanced survival to an extent equal to pentobarbital. As thiopental is a more
commonly utilized barbiturate in human anesthesia, this implies greater potential
applicability clinically.
Investigation of the mechanism of the radioprotective effect of pentobarbital
in the rodent model. In spite of continued investigation of this phenomenon and
documentation of its reproducibility, the mechanism of the effect remains to be
delineated. An investigation utilizing simultaneous quantitative autoradiographic
analysis of cerebral blood flow and glucose utilization in the irradiated rat is
currently underway.
Investigation of the radioprotective effect of pentobarbital in a primate model
of cerebral radiation toxicity. A concern remains as to the applicability of a model of
acute radiation toxicity in a rodent to the radiation toxicity seen in humans with
cerebral radiation injury due to treatment of brain tumors. A primate model was
designed to better approximate the injury seen in humans, which is more delayed in
onset. Two groups of animals were utilized. One received 15 Gy of whole-brain, X-
irradiation while lightly sedated with ketamine, an anesthetic having
radioprotective effect in the rat model. The second group underwent radiation
after a dose of pentobarbital which results in 30 to 60 second EEG burst suppression.
Baseline studies priorto X-irradiation included anatomic studies utilizing MRI
without and with gadolinium, physiologic studies utilizing position emission
tomography with i8f|uoro-deoxyglucose (to evaluate neuronal and glial integrity)
and 68Ga-EDTA (to evaluate blood brain barrier and microvascular integrity), and
neuroendocrinologic studies utilizing insulin, L-dopa, arginine, thyroid-releasing
hormone, and gonadotropin-releasing hormone as stimulants to measure glucose,
Cortisol, growth hormone, prolactin, thyroid-stimulating hormone, follicle-
stimulating hormone, and luteinizing hormone responses. MRI studies were
subsequently planned for 1 week, 1 month, 6 months, 12 months, 18 months and 24
months after X-irradiation. PET studies were at 1 week, 1 month, 1 year and 2 years
6SNB/DIR
after X-irradiation. Endocrinologic studies are carried out 1 week, 1 month, 3
months, 6 months, 12 months, 18 monthsand 2 yearsafter irradiation. The animals
are sacrificed 2 years after X-irradiation and histologic exam performed.
The primate study is long-term and is ongoing at this time. Four animals have
been enrolled in each group. All animals surviving beyond 6 months have
demonstrated white matter abnormalities to varying degrees, regardless of the
group to which they belong. The supports the efficacy of the method of radiation
injury. Intergroup differences in white matter injury as assessed by MRI await
quantitation at the completion of the study.
Endocrinologic studies reveal that animals irradiated under the influence of
ketamine have evidence of early hypothalamic/pituitary functional abnormalities.
After 3 months, the peak luteinizing hormone response in the control group was
significantly less (mean, 23.1 ng/ml [SD, 3.9 ng/ml]) than in the pentobarbital group
(mean, 31.7 ng/ml [SD, 4.4 ng/ml]). The prolactin level 30 minutes after L-dopa
administration rose progressively after 3 and 6 months in control monkeys as
compared with those treated with pentobarbital. Peak thyroid-stimulating
hormone response to thyrotropin-releasing hormone in the control group (mean,
13.7iiU/ml [SD, 4.1 iiU/ml]) was less than that of the pentobarbital group (22.6 uU/ml
[SD 5.0 pU/ml]). No difference was noted between the treated and control groups
when measuring growth hormone, follicle-stimulating hormone, or Cortisol
responses.
Two of the four animals radiated with ketamine for anesthesia succumbed to
radiation toxicity as of 6 months following treatment. The other two animals remain
intact 1 to 2 years following this treatment. One of the four animals radiated under
deep pentobarbital anesthesia succumbed 7 months after treatment. The other
three animals remain alive, well and growing, 12 to 18 months after treatment. This
substantiates the degree of toxicity imparted by the radiation dose utilized. The
protective effect imparted by pentobarbital as seen in the original rat model of
cerebral injury is supported in the findings with the primate model thus far. As seen
in the rat model, this protection can be overwhelmed by high doses of radiation.
Importantly, there appears to be protection of the hypothalamic/pituitary axis, an
area frequently injured in children undergoing brain radiation.
In an additional primate study the potential radiation protective effect of
pentobarbital is being examined with multiple fractions of radiotherapy,
fractionation analogous to that used clinically. Information on the basic
mechanism(s) of radiation damage of the brain and hypothalamic-pituitary axis will
also be obtained.
2. Clinical use of immunotoxins to treat CNS tumors
A Phase I clinical trial was begun to investigate the clinical use of immunotoxins
in the treatment of leptomeningeal carcinomatosis. Immunotoxin, a conjugate of a
human antitransferrin receptor antibody with ricin A chain, is being administered in
single injections in patients who have leptomeningeal carcinomatosis refractory to
current therapies. Five patients have been treated, no toxicity has been seen.
Efficacy remains to be established.
Methods are being investigated for direct injection into the brain and into CNS
tumors, to achieve a homogeneous distribution of antiglioma agents throughout
7SNB/DIR
the tumor volume by direct parenchymal infusion. At present this study is limited to
studing the spatial and temporal distribution, using quantitative autoradiography,
of representative agents infused into the feline cerebrum.
3. Phase II trial of Suramin to treat patients with malignant gliomas
The efficacy of suramin combined with hydrocortisone therapy in the
treatment of patients with malignant gliomas which occur after radiation therapy is
being performed. In vitro data indicate that this combination inhibits angiogenesis
and interrupts growth of glioma cells by binding certain growth factors. This new
type of systemic chemotherapeutic agent has been demonstrated to be effective in
patients with adrenal cortical cancer but has not previously been evaluated in
patients with brain tumors.
4. Intraarterial chemotherapy
One of the basic tenets of anticancer chemotherapy is that increased tumor
exposure to a drug should result in increased tumor response. One method currently
being used to increase drug exposure of malignant brain tumors is by intracarotid
infusion. Our research results have demonstrated the following :
Drug streaming during intracarotid delivery results in maldistribution of the
infused agent with the potential of delivering very high (toxic) concentrations of
drug to some regions of the brain, while other areas receive minimal drug.
The presence of streaming, and its elimination by phased, diastolic infusion,
was investigated in 9 glioma patients in vivo using PET. We demonstrated that
standard infusion techniques (continuous infusion of 1-4 cc/min) of H215 O-labeled
water into the supra-ophthalmic segment of the internal carotid artery caused
extremely heterogeneous delivery of the inf usate to the brain in the distribution of
the internal carotid artery. When the infusions were done with a phased, diastolic
infusion, heterogeneous delivery was eliminated. These findings confirm our data in
other species, and will lead to further clinical studies with intracarotid drug infusions
to improve drug delivery, limit toxicity, and potentially improve survival of glioma
patients.
We completed two projects which quantified permeability of the blood-
brainbarrier in the primate brain before and after barrier disruption by intracarotid
mannitol using PET scanning. In a study of baboons the permeability across normal
brain vessels in the gray and white matter was measured repeatedly and the
duration and extent of attention in permeability was determined. A similar study in
humans permitted quantification of vascular permeability in primary brain tumors,
brain around tumor and normal brain. The results of these studies will permit
rational designs of approaches to deliver drugs of various types to the brain using
this technique.
5. Studies of patients with von Hippel-Lindau Disease
Von Hippel-Lindau's (VHL) disease is an inherited disorder in which patients
suffer from hemangioblastomasof the retina, cerebellum and spinal cord as well as
renal cell carcinomas, pheochromocytomas, and visceral cysts. The disease is passed
8SNB/DIR
in an autosomal dominant pattern which has been linked by recombinant fragment
length polymorphism analysis to the short arm of the third chromosome. The
defective gene is in the 3p 14 region.
In collaboration with Drs. Berton Zbar and Marston Linehan of the NCI, we
studied 1 3 separate tumors from 5 patients, analyzing them for loss of alleles on the
third chromosome. These tumors demonstrate loss of one copy of the vHL allele.
However, in the tumor cells, the loss is of the normal, wild type allele inherited from
the normal parent. This loss of the balancing wild type allele leaves the tumor with
only the abnormal allele inherited from the parent carrying von Hippel-Lindau
disease. This supports the theory that the tumors in VHL disease are caused by the
loss of a tumor suppressor gene similar to that seen with retinoblastoma and type II
neurofibromatosis; the affected parent contributes a defective copy of the gene
allowing cells at risk to develop tumors when the other copy, the balancing wild-
type gene from the normal parent, is lost or rendered nonfunctional.
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B. Pituitary Tumors
Venous sampling to establish the diagnosis and location of hormone-secreting
pituitary microadenomas
We have now performed bilateral and simultaneous inferior petrosal sinus
sampling in over 320 patients with Cushing's syndrome. The results are used (1) to
confirm the diagnosis of Cushing's disease preoperatively, and (2) to determine the
half of the pituitary gland in which a microadenoma resides. The study has been
particularly rewarding and has demonstrated the following: (1) sampling from both
inferior petrosal sinuses simultaneously with CRH injection consistently distinguishes
patients with Cushing's disease from those with ectopic ACTH syndrome (100%
accuracy); (2) sampling from a single inferior petrosal sinus, as has previously been
general practice, to establish the diagnosis of Cushing's syndrome, may be
misleading and could result in an incorrect assumption of the source of excess ACTH
secretion in as many as 43% of patients with Cushing's syndrome; and (3)
preoperative sampling for ACTH concentrations in the inferior petrosal sinuses
determines the site of ACTH- secreting microadenomas within the pituitary gland
with about 85% accuracy. Therefore, bilateral sampling permits the surgeons' search
for small microadenomas to be focused to one side of the gland, which should be
helpful in finding smallertumors. If no tumor is found, the half of the gland
containing the microadenoma can be removed. This technique is now being widely
employed in the evaluation of patients with Cushing's syndrome.
Radiologic imaging and Cushing's syndrome
To evaluate the MRI scan as a means of aiding the differential diagnosis of
patients with Cushing's syndrome and as a mechanism for tumor localization pre-
operatively, we prospectively evaluated the results of CT scanning in 67 patients with
Cushing's syndrome. The diagnostic accuracy of MRI scanning with gadolinium -
DTPA contrast was only 55%. The conclusion of this study was that MRI scanning is
only occasionally helpful in locating, pre-operatively, the site of an ACTH-secreting
pituitary microadenoma, and is therefore only rarely beneficial as a diagnostic aid in
the differential diagnosis of patients with Cushing's syndrome.
Autopsy studies have indicated an incidence of pituitary adenomas in
asymptomatic patients to be 7-33%. To establish the incidence of MRI abnormalities
in the general population consistent with a diagnosis of a pituitary adenoma we
studied 100 normal subjects with and without gadolinium enhanced MRI scans. The
results of this study indicate that 15% of the normal population have pituitary
lesions that appearto be pituitary adenomas by MRI scanning. These results indicate
that an appreciable percentage of normal adults have pituitary adenomas and
indicate that positive findings on an MRI in a patient with endocrinopathy do not
necessarily establish that the source of the endocrinopathy is in the pituitary gland.
Repeat pituitary surgery in patients with Cushing's disease despite previous
treatment
Since the ACTH-secreting pituitary tumors that cause Cushing's disease respond
to negative feedback by glucocorticoids, but have a higher threshold for
glucocorticoid suppression than normal corticotrophs, a potential abnormality in the
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tumor cells accounting for hypercortisolism in these patients is that the
glucocorticoid receptor is abnormal. Pituitary adenomas from 8 patients with
Cushing's disease were investigated to determine if the glucocorticoid receptor in
the adenomas in Cushing's disease was normal or abnormal as determined by
restriction fragment length polymorphism analysis. Results of this study indicated
no abnormality in the adenomas compared to normal tissues. It appears unlikely
that a genetic abnormality in the glucocorticoid receptor is the sole explanation of
the resistances to negative feedback by steroids in these tumors.
Origin of ACTH-secretinq pituitary microadenomas in Cushing's disease
Because of the female preponderance (90%) in Cushing's disease, it has been
thought that these tumors may be induced by endocrine factors. In experimental
models in animals induction of pituitary tumors by hormonal manipulation
(thyroidectomy or treatment with high-dose estrogen) initially produces areas of
pituitary hyperplasia. Small focal pituitary adenomas arise several weeks later. To
assess whether ACTH-secreting tumors in Cushing's disease originated from a single
cell or from multiple cells we used the X-chromosomal inactivation scheme. The
results indicated that 3 of the tumors originated from multiple cells whereas 6
originated from a single cell (see Molecular Biology Units of this portion of this
annual report for more detail presentation).
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C. Surgical Treatment of Medically Intractable Epilepsy
The aim of the surgical arm of the NINDS epilepsy program is to develop
surgical techniques that allow more accurate localization and safer resection of
epileptogenic foci than can be achieved with methods now available. The
development and implementation of surgical treatments for patients whose seizures
are intractable to currently available medical and surgical therapies is the immediate
and long-term goal of this program.
Special subdural surface electrodes designed and built at NIH in collaboration
with the BEI Branch of DRS are now being implanted in selected patients so that EEG
recordings can be obtained directly from the cortical surface for much longer periods
than is possible during intraoperative electrocorticography. During the period of
implantation these electrodes are also utilized for focal cortical stimulation to
discriminate areas that can be safely resected from areas critical for motor, sensory,
language, memory-related and other functions. Such discrimination is crucial in the
topographic identification of overlap between critical cortical areas and epileptic
foci during surgical procedures.
A subset of patients with epileptogenic foci originating in the language-
dominate hemisphere are undergoing implantation of a new type of subdural
electrode designed and built here at NIH. Recordings and functional stimulation
mapping utilizing these electrodes during a period preceding resective surgery
should allow maximum, and safer, surgical resection to be performed under general
anesthesia in these patients who are difficult or impossible to cure using current
approaches. During the past year a novel language area has been identified and
located within the cortex of the basal temporal lobe. To our knowledge, we are the
first group to confirm this observation and are working to anatomically and
functionally define this basal temporal language area.
Analysis of data from several recently developed methods for localizing
epileptic foci is being performed to determine comparative sensitivity and reliability
of the various techniques. During surgery for focal epilepsy, depth and special
subdural (surface cortical) electrodes are being used to record from deep structures
inaccessible by routine electrocorticography to identify and confirm areas of
potential epileptogenic activity suggested by the preoperative investigational
methods of PET scanning, magnetoencephalography, MRI and magnetic resonance
spectroscopy (MRS).
MEG offers the possibility for localization of abnormal seizure-related
electromagnetic phenomena in 3 dimensions. No other methodology currently
available can noninvasively acquire this type of information, which is highly
desirable for identifying epileptogenic foci for surgical excision and to study basic
mechanisms in epilepsy. In several patients we were able to predict correctly the
localization of foci causing complex partial seizures using MEG data in patients
whose preoperative EEG (in retrospect) gave misleading and/or false localizing
information. These foci, which were successfully extirpated, would have been
missed if a standard temporal lobectomy had been performed. The new 7-channel
MEG, now in place, promises even greater 3-dimensional localizing capability, and
we are now studying focal motor seizures as well as complex partial and generalized
tonic-clonic seizures with this technique.
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In selected patients, subdural electrodes are modified so that in addition to
their recording capability, they can be utilized to create a current dipole. This dipole
can then be detected by the MEG technique, allowing, for the first time, direct proof
of the ability of the MEG to accurately and precisely predict the location of a current
dipole source within the three-dimensional space of the human cranium.
Electrocorticography (ELOG) under general anesthesia at resective surgery
often generates data of uncertain significance. An ongoing study is carefully
evaluating the effect of different anesthetic agents on interictal spikes. Background
data gathered from the first group of patients studied under this protocol reveal
that isoflurane suppresses the number of abnormal spikes during surgical ELOG
when compared to recordings immediately before and after its use in the same
patient. Enflurane under similar conditions increases the number of abnormal spikes
and produces paroxysms of synchronous high-voltage spikes. Isoflurane can
suppresses epileptogenic foci and that both isoflurane and enflurane can distort the
ECOG, confounding accurate identification of the seizure foci during surgery. When
used judiciously, however, enflurane may be a potent synchronizer and activator of
the epileptogenic focus, permitting easier identification.
An approach that should yield information about the chemical changes that
occur in tissues in which seizures arise is magnetic resonance spectroscopy (in vivo
NMR spectroscopy). Under a new protocol, pH and lactate measurements are taken
from specific regions of interest in seizure patients and are compared with
measurements from similar regions in the opposite hemisphere and with
measurements in normal volunteers. This study is being done in collaboration with
Drs. Giovanni Di Chiro and Jeffrey Alger, NIS.
One of the most severe postoperative deficits suffered by patients undergoing
temporal lobectomy is unpredictable recent memory loss. It is presumed that this
memory deficit results from poor memory reserve in the temporal lobe contralateral
to the resection. Routine preoperative amytal (WADA) testing has been only
minimally successful in predicting post-operative memory deficits in patients who
receive unilateral hippocampectomy for seizures. Therefore, a study of super
selective posterior cerebral artery amytal testing began in collaboration with the
Radiology Department, CC. This test as proposed should selectively block
hippocampal function unilaterally in the awake patient allowing predictions for the
state of postoperative memory function after surgery.
