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Serotonin inhibits the depolarization-evoked release of endogenous glutamate from rat cerebellar nerve endings
218
Neuroscience Letters, 67 (1986) 218 22, Elsevier Scientific Publishers Ireland Ltd.
NSL 03992
SEROTONIN INHIBITS THE DEPOLARIZATION-EVOKED RELEASE OF ENDOGENOUS GLUTAMATE FROM RAT CEREBELLAR NERVE ENDINGS
GUIDO MAURA, ANNAMARIA RICCHETTI and MAURIZIO RAITERI* lstituto di Farmaeologia e Farmacognosia, Universitgt di Genova, Viale Cembrano 4, 1-16148 Genova ( ItaO') (Received November 1lth, 1985; Accepted January 8th, 1986)
Key words: glutamate release serotonin - rat cerebellum - serotonin (5-HT0 receptor - superfused synaptosome methiothepin cinanserin - ketanserin
Glutamic acid (Glu) has been proposed as the neurotransmitter of cerebellar granule cells. 5-Hydroxytryptamine (5-HT) afferents project to the cerebellar cortex. The possible interaction between 5-HT and Glu was investigated by studying the effect of 5-HT on Glu release. The Ca2+-dependent depolarizationevoked release of endogenous Glu from superfused rat cerebellar synaptosomes was potently inhibited by 5-HT. Methiothepin, but not ketanserin, cinanserin or methysergide, antagonized 5-HT. It is concluded that the release of Glu can be modulated by 5-HT through receptors sited on Glu terminals. These receptors belong to the 5-HTrtype.
Evidence has accumulated to suggest thatglutamic acid (Glu) has a neurotransmitter role in the cerebellum. In particular, it has been proposed that this excitatory amino acid could be the transmitter of the granule cells released at the terminals of the parallel fibres [7, 10, 12, 14, 18, 20]. Serotonergic afferents from the raphe nuclei reach the cerebellar cortex [2]. Some of these fibers terminate with fine varicosities in the granular cell layer and others in the molecular layer [16]. Consistent with a possible anatomical relationship between 5-hydroxytryptamine (5-HT) and Glu neurons is the observation that 5-HT could inhibit the release of Glu evoked by high K + in rat cerebellar slices [5]. The inhibitory action of 5-HT on the release of Glu could be mediated by 5-HT receptors localized on the soma and/or dendrites of granule cells. In fact, the serotonergic projections to the granular layer appear to synapse with the granule cell dendrites [4], However, since serotonergic fibers also reach the molecular layer, an alternative or additional possibility is that 5-HT regulates Glu release by activating presynaptic 5-HT receptors located on the parallel fiber Glu terminals. In the present work we have investigated the latter possibility by studying the ac-
*Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
219
tion of 5-HT on the release of endogenous Glu from nerve terminals isolated from the rat cerebellum. Crude synaptosomal fractions were prepared from adult male Sprague-Dawley rats essentially according to Gray and Whittaker [8]. The synaptosomal pellet obtained after centrifugation at 12,000 g was resuspended in a physiological salt medium having the following composition (mM): NaCI 125, KC1 3, MgSO4 1.2, CaCI2 1.2, NaH2PO4 1.0, NaHCO3 22, glucose 10 (aeration with 5% CO2 in O~); pH 7.2-7.4. The synaptosomal suspension was incubated 15 min at 37'~C in standard medium: identical aliquots were then distributed in a set of parallel superfusion chambers thermostated at 37'C [13] and superfusion was started, at a rate of 0.6 ml/min, with medium aerated with 5% CO2 in 02. After 20 min of equilibration, collection of superfusate fractions was started (t = 0) according to the following scheme: one single fraction from t = 0 to t = 17; then one fraction of 2 min from t = 17 to t = 19 (basal release) and one fraction of 2 min from t = 20 to t = 22, corresponding to the maximal release observed during depolarization with high K + (15 mM KC1 was introduced at t = 18). Exogenous 5-HT was added concomitantly with high K + and the 5-HT antagonists 8 min before depolarization. Endogenous Glu was determined by highperformance liquid chromatography with fluorescent detection following precolumn derivatization with o-phthalaldeyde, essentially according to Tonnaer et al. [17]. The
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BASAL RELEASE E~ l ~
15ram KCI EVOKED RELEASE 15r,,M KCI EVOKED RELEASE (Ca 2" free medlLirn]
T
.o_
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4i
-~- 20
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o
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100
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Serotonin concentration (nM) Fig. 1. Depolarization-evoked release of endogenous Glu from superfused rat cerebellar synaptosomes and its calcium-dependence. Synaptosomes were depolarized with 15 mM KCI in the presence or in the absence of Ca ~ ' (omitted from the medium 8 rain before depolarization). Means ± S.E.M. of 3 6 experiments in triplicate are presented. Fig. 2. Inhibition of depolarization-evoked Glu release by 5-HT and antagonism by methiotbepin. Q) - C), 5-HT; • O , 5-HT + 6 n M methiothepin. The data reported are means + S.E.M. of 5 8
experiments in triplicate.
