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Voltage-dependency of the responses of cerebellar Purkinje cells to excitatory amino acids
Brain Research, 419 (1987) 379-382 Elsevier
379
BRE 22459
Voltage-dependency of the responses of cerebellar Purkinje cells to excitatory amino acids Brigitte Hamon, Francis Crepel and Marc Debono Laboratoire de Neurobiologie et Neuropharmacologie du D~veloppement, Centre d'Orsay, Orsay (France) (Accepted 26 May 1987) Key words: Purkinje cell; Excitatory amino acid; Voltage clamp
The voltage-dependency of the responses of Purkinje cells to excitatory amino acids was examined in rat cerebellar slices, using intrasomatic recordings with the single electrode voltage-clamp. In standard perfusion medium, the depolarizations evoked in these neurones by ionophoretic pulse applications (less than 300 ms) of L-glutamate, L-aspartate and quisqualate in their dendritic fields had underlying inward currents which did not increase or even decreased, as the holding potential was shifted to values more negative than -65 mV. This 'abnormal' voltage-dependency was still present in Mg2+ -free solution but was abolished in the presence of CsCI~ (10 mM) in the perfusion medium. When TTX (5/~M) and CdC12(0.1 mM) were further added to the bath in order to block regenerative conductances, thus broadening the range of the clamp voltages to more positive values than -50 mV, the current-voltage relation between -80 and 0 mV for responses to L-glutamate and L-aspartate was almost linear. Our results support the view that low doses of both amino acids act on Purkinje cells essentially via the activation of receptors which are not of the N-methyl-D-aspartate type.
In the adult CNS, many functionally well-defined neurones bear the 3 different subtypes of receptors to excitatory amino acids ( E A A s ) defined by McLennan et al. 9 and by Watkins 15, i.e. activated preferentially by N-methyl-D-aspartate ( N M D A ) , by quisqualate and by kainate. Cerebellar Purkinje cells (PCs) seem to be peculiar in this respect since Crepel et al. 1'2, then Kimura et al. 5, have shown, in vitro, that these cells possess very few activatable receptors of the N M D A subtype. However, Quinlan and Davies 14, on the basis of ionophoretic experiments in vivo, recently concluded that well developed N M D A receptors are represented on PCs. This discrepancy led us to complete our observations on the nature of PC receptors to L-glutamate (L-GIu) and L-aspartate (L-Asp) in adult rat cerebellar slices. This was done by examining the voltage-dependency of the responses of PCs to E A A s , since the transmembrane currents evoked by the activation of N M D A receptors display an abnormal voltage-dependency below - 4 0 mV in the presence of Mg 2+ in the extracellular
space, which is not observed for responses mediated by other receptor types 1°-13. Experiments were performed in 400 ~tm thick sagittal cerebellar slices of adult Wistar rats. Methods for preparing the slices and the recording chamber were the same as previously described 4. The standard perfusion medium contained (in mM): NaC1 124, KC1 5, KH2PO 4 1.15, MgSO 4 1.15, CaC12 2.5, N a H C O 3 25, glucose 10. Purkinje cells were recorded intracellularly at the somatic level with glass microelectrodes filled with 3 M KC1 (resistance 60-80 Mf~), with the use of a single electrode voltage-clamp circuit (Dagan 8100). Three-barrelled micropipettes were used to deliver Na-L-Asp (0.5 M, pH 6) and Na-L-Glu (0.5 M, pH 6) in the lower third of the dendritic field of the recorded PCs by the use of short ionophoretic pulses (10-300 ms in duration, 20-80 n A in amplitude) given with a Neurophore Unit (Digitimer), with one barrel filled with NaC1 3 M and used for current compensation. In the current-clamp mode and in the presence of
Correspondence: F. Crepel, Laboratoire de Neurobiologie et Neuropharmacologie du D6veloppement, Centre d'Orsay, Bat. 440, 91405 Orsay Cedex, France. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
380 1.15 Mg 2+ in the perfusion medium (n = 10), L-Glu and L-Asp applications elicited a transient and dosedependent depolarization of PCs (Fig. 1A, B) which could reach the firing threshold of the recorded cells when they were held at their resting potential, i.e. between - 6 0 and - 6 5 m V (Fig. 1A). W h e n the cells were hyperpolarized by a steady negative current, the amplitude of these responses either did not increase (4 cells) or even decreased for both substances (6 cells), as illustrated in Fig. 1A and B. Therefore, this was reminiscent of the abnormal voltage-depen-
