Amino Acid Neurotransmitters and Dopamine in Brain and Pituitary of the Goldfish: Involvement in the Regulation of Gonadotropin Secretion

An isocratic high-performance liquid chromatographic technique was developed to measure levels of gamma-aminobutyric acid (GABA), glutamate, and taurine in the brain and pituitary of goldfish. Accuracy of this procedure for quantification of these compounds was established by evaluating anesthetic and postmortem effects and by selectively manipulating GABA concentrations by intraperitoneal administration of the glutamic acid decarboxylase (GAD) inhibitor 3-mercaptopropionic acid or the GABA transaminase inhibitor gamma-vinyl GABA. The technique provided a simple, rapid, and reliable method for evaluating the concentrations of these amino acids without the use of complex gradient chromatographic systems. To investigate the relationship between neurotransmitter amino acids and the control of pituitary secretion of gonadotropin, the effects of injection of taurine, GABA, or monosodium glutamate on GABA, glutamate, taurine, and, in some instances, monoamine concentrations in the brain and pituitary were evaluated and related to serum gonadotropin levels. Injection of taurine caused an elevation in serum gonadotropin concentrations. In addition, injection of the taurine precursor hypotaurine but not the taurine catabolite isethionic acid elevated serum gonadotropin levels. Intracerebroventricular injection of either GABA or taurine also elevated serum gonadotropin concentrations. Pretreatment of recrudescent fish with alpha-methyl-p-tyrosine reduced pituitary dopamine concentrations and also potentiated the serum gonadotropin response to taurine. Injection of monosodium glutamate caused an increase of glutamate content in the pituitary at 24 h; this was followed by a decrease at 72 h after administration. Pituitary GABA, taurine, and dopamine concentrations underwent a transient depletion after monosodium glutamate administration, and this was associated with an elevation of serum gonadotropin content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Huntington's disease: studies on brain free amino acids

We studied the levels of free amino acids in putamen and Brodmann's area 10 of 12 patients who died with Huntington's disease and 13 non-neurologic controls. GABA, glutamate and alpha-amino-n-butyric acid concentrations were found to be reduced in putamen of Huntington's disease patients. In Brodmann's area 10 the levels of glutamate, histidine and lysine were decreased, but the content of aspartate, GABA, glycine, serine and taurine was increased in the same group of patients.     

Effect of Impact Trauma on Neurotransmitter and Nonneurotransmitter Amino Acids in Rat Spinal Cord

N-Methyl-D-aspartate (NMDA) administration exacerbates neurological dysfunction after traumatic spinal cord injury in rats, whereas NMDA antagonists improve outcome in this model. These observations suggest that release of excitatory amino acids contributes to secondary tissue damage after traumatic spinal cord injury. To further examine this hypothesis, concentrations of free amino acids were measured in spinal cord samples from anesthetized rats subjected to various degrees of impact trauma to the T9 spinal segment. Levels of excitatory and inhibitory neurotransmitter amino acids [gamma-aminobutyric acid (GABA), glutamate, aspartate, glycine, taurine] and levels of nonneurotransmitter amino acids (asparagine, glutamine, alanine, threonine, serine) were determined at 5 min, 4 h, and 24 h posttrauma. Uninjured surgical (laminectomy) control animals showed modest but significant declines in aspartate and glutamate levels, but not in other amino acids, at all time points. In injured animals, the excitatory amino acids glutamate and aspartate were significantly decreased by 5 min posttrauma, and remained depressed at 4 h and 24 h as compared with corresponding laminectomy controls. In contrast, the inhibitory amino acids, glycine, GABA, and taurine, were decreased at 5 min postinjury, had partially recovered at 4 h, and were almost fully recovered at 24 h. The nonneurotransmitter amino acids were unchanged at 5 min posttrauma and significantly increased at 4 h, with partial recovery at 24 h. At 4 h postinjury, severe trauma caused significantly greater decreases in aspartate and glutamate than did either mild or moderate injury. These findings are consistent with the postulated role of excitatory amino acids in CNS trauma.     