220 amount of Glu released into each fraction was expressed as a percentage of the total synaptosomal Glu content at the onset of the fraction considered. The K +-evoked release was obtained by subtracting the basal release from the postdepolarization fraction having the highest Glu content. Depolarization of cerebellar synaptosomes with 15 m M KC1 caused strong stimulation of Glu release (about 6 times the basal release). Omission of Ca 2÷ from the superfusion medium totally prevented the K+-induced stimulation of the release of the amino acid (Fig. 1). Fig. 2 illustrates the action of 5-HT on Glu release. When 5-HT was added to the superfusion medium, a concentration-dependent inhibition of the K +-evoked Glu release was observed. Serotonin was extremely potent in its inhibitory action and caused a significant (15-20 %) inhibition already at a concentration of 0.01 nM. The action of 5-HT was antagonized by methiothepin which shifted the concentration-response curve of 5-HT to the right in a parallel way (Fig. 2) suggesting a competitive antagonism. In contrast, other 5-HT receptor antagonists (ketanserin, methysergide, cinanserin) were ineffective when used at concentrations 100 times higher than that of the agonist (Table I). The first result of the present investigation indicates that in rat cerebellum 5-HT inhibited the depolarization-evoked release of Glu by activating presynaptic receptors localized on Glu nerve endings. During the preparation of synaptosomes, the cellular integrity is destroyed and any effect on Glu release from nerve terminals caused by 5-HT acting on dendritic and/or somatic receptors is therefore prevented. That the Glu released during depolarization under our experimental conditions originated from Glu terminals is very likely since we measured release of the endogenous amino acid from synaptosomes. In fact it was reported that the K+-evoked release of radioactive GIu previously taken up into cerebellar synaptosomes was only modestly Ca: ' -dependent, whereas the release of endogenous Glu showed an absolute Ca 2+ requirement [9, 10] (Fig. l). According to Levi et al. [10] the radioactive amino acid labels not only the Glu synaptosomes, but also the glial fragments (glioTABLE I EFFECT OF 5-HT ANTAGONISTS ON THE INHIBITION BY 5-HT OF THE K+-EVOKED RELEASE OF ENDOGENOUS Glu FROM CEREBELLUM SYNAPTOSOMES The data reported represent the percent changes of the K +-evokedrelease. Each value represents the mean _+ S.E.M. of several experiments(number in parentheses) performed in triplicate. Drugs
10 nM 5-HT 10 nM 5-HT + 10 nM 5-HT + 10 nM 5-HT + 10 nM 5-HT +
K +-evoked release of Glu (% inhibition)
10 nM methiothepin 1000nM ketanserin I000 nM cinanserin 1000 nM methysergide
51.5+3.12 12.0+0.31 48.0+ 1.20 50.5+3.10 49.0+2.90
(6) (4) (3) (3) (4)
221