A
61u
dency described for responses to E A A s via N M D A receptors. However, since similar results were obtained in 4 experiments performed in a Mg2+-free solution (not illustrated), another interpretation might be that the depolarization due to L-Asp and k-Glu activates voltage-dependent Na and Ca currents 7''~, which could then increase the amplitude of the responses and thus at least partially explain the apparent anomalous voltage-dependency of the responses of PCs to E A A s . As shown in the Fig. 1C, the counterpart of the de-
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Fig. 1. Responses of PCs to dendritic application of excitatory amino acids, recorded during standard medium perfusion, under current-clamp (A and B) and voltage-clamp (C and D) conditions. In this and the following figure, the values of these potentials are indicated alongside the responses and 15 sweeps have been averaged for each trace. A: depolarizations (all responses) and action potentials (upper responses) induced by ionophoretic pulse application of L-Glu and L-Asp at the resting potential (upper responses) and during steady hyperpolarizations (middle and lower responses); the ionophoretic currents are shown in the last trace. B: depolarizations evoked in another cell by L-GIu (35 hA, 180 ms) and L-Asp (20 hA, 200 ms) recorded at 2 levels of hyperpolarization. C: currents induced in a voltage-clamped PC by L-GIu and L-Asp a t different holding potentials. D: same protocole as in C for another PC excited by L-Asp and qulsqualate. In C and D, note that, when the cell membrane was hypcrpolarized, the amplitude of the clamp currents did not increase or even diminished slightly (right responses in C).
381
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Fig. 2. Voltage-clamp analysis of PC responses to L-GIuand L-Asp during the blockade of voltage-dependent conductances. A: inward currents elicited in the same neurone as in Fig. 1C by both amino acids after addition of 10 mM CsCI 2 to the perfusion medium. B: curves of the current-voltage relations for the responses presented in A (O, L-Glu; O, L-Asp), and those evoked in control conditions by L-Glu (A) and L-Asp (A) (recordings shown in Fig. 1C), C: current responses to both amino acids, recorded at different holding potentials between 0 and -90 mV, in the presence of 10 mM CsCl2, 5/~M "I'TXand 0.1 mM CdCI2. D: curves of the currentvoltage relations for the signals elicited by L-Glu (O) and L-Asp (0) illustrated in C. Note that, under these conditions, the curves were almost linear between -80 and 0 inV.
polarization induced by L-Glu and L-Asp during the voltage-clamp of PCs at their resting potential consisted of a transient and well developed inward current. In such conditions, the residual depolarization
in the voltage records was no more than 5 - 1 0 % of that recorded in the current-clamp mode. In standard perfusion medium, the amplitude of this current did not increase, or even became smaller, when the hold-
382 ing potential of the cells reached more negative values than its initial one o f - 6 5 mV, which is in keeping with the results obtained in the current-clamp mode (Fig. 1). However, one knows that the hyperpolarization of PCs below their resting potential induces an inward rectification 3 which might produce an artefactual decrease of the currents induced by LGlu and L-Asp in the dendrites when recorded at the somatic level, due to the limits of the space clamp (see above). This may well explain why the responses of PCs to quisqualate also showed an abnormal voltage-sensitivity in standard perfusion medium (Fig. 1D), while this agonist is expected to activate only receptors of the quisqualate subtype, i.e. with a normal voltage-dependency. Indeed, when the inward rectification was abolished by 10 mM Cs in the bath, while this cation is not known to interfere with L-GIu and LAsp receptor-coupled channels 6A°-13, the currents produced by L-GIu and L-Asp (6 cells) now increased linearly as the cell m e m b r a n e was hyperpolarized from its initial value (Fig. 2A,B). Finally, in 4 experiments, the study of the voltagedependency of PC responses to E A A s was extended at potentials less negative than - 5 0 mV by blocking regenerative Na and Ca conductances. This was achieved by replacing Ca 2÷ by 0.1 m M of the Ca 2÷
