Forskolin attenuates the evoked release of [H]GABA from striatal neurons in primary culture

The actions of the diterpene forskolin, and cyclic AMP analogues, on the evoked release of [³H]GABA (γ-aminobutyric acid) was examined in intact striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to forskolin (100 μM) resulted in a 40–55% attenuation of [³H]GABA release evoked by either KCl (30 mM) or veratrine (2 μg/ml), while baseline levels of release were unaffected. The dose-dependence for forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable identical to the dose-dependent elevation of cyclic AMP levels by forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable analogues of cyclic AMP, such as p-chlorophenylthio cyclic AMP (0.5 mM) and dibutyryl cyclic AMP (1 mM), resulted in a 25 and 26% attenuation of [³H]GABA release, respectively; dibutyryl cyclic GMP (1 mM) was without effect. The similarity between the actions of forskolin and the cyclic AMP analogues suggests that, in striatal neurons in primary culture, the elevation of cyclic AMP levels results in the attenuation of the evoked release of [³H]GABA. The greater effectiveness of forskolin, compared to the cyclic AMP analogues, may be related to the recently reported, additional direct actions of forskolin on neuronal membrane ion channels.     

Excitatory amino acid-evoked release of [H]GABA from hippocampal neurons in primary culture

The novel glutamate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited glutamate stimulated [³H]GABA release from cortical neurons in vitro. Kainate-induced release was blocked in a competitive fashion butN-methyl-d-aspartate (NMDA)-induced release was blocked non-competitively by CNQX. 7-Chlorokynurenate (7-CK) also inhibited NMDA evoked [³H]GABA release non-competitively, but had no effect on kainate induced release. The effects of both CNQX and 7-CK on NMDA-induced release were reversed by addition of exogenous glycine but the effects of CNQX on kainate-induced release were not altered by glycine. This suggests that both CNQX and 7-CK may interact with the glycine regulatory site of the NMDA receptor.     

The effects of excitatory amino acids on intracellular calcium in single mouse striatal neurons in vitro

Using microspectrofluorimetry and the calcium-sensitive dye fura-2, we examined the effect of excitatory amino acids on [Ca2+]i in single striatal neurons in vitro. N-methyl-D-aspartic acid (NMDA) produced rapid increases in [Ca2+]i. These were blocked by DL-2-amino-5-phosphonovaleric acid (AP5), by Mg2+, by phencyclidine, and by MK801. The block produced by Mg2+ and MK801 could be relieved by depolarizing cells with veratridine. When external Ca2+ was removed, NMDA no longer increased [Ca2+]i. Furthermore, the effects of NMDA were not blocked by concentrations of La3+ that blocked depolarization induced rises in [Ca2+]i. Substitution of Na+o by Li+ did not block the effects of NMDA. Concentrations of L-glutamate greater than or equal to 10(-6) M also increased [Ca2+]i. The effects of moderate concentrations of glutamate were blocked by AP5 but not by La3+ or by substitution of Na+ by Li+. The effects of glutamate were blocked by removal of external Ca2+ but were not blocked by concentrations of Mg2+ or MK801 that completely blocked the effects of NMDA. The glutamate analogs kainic acid (KA) and quisqualic acid also increased [Ca2+]i. The effects of KA were blocked by removal of external Ca2+ but not by La3+, Mg2+, MK801, or replacement of Na+ by Li+. Although AP5 was able to block the effects of KA partially, very high concentrations were required. These results may be explained by considering the properties of glutamate-receptor-linked ionophores. Excitatory amino acid induced increases in [Ca2+]i are consistent with the possibility that Ca2+ mediates excitatory amino acid induced neuronal degeneration.     

Excitatory amino acid-evoked release of [3H]GABA from hippocampal neurons in primary culture

