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Spider toxin (JSTX) blocks glutamate synapse in hippocampal pyramidal neurons
Brain Research, 346 (1985) 397-399 Elsevier
397
BRE 21138
Spider toxin (JSTX) blocks glutamate synapse in hippocampal pyramidal neurons MITSUYOSHI SAITOl, NOBUFUMI KAWAI1, AKIKO MIWA1, HIDEMITSU PAN-HOU2 and MASANORI YOSHIOKA2
1Tokyo Metropolitan Institutefor Neurosciences, Department of Neurobiology 2-6, Musashidai, Fuchu-City, Tokyo, 183 and 2Faculty of Pharmaceutical Sciences, Setsunan University, 45-1, Nagaotoge, Hirakata-City, Osaka, 573-01 (Japan) (Accepted June 11th, 1985)
Key words: glutamate receptor - - spider toxin - - JSTX - - hippocampus - - pyramidal cell
Effects of a spider toxin (JSTX) - - a specific blocker of glutamate receptors - - on single pyramidal neurons of the hippocampus were studied using tissue slices in vitro. JSTX blocked the synaptic response in CA, pyramidal cells evoked by Schaffer collateral stimulation without affecting the antidromic spike potential. The toxin suppressed glutamate-induced cell firings whereas it had little effect on aspartate-induced responses. The results suggest that glutamate is a neurotransmitter of the Schaffer collateral input to CA] pyramidal neurons. The hippocampus is one of the best studied systems in the brain with respect to the role of acidic amino acids. Many physiological as well as biochemical data have suggested glutamate and/or aspartate as the neurotransmitter in this system3, 5. However, recent results favour glutamate as a neurotransmitter in C A 1 neurons. For example, pyramidal cells in the C A 1 region are activated by iontophoretically applied glutamate into a highly sensitive region of the dendrites4,13. Biochemical data using selective stimulation of C A 1 excitatory inputs suggest that L-glutamate is a neurotransmitter of the Schaffer collaterals of the C A 3 neuron 11. Furthermore, Hablitz and Langmoen6 showed a close resemblance between the reversal potential for the glutamate potential and that for the excitatory postsynaptic potential (EPSP) of C A 1 cells. To strengthen evidence for L-glutamate as the neurotransmitter the use of a receptor blocker with a high degree of specificity m a y b e most helpful. In previous studies we reported that a spider toxin (JSTX) - - a blocker of glutamate receptors in the lobster neuromuscular synapse1, 7 - suppressed postsynaptic potentials in the brain slice of the hippocampus 8,9. In that study we recorded extracellular field potentials of pyramidal neurons of C A 3 and found that much higher concentrations of the toxin are needed to suppress the postsynaptic potentials than
those used in the crustacean neuromuscular synapse. The present study was undertaken to determine more precisely the effect of the purified toxin on a single pyramidal neuron activated by selective stimulation of Schaffer collaterals. The procedures for making hippocampal slices of guinea-pig brain was similar to that described previously16A 7. Stimulating electrodes made of platinum-iridium were placed at the stratum radiatum or alveus to deliver orthodromic or antidromic stimulation, respectively. Radiatum stimulation was controlled so that the currents activated exclusively Schaffer collaterals 2. Glass microelectrodes for intracellular recording were filled with 4 M potassium acetate and for extracellular recording, with saline or 2 M NaC1. Double- or triple-barrel electrodes were used for iontophoretic application of glutamate or aspartate (1 M L-sodium glutamate or 1 M L-sodium aspartate adjusted to p H 8.0). To check non-specific activation of the neuron by the currents the same strength of currents as used to eject glutamate or aspartate were applied from the other barrel filled with 2 M NaCI. Spiders (Nephila clavata, Joro spider) were collected in central Japan. Spider toxin (JSTX) was purified by the procedures of gel filtration, ionexchange column chromatography followed by fast performance liquid chromatography (in prepara-
Correspondence: N. Kawai, Tokyo Metropolitan Institute for Neurosciences, 2-6 Musashidai, Fuchu-City, Tokyo 183, Japan. 0006-8993/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
398 tion). The purified toxin was diluted with saline, suctioned into the glass pipette tip and e j e c t e d by air pressure (Picosplitzer II, G e n e r a l Valve). The procedure enabled us to apply a small a m o u n t of toxin in a localized area. In most cases we used 5 - 1 0 nl of toxin solution which contains ca. 10 -3 to 10 -4 U (1 U corresponds to the amount of purified toxin that is in one venom gland). Fig. 1 A shows a typical example of the blocking action of JSTX on the C A 1 pyramidal neuron. In response to activation of the stratum radiatum, excitatory postsynaptic potentials (EPSPs) followed by the action potentials were evoked. A f t e r the JSTX solution was applied to the outer part of the stratum radiatum, with a delay of about a minute the rising phase of EPSP slowed and then the action potentials were abolished, leaving only EPSPs. The remaining EPSPs were suppressed gradually and by 5 min they had disappeared. The volume of toxin solution required for the block of EPSPs ranged from 1 to 10 nl (=< 10 -3 U) which was reasonably low c o m p a r e d to that n e e d e d in the crustacean neuromuscular synapse 1.7. W h e n less toxin was applied, m o r e time was n e e d e d to block the action potential and the EPSPs were reduced in size. Frequently the EPSPs were not abolished and continued to be e v o k e d with r e d u c e d A
B
Orth.