Release of Endogenous Glutamate,Aspartate, GABA,and Taurine from Hippocampal Slices from Adult and Developing Mice under Cell-Damaging Conditions

The releases of endogenous glutamate, aspartate, GABA and taurine from hippocampal slices from 7-day-, 3-, 12-, and 18-month-old mice were investigated under cell-damaging conditions using a superfusion system. The slices were superfused under hypoxic conditions in the presence and absence of glucose and exposed to hydrogen peroxide. In the adult hippocampus under normal conditions the basal release of taurine was highest, with a response only about 2-fold to potassium stimulation (50 mM). The low basal releases of glutamate, aspartate, and GABA were markedly potentiated by K⁺ ions. In general, the release of the four amino acids was enhanced under all above cell-damaging conditions. In hypoxia and ischemia (i.e., hypoxia in the absence of glucose) the release of glutamate, aspartate and GABA increased relatively more than that of taurine, and membrane depolarization by K⁺ markedly potentiated the release processes. Taurine release was doubled in hypoxia and tripled in ischemia but K⁺ stimulation was abolished. In both the mature and immature hippocampus the release of glutamate and aspartate was greatly enhanced in the presence of H₂O₂, that of aspartate particularly in developing mice. In the immature hippocampus the increase in taurine release was 10-fold in hypoxia and 30-fold in ischemia, and potassium stimulation was partly preserved. The release processes of the four amino acids in ischemia were all partially Ca²⁺-dependent. High concentrations of excitatory amino acids released under cell-damaging conditions are neurotoxic and contribute to neuronal death during ischemia. The substantial amounts of the inhibitory amino acids GABA and taurine released simultaneously may constitute an important protective mechanism against excitatory amino acids in excess, counteracting their harmful effects. In the immature hippocampus in particular, the massive release of taurine under cell-damaging conditions may have a significant function in protecting neural cells and aiding in preserving their viability.     

Taurine, glutamate and GABA modulate the outgrowth from goldfish retinal explants and its concentrations are affected by the crush of the optic nerve

Summary The amino acid taurine plays an important trophic role during development and regeneration of the central nervous system. Other amino acid systems, such as those for glutamate and gamma-aminobutyric acid (GABA), are modified during the same physiological and pathological processes. After crushing the optic nerve, goldfish retinal explants were plated in the absence and in the presence of different amino acids and amino acid receptor agonists. The length and the density of the neurites were measured at 5 days in culture. Taurine increased the length and the density of neurites. Glutamate and glycine increased them at low concentration, but were inhibitors at higher concentration. The combination of N-methyl-D-aspartate (NMDA) and glycine produced a greater inhibitory effect than NMDA alone. NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) added simultaneously with taurine impaired the stimulatory effect of the latter. GABA stimulated the emission of neurites in a concentration dependent manner. Hypotaurine also elevated the length of neurites, but cysteinesulfinic acid did not produce a significant effect. The concentrations of taurine, glutamate and GABA were determined by HPLC with fluorescent detection in the retina of goldfish at various days post-crushing the optic nerve. The levels of taurine were significantly increased at 48 h after the crush, and were elevated up to 20 days. Glutamate level decreased after the lesion of the optic nerve and was still low at 20 days. GABA concentration was not significantly different from the control. The interaction of these amino acids during the regenerative period, especially the balance between taurine and glutamate, may be a determinant in restoring vision after the crush.Abbreviations AMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - GABA gamma-aminobutyric acid - NMDA N-methyl-D-aspartate     

Changes in the Extracellular Concentrations of Amino Acids in the Rat Striatum During Transient Focal Cerebral Ischemia

Abstract: Although considerable evidence supports a role for amino acids in transient global cerebral ischemia and permanent focal cerebral ischemia, effects of transient focal cerebral ischemia on the extracellular concentrations of amino acids have not been reported. Accordingly, our study was undertaken to examine the patterns of changes of extracellular glutamate, aspartate, GABA, taurine, glutamine, alanine, and phosphoethanolamine in the striatum of transient focal cerebral ischemia, as evidence to support their pathogenic roles. Focal ischemia was induced using the middle cerebral artery occlusion model, with no need for craniotomy. Microdialysis was used to sample the brain's extracellular space before, during, and after the ischemic period. One hour of middle cerebral artery occlusion followed by recirculation caused neuronal damage that was common in the frontoparietal cortex and the lateral segment of the caudate nucleus. During 1 h of ischemia, the largest increase occurred for GABA and moderate increases were observed for taurine, glutamate, and aspartate. Alanine, which is a nonneuroactive amino acid, increased little. After recirculation, the levels of glutamate and aspartate reverted to normal baseline values right after reperfusion. Despite these rapid normalizations, neuronal damage occurred. Therefore, uptake of excitatory amino acids can still be restored after 1 h of middle cerebral artery occlusion, and tissue damage occurs even though high extracellular levels of glutamate are not maintained.     

