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The association of [3H]d -aspartate binding and high-affinity glutamate uptake in the human brain
Neuroscienee Letters, 63 (1986) 121 124
121
Elsevier Scientific Publishers Ireland Ltd. NSL 03706
THE A S S O C I A T I O N OF [3H]D-ASPARTATE BINDING A N D HIGH-AFFINITY G L U T A M A T E UPTAKE IN THE H U M A N BRAIN
A.J. CROSS*. W.J. SKAN and P. SLATER D~7~artment ~1' Phy.~iology, University ~/ Manchester. Manchester M 13 9PT ( U.K. )
(Received July 291h, 1985; Revised version received September 29th, 1985; Accepted October Ist, 1985)
Key word~: amino acid
L-glutamate D-aspartate ~uptake nerve terminal
human brain
The binding of [3H]D-aspartate ([all]D-Asp) to human cerebellum homogenate was compared with the uptake of [qt]glutamate ([~H]Glu) by homogenates prepared from rapidly frozen human cerebral c o r l c x . There was a close correlation between the potencies of a range of drugs for inhibiting the binding of [3HIDAsp and the uptake of [~H]L-Glu. Compounds selective for postsynaptic Glu receptors were inactive. The tindings arc consislent with the labelling of high-affinity Glu uptake sites by [3H]D-Asp, which may be a valuable ligand for studying excitatory amino acid terminals in human brain.
The d i c a r b o x y l i c a m i n o acids l,-glutamate (L-GIu) a n d t,-aspartate (L-Asp) m a y together constitute the m a j o r e x c i t a t o r y n e u r o t r a n s m i t t e r s in the m a m m a l i a n C N S (reviewed in ref. 3). H o w e v e r , due to the u b i q u i t o u s n a t u r e o f these a m i n o acids in brain, it has been difficult to s t u d y m a n y aspects o f their n e u r o t r a n s m i t t e r functions. The presence o f a specific, high-affinity u p t a k e system for L-GIu has nonetheless p r o vided a simple a n d reliable m a r k e r o f g l u t a m a t e r g i c terminals in brain. This system has been used not only to assess the integrity o f e x c i t a t o r y a m i n o acid ( E A A ) terminals in b r a i n s o f e x p e r i m e n t a l a n i m a l s [14] but also to p r o v i d e a m a r k e r o f E A A nerve terminals in n e u r o a n a t o m i c a l e x p e r i m e n t s (for e x a m p l e see ref. 12). M u c h interest is c u r r e n t l y focussed on the role o f E E A s in the p a t h o l o g y o f several n e u r o p s y c h i a t r i c diseases, p a r t i c u l a r l y H u n t i n g t o n ' s disease [7], A l z h e i m e r - t y p e dem e n t i a [9] and epilepsy [8]. A n u m b e r o f studies have d e m o n s t r a t e d the presence o f a high-affinity L-Glu u p t a k e system in h u m a n b r a i n [6, 1 1]. W h i l s t this activity can be detected in b r a i n s o b t a i n e d at a u t o p s y a n d stored frozen, the activity is c o n s i d e r a bly reduced when c o m p a r e d with fresh or r a p i d l y frozen tissue [1 1]. Thus, I : G l u u p t a k e m a y not be a reliable index o f g l u t a m a t e r g i c terminals in h u m a n brain o b t a i n e d at p o s t m o r t e m e x a m i n a t i o n . Recently, ['H]D-Asp, a p o t e n t s u b s t r a t e o f the L-GIu u p t a k e system, has been p r o posed as a suitable ligand for use in b i n d i n g studies o f L-GIu u p t a k e sites in rat brain [10]. In view o f the p o t e n t i a l usefulness o f this technique, we have, in the present
*Author l\)r correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
122
study, compared the properties of [3H]D-Asp binding with [3H]L-Gtu uptake in human brain. Brains were obtained at autopsy from 3 subjects (with no known history of neuropsychiatric disease) and frozen at -40~'C. The delay between death and postmortem was 4 0 + 7 h. The cerebellar cortex was removed and homogenized in 100 vols. of 50 mM Tris-HC1, pH 7.4. The homogenate was centrifuged at 15,000 g for 10 rain and the resulting pellet washed once by rehomogenization and centrifugation. The pellet was resuspended finally in 100 vols. of 50 mM Tris-HC1, pH 7.4, containing 300 mM NaC1. Portions of this preparation (160 #1) were routinely incubated with 50 nM [3H]D-Asp (t5 Ci/mmol, Amersham, U.K.), in a total volume of 200/tl, tbr 30 min at room temperature. For saturation analysis, a range of 8 [3H]D-Asp concentrations between 25 and 4000 nM were used. Bound ligand was separated by filtration and washing with 50 mM Tris-HC1, pH 7.4 [5]. Non-specific binding was defined as that not displaced by 100 #M asparate-/~-hydroxamate. For ['H]L-Glu uptake samples of human cerebral cortex, which had been obtained within 5 h of death and rapidly frozen in isopentane at - 3 5 " C , were used. Tissue was homogenized in 10 vols. of 0.25 M sucrose+50 mM Tris-HCl, pH 7.4, and the uptake of [-~H]L-GIu (50 nM) studied as described previously [4]. [ 3 H ] D - A s p binding to human cerebellum was saturable, with a Kd of 1.0±0.3 llM and a B...... of 7.5_+ 1.1 pmol/mg protein (mean +S.E.M.; n = 3 ) . These values arc comparable to those obtained in rat brain [10]. The binding of [3HID-Asp was totally dependent on the presence of Na ~ ions (Fig. 1). Sodium chloride stimulated [3H]DAsp binding with an ED~ of 25 mM, which is similar to the effect in rat brain on [3H]Glu uptake [1] and sodium-dependent [:~H]GIu binding [13]. [3H]D-Asp binding reached equilibrium by 20 min either at 2 0 C or 2'C. The association rate constant (at 20 C ) was 0.188 /~M/min. The dissociation of [3HID-Asp at 2 0 C was rapid ( t = 2 min) with a dissociation rate constant of 0.274/min. The K,~ calculated from these kinetic constants was 1.46 #M, which agrees well with that determined t¥om equilibrium data. The potencies of a range of dicarboxylic amino acids and analogues in AI.2 o E 'ID
c 0.8
Q.
Q~ 0.4
o
5'o
,oo'
NaCl conc. (raM) Fig. 1. Dependence of [3HID-Asp binding on NaCI concentration. Membranes were incubated with increasing concentrations of NaCI, with the appropriate a m o u n t of choline chloride to maintain osmolarity. Mean of two determinations, which differed by less than 15",~.
123 TABLE 1 INHIBITION OF pH]D-Asp BINDING AND [3H]Glu U P T A K E BY DICARBOXYLIC ACIDS AND ANALOGUES ]C~, values were determined from Hill plots using at least 5 concentrations of inhibitor. Values are means of duplicate or triplicate (_+ S.E.M.) determinations. Compounds producing less than 50% inhibition at 500 I~M were: ibotenate, 2-amino-5-phosphonovalerate, aminophosphonoheptanoate, aminophosphonobutyrate, glutamic acid diethyl ester, D-c~-aminoadipate, D-c~-aminosuberate. Compound
IC5o (/IM)
( 1) ('ysteine sulfinate (2) D-Asp (3) L-Asp (4) I.-Glu (5) Aspartatc-fl-hydroxamate (6) N-Methyl-D-asparlate (7) Aminophosphonopropionate (8) N-Acetylaspartate
[3H]D-Asp
[~H]Glu
4.8_+2.3 8.9±4.2 7.8_+3.2 7.6_+3.2 15.6_+2.1 45 160 100
1.1 4 8 10 45 250 400 500
inhibiting pH]D-Asp binding and [3H]Glu uptake are given in Table I. As in rat brain systems, cysteine sulfinic acid and D-Asp were potent inhibitors along with aspartatefi-hydroxamate. Whilst several analogues of Asp were less potent inhibitors (Table [), compounds selective for postsynaptic Glu receptors were inactive. N-Acetylaspartate, a potent inhibitor of L-Asp binding human brain [2], was a weak inhibitor of pH]D-Asp binding. A highly significant correlation was observed between drug potencies in pH]D-Asp binding and pH]L-Glu uptake (Fig. 2). However, it is noteworthy that the slope of the line of best fit was considerably less than unity, possibly reflecting differences in tissue concentration and preparation between the two assays. No significant correla7
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~
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3 4 5 6 pICso [3HJL-Asp binding
I
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Fig. 2. Relationship belween drug potencies in pH]n-Asp binding and pH]Glu uptake (A) and ptt][ -Asp binding (B) in human brain. A: r - 0 . 8 4 : P<0.01; slope-0.71; and B: r - 0 . 2 2 : P>0.1: slope-0.43. The values Ii~r pit]L-Asp binding inhibition in human cerebellum are taken from ref. 2. The numbers refcr Io the compounds listed in Table I.