Anti.
1 Control JSTX
Control
j~_
jL,,,._
_
A
I
JSTX
1.5-J " - ~
4.5-_~---6 - ~
120___.mY Wash IOtas
,J
~\
120mV I0 ms
Fig. 1. Block of EPSPs in CA l pyramidal cells of the hippocampus by a spider toxin (JSTX). A: intracellular records from a pyramidal cell in response to activation by Schaffer collaterals. The numerals on the left indicate the time in min after application of JSTX (6.5 x 10-4 U ) . B: another pyramidal cell was activated by Schaffer collateral (Orth.) followed by alveus (Anti.) stimulation. Upper trace, control. Middel trace, 5 min after applying JSTX (8 x 10_4 U). Lower traces, after rinsing the prep-" aration with normal saline. No recovery of EPSPs was found.
GILl 2
GIu 4
3
8
Asp4
5 nA
8 nA
2
4
Glu4 8
6
8 nA
Asp 4
8 nA
Fig. 2. Suppression of glutamate-induced firings of the pyramidal cells by JSTX. A: cell firings produced by iontophoretically applied glutamate in the absence (left) and presence (right) of JSTX (1 x 10-3 U). B: discharges induced by glutamate and aspartate before and after JSTX (1 x 10-3 U) application. A and B are taken from different cells. In both cases cell firings were recorded 10 min after toxin application. Vertical calibration indicates 10 Hz. amplitude. In either case, once EPSPs were suppressed no sign of recovery was found even though the p r e p a r a t i o n was peffused with normal Ringer for more than 1 h. The resting m e m b r a n e potential and the input resistance of the neuron were not changed before and after the toxin application. Insensitivity of non-synaptic portions of soma m e m b r a n e to the toxin was shown by the antidromic stimulation (Fig. 2B). CA~ neurons were activated by stimulating the stratum radiatum followed by the alveus. On applying JSTX, only the orthodromically e v o k e d action potentials were blocked, whereas the antidromic action potentials were little changed. This suggests that JSTX did not affect the soma m e m b r a n e which is responsible for the generation of the spike. CA~ neurons were activated also by iontophoretic application of L-glutamate. W h e n the glutamate pipette was suitably positioned at the apical dendrites in the stratum radiatum, spike discharges were recorded from the pyramidal neurons (Fig. 2). By raising the current more frequent discharges were e v o k e d from the b r o a d e r area of the dendrites. H o w e v e r , strong currents sometimes directly activate neurons because the same strength of currents delivered from another barrel containing 2 M NaC1 p r o d u c e d firing of neurons. W e therefore used considerably lower current strengths (less than 10 n A ) which only activate a well-localized dendrite area 5,13. In Fig. 2A, with an increase in glutamate currents from 2 to 5 nA, the frequency of spike discharges increased. After JSTX was applied to the dendritic area within 50
399 /~m of the current p i p e t t e , spike discharges were greatly suppressed. Pyramidal neurons were also activated by L-aspartic acid with similar current strengths to L-glutamate (Fig. 2B). Cell firings induced by L-glutamate were substantially suppressed in the presence of JSTX whereas the responses to L-aspartate were barely affected. W e found also that quisqualic acid and kainic acid, glutamate agonists, activated p y r a m i d a l neurons. JSTX suppressed quisqualate-induced spike discharges. Suppression was also evident in kainate-induced discharges but to a lesser degree than quisqualate. The above results are in a g r e e m e n t with data obtained in the crustacean n e u r o m u s c u l a r synapsO, 7