We investigated the release of gamma-[2,3-3H(N)]aminobutyric acid ([3H]GABA) from hippocampal neurons in primary cell culture. [3H]GABA release was stimulated by the excitatory amino acid neurotransmitter glutamate as well as by N-methyl-D-aspartate (NMDA) and kainate. Cell depolarization induced by raising [K+]o or by veratridine also stimulated [3H]GABA release. NMDA-induced release was completely blocked by 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP+), Mg2+ and Zn2+ whereas the release induced by glutamate and kainate was much less susceptible to inhibition by these substances. Furthermore, removal of external Ca2+ inhibited NMDA-induced release, but not that induced by glutamate, kainate, veratridine or 50 mM K+. Removal of external Na+ reduced [3H]GABA release evoked by all stimuli, but to different extents. All of the excitatory amino acids tested increased [Ca2+]i within hippocampal neurons as assessed by fura-2 based microspectrofluorimetry. This increase in [Ca2+]i was completely dependent on the presence of external Ca2+. These results suggest that Ca2+-dependent and -independent forms of GABA release from hippocampal interneurons may occur. [3H]GABA release evoked by glutamate, kainate, veratridine or 50 mM K+, appeared to be mediated by the reversal of electrogenic, Na+-coupled GABA uptake. Release was inhibited by nipecotic acid, an inhibitor of the Na+-coupled GABA uptake system. However, release induced by NMDA may also include a Ca2+-dependent component.     

GABA release from mouse striatal neurons in primary culture as a test for the functional activity of N-methyl-D-aspartate (NMDA) antagonists.

N-Methyl-D-aspartate (NMDA)-induced release of [3H]GABA from mouse striatal neurons in primary culture has been evaluated as a screening method for demonstrating the functional activity of potential NMDA antagonists with respect to a cellular response. Antagonists were chosen for their specificity towards each of the three principal binding sites which have been characterised on the NMDA-receptor complex: the glutamate site, the ion-channel and in particular the glycine regulatory site where several novel halogenated derivatives of kynurenic acid have been tested. All the compounds were effective in blocking [3H]GABA release and their activity was related to their potency in displacing the binding of specific ligands for each of the three sites in rat cortex membrane preparations. This was confirmed by a correlation curve for the series of kynurenate derivatives (correlation coefficient r = 0.96). The specificity of these latter compounds for the glycine site was demonstrated by the addition of excess glycine which totally reversed their inhibition but not that of antagonists acting at the glutamate or ion-channel sites. Within the kynurenate series the 5,7-dichloro derivative was shown to be more active than the 7-chloro derivative, the most active glycine antagonist previously described. These results show that this is a simple and reliable system for demonstrating a functional effect of NMDA antagonists.     

Apoptosis induced by excitatory sulfur amino acids in primary cultured rat immature cerebral neurons]

In the neural tissue of patients with amyotrophic lateral sclerosis (ALS), the elevation of taurine, the final product of the metabolic pathway of sulfur amino acids (SAAs), has been reported, suggesting that excitatory SAAs, the intermediates of this pathway, could also be increased. This study was undertaken to evaluate whether excitatory SAAs have the ability to inhibit cystine uptake. Since immature neurons have not yet expressed the receptor channels, they are not susceptible to excitotoxicity. Inhibition of cystine transport leads to a depletion of glutathione, and results in cell death due to oxidative stress. Cell cultures were obtained from the cerebral cortex of fetal Wistar rats. Cytotoxicity studies were performed 48 hours after plating by addition of the culture medium containing SAAs; cysteine sulfinic acid, cysteic acid, homocysteine sulfinic acid (HCSA), homocysteic acid (HCA) and S-sulfocysteine. Cell death was quantified by the release of the cytosolic enzyme lactate dehydrogenase, and single cell assessment of apoptosis was carried out by staining cells with acridine orange, DNA isolation and agarose gel electrophoresis were also performed. Protection of cycloheximide, a protein synthesis inhibitor, against non-receptor mediated excitatory SAA cytotoxicity was also assessed. HCA and HCSA showed cytotoxicity, the morphology and biochemistry of which were compatible to apoptosis. It will be a subject for future study to examine whether this mechanism of cell death is primarily present in ALS.     

Regulation of striatal serotonin release by the lateral habenula-dorsal raphe pathway in the rat as demonstrated by in vivo microdialysis: role of excitatory amino acids and GABA