124
tion existed between drug potencies in [3H]D-Asp b i n d i n g a n d those observed previously for [3H]L-Asp b i n d i n g (Fig. 2). It is unlikely that [3HID-Asp b i n d i n g included u p t a k e into vesicles which may be present in crude m e m b r a n e preparations, as the Kd d e t e r m i n e d from e q u i l i b r i u m data was similar to that d e t e r m i n e d from kinetic experiments. M o r e o v e r [3H]D-Asp b i n d ing occurred at 2 C , at which t e m p e r a t u r e u p t a k e processes are inhibited [1,4]. The results of the present study are consistent with the labelling o f high-affinity G l u u p t a k e sites by [3H]D-Asp. M o r e o v e r it would a p p e a r that the pharmacological specificity of these sites in h u m a n b r a i n is similar to that of rat brain. As these sites m a y be located specifically o n the terminals o f E A A c o n t a i n i n g n e u r o n s , [3H]D-Asp b i n d i n g m a y prove to be a useful tool in the study of these n e u r o n s in p o s t m o r t e m brains of n e u r o p s y c h i a t r i c patients. This work was s u p p o r t e d by a g r a n t from the Wellcome Trust. 1 Balcar, V.J. and Johnston, G.A.R., The structural specificityof the high-affinityuptake of L-glutamate and L-aspartate by rat brain slices, J. Neurochem., 19 (1972) 2657-2666. 2 Cross, A.J., Skan, W.J. and Slater, P., Excitatory amino acid binding sites in human cerebellum, Br. J. Pharmacol., in press. 3 Fonnum, F., Glutamate: a neurotransmitter in mammalian brain, J. Neurochem., 42 (1984) 1 1I. 4 Fonnum, F., Walaas, I. and Iversen, E., Localisation of GABAergic, cholinergic and aminergic structures in the mesolimbic system, J. Neurochem., 29 (t977) 221-230. 5 Hall, M. and Thor, L., Evaluation of a semi-automated filtration technique for receptor binding studies, Life Sci., 24 (1979) 2293-2300. 6 Hardy, J.A., Dodd, P.R., Oakley, A.E., Kidd, A.M., Perry, R.H. and Edwardson, J.A., Use of postmortem human synaptosomes for studies of metabolism and transmitter amino acid release, Neurosei. Lett., 33 (1982) 317-322. 7 McBean, G.J. and Roberts, P.J., Chronic infusion of L-glutamatecauses neurotoxicity in rat striatum, Brain Res., 290 (1984) 372-375. 8 Meldrum, B.S., Amino acid neurotransmitters and new approaches to anticonvulsant drug action, Epilepsia, 25 (1984) S140-149. 9 Moroni, F., Lombardi, G., Moneti, G. and Aldino, C., The excitotoxin quinolinic acid is present in the brain of several mammals and its cortical content increases during the aging process, Neurosci. Lett., 47 (1984) 51 55. 10 Parsons, B. and Rainbow, T.C., Quantitative autoradiography of sodium-dependent [3H]D-aspartatebinding sites in rat brain, Neurosci. Lett., 36 (1983) 9-12. 11 Schwarcz, R. and Whetsell, N.O., Post-mortem high-affinityglutamate uptake in human brain, Neuroscience, 7 (1982) 1771-1778. 12 Taxt, T. and Storm-Matthisen, J., Uptake of o-aspartate and L-glutamatein excitatory axon terminals in hippocampus: autoradiographic and biochemical comparison with y-aminobutyrate and other amino acids in normal rats and in rats with lesions, Neuroscience, 11 (1984) 79-100. 13 Vincent, S.R. and McGeer, E.G., A comparison of sodium-dependent glutamate binding with highaffinityglutamate uptake in rat striatum, Brain Res., 184 (1980) 99-108. 14 Young, A.B., Oster-Granite, M.L., Merndon, R.M. and Snyder, S.H., Glutamic acid: selectivedepletion by viral induced granule cell loss in hamster cerebellum, Brain Res., 73 (1974) 1-13.