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II. BIOCHEMISTRY SECTION
Richard J. Youle, Ph.D. - Chief
A. Monoclonal Antibody Mediated Killing of Tumor Cells
The Section of Biochemistry, headed by Dr. Richard Youle, is studying the use of
monoclonal antibodies to kill disease-causing cells. Monoclonal antibodies which
selectively bind tumor cells can be generated, but alone are usually not cytotoxic to
the tumor. Toxic proteins such as ricin and diphtheria toxin can be chemically linked
to monoclonal antibodies. The new hybrid molecules bind tumor cells via the
antibody moiety and then kill the cells via the toxin moiety. The toxins used are
enzymes that catalytically inactivate protein synthesis in target cells with only one or
two molecules in the cytoplasm killing a cell. However, the non-target cell toxicity of
the toxins must be blocked with excess lactose to prevent toxin binding and this
currently limits this approach to in vitro applications. The cell-type-specific reagents
have immediate clinical application in vitro in bone marrow transplantation where
T-cell depletion improves allogeneic transplantation. The Section of Biochemistry is
collaborating in clinical trials in bone marrow transplantation at the University of
Minnesota. Twenty-four patients have now been treated with immunotoxin purged
bone marrow as the sole prophylaxis for graft-versus-host disease. Comparing the
outcomes with historic controls treated post-transplant with methotrexate, several
conclusions can be drawn. The patients had a milder course as evidenced by a
significantly shorter hospitalization. Engraftment of donor marrow occurred within
a shor r time until leucocyte generation and no severe graft-versus-host disease
was seen.
The major goal of the laboratory is to develop immunotoxins which will
selectively kill tumor cells in vivo. Currently the limiting steps for antibody-toxin
hybrids are (1) the entry of the toxin molecule into the cell; and (2) in vivo access of
the drug to the tumor cells. To promote access of monoclonal antibody-toxin
conjugates to tumors we have focused on tumors localized in body compartments
such as the brain and the peritoneal cavity. The brain may be an optimal
compartment for antibody modulation of cell function.
The discovery of point mutants of diphtheri toxin (DT) that increase
immunotoxin selectivity in FY 88 and the demonstration of immunotoxin activity in
animal models of leptomeningeal cancer in FY88 have been moved into clinical trials
in FY 89. Extensive toxicity testing has shown that a 100-10000fold therapeutic
window exists between the concentration toxic to tumor cells and the dose tolerable
in rats, guinea pigs and monkeys. Based upon our thorough preclinical testing we
determined that immunotoxins may offer a therapeutic benefit to patients with
leptomeningeal carcinomatsis and medulloblastoma and we have begun clinical
trials. We have begun trials with a monoclonal antibody against the human
transferrin receptor linked to recombinant ricin A chain supplied by Cetus Corp. We
have treated 5 patients so far in a dose escalation that has spanned a 100-fold
increase in drug injected. As yet we have not reached dose limiting toxicity. We
have examined the immunotoxin pharmacology, and immunogenecity. At this early
stage we cannot establish efficacy of the drug to the patients. We will continue our
dose escalation with this reagent as well as prepare second generation
immunotoxins that may offer greater potency for clinical trials.
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To improve entry of immunotoxins into cells we have modified toxins
chemically, molecular biologically and with monoclonal antibodies. We are studying
the structure-function relations of the toxin molecule and the cell biology of toxin
internalization and membrane penetration.
Toxins may be best adapted for tumor specific toxicity by alterations
of amino acid sequence at the gene level. To begin improving immunotoxins at the
gene level we have worked with the prokaryotic toxin, diphtheria toxin. Intact
diphtheria toxin was linked to a monoclonal antibody specific for human T cells and
was found to specifically kill target cells at 10-12M. The rate of specific killing was 10-
fold faster than previously reported immunotoxins. This model system was then
used to study cloned fragments of diphtheria toxin. In collaboration with Dr. Larry
Greenfield, who has cloned DT, we deleted the C-terminal 1 5kD region shown to
include the cell surface binding site. This left the toxin Asubunit plus a 17 kD
fragment of DT B-chain thought to facilitate transmembrane transport. When
linked to monoclonal antibodies, this truncated DT was 100-fold more toxic than DT
A-chain linked to antibody, and the toxicity was blocked by excess antibody. The
cloned DT fragment was 1000 times less toxic to guinea pigs than the native toxin.
Therefore, the fragment of DT B-chain included by cloning increased target cell
toxicity more than non-target cell toxicity indicating that separation of B-chain entry
functions from binding was accomplished to some degree. We compared intact DT
linked to monoclonal antibody with the cloned fragment of DT, which showed the
C-terminal fragment of DT further increased the antibody mediated toxicity 100-
fold.
We have identified point mutations in the receptor binding site of diphtheria
toxin. CR.M103 has a single point mutation at amino acid 508 and is 1000 times less
toxic to cells than native diphtheria toxin. CRM107 has a single point mutation at
amino acid 525 and is 10,000 times less toxic than native diphtheria toxin. Upon
linking either of these two mutants to monoclonal antibodies, full potency,
indistinguishable from that of the 10 12M potency of the native toxin linked to
antibodies, was achieved. These mutations offer an improved therapeutic window
from other immunotoxins. Most recently we have combined these two beneficial
mutants into one new toxin called CTM 1 . We are currently studying whether or not
this mutant will have an even greater therapeutic window than either of the single
point mutants.
In collaboration with the biomedical engineering branch we have studied
immunotoxin pharmacology. We have found that immunotoxins specifically bind
tumors in vivo and have developed a new model that incorporates the number of
receptor binding sites per cell times the affinity constant. We have also initiated
studies of direct infusion of immunotoxins into brain and solid brain tumors.
We have recently identified point mutations in a hydrophobic domain of
diphtheria toxin B-chain that inhibit membrane translocation activity 90%. This
results shows a role of protein 309 in the translocation activity of diphtheria toxin.
A recent paper in Science reports that diphtheria toxin is a DNase. We have
found that, although DNase activity was detectable in commercial samples of
diphtheria toxin, recombinant diphtheria toxin from Cetusand the CRM107 mutant
purified by collaborator at the NIH had very little DNase activity. Upon HPLC
purification of the commercial DT, the DNase activity was separated from the DT
activity indicating that the initial report was wrong.
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We have found ways to block the immune response animals generate against
immunotoxins. Injection of a monoclonal antibody against the helperT-cell CD4
antigen completely prevent the primary immune response to weekly injections of
immunotoxins for 3 months or more.
In an effort to develop immunotoxins to treat AIDS we have collaborated with
Biogen to create CD4-toxin conjugates that specifically kill HIV-infected cells. This
project has just begun and the bioactivity of these new toxins is not yet known.
During investigation of the clinical potential for immunotoxins injected
intrathecal^ we noticed that animals became atoxic. Thorough analysis of this
revealed that immunotoxins specifically kill up to 70% of Purkinje cells in animals
with no detectable toxicity to other neurons or cells. This affords a new animal
model of Purkinje cell loss, yields a valuable clue to the potential dose limiting
toxicity of immunotoxins in clinical trials, and points to a new receptor-mediated
uptake pathway in the CNS.
B. Programmed Cell Death in the Nervous and Immune System
Our laboratory has begun a project to study the mechanism and physiologic
role of programmed cell death. In both the nervous system and the immune system,
massive numbers of cells die during normal development. In the spinal cord, for
example, over half the neuronsdie during fetal development. The immune system
also has large cell loss at precise stages in development such as thymocyte death and
thymus involution at puberty. The physiologic role and the biochemical mechanism
of programmed cell deaths is unknown. Understanding the mechanism and
regulation of such physiologic cell deaths may shed light on neurodegenerative
diseases and immunodeficiency disorders.
In our initial studies we found that thymocytes could be induced to die by the
same signal that stimulates mature T-cel Is to proliferate. RNA and protein synthesis
inhibitors block new gene expression as well as the programmed cell death. In
addition, we found that prolactin can block the glucocorticoid-induced cell death of
thymocytes. This result has significance for clinical immunosuppression and
autoimmune disorders.
In conjunction with our studies studies on immunotoxins we have made now
animal models of Purkinje cell death. Injection of immunotoxins can lead to 70-80%
loss of Purkinje cells without affecting other neurons. This discovery not only
permits generation of a new animal model but also points toward the possible
mechanism of Purkinje cell loss in a variety of neurological disorders. Purkinje cell
loss in inherited metabolic diseases may be a direct consequence of the high
endocytosis rate expressed by these cells.
We are extending our studies into the nervous system by examining the
mechanism of thymocyte death in various ataxic mutant mice and by examining the
mechanism of glutamate-induced neuron death in vitro and in vivo. We have
found that thymocyte-programmed cell death can be followed morphologically with
Nromarski optics and that the thymocyte death resembles the neuronal cell death.
Morphologic analysis of nuclear disintegration has allowed us to test whether cell
death is due to production of a toxic factor or due to the loss of a protective factor.
We found that the cell death mechanism is a dominant, trans-acting death induction
in cell-cell hybrids. We are now testing the transacting death signal in neuronal cells.
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III. Central Nervous System Implantation Unit
Robert J. Plunkett, M.D., Head
1. Tissue implantation in Parkinsonian animal models
The effect of tissue implants into the caudate nucleus of Parkinsonian animals
is being studied from behavioral, biochemical, and histological viewpoints. The
models utilized are the hemiparkinsonian monkey and rat model developed in our
lab. Our previous work showed that grafts of fetal mesencephalon lead to rapid,
near complete recovery from the motor deficits in the monkeys, while
nondopaminergic fetal grafts lead to moderate motor recovery. In contrast, adrenal
allografts, adrenal autografts, nondopaminergic adult tissues, and surgical
cavitation alone all lead to modest behavioral recovery which occurs over many
months. One phenomenon which we have observed to varying degrees after all
implants and after cavitation alone is sprouting of intact host dopaminergic fibers,
presumably from the mesolimbic dopamine system. Efforts to trace the sprouted
fibers using retrograde and anterograde tracers have thus far not been successful,
but further work continues. We have performed cell counts of dopaminergic
neurons in the midbrain of normal and hemiparkinsonian monkeys; the pattern of
cell loss after MPTP suggests that some of the nuclei included in the mesolimbic
system are susceptible to MPTP and are functionally related to the substantia nigra.
To further investigate this sprouting, we implanted fetal monkey amnion
(which produces a neurite-promoting factor in vitro) into hemiparkinsonian
monkeys. We observed significant behavioral improvement (almost as good as seen
after fetal dopaminergic implants). We are studying these animals with a variety of
techniques including 6-fluorodopa PET, receptor autoradiography, and
immunohistochemistry. Term amnion, which is much more readily accessible than
fetal amnion, has also been implanted, but the behavioral response has been
minimal. We have also developed an in vivo microdialysis system to study
biochemical changes in the implanted monkeys. The system is useful to compare the
normal and hemiparkinsonian sides and to detect increased dopaminergic activity
after implantation.
Since many of the cells or tissues which may be used for implantation are
allografts, we have established the appropriate immunohistochemical techniques to
characterize the immune or inflammatory response to grafting. Using these
techniques, we have determined that rejection did occur in one of the animals
receiving a fetal dopaminergic implant. In addition, in adrenal autografts or at the
site of cavitation alone, there are macrophages and T lymphocytes found from
weeks to more than a year after grafting. This persistence of inflammatory cells
(some expressing the IL-2 receptor, a sign of activation) suggests a role for these cells
in the host regenerative sprouting response. We have performed two experiments
in which an allograft or a xenograft was placed in monkeys, and peripheral blood
examined for the IL-2 receptor and the lymphocytic response. This system is not yet
sensitive enough to detect rejection peripherally.
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2. Neurite-promotinq activity from damaged central nervous system and other
tissues
We have previously established that there is neurite-promoting activity at the
site of tissue injury in the cortex of rats or monkeys, and in the caudate of monkeys.
The bioassay used is based on the chick dorsal root ganglion and is qualitative, with
nerve growth factor (NGF) used as the standard. In addition, two other assays have
been developed : the rat superior cervical ganglion and the fetal mesencephalon of
rat. These are closer to the in vivo situation in Parkinsonian models. We have
studied several other rat, monkey, and human tissues using these assays and have
found significant neurite-promoting activity in fetal kidney and amnion and less
activity in term amnion. Current efforts are directed at trying to fractionate the
proteins in buffer from term amnion and to assay the fractions, and then to purify
the protein with neurite-promoting activity. We are also studying the RNA from
amnion with molecular probes for known growth factors.
3. Cellular implantation in a new rat model of parkinsonism
In order to have a small animal model to perform implantation experiments,
we modified the standard hemiparkinsonian rat model. The changes we made allow
destruction of the substantia nigra while sparing the ventral tegmental area (this
model is analogous to the MPTP-hemiparkinsonian monkey model). In this model
there is some tyrosine hydroxylase activity in the medial caudate, but very consistent
and stable turning in response to amphetamine. We chose to examine cellular
implants delivered stereotaxically to see if they would influence the turning
behavior.
Three cell populations have been examined in this model. Activated
inflammatory cells from the peritoneal cavity of the rat implanted into the
denervated caudate lead to significant motor improvement which is not seen in
sham-implanted or inactivated cell implanted hemiparkinsonian rats. There is some
evidence that the improvement is mediated through a dopaminergic mechanism.
Implantation of cultured rat microglial also leads to improvement in this model.
Finally, cell suspension grafts of amnion lead to moderate recovery which is not seen
when killed amnion cells are implanted. We are currently looking at
autoradiography for the blood-brain barrier, dopamine uptake, and dopamine
receptors to try and determine the mechanism(s) of improvement. The results with
inflammatory cells and microglia may reflect a cytokine/glia/neuronal interaction
which leads to increased turnover of dopamine or to sprouting of new dopaminergic
fibers. It is possible that direct implantation of fetal astrocytes would also lead to
recovery and this experiment is planned.
4. Fetal pituitary and pituitarv/hypothalamic transplants
We have developed a successful model of transplantation of fetal rat pituitary
+ hypothalamus into hypophysectomized rats. We use small tissue fragments
delivered stereotaxically to the region of the median eminence. There are peripheral
plasma levels of some anterior pituitary hormones post-transplantation, and
immunohistochemical evidence of surviving anterior pituitary cells. There is some
cellular infiltrate in the implants; we are currently characterizing these cells using
specific monoclonal antibodies. Grafted animals have been tested for evidence of
integration of the graft into the hypothalamic/pituitary/peripheral organ feedback
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loop The grafts do respond to direct administration of releasing factors, e.g. CRF,
but not to indirect stimulation, e.g. insulin stress. Further work is underway to
determine if cell suspension grafts of pituitary, or earlier grafting after
hypophysectomy can lead to better integration into the host. We are also
collaborating in efforts to transfect fibroblasts with releasing factor genes.
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IV. Tumor Biology Unit
Marsha J. Merrill, Ph.D., Head
A. Vascular Permeability Factor (VPF) Produced by Glioma Cells
Cerebral edema is a significant cause of the neurologic deficits and elevated
intracranial pressure associated with malignant brain tumors, and is an important
challenge in the clinical management of patients with this disease. We have
determined that medium conditioned by cultured human glioma cells contains a
substance capable of increasing vascular permeability. This substance is also found
in cyst fluid from brain tumor patients. This VPF is probably at least partially
responsible for the cerebral edema associated with brain tumors. VPF is an
angiogenic endothelial cell mitogen which also increases capillary permeability. It is
a 40 kDa cationic polypeptide with some homology to PDGF. Recent advances in the
field have determined that VPF is the same molecule as pituitary-derived vascular
endothelial growth factor, a potent mitogen selective for endothelial cells. Evidence
to date suggests that the mechanism of action of VPF involves binding to endothelial
cells through a specific receptor on the cell surface and subsequent influx of Ca^ + .
Glucocorticoids are a useful tool in the clinical management of brain tumor-
associated edema. Our studies include investigations into the mechanisms of action
of glucocorticoids both at the level of regulation of expression of VPF in brain tumor
cells, and at the level of the effects of VPF on the target endothelial cells. Through
these studies we hope to gain further insight into the mechanism of action of VPF,
the role of this factor in brain tumor-associted edema and angiogenesis, and the
possibility of improved therapeutic intervention.
B. The Role of Insulin and Insulin-Like Growth Factors in Glioma Cells
Insulin and insulin-like growth factor (IGF) receptors have been detected in the
human central nervous system. The role of IGFs in the nervous system is not well
understood although IGF-1 may be involved in the growth of the peripheral nervous
system, and IGF-2 may play a role in glial cell growth and maturation in the brain.
We initiated studies to determine the nature of insulin and IGF receptors on human
glioma cellsderived from surgical specimens. Our results demonstrate (1) high levels
of IGF-1 receptors on some gliomas; (2) the presence of receptors for IGF-1 and IGF-
2, but not for insulin on cultured cellsderived from human gliomas; and (3) IGF-1
receptors on cultured glioma cells are functional, as determined by the ability of IGF-
1 to stimulate autophosphorylation of the receptor and DNA synthesis. In addition,
high levels of high affinity IGF-binding proteins (IGF BP) are produced by glioma cells
and associated with some glioma surgical specimens. Certain characteristics of this
IGF BP suggest it may be distinct from previously described IGF BPs and thus may
have a function specific for glioma cells. These results suggest a role for IGF-1 , its
receptor and binding protein in the regulating growth in some gliomas.
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V. Molecular Biology Unit
Iqbal U. AM, Ph.D., Head
The Molecular Biology Unit of the Surgical Neurology Branch is studying the
genetic abnormalities of various brain disorders, especially brain cancers. During the
past 12-15 months we have focused on molecular genetic analyses of two CNS
tumors, glioblastomas and pituitary adenomas.
Glioblastomas
Extensive studies of the molecular origins of various human cancers done over
the past several years have implicated dominantly acting proto-oncogenes in various
malignancies. These proto-oncogenes appear to have essential roles in normal
cellular physiology and are probably involved in regulatory functions. Several
mechanisms, such as point mutations, rearrangement, amplification and/or elevated
expression, are responsible for the conversion of these normal genes into
oncogenes. Recently, yet another mechanism, homozygous loss of gene function, is
believed to be one of the genetic events involved in the development of certain
neoplasms.