somes) which contaminate the preparation of cerebellar synaptosomes and from which Glu is not released in a CaZ+-dependent way. Although the presence of 5-HT receptors on Glu terminals appears to be likely, the determination of the anatomical releationships between 5-HT afferent fibers and Glu terminals requires further investigation. The observation that in rat cerebellum only 3 % of the varicosities labeled by [3H]5-HT exhibited active zones in single thin sections ofcerebellar cortex [1] makes it unlikely that 5-HT varicosities form frequent axo-axonic synapses with the parallel fiber terminals. On the other hand, the idea of a non-synaptic or parasynaptic transmission in which modulatory or informational substances reach specific target cell receptors by diffusion from release points through extracellular fluids is gaining increasing acceptance [6, 15, 19]. Thus, in rat cerebellum, 5-HT may modulate the release of the primary transmitter Glu in such a way. The second relevant result concerns the type of 5-HT receptor involved in the inhibitory action of 5-HT on Glu release. As previously mentioned, 5-HT also inhibited the release of endogenous Glu evoked by high K+ in cerebellar slices; however, in this system, the action of 5-HT was antagonized by cinanserin [5], while this drug was inefl'ective in synaptosomes (Table I). This result suggests that 5-HT may act both at the level of the granule cell dendrites and on the parallel fiber terminal boutons, but through different types of 5-HT receptors. The finding that in isolated nerve endings 5-HT could not be blocked by the 5-HT2 antagonists ketanserin, methysergide or cinanserin indicates that the receptors involved are of the 5-HT~-type. This view is corroborated by the strong antagonism shown by methiothepin, a non-selective 5-HT antagonist which has pronounced 5-HT~ blocking activity [3, 11]. Interestingly, a pharmacological analysis with a number of selective 5-HTt agonists and antagonists has recently led to the conclusion that the 5-HT receptor sited on Glu terminals in rat cerebellum may represent a novel subtype of the 5-HT~ receptor (Raiteri et al., submitted for publication). This work was supported by grants from the Italian Ministry of Education and from the Italian C.N.R. The authors are grateful to Mrs. Maura Agate for her excellent preparation of this manuscript. 1 Beaudet, A. and Sotelo, C., Synaptic remodeling of serotonin axon terminals in rat agranular cerebellum, Brain Res., 206 (1981)305-329. 2 Bobillier, P., Seguin, S., Petitjean, F., Salvert, D., Touret, M. and Jouvet, M., The raphe nuclei of the cat brain stem: a topographical atlas of their efferent projections as revealed by autoradiography, Brain Res., 113 (1976) 449 486. 3 ('errito, F. and Raiteri, M., Serotonin release is modulated by presynaptic autoreceptors, Eur. J. Pharmacol., 57 (1979) 4274-30. 4 Chan-Palay, V., Cerebellar Dentate Nucleus Organization, Cytology and Transmitters, Springer, Berlin, 1977, pp. 390-454. 5 Davies, J.A. and Leighton, G.E., Effect of cinanserin on the release of glutamate from rat cerebellar slices, Br. J. Pharmacol., 82 (1984) 271P. 6 Dismukes, R.K., New concepts of molecular communication a m o n g neurons, Behav. Brain Sci., 2 (1979) 409 448.