Striatal extracellular levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were monitored with the microdialysis technique during electrical stimulation of the lateral habenula-dorsal raphe (LHb-NRD) pathway in halothane anaesthetized rats. A new double-loop probe, with an improved recovery factor, was implanted into the head of the caudate-putamen and perfused with Ringer solution containing 1 microM of the 5-HT uptake blocker indalpine. Samples were collected every 15 min and analyzed with HPLC coupled to fluorimetric detection. Low frequency stimulation of the LHb (1.5 and 3 Hz, 0.5 mA) produced no detectable changes in striatal indole levels, whereas 15 Hz stimulation induced a 70% increase in 5-HT release. This effect was most likely mediated by a direct LHb-NRD link, since it persisted after ibotenic acid lesions of the interpeduncular nucleus (which is the major projection area for the medial habenular nucleus), but was completely abolished after transection of the fasciculus retroflexus, which carries the axons of the LHb-NRD pathway. The possible identity of the transmitter operating in the LHb-NRD pathway was investigated by NRD injections of kynurenic acid, a potent blocker of excitatory amino acid transmission, and by NRD injections of the GABA antagonist bicuculline. Kynurenic acid (300 nl, 50 mM) did not by itself induce any detectable changes in spontaneous indole output, but completely blocked the effect of LHb stimulation. Injection of bicuculline (300 nl, 2 mM) increased the striatal 5-HT output by about 70%, and potentiated the effect of LHb stimulation by a further 50%. In none of the experiments performed in this study were there any significant changes in striatal 5-HIAA output. These data are compatible with the idea that excitatory amino acids in the LHb-NRD pathway are involved in the regulation of striatal 5-HT release, and that this influence is modulated by GABAergic synaptic activity at the level of the NRD.     

Activation by nicotine of striatal neurons receiving excitatory input from the substantia nigra via dopamine release

An electrophysiological study was performed to elucidate the role of nicotinic receptors in the striatal neurons in chloral hydrate-anesthetized rats. The effects of microiontophoretic application of nicotine and other drugs were examined on the caudate nucleus (CN) neurons activated monosynaptically by stimulation of the substantia nigra pars compacta (SN). Application of nicotine facilitated spontaneous firing. The nicotine-induced firing of the CN neurons was inhibited by concomitant application of domperidone or hexamethonium. These findings suggested that nicotine enhances dopamine release from the SN-derived dopaminergic nerve terminals by activating the neurons via D2 receptors.     

Differential induction of immediate early genes by excitatory amino acid receptor types in primary cultures of cortical and striatal neurons.

In primary cultures of neurons from cerebral cortex and striatum, 30 s stimulation with the excitatory amino acid glutamate elicited a 5 to 9-fold increase in immediate early gene (IEG) mRNAs. Glutamate increased c-fos, c-jun, jun-B, and NGFI-A (zif/268) mRNAs by binding to both alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor types, and increased c-fos, jun-B, and NGFI-A mRNAs by binding to the metabotropic receptor. NMDA receptor activation elicited IEG expression by a transmembrane calcium influx; AMPA receptor-induced depolarization played a permissive role for the opening of the NMDA receptor channel. The protein kinase C (PKC) inhibitor H-7 (but not inhibitors of cyclic nucleotide-dependent and calcium/calmodulin-dependent protein kinases) partially blocked IEG expression induced by glutamate.     

Messenger RNAs encoding glutamate-decarboxylases are differentially affected by nigrostriatal lesions in subpopulations of striatal neurons.

Dopaminergic nigrostriatal neurons constitute one of the major inputs to the striatum, and play a role in the regulation of gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD), the GABA-synthesizing enzyme, in striatal neurons. The effect of nigrostriatal lesions on the level of expression of messenger RNAs encoding two distinct isoforms of glutamate decarboxylase was examined at the single cell level with in situ hybridization histochemistry. Rats received a unilateral injection of the neurotoxin 6-hydroxydopamine in the substantia nigra and were sacrificed 2 or 3 weeks later. Sections of the striatum were processed for in situ hybridization histochemistry with radiolabeled RNA probes selective for mRNAs encoding glutamate decarboxylase with molecular weights of 65,000 and 67,000, respectively. In addition, immunohistochemistry with a monospecific antibody for the latter glutamate decarboxylase isoform was performed. In agreement with previous reports, we observed increased labeling for the messenger RNA encoding glutamate decarboxylase (M(r) 67,000) in a population of medium-sized striatal efferent neurons normally expressing low levels of this messenger RNA. We now show that this effect occurred in two striatal compartments, the striosomes and the extrastriosomal matrix, and was accompanied by increased immunostaining for the corresponding protein with a monospecific antibody. In contrast, labeling for messenger RNA encoding GAD (M(r) 67,000) was decreased in a population of medium-sized neurons normally expressing high levels of this messenger RNA and corresponding to GABAergic interneurons. Labeling for messenger RNA encoding glutamate decarboxylase (M(r) 65,000) was not modified in the dopamine-depleted striatum. The results show that dopamine depletion differentially affects gene expression for different isoforms of glutamate decarboxylase in distinct subpopulations of striatal neurons in rat.     