1 Cr6pel, F., Dhanjal, S.S. and Sears, T.A., Effect of glutamate, aspartate and related derivatives on cerebeUar Purkinje cell dendrites in the rat: an in vitro study, J. Physiol. (London), 329 (1982) 297-317. 2 Cr6pel, F., Dupont, J.L. and Gardette, R., Voltage-clamp analysis of the effect of excitatory amino acids and derivatives on Purkinje cell dendrites in rat cerebellar slices maintained in vitro, Brain Research, 279 (1983) 311-315. 3 Cr6pel, F. and P6nit-Soria, J., Inward rectification and low threshold calcium conductances in rat cerebellar Purkinje cells. An in vitro study, J. Physiol. (London), 372 (1986) 1-23. 4 Curtis, D.R., The action of amino acids upon mammalian neurones. In D.R. Curtis and A.K. McIntyre (Eds.), Studies in Physiology, Springer, New York, 1965, pp. 34-42. 5 Kimura, H., Okamoto, K. and Sakai, Y., Pharmacological characterization of postsynaptic receptors for excitatory amino acids in Purkinje cell dendrites in the guinea pig cerebellum, J. Pharmacobio-Dyn., 8 (1985) 120-128. 6 Kimura, H., Okamoto, K. and Sakai, Y., Pharmacological evidence for L-aspartate as the neurotransmitter of cerebellar climbing fibres in the guinea-pig, J. Physiol. (London), 365 (1985) 103-119. 7 Llinas, R. and Sugimori, M., Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices, J. Physiol. (London), 305 (1980) 171-195. 8 Llinas, R. and Sugimori, M., Eleetrophysiological properties of in vitro Purkinje cell dendrites in mammalian cere-
channel blocker Cd and by adding 5 # M T T X to the Cs-containing perfusing solution. In these 4 cells, the relationship between the amplitude of the L-Asp- and L-Glu-induced currents and the holding potential was rather linear between - 8 0 and 0 mV~ the limitation in the clamp system preventing to pass current enough to further depolarize neurones beyond 0 mV (Fig. 2C,D). The present experiments show that, when most voltage-dependent conductances of PCs are blocked, the amplitude of the inward currents produced by the ionophoretic application of L-Asp and L-Glu have a voltage-sensitivity close to a linear function of the membrane potential and this, even in the presence of Mg 2+ in the peffusion medium. This is characteristic of responses mediated by the activation of receptors which are not preferentially activated by N M D A 1°-13. Indeed, one might argue that blockers used to abolish the voltage-dependent conductances also block N M D A receptor-coupled channels, thus rendering linear the current-voltage relationship. So far, this is unlikely for Cs 6'1°-13 as well as for Cd, at least at the concentration used 1°'12 Therefore, this fully confirms that L-Asp and L-Gtu mainly act on PCs in in vitro slices by activating non N M D A receptors.
bellar slices, J. Physiol. (London), 305 (1980) 197-2 t 3. 9 McLennan, H., Hicks, T.P. and Hall, J.G., Receptors for the excitatory amino acids. In F.W. De Feudis and P. Mandel (Eds.), Advances in Biochemical Psychopharmacology, Vol. 29, Amino Acid Neurotransmitters, Raven, New York, 1981, pp. 213-229. 10 Mayer. M.L. and Westbrook, G.L., The action of N-methyl-D-aspartic acid on mouse spinal neurones in culture, J. Physiol. (London), 361 (1985) 65-90. l l Mayer. M.L. and Westbrook, G.L.. Mixed agonist action of excitatory amino acids on mouse spinal cord neurones m culture. J. Physiol. (London), 361 (1985) 65 -90 12 Mayer. M.L. and Westbrook, G.L. and Guthrie, P.B.. Voltage-dependent block by Mg2+ of the NMDA responses in spinal cord neurones. Nature (London), 309 (1984) 261-263. 13 Nowak, L., Bregestovski, P.. Ascher, P., Herbet, A. and Prochiantz, A., Magnesium gates glutamate-activated channels in mouse central neurones, Nature (London), 307 (1984) 462-465. 14 Quinlan. J.E. and Davies, J.. Excitatory and inhibitory responses of Purkinje cells, in the rat cerebellum m vivo. reduced by excitatory amino acids. Neurosci. Lett., 60 (1985) 39-46. 15 Watkins, J.C., Pharmacology of excitatory amino acid transmitters. In F.V. De Feudis and P. Mandel (Eds.), Amino Acid Neurotransmitters. Raven, New York. 1981. pp. 205-212
379
BRE 22459
Voltage-dependency of the responses of cerebellar Purkinje cells to excitatory amino acids Brigitte Hamon, Francis Crepel and Marc Debono Laboratoire de Neurobiologie et Neuropharmacologie du D~veloppement, Centre d'Orsay, Orsay (France) (Accepted 26 May 1987) Key words: Purkinje cell; Excitatory amino acid; Voltage clamp