Glial tumors, which account for the vast majority of primary tumors of the CNS,
are exceptionally diverse in origin location, histology and biologic behavior.
Malignant gliomas, with a whole spectrum of increasing anaplasia starting from
well-differentiated astrocytoma to anaplastic glioblastoma multiforme, offer an
excellent model system to study molecular mechanisms that confer upon these cells
an increased ability to grow, interact with the environment and eventually
metastasize. A variety of genetic lesions may therefore be expected to contribute to
the expression of neoplastic phenotype of gliomas. We are studying primary glial
tumors as well as tissue culture cell lines derived from glioblastomas. We have taken
the following approaches to understand the genetic aberrations responsible for the
expression of malignant phenotype in glial tumors, which probably evolve through a
series of genetic lesions.
1. We have analyzed the genomic integrity and organization of several genes
that are related to growth factors or growth factor receptors for e.g. bFGF,
TGFa, TGF{3, c-sis, c-erbB2, c-erbA2, and int-2 in a panel of 20 primary glial
tumors and eight glioblastoma cell lines. In addition, we have also screened
these tumors and cell lines for possible abnormalities of those proto-oncogenes
that have been found to be frequently altered in various other human
malignancies for ras and myc. Our results did not show any gross
rearrangements and/or amplifications of these genes in primary tumors or
tumor-derived cell lines.
2. One of the characteristic features of glioblastoma is vascular proliferation
suggesting a response to angiogenic factors. A wide variety of factors that
share structure characteristic of signal-transmitting molecules seem to have
both growth promoting the angiogenic activities. These include acidic and
basic FGFs, FGF-5, int-2, hst-1, c-sea, TGFs, and int-1. We analyzed several
glioblastoma cell lines for the expression of various growth factor genes and
proto-oncogenes. These include bFGF, TFGa, TFG(J, c-sis (which encodes for the
P chain of platelet derived growth factor), c-erbB2 (which is related to the EGF
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receptor), N-ras, H-ras and c-myc genes. Elevated expression of bFGF, TFGa,
TGFJ3, and N-ras genes was observed in most glioblastoma cell lines compared
to the normal brain and other human tumor cell lines.
3. The loss of heterozygosity of specific genes, as detected by restriction fragment
length polymorphism analysis (RFLP), suggests the presence of oncogenetic
mutations. However, it may also represent somatic events associated with
tumor progression. Molecular genetic analyses have identified allelic losses on
several chromosomes in human lung cancers, colorectal tumors, and primary
breast carcinoma. We have so far analyzed 30 astrocytomas and glioblastomas
and their matched lymphocytes for possible allelic deletions of genes on
various chromosomes and have identified loss of heterozygosity of genes on
chromosomes 10 and 17 in a significant number of tumors. These deletions
identify the map positions of genes with possible roles in the developmental
and regulatory processes of glial cells.
Pituitary Adenomas
Corticotroph adenomas are ACTH (adrenocorticotropic hormone) producing
tumors that generally originate in the anterior lobe corticotrophs of the pituitary
gland. These tumors cause increased ACTH secretion and consequent hypersecretion
of Cortisol, a condition first described by Cushing half a century ago and therefore
named after him. Corticotroph adenomas in association with Cushing's disease are
often microadenomas which do not grow over several years. However, in some
patients, with a condition called Nelson's syndrome, adrenalectomy gives rise to
hypersecretion of ACTH by pituitary adenomas which are usually large, aggressive,
and rapidly growing neoplasms. Hypersecretion of ACTH can some times also be
caused by ectopic nonpituitary tumors.
We have initially taken two approaches to study different kinds of ACTH-
secretion adenomas: 1) analysis of the clonal composition, and 2) mutations of the
ras gene family.
Analysis of the Clonal Composition
Somatic mutation theory of carcinogenesis implies that most cancers develop
as a consequence of an extremely rare somatic mutation in a single cell giving rise to
tumors of monoclonal origin. Polyclonal composition, on the other hand, may be
expected in hereditary tumors or endocrine tumors where the stimulus can act on
large population of cells in the target tissue. We have studied the clonal
composition of ACTH-producing adenomas by using X-chromosome inactivation
analysis. The method exploits RFLPs of X-chromosome linked genes in female cells
which undergo random inactivation of one X-chromosome during embryogenesis
and stably transfer the pattern to the progeny. Since active genes undergo changes
in methylation patterns, methylation-sensitive enzymes are used to distinguish
between active and inactive genes. Out of 27 tumors analyzed, nine showed
heterozygosity at hypoxanthine phosphoribosyl transferase (HPRT) or
phosphoglycerate kinase (PGK) genes. Six tumors showed a single active allele of the
X chromosome linked genes and were monoclonal in nature. The other three
tumors, including one microadenoma from a patient with Nelson's syndrome,
revealed a polyclonal pattern. Our results demonstrate that pituitary corticotroph
22 SNB/DIR
adenomas can be monoclonal or polyclonal, thus emphasizing the complexity of
genetic events with fundamentally different mechanisms involved in oncogenesis.
Mutation of the ras genes
Mutations leading to the activation of the transforming potential of cellular
ras genes have been implicated in the development of many human tumors of
diverse origin. Mostly ras gene mutation is associated with the progression of the
disease, but it can also be an early event, possibly even an initiating event such as in
colorectal adenomas. Analysis of a large number of activated ras proto-oncogenes
derived from a variety of tumors showed that there are three mutational hot spots
(codons 12, 13, and 61) in all three ras genes i.e., H-ras, K-ras, and N-ras.
We have amplified the regions encompassing codons 12 and 13 in exon 1 and
codon 61 in exon 2 of all three ras genes by the polymerase chain reaction (PCR) from
several pituitary corticoph adenomas. We are now sequencing these amplified
regions for the presence of possible mutations.
23SNB/DIR
VI. Brain Imaging Unit
Linda J. Porrino, Ph.D. - Head
The Unit on Brain Imaging employs autoradiographic methods to study the
effects of dopaminergic denervation in primates and rodents as as model of
parkinsonism. These methods are also used to study the effects of the acute and
chronic administration of a variety of pharmacological agents. A variety of methods
are utilized including measurement of rates of local cerebral glucose utilization,
determination of the topographic distribution and the density of receptors,
measurement of local rates of protein synthesis, and autoradiographic
determination of alterations in the blood brain barrier.
Studies of animal models of parkinsonism
In our previous work we have characterized the alterations in the anatomic
distribution of both Di and D2 receptor binding sites in the primate model of
hemiparkinsonism provided by the intracarotidfinfusion of the neurotoxin, 1-
methyl-4-phenyl-1 ,2,3,6-tetrahydropyridine (MPTP) which produces destruction of
the dopaminergic cells of the substantia nigra parscompacta. We have extended
these studies to include other receptor populations. Quantitative autoradiography
after in vitro radioligand binding with [3H]naloxone (opiate antagonist) was used to
determine the distribution of opiate receptor sites in MPTP-induced
hemiparkinsonian monkeys. An increased density of opiate sites was observed in the
striatum on the side of dopaminergic denervation. The anatomic distribution of
these sites resembled the distribution of dopaminergic D1 binding sites in the
striatum, suggesting potential interactions between dopamine and opiate peptides
in the striatum.
In other studies the 2-p4c]deoxyglucose (2-DG) method is being applied to
animals in which unilateral lesions of the substantia nigra pars compacta have been
made in order to map the neural circuits involved in the symptoms of parkinsonism
and its treatment with dopaminergic agonist drugs. We have completed our studies
of glucose utilization in hemiparkinsonian monkeys following treatment with
apomorphine and L-DOPA, and have now begun studies of the effects of lesions of
the substantia nigra pars compacts produced by the local infusion of 6-
hydroxydopamine. Our lesions have been relatively small and localized to either the
medial or lateral portions of the substantia nigra. We have shown that the loci of
the damage to dopaminergic cells along the medial-lateral axis of the substantia
nigra is important in determining the resulting pattern of behavioral effects.
Furthermore, the level of functional activity in the substantia nigra pars reticulata
determines the direction of turning behavior elicited by the administration of
dopaminergic drugs such as apomorphine.
Pharmacologic Studies
In these experiments we have used metabolic mapping methods to examine
different aspects of the behavioral and pharmacologic actions of abused drugs, i.e.,
cocaine, methamphetamine, and morphine. In one set of experiments the
quantitative 2-DG method was used to map the distribution of alterations in local
rates of cerebral glucose metabolism associated with the self-administration of
24SNB/DIR
cocaine in rats. Triads of male littermate rats were studied. One rat of each triad
was allowed to self-administer cocaine, another received identical yoked infusions of
cocaine, while the third received yoked saline infusions. We demonstrated that the
metabolic effects of the contingent presentation of cocaine are different from the
effects of noncontingent presentation. These differences may be directly related to
the reinforcement associated with cocaine self-administration. Contingent self-
administration was associated with increased rates of local cerebral glucose
utilization in the mesocorticolimbic system including the prefrontal cortex, nucleus
accumbens, olfactory tubercle, and amygdala, when compared to yoked cocaine or
saline controls. These neural circuits appear to be central to the mediation of
positive reinforcement.
Experiments to compare the sites of action of the reward-enhancing effects of
morphine as measured in self-stimulation paradigms with the sites of action of the
antinociceptive properties of morphine have also been completed this year. Our
data have identified the dorsal raphe and lateral habenula as important substrates
of the analgesic effects of morphine, whereas the euphorigenic properties of
morphine appear to be mediated by portions of the mesocorticolimbic system. The
brain regions involved in the rewarding effects of morphine appear to be quite
different from those involved in analgesia.
25SNB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02454-10 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Studies of Human Pituitary Tumors
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
Edward Oldfield, M.D. Chief, SNB, Principal Investigator, NINDS
COOPERATING UNITS Of an,)
Developmental Endocrinology Branch, NINDS
Diagnostic Radiology, CC
LAB/BRANCH
Surgical Neurology Branch
SECTION
Clinical Neurosurgery Section, CNP
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL: Q g
OTHER: QQ
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects [T] (b) Human tissues ] (c) Neither
] (a1) Minors
] (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
We continue to investigate venous samplings of the pituitary venous drainage to aid in the diagnosis and
treatment of patients with Cushinq's syndrome Over 300 patients have now received bilateral
simultaneous inferior petrosal sinus (IPS) sampling The results indicate that 1) the procedure can be
performed successfully in all patients with Cushing's syndrome (successful sampling has been performed
in over 99% of the patients in whom it has been attempted); 2) the procedure distinguishes patients
with ectopic ACTH secretion from those with pituitary adenomas with 100% accuracy; 3) IPS sampling
successfully determines which side of the pituitary gland microadenomas reside in patients with
Cushing's disease with 75% accuracy; and 4) unilateral inferior petrosal sinus sampling, which is
commonly used clinically, is frequently misleading.
Repeat transsphenoidal surgery is successful in elimmating the hypercortisolism of Cushing's
disease in about 70% of patients This therapy for patients with Cushing's disease after previous
pituitary surgery had not previously been examined.
The CRF stimulation test and the dexamethasone suppression test are equally accurate in
determining and distinguishing patients with Cushing's disease from those with ectopic ACTH secretion
in Cushing's syndrome Both have approximately 10-15% diagnostic inaccuracy, when used alone, in our
experience
MRI scanning with and without gadolinium-EDTA was used to evaluate patients with Cushing's
disease preoperatively. This technique permitted identification of the adenoma in about 55% of those
patients with surgically proven microadenomas Proper timing of the MRI after the administration of
gadolinium EDTA was critical in the optimal use of the technigue. Pituitary adenomas were detected in
15% of 100 normal subjects with MRI scanning with contrast.
PHHMOHr. 1 sj 27 SNB DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02813-01 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Pharmacokinetics of Direct Brain Infusion
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
R. Hunt Bobo, M.D. Senior Staff Fellow, NINDS
Edward Oldfield, M.D. Chief, Surgical Neurology Branch, NINDS
Aytac Akbasak, M.D. Visiting Associate, NINDS
COOPERATING UNITS (it any)
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Clinical Neurosurgery Section
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS: - ,
PROFESSIONAL: Q 3
OTHER: QQ
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects ] (b) Human tissues [V] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The temporal and spatial distribution (pharmacokinetics) of potential anti-glioma agents administered
into the feline brain by direct infusion are being studied. Reasons for direct infusion are avoidance of
the blood brain barrier, decreased systemic exposure, and avoidance of systemic metabolic and
immunologic interaction with the agent. The aim of the project is to develop the methods to administer
antiglioma agents by direct infusion, and achieve an homogeneous distribution throughout the tumor
volume, in a clinical setting. Agents for study will be analogous to immunotoxins, 1-125 labeled
iododeoxyuridine, and commercially available antineoplastic agents.
PHS&MOIRe. 114 ?8 SNB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02812-01 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PRO J E CT (80 characters or loss. Titlo musl fit on one line between the borders.)
Pentobarbital Effects on Damage of the Primate Brain by Fractionated Whole Brain Radiation
PRINCIPAL IN VE STIGATOR (List other professional personnel below the Principal Investigator.) (htame. title, laboratory, and institute affiliation)
R. Hunt Bobo, M.D. Senior Staff Fellow, SNB
Tom Goffman, M.D
KathrynOrr, R.N.
Edward Oldfield, MD
Radiation Oncology Branch, NCI
Radiation Oncology Branch, NCI
Surgical Neurology Branch, NINDS
COOPERATING UNITS of an,)
Radiation Oncology Branch, NCI
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Clinical Neurosurgery Section
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS.
0.4
PROFESSIONAL:
0.4
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
1 | (a) Human subjects
] (a1) Minors
J (a2) Interviews
J (b) Human tissues | « | (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
After baseline MRI scans of the brain and neuroendocrine testing, primates undergo whole brain
radiotherapy in 10 daily fractions, 340 rads each. Six of these primates will be anesthetized with
pentobarbital during the irradiation, six will not receive any pentobarbital. Neuroendocrine testing and
MRI scans will be done at 3, 6, 12, 18 and 24 months after radiation. Quantitative histology will be done
on the capillary bed, glial, and neuronal populations after sacrifice. The purpose of this study is to
determine if pentobarbital has protective effects on multiple fractionated whole brain irradiation and to
further elucidate the microanatomic substrate of cerebral radiation damage.
PHSMMOfRev 1 M
29 SNB DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 0281 1-01 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ ECT (80 characters or less. Title must fit on one line between the borders.)
Phase II Clinical Trial of Suramin and Hydrocortisone in the Therapy of Current Malignant Gliomas
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator .) (Name, title, laboratory, and institute affiliation)
Edward H. Oldfield, M.D. Chief, SNB, Principal Investigator, NINDS
R. HuntBobo, M.D.
Hetty DeVroom, R.N.
RV LaRocca, M.D., M. Cooper,
David Kohler, Pharm.D.
Barry Goldspiel, Pharm.D.
M.D..CE Myers, M.D.
Senior Staff Fellow, Associate Investigator, NINDS
Clinical Nurse, NINDS
National Cancer Institute
Pharmacy Department, CC
Pharmacy Department, CC
COOPERATING UNITS (if any)
Radiation Oncology
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Clinical Neurosurgery Section
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
0.4
PROFESSIONAL:
0.4
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
I x | (a) Human subjects
] (a1) Minors
J (a2) Interviews
J (b) Human tissues J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This is a phase II study of the combination of suramin (a polysulfonated naphthylurea) and
hydrocortisone in the treatment of malignant gliomas, recurrent after radiation therapy. Both efficacy
and toxicity will be monitored. Tumor volumes are measured on MRI scans to judge radiographic
respone or progression. Fourteen patients will be treated the first year, followed by 10 more the
following year, if at least one partial response is obtain during the first year.
PHS 6040 (Rtv. 1/84
30 SNB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02697-06 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Protection of the Brain Against Injury by Ionizing Radiation with Pentobarbital
PRINCIPAL INVESTIGATOR (tut other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Jeffrey J. Olson, M.D. Principal Investigator, SNB, NINDS
Edward H. Oldfield, M.D. Chief, SNB , NINDS
Craig Shelley, M.D. Medical Staff Fellow, SNB
Calvin Hawkins Bio Lab Technician, SNB
COOPERATING UNITS Of any)
National Cancer Institute, Radiation Oncology Branch
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Clinical Neurosurgery Section
INSTITUTE AND LOCATION
NINDS, National Institutes of Health Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
LJ (a) Human subjects [7] (b) Human tissues | | (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The efficacy of radiation therapy in the treatment of brain tumors is limited by the toxicity of ionizing
radiation to the surrounding normal tissue
In the rat model of cerebral radiation injury, pentobarbital has been shown to enhance overall
survival in a dose-dependent manner. Evaluation of alternative barbiturates reveals that thiopental is of
equivalent radioprotective value to pentobarbital.
The rodent model of radiation injury does not parallel that of human injury. A primate model was
designed to assess the role of pentobarbital in circumstances more applicable to the human situation
This ongoing study has thus far revealed the ability of pentobarbital to limit the toxicity of the radiation
utilized. Neuroendocrinoloqic evaluation has revealed early dysfunction of thyroid-stimulating
hormone, luteinizing hormone, and prolactin responses to stimulatory testing in the animals irradiated
while anesthetized with ketamine. Significantly less abnormalities have occurred in the pentobarbital
group.