Stimulus-coupled release of amino acids from cerebellar granule cells in culture

Cerebellar cultures greatly enriched in excitatory granule neurones were depolarized by exposure to either elevated K⁺ or veratrine. Stimulation of the release of not only Glu, but also of certain amino acids, including Gly, Ala and Ser, was observed. The effect was specific, as depolarization did not induced the release of all the estimated amino acids or of lactate dehydrogenase. In comparison with the characteristics of the evoked release of Glu, those of the responsive neutral amino acids were similar in terms of Ca²⁺-dependence, but differences were also noted. Thus, upon stimulation, the relative rise was smaller than for Glu and the degree of depolarization causing maximal release was lower. The questions of whether stimulus-coupled release of the non-transmitter amino acids from granule cells may play a neuromodulatory role in the cerebellum is discussed.     

Peripheral induction of burst firing in locus coeruleus neurons by nicotine mediated via excitatory amino acids

The effect of systemic nicotine administration (50 micrograms kg-1 i.v.) on the activity of brain noradrenaline neurons in the locus coeruleus (LC) of chloral hydrate-anesthetized rats was analyzed with single cell recording techniques and quantitative computer assessment of firing rate, degree of bursting, and regularity of firing. Nicotine caused an increased firing rate of the cells, with an average time of onset of 1.7 s. An increase in burst activity was observed, as well as deregularization of the firing pattern. Intraventricularly administered kynurenic acid (1 mumol), an antagonist of excitatory amino acids (EAA), did not change the firing rate of the LC cells, but did induce a marked regularization of their firing pattern into a pacemaker-like activity and completely abolish burst firing. The EAA antagonist also blocked all of the above effects of nicotine on the LC neurons as well as their typical burst-activation response to a peripheral, noxious stimulus such as paw-pinch. Since the circulation time in the rat is about 20 s, these results provide unequivocal evidence for a peripheral site of origin for the rapid LC activation induced by systemic nicotine administration. The data also allow the conclusion that the nicotine-induced LC activation is indirect and dependent on EAA in brain. Our results provide evidence for a tonically active EAA input to the LC, being of importance for induction of changes in the spontaneous, pacemaker activity of LC neurons into burst firing or more irregular firing patterns. It is suggested that the LC activation by nicotine may be significant in relation to tobacco dependence.     

Glia protect fetal midbrain dopamine neurons in culture from L-DOPA toxicity through multiple mechanisms

Summary Mesencephalic glia produce soluble factors that protect dopamine neurons from L-DOPA toxicity. The chemical composition of these soluble factors is unknown. We investigated the protective effect against L-DOPA neurotoxicity in midbrain dopamine neurons of fractions of different molecular size of glia conditioned medium and candidate neuroprotective agents produced by glia including neurotrophic factors and antioxidants. Protective effects were evaluated according to the number of tyrosine hydroxylase immunoreactive cells, high affinity dopamine uptake and levels of quinones. Both fractions of glia conditioned medium, smaller and larger than 10kD, protected against L-DOPA, but the fraction of smaller molecular size, that contains small free radical scanvenger molecules, was more effective than the fraction of larger molecular size, that contains large neurotrophic peptides. Among the neurotrophic factors GDNF and BDNF totally prevented L-DOPA neurotoxicity, while NGF and bFGF were less effective. However, only NGF significantly reduced the elevation of quinones induced by L-DOPA. Ascorbic acid, at the concentration found in glia conditioned medium, provided partial protective effect against L-DOPA toxicity. Glutathione, had neurotrophic effects on untreated midbrain dopamine neurons and prevented the effect of L-DOPA. In conclusion, the protective effect against L-DOPA neurotoxicity by glia conditioned medium is mediated by several compounds including neurotrophic factors and small antioxidants.     