1 Abe, T., Kawai, N. and Miwa, A., Effects of a spider toxin on the glutaminergic synapse of lobster muscle, J. Physiol. (London), 339 (1983) 243-353. 2 Andersen, P., Silfvenius, H., Sundberg, S.H. and Sveen, O., A comparison of distal and proximal dendritic synapses on CA 1 pyramids in guinea-pig hippocampal slices in vitro, J. Physiol. (London), 307 (1980) 273-299. 3 Curtis, D.R. and Johnston, G.A.R., Amino acid transmitters in the mammalian central nervous system, Ergebn. Physiol., 69 (1974) 94-188. 4 Dudar, J.D., In vitro excitation of hippocampal pyramidal cell dendrites by glutamic acid, Neuropharmacology, 13 (1974) 1083-1089. 5 Fagg, G.D. and Foster, A.C., Amino acid neurotransmitters and their pathways in the mammalian central nervous system, Neuroscience, 9 (1983) 701-719. 6 Hablitz, J.J. and Langmoen, I.A., Excitation of hippocampal pyramidal cells by glutamate in the guinea-pig and rat, J. Physiol. (London), 325 (1982) 317-331. 7 Kawai, N., Niwa, A. and Abe, T., Spider venom contains specific receptor blocker of glutaminergic synapses, Brain Research, 247 (1982) 169-171. 8 Kawai, N., Niwa, A. and Abe, T., Effect of a spider toxin on glutaminergic synapses in the mammalian brain, Bioreed. Res., 3 (1982) 353-355. 9 Kawai, N., Miwa, A. and Abe, T., Block of glutamate receptors by a spider toxin. In P. Mandel and F.V. DeFeudis (Eds.), CNS Receptors -- From Molecular Pharmacology to Behavior, Raven, New York, 1983, pp. 221-227.
and squid giant synapse 10 in respect to the differential effect of JSTX on glutamate and aspartate. It is generally accepted that the responses of v e r t e b r a t e neurons' to glutamate involve at least 3 r e c e p t o r types, which have been characterized as N-methyl-D-aspartate ( N M D A ) , kainate and quisqualate preferring 12,~5. In this connection, the present results indicate that JSTX m a y preferentially block quisqualate type receptors in CA1 p y r a m i d a l neurons. The authors thank Dr. V. R. E d g e r t o n for reading the manuscript. This work is s u p p o r t e d by Grants-inaid 57480123, 59216020 and 59223017 from the Ministry of Science.
10 Kawai, N., Yamagishi, S., Saito, M. and Furuya, K., Blockade of synaptic,transmission in the squid giant synapse by a spider toxin (JSTX), Brain Research, 278 (1983) 346-349. 11 Malthe-S~renssen, D., Skrede, K.K. and Fonnum F., Calcium-dependent release of o-[3H]aspartate evoked by selective electrical stimulation of excitatory afferent fibres to hippocampal pyramidal cells in vitro, Neuroscience, 4 (1979) 1255-1263. 12 Mclennan, H., Hicks, T.P. and Hall, J.G., Receptors for the excitatory amino acids. In F.V. Defeudis and P. Mandel (Eds.), Amino Acid Neurotransmitters, Raven, New York, 1§81, pp. 213-221. 13 Schwartzkroin, P.A. and Andersen, P., Glutamic acid sensitivity of dendrites in hippocampal slices in vitro, Adv. Neurol., 12 (1975) 45-51. 14 Storm-Mathisen, J., Localization of transmitter candidates in the brain: the hippocampal formation as a model, Prog. Neurobiol., 8 (1977) 119-181. 15 Watkins, J.C., Pharmacology of excitatory amino acid transmitter. In F.V. Defeudis and P. Mandel (Eds.), Amino Acid Neurotransmitters, Raven, New York, 1981, pp. 205-212. 16 Yamamoto, C., Activation of hippocampal neurons by mossy fiber stimulation in thin brain sections in vitro, Exp. Brain Res., 14 (1972) 423-435. 17 Yamamoto, C. and Kawai, N., Seizure discharges evoked in vitro in thin sections from guinea pig hippocampus, Science, 155 (1967) 341-342.