The voltage-dependency of the responses of Purkinje cells to excitatory amino acids was examined in rat cerebellar slices, using intrasomatic recordings with the single electrode voltage-clamp. In standard perfusion medium, the depolarizations evoked in these neurones by ionophoretic pulse applications (less than 300 ms) of L-glutamate, L-aspartate and quisqualate in their dendritic fields had underlying inward currents which did not increase or even decreased, as the holding potential was shifted to values more negative than -65 mV. This 'abnormal' voltage-dependency was still present in Mg2+ -free solution but was abolished in the presence of CsCI~ (10 mM) in the perfusion medium. When TTX (5/~M) and CdC12(0.1 mM) were further added to the bath in order to block regenerative conductances, thus broadening the range of the clamp voltages to more positive values than -50 mV, the current-voltage relation between -80 and 0 mV for responses to L-glutamate and L-aspartate was almost linear. Our results support the view that low doses of both amino acids act on Purkinje cells essentially via the activation of receptors which are not of the N-methyl-D-aspartate type.
In the adult CNS, many functionally well-defined neurones bear the 3 different subtypes of receptors to excitatory amino acids ( E A A s ) defined by McLennan et al. 9 and by Watkins 15, i.e. activated preferentially by N-methyl-D-aspartate ( N M D A ) , by quisqualate and by kainate. Cerebellar Purkinje cells (PCs) seem to be peculiar in this respect since Crepel et al. 1'2, then Kimura et al. 5, have shown, in vitro, that these cells possess very few activatable receptors of the N M D A subtype. However, Quinlan and Davies 14, on the basis of ionophoretic experiments in vivo, recently concluded that well developed N M D A receptors are represented on PCs. This discrepancy led us to complete our observations on the nature of PC receptors to L-glutamate (L-GIu) and L-aspartate (L-Asp) in adult rat cerebellar slices. This was done by examining the voltage-dependency of the responses of PCs to E A A s , since the transmembrane currents evoked by the activation of N M D A receptors display an abnormal voltage-dependency below - 4 0 mV in the presence of Mg 2+ in the extracellular
space, which is not observed for responses mediated by other receptor types 1°-13. Experiments were performed in 400 ~tm thick sagittal cerebellar slices of adult Wistar rats. Methods for preparing the slices and the recording chamber were the same as previously described 4. The standard perfusion medium contained (in mM): NaC1 124, KC1 5, KH2PO 4 1.15, MgSO 4 1.15, CaC12 2.5, N a H C O 3 25, glucose 10. Purkinje cells were recorded intracellularly at the somatic level with glass microelectrodes filled with 3 M KC1 (resistance 60-80 Mf~), with the use of a single electrode voltage-clamp circuit (Dagan 8100). Three-barrelled micropipettes were used to deliver Na-L-Asp (0.5 M, pH 6) and Na-L-Glu (0.5 M, pH 6) in the lower third of the dendritic field of the recorded PCs by the use of short ionophoretic pulses (10-300 ms in duration, 20-80 n A in amplitude) given with a Neurophore Unit (Digitimer), with one barrel filled with NaC1 3 M and used for current compensation. In the current-clamp mode and in the presence of
Correspondence: F. Crepel, Laboratoire de Neurobiologie et Neuropharmacologie du D6veloppement, Centre d'Orsay, Bat. 440, 91405 Orsay Cedex, France. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
380 1.15 Mg 2+ in the perfusion medium (n = 10), L-Glu and L-Asp applications elicited a transient and dosedependent depolarization of PCs (Fig. 1A, B) which could reach the firing threshold of the recorded cells when they were held at their resting potential, i.e. between - 6 0 and - 6 5 m V (Fig. 1A). W h e n the cells were hyperpolarized by a steady negative current, the amplitude of these responses either did not increase (4 cells) or even decreased for both substances (6 cells), as illustrated in Fig. 1A and B. Therefore, this was reminiscent of the abnormal voltage-depen-
A
61u
dency described for responses to E A A s via N M D A receptors. However, since similar results were obtained in 4 experiments performed in a Mg2+-free solution (not illustrated), another interpretation might be that the depolarization due to L-Asp and k-Glu activates voltage-dependent Na and Ca currents 7''~, which could then increase the amplitude of the responses and thus at least partially explain the apparent anomalous voltage-dependency of the responses of PCs to E A A s . As shown in the Fig. 1C, the counterpart of the de-
B
lisp
,
nlu :.