Osmolyte contents of cultured astrocytes grown in hypoosmotic medium

Primary rat cerebral astrocyte cultures were grown for 2 weeks in isoosmotic medium (305 mosmol) and then placed in similar medium with a reduced NaCl concentration. During the first hour of growth in this moderately hypoosmotic medium (240 mosmol), the cells lose 88% of their taurine contents, 62% of their alanine contents, and 54% of their aspartate contents while regaining normal volume. Loss of these amino acids accounts for 43% of observed volume regulation. Contents of these amino acids remain decreased during 24 h of growth in hypoosmotic medium. In contrast, potassium, glutamate, glutamine, and asparagine contents are not changed, relative to cells in isoosmotic medium, at time points between 1 h and 24 h of hypoosmotic exposure. The data suggest astrocytes contribute to net loss of amino acids, but not potassium, from brains exposed to hypoosmotic conditions in situ.     

Disturbances of Amino Acids from Temporal Lobe Synaptosomes in Human Complex Partial Epilepsy

We have studied the levels of neuroactive amino acids in synaptosomes (P₂ fraction) isolated from brain tissue of ten patients with medically intractable epilepsy who were undergoing temporal lobectomy. First, lateral temporal tissue (nonfocal) was removed followed by medial temporal tissue (focal). A synaptosomal fraction (P₂) was immediately prepared from each tissue and analyzed for free amino acid concentrations. Statistically significant reductions were seen in glutamine and GABA concentrations in focal tissue compared to nonfocal tissue. The ratio of excitatory amino acids (aspartate and glutamate) to inhibitory amino acids (taurine and GABA) was significantly higher in focal tissue compared to nonfocal. The glutamine/glutamate ratio was significantly reduced. These data support the hypothesis that alterations in the balance between excitatory and inhibitory amino acids may be involved in the expression of epilepsy.     

Extracellular Levels of Amino Acids and Choline in Human High Grade Gliomas: An Intraoperative Microdialysis Study

The concentrations of endogenous amino acids and choline in the extracellular fluid of human cerebral gliomas have been measured, for the first time, by in vivo microdialysis. Glioblastoma growth was associated with increased concentrations of choline, GABA, isoleucine, leucine, lysine, phenylalanine, taurine, tyrosine, and valine. There was no difference between grade III and grade IV tumors in the concentrations of phenylalanine, isoleucine, tyrosine, valine, and lysine, whereas the concentrations of choline, aspartate, taurine, GABA, leucine, and glutamate were significantly different in the two tumor-grade subgroups. In contrast to the other compounds, the concentration of glutamate was decreased in glioma. The parenchyma adjacent to the tumor showed significant changes only in the extracellular concentration of glutamate, isoleucine, and valine. The concentrations of choline and the amino acids, glutamate, leucine, taurine, and tyrosine showed significant positive correlations with the degree of cell proliferation. Epilepsy, which is relatively common in subjects with gliomas, was shown to be a significant confounding variable when the extracellular concentrations of aspartate, glutamate and GABA were considered.     

Possible role of glutamate, aspartate, glutamine, GABA or taurine on cadmium toxicity on the hypothalamic pituitary axis activity in adult male rats

This work was designed to evaluate the possible changes in glutamate, aspartate, glutamine, GABA and taurine within various hypothalamic areas the striatum and prefrontal cortex after oral cadmium exposure in adult male rats, and if these changes are related to pituitary hormone secretion. The contents of glutamine, glutamate, aspartate, GABA and taurine in the median eminence, anterior, mediobasal and posterior hypothalamus, and in prefrontal cortex in adult male rats exposed to 272.7 mol l^–1 of cadmium chloride (CdCl₂) in the drinking water for one month. Cadmium diminished the content of glutamine, glutamate and aspartate in anterior hypothalamus as compared to the values found in the untreated group. Besides, there is a decrease in the content of glutamate, aspartate and taurine in the prefrontal cortex. The amino acids studied did not change in median eminence, mediobasal and posterior hypothalamus or the striatum by cadmium treatment. Plasma prolactin and LH levels decreased in rats exposed to the metal. These results suggest that (1) cadmium differentially affects amino acid content within the brain region studied and (2) the inhibitory effect of cadmium on prolactin and LH secretion may be partially explained by a decrease in the content of both glutamate and aspartate in anterior hypothalamus, but not through changes in GABA and taurine.     