222 7 Gallo. V.. Ciotti. M.T.. Coletti, A., Aloisi. F. and Levi. G., Selective release of glutamate from cerebellar granule cells differentiating in culture, Prec. Natl. Acad. Sci. USA, 79 (1982) 7919-7923. 8 Gray, E.G. and Whittaker. V.P., The isolation of nerve endings from brain: an electron microscope st udy of cell fragments derived by homogenization and centrifugation. J. Anat.. 96 (1962) 79-87. 9 Levi. G.. Gallo. V. and Raiteri, M., GABA potentiates the depolarization-induced release of glutamate from cerebetlar nerve endings. In G. Di Chiara and G.L. Gessa (Eds.), Glutamate as a Neurotransmitter, Raven Press. New York. 1981. pp. 127-137. 10 Levi. G.. Gordon, R.D., Gallo. V., Wilkin, G.P. and Balazs. R.. Putative acidic amino acid transmitters in the cerebellum. I. Depolarization-induced release, Brain Res.. 239 (1982) 425-445. I I Martin. L.L. and Sanders-Bush. E., Comparison of the pharmacological characteristics of 5-HTt and 5-HT2 binding sites with those of serotonin autoreceptors which modulate serotonin release, NaunynSchmiedeberg's Arch. Pharmacol.. 321 t 1982) t 65-170. 12 McBride. A.J.. Nadi. N.S.. Altman. J and Aprison. M.H.. Effects of selective doses of X-irradiation on the levels of several amino acids in the cerebellum of the rat. Neurochem. Res., 1 (1976) 141-152. 13 Raiteri. M.. Angelini. F. and Levi. G.. A simple apparatus for studying the release of neurotransmitters from synaptosomes. Eur. J. Pharmacol.. 25 (1974) 411-414. 14 Sandoval. M.E. and Cotman. C.W.. Evaluation of glutamate as a neurotransmitter of cerebellar parallel fibers. Neuroscience. 3 (1978) 199-206. 15 Schmitt. F.O.. Molecular regulators of brain function: a new view. Neuroscience, 13 (1984) 991-1001. 16 Takeuchi. Y.. Kimura. H. and Sano. Y.. lmmunohistochemical demonstration of serotonin-containing nerve fibers in the cerebellum. Cell Tissue Res.. 226 (1982) 1-12. 17 Tonnaer. J.A.D.M.. Engels, G.M.H.. Wiegant, V.M.. Burbach. J.P.H.. De Jong, W. and De Wied, D.. Proteolytic conversion angiotensins in rat brain tissue, Eur. J. Biochem., 131 (t983) 415-421. 18 Valcana, T.. Hudson. D. and Timiras. P.S., Effects of X-irradiation on the content of amino acids in the developing rat cerebellum, J, Neurochem.. 19 (1972) 2229--2232. 19 Vizi. E.S.. Non-Synaptic Interactions between Neurons: Modulation of Neurochemical Transmission, John Wiley & Sons. Chichester, New York. Brisbane. Toronto. singapore, 1984. 20 Young, A.B.. Oster-Granite. M.L.. Herndon. R.M. and Snyder. S.H.. Glutamic acid: selective depletion by viral-induced granule cell loss in hamster cerebellum, Brain Res.. 73 (1974) 1-13.
Neuroscience Letters, 67 (1986) 218 22, Elsevier Scientific Publishers Ireland Ltd.
NSL 03992
SEROTONIN INHIBITS THE DEPOLARIZATION-EVOKED RELEASE OF ENDOGENOUS GLUTAMATE FROM RAT CEREBELLAR NERVE ENDINGS
GUIDO MAURA, ANNAMARIA RICCHETTI and MAURIZIO RAITERI* lstituto di Farmaeologia e Farmacognosia, Universitgt di Genova, Viale Cembrano 4, 1-16148 Genova ( ItaO') (Received November 1lth, 1985; Accepted January 8th, 1986)
Key words: glutamate release serotonin - rat cerebellum - serotonin (5-HT0 receptor - superfused synaptosome methiothepin cinanserin - ketanserin
Glutamic acid (Glu) has been proposed as the neurotransmitter of cerebellar granule cells. 5-Hydroxytryptamine (5-HT) afferents project to the cerebellar cortex. The possible interaction between 5-HT and Glu was investigated by studying the effect of 5-HT on Glu release. The Ca2+-dependent depolarizationevoked release of endogenous Glu from superfused rat cerebellar synaptosomes was potently inhibited by 5-HT. Methiothepin, but not ketanserin, cinanserin or methysergide, antagonized 5-HT. It is concluded that the release of Glu can be modulated by 5-HT through receptors sited on Glu terminals. These receptors belong to the 5-HTrtype.
