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Fine structural changes in the rat striatum after local injections of kainic acid
174
Brain Research, ~29 (i 977) 174-180 (:~ Elsevier/North-Holland Biomedical Press
Fine structural changes in the rat striatum after local injections of kainic acid
T.
HATTORI and
E. G. McGEER
Division of Neurological Sciences, Department of Psychiatry, University oJ British Columbia. Vancouver, B.C. V6T I W5 (Canada)
(Accepted March 10th, 1977)
The stereotaxic injections of kainic acid into the striatum have recently been shown to cause the depletion of two neurotransmitter synthetic enzymes, choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD), while tyrosine hydroxylase (TH) activity was actually increased 1,5. Light microscopic analysis o f the affected area of the striatum showed a profound loss of neurons, with the space being largely occupied by increased glial elements 1.5. Similar morphological changes have been described in the arcuate nucleus, where kainic acid 9 or monosodium glutamate s preferentially destroyed the perikarya and dendrites, while axons passing through or terminating in the region seemed to be unaffected. The present study describes fine structural changes of various neurona~ components which occur as a function of time after local injections of kainic acid into the striatum. Kainic acid 1.25-5 nmoles (obtained from Sigma Chemical Co. l, dissolved in 0.5 /~1 isotonic saline, was injected stereotaxically into the rat striaturn, coordinates: AP 8.38, L 2.8, DV ? 0.2 according to the K6nig and Klippel atlas 3. At intervals of 5, 10. 24, 48 and 72 h each, two animals were fixed by perfusion with 4".~, paraformaldehyde, 0.5°o glutaraldehyde and 0.54'}~i glucose in 0.01 M phosphate buffer (pH 7.41. Both the target area and the rostral striatum most remote from the injection site were dissected out, briefly rinsed in buffer and postfixed with I 'Yo osmium tetroxide. The sections were stained with uranyl acetate and lead citrate and processed for electron microscopic examination. At 5 h after the local injection of 1.25 nmoles of kainic acid into the striatum, a considerable number of degenerating cell soma could be detected in the injection area. They were characterized by a considerable number of vacuoles distributed in the electron dense cytoplasm. Dilatation of endoplasmic reticulum was also observed (Fig. 1). Major dendrites did not show much sign of degeneration at this stage. By tO h after the injection, many dendrites had become electron-dense, containing many vacuoles in their cytoplasm (Fig. 2A). In some soma and dendrites, where vacuolization had proceeded to its maximal level, all the interior materials had disappeared to leave empty, swollen structures (Fig. 2B). At both 5 and 10 h, there was no detectable
175
Fig. 1. Degeneration of cell soma 5 h after injection of 1.25 nmoles kainic acid into the striatum. Many vacuoles and dilated endoplasmic reticu[um (arrows) can be noticed. Magnification ~ 8100.
degeneration except within 2-3 m m o f the injection site, although at further distances astrocytic elements seemed to occupy more space than in the non-injected contralateral striatum. Unmyelinated axons and terminal boutons showed no signs of degeneration even at the injection site. At 24-48 h, degeneration seemed to involve the whole striatum. The swelling and vacuolization o f soma and dendrites had reached the maximum level and many dendrites were either empty or contained diffusely distributed debris. They were surrounded by watery astrocytes, which now occupied a very large percentage o f the total area. At 48 h after the injection dark-type degeneration was first seen in unmyelinated axons as well as in some small myelinated axons. After 72 h, degenerating soma and dendrites were very rarely encountered and instead a significant number of degenerating terminal boutons could be detected. They were mostly small (0.5-0.8/~m) and made asymmetrical contacts with dendritic spines (Fig. 3A). Some clear symmetrical synapses were also seen to degenerate (Fig. 3B).
176
Fig. 2. Degeneration of soma and dendrites 10 h after intrastriatal injection of 1.25 nmoles kainic acid. A: electron-dense dendrite containing many vacuoles. Magnification -: 22,140. B: maximum state of vacuolization in soma, which contains only cell debris and membrane structures. Arrow indicates synaptic contact on the swollen dendrite. Magnification x 37,270.