In vivo release patterns and cardiovascular properties of inhibitory and excitatory amino acids in the hypothalamus

Summary The posterior hypothalamus of conscious, freely moving rats was superfused with artificial cerebrospinal fluid through a push-pull cannula and the release of amino acids was determined in the superfusate. Under basal conditions, the release rates of taurine, GABA and glutamate fluctuated according to ultradian rhythms with different frequencies. Hypothalamic superfusion with veratridine or high concentrations of potassium choride enhanced the release rates of taurine, GABA and glutamate in a concentration-dependent way. Tetrodotoxin decreased the basal release rates of the three amino acids. The release of arginine was not influenced significantly by these compounds. A fall of blood pressure elicited by intravenous infusion of nitroprusside decreased the release rates of GABA and taurine and enhanced the release of glutamate. Infusion of noradrenaline increased blood pressure and release rates of GABA and taurine, while the release of glutamate was not influenced. Neither the pressor, nor the depressor responses to drugs influenced the release of arginine in the hypothalamus. It is concluded that the inhibitory amino acids taurine and GABA released from hypothalamic neurons possess a tonic hypotensive function. The excitatory amino acid glutamate, released from glutamatergic neurons of the hypothalamus, seems to possess a hypertensive function in counteracting a fall of blood pressure.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung. These results were presented at the Third International Congress on Amino Acids, Vienna, August 1993     

EFFECT OF ELECTRICAL STIMULATION AND CHLORPROMAZINE ON THE UPTAKE AND RELEASE OF TAURINE, γ-AMINOBUTYRIC ACID AND GLUTAMIC ACID IN MOUSE BRAIN SYNAPTOSOMES

—The concentrations of taurine and GABA were determined in isolated mouse brain synaptosomes incubated in Krebs-Ringer phosphate medium (pH 7·4). The concentration of GABA gradually decreased during incubation, but that of taurine remained approximately unchanged. In the presence of chlorpromazine the amount of GABA in the synaptosomes increased, but the efflux and influx of GABA were slightly reduced. The content and efflux of both taurine and GABA increased in electrically stimulated synaptosomes, and the influx of taurine, GABA and glutamate into the synaptosomes similarly increased. All three amino acids are taken up by the synaptosomes through at least two mechanisms: low-affinity and high-affinity. In the high-affinity system the K_m values were 33 μm for taurine, 24 μm for GABA and 68 μm for glutamate, and in the low-affinity one 1·1 mil, 0·9 mm and 1·2mm , respectively. The influx capacity (V_max) was highest for glutamate, second highest for GABA and lowest for taurine.     

Potassium-Induced Release of Endogenous Amino Acids in the Guinea Pig Cochlea

Guinea pig cochleae were perfused with high-potassium solutions to depolarize hair cells artificially and induce the release of afferent neurotransmitter. Sequential injections of artificial perilymph containing 5 mM KCl, then 50 mM KCl, and finally 5 mM KCl were made into the scala tympani. This injection sequence was conducted under either normal divalent-cation conditions (2.0 mM CaCl2, 1.0 mM MgCl2) or calcium-deficient conditions intended to antagonize evoked transmitter release (0.1 mM CaCl2, 20.0 mM MgCl2). The levels of 21 endogenous primary amines in effluent collected from the scala vestibuli were determined by gradient-elution, reverse-phase HPLC using o-phthaldialdehyde-thiol adducts with fluorescence detection. Analyses indicated effluent concentrations of glutamate, taurine, and a coeluting taurine-gamma-aminobutyrate (GABA) fraction (but not GABA alone) increased significantly after exposure to 50 mM KC1 and returned to baseline levels after reintroduction of 5 mM KC1 under normal divalent-cation conditions. Correspondent changes in the release of these constituents were significantly attenuated under calcium-deficient conditions. This was not the case for potassium-induced changes in the release of arginine, aspartate, and isoleucine. These data are consistent with the hypothesis that the receptoneuronal transmitter is glutamate and further suggest a calcium-dependent mechanism involving taurine.     