Evidence has accumulated to suggest thatglutamic acid (Glu) has a neurotransmitter role in the cerebellum. In particular, it has been proposed that this excitatory amino acid could be the transmitter of the granule cells released at the terminals of the parallel fibres [7, 10, 12, 14, 18, 20]. Serotonergic afferents from the raphe nuclei reach the cerebellar cortex [2]. Some of these fibers terminate with fine varicosities in the granular cell layer and others in the molecular layer [16]. Consistent with a possible anatomical relationship between 5-hydroxytryptamine (5-HT) and Glu neurons is the observation that 5-HT could inhibit the release of Glu evoked by high K + in rat cerebellar slices [5]. The inhibitory action of 5-HT on the release of Glu could be mediated by 5-HT receptors localized on the soma and/or dendrites of granule cells. In fact, the serotonergic projections to the granular layer appear to synapse with the granule cell dendrites [4], However, since serotonergic fibers also reach the molecular layer, an alternative or additional possibility is that 5-HT regulates Glu release by activating presynaptic 5-HT receptors located on the parallel fiber Glu terminals. In the present work we have investigated the latter possibility by studying the ac-
*Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
219
tion of 5-HT on the release of endogenous Glu from nerve terminals isolated from the rat cerebellum. Crude synaptosomal fractions were prepared from adult male Sprague-Dawley rats essentially according to Gray and Whittaker [8]. The synaptosomal pellet obtained after centrifugation at 12,000 g was resuspended in a physiological salt medium having the following composition (mM): NaCI 125, KC1 3, MgSO4 1.2, CaCI2 1.2, NaH2PO4 1.0, NaHCO3 22, glucose 10 (aeration with 5% CO2 in O~); pH 7.2-7.4. The synaptosomal suspension was incubated 15 min at 37'~C in standard medium: identical aliquots were then distributed in a set of parallel superfusion chambers thermostated at 37'C [13] and superfusion was started, at a rate of 0.6 ml/min, with medium aerated with 5% CO2 in 02. After 20 min of equilibration, collection of superfusate fractions was started (t = 0) according to the following scheme: one single fraction from t = 0 to t = 17; then one fraction of 2 min from t = 17 to t = 19 (basal release) and one fraction of 2 min from t = 20 to t = 22, corresponding to the maximal release observed during depolarization with high K + (15 mM KC1 was introduced at t = 18). Exogenous 5-HT was added concomitantly with high K + and the 5-HT antagonists 8 min before depolarization. Endogenous Glu was determined by highperformance liquid chromatography with fluorescent detection following precolumn derivatization with o-phthalaldeyde, essentially according to Tonnaer et al. [17]. The
70 6o
BASAL RELEASE E~ l ~
15ram KCI EVOKED RELEASE 15r,,M KCI EVOKED RELEASE (Ca 2" free medlLirn]
T
.o_
.~
so
.~_ o
4(]
a~
~ 3o -'~
4i
-~- 20
m
o
'
z
(9
IIIIII
® i
0.()1
01
f
10
100
100(]
Serotonin concentration (nM) Fig. 1. Depolarization-evoked release of endogenous Glu from superfused rat cerebellar synaptosomes and its calcium-dependence. Synaptosomes were depolarized with 15 mM KCI in the presence or in the absence of Ca ~ ' (omitted from the medium 8 rain before depolarization). Means ± S.E.M. of 3 6 experiments in triplicate are presented. Fig. 2. Inhibition of depolarization-evoked Glu release by 5-HT and antagonism by methiotbepin. Q) - C), 5-HT; • O , 5-HT + 6 n M methiothepin. The data reported are means + S.E.M. of 5 8
experiments in triplicate.