PHS 6M0 (Rev 1 84 31 SNB'DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02728-04 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ ECT (80 characters or less, rule must fit on one line between the borders.)
Studies on the Heterogeneity of Drug Delivery During Intracarotid Chemotherapy
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Stephen Saris, M.D. Senior Staff Fellow, Principal Investigator, SNB
Robert Lutz, Ph.D.
Ron Blasberg, M.D.
Donald Wright, M.D.
Edward H.OIdfield, M.D.
Staff Fellow
Senior Investigator, Department of Nuclear Medicine
Medical Officer, SNB, NINDS
Chief, SNB, NINDS
COOPERATING UNITS (if any)
Department of Nuclear Medicine
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Clinical Neurosurgery Section
INSTITUTE AND LOCATION
NINDS, National Institutes of Health Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.0
PROFESSIONAL:
1.0
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
| x | (a) Human subjects
! ] (a1) Minors
] (a2) Interviews
] (b) Human tissues Q (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Project was completed in July, 1990.
PHS 6040 (H»v I'M
32 SNBTJIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02778-03 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders t
Adoptive Immunotherapy of Brain Tumors
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute atiiliation)
Stephen Saris, M.D.
Aytac Akbasak, M.D.
Ikejiri, Barbara
Edward Oldfield, MD.
Principal Investigator, SNB, NINDS
Special Volunteer, SNB, NINDS
Biologist, SNB, NINDS
Chief, SNB, NINDS
COOPERATING UNITS (it any)
Surgical Oncology Branch, NCI
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Clinical Neurosurgery Section
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.0
PROFESSIONAL:
10
OTHER:
00
CHECK APPROPRIATE BOX(ES)
I x | (a) Human subjects
] (a1) Minors
J (a2) Interviews
j (b) Human tissues J (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
There are 10-15,000 malignant brain tumors diagnosed each year The most common of these are
glioblastomas. Numerous treatments involving surgery, chemotherapy, and radiation are of palliative
benefit only. We study the adoptive immunotherapy of malignant brain tumors with interleukin-2 (IL-2),
lymphokine-activated killer (LAK) cells, and tumor infiltrating lymphocytes (TILs) in animal models and in
patients. In the 9L gliosarcoma rat model, we studied the efficacy of parenteral IL-2 and its effect on the
blood-brain barrier of normal and neoplastic tissue, and in the C3H mouse we studied the cytolytic
activity of LAK against metastases in the brain. In patients with extracranial cancer, we examined the
kinetics of IL-2 in the cerebrospinal fluid as compared to serum, and in patients with gliomas we
investigated the cerebral toxicity of parenteral IL-2. We are currently completing our studies with IL-2
and LAK cells, and are expanding our efforts with TILs In animals models of primary and metastatic
brain tumors, we have raised TILs against tumors in the subcutaneous space; these cells will then be used
to treat animals with intracerebral tumors and to study trafficking across the blood-brain barrier with
111lndium labeled cells. In patients, we are raising TILs from brain tumors grown in the subcutaneous
space. These have been expanded in vitro with IL-2, and injected parenterally into mice to study
lymphocyte trafficking and efficacy against intracerebral tumors In patients we have raised TILs from
fresh specimens sent from the operating room This creates the possibility of proceeding with Phase I
clinical trials based on the final outcome of our laboratory efforts. Lastly, we are investigating the
regulation of major histocompatability antigens of brain tumors in vitro by interleukins, interferons, and
tumor factor.
PHSMMOISe. 1 8J
33SNBT5IR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02673-06 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Monoclonal Antibodies Linked to Ricin for Use in Human Bone Marrow Transplantation
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Richard J. Youle, Ph.D. Principal Investigator, SNB, NINDS
Pat Johnson
Virginia Johnson, Ph.D.
Bio Laboratory Technician, SNB, NINDS
Senior Staff Fellow, SNB, NINDS
COOPERATING UNITS Of any)
American Red Cross; Washington Children's Hospital; National Cancer Institute, Immunology Branch,
DCBD.
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Biochemistry Section
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.5
PROFESSIONAL:
1.5
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
[ x | (a) Human subjects
] (a1) Minors
J (a2) Interviews
I x I (b) Human tissues j (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
This project was completed July, 1990.
f>HS6M0(R*». VM
34SNBT)IR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02823-01 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Antibody-Toxin Conjugates for the Treatment of Human Brain Tumors
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Richard J. Youle, Ph.D. Chief, Biochemistry Section, SNB, NINDS
Deborah Wilson, B.S Biologist, SNB, NINDS
Karin M. Muraszko, M.D. University of Michigan, Section of Neurosurgery
Edward H. Oldfield, M.D. Chief, Surgical Neurology Branch, NINDS
David Katz, M.D. NINDS
Cynthia Sung, Ph.D. BEIB
Robert Dedrick, Ph.D. BEIB
COOPERATING UNITS Of *ny)
Diagnostic Radiology; Nuclear medicine Department; National Cancer Institute
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Biochemistry Section
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL: .. r
OTHER:
00
CHECK APPROPRIATE BOX(ES)
[7] (a) Human subjects ] (b) Human tissues ] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The purpose of this study is to investigate the use of immunotoxins in the treatment of primary brain
tumors and meningeal carcinomatosis Immunotoxins are molecular hybrids composed of monoclonal
antibodies covalently attached to peptide toxins. The monoclonal antibody targets the conjugate to the
surface of tumor cells bearing the appropriate antigen The toxin moiety then penetrates the cell
membrane and inactivates protein synthesis. The compound 454A12MAB-rRA is a conjugate of an anti-
human transferrin receptor antibody (454A12) and the recombinant A chain portion of the toxin ricin
We have shown that 454A1 2MAB-rRA kills various cell lines of tumor that spread through the CSF
pathways. In an animal model of intrathecal leukemia, immunotoxin therapy killed 90-99 9% of the
tumor cells in the CSF and a single injection of immunotoxins prolonged survival of the guinea pigs At
present there is a Phase l/ll trial with 454A12MAB-rRAforthe treatment of refractory carcinomatous
meningitis. It is ongoing in the Surgical Neurology Branch. The purpose of the study is to determine the
toxicity of 454A12MAB-rRA administered intrathecal I y in a limited escalation dosage schedule The CSF
pharmacokinetics of intraventricular^ administered 454A12MAB-rRA are determined and will be
compared to the pharmacokinetics that have been previously obtained from primate studies using the
same immunotoxin. In addition, we will determine the formation of antibodies to 454A1 2MAB-rRA
after intrathecal administration. Finally we will examine the potential therapeutic efficacy of this
intrathecal immunotoxin in ieptomeninqeai meningitis
To date, 5 patients have been treated with immunotoxin injections into the intrathecal space and
pharmacokinetic studies have been performed on each of these patients. No toxicity has been found at
this time related to the immunotoxin. The maximum dose administered to date is 120 mg of immuno-
toxin into the intrathecal space There have been 3 deaths within the study group, none of which was
related to the administration of immunotoxin. Two of these patients have undergone autopsy and no
toxicity has been appreciated on histologic analysis of their tissues after immunotoxin injection
Pharmacokinetic data of the immunotoxin suggests that it circulates well within the CSF, that the
immunotoxin is stable within the CSF and that further dose escalation will be performed We plan to
proceed with further dose escalation in these patients and to continue to monitor them both for the
pharmacokinetic data within the CSF as well as for efficacy as judged both clinically and by CSF cytology
PHS6M0{Rev 184 35 SNB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02674-06 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Monoclonal Antibody-Toxin Conjugates for Tumor Therapy In Vivo
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Richard J. Youle, Ph.D. Principal Investigator, SNB, NINDS
Virginia Johnson, Ph.D. Senior Staff Fellow, SNB, NINDS
Debbie Wilson Bio Lab Technician, SNB, NINDS
Dianne Newton Special Volunteer, SNB, NINDS
Susanna Rybak, Ph.D. Special Expert, SNB, NINDS
Peter Nicholls, Ph.D. Visiting Fellow, SNB, NINDS
COOPERATING UNITS (if any)
Biogen
Cetus Corporation
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Biochemistry Section
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
I x I (a) Human subjects
] (a1) Minors
J (a2) Interviews
I x | (b) Human tissues
(c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Monoclonal antibodies selectively bind tumor cell differentiating antigens in vitro and in vivo. Natural
effector mechanisms often do not mediate killing of monoclonal antibody bound cells so we have
devised methods of linking extremely toxic proteins to the antibodies to selectively kill tumor cells.
Two methods of coupling toxic proteins, like ricin to antibodies, have been used to kill antigen positive
cells in vitro. Ricin has two subunits, the A subunit blocks protein synthesis when in the cytosol and the B
subunit binds galactose groups on all cell surfaces but also facilitates the transport of ricin A chain to the
cytosol. 1) Linkage of the ricin A chain to antibodies yields reagents with low non-target toxicity but
target cell toxicity too slow for in vivo applications; 2) Linkage of intact ricin to antibodies results in very
potent target cell toxicity but the non-target cell killing must be prevented by a ligand which blocks ricin
B chain binding to cells. This has limited its application to in vitro situations where 100 mM lactose can
block ricin binding.
We have succeeded in developing several new approaches to apply immunotoxins in vivo. 1) Cloning of
toxins then altering their structure at the gene level to decrease non-target cell toxicity; 2) intrathecal
administration of immunotoxins for therapy of brain tumors that kill 2-5 logs of tumor cells in animal
models; 3) preparation of genetically engineered immunotoxins for clinical trials of human brain tumor
patients; 4) prevention of immune response against immunotoxin with anti-CD4 antibodies; 5)
construction of HIV infected cell immunotoxins; and 6) specific deletion of Purkinje cells in rats, guinea
pigs and rhesus monkeys.
CHS 6040 (Rev 1/84
36 SNB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02781-03 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders t
Tissue Implantation in Parkinsonian Models
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Wame. title, laboratory, and institute affiliation)
Robert Plunkett, M.D. Principal Investigator, SNB
Kris Bankiewicz, M.D. - Visiting Fellow, SNB JG Sheng, M.D. - Visiting Fellow, SNB
Mark Luciano, M.D. - Medical Staff Fellow, SNB Bernhard Zunkeler, M.D. - Visiting Fellow, SNB
Ian McCutcheon, M.D. - Medical Staff Fellow, SNB Jin Wang, M.D - Visiting Fellow, SNB
Scott Ewing, M.D. - Visiting Medical Student, SNB Kap Holloway, M.D. - Special Volunteer, SNB
Jim Kim, M.D. - Visiting Fellow, SNB
COOPERATING UNITS (,f an,)
Meg Palmatier, Ph.D. - Staff Fellow, Clinical Neuroscience Branch; R. Gress, M.D., Scientist, NCI
RJ Weber, Ph.D., Scientist, NIDDK; Peter Basser, Ph.D., Biomedical Engineering, NIH
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Clinical Neurosurgery Section, Central Nervous System Implant Unit
INSTITUTE AND LOCATION
NINDS, National Institutes of Health Bethesda, Maryland
TOTAL MAN-YEARS: , c
0.3
PROFESSIONAL: fi 5
OTHER: QQ
CHECK APPROPRIATE BOX(ES)
[xj (a) Human subjects [7] (b) Human tissues ] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard type. Do not exceed the space provided.)
The behavioral, biochemical, and histological effects of tissue implants in rodent and primate models of
parkinsonism is being studied The grafts which have been examined include fetal and adult
dopaminergic and nondopaminergic tissues. There is some behavioral improvement with any operative
trauma to the caudate, whether a graft is placed or not Generally, fetal tissue grafts (dopaminergic or
nondopaminergic) lead to a much greater degree of recovery than adult tissue grafts or trauma alone.
The histologic observation of dopaminergic fiber ingrowth (sprouting) in all these animals suggests that
the improvement is mediated through a neurotrophic interaction. We are trying to determine the cell-
to-cell interaction which leads to new growth of fibers from an adult neuron, using in vivo and in vitro
methods. Two major areas of emphasis are: what cascade of events in the host after trauma leads to
sprouting and why does fetal tissue enhance the recovery (even nondopaminergic tissue)
The current experiments include implantation of term amnion into hemiparkinsoman monkeys (solid
tissue into preformed cavities), cell suspension implants of term amnion into rats, and biochemical and
molecular analysis of the neurotrophic factor(s) produced by amnion cells We are also incorporating
laminin into a slow-release polymer and implanting this in hemiparkinsonian rats To further investigate
the host response to tissue trauma, we are implanting inflammatory cells in the denervated caudate.
Macrophages, T-cells, and microglia, alone or combined, and their secretory products such as IL-1 are
being studied. We are now using autoradiographic techniques to study the blood brain barrier, receptor
binding and dopamine uptake in the inflammatory cell implant model
PHS 6040 <Re«. I M 37 SNB DIP
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02729-04 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (30 characters or less. Title must fit on one line between the borders.)
Adrenal Medullary Autografts in Parkinsonian Patients
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Robert J. Plunkett, M.D. Principal Investigator, SNB
Kris Bankiewicz, M.D. Visiting Associate, SNB
Jeff Norton, M.D. Senior Staff Fellow, NCI
Hetty DeVroom, R.N. Clinical Nurse, SNB, NINDS
Robert Miletich, M.D., Ph.D. Senior Staff Fellow, NIS, NINDS
Edward Oldfield, M.D. Chief, SNB
COOPERATING UNITS (if any)
Surgery Branch, National Cancer Institute
Neuroimaging Section, NINDS
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Clinical Neurosurgery Section, Central Nervous System Implantation Unit
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL: . c
OTHER: 00
CHECK APPROPRIATE BOX(ES)
|T] (a)Human subjects □ (b) Human tissues □ (c) Neither
J (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard type. Do not exceed the space provided.)
This study is designed to assess the safety and efficacy of adrenal medullary autografts and fetal
mesencephalic grafts in patients with severe Parkinson's disease The patients selected will have Stage IV
disease, but still show some response to oral dopamine replacement therapy. The patient will undergo
extensive testing pre-operatively including gait, posture, reaction time, speech, and neuropsychiatric
assessment. Cavities will be created in the right caudate nucleus via a transcallosal approach, and a
ventricular reservoir placed. Two weeks later one adrenal gland will be removed, and placed into the
preformed cavity (or fetal mesencephalic tissue will be placed in the cavity and no adrenalectomy
performed). The preoperative testing will be repeated at regular intervals after implantation. CSF
biochemistry and 6-fluorodopa PET studies will also be part of the testing carried out.
One patient has received an adrenal medullary autograft. He has shown mild improvement with
reduction of the severity of his on/off fluctuations and improved mobility during his "off" time. In
addition, he has had a lessening of the dyskinesias which limit his tolerance for oral therapy. The CSF
studies have revealed an elevation of enkephalin in the first few months compared to pre-operatively,
but no long-lasting elevation of enkephalin or elevation of catecholamines. No patients have received
fetal mesencephalic grafts due to a ban on such work by the Department of Health and Human Services.
PHS6M0 (Rev 1,84 38 SN8/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02708-05 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less Title must fit on one line between the borders.)
Vascular Permeability Factor Produced by Human Glioma Cells
PRINCIPAL INVESTIGATOR tList other professional personnel below the Principal Investigator I (Name, title, laboratory, and institute affiliation)
Marsha Merrill, Ph.D. Principal Investigator, SNB, NINDS
Nancy Edwards, B. A Biologist, SNB
Calvin Hawkins Technician, SNB
Lou Rosa, M.D Senior Staff Fellow, SNB
Edward Oldfield, M.D. Chief, SNB
COOPERATING UNITS (.» any)
Jack Knightly, M.D., National Naval Medical Center, Bethesda, Maryland
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Clinical Neurosurgery Section, Tumor Biology Unit
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland
TOTAL MAN-YEARS: ?
PROFESSIONAL: . c
OTHER. 1 q
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects [7] (b) Human tissues | | (c) Neither
] (a1) Minors
] (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The cerebral edema associated with malignant brain tumors causes neurologic deficits and increased
intracranial pressure, and contributes to the morbidity and mortality associated with the neoplasm. We
have determined that the conditioned medium of glioblastoma-derived cell cultures contains a
substance capable of increasing the vascular permeability in a bioassay which measures the induction of
capillary permeability in normal skin. This substance has been identified as vascular permeability factor
(VPF) and may be partially responsible for the cerebral edema associated with brain tumors. VPF is a 40
kDa cationic polypeptide with some homology to PDGF. In addition to increasing capillary permeability,
VPF is also an angiogenic, endothelial cell mitogen. Evidence to date suggests that the mechanism of
action of VPF involves binding to endothelial cells through a specific receptor on the cell surface and
subsequent influx of Ca2 + . Glucocorticoids are a useful tool in the clinical management of brain tumor-
associated edema. Ou studies indicate that glucocorticoids act both at the level of the regulation of
expression of VPF in brain tumor cells and on the target endothelial cells Through these studies we hope
to gain further insight into the mechanism of action of VPF, the role of this factor in brain tumor-
associated edema and angiogenesis, and the possibility of improved therapeutic intervention.
pms two («*» i 8« 39SNBDIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02707-05 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
The Role of Insulin and Insulin-like Growth Factors in Glioma Cells
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Marsha Merrill, Ph.D. Principal Investigator, SNB
Nancy Edwards Biologist, SNB
Jeffrey Olson, M.D. Senior Staff Fellow, SNB
COOPERATING UNITS (if any)
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Clinical Neurosurgery Section, Tumor Biology Unit
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL: .. n
OTHER:
0.5
CHECK APPROPRIATE BOX(ES)
□ (a) Human subjects [7] (b) Human tissues ] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Insulin and insulin-like growth factors (IGF-1 and -2 or somatomedins) are anabolic effectors in many
tissues and cultured cells including astrocytes and neurons. Receptors for insulin and IGFsare found
throughout the human brain. Receptors for several growth factors are increased in tumor vs. normal
tissue and this discrepancy may have therapeutic value in targeting toxic agents to tumor cells. To
evaluate the potential role of IGFs in human CNS tumors, we examined the level of insulin and IGF
receptors in tumors (astrocytomas and glioblastomas) and in normal brain. Although all surgical
specimens contain receptors for all three growth factors, the highest values were observed with IGF-1
binding to glioma specimens. The IGF-1 receptor in tumor is the same size (1 18 kDa a-subunit) as the
receptor in normal brain, confirming the neural origin of the tumor cells expressing the IGF-1 receptor.