F r o m those d a t a it can be c o n c l u d e d that the striatal degeneration, which is i n d u c e d by kainic acid, occurs first in cell soma. It proceeds to the dendritic elements within 10 h, to small axons at 24-48 h and finally reaches t e r m i n a l b o u t o n s at 72 h W i t h a n increasing dose o f kainic acid (2.5-5 nmoles), the rate o f degeneration was accelerated, resulting in t o t a l f r a g m e n t a t i o n o f the whole injected a r e a within 10 h. Consequently, the s t r i a t u m h a d the a p p e a r a n c e o f very p o o r l y fixed tissue. P o o r
177
Fig. 3. Degeneration of terminal boutons 72 h after injection of 1.25 nmoles kainic acid. A: degenerating axospinous asymmetrical synapse (arrowhead). Magnification x 38,270. B: engulfed symmetrical synapse in astrocyte. Arrow indicates synaptic contact. Magnification x 38,270.
178 TABLE I G A D aml CA T in striatum injected with kainic acid as percent o1' control
Enzyme activities were determined on aliquots of 1 : 10 (w/v) 0.32 M sucrose homogeaates of the striata by previously reported radioactive methods ~,7. Protein was determined by the method of Lowry et al. 4. Results were adjusted to a protein basis and then the activity in the injected striatum expressed as a percentage of that in the contralateral (control) striatum. Control values were not significantly different from those found in untreated rats. Time between injection and sacrifice
No. ~?[' animals
GAD ( mean :~: S.E. )
CAT (mean :i S.E.)
84:1 86 67 ~ 50 ~ 31 :i 38 :-k 28 2: 33 :=
(a) 5 nmoles in 1 /tl 9.5h 12 h I day 2days 3 days 4days 9days 40 days
3 5 9 5 9 5 6 4
71 86 62 55 37 42 41 35
(b) 3 nmoles in 1 / d 3days 7 days
II 4
67 2 6% 62 ~: 11 741
49 ~ 5% 52 f 2%
63 t 8% 69 :[ 4'~o
57 ~ 6"/0 65 ~: 7%
(c) 2.5 nmoles in 1 /fl 3 days 7 days
8 3
~ 5% :+~: 10%;
± 6% - 7% i 4% i 7% : 9% ~: 4%
3% 9% 6% 8% 6% 13% 6% 4%
fixation was not the cause, however, since the fine structure o f the contralateral s t r i a t u m was intact. T a b l e Ia indicates the time course o f activity changes o f choline acetyltransferase ( C A T ) a n d glutamic acid d e c a r b o x y l a s e ( G A D ) after striatal injections o f 5 nmoles kainic acid. There was a b o u t 15 o.~ d r o p in the activity o f these two enzymes at 12 h time period. The activities were only a b o u t 35 ~ o f c o n t r o l at 72 h, a n d this residual level o f enzymic activities was m a i n t a i n e d even 40 days after injection. Thus, loss o f enzymic activities seemed to be c o m p l e t e by 72 h after the local injection, and this also a p p e a r e d to be true if lesser a m o u n t s (2.5-3 nmoles) were injected (Tables I b a n d l c). F u r t h e r m o r e , there a p p e a r e d to be some shift in the p r o p o r t i o n o f enzymic activities f o u n d in the 34,000 x g s u p e r n a t a n t as indicated in T a b l e II. A l t h o u g h very few rats were studied in this regard, the results f r o m rat to rat were quite consistent. The p r o d u c t i o n o f significant enzymic losses in the s t r i a t u m as a whole requires larger doses o f kainic acid t h a n those needed to p r o d u c e electron microscopically detectable changes in the fine structure, but it is interesting t h a t the t i m e p e r i o d (72 h) by which enzymic losses were substantially c o m p l e t e is the same as t h a t by which all the c o n c e r n e d n e u r o n a l c o m p a r t m e n t s h a d shown degenerative changes. The serial o r d e r o f d e s t r u c t i o n o b s e r v e d ( s o m a -+ dendrites -+ axons ~ b o u t o n s ) suggests t h a t the axons a n d b o u t o n s are less susceptible t h a n are s o m a a n d dendrites to the direct effects
179 TABLE il Enzymic activities in supernatant fractions from the striatum
Striata were weighed and homogenized in 0.32 M sucrose and centrifuged at 34,000 ~, g/60 min to obtain the supernatant and P2 pellet fractions. The P2 pellet was resuspended in the supernatant volume of 0.32 M sucrose and enzyme determinations done on the initial homogenates and fractions. Per cent of total (mean ± S.E.)