HAP1 facilitates effects of mutant huntingtin on inositol 1,4,5-trisphosphate-induced Ca release in primary culture of striatal medium spiny neurons

Huntington's disease is caused by polyglutamine expansion (exp) in huntingtin (Htt). Htt-associated protein-1 (HAP1) was the first identified Htt-binding partner. The type 1 inositol (1,4,5)-trisphosphate receptor (InsP3R1) is an intracellular Ca2+ release channel that plays an important role in neuronal function. Recently, we identified a InsP3R1-HAP1A-Htt ternary complex in the brain and demonstrated that Httexp, but not normal Htt, activates InsP3R1 in bilayers and facilitates InsP3R1-mediated intracellular Ca2+ release in medium spiny striatal neurons [MSN; T.-S. Tang et al. (2003) Neuron, 39, 227-239]. Here we took advantage of mice with targeted disruption of both HAP1 alleles (HAP1 -/-) to investigate the role of HAP1 in functional interactions between Htt and InsP3R1. We determined that: (i) HAP1 is expressed in the MSN; (ii) HAP1A facilitates functional effects of Htt and Htt(exp) on InsP3R1 in planar lipid bilayers; (iii) HAP1 is required for changes in MSN basal Ca2+ levels resulting from Htt or Htt(exp) overexpression; (iv) HAP1 facilitates potentiation of InsP3R1-mediated Ca2+ release by Htt(exp) in mouse MSN. Our present results indicate that HAP1 plays an important role in functional interactions between Htt and InsP3R1.     

Neurotransmitters, KCl and antioxidants rescue striatal neurons from apoptotic cell death in culture

Striatal neurons grown in low density culture on serum-free media and in the absence of glia die within 3 days of plating. In this study, we sought to determine the mechanism of cell death (e.g., apoptosis) and whether trophic influences, such as, growth factors, neurotransmitters, antioxidants or KCl-mediated depolarization could improve their survival. We found that striatal neurons grown in this manner die via apoptosis unless treated with one of several different rescuing agents. One way to prevent the death of most striatal neurons was continual treatment with 5–20 μM dopamine (DA) or other monoamines. Although the survival effect of DA was mimicked by the specific D1 receptor agonist, SKF38393, no D1 or D2 receptor antagonists blocked the effect. As with DA, chronic depolarization with KCl (12–39 mM) or treatment with antioxidants, such as the vitamin E analog, Trolox (10–10–500 μM), or the hormone, melatonin (10–10–500 μM) also rescued striatal neurons from impending cell death. Surprisingly, growth factors, such as BDNF, bFGF, GDNF, NGF, NT3 and EGF, demonstrated no ability to rescue striatal neurons in this model, suggesting that death was not solely caused by the absence of essential trophic factors. We conclude that a variety of agents, but not growth factors, can prevent the demise of striatal neurons, presumably by neutralizing damage at one or more steps in the death cascade.     

Effects of excitatory amino acids,and of their agonists and antagonists on the release of neurotransmitters from the chick retina

Effects of glutamic, aspartic, and cysteic acid, and of kainic acid and N-methyl aspartate on the release of labeled GABA, glycine, and taurine were examined in isolated, perfused chick retina. Glutamic acid (0.5-2 mM), increased the release of ³H-GABA by more than four times and that of ¹⁴C-glycine by about two times. The release of GABA decreased 50% and that of glycine 95% in the presence of the antagonist of glutamic acid receptors, glutamate diethyl ester (300 m?M). N-methyl aspartate, used as an agonist of aspartic acid receptors, preferentially increased the release of GABA (seven times) over that of glycine (three times). The stimulatory effect of N-methyl aspartate was antagonized by D-α aminoadipate and by Mg. Kainic acid (10 m?M) induced the release of glycine but not that of GABA. Cysteic acid failed to modify the release of any of the amino acids examined. The efflux of labeled taurine was practically unaffected by all the compounds utilized. The release of GABA by the excitatory amino acids and agonists was Ca-independent but Na-dependent, whereas the release of glycine was markedly Ca-dependent. The evidence presented here suggests that experimental conditions activating receptors of excitatory amino acids differently affect the release of inhibitory amino acids.     

GABA binding and bicuculline in spinal cord and cortical membranes from adult rat and from mouse neurons in cell culture