121
Elsevier Scientific Publishers Ireland Ltd. NSL 03706
THE A S S O C I A T I O N OF [3H]D-ASPARTATE BINDING A N D HIGH-AFFINITY G L U T A M A T E UPTAKE IN THE H U M A N BRAIN
A.J. CROSS*. W.J. SKAN and P. SLATER D~7~artment ~1' Phy.~iology, University ~/ Manchester. Manchester M 13 9PT ( U.K. )
(Received July 291h, 1985; Revised version received September 29th, 1985; Accepted October Ist, 1985)
Key word~: amino acid
L-glutamate D-aspartate ~uptake nerve terminal
human brain
The binding of [3H]D-aspartate ([all]D-Asp) to human cerebellum homogenate was compared with the uptake of [qt]glutamate ([~H]Glu) by homogenates prepared from rapidly frozen human cerebral c o r l c x . There was a close correlation between the potencies of a range of drugs for inhibiting the binding of [3HIDAsp and the uptake of [~H]L-Glu. Compounds selective for postsynaptic Glu receptors were inactive. The tindings arc consislent with the labelling of high-affinity Glu uptake sites by [3H]D-Asp, which may be a valuable ligand for studying excitatory amino acid terminals in human brain.
The d i c a r b o x y l i c a m i n o acids l,-glutamate (L-GIu) a n d t,-aspartate (L-Asp) m a y together constitute the m a j o r e x c i t a t o r y n e u r o t r a n s m i t t e r s in the m a m m a l i a n C N S (reviewed in ref. 3). H o w e v e r , due to the u b i q u i t o u s n a t u r e o f these a m i n o acids in brain, it has been difficult to s t u d y m a n y aspects o f their n e u r o t r a n s m i t t e r functions. The presence o f a specific, high-affinity u p t a k e system for L-GIu has nonetheless p r o vided a simple a n d reliable m a r k e r o f g l u t a m a t e r g i c terminals in brain. This system has been used not only to assess the integrity o f e x c i t a t o r y a m i n o acid ( E A A ) terminals in b r a i n s o f e x p e r i m e n t a l a n i m a l s [14] but also to p r o v i d e a m a r k e r o f E A A nerve terminals in n e u r o a n a t o m i c a l e x p e r i m e n t s (for e x a m p l e see ref. 12). M u c h interest is c u r r e n t l y focussed on the role o f E E A s in the p a t h o l o g y o f several n e u r o p s y c h i a t r i c diseases, p a r t i c u l a r l y H u n t i n g t o n ' s disease [7], A l z h e i m e r - t y p e dem e n t i a [9] and epilepsy [8]. A n u m b e r o f studies have d e m o n s t r a t e d the presence o f a high-affinity L-Glu u p t a k e system in h u m a n b r a i n [6, 1 1]. W h i l s t this activity can be detected in b r a i n s o b t a i n e d at a u t o p s y a n d stored frozen, the activity is c o n s i d e r a bly reduced when c o m p a r e d with fresh or r a p i d l y frozen tissue [1 1]. Thus, I : G l u u p t a k e m a y not be a reliable index o f g l u t a m a t e r g i c terminals in h u m a n brain o b t a i n e d at p o s t m o r t e m e x a m i n a t i o n . Recently, ['H]D-Asp, a p o t e n t s u b s t r a t e o f the L-GIu u p t a k e system, has been p r o posed as a suitable ligand for use in b i n d i n g studies o f L-GIu u p t a k e sites in rat brain [10]. In view o f the p o t e n t i a l usefulness o f this technique, we have, in the present
*Author l\)r correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
122
study, compared the properties of [3H]D-Asp binding with [3H]L-Gtu uptake in human brain. Brains were obtained at autopsy from 3 subjects (with no known history of neuropsychiatric disease) and frozen at -40~'C. The delay between death and postmortem was 4 0 + 7 h. The cerebellar cortex was removed and homogenized in 100 vols. of 50 mM Tris-HC1, pH 7.4. The homogenate was centrifuged at 15,000 g for 10 rain and the resulting pellet washed once by rehomogenization and centrifugation. The pellet was resuspended finally in 100 vols. of 50 mM Tris-HC1, pH 7.4, containing 300 mM NaC1. Portions of this preparation (160 #1) were routinely incubated with 50 nM [3H]D-Asp (t5 Ci/mmol, Amersham, U.K.), in a total volume of 