Differential Calcium Dependence of