Cultured cells derived from glioma specimens also express IGF-1 receptors, and many of these lines
demonstrate a functional receptor as indicated by stimulation of DNA synthesis and receptor
autophosphorylation in response to IGF-1. This demonstration of functional IGF-1 receptors in glioma
cells suggests a role for this receptor in the regulation of glioma cell growth. In addition, high levels of
high affinity IGF binding proteins (IGF BP) are produced by glioma cells. Certain characteristics of this IGF
BP suggest it may be distinct from previously described IGF BPs, and thus may have a function specific for
glioma cells.
phs M40 (Rev i 84 40 SNB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02814-01 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less- Title must fit on one line between the borders.)
Genetic Abnormalities in Primary Glial Tumors
(jenetic ADnormanties in primary unai I umors
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Iqbal U. Ali, Ph. D. Principal Investigator, SNB, NINDS
Abha Saxena, Ph.D. Visiting Fellow, SNB, NINDS
Craig Shelley, M.D. Medical Staff Fellow, SNB, NINDS
William C. Reinhold Molecular Biologist, SNB, NINDS
Edward H. Oldfield, M.D. Chief, SNB, NINDS
COOPERATING UNITS Of any)
St. Judes Hospital, Memphis, Tennessee
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Molecular Biology Unit
INSTITUTE AND LOCATION
NINDS, National Institutes of Health Bethesda, Maryland
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER: nn
CHECK APPROPRIATE BOX(ES)
□ (aJHuman subjects [7] (D) Human tissues H (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Gliomas are the most common primary brain tumors ranging from benign low-grade astrocytomas to
highly malignant glioblastoma multiforme. The classification of primary glial tumors reflecting their
biological aggressiveness is based upon the histopathologic characteristics We have taken three
approaches to understand and identify, at a molecular level, the underlying mechanisms that translate
into the phenotypic differences of various grades of gliomas.
1 . The genomic organization of H-ras, N-ras, c-myc, bFGF, TGFa, TGFfJ, c-sis, c-erbB2, c-erbA2, and int-2
was analyzed and found to be normal in 20 primary tumors and eight glioblastoma cell lines.
2. The transcription of bGFG, TGFa, TGF0, and N-ras was generally elevated in most of the glioblastoma
cell lines tested as compared to normal brain, an SVuo transformed human astroglial cell line, human
breast cancer cell lines, a bladder carcinoma cell line, T24, and a hematopoetic cell line K562. An
increased level of N-ras and c-myc protooncogenes was observed in all human tumor cell lines
examined, whereas c-erbB2 expression was relatively increased in breast cancer cell lines.
3. Recessive mutations that predispose to cancer are unmasked in several human cancers by the loss of
normal alleles. Restriction fragment length polymorphism analysis (RFLP) was used to compare the
constitutional and tumor genotypes in a panel of 30 astrocytomas and glioblastomas. Loss of
heterozygosity of several markers on chromosomes 17 and 10 was detected in a significant number
of astrocytomas and glioblastoma multiforme respectively. Deletion mapping studies are being
carried out to more precisely define the position of the gene(s) whose deletion is probably important
in the genesis of these tumors
PHS 6040 (Kev 1/84 41 SNB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02815-01 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders-)
Molecular Genetics of Pituitary Conticotroph Adenomas
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
Iqbal U. AM, Ph.D. Principal Investigator, SNB, NINDS
HeinrichSchulte, M.D. Visiting Associate, SNB, NINDS
Salvatore Aguanno, Ph.D. Visiting Fellow, SNB, NINDS
David Katz, M.D. Special Expert, Neuropathologist, NINDS
Barbara Ikejiri Biologist, SNB, NINDS
Edward Oldfield, M.D. Chief, SNB, NINDS
COOPERATING UNITS {if any)
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Molecular Biology Unit
INSTITUTE AND LOCATION
NINDS, National Institutes of Health Bethesda, Maryland
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER: 0Q
CHECK APPROPRIATE BOX(ES)
\*~\ (a) Human subjects [T] (b) Human tissues ] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Cushinq's disease is caused by the pituitary hypersecretion of ACTH and occurs predominately in women.
Patients are cured by surgical removal of an ACTH-producing adenoma, suggesting evolution and
expansion of a genetically aberrant cell. However, hypothalamic dysfunction and excessive stimulation
of anterior pituitary corticotrophs by one or more neurotransmitter substances may also lead to the
development of corticortropic adenomas.
To study the clonal composition of ACTH-producing pituitary adenomas, we used restriction
fragment length polymorphism (RFLP) of two X-chromosome linked genes, hypoxanthine
phosphoribosyl transferase (HPRT) and phosphoglycerate kinase (PGK), in conjunction with their
methylation patterns. Tumors from female patients only were used because in all female cells only one
X-chromosome remains active which can e detected by the methylation pattern. Out of 27 tumors
analyzed, nine were heterozygous at the HPRT or PGK loci The clonal analysis demonstrated a
monoclonal pattern in six of these tumors, whereas a polyclonal pattern was observed in three tumors
including a pituitary adenoma from a patient with the Nelson's syndrome. There was no correlation
between the clonal composition and the clinical features of the tumors.
Studies are also in progress to search for possible mutations in the members of the rasqene family.
Specific regions of the three ras genes, H-, K-, and N-ras, including the mutational hot spots in codons 12,
13, and 61 were amplified by the polymerase chain reaction chain reaction, PCR, and will be directly
sequenced using the dideoxynucleotide chain termination method.
PHS 6040 (Rev 184 42 SNB DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02760-03 SN'
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or /ess. Title must fit on one line between the borders )
Metabolic Mapping of the Brain During Rewarding Brain Stimulation
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
Linda Porrino, PhD Principal Investigator, Research Psychologist, SNB, NINDS
Francesco Pontieri, M.D.
Conan Kornetsky, Ph.D.
Lewis Seiden, Ph D
Mark Kleven, Ph.D.
Visiting Fellow, NIMH
Boston University
University of Chicago
University of Chicago
COOPERATING UNITS Of any)
Laboratory of Cerebral Metabolism, NIMH; Behavioral Pharmacology Laboratory, Boston University
School of Medicine; Department of Pharmacology, University of Chicago
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Brain Imaging Unit
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
1.1
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
J (a) Human subjects
] (a1) Minors
J (a2) Interviews
J (b) Human tissues | x | (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The deoxyglucose method is being used to study the alterations in local cerebral metabolic activity
resulting from both rewarding and aversive electrical brain stimulation to discrete brain sites as well as
resulting from the administration of drugs of abuse By mapping metabolic activity in rats under these
conditions, information can be obtained about these areas of the brain involved in motivation and
reinforcement. Furthermore, the functional consequences of chronic drug used can be assessed.
'Formally CNB - transferred to SNB 1/14/90
PHS&M0(N«» 1 M
43 SNB DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02762-03 SNB*
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (BO characters or less. Title must fit on one line between the borders.)
Quantitative Autoradiographic Determination of Dopamine Receptor Distribution in Primates
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Linda Porrino, Ph.D. Principal Investigator, Research Psychologist, SNB, NINDS
Francesco Pontieri, Ph.D. Visiting Fellow, NIMH
Massako Kadekaro, Ph.D. University of Texas Medical Branch
COOPERATING UNITS (if any)
Laboratory of Cerebral Metabolism, NIMH
Department of Neurosurgery, University of Texas Medical Branch - Gal venston
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Brain Imaging Unit
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
.2
OTHER:
CHECK APPROPRIATE BOX(ES)
] (a) Human subjects
] (a1) Minors
J (a2) Interviews
j (b) Human tissues (Vj (c) Neither
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The primate model of hemiparkinsonism produced by theintracarotid infusion of the neurotoxin,^
methyl-4-phenvl-1,2,3,6-tetrahvdropyridine (MPTP), allows the study of neurochemical changes
associated with lesions of the substantia nigra parscompacta Quantitative autoradiography after in
vitro radioligand binding with PH]naloxone (opiate antagonist) was used to determine the distribution
of opiate receptor sites in MPTP-induced hemiparkinsonian moneys. An increased density of opiate sites
was observed in the striatum on the side of dopaminergic denervation. The anatomic distribution of
these sites resembled the distribution of dopaminergic Di binding sites in the striatum, suggesting
potential interactions between dopamine and opioid peptides in the striatum.
•Formally CNB - transferred to SNB 1/14/90
PHS 6040 (Rev 1 M
44 SNB D1R
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02761-03 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJ ECT (BO characters or less. Title must fit on one line between the borders.)
Metabolic Mapping of Animal Models of Parkinsonism
PRINCIPAL INVESTIGATOR ttist other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)
Linda Porrino, Ph.D. Principal Investigator, Research Psychologist, SNB, NINDS
Francesco Pontieri Visiting Fellow, NIMH
COOPERATING UNITS i.tany)
Laboratory of Cerebral Metabolism, NIMH
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Brain Imaging Unit
INSTITUTE AND LOCATION
NINDS, National Institutes of Health, Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL:
OTHER:
CHECK APPROPRIATE BOX(ES)
Q (a) Human subjects ] (b) Human tissues \~*~\ (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Neurotoxic destruction of the dopaminergic cells of the substantia nigra parscompacta of primates and
rodents produces useful models of parkinsonism The 2-[14C]deoxyqlucose method is being applied to
animals in which unilateral lesions of the substantia nigra pars compacta have been made in order to
map the neural circuits involved in the symptoms of parkinsonism and its treatment with dopaminergic
agonist drugs. Metabolic studies in rats with anatomically specific lesions of the substantia nigra pars
compacta have demonstrated the significance of the loci of the damage to dopaminergic cells along the
medial-lateral axis of the nigra in determining the resulting pattern of behavioral effects. Furthermore,
the level of functional activity in the substantia nigra pars reticulata has been shown to be the
determinant of the directions of turning behavior elicited by the administration of dopaminergic drugs
* Formal ly CNB - Transferred to SNB 1-1 4-90
PHS6M0(Rev 1/84 45 SNB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02739-04 SN
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)
Clinical and Laboratory Investigation of Central Nervous System Vascular Disorders
PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
Edward H. Oldfield, M.D. Chief, SNB, Principal Investigator, NINDS
David Barba, M.D. Senior Staff Fellow, SNB
Michele Filing-Katz, M.D. SeniorStaff Fellow, ETB, NINDS
Marston Linehan, M.D. Surgical Branch, NCI
Berton Zbar, M.D. Senior Investigator, NCI
COOPERATING UNITS (if any)
Diagnostic Radiology Department, CC, Experimental Therapeutics Branch, NINDS
Surgery Branch, National Cancer Institute
LAB/BRANCH
Surgical Neurology Branch, CNP
SECTION
Clinical Neurosurgery Section
INSTITUTE AND LOCATION
NINDS, National Institutes of Health Bethesda, Maryland 20892
TOTAL MAN-YEARS:
PROFESSIONAL: y 5
OTHER:
0.0
CHECK APPROPRIATE BOX(ES)
[V] (a) Human subjects [T] (b) Human tissues ] (c) Neither
] (a1) Minors
J (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The clinical features, arteriographic findings, and treatment of 81 patients with spinal arteriovenous
malformations (AVSs) demonstrated that there are distinguishing clinical features in patients with
arteriovenous malformations of the spinal cord compared to those of patients with arteriovenous (AV)
fistulas of the spinal dura. The findings indicate that spinal arteriovenous fistulas are acquired lesions,
and not congenital, as was previously thought, and support arteriovenous malformations of the spinal
cord as congenital lesions. The findings also indicate that AVMs of the spinal cord produce myelopathy
as a result of high blood flow, but that patients with spinal dural AV fistulas develop myelopathy as a
result of increased venous pressure in the spinal cord.
Magnetic resonance imaging permits demonstration of the presence and site of AVMs of the spinal
cord and therefore is a valuable and safe, noninvasive technique to investigate patients suspected of
having spinal AVMs.
Foix-Alajounine syndrome was demonstrated to be due to venous congestion, and not venous
thombosis, and therefore, amenable to reversal by treatment of the spinal AVM. Spinal AVMs were
shown to recanalize consistently after embolic occlusion.
Patients with Von Hippel-Lindau disease were investigated and the following were shown:
investigation of the molecular biology of the hemanqioblastomas of the central nervous system revealed
a homozygous deletion of a portion of the short arm of the third chromosome, demonstrating that these
tumors are probably caused by the absence of a tumor suppressing gene, as are familial retinoblastomas.
MRI with gadolinium EDTA contrast enhancement was shown to be a sensitive technique of detection
for small hemangioblastomas of the central nervous system Excision of the tumors alone was shown to
result in resolution of syringomyelia associated with spinal cord hemangioblastomas. Therefore, the
tumor-associated syrinx does not need separate treatment
PHS 6040 (Rev 184 46SNBOIR
>
00
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ANNUAL REPORT
October 1, 1989 through September 30, 1990
Clinical Neuroscience Branch
National Institute of Neurological Disorders and Stroke
Table of Contents
RESEARCH SUMMARY 1-12
RESEARCH REPORTS
Biochemical Indices of Adrenergic Function in Humans
Z01 NS02115-17CNB 13
Clinical, Genetic and Biochemical Studies of Familial
Alzheimer's Disease
Z01 NS 02630-07 CNB 14
Neurotoxins and Animal Models of Neurological Diseases
Z01 NS 02716-04 CNB 15
Biochemical Evaluation of Aminergic Function During
Responses to Stress and in Disease States
Z01 NS 02717-05 CNB 16
Regulation of Synthesis and Expression of Neurotrophic
Agents and Neuropeptides
Z01 NS 02752-03 CNB 17
ANNUAL REPORT
October 1, 1989 through September 30, 1990
Clinical Neuroscience Branch
Clinical Neurosciences Program, DIR
National Institute of Neurological Disorders and Stroke
Irwin J. Kopin, M.D., Chief
The Clinical Neuroscience Branch (CNB) conducts research on the role of
neurotransmitters in brain and in peripheral autonomic function; the effects of
abnormalities in neurotransmitter metabolism and receptor activation and their
occurrence in neurologic disorders; and the effects of drugs and other therapies on
regulation of neurotransmitter release and actions. At present the Branch is divided
into two Sections, with a third section planned. Investigators in the Section on
Clinical Neuropharmacology examine neurotransmitter function in patients with
autonomic insufficiency or Alzheimer's disease. In the Section on Aminergic
Mechanisms (SAM), studies in animals and animal models of human neurologic
disorders are designed to examine the role of neurotransmitters and their receptors
in pathogenesis and treatment of neurologic diseases, with particular emphasis on
biogenic amines. A new Section on Sympathetic Function under the leadership of
Dr. David Goldstein will be developed during the next year. The clinical research
efforts of this Section will complement the studies in animals performed in the SAM;
studies with new 18F-Iabelled compounds appropriate for Positron Emission
Tomography (PET) imaging are planned and will be performed in collaboration with
investigators in the Neuroimaging Branch.
There is also a Unit on Growth Factors in CNB which, in addition to independent
research on regulation of growth factors, provides support for studies related to the
development or regeneration and the function in vivo of specific neuronal systems.
Much of this work is performed in collaboration with investigators in the Surgical
Neurology Branch.
CLINICAL NEUROPHARMACOLOGY SECTION
The Clinical Neuropharmacology Section continues to develop biochemical and
pharmacologic methods to assess neurotransmitter function and metabolism in man.
These strategies have been applied in the investigation and treatment of patients
with autonomic nervous systems disorders. Investigation of patients with lesion(s) of
the autonomic nervous system provides an opportunity to examine the interaction
between autonomic and other hormonal/peptide control systems. Two distinct
disorders remain the main focus of these efforts: autonomic failure may occur alone
(pure autonomic failure, PAF) or in association with (central nervous system, CNS)
degeneration (multiple system atrophy, MSA).
Our studies primarily focus on the noradrenergic system because norepinephrine
(NE) is released by most postganglionic sympathetic neurons and also is important in
central pathways concerned with blood pressure regulation. High performance
liquid chromatography, liquid scintillation spectrometry, and gas chromatography-
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mass spectroscopy are used to measure neurotransmitter and metabolite levels in
various biologic fluids. Radioenzymatic methods are employed to determine the
activities of synthetic and degradation enzymes related to neurotransmitter
metabolism. Peptide and hormonal levels are analyzed by radioimmunoassay.
These substances are measured under basal conditions and after a variety of stimuli
have been applied to evaluate the responsiveness of the particular system under
investigation.
Previously, we pharmacologically distinguished PAF and MSA patients based on their
blood pressure responses to NE. Patients with PAF manifest low basal plasma NE
levels and a shift-to-the-left of their blood pressure dose-response curve to NE,
consistent with noradrenergic receptor supersensitivity. The increase in slope
without a shift of the dose-response curve in MSA suggests a preganglionic lesion.