200/tl, tbr 30 min at room temperature. For saturation analysis, a range of 8 [3H]D-Asp concentrations between 25 and 4000 nM were used. Bound ligand was separated by filtration and washing with 50 mM Tris-HC1, pH 7.4 [5]. Non-specific binding was defined as that not displaced by 100 #M asparate-/~-hydroxamate. For ['H]L-Glu uptake samples of human cerebral cortex, which had been obtained within 5 h of death and rapidly frozen in isopentane at - 3 5 " C , were used. Tissue was homogenized in 10 vols. of 0.25 M sucrose+50 mM Tris-HCl, pH 7.4, and the uptake of [-~H]L-GIu (50 nM) studied as described previously [4]. [ 3 H ] D - A s p binding to human cerebellum was saturable, with a Kd of 1.0±0.3 llM and a B...... of 7.5_+ 1.1 pmol/mg protein (mean +S.E.M.; n = 3 ) . These values arc comparable to those obtained in rat brain [10]. The binding of [3HID-Asp was totally dependent on the presence of Na ~ ions (Fig. 1). Sodium chloride stimulated [3H]DAsp binding with an ED~ of 25 mM, which is similar to the effect in rat brain on [3H]Glu uptake [1] and sodium-dependent [:~H]GIu binding [13]. [3H]D-Asp binding reached equilibrium by 20 min either at 2 0 C or 2'C. The association rate constant (at 20 C ) was 0.188 /~M/min. The dissociation of [3HID-Asp at 2 0 C was rapid ( t = 2 min) with a dissociation rate constant of 0.274/min. The K,~ calculated from these kinetic constants was 1.46 #M, which agrees well with that determined t¥om equilibrium data. The potencies of a range of dicarboxylic amino acids and analogues in AI.2 o E 'ID
c 0.8
Q.
Q~ 0.4
o
5'o
,oo'
NaCl conc. (raM) Fig. 1. Dependence of [3HID-Asp binding on NaCI concentration. Membranes were incubated with increasing concentrations of NaCI, with the appropriate a m o u n t of choline chloride to maintain osmolarity. Mean of two determinations, which differed by less than 15",~.
123 TABLE 1 INHIBITION OF pH]D-Asp BINDING AND [3H]Glu U P T A K E BY DICARBOXYLIC ACIDS AND ANALOGUES ]C~, values were determined from Hill plots using at least 5 concentrations of inhibitor. Values are means of duplicate or triplicate (_+ S.E.M.) determinations. Compounds producing less than 50% inhibition at 500 I~M were: ibotenate, 2-amino-5-phosphonovalerate, aminophosphonoheptanoate, aminophosphonobutyrate, glutamic acid diethyl ester, D-c~-aminoadipate, D-c~-aminosuberate. Compound
IC5o (/IM)
( 1) ('ysteine sulfinate (2) D-Asp (3) L-Asp (4) I.-Glu (5) Aspartatc-fl-hydroxamate (6) N-Methyl-D-asparlate (7) Aminophosphonopropionate (8) N-Acetylaspartate
[3H]D-Asp
[~H]Glu
4.8_+2.3 8.9±4.2 7.8_+3.2 7.6_+3.2 15.6_+2.1 45 160 100
1.1 4 8 10 45 250 400 500
inhibiting pH]D-Asp binding and [3H]Glu uptake are given in Table I. As in rat brain systems, cysteine sulfinic acid and D-Asp were potent inhibitors along with aspartatefi-hydroxamate. Whilst several analogues of Asp were less potent inhibitors (Table [), compounds selective for postsynaptic Glu receptors were inactive. N-Acetylaspartate, a potent inhibitor of L-Asp binding human brain [2], was a weak inhibitor of pH]D-Asp binding. A highly significant correlation was observed between drug potencies in pH]D-Asp binding and pH]L-Glu uptake (Fig. 2). However, it is noteworthy that the slope of the line of best fit was considerably less than unity, possibly reflecting differences in tissue concentration and preparation between the two assays. No significant correla7
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4 5 6 p]Cso [aHiGlu uptake
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3 4 5 6 pICso [3HJL-Asp binding
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Fig. 2. Relationship belween drug potencies in pH]n-Asp binding and pH]Glu uptake (A) and ptt][ -Asp binding (B) in human brain. A: r - 0 . 8 4 : P<0.01; slope-0.71; and B: r - 0 . 2 2 : P>0.1: slope-0.43. The values Ii~r pit]L-Asp binding inhibition in human cerebellum are taken from ref. 2. The numbers refcr Io the compounds listed in Table I.