Control 1 day post 5 nmoles k.a. 3 days post 5 nmoles k.a.
GAD
CA T
TH
N
37.0 ± 1.8~ 24.7 ± 1.5~* 30.3 ± 1.8~**
20.5 ~ 1.2% 16.3 ± 1.2~** 21.7 ± 0.3%
15.2 ± 0.5~ 17.7 ± 2.2 24.0 ± 1.7"
6 3 3
Significant differences between test and control groups: * P < 0.001 ; ** P < 0.05.
(possibly excitotoxic) o f kainic acid and m a y therefore be m o r e c a p a b l e o f a c c o m m o d a t i n g to p r o l o n g e d repetitive stimulation. If one hypothesizes that cell s o m a enzymic activity is largely s u p e r n a t a n t while most o f the nerve ending activity precipitates with the P,,, pellet, the a p p a r e n t shift t o w a r d s less s u p e r n a t a n t C A T a n d G A D at 24 h after kainic acid injections is consistent with earlier destruction o f cell s o m a as c o m p a r e d with nerve endings. A t 3 days, when the degeneration o f affected nerve endings in the s t r i a t u m is p r o b a b l y substantially complete, the percentage o f C A T f o u n d in the s u p e r n a t a n t is the same as in controls while the percentage o f G A D in the s u p e r n a t a n t is still less t h a n control but a p p a r e n t l y greater t h a n f o u n d at one day. These results are consistent with the affected C A T - c o n t a i n i n g neurons being almost entirely intrinsic while both intrinsic a n d extrinsic G A D - c o n t a i n i n g neurons are destroyed. D e s t r u c t i o n o f extrinsic g a b a n e r g i c neurons had previously been suggested on the basis o f losses in G A D in the substantia nigra following striataI injections o f kainic acid 1. The a p p a r e n t shift at 3 days in tyrosine h y d r o x y l a s e activity f r o m the particulate to the s u p e r n a t a n t fraction is m o r e difficult to explain but m a y reflect some change in enzyme p r o p e r t i e s occurring in response to loss o f f e e d b a c k f r o m the p o s t - s y n a p t i c neurons. T w o distinct types o f terminal b o u t o n s were affected by kainic acid; one type being small a s y m m e t r i c a l and the other symmetrical boutons. These c o r r e s p o n d generally to the types o f m o r p h o l o g y previously suggested for cholinergic and g a b a n e r g i c b o u t o n s o f the striatum z and other brain areas 10, a l t h o u g h the degenerative processes rendered absolute identification difficult and other, chemically unidentified types o f neurons, m a y well be affected by the kainic acid injections.
1 Coyle, J. T. and Schwarcz, R., Lesion of striatal neurons with kainic acid provides a model for Huntington's chorea, Nature (Lond.), 263 (1976) 244 246. 2 Hanori, T., Singh, V. K., McGeer, E. G. and McGeer, P. L., lmmunohistochemical localization of choline acetyltransferase containing neostriatal neurons and their relationship with dopaminergic synapses, Brain Research, 102 (1976) 164-173. 3 K6nig, J. F. R. and Klippel, R. A., The Rat Brain, Robert E. Krieger Publishing Co. Inc., Huntington, New York, 1967.
180 4 Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, E. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265 275. 5 McGeer, E. G. and McGeer, P. L., Duplication of biochemical changes of Huntington's chorea by intrastriatal injections of glutamic and kainic acids, Nature (Loud.), 263 (1976) 517- 519. 6 McGeer, P. L. and McGeer, E. G., Cholinergic enzyme systems in Parkinson's disease, Ar~h. Neurol. (Chic.), 25 (1971) 265-268. 7 McGeer, P. L. and McGeer, E. G., Evidence for glutamic acid decarboxylase containing interneurons in the neostriatum, Brain Res'earch, 91 (1975) 331 335. 8 0 l n e y , J. W. and Sharpe, L. G., Brain lesions in an infant rhesus monkey treated with monosodium glutamate, Science, 166 (1969) 386 388. 9 0 l n e y , J. W., Sharpe, L. G. and de Gubareff, Y., Excitotoxic amino acids, Net#osci. Ahstr.~ 5 (1975) 371. 10 Ribak, C. E., Vaughn, J. E., Saito, K., Barber, R. and Roberts, E., Inamunocytochemical localization of glutamate decarboxylase in rat substantia nigra, Brain Researeh, 116 (1976) 287--298.