220 amount of Glu released into each fraction was expressed as a percentage of the total synaptosomal Glu content at the onset of the fraction considered. The K +-evoked release was obtained by subtracting the basal release from the postdepolarization fraction having the highest Glu content. Depolarization of cerebellar synaptosomes with 15 m M KC1 caused strong stimulation of Glu release (about 6 times the basal release). Omission of Ca 2÷ from the superfusion medium totally prevented the K+-induced stimulation of the release of the amino acid (Fig. 1). Fig. 2 illustrates the action of 5-HT on Glu release. When 5-HT was added to the superfusion medium, a concentration-dependent inhibition of the K +-evoked Glu release was observed. Serotonin was extremely potent in its inhibitory action and caused a significant (15-20 %) inhibition already at a concentration of 0.01 nM. The action of 5-HT was antagonized by methiothepin which shifted the concentration-response curve of 5-HT to the right in a parallel way (Fig. 2) suggesting a competitive antagonism. In contrast, other 5-HT receptor antagonists (ketanserin, methysergide, cinanserin) were ineffective when used at concentrations 100 times higher than that of the agonist (Table I). The first result of the present investigation indicates that in rat cerebellum 5-HT inhibited the depolarization-evoked release of Glu by activating presynaptic receptors localized on Glu nerve endings. During the preparation of synaptosomes, the cellular integrity is destroyed and any effect on Glu release from nerve terminals caused by 5-HT acting on dendritic and/or somatic receptors is therefore prevented. That the Glu released during depolarization under our experimental conditions originated from Glu terminals is very likely since we measured release of the endogenous amino acid from synaptosomes. In fact it was reported that the K+-evoked release of radioactive GIu previously taken up into cerebellar synaptosomes was only modestly Ca: ' -dependent, whereas the release of endogenous Glu showed an absolute Ca 2+ requirement [9, 10] (Fig. l). According to Levi et al. [10] the radioactive amino acid labels not only the Glu synaptosomes, but also the glial fragments (glioTABLE I EFFECT OF 5-HT ANTAGONISTS ON THE INHIBITION BY 5-HT OF THE K+-EVOKED RELEASE OF ENDOGENOUS Glu FROM CEREBELLUM SYNAPTOSOMES The data reported represent the percent changes of the K +-evokedrelease. Each value represents the mean _+ S.E.M. of several experiments(number in parentheses) performed in triplicate. Drugs
10 nM 5-HT 10 nM 5-HT + 10 nM 5-HT + 10 nM 5-HT + 10 nM 5-HT +
K +-evoked release of Glu (% inhibition)
10 nM methiothepin 1000nM ketanserin I000 nM cinanserin 1000 nM methysergide
51.5+3.12 12.0+0.31 48.0+ 1.20 50.5+3.10 49.0+2.90
(6) (4) (3) (3) (4)
221
somes) which contaminate the preparation of cerebellar synaptosomes and from which Glu is not released in a CaZ+-dependent way. Although the presence of 5-HT receptors on Glu terminals appears to be likely, the determination of the anatomical releationships between 5-HT afferent fibers and Glu terminals requires further investigation. The observation that in rat cerebellum only 3 % of the varicosities labeled by [3H]5-HT exhibited active zones in single thin sections ofcerebellar cortex [1] makes it unlikely that 5-HT varicosities form frequent axo-axonic synapses with the parallel fiber terminals. On the other hand, the idea of a non-synaptic or parasynaptic transmission in which modulatory or informational substances reach specific target cell receptors by diffusion from release points through extracellular fluids is gaining increasing acceptance [6, 15, 19]. Thus, in rat cerebellum, 5-HT may modulate the release of the primary transmitter Glu in such a way. The second relevant result concerns the type of 5-HT receptor involved in the inhibitory action of 5-HT on Glu release. As previously mentioned, 5-HT also inhibited the release of endogenous Glu evoked by high K+ in cerebellar slices; however, in this system, the action of 5-HT was antagonized by cinanserin [5], while this drug was inefl'ective in synaptosomes (Table I). This result suggests that 5-HT may act both at the level of the granule cell dendrites and on the parallel fiber terminal boutons, but through different types of 5-HT receptors. The finding that in isolated nerve endings 5-HT could not be blocked by the 5-HT2 antagonists ketanserin, methysergide or cinanserin indicates that the receptors involved are of the 5-HT~-type. This view is corroborated by the strong antagonism shown by methiothepin, a non-selective 5-HT antagonist which has pronounced 5-HT~ blocking activity [3, 11]. Interestingly, a pharmacological analysis with a number of selective 5-HTt agonists and antagonists has recently led to the conclusion that the 5-HT receptor sited on Glu terminals in rat cerebellum may represent a novel subtype of the 5-HT~ receptor (Raiteri et al., submitted for publication). This work was supported by grants from the Italian Ministry of Education and from the Italian C.N.R. The authors are grateful to Mrs. Maura Agate for her excellent preparation of this manuscript. 1 Beaudet, A. and Sotelo, C., Synaptic remodeling of serotonin axon terminals in rat agranular cerebellum, Brain Res., 206 (1981)305-329. 2 Bobillier, P., Seguin, S., Petitjean, F., Salvert, D., Touret, M. and Jouvet, M., The raphe nuclei of the cat brain stem: a topographical atlas of their efferent projections as revealed by autoradiography, Brain Res., 113 (1976) 449 486. 3 ('errito, F. and Raiteri, M., Serotonin release is modulated by presynaptic autoreceptors, Eur. J. Pharmacol., 57 (1979) 4274-30. 4 Chan-Palay, V., Cerebellar Dentate Nucleus Organization, Cytology and Transmitters, Springer, Berlin, 1977, pp. 390-454. 5 Davies, J.A. and Leighton, G.E., Effect of cinanserin on the release of glutamate from rat cerebellar slices, Br. J. Pharmacol., 82 (1984) 271P. 6 Dismukes, R.K., New concepts of molecular communication a m o n g neurons, Behav. Brain Sci., 2 (1979) 409 448.