397
BRE 21138
Spider toxin (JSTX) blocks glutamate synapse in hippocampal pyramidal neurons MITSUYOSHI SAITOl, NOBUFUMI KAWAI1, AKIKO MIWA1, HIDEMITSU PAN-HOU2 and MASANORI YOSHIOKA2
1Tokyo Metropolitan Institutefor Neurosciences, Department of Neurobiology 2-6, Musashidai, Fuchu-City, Tokyo, 183 and 2Faculty of Pharmaceutical Sciences, Setsunan University, 45-1, Nagaotoge, Hirakata-City, Osaka, 573-01 (Japan) (Accepted June 11th, 1985)
Key words: glutamate receptor - - spider toxin - - JSTX - - hippocampus - - pyramidal cell
Effects of a spider toxin (JSTX) - - a specific blocker of glutamate receptors - - on single pyramidal neurons of the hippocampus were studied using tissue slices in vitro. JSTX blocked the synaptic response in CA, pyramidal cells evoked by Schaffer collateral stimulation without affecting the antidromic spike potential. The toxin suppressed glutamate-induced cell firings whereas it had little effect on aspartate-induced responses. The results suggest that glutamate is a neurotransmitter of the Schaffer collateral input to CA] pyramidal neurons. The hippocampus is one of the best studied systems in the brain with respect to the role of acidic amino acids. Many physiological as well as biochemical data have suggested glutamate and/or aspartate as the neurotransmitter in this system3, 5. However, recent results favour glutamate as a neurotransmitter in C A 1 neurons. For example, pyramidal cells in the C A 1 region are activated by iontophoretically applied glutamate into a highly sensitive region of the dendrites4,13. Biochemical data using selective stimulation of C A 1 excitatory inputs suggest that L-glutamate is a neurotransmitter of the Schaffer collaterals of the C A 3 neuron 11. Furthermore, Hablitz and Langmoen6 showed a close resemblance between the reversal potential for the glutamate potential and that for the excitatory postsynaptic potential (EPSP) of C A 1 cells. To strengthen evidence for L-glutamate as the neurotransmitter the use of a receptor blocker with a high degree of specificity m a y b e most helpful. In previous studies we reported that a spider toxin (JSTX) - - a blocker of glutamate receptors in the lobster neuromuscular synapse1, 7 - suppressed postsynaptic potentials in the brain slice of the hippocampus 8,9. In that study we recorded extracellular field potentials of pyramidal neurons of C A 3 and found that much higher concentrations of the toxin are needed to suppress the postsynaptic potentials than
those used in the crustacean neuromuscular synapse. The present study was undertaken to determine more precisely the effect of the purified toxin on a single pyramidal neuron activated by selective stimulation of Schaffer collaterals. The procedures for making hippocampal slices of guinea-pig brain was similar to that described previously16A 7. Stimulating electrodes made of platinum-iridium were placed at the stratum radiatum or alveus to deliver orthodromic or antidromic stimulation, respectively. Radiatum stimulation was controlled so that the currents activated exclusively Schaffer collaterals 2. Glass microelectrodes for intracellular recording were filled with 4 M potassium acetate and for extracellular recording, with saline or 2 M NaC1. Double- or triple-barrel electrodes were used for iontophoretic application of glutamate or aspartate (1 M L-sodium glutamate or 1 M L-sodium aspartate adjusted to p H 8.0). To check non-specific activation of the neuron by the currents the same strength of currents as used to eject glutamate or aspartate were applied from the other barrel filled with 2 M NaCI. Spiders (Nephila clavata, Joro spider) were collected in central Japan. Spider toxin (JSTX) was purified by the procedures of gel filtration, ionexchange column chromatography followed by fast performance liquid chromatography (in prepara-
Correspondence: N. Kawai, Tokyo Metropolitan Institute for Neurosciences, 2-6 Musashidai, Fuchu-City, Tokyo 183, Japan. 0006-8993/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
398 tion). The purified toxin was diluted with saline, suctioned into the glass pipette tip and e j e c t e d by air pressure (Picosplitzer II, G e n e r a l Valve). The procedure enabled us to apply a small a m o u n t of toxin in a localized area. In most cases we used 5 - 1 0 nl of toxin solution which contains ca. 10 -3 to 10 -4 U (1 U corresponds to the amount of purified toxin that is in one venom gland). Fig. 1 A shows a typical example of the blocking action of JSTX on the C A 1 pyramidal neuron. In response to activation of the stratum radiatum, excitatory postsynaptic potentials (EPSPs) followed by the action potentials were evoked. A f t e r the JSTX solution was applied to the outer part of the stratum radiatum, with a delay of about a minute the rising phase of EPSP slowed and then the action potentials were abolished, leaving only EPSPs. The remaining EPSPs were suppressed gradually and by 5 min they had disappeared. The volume of toxin solution required for the block of EPSPs ranged from 1 to 10 nl (=< 10 -3 U) which was reasonably low c o m p a r e d to that n e e d e d in the crustacean neuromuscular synapse 1.7. W h e n less toxin was applied, m o r e time was n e e d e d to block the action potential and the EPSPs were reduced in size. Frequently the EPSPs were not abolished and continued to be e v o k e d with r e d u c e d A
B
Orth.