Since the intermediolateral column (ILC) innervates sympathetic ganglia and the
number of ILC neurons is substantially lower than normal in MSA, we measured the
plasma NE response to intravenous acetylcholine (Ach) in patients with autonomic
failure to assess ganglionic responsiveness. All subjects were pretreated with
glycopyrrolate to block peripheral muscarinic effects of Ach. Mean blood pressure
and heart rate did not change during the infusion in any group. Normal subjects
increased plasma NE proportional to the rate of Ach infusion. Patients with PAF
failed to significantly increase plasma NE, consistent with post-ganglionic
sympathetic neuronal dysfunction. Patients with MSA can be subdivided into
nonresponders and responders based on their plasma NE response at low doses of
Ach. The nonresponders do not increase their plasma NE, similar to PAF patients.
The remaining MSA patients increase their plasma NE significantly greater than
normal at low doses, followed by a return to basal levels as the infusion rate was
increased. This biphasic response suggests that some patients with MSA may have
cholinergic supersensitivity since the increase in plasma NE levels at low doses is
more pronounced than in normal subjects. The subsequent reduction in plasma NE
could result from increased removal from plasma, enhanced neuronal uptake, or
decreased NE release. Increased clearance from plasma is not likely since blood
pressure and heart rate did not change. The most plausible mechanism is the
development of depolarization blockade at higher Ach infusion rates, consistent
with ganglionic supersensitivity. Those MSA patients who lacked a NE response
might have postganglionic involvement although it is possible that extreme
supersensitivity might have produced blockade at even the lowest doses employed
in this study. We plan to use other neurochemical (co-release of peptides,
measurement of NE metabolites) and electrophysiologic methods to further clarify
the mechanism of this abnormality. When it becomes technically feasible, the use of
i8F-dopamine PET scanning might be employed to examine NE turnover in these
patients.
Much of the research on patients with autonomic failure is concerned with a-
adrenergic function. However, our previous work demonstrating abnormal
circadian patterns of melatonin secretion and alterations in the release of gastrin
during hypoglycemia suggest that ^-adrenergic function is also impaired in patients
with PAF and MSA. We examined cardiovascular, plasma NE, and renin (PRA)
responses to isoproterenol infusion in patients with autonomic failure and in normal
subjects. Increased slopes of the blood pressure dose-responses curves in the
patients are consistent with impaired baroreflex modulation. A leftward shift in
patients with PAF suggests ^-adrenergic receptor supersensitivity. Plasma NE and
PRA increased proportional to the log of the plasma isoproterenol level in normal
subjects. These normal responses may be due to reflexive cardiovascular and renal
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mechanisms since neither patient group manifested increases in NE or PRA during
isoproterenol infusion despite a reduction in mean blood pressure.
In addition to abnormal cardiovascular control, patients with autonomic dysfunction
also develop symptoms related to disruption of the innervation to other organs.
Although diminished or absent sweating occurs in most patients, there have not
been any systematic pharmacologic investigations of sudomotor function. We
measured sweat production to direct gland stimulation with intradermal
methacholine and also assessed the sympathetic skin response to electrical
stimulation. Sweat production was significantly less than normal in PAF and MSA.
There was no correlation between the sympathetic skin response and sweat induced
by methacholine. The diminished sweat production in response to intradermal
methacholine suggests that human sweat glands do not develop denervation
supersensitivity.
Clinical distinction between MSA and PAF may be difficult in the early stages prior to
the development of central neurologic signs. Our previous experience indicates that
it is necessary to wait at least 5 years from the onset before making a definitive
diagnosis of PAF. We have analyzed the results of neuroimaging studies in 43
patients using CT, MRI, and PET scanning. In contrast to normal findings in PAF, the
main abnormalities in patients with MSA were atrophy of the cerebellar
hemispheres and vermis, MRI signal hypointensity in the posterolateral putamen,
and decreased glucose utilization in the cerebellum and most supratentorial
structures. Thus, neuroimaging studies facilitate distinction between MSA and PAF.
Neuropathology examination of post-mortem tissue in MSA reveals neuronal loss
and gliosis in a number of central nervous system areas. The nonspecific nature of
these changes does not yield any clues to trie underlying cause of MSA Immune
processes have been described in a variety of neurologic disease states. We used
immunocytochemical methods to search for CSF antibodies against specific regions
in the rat brain that parallel the known areas affected by degeneration in MSA. CSF
immunoreactivity to rat locus coeruleus occurred in a significantly greater number of
samples from MSA patients compared to control subjects or patients with PAF.
Other brain regions infrequently showed immunoreactivity. Degeneration in MSA
may release antigen(s) that induce antibodies against locus coeruleus neurons. The
specificity of this abnormality is particularly interesting since immunoreactivity
towards other brain regions known to degenerate was not observed. Furthermore,
patients with Parkinson's disease have a CSF antibody that reacts with the substantia
nigra but not the locus coeruleus. This finding increases the diagnostic potential of
the immunoreactivity in patients with MSA. Further characterization of this
immunologic abnormality is in progress to determine whether it has pathogenetic
significance. Lack of CSF immunoreactivity in PAF is consistent with primarily
peripheral involvement.
We also continue to collect psychosocial histories and pedigrees on all autonomic
failure patients to determine whether social, environmental, or familial factors
contribute to the development and progression of these disorders. A more detailed
analysis of our preliminary observation of increased exposures to toxic substances
has been completed in collaboration with the Environmental Epidemiology Branch,
NCI. MSA patients had significantly more exposures to metal dusts and fumes,
plastic monomers and additives, organic solvents, and pesticides than the control
population. The exposures were determined in most subjects by their reported usual
occupations. These toxic exposures in the patients and the increased incidence of
autonomic and neurologic symptoms in their first-degree relatives suggests a
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multifactorial etiology. There may be a genetically determined, selective
vulnerability that provides targets for the toxic insults resulting in neuronal death
within specific systems. These studies will be expanded to further address the
questions raised by these data.
Treatment and emotional support are provided to all patients with MSA and PAF.
Orthostatic hypotension is the primary focus for therapeutic trials. Fludrocortisone
and ibuprofen remain the most effective pharmacologic agents for raising blood
pressure. Although we are using guanfacine to assess peptide responses mediated
by central noradrenergic pathways, a treatment phase has been initiated.
Preliminary therapeutic efforts with guanfacine have not been encouraging. We
plan to re-evaluate the use of L-threo-DOPS, an unnatural NE precursor, for treating
patients with MSA and PAF. It has now been shown that the drug increases brain NE,
even when the normal synthetic pathway is available. This agent may be useful for
treating the peripheral and central noradrenergic deficiencies we have observed in
these patients. Use of a somatostatin analog may also help patients who fail more
conventional approaches. During the past 6 months new noninvasive technology
has become commercially available and may be adapted as the sensor arm of the
sympathetic neural prosthesis. This would allow accurate control of blood pressure
without invasive monitoring. Pending availability of funds, we hope to extend the
contract for an additional 4 months in order to realize the desired outcome from this
project. It should be possible to test the device within the next year pending
approval by the U.S. Food and Drug Administration.
Many adjustments in lifestyle are necessary for patients and their families to
successfully cope with a progressive degenerative disorder. Of major concern to this
patient population are the increasing cost of chronic care, lack of resources for care
(home care nursing), and quality of life decisions as the disease progresses
(tracheostomy, gastric tubes). In an effort to increase support, a meeting was held to
organize a national support group. This facilitated dissemination of information on
research resources and provided opportunity to share our experience in dealing with
these chronic disorders.
A number of other studies are currently in progress:
1. Peptide pathways in sympathetic and dorsal root ganglia as well as spinal cord
are being studied in collaboration with Dr. Margaret Matthews, Oxford
University. Preliminary results differentiate PAF and MSA.
2. The radioimmunoassay for neuropeptide Y has not been satisfactory. We are
currently evaluating a new antibody. We hope to use colocalization of peptides
as a strategy for evaluating mechanisms involved with release of
neurotransmitters.
3. Modification of several peptide assays for cerebrospinal fluid CSF are in progress.
These include CRF, substance P, calcitonin gene-related peptide, brain natriuretic
peptide, and endothelin.
4. Several protocols have been initiated commensurate with the arrival of a new
Medical Staff Fellow. It was necessary to delay these projects in order to facilitate
completion of other protocols. These studies include: (1) evaluation of the
sympathetic response to hypercarbia, (2) measurement of physiologic and
neurochemical effects of ANP infusion, and (3) sleep studies.
5. Detailed studies of excitatory amino acid receptors and subtypes of GABA
receptors in the cerebellum of post-mortem brain specimens from MSA patients is
being carried out in collaboration with Dr. Anne B. Young, University of
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Michigan. Four amino acid receptors will be measured by autoradiography: (1)
NMDA, (2) AMPA, (3) kainate, and (4) metabotropic receptor. The high and low
affinity forms of the GABAAand GABAb receptors will also be evaluated.
6. We have studied p-adrenergic responses and ACTH responses to arecoline in two
patients with multiple sclerosis. The data will be analyzed after 5 patients have
been investigated. It has been suggested that autonomic lesions may play a role
in the progression of this demyelinating disease.
7. A follow-up study was initiated to evaluate all twins in North Carolina who
developed paralytic polio between 1940-1948. Detailed family studies will be
obtained. This work has relevance to our study of MSA patients since 4 of the
MSA cases had a history of paralytic polio.
The Clinical Neuropharmacology Section has continued the study of familial
Alzheimer's disease (AD) as a major priority within the scope of its research efforts.
AD remains a major medical and social problem since it is the most common cause of
irreversible, chronic dementia. Clinical and therapeutic studies in AD are
significantly limited by accuracy and timing of diagnosis. Thus, the main justification
for studying the more uncommon, autosomal dominant subgroup of familial cases
lies in the accuracy of diagnosis that may be inferred through post-mortem
examination of other affected family members. Our two major research directions
the primary, underlyi
of AD, and (2) longitudinal investigation of clinical and biochemical characteristics of
are: (1) genetic linkage to identify the primary, underlying molecular pathogenesis
the disease to provide clues for earlier, accurate diagnosis and more rational
therapeutic strategies.
Additional samples have been submitted to the Coriell Institute for Medical
Research, Camden, New Jersey, for the establishment of skin fibroblast and
peripheral blood lymphoblast cultures under an intra-agency agreement between
the NINDS and NIA. Currently, we are working with 20 families that have an
autosomal dominant inheritance pattern. Samples have been submitted from four
categories of family members who undergo neurologic examination: affected, at-
risk (first-degree), escapees, spouses. This is also part of an international
collaboration organized and coordinated by our Section. Families from the U.S.,
Canada, Italy, France, and Germany are participating in this effort. Identification of
new branches of these families and continued follow-up with the families to verify
clinical status of at-risk members increases their value for genetic linkage studies.
The cultures continue to serve as a renewable source of DNA and cells for basic
research in AD.
Further investigation of clinical genetics and linkage analysis support the concept of
heterogeneity. Only early onset families appear to be linked to markers on
chromosome 21, although a significant lod score has still not been obtained using a
single marker in an individual family. We plan to compare phenotypic characteristics
between various pedigrees to identify parameters that may allow selection of more
homogeneous subgroups. In addition, we continue to ascertain new pedigrees,
expand the number of participating members of the current families, and update
the clinical status to provide the maximum amount of accurate data for the linkage
analysis. Attempts to identify additional branches of the large Canadian pedigree
have not been successful. Efforts have also continued to search for additional
informative markers. Emphasis will be given to VNTR (variable number tandem
repeat) probes, probes containing mini-satellite polymorphisms, and probes
showing sequence variability. A search will also be made to identify coding
sequences in AD brain. Long-range physical mapping will be used to search for and
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clone structural chromosomal rearrangements in familial AD cases or in other AD
cases that have chromosome 21 translocations since these could be the sight of the
gene defect. We will also search for additional loci on other chromosomes,
especially 19 which was recently found linked in late onset cases. In collaboration
with several other institutions, we participated in an investigation of transmission
and age-at-onset patterns in 70 kindreds with familial AD. The age-at-onset among
the families showed a bimodal distribution; a mean onset age of 58 years
distinguished early from late onset families. The lifetime AD risk for at-risk offspring
in early onset cases was 53%, significantly less than the risk of 86% in late onset
cases. The results in early onset families are consistent with an autosomal dominant
transmission model whereas the late onset form appears to have at least 2
etiologies.
Our longitudinal investigations have continued and psychologicsupport and genetic
counseling have been given. Measurement of monoamine metabolites,
acetylcholinesterase, and somatostatin provide further confirmation of our
preliminary observations. A third at-risk subject, among the subgroup who have low
CSF MHPG and somatostatin, has developed dementia. These newly affected AD
patients highlight the significance of these neurochemical changes. Studies of other
neuropeptides including corticotropin releasing factor are in progress. We have also
used our longitudinal collection of CSF samples in a collaborative search for anti-
neuronal antibodies. Previous investigations demonstrated the presence of
antibodies in the CSF from AD patients; these antibodies recognize specific neuronal
populations in the adult rat CNS. In the developing rat CNS different types of
antigens were recognized. One of the most remarkable differences was the
recognition of amoeboid microglial cells. Electron microscopy revealed that the AD-
CSF antibody recognizes specific membrane receptors in the macrophagic microglia.
These results further support the concept that inflammation and similar immune
mechanisms may play a role in AD.
Cerebral glucose metabolism has been studied in 8 affected patients and 20 first-
degree at-risk subjects. The demented patients had significant hypometabolism in
the parietotemporal regions, with some involvement of the occipital and temporal
lobes. Overall, metabolism in the at-risk group did not differ from normal subjects.
Repeat scans were performed on three at-risk subjects who subsequently developed
dementia; these studies showed a 10-17% decline in relative parietal lobe
metabolism. In one of these subjects, it appears that an isolated regional change in
cerebral glucose metabolism may have preceded clinical expression of the disease. A
more detailed analysis of these preliminary findings is in progress.
Additional studies that have been continued or in progress include:
1. A study of ACTH responses to arecoline is currently being conducted. This may
serve as a neuroendocrine assessment of a central cholinergic pathway. More
patients will be studied before the results are analyzed.
2. Two approaches are being employed to investigate noradrenergic function:
evaluation of blood pressure responses to intravenous NE and measurement of
urinary NE metabolite patterns. We anticipate that abnormalities may reflect
locus coeruleus involvement which occurs in most AD cases. The results are
currently being analyzed.
3. In collaboration with Dr. Jesse Sisken, University of Kentucky, fibroblast cultures
are being used to determine whether alterations in calcium metabolism are
present in fibroblasts from patients with familial AD. The photoprotein,
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aequorin, is used to probe for transient changes in free intracellular calcium
levels in cells that have been serum deprived and then stimulated with either
serum or bradykinin. Preliminary results indicate that all FAD cells respond to the
stimulation but the size of the calcium transient appears to be smaller in the
patients compared to controls. It is necessary to confirm these findings in a larger
series prior to investigating at-risk subjects.
4. A collaborative study of DNA repair is being conducted with Dr. W. Clark
Lambert, UMDNJ. An attempt is being made to further elucidate the mechanisms
involved in producing the defect in DNA repair which was observed previously by
other collaborators. Preliminary results reveal decreased viability of lymphoblasts
following exposure to methyl-methanesulfonate. Unscheduled DNA synthesis is
reduced in a sporadic AD case; investigation of familial AD lines is currently in
progress.
5. Quantitative assessment of smell is being carried out in affected and at-risk
members of these families with dominantly inherited AD. This study will attempt
to identify a clinical correlate of the neuropathology abnormality recently
described in olfactory neurons.
SECTION ON AMINERGIC MECHANISMS
Studies in this Section have focussed on biochemical evaluation of aminergic
mechanisms and in part continue to be collaborative efforts with investigators in the
NHLBI for examination of peripheral autonomic function and in the Surgical
Neurology Branch, NINDS, for evaluation of brain aminergic function.
The studies on the autonomic nervous system have used measurements of levels of
NE, dopamine (DA), 3,4-dihydroxyphenylalanine (DOPA) and their metabolites in
body fluids to assess sympathetic neuronal function. The results of these studies have
shown that DOPA, the amino acid precursor of catecholamines, is derived mainly
from peripheral sympathetic nerves, but is taken up and retained in skeletal muscle
and released into blood during muscular contraction. The amounts of DOPA in
plasma reflect catecholamine synthesis in peripheral sympathetic nerves and levels
of the amino acid increase during acute stress. There is a dissociation, however,
between the tissue levels of tyrosine hydroxylase and the rate of tyrosine
hydroxylation. During acute stress, tyrosine hydroxylation increases without any
significant alteration in the enzyme levels, whereas after repeated stress, levels of
the enzyme are increased in some tissues, but without stress-induced sympathetic
activation, the rate of tyrosine hydroxylation does not appear to be increased.
Although DOPA is also the precursor of melanin, this source does not contribute
significantly to plasma DOPA. The intraneuronal metabolite of norepinephrine, 3,4-
dfhydroxyphenylglycol (DHPG), is an index of sympathetic neuronal integrity which
in combination with plasma NE levels provides information about sympathetic
neuronal function. DHPG is formed mainly from NE which "leaks' into the
cytoplasm from storage vesicles, but some is formed from NE which is recaptured
after release into the synapse.
The venous-arterial differences in radiolabeled and unlabelled NE and blood flow
provide a means of assessing the rate of overflow of NE from the sympathetic nerves
in a tissue. Overflow of this amine generally correlates with sympathetic nerve
activity, as does release of DOPA and DHPG into the circulation. DHPG clearance,
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however, may be altered by changes in the distribution of blood flow so that its
plasma leveis may be elevated even when net release is diminished.