In Vivo Studies on the Extracellular, and Veratrine-Releasable, Pools of Endogenous Amino Acids in the Rat Striatum: Effects of Corticostriatal Deafferentation and Kainic Acid Lesion

The effects of corticostriatal deafferentation (decortication) and destruction of intrinsic neurons (intrastriatal kainate injection) on the extracellular concentration, and veratrine-releasable pools, of endogenous amino acids in the rat striatum were examined using the in vivo brain dialysis technique. Intracellular amino acid content was also determined. Decortication reduced selectively intra- and extracellular levels of glutamate (Glu) and aspartate (Asp). Extracellular changes were more pronounced than those in tissue content. gamma-Aminobutyric acid (GABA), taurine (Tau), and phosphoethanolamine (PEA) levels were not affected, whereas nonneuroactive amino acids were increased at 1 week but not at 1 month post-lesion. The intracellular pool of Glu and Asp was also reduced in kainate-lesioned striata. However, extracellular levels of these compounds were not affected significantly by this treatment. The tissue content of all other amino acids was decreased, the most prominent change being in the concentration of GABA. Extracellular GABA concentration was also reduced dramatically, whereas the concentrations of noneuroactive amino acids were increased to varying degrees. These data suggest that transmitter pools of neuroactive amino acids are an important supply for their extracellular pools. Lesion-induced alterations in nonneuroactive amino acids are discussed with regard to the loss of metabolic pools, glial reactivity, and changes in blood-brain barrier transport. Veratrine induced a massive release of neuroactive amino acids such as Glu, Asp, GABA, and Tau into the extracellular fluid, and a delayed increase in PEA. Extracellular levels of neuroactive amino acids were raised slightly. Decortication reduced, selectively, the amounts of Glu and Asp released by veratrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentrations of Amino Acids in Extracellular Fluid After Opening of the Blood-Brain Barrier by Intracarotid Infusion of Protamine Sulfate

Abstract: This article evaluates the influence of an opening of the blood-brain barrier (BBB) on compounds in brain extracellular fluid. The concentrations of amino acids and some other primary amines were determined in dialysates sampled from the right parietal cortex of rats before and after an intracarotid infusion of protamine sulfate. Extravasated plasma proteins were visualized by Evans blue/albumin and immunohistochemistry. CSF albumin— an indicator of blood-CSF barrier opening—was quantified with immunoelectrophoresis. The brains were macroscopically edematous after 10 mg but not after 5 mg of protamine sulfate. The higher dose led to a 50% death rate. The concentrations of amino acids did not change 10 min after the BBB opening. No significant alterations in the amino acid concentrations were observed after the lower dose. The concentrations of glutamate, aspartate, GABA, glycine, taurine, and phosphoethanolamine increased significantly within 50–80 min after the infusion of 10 mg of protamine sulfate. CSF albumin levels were significantly increased 1 h after infusion. We conclude that a dysfunction of the BBB, of a degree known to induce brain edema (10 mg of protamine sulfate), significantly increases the extracellular concentration of excitatory amino acids, GABA, taurine, and phosphoethanolamine in the extracellular space.     

Amino Acids in the Substantia Nigra of Rats with Striatal Lesions Produced by Kainic Acid

In an attempt to estimate the pool size of glutamate and other amino acids in γ-aminobutyric acid (GABA)-containing neurons, we determined the content of 12 amino acids in the bilateral substantia nigra of rats, in which unilateral striatal lesions had been made with kainic acid two weeks earlier. The assay of the amino acids (including glutamate, aspartate, glutamine, asparagine, glycine, and GABA) and ethanolamine was based on HPLC and fluorimetric detection after precolumn derivatization with o-phthaldialdehyde. The levels of all measured amino acids (except those of tyrosine, threonine, and ethanolamine) were decreased in the affected striatum, but only the levels of aspartate, taurine, and GABA were lowered in the ipsilateral substantia nigra. These results indicate that the pool size of the various amino acids in the striatonigral GABAergic pathway is small compared to their nigral content, and that in addition to GABA a significant fraction of aspartate and taurine may be confined to nerve terminals in the substantia nigra.     