Brain Research, ~29 (i 977) 174-180 (:~ Elsevier/North-Holland Biomedical Press
Fine structural changes in the rat striatum after local injections of kainic acid
T.
HATTORI and
E. G. McGEER
Division of Neurological Sciences, Department of Psychiatry, University oJ British Columbia. Vancouver, B.C. V6T I W5 (Canada)
(Accepted March 10th, 1977)
The stereotaxic injections of kainic acid into the striatum have recently been shown to cause the depletion of two neurotransmitter synthetic enzymes, choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD), while tyrosine hydroxylase (TH) activity was actually increased 1,5. Light microscopic analysis o f the affected area of the striatum showed a profound loss of neurons, with the space being largely occupied by increased glial elements 1.5. Similar morphological changes have been described in the arcuate nucleus, where kainic acid 9 or monosodium glutamate s preferentially destroyed the perikarya and dendrites, while axons passing through or terminating in the region seemed to be unaffected. The present study describes fine structural changes of various neurona~ components which occur as a function of time after local injections of kainic acid into the striatum. Kainic acid 1.25-5 nmoles (obtained from Sigma Chemical Co. l, dissolved in 0.5 /~1 isotonic saline, was injected stereotaxically into the rat striaturn, coordinates: AP 8.38, L 2.8, DV ? 0.2 according to the K6nig and Klippel atlas 3. At intervals of 5, 10. 24, 48 and 72 h each, two animals were fixed by perfusion with 4".~, paraformaldehyde, 0.5°o glutaraldehyde and 0.54'}~i glucose in 0.01 M phosphate buffer (pH 7.41. Both the target area and the rostral striatum most remote from the injection site were dissected out, briefly rinsed in buffer and postfixed with I 'Yo osmium tetroxide. The sections were stained with uranyl acetate and lead citrate and processed for electron microscopic examination. At 5 h after the local injection of 1.25 nmoles of kainic acid into the striatum, a considerable number of degenerating cell soma could be detected in the injection area. They were characterized by a considerable number of vacuoles distributed in the electron dense cytoplasm. Dilatation of endoplasmic reticulum was also observed (Fig. 1). Major dendrites did not show much sign of degeneration at this stage. By tO h after the injection, many dendrites had become electron-dense, containing many vacuoles in their cytoplasm (Fig. 2A). In some soma and dendrites, where vacuolization had proceeded to its maximal level, all the interior materials had disappeared to leave empty, swollen structures (Fig. 2B). At both 5 and 10 h, there was no detectable
175
Fig. 1. Degeneration of cell soma 5 h after injection of 1.25 nmoles kainic acid into the striatum. Many vacuoles and dilated endoplasmic reticu[um (arrows) can be noticed. Magnification ~ 8100.
degeneration except within 2-3 m m o f the injection site, although at further distances astrocytic elements seemed to occupy more space than in the non-injected contralateral striatum. Unmyelinated axons and terminal boutons showed no signs of degeneration even at the injection site. At 24-48 h, degeneration seemed to involve the whole striatum. The swelling and vacuolization o f soma and dendrites had reached the maximum level and many dendrites were either empty or contained diffusely distributed debris. They were surrounded by watery astrocytes, which now occupied a very large percentage o f the total area. At 48 h after the injection dark-type degeneration was first seen in unmyelinated axons as well as in some small myelinated axons. After 72 h, degenerating soma and dendrites were very rarely encountered and instead a significant number of degenerating terminal boutons could be detected. They were mostly small (0.5-0.8/~m) and made asymmetrical contacts with dendritic spines (Fig. 3A). Some clear symmetrical synapses were also seen to degenerate (Fig. 3B).
176
Fig. 2. Degeneration of soma and dendrites 10 h after intrastriatal injection of 1.25 nmoles kainic acid. A: electron-dense dendrite containing many vacuoles. Magnification -: 22,140. B: maximum state of vacuolization in soma, which contains only cell debris and membrane structures. Arrow indicates synaptic contact on the swollen dendrite. Magnification x 37,270.