Simultaneous estimation of urinary excretion rates on plasma levels of homovanillic
acid (HVA) (the major metabolite of dopamine) and of MHPG and VMA (the major
metabolites of NE) before and during administration of debrisoquin are used to
estimate the rate of brain DA metabolism. This method, which had been verified
using animals treated with MPTP to eliminate brain dopaminergic neurons, is now
being used to study DA metabolism in humans. Patients with early, untreated
Parkinson's disease have been found to have low levels of HVA formation in brain,
consistent with a deficit in available DA. Although in has been reported that brains
of elderly persons brains have low levels of DA, the rate of formation of brain HVA
does not appear to differ with age, suggesting that the turnover rate of brain DA
increases with age to compensate for the diminished DA content. A surprising
preliminary finding of a sex difference in brain DA is awaiting confirmation in a
larger number of subjects.
18F-labelled DA has been shown to be a useful compound for imaging sympathetic
nerves and their activity in vivo in experimental animals. In dogs, denervation of the
salivary gland by removal of one superior cervical ganglion results in loss of uptake
and retention of the 18F-Iabel. Furthermore, the 18F accumulated in the heart
declines more slowly when sympathetic nerve traffic is diminished and more rapidly
during pharmacolgically induced NE release. Thus, 18F-DA appears to be converted
to 18F-NE in sympathetic nerves and, as we had previously shown in rats, can be used
to reflect NE turnover.
Another major research endeavor is centered on neurotoxins which may produce
neurologic disorders. Current investigations centered on two toxins, MPTP (1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and BMAA (2-amino-3-(methylamino)-
propanoic acid). MPTP toxicity results in a movement disorder in primates, including
humans, which closely resembles Parkinson's disease. When administered into one
carotid artery, this toxin produces in monkeys a hemiparkinsonian syndrome which
we have used to examine biochemical and metabolic changes, behavioral effects and
therapeutic potential of drugs and tissue implants.
Local cerebral glucose utilization (LCGU) in monkeys with MPTP hemiparkinsonism
was examined using quantitative 2-[i4C]deoxyglucose autoradiography. These
monkeys display rigidity, bradykinesia, and tremor of the limbs contralateral to the
side of MPTP infusion; during spontaneous activity they turn toward the side of the
lesion. Of 82 brain areas examined, statistically significant metabolic changes were
confined mainly to basal ganglia structures ipsilateral to the side of the lesion. LCGU
was reduced in the substantia nigra pars compacta and ventral tegmental area, i.e.,
in the areas of cell loss, where as increases in LCGU were found in regions normally
innervated by the lesioned area, e.g., the post-commissural portions of the putamen
and dorsolateral caudate. Other structures showing statistically significant metabolic
changes were the external segment of the globus pallidus ( + 40%), subthalamic
nucleus (-17%), and pedunculopontine nucleus (+15%). There were also smaller
changes in portions of the thalamus (ventral anterior nucleus, parafascicular
nucleus) and premotor cortex. All significant metabolic changes were confined to
the side of the substantia nigra lesion and were essentially restricted to regions
involved in the production of movement or maintenance of posture.
We have previously reported that ethanol and acetaldehyde potentiate MPTP
neurotoxicity in mice, enhancing DA depletion in the striatum. This enhancement of
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neurotoxicity was found to be specific for the nigrostriatal DA pathway, but there
were differences in vulnerability to the toxin with age. In 5-week-old mice
acetaldehyde treatment did not enhance DA depletion, whereas in 8-week-old
animals, acetaldehyde or ethanol given with MPTP decreased striatal DA content to
about 10% of controls in contrast to 43% of controls when MPTP was given alone. In
acetaldehyde or ethanol and MPTP-treated mice, changes in DA levels were
observed only in the striatum. DA contents in the hypothalamus, olfactory bulb and
frontal cortex were similar to that in controls. Contents of NE and serotonin in
striatum, hypothalamus, olfactory bulb and cerebral cortex were not affected by any
of the treatments. Three months after MPTP alone, striatal DA recovered to 74% of
controls in 8-week-old mice, whereas no recovery occurred in acetaldehyde- MPTP-
treated mice. Moreover, both tyrosine hydroxylase immunocytochemistry and Cresyl
violet staining showed an extensive and selective cell loss in the parscompacta of the
substantia nigra of the mice treated with acetaldehyde or ethanol and MPTP,
whereas MPTP alone caused only a limited cell degeneration.
Accumulation of the MPTP toxic metabolite, 1-methyl-4-phenylpyridinium ion
(MPP + ) in both striatum and whole brain after MPTP alone or after combined
treatments with ethanol or acetaldehyde did not affect MPTP metabolism. The
results from the in vivo experiments indicated that relatively low doses of ethanol or
acetaldehyde directly potentiate MPP+ toxicity, apparently without interferring
with its pharmacokinetics. However, higher doses of these drugs decreased MPP +
clearance from the striatum. Acetaldehyde also increased initial MPTP accumulation
in the whole brain but failed to enhance MPP+ levels, indicating that MPTP
metabolism was unaffected. In vitro studies confirmed that acetaldehyde does not
modify MPTP metabolism in striatal or mesencephalic astrocytes in culture. In
mesencephalic neuronal cultures, acetaldehyde does not interfere with the
conversion of MPTP to MPP+ by glial cells and does not change the levels of MPP +
uptake nor its spontaneous release. These results indicate that the slower MPP +
clearance from the stratum after acetaldehyde is not related to a direct effect of
acetaldehyde on DA neurons or astrocytes.
Cerebellar granule cells in enriched primary culture are also susceptible to the
neurotoxic effects of MPP + . Relatively high MPP+ concentrations are required to
elicit neurotoxic effects at early culture times, but lower concentrations of MPP +
produce comparable neurotoxic effects at later culture times. Under identical
culture conditions, MPTP is not neurotoxic. Preincubation with the glutamate
uptake blockers, DL-threo-3-hydroxyaspartic acid or dihydrokainate, or the
dopaminergic uptake blocker mazindol, protects the granule cells from the cytotoxic
effects of MPP + . Although MPTP is not neurotoxic in an enriched granule cell
culture, in coculture with cerebellar astrocytes MPTP is toxic to granule cells,
presumably because it is converted in astrocytes to MPP + . Cerebellar astrocytes
remain confluent and viable. The addition of pargyline to the coculture abolishes
the neurotoxicity consistent with a role of MAO B in bioactivation of MPTP. The
concentration of MPP+ in the coculture medium (13 uM) was less than that required
for the toxic effect in enriched neuronal cultures at earlier culture times, suggesting
that an astroglial-neuronal interaction, perhaps by proximity, enhances the
neurotoxicity of MPP + . These results might explain the reported effects of MPTP on
some cerebellar cells in mice.
The second toxin, BMAA, was selected for study because it had been implicated in
the unusually high incidence of amyotrophic lateral sclerosis-parkinsonism-dementia
(ALS-PD) disorder among the Chamorros native to the island of Guam. The disease
had been attributed to consumption of flour prepared from the seeds of Cycas
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circinalis L, the false sago palm. The disease occurs also in other regions of the South
Pacific where cycad seeds have been implicated because of their medicinal use on
open wounds or sores. In support of this relationship it was recently shown that,
when given in large doses to primates, BMAA, a minor cycad component, can cause
selective degeneration of upper and lower motor neurons in the spinal cord and
clinical features similar to those of ALS. In order to test the relationship between ALS
and cycads, we developed a sensitive and precise gas chromatography/mass
spectrometric (GC/MS) assay for BMAA which allows direct assessment of the BMAA
content in foods and is directly applicable to the assay of BMAA in biologic tissues
and fluids. BMAA was found in the endosperm of a range of cycad seeds collected
from Guam at levels of approximately 1 g/g (dry weight).
The levels of the neurotoxin in cycad flour, however, indicated that over 90% of the
total BMAA content was removed during processing. Furthermore, in one-half the
samples almost all (>99%) of the total BMAA was removed. There were no
significant regional differences in the BMAA content of flour prepared from cycad
seeds collected from several villages on Guam. The processed cycad flour as prepared
by the Chamorros of Guam and Rota contains extremely low levels of BMAA, which
are in the order of only 0.005% by weight (mean values for all samples). Thus, even
when cycad flour is a dietary staple andeaten regularly, it seems unlikely that these
low levels could cause the delayed and widespread neurofibrillary degeneration of
nerve cells observed in ALS-PD of Guam.
Another possibility, however, is that the toxin is accumulated selectively in specific
sites whereafter chronic exposure, concentrations of BMAA become sufficiently high
to cause toxic effects. To examine this possibility, BMAA bioavailability, uptake,
storage and penetration into brain were studied in rats. The results of this study
showed that only a small fraction of injected BMAA, whether adminstered as a
single acute i.v. injection or infused s.c. continuously over a 2-week interval, reach
the brain and only low concentrations (average <300 uM) are found. The cortex and
spinal cord, which are most affected in ALS-PD, have among the lowest levels of the
toxin.
When BMAA toxicity to cerebellar granule cells in tissue culture was compared to the
toxicity of cycad flour obtained from various sources in Guam, there was no
relationship between toxic effects and BMAA content. Although BMAA was
nontoxic in concentrations greater than found in the flour samples, some of the
samples were toxic to these cultured cells. This led us to characterize the substance
which appeared to be responsible for the toxicity. Because of its physical properties
and stability, metals were implicated; analysis for metals revealed high
concentrations of zinc in only the few toxic flour samples. The concentrations found
were sufficient to account for toxicity in the tissue culture and would result in
dietary intake of zinc in excess of one hundred-fold greater than normal. The source
of the zinc may have been galvanized containers used for soaking the cycad seeds
during the preparation of the flour. This observation provides a link between the
two major alternative hypotheses (cycad and trace metal imbalance) for the etiology
of ALS-PD.
Investigators in the unit on growth factors have been examining the regulation of
synthesis and expression of neurotrophic agents and neuropeptides. Nerve growth
factor (NGF) isolated from the mouse submaxillary gland bv Levi-Montalcini, was the
first discovered of a number of such protein factors which are required by neurons
for survival and maintenance of function. Recent evidence suggests that
many"nerve growth factors" may exist, each specific for a particular population of
10-CNB/DIR
neurons in either the CNS or the the peripheral autonomic nervous systems. In
addition, neuropeptides may play a role as neurotrophic factors.
Several animal models of neurologic disease changes in the synthesis or tissue
content of factors appear to be associated with the response to CNS injury or
involved in recovery. The Parkinsonian disorder with destruction of dopaminergic
neurons of the nigrostiatal pathway which appears in mice or monkeys after
systemic treatment with the neurotoxin MPTP, partial ablation of cortex in rats,
"injury" genetically programmed death of cerebellar Purkinje cells between 20-50
days after birth in an inbred mouse strain, the pcd mutant, are all models which can
be used to determine whether proliferating astrocytes produce trophic factors. The
reactive astrocytes can be cultured and growth factor activity sought in the medium.
Neurotrophic activity is assessed by bioassays which utilize chick dorsal root ganglia
neurons, rat superior cervical ganglion or CNS neurons, as well as by a 2-site assay for
NGF. In addition, mRNA is prepared and analyzed for NGF mRNA as well as cross-
hybridizing species (under low stringency). These blots are also being examined for
changes in neuropeptide and neurotransmitter synthetic enzyme mRNAs. Some of
the mRNA has been used in the preparation of cDNA libraries. Molecular
mechanisms for regulating the synthesis of NGF and other neurotrophic factors,
primary cultures of astrocytes are being studied as model systems. NGF mRNA has
been detected in primary cultures of rat cortical, cerebellar and striatal astrocytes,
and Schwann cells. NGF synthesis can be increased in astrocytes by both forskolin
and isoproterenol, supporting the idea that in these cells, cyclic AMP regulates
transcription of the NGF gene. Isoproterenol has been shown to work through the
classic ^-adrenergic receptor: the effect is blocked by ^-antagonists. The presence of
the ^-receptor on astrocytes suggests a mode for neuronal-glial communications. In
addition, VIP (vasoactive intestinal peptide) can stimulate NGF synthesis in
astrocytes: whether this occurs via activation of adenylate cyclase has yet to be
determined. This finding is of particular interest since in spinal cord cultures, VIP acts
on astrocytes to release neurotrophic factors.
Astrocytes have recently been found to express certain neuropeptide genes, with
both gene- and brain region-specificity. Astrocytes from cortex, cerebellum and
striatum all express the proenkephalin (PE) gene. The cells synthesize and process
the precursor to free enkephalin peptides, although the pattern of processing
appears to vary with the brain region and cerebellar cells contain a C-terminally
extended form of enkephalin. Only cerebellar astrocytes express somatostatin (SS)
mRNA and peptide: none express cholecystokinin, substance P or the other opioid
peptide precursors. Neuropeptide expression is developmentally regulated, with
peak expression in astrocytes prepared from embryonic day 20 to postnatal day 3
animals; levels decrease sharply in cells prepared from 8-day-old animals and are
almost undetectable in cells prepared from adult animals. These results suggest the
possibility that enkephalins or SS may act as astrocyte-derived trophic factors for
neurons early in development, consistent with results previously obtained on PE
gene expression in striatum, as well as analyses of proenkephalin (PE) and SS gene
expression in cortex and cerebellum during development. The developmental time
course of expression of PE mRNA in striatum is of particular interest, since it shows a
biphasic curve. The first peak before synapses have yet formed, but when astrocyte
expression is maximal, suggesting that enkephalins may serve a trophic-like function
early in development. Similar observations have been made regarding other
neuropeptides including vasoactive intestinal peptide (VIP). The second peak in PE
mRNA occurs at the time of active synapse formation and presumably represents the
expression of enkephalins as neurotransmitters/neuromodulators. These studies are
being complemented by situ hybridization analysis of changes in neuropeptide and
11 -CNB/DIR
neurotransmitter mRNAs at the single cell level. This information will be used to
determine if in vivo PE and SS gene expression occurs in astrocytes early in
development. Current studies are directed toward determining if expression of the
neuropeptide genes turn on again in reactive astrocytes and if exposure of either
neurons or glia in culture to peptides alter either morphology or specific
neurochemical markers.
Using a method previously developed in our laboratory, changes in the turnover rate
of neuropeptides are being examined in pharmacologic studies on rat brain. The
method is based on measurement of precursor mRNA, precursor proteins, and the
biologically active peptides: the combination of these measurements is used to
determine whether a drug affects neuropeptide levels by changing the transcription
of the gene, the rate of translation to or processing of the precursor, or the
utilization of the peptide itself. The results have demonstrated that certain
neuroactive drugs, such as morphine, can affect gene expression and thereby alter
peptide levels, whereas others affect peptide levels independent of an action on the
rate of biosynthesis. We have now analyzed changes in enkephalin biosynthesis and
turnover following MPTP administration to mice, and find that depletion of DA
affects enkephalinergic neurons differently in striatum and olfactory tubercle or
cortex. These studies contribute to understanding the role which neurotrophic
factors may play in neurologic disease, the effect of their absence on
neuropeptide/neurotransmitter function, the molecular mechanisms by which
synthesis of these agents may be regulated, and the possible neurotrophic functions
of neuropeptidesearly in CNS development.
12-CNB/DIR
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECTNUMBER
Z01 NS 02115- 17CNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT ($0 characters or less. Title must fit on one line between the borders.)
BIOCHEMICAL INDICES OF ADRENERGIC FUNCTION IN HUMANS
PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)
P.I.: R. J. POUNSKY, M.D., CHIEF, CNS, CNB, NINDS
OTHERS: (CNS) - S.M. BASER, M.D., MEDICAL STAFF FELLOW; A.M. MARINI, M.D., SENIOR STAFF
FELLOW; (OCD) - LE. NEE, M.S.W., SOCIAL SCIENCE ANALYST; (MNB) - M. HALLETT, M.D., CHIEF;
(NIS) - M. FULHAM, M.D., VISITING ASSOCIATE; G. DICHIRO, M.D., CHIEF; (NIB) - H. McFARLAND, M.D.,
DEPUTY CHIEF; (CNB) - I.J. KOPIN, M.D., CHIEF
COOPERATING UNITS (if an,)
DEPT. OF HISTOLOGY, UNIV OF GOTEBORG, SWEDEN (A. McRAE, A. DAHLSTR6M ); NATIONAL CANCER INSTITUTE (M. GOMEZ,
S BALE); DEPT. OF HUMAN ANATOMY, UNIV OF OXFORD (M. MATTHEWS); UNIV OF MICHIGAN (A. YOUNG)
LAB/BRANCH
CLINICAL NEUROSCIENCE BRANCH, CNP, DIR, NINDS
SECTION
CLINICAL NEUROPHARMACOLOGY SECTION
INSTITUTE AND LOCATION
NINDS, NIH, BETHESDA, MP 20892
TOTAL MAN-YEARS: , j.
PROFESSIONAL: 2 5
OTHER:
1.0
CHECK APPROPRIATE BOX(ES)
|T] (a) Human subjects Q (b) Human tissues I I (c) Neither
] (a1) Minors
] (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Autonomic nervous system activity is essential for maintaining circulatory and metabolic homeostasis. In
order to study sympathetic nervous system function and its relationship to other neuroendocrine
systems, it is necessary to measure neurotransmitter, hormonal, and peptide levels in response to various
stimuli. The levels of norepinephrine, epinephrine, and dopamine and their metabolites in various body
fluids reflect the activity of the neurones from which these neurotransmitters are released.