Sodium-independent [³H]GABA and [³H]muscimol binding was determined in adult rat cerebral cortical and spinal cord membranes and in membranes from fetal mouse cortical and spinal cord neurons in primary dissociated cell culture. In adult rat cerebral cortical membranes, [³H]GABA bound to two sites (K_d=8nM,B_max=0.62pmol/mg protein; K_d=390nM,B_max=3.9pmol/mg protein) whereas the GABA agonist, [³H]muscimol, bound only to a high affinity site (K_d=5.6nM,B_max=1.9pmol/mg protein). In adult rat spinal cord, only a low affinity site was seen with [³H]GABA (K_d=340nM,B_max=9.8pmol/mg protein) and only a high affinity site was seen with [³H]muscimol (K_d=5.6nM,B_max=0.25pmol/mg protein). The inability to measure a high affinity [³H]GABA binding site in spinal cord probably reflects the high ratio of low to high affinity sites in spinal cord (39:1). In membranes from mouse neurons in cel; culture, [³H]GABA bound to two sites on cortical neurons (K_d=9nM,B_max=0.24pmol/mg protein; K_d=510nM,B_max=1.3pmol/mg protein) and spinal cord neurons (K_d=13nM,B_max=0.12pmol/mg protein; K_d=640nM,B_max=3.2pmol/mg protein). Again, the ratio of low to high affinity sites in cultured mouse spinal cord neurons was high (27:1).The effects of the potent GABA antagonist, (+)bicuculline, on both low and high affinity [³H]GABA binding was determined. Bicuculline appeared to inhibit binding to both sites competitively but theK_i for inhibiting the high affinity site was 5 μM and for inhibiting the low affinity site was 115 μM. Bicuculline inhibited [³H]muscimol binding in both brain and spinal cord competitively withK_is of 4μM and 10 μM respectively. Bicuculline inhibition of [³H]muscimol binding in cultured neuronal membranes was similar to that in adult rat membranes.The binding of the potent GABA agonist, muscimol, only to the high affinity site in both adult rat and cultured mouse neuronal membranes suggests that the high affinity site is the physiologically relevant postsynaptic GABA receptor. The fact that bicuculline inhibits the high affinity site (but not the low affinity site) in concentrations similar to those needed to block GABA-responses in physiological experiments²⁸ supports this hypothesis.     

Membranes from pituitary intermediate lobe cells enhance differentiation of fetal hypothalamic dopaminergic neurons in primary culture

Coculture of adult pituitary intermediate lobe (IL) cells, a target for hypothalamic dopaminergic neurons, with fetal rat hypothalamic cells accelerate differentiation of dopaminergic neurons. This involves long range diffusible as well as additional factors which may be membrane-bound. To determine whether IL membrane-bound factors contribute to the differentiating effect of IL cells, IL membranes were added to dispersed fetal hypothalamic neurons. This stimulated the outgrowth of dopaminergic neurites and elevated TH levels. Limited trypsin proteolysis of IL cell surface abolished the effect on TH levels. Addition of adenohypophyseal membranes was ineffective. Joint treatment with IL membranes, and medium conditioned (CM) over IL cells, produced the same effect on TH levels as did coculture with the same number of IL cells. The results demonstrate that IL cells express on their surface a membrane-bound factor promoting differentiation of fetal dopaminergic neurons in vitro; this factor acts in addition to diffusible activities.     

Inhibitory and excitatory amino acids in CSF of patients suffering from complex partial seizures during chronic treatment with gamma-vinyl GABA (vigabatrin)

The effect of chronic administration of gamma-vinyl GABA (GVG; vigabatrin) on levels of neurotransmission-related amino compounds was studied in lumbar cerebrospinal fluid of 65 patients with complex partial epilepsy. The first sample of cerebrospinal fluid was taken before a 3-month period of treatment with 3 g gamma-vinyl GABA/day, and the second was taken afterwards. From patients who showed a greater than 50% reduction in seizures (responders) or marked improvement in global performance, a third sample was taken at the end of the next 3-month phase, during which 3 g or 1.5 g gamma-vinyl GABA had been administered daily. During treatment with 3 g gamma-vinyl GABA/day, 55% of the patients showed more than 50% reduction in complex partial seizures; and at the same time free GABA, total GABA, homocarnosine, and glycine concentrations in the cerebrospinal fluid increased by 104%, 151%, 194% and 16%, respectively. After reduction of the daily dose to 1.5 g, the levels of free GABA, total GABA and homocarnosine were still increased by 65%, 115% and 102%, respectively. gamma-Vinyl GABA correlated with the levels of free GABA (P less than 0.002) and glycine (P less than 0.001). Concentrations of homocarnosine at baseline and homocarnosine and total GABA during gamma-vinyl GABA treatment were lower (P less than 0.005) in the group of non-responders than in the responder group. Glutamic acid, glutamine, aspartic acid, asparagine, and taurine levels did not change during gamma-vinyl GABA treatment. In conclusion, administration of gamma-vinyl GABA reduces epileptic seizures and produces dosage-dependent increases in levels of free GABA, GABA-containing peptides and of glycine in cerebrospinal fluid, without concomitant change in levels of excitatory amino acids.