F r o m those d a t a it can be c o n c l u d e d that the striatal degeneration, which is i n d u c e d by kainic acid, occurs first in cell soma. It proceeds to the dendritic elements within 10 h, to small axons at 24-48 h and finally reaches t e r m i n a l b o u t o n s at 72 h W i t h a n increasing dose o f kainic acid (2.5-5 nmoles), the rate o f degeneration was accelerated, resulting in t o t a l f r a g m e n t a t i o n o f the whole injected a r e a within 10 h. Consequently, the s t r i a t u m h a d the a p p e a r a n c e o f very p o o r l y fixed tissue. P o o r
177
Fig. 3. Degeneration of terminal boutons 72 h after injection of 1.25 nmoles kainic acid. A: degenerating axospinous asymmetrical synapse (arrowhead). Magnification x 38,270. B: engulfed symmetrical synapse in astrocyte. Arrow indicates synaptic contact. Magnification x 38,270.
178 TABLE I G A D aml CA T in striatum injected with kainic acid as percent o1' control
Enzyme activities were determined on aliquots of 1 : 10 (w/v) 0.32 M sucrose homogeaates of the striata by previously reported radioactive methods ~,7. Protein was determined by the method of Lowry et al. 4. Results were adjusted to a protein basis and then the activity in the injected striatum expressed as a percentage of that in the contralateral (control) striatum. Control values were not significantly different from those found in untreated rats. Time between injection and sacrifice
No. ~?[' animals
GAD ( mean :~: S.E. )
CAT (mean :i S.E.)
84:1 86 67 ~ 50 ~ 31 :i 38 :-k 28 2: 33 :=
(a) 5 nmoles in 1 /tl 9.5h 12 h I day 2days 3 days 4days 9days 40 days
3 5 9 5 9 5 6 4
71 86 62 55 37 42 41 35
(b) 3 nmoles in 1 / d 3days 7 days
II 4
67 2 6% 62 ~: 11 741
49 ~ 5% 52 f 2%
63 t 8% 69 :[ 4'~o
57 ~ 6"/0 65 ~: 7%
(c) 2.5 nmoles in 1 /fl 3 days 7 days
8 3
~ 5% :+~: 10%;
± 6% - 7% i 4% i 7% : 9% ~: 4%
3% 9% 6% 8% 6% 13% 6% 4%
fixation was not the cause, however, since the fine structure o f the contralateral s t r i a t u m was intact. T a b l e Ia indicates the time course o f activity changes o f choline acetyltransferase ( C A T ) a n d glutamic acid d e c a r b o x y l a s e ( G A D ) after striatal injections o f 5 nmoles kainic acid. There was a b o u t 15 o.~ d r o p in the activity o f these two enzymes at 12 h time period. The activities were only a b o u t 35 ~ o f c o n t r o l at 72 h, a n d this residual level o f enzymic activities was m a i n t a i n e d even 40 days after injection. Thus, loss o f enzymic activities seemed to be c o m p l e t e by 72 h after the local injection, and this also a p p e a r e d to be true if lesser a m o u n t s (2.5-3 nmoles) were injected (Tables I b a n d l c). F u r t h e r m o r e , there a p p e a r e d to be some shift in the p r o p o r t i o n o f enzymic activities f o u n d in the 34,000 x g s u p e r n a t a n t as indicated in T a b l e II. A l t h o u g h very few rats were studied in this regard, the results f r o m rat to rat were quite consistent. The p r o d u c t i o n o f significant enzymic losses in the s t r i a t u m as a whole requires larger doses o f kainic acid t h a n those needed to p r o d u c e electron microscopically detectable changes in the fine structure, but it is interesting t h a t the t i m e p e r i o d (72 h) by which enzymic losses were substantially c o m p l e t e is the same as t h a t by which all the c o n c e r n e d n e u r o n a l c o m p a r t m e n t s h a d shown degenerative changes. The serial o r d e r o f d e s t r u c t i o n o b s e r v e d ( s o m a -+ dendrites -+ axons ~ b o u t o n s ) suggests t h a t the axons a n d b o u t o n s are less susceptible t h a n are s o m a a n d dendrites to the direct effects
179 TABLE il Enzymic activities in supernatant fractions from the striatum
Striata were weighed and homogenized in 0.32 M sucrose and centrifuged at 34,000 ~, g/60 min to obtain the supernatant and P2 pellet fractions. The P2 pellet was resuspended in the supernatant volume of 0.32 M sucrose and enzyme determinations done on the initial homogenates and fractions. Per cent of total (mean ± S.E.)
Control 1 day post 5 nmoles k.a. 3 days post 5 nmoles k.a.