Anti.
1 Control JSTX
Control
j~_
jL,,,._
_
A
I
JSTX
1.5-J " - ~
4.5-_~---6 - ~
120___.mY Wash IOtas
,J
~\
120mV I0 ms
Fig. 1. Block of EPSPs in CA l pyramidal cells of the hippocampus by a spider toxin (JSTX). A: intracellular records from a pyramidal cell in response to activation by Schaffer collaterals. The numerals on the left indicate the time in min after application of JSTX (6.5 x 10-4 U ) . B: another pyramidal cell was activated by Schaffer collateral (Orth.) followed by alveus (Anti.) stimulation. Upper trace, control. Middel trace, 5 min after applying JSTX (8 x 10_4 U). Lower traces, after rinsing the prep-" aration with normal saline. No recovery of EPSPs was found.
GILl 2
GIu 4
3
8
Asp4
5 nA
8 nA
2
4
Glu4 8
6
8 nA
Asp 4
8 nA
Fig. 2. Suppression of glutamate-induced firings of the pyramidal cells by JSTX. A: cell firings produced by iontophoretically applied glutamate in the absence (left) and presence (right) of JSTX (1 x 10-3 U). B: discharges induced by glutamate and aspartate before and after JSTX (1 x 10-3 U) application. A and B are taken from different cells. In both cases cell firings were recorded 10 min after toxin application. Vertical calibration indicates 10 Hz. amplitude. In either case, once EPSPs were suppressed no sign of recovery was found even though the p r e p a r a t i o n was peffused with normal Ringer for more than 1 h. The resting m e m b r a n e potential and the input resistance of the neuron were not changed before and after the toxin application. Insensitivity of non-synaptic portions of soma m e m b r a n e to the toxin was shown by the antidromic stimulation (Fig. 2B). CA~ neurons were activated by stimulating the stratum radiatum followed by the alveus. On applying JSTX, only the orthodromically e v o k e d action potentials were blocked, whereas the antidromic action potentials were little changed. This suggests that JSTX did not affect the soma m e m b r a n e which is responsible for the generation of the spike. CA~ neurons were activated also by iontophoretic application of L-glutamate. W h e n the glutamate pipette was suitably positioned at the apical dendrites in the stratum radiatum, spike discharges were recorded from the pyramidal neurons (Fig. 2). By raising the current more frequent discharges were e v o k e d from the b r o a d e r area of the dendrites. H o w e v e r , strong currents sometimes directly activate neurons because the same strength of currents delivered from another barrel containing 2 M NaC1 p r o d u c e d firing of neurons. W e therefore used considerably lower current strengths (less than 10 n A ) which only activate a well-localized dendrite area 5,13. In Fig. 2A, with an increase in glutamate currents from 2 to 5 nA, the frequency of spike discharges increased. After JSTX was applied to the dendritic area within 50
399 /~m of the current p i p e t t e , spike discharges were greatly suppressed. Pyramidal neurons were also activated by L-aspartic acid with similar current strengths to L-glutamate (Fig. 2B). Cell firings induced by L-glutamate were substantially suppressed in the presence of JSTX whereas the responses to L-aspartate were barely affected. W e found also that quisqualic acid and kainic acid, glutamate agonists, activated p y r a m i d a l neurons. JSTX suppressed quisqualate-induced spike discharges. Suppression was also evident in kainate-induced discharges but to a lesser degree than quisqualate. The above results are in a g r e e m e n t with data obtained in the crustacean n e u r o m u s c u l a r synapsO, 7