Asp
" k
f.
•
t
.
-85
~:
I
:.,
,..
'
m 75
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Fig. 1. Responses of PCs to dendritic application of excitatory amino acids, recorded during standard medium perfusion, under current-clamp (A and B) and voltage-clamp (C and D) conditions. In this and the following figure, the values of these potentials are indicated alongside the responses and 15 sweeps have been averaged for each trace. A: depolarizations (all responses) and action potentials (upper responses) induced by ionophoretic pulse application of L-Glu and L-Asp at the resting potential (upper responses) and during steady hyperpolarizations (middle and lower responses); the ionophoretic currents are shown in the last trace. B: depolarizations evoked in another cell by L-GIu (35 hA, 180 ms) and L-Asp (20 hA, 200 ms) recorded at 2 levels of hyperpolarization. C: currents induced in a voltage-clamped PC by L-GIu and L-Asp a t different holding potentials. D: same protocole as in C for another PC excited by L-Asp and qulsqualate. In C and D, note that, when the cell membrane was hypcrpolarized, the amplitude of the clamp currents did not increase or even diminished slightly (right responses in C).
381
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Fig. 2. Voltage-clamp analysis of PC responses to L-GIuand L-Asp during the blockade of voltage-dependent conductances. A: inward currents elicited in the same neurone as in Fig. 1C by both amino acids after addition of 10 mM CsCI 2 to the perfusion medium. B: curves of the current-voltage relations for the responses presented in A (O, L-Glu; O, L-Asp), and those evoked in control conditions by L-Glu (A) and L-Asp (A) (recordings shown in Fig. 1C), C: current responses to both amino acids, recorded at different holding potentials between 0 and -90 mV, in the presence of 10 mM CsCl2, 5/~M "I'TXand 0.1 mM CdCI2. D: curves of the currentvoltage relations for the signals elicited by L-Glu (O) and L-Asp (0) illustrated in C. Note that, under these conditions, the curves were almost linear between -80 and 0 inV.
polarization induced by L-Glu and L-Asp during the voltage-clamp of PCs at their resting potential consisted of a transient and well developed inward current. In such conditions, the residual depolarization
in the voltage records was no more than 5 - 1 0 % of that recorded in the current-clamp mode. In standard perfusion medium, the amplitude of this current did not increase, or even became smaller, when the hold-
382 ing potential of the cells reached more negative values than its initial one o f - 6 5 mV, which is in keeping with the results obtained in the current-clamp mode (Fig. 1). However, one knows that the hyperpolarization of PCs below their resting potential induces an inward rectification 3 which might produce an artefactual decrease of the currents induced by LGlu and L-Asp in the dendrites when recorded at the somatic level, due to the limits of the space clamp (see above). This may well explain why the responses of PCs to quisqualate also showed an abnormal voltage-sensitivity in standard perfusion medium (Fig. 1D), while this agonist is expected to activate only receptors of the quisqualate subtype, i.e. with a normal voltage-dependency. Indeed, when the inward rectification was abolished by 10 mM Cs in the bath, while this cation is not known to interfere with L-GIu and LAsp receptor-coupled channels 6A°-13, the currents produced by L-GIu and L-Asp (6 cells) now increased linearly as the cell m e m b r a n e was hyperpolarized from its initial value (Fig. 2A,B). Finally, in 4 experiments, the study of the voltagedependency of PC responses to E A A s was extended at potentials less negative than - 5 0 mV by blocking regenerative Na and Ca conductances. This was achieved by replacing Ca 2÷ by 0.1 m M of the Ca 2÷