124
tion existed between drug potencies in [3H]D-Asp b i n d i n g a n d those observed previously for [3H]L-Asp b i n d i n g (Fig. 2). It is unlikely that [3HID-Asp b i n d i n g included u p t a k e into vesicles which may be present in crude m e m b r a n e preparations, as the Kd d e t e r m i n e d from e q u i l i b r i u m data was similar to that d e t e r m i n e d from kinetic experiments. M o r e o v e r [3H]D-Asp b i n d ing occurred at 2 C , at which t e m p e r a t u r e u p t a k e processes are inhibited [1,4]. The results of the present study are consistent with the labelling o f high-affinity G l u u p t a k e sites by [3H]D-Asp. M o r e o v e r it would a p p e a r that the pharmacological specificity of these sites in h u m a n b r a i n is similar to that of rat brain. As these sites m a y be located specifically o n the terminals o f E A A c o n t a i n i n g n e u r o n s , [3H]D-Asp b i n d i n g m a y prove to be a useful tool in the study of these n e u r o n s in p o s t m o r t e m brains of n e u r o p s y c h i a t r i c patients. This work was s u p p o r t e d by a g r a n t from the Wellcome Trust. 1 Balcar, V.J. and Johnston, G.A.R., The structural specificityof the high-affinityuptake of L-glutamate and L-aspartate by rat brain slices, J. Neurochem., 19 (1972) 2657-2666. 2 Cross, A.J., Skan, W.J. and Slater, P., Excitatory amino acid binding sites in human cerebellum, Br. J. Pharmacol., in press. 3 Fonnum, F., Glutamate: a neurotransmitter in mammalian brain, J. Neurochem., 42 (1984) 1 1I. 4 Fonnum, F., Walaas, I. and Iversen, E., Localisation of GABAergic, cholinergic and aminergic structures in the mesolimbic system, J. Neurochem., 29 (t977) 221-230. 5 Hall, M. and Thor, L., Evaluation of a semi-automated filtration technique for receptor binding studies, Life Sci., 24 (1979) 2293-2300. 6 Hardy, J.A., Dodd, P.R., Oakley, A.E., Kidd, A.M., Perry, R.H. and Edwardson, J.A., Use of postmortem human synaptosomes for studies of metabolism and transmitter amino acid release, Neurosei. Lett., 33 (1982) 317-322. 7 McBean, G.J. and Roberts, P.J., Chronic infusion of L-glutamatecauses neurotoxicity in rat striatum, Brain Res., 290 (1984) 372-375. 8 Meldrum, B.S., Amino acid neurotransmitters and new approaches to anticonvulsant drug action, Epilepsia, 25 (1984) S140-149. 9 Moroni, F., Lombardi, G., Moneti, G. and Aldino, C., The excitotoxin quinolinic acid is present in the brain of several mammals and its cortical content increases during the aging process, Neurosci. Lett., 47 (1984) 51 55. 10 Parsons, B. and Rainbow, T.C., Quantitative autoradiography of sodium-dependent [3H]D-aspartatebinding sites in rat brain, Neurosci. Lett., 36 (1983) 9-12. 11 Schwarcz, R. and Whetsell, N.O., Post-mortem high-affinityglutamate uptake in human brain, Neuroscience, 7 (1982) 1771-1778. 12 Taxt, T. and Storm-Matthisen, J., Uptake of o-aspartate and L-glutamatein excitatory axon terminals in hippocampus: autoradiographic and biochemical comparison with y-aminobutyrate and other amino acids in normal rats and in rats with lesions, Neuroscience, 11 (1984) 79-100. 13 Vincent, S.R. and McGeer, E.G., A comparison of sodium-dependent glutamate binding with highaffinityglutamate uptake in rat striatum, Brain Res., 184 (1980) 99-108. 14 Young, A.B., Oster-Granite, M.L., Merndon, R.M. and Snyder, S.H., Glutamic acid: selectivedepletion by viral induced granule cell loss in hamster cerebellum, Brain Res., 73 (1974) 1-13.