222 7 Gallo. V.. Ciotti. M.T.. Coletti, A., Aloisi. F. and Levi. G., Selective release of glutamate from cerebellar granule cells differentiating in culture, Prec. Natl. Acad. Sci. USA, 79 (1982) 7919-7923. 8 Gray, E.G. and Whittaker. V.P., The isolation of nerve endings from brain: an electron microscope st udy of cell fragments derived by homogenization and centrifugation. J. Anat.. 96 (1962) 79-87. 9 Levi. G.. Gallo. V. and Raiteri, M., GABA potentiates the depolarization-induced release of glutamate from cerebetlar nerve endings. In G. Di Chiara and G.L. Gessa (Eds.), Glutamate as a Neurotransmitter, Raven Press. New York. 1981. pp. 127-137. 10 Levi. G.. Gordon, R.D., Gallo. V., Wilkin, G.P. and Balazs. R.. Putative acidic amino acid transmitters in the cerebellum. I. Depolarization-induced release, Brain Res.. 239 (1982) 425-445. I I Martin. L.L. and Sanders-Bush. E., Comparison of the pharmacological characteristics of 5-HTt and 5-HT2 binding sites with those of serotonin autoreceptors which modulate serotonin release, NaunynSchmiedeberg's Arch. Pharmacol.. 321 t 1982) t 65-170. 12 McBride. A.J.. Nadi. N.S.. Altman. J and Aprison. M.H.. Effects of selective doses of X-irradiation on the levels of several amino acids in the cerebellum of the rat. Neurochem. Res., 1 (1976) 141-152. 13 Raiteri. M.. Angelini. F. and Levi. G.. A simple apparatus for studying the release of neurotransmitters from synaptosomes. Eur. J. Pharmacol.. 25 (1974) 411-414. 14 Sandoval. M.E. and Cotman. C.W.. Evaluation of glutamate as a neurotransmitter of cerebellar parallel fibers. Neuroscience. 3 (1978) 199-206. 15 Schmitt. F.O.. Molecular regulators of brain function: a new view. Neuroscience, 13 (1984) 991-1001. 16 Takeuchi. Y.. Kimura. H. and Sano. Y.. lmmunohistochemical demonstration of serotonin-containing nerve fibers in the cerebellum. Cell Tissue Res.. 226 (1982) 1-12. 17 Tonnaer. J.A.D.M.. Engels, G.M.H.. Wiegant, V.M.. Burbach. J.P.H.. De Jong, W. and De Wied, D.. Proteolytic conversion angiotensins in rat brain tissue, Eur. J. Biochem., 131 (t983) 415-421. 18 Valcana, T.. Hudson. D. and Timiras. P.S., Effects of X-irradiation on the content of amino acids in the developing rat cerebellum, J, Neurochem.. 19 (1972) 2229--2232. 19 Vizi. E.S.. Non-Synaptic Interactions between Neurons: Modulation of Neurochemical Transmission, John Wiley & Sons. Chichester, New York. Brisbane. Toronto. singapore, 1984. 20 Young, A.B.. Oster-Granite. M.L.. Herndon. R.M. and Snyder. S.H.. Glutamic acid: selective depletion by viral-induced granule cell loss in hamster cerebellum, Brain Res.. 73 (1974) 1-13.