Cerebrospinal fluid levels of monoamine metabolites, enzymes related to neurotransmitter synthesis and
degradation, and peptides can be used to assess central nervous system neurotransmitter/neuropeptide
function and metabolism. It is necessary to consider the origin of these metabolites to make appropriate
corrections for valid interpretations of the data. These strategies have been used to study patients with
neurogenic orthostatic hypotension and other disorders in which abnormal adrenergic function is
suspected. Plasma NE increases following intravenously administered acetylcholine in normal subjects.
Lack of an increase in patients with pure autonomic failure (PAF) is consistent with post-ganglionic
sympathetic neuronal dysfunction. Patients with multiple system atrophy (MSA) may develop
depolarization blockade due to cholinergic receptor supersensitivity since they manifest an exaggerated
response at low doses followed by a return of plasma NE to baseline levels. Only patients with PAF have
(3-adrenergic receptor supersensitvity. Increases in plasma norepinephrine and renin activity following
isoproterenol administration in normal subjects may result from reflex cardiovascular and renal
mechanisms. Diminished sweat production to intradermal methacholine in patients with autonomic
failure suggests that human sweat glands do not develop denervation supersensitivity. Cerebrospinal
fluid immunoreactivity to rat locus coeruleus suggests that degeneration in MSA may release antigens
that induce antibodies against neurons in selected brain regions Structural and metabolic brain
abnormalities defined by neuroimaging studies facilitate clinical distinction between PAF and MSA.
Improved diagnostic accuracy and a more thorough understanding of the underlying mechanisms
involved in producing abnormalities of neurotransmitter metabolism and peptide function in these
clinical disorders leads to more rational therapeutic approaches.
13-CND/DIR
PHS6M0 [Rev 1 84)
utKAKiMtNT OF HEALTH AND HUMAN SERVICES -PUBLIC HEALTHS, :
NOTICE OF INTRAMURAL RESEARCH PROJECT
PERIOD COVERED
October 1, 1989 through September 30, 1990
PROJECl NUMBER
ZOI NS 02630-07 CNB
TITLE OF PROJECT (so chtncfrs or ttu. THIimun fit on on* lint bmf¥mtnih»bord»rs.)
CLINICAL, GENETIC, AND BIOCHEMICAL STUDIES OF FAMILIAL ALZHEIMER'S DISEASE
PRINCIPAL INVESTIGATOR (L,sx othor pror^suontl p,ry>n^l bolow <h, Pr.naotl Inving.ior.) <Artm«. Ml: ItborKory, tnd Institul, tmi*„on)
PI-: R. j. POUNSKY, M.D., CHIEF, CNS, CNB, NINDS
OTHERS: L.E. NEE, M.S.W SOCIAL SCIENCE ANALYST OCD, CNP, NINDS
S.M. BASER, M.D. MEDICAL STAFF FELLOW CNS, CNB, NINDS
A.M. MARIN!, M.D. SENIOR STAFF FELLOW CNS, CNB, NINDS
G. DICHIRO.M.D. CHIEF N IS, OCD,' NINDS
J. GRAFMAN, PH.D. PSYCHOLOGIST CNU, MNB, NINDS
COORPERATING UNITS (it ,n,)
GENETICS UNIT. MASS. GEN HOSP 'J. GUSELLA. P. HYSLOP) ). 5 ILLINOIS UNIV .. ( R. STRUBLE). SMID-SUD. ITALY (A BRUNI). KUNIK BAVARIA (P FROMMELT); UNIV OF
KV(j SISKEN). UNIV OFCA(0 COX. V SHEFFIELD). UMONj (W C LAMBERT). UNIV OF GOTEBORG. SWEDEN (A MCRAE. A. OAMLSTROM). CEDARS-SINAI, CA (S PULST)
LAB/BRANCH
CLINICAL NEUROSCIENCE BRANCH, CNP, DIR, NINDS
SECTION
CLINICAL NEUROPHARMACOLOGY SECTION
INSTITUTE AND LOCATION
NINDS. NIH, BETHESDA, MP 20892
TOTAL MAN-YEARS:
4.0
PROFESSIONAL: 3 Q
OTHER: 1 q
CHECK APPROPRIATE BOX(ES)
[I] (a)Human subjects H (b) Human tissues I I (c) Neither
I x | (a1) Minors
|x| (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Alzheimer's disease (AD) is the most common cause of irreversible, chronic dementia.
Although AD may be familial in only one-third of all cases, the main justification for
studying autosomal dominant cases lies in the accuracy of diagnosis which may be
inferred through post-mortem examination of other affected family members. More
than 250 members of 20 pedigrees with an autosomal dominant form of AD have had
skin fibroblast and peripheral blood lymphoblast cultures established. These cultures
serve as a renewable source of DNA and cell lines for genetic linkage, viability, and
biochemical studies. Recombinant DNA technology has been applied to perform
genetic linkage studies in these families with inherited AD. Additional families have
provided furtner confirmation of our earlier finding of a region of chromosome 21 that
is linked to AD in early onset cases; however, a single marker has not been identified.
In a collaborative study of 70 families, the age-at-onset showed a bimodal distribution.
The lifetime AD risk for at-risk offspring in early onset cases was 53%, significantly
lower than 86% in late onset AD. Transmission pattern and age-at-onset provide
further evidence for heterogeneity. Longitudinal investigation of
neurotransmitters/neuropeptides in crebrospinal fluid (CSF) continues to confirm our
previous findings; three at-risk subjects, among the subgroups with low CSF MHPG and
somatostatin, have developed dementia. Anti-neuronal antibodies have also been
detected in the CSF from AD patients. These antibodies recognize specific neuronal
populations in the adult rat central nervous system. In developing rat brain these
antibodies recognize specific membrane receptors ir. macropnagic microglia.
Inflammation and similar immune mechanisms may play a pathogenetic role in AD.
Cerebral glucose metabolism is significantly reduced in the parietotemporal regions
with some occipital and temporal involvement. A mild (10-17%) reduction was
observed in three at-risk subjects who later developed AD.
14-CNB/DIR
PHS6040IP-X 1/B4I
ucrMi\ i men i ur i-icml in <ii»^ ■ iui«im« jtn ni.u - ■ w — —
Z01 NS 02716-05
NOTICE OF INTRAMURAL RESEARCH PROJECT
CNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (so c/» ncimr,oi mi Till* mutt fit on ont lint btl-tmn tnt bordtrl.)
Neurotoxins and Animal Models of Neurological Diseases
PRINCIPAL INVESTIGATOR am oci«<pro»»jwo/M' w'ionn./ixio* f*. *inupj/(n««jt.0i(<vj <*,m« Mil, utborttory. tnd innttuffftlittion)
PI: Irwin J. Kopin.M.D., Chief, CNB, Director, DIR, NINDS
Others (CNB, NINDS):
Virginia Weise, Chemist; AlessandroZuddas, M.D., Visiting Fellow; Linda Porrino, Ph.D., Research
Psychologist; Mark Duncan, Ph.D., Visiting Fellow; Ann M. Marini, M.D., Sr. Staff Fellow: Margaret
Palmatier, Ph.D., Staff Fellow; Judith Harvey-White, B.S., Technician.
Sanford P. Markey, Ph.D., Pharmacologist, LCS, NIMH
Krzysztof S. Bankiewicz, M.D., Visiting Fellow, SNB
COORPERATING UNITS „!,»,)
Surgical Neurology Branch, NINDS
Laboratory of Clinical Science, NIMH
LAB/BRANCH
Clinical Neuroscience Branch, CNP
SECTION
Aminerqic Mechanisms
INSTITUTE ANO LOCATION
NINDS, NIH, Bethesda, MP 20892
TOTAL MAN-YEARS: --
PROFESSIONAL: 2 2
OTHER: 1 0
CHECK APPROPRIATE BOX(ES)
LJ (a) Human subjects Q (b) Human tissues l"x~1 (c) Neither
^ (a1) Minors
(a2) Interviews
□
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The objectives of this project are to identify and characterize toxins which target specific neuronal
systems, determine the mechanisms of action of these toxins, and use them to produce animal models of
human neurologic disorders. At present the main focus is on two toxins, 1-methyl-4-phenyl-1, 2,3,6-
tetrahydropyridme (MPTP) and beta-methylaminoalanine (BMAA).
MPTP is administered via the internal carotid artery to produce hemiparkinsonism in monkeys. These
animals are used to evaluate changes cerebral metabolism (as indicated by autoradiographic
determination of the uptake of 14C-2-deoxyglucose) as a result of the toxin-induced neurologic disorder
and to examine the efficacy of tissue implants in alleviating the neurologic deficits. Biochemical and
behavioral abnormalities are examined to evaluate the efficacy of tissue implants to replace destroyed
neurons or enhance sprouting of surviving neurons. In other studies, mice are treated with various doses
of MPTP and with substances which may potentiate its toxic effects. Fluid obtained from gelfoam placed
in MPTP and surgically damaged brain is examined for substances which stimulate growth of neurons in
tissue culture. Enhancement of MPTP toxicity by low doses of ethanol or acetaldehyde is not due to any
effect on MPTP metabolism or retention of its metabolite, MPPVThis enhancement of toxicity has now
been shown to be negligible in young (7-day-old) mice, but marked in older (8-week-old) animals.
Furthermore, ethanol or acetaldehyde appears to enhance the specificity of the toxin in targeting
specific dopaminergic neurons. Gel foam plugs removed from cavities made in the caudate nucleus of
MPTP parkinsonian monkeys contains soluble substance(s) which promotes neurite outgrowth in tissue
cultured neurons. Similar substances have been found in fetal tissues, including amnion.
BMAA has been implicated as responsible for the putative toxicity of flour produced from cycas circinalis
and cited as one etiologic factor in the Amytrophic lateral sclerosis-parkinsonism-dementia complex of
Guam (Guamanian ALS-PD). Samples of a flour from Guam as well as cycad plants from various sources
have been assayed (by GC-MS) Processing of the cycad to produce the flour removes greater than 80%
of the BMAA (usually over 95%) and the residual BMAA content is not sufficient to reach even a small
fraction of the amounts reported to be toxic in monkeys. The BMAA contents of flour from various
sources have been compared with toxicity elicited in vitro using granule cell tissue cultures. At the
concentrations in flour, BMAA is not toxic to these cells, but some of the flour samples were toxic High
concentrations of zinc were found in only toxic samples. Galvanized metal containers might have been
used in preparation of these samples of flour, implicating zinc as a toxic metal related to Guamanian
ALS-PD
15-CNB/DIR
Phs(,W0(R«. I Ml
DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HtALI H btKVILt
NOTICE OF INTRAMURAL RESEARCH PROJECT
r(\u^v. i m w.'
Z01 NS 02717-05
CNB
PFRI0DC0VERED
October 1 , 1 989 through September 30, 1 990
TITLE OF PROJECT (SO ctunatrt or Itsi- m* must fh on on* lint Mmm Itn bordtrt.)
Biochemical Evaluation of Aminergic Function During Responses to Stress and in Disease States
PRINCIPAL INVESTIGATOR (Unolhtr proftsaontl otrsonntl btlonr thi Frlnctp*! tnvsiigt'or.) (hltm*. tnlt. KbwctOfy. tndinstituf Mffilmlon)
PI: Irwin J. Kopin, M.D., Chief, CNB, Director, DIR, NINDS
Others (CNB, NINDS):
Graeme Eisenhofer, Ph.D., Visiting Associate, CNB, DIR, NINDS; Katalin Szemeredi, Ph.D., Visiting Fellow,
CNB, DIR, NINDS; Anna Starosta, M.D., Visiting Fellow, CNB, DIR, NINDS
Ronald Polinsky, M.D., Chief, Clinical Neuropharmacology Section
Virginia Weise, Chemist, CNB, NINDS; Judith Harvey-White, B.S., Technician., CNB, NINDS
David Goldstein, M.D., Ph.D., Medical Officer, HEB, NHLBI
foWftWflUKte
Laboratory of Clinical Science, NIMH
LAB/BRANCH
Clinical Neuroscience Branch, CNP
SECTION
Aminerqic Mechanisms
INSTITUTE AND LOCATION
NINDS. NIH. Bethesda. MP 20892
TOTAL MAN-YEARS: 3J PROFESSIONAL: 22
OTHER:
1.0
CHECK APPROPRIATE BOX(ES)
□ (a)Human subjects ] (b) Human tissues IT] (c) Neither
J (a1) Minors
II (a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
The main objectives of this project are (1) to determine the physiologic significance of alterations in
plasma levels and urinary excretion rates of the catecholamines, their metabolites and their precursor,
DOPA; (2) to develop in animals and humans clinically useful methods for assessing catecholaminergic
function; and (3) to apply these methods to examine function of catecholaminergic neurons in the
peripheral sympathetic nervous system and in brain.
Changes in tissue or plasma levels or urinary excretion rates of catecholamines and their metabolites,
and tissue tyrosine hydroxylase levels are evaluated, during and after stress, in response to
pharmacologic treatments, or in disease states in animals, in normal subjects, and in patients with
various neurologic or related disorders (autonomic dysfunction, Parkinson's disease, hypertension, etc).
Use of !8.f labelled dopamine has been developed for imaging by positron emission tomography (PET)
peripheral sympathetic activity in vivo in dogs and is awaiting application to humans.
In rats, plasma levels of DOPA parallel pharmacologic or stress-induced enhancement of norepinephrine
release, even after adrenalmedullectomy. Tyrosine hydroxylase levels in tissues do not reflect the rate of
catecholamine production, indicating that tyrosine hydroxylation, can be regulated independently of
the levels of tyrosine hydroxylase. Chronic elevation of plasma glucocorticoid levels elevates rat blood
pressure but depresses plasma norepinephrine levels. Baroreceptor sensitivity is unaltered and pressor
responses to phenylephrine are normal. There is, however, an increase in the tachycardia induced by
isoproterenol, indicating that cardiac beta-ad renoceptors are hyperresponsive. The mechanism of
hypertension attending chronic elevation of corticosteriods differs from that in other forms of
hypertension.
Most patients with pure autonomic failure were found to have low plasma levels of DOPA as well as of
norepinephrine, DHPG and DOPAC, consistent with loss of peripheral sympathetic neurons, whereas in
most patients with multiple system atrophy, levels of DOPA, DHPG and DOPAC are generally normal; this
is consistent with normal catecholamine biosynthesis in these patients who are believed to have intact
peripheral sympathetic neurons but diminished nerve impulse traffic from the central nervous system.
16-CNB/DIR
PHS *M0 (Rtv. 1«l
DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT
PROJECT NUMBER
Z01 NS 02752-03
CNB
PERIOD COVERED
October 1, 1989 through September 30, 1990
TITLE OF PROJECT (»ch»r»<t«rj or ten. Tit (• nvn fir on on* lint bttw—rt tfx borrtt'l J
Regulation of Synthesis and Expression of Neurotrophic Agents and Neuropeptides
PRINCIPAL INVESTIGATOR fUn othtr provisional ptrsonr— I bltow 1h* Principal trtvmrtiOHOr.) fNama. titlt. laboratory, and inttrtuta afiltation)
Joan P. Schwartz, Ph.D., Research Chemist, CNB, NINDS
Others:
Kunihiko Mitsuo, Ph.D., Visiting Fellow, CNB, NINDS
Margaret Palmatier, Ph.D., Staff Fellow, CNB, NINDS
COOPERATING UNITS (Hany)
LAB/BRANCH
Clinical Neuroscience Branch, CNP
SECTION
Section on Aminerqic Mechanisms
INSTITUTE AND LOCATION
NINDS, NIH, Bethesda. MP 20892
TOTAL MAN-YEARS: -
PROFESSIONAL: , 1
OTHER: 1 q
CHECK APPROPRIATE BOX(ES)
O (a)JHuman subjects □ (b) Human tissues ["*! (c) Neither
(a1) Minors
(a2) Interviews
SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)
Evidence suggests that parallel biochemical and regulatory processes occur during normal development
and following various forms of CNS injury. Among these areas of particular interest are: (1)
identification of CNS neurotrophic factors, and (2) the analysis of the regulation of neuropeptide gene
expression during development and in response to injury. Studies are underway to identify trophic
factors produced in specific model systems, since recent evidence suggests that a family of nerve growth
factors (NGF) exists, each specific for certain populations of neurons. An NGF-like factor increases in the
cerebellum of the pcd mutant mouse as the Purkinje cells die out and astrocytes proliferate. In another
injury paradigm, multiple cortical lesions are made in rat brain: one week later, RNA is prepared from
various brain regions to be analyzed for any change in NGF-like mRNAs relative to unlesioned brain.
MPTP-lesioned animals (both mice and monkeys) represent a Parkinson-like model in which changes in
NGF-like molecules are being examined. Since astrocytes can synthesize NGF, primary cultures of
astrocytes are being used to determine factors which regulate NGF gene transcription as well as to assess
production of other potential trophic factors. Reactive astrocytes are prepared from regions affected by
the various injuries and their production of trophic factors compared to that of control astrocytes
Potential neurotrophic functions for neuropeptides in early CNS development are being explored in
several model culture systems.
At the same time, these injury models can be evaluated for changes in neuropeptide and/or
neurotransmitter synthesis occurring in response to the lesions. One can derive an estimate of peptide
turnover by combining measurements of the precursor mRNA, the precursor, and the peptide. Our
studies have demonstrated that peptides are differentially regulated by such chronic drug treatments as
reserpine, haloperidol, 6-hydroxydopamine or 5,7-dihydroxytryptamine. Work is in progress to
determine the effects of CNS injury and recovery, including MPTP treatment, on various neuropeptides
as well as such neurotransmitter synthetic enzymes as tyrosine hydroxylase and GAD, and the dopamine
D2 receptor.
17-CNB/DIR
PHS6W0(R«v 1*4)
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