1 Cr6pel, F., Dhanjal, S.S. and Sears, T.A., Effect of glutamate, aspartate and related derivatives on cerebeUar Purkinje cell dendrites in the rat: an in vitro study, J. Physiol. (London), 329 (1982) 297-317. 2 Cr6pel, F., Dupont, J.L. and Gardette, R., Voltage-clamp analysis of the effect of excitatory amino acids and derivatives on Purkinje cell dendrites in rat cerebellar slices maintained in vitro, Brain Research, 279 (1983) 311-315. 3 Cr6pel, F. and P6nit-Soria, J., Inward rectification and low threshold calcium conductances in rat cerebellar Purkinje cells. An in vitro study, J. Physiol. (London), 372 (1986) 1-23. 4 Curtis, D.R., The action of amino acids upon mammalian neurones. In D.R. Curtis and A.K. McIntyre (Eds.), Studies in Physiology, Springer, New York, 1965, pp. 34-42. 5 Kimura, H., Okamoto, K. and Sakai, Y., Pharmacological characterization of postsynaptic receptors for excitatory amino acids in Purkinje cell dendrites in the guinea pig cerebellum, J. Pharmacobio-Dyn., 8 (1985) 120-128. 6 Kimura, H., Okamoto, K. and Sakai, Y., Pharmacological evidence for L-aspartate as the neurotransmitter of cerebellar climbing fibres in the guinea-pig, J. Physiol. (London), 365 (1985) 103-119. 7 Llinas, R. and Sugimori, M., Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices, J. Physiol. (London), 305 (1980) 171-195. 8 Llinas, R. and Sugimori, M., Eleetrophysiological properties of in vitro Purkinje cell dendrites in mammalian cere-
channel blocker Cd and by adding 5 # M T T X to the Cs-containing perfusing solution. In these 4 cells, the relationship between the amplitude of the L-Asp- and L-Glu-induced currents and the holding potential was rather linear between - 8 0 and 0 mV~ the limitation in the clamp system preventing to pass current enough to further depolarize neurones beyond 0 mV (Fig. 2C,D). The present experiments show that, when most voltage-dependent conductances of PCs are blocked, the amplitude of the inward currents produced by the ionophoretic application of L-Asp and L-Glu have a voltage-sensitivity close to a linear function of the membrane potential and this, even in the presence of Mg 2+ in the peffusion medium. This is characteristic of responses mediated by the activation of receptors which are not preferentially activated by N M D A 1°-13. Indeed, one might argue that blockers used to abolish the voltage-dependent conductances also block N M D A receptor-coupled channels, thus rendering linear the current-voltage relationship. So far, this is unlikely for Cs 6'1°-13 as well as for Cd, at least at the concentration used 1°'12 Therefore, this fully confirms that L-Asp and L-Gtu mainly act on PCs in in vitro slices by activating non N M D A receptors.
bellar slices, J. Physiol. (London), 305 (1980) 197-2 t 3. 9 McLennan, H., Hicks, T.P. and Hall, J.G., Receptors for the excitatory amino acids. In F.W. De Feudis and P. Mandel (Eds.), Advances in Biochemical Psychopharmacology, Vol. 29, Amino Acid Neurotransmitters, Raven, New York, 1981, pp. 213-229. 10 Mayer. M.L. and Westbrook, G.L., The action of N-methyl-D-aspartic acid on mouse spinal neurones in culture, J. Physiol. (London), 361 (1985) 65-90. l l Mayer. M.L. and Westbrook, G.L.. Mixed agonist action of excitatory amino acids on mouse spinal cord neurones m culture. J. Physiol. (London), 361 (1985) 65 -90 12 Mayer. M.L. and Westbrook, G.L. and Guthrie, P.B.. Voltage-dependent block by Mg2+ of the NMDA responses in spinal cord neurones. Nature (London), 309 (1984) 261-263. 13 Nowak, L., Bregestovski, P.. Ascher, P., Herbet, A. and Prochiantz, A., Magnesium gates glutamate-activated channels in mouse central neurones, Nature (London), 307 (1984) 462-465. 14 Quinlan. J.E. and Davies, J.. Excitatory and inhibitory responses of Purkinje cells, in the rat cerebellum m vivo. reduced by excitatory amino acids. Neurosci. Lett., 60 (1985) 39-46. 15 Watkins, J.C., Pharmacology of excitatory amino acid transmitters. In F.V. De Feudis and P. Mandel (Eds.), Amino Acid Neurotransmitters. Raven, New York. 1981. pp. 205-212