GAD
CA T
TH
N
37.0 ± 1.8~ 24.7 ± 1.5~* 30.3 ± 1.8~**
20.5 ~ 1.2% 16.3 ± 1.2~** 21.7 ± 0.3%
15.2 ± 0.5~ 17.7 ± 2.2 24.0 ± 1.7"
6 3 3
Significant differences between test and control groups: * P < 0.001 ; ** P < 0.05.
(possibly excitotoxic) o f kainic acid and m a y therefore be m o r e c a p a b l e o f a c c o m m o d a t i n g to p r o l o n g e d repetitive stimulation. If one hypothesizes that cell s o m a enzymic activity is largely s u p e r n a t a n t while most o f the nerve ending activity precipitates with the P,,, pellet, the a p p a r e n t shift t o w a r d s less s u p e r n a t a n t C A T a n d G A D at 24 h after kainic acid injections is consistent with earlier destruction o f cell s o m a as c o m p a r e d with nerve endings. A t 3 days, when the degeneration o f affected nerve endings in the s t r i a t u m is p r o b a b l y substantially complete, the percentage o f C A T f o u n d in the s u p e r n a t a n t is the same as in controls while the percentage o f G A D in the s u p e r n a t a n t is still less t h a n control but a p p a r e n t l y greater t h a n f o u n d at one day. These results are consistent with the affected C A T - c o n t a i n i n g neurons being almost entirely intrinsic while both intrinsic a n d extrinsic G A D - c o n t a i n i n g neurons are destroyed. D e s t r u c t i o n o f extrinsic g a b a n e r g i c neurons had previously been suggested on the basis o f losses in G A D in the substantia nigra following striataI injections o f kainic acid 1. The a p p a r e n t shift at 3 days in tyrosine h y d r o x y l a s e activity f r o m the particulate to the s u p e r n a t a n t fraction is m o r e difficult to explain but m a y reflect some change in enzyme p r o p e r t i e s occurring in response to loss o f f e e d b a c k f r o m the p o s t - s y n a p t i c neurons. T w o distinct types o f terminal b o u t o n s were affected by kainic acid; one type being small a s y m m e t r i c a l and the other symmetrical boutons. These c o r r e s p o n d generally to the types o f m o r p h o l o g y previously suggested for cholinergic and g a b a n e r g i c b o u t o n s o f the striatum z and other brain areas 10, a l t h o u g h the degenerative processes rendered absolute identification difficult and other, chemically unidentified types o f neurons, m a y well be affected by the kainic acid injections.
1 Coyle, J. T. and Schwarcz, R., Lesion of striatal neurons with kainic acid provides a model for Huntington's chorea, Nature (Lond.), 263 (1976) 244 246. 2 Hanori, T., Singh, V. K., McGeer, E. G. and McGeer, P. L., lmmunohistochemical localization of choline acetyltransferase containing neostriatal neurons and their relationship with dopaminergic synapses, Brain Research, 102 (1976) 164-173. 3 K6nig, J. F. R. and Klippel, R. A., The Rat Brain, Robert E. Krieger Publishing Co. Inc., Huntington, New York, 1967.
180 4 Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, E. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265 275. 5 McGeer, E. G. and McGeer, P. L., Duplication of biochemical changes of Huntington's chorea by intrastriatal injections of glutamic and kainic acids, Nature (Loud.), 263 (1976) 517- 519. 6 McGeer, P. L. and McGeer, E. G., Cholinergic enzyme systems in Parkinson's disease, Ar~h. Neurol. (Chic.), 25 (1971) 265-268. 7 McGeer, P. L. and McGeer, E. G., Evidence for glutamic acid decarboxylase containing interneurons in the neostriatum, Brain Res'earch, 91 (1975) 331 335. 8 0 l n e y , J. W. and Sharpe, L. G., Brain lesions in an infant rhesus monkey treated with monosodium glutamate, Science, 166 (1969) 386 388. 9 0 l n e y , J. W., Sharpe, L. G. and de Gubareff, Y., Excitotoxic amino acids, Net#osci. Ahstr.~ 5 (1975) 371. 10 Ribak, C. E., Vaughn, J. E., Saito, K., Barber, R. and Roberts, E., Inamunocytochemical localization of glutamate decarboxylase in rat substantia nigra, Brain Researeh, 116 (1976) 287--298.