Regional alterations in brain amino acids during the estrous cycle of the rat

Concentrations of 11 amino acids, including the neurotransmitters GABA, glutamate, aspartate, glycine and taurine, were determined in 12 brain regions of female rats during different stages of the estrous cycle. In addition, amino acids and sex hormone levels were determined in plasma. All sample collections were done in the forenoon between 9 and 11 a.m. Most regional amino acid levels measured did not change signficantly during estrous cycle, but significant alterations were found for GABA and glutamate in hypothalamus. Both amino acids were slightly decreased in hypothalamus during proestrus, which might reflect an alteration of GABA turnover in response to the high estrogen levels during this stage. A decreased glutamate level during proestrus was also found in thalamus, while both glutamate and GABA did not vary throughout estrous cycle in any of the other examined regions, including substantia nigra, amygdala, striatum, cortex and hippocampus. When diestrus was subdivided according to progesterone levels, high levels of this hormone seemed to be associated with effects on metabolism of certain amino acids, including glycine in substantia nigra, alanine in thalamus and threonine in pons/medulla. However, the few changes in regional amino acid concentrations found during the estrous cycle were so small that the functional significance of these changes cannot be ascertained without further determination of the cellular or subcellular compartments of brain tissue involved.     

The use of microdialysis for studying the regional effects of pharmacological manipulation on extracellular levels of amino acids--some methodological aspects.

The purpose of this study was to examine and validate the use of microdialysis for sampling and pharmacologically manipulating extracellular amino acids in the brain. Repeated use of microdialysis probes in acute intracerebral experiments did not significantly alter the relative recovery in vitro for the amino acids quantitated (GABA, aspartate, glutamate, glycine, taurine, and alanine). Regional differences in basal levels of some of the amino acids were detected in dialysates collected from the dorsomedial hypothalamus, striatum, and frontal cortex. The percent in vitro recoveries for the amino acids from the probes used in the three regions were not significantly different suggesting that the regional differences in basal levels of amino acids were functionally derived and not a consequence of variations in probe recovery. Perfusion with nipecotic acid, an inhibitor of GABA uptake, resulted in selective elevations in extracellular GABA in the three regions studied. Conversely, perfusion with high-potassium, a depolarizing agent, resulted in significant elevations in not only extracellular GABA but also aspartate, glutamate, and taurine. Thus, microdialysis is a method which can be employed to assess and to pharmacologically manipulate extracellular amino acids in the rat brain.     

Amino Acid Content of Nerve Endings (Synaptosomes) in Different Regions of Brain: Effects of Gabaculine and Isonicotinic Acid Hydrazide

Abstract: The amino acid content of synaptosomes was determined in six regions of rat brain, and in all regions the five predominant amino acids were glutamate, glutamine, aspartate, taurine, and GABA (γ-aminobutyrate). However, the proportions of the individual amino acids varied considerably from one region to another, the GABA content being particularly high and the taurine content low in synaptosomes from the diencephalon and mesencephalon. Administration of isonicotinic acid hydrazide to rats lowered the synaptosomal GABA level by similar amounts in all brain regions, but the administration of gabaculine resulted in a particularly long-acting elevation in GABA levels in the nerve endings of the diencephalon and mesencephalon. The possibility is raised that the high GABA levels in the nerve terminals of the diencephalon may be involved in the gabaculine-induced lowering of the body temperature of the rats. A constancy in the amount of the synaptosomal pool of "aspartate + glutamate + glutamine + GABA" was observed despite large changes in the relative amounts of the four amino acids brought about by gabaculine.     

Glutamate release from cerebellar granule cells differentiating in culture: Modulation of the K+-stimulated release by inhibitory amino acids

The properties of l-[³H]glutamate release with an emphasis on the modulation by inhibitory amino acids of the potassium-induced release were studied with cerebellar granule cells from 7-day-old rats cultured for 7 or 14 days. Spontaneous glutamate release from cells grown for 7 days was fast, being slightly enchanced in Na⁺-free medium. l-Glutamate, kainate and quisqualate stimulated the release whereas N-methyl-d-aspartate and taurine were without any effect. The potassium-evoked glutamate release was Ca²⁺-dependent and potentiated by l-glutamate and quisqualate. Stimulated release was strongly depressed by glutamatediethylester. This inhibition was antagonized by GABA but not by taurine. GABA and its structural analogues taurine, hypotaurine, β-alanine and glycine were all equally effective in depressing stimulated glutamate release. The inhibition by GABA could be blocked by GABA antagonist. Both K⁺-evoked release and the kainate-induced release of glutamate were significantly greater in 14-day-old than in 7-day-old cultures, but the other properties of release were similar. The demonstration of calcium-dependent and potassium-stimulated glutamate release from cerebellar granule cells is consonant with the proposed neurotransmitter role of glutamate in these cells. The release could be modulated by both glutamatergic substances and inhibitory amino acids, the effect of GABA probably being mediated by GABAergic receptors.