1 Abe, T., Kawai, N. and Miwa, A., Effects of a spider toxin on the glutaminergic synapse of lobster muscle, J. Physiol. (London), 339 (1983) 243-353. 2 Andersen, P., Silfvenius, H., Sundberg, S.H. and Sveen, O., A comparison of distal and proximal dendritic synapses on CA 1 pyramids in guinea-pig hippocampal slices in vitro, J. Physiol. (London), 307 (1980) 273-299. 3 Curtis, D.R. and Johnston, G.A.R., Amino acid transmitters in the mammalian central nervous system, Ergebn. Physiol., 69 (1974) 94-188. 4 Dudar, J.D., In vitro excitation of hippocampal pyramidal cell dendrites by glutamic acid, Neuropharmacology, 13 (1974) 1083-1089. 5 Fagg, G.D. and Foster, A.C., Amino acid neurotransmitters and their pathways in the mammalian central nervous system, Neuroscience, 9 (1983) 701-719. 6 Hablitz, J.J. and Langmoen, I.A., Excitation of hippocampal pyramidal cells by glutamate in the guinea-pig and rat, J. Physiol. (London), 325 (1982) 317-331. 7 Kawai, N., Niwa, A. and Abe, T., Spider venom contains specific receptor blocker of glutaminergic synapses, Brain Research, 247 (1982) 169-171. 8 Kawai, N., Niwa, A. and Abe, T., Effect of a spider toxin on glutaminergic synapses in the mammalian brain, Bioreed. Res., 3 (1982) 353-355. 9 Kawai, N., Miwa, A. and Abe, T., Block of glutamate receptors by a spider toxin. In P. Mandel and F.V. DeFeudis (Eds.), CNS Receptors -- From Molecular Pharmacology to Behavior, Raven, New York, 1983, pp. 221-227.
and squid giant synapse 10 in respect to the differential effect of JSTX on glutamate and aspartate. It is generally accepted that the responses of v e r t e b r a t e neurons' to glutamate involve at least 3 r e c e p t o r types, which have been characterized as N-methyl-D-aspartate ( N M D A ) , kainate and quisqualate preferring 12,~5. In this connection, the present results indicate that JSTX m a y preferentially block quisqualate type receptors in CA1 p y r a m i d a l neurons. The authors thank Dr. V. R. E d g e r t o n for reading the manuscript. This work is s u p p o r t e d by Grants-inaid 57480123, 59216020 and 59223017 from the Ministry of Science.
10 Kawai, N., Yamagishi, S., Saito, M. and Furuya, K., Blockade of synaptic,transmission in the squid giant synapse by a spider toxin (JSTX), Brain Research, 278 (1983) 346-349. 11 Malthe-S~renssen, D., Skrede, K.K. and Fonnum F., Calcium-dependent release of o-[3H]aspartate evoked by selective electrical stimulation of excitatory afferent fibres to hippocampal pyramidal cells in vitro, Neuroscience, 4 (1979) 1255-1263. 12 Mclennan, H., Hicks, T.P. and Hall, J.G., Receptors for the excitatory amino acids. In F.V. Defeudis and P. Mandel (Eds.), Amino Acid Neurotransmitters, Raven, New York, 1§81, pp. 213-221. 13 Schwartzkroin, P.A. and Andersen, P., Glutamic acid sensitivity of dendrites in hippocampal slices in vitro, Adv. Neurol., 12 (1975) 45-51. 14 Storm-Mathisen, J., Localization of transmitter candidates in the brain: the hippocampal formation as a model, Prog. Neurobiol., 8 (1977) 119-181. 15 Watkins, J.C., Pharmacology of excitatory amino acid transmitter. In F.V. Defeudis and P. Mandel (Eds.), Amino Acid Neurotransmitters, Raven, New York, 1981, pp. 205-212. 16 Yamamoto, C., Activation of hippocampal neurons by mossy fiber stimulation in thin brain sections in vitro, Exp. Brain Res., 14 (1972) 423-435. 17 Yamamoto, C. and Kawai, N., Seizure discharges evoked in vitro in thin sections from guinea pig hippocampus, Science, 155 (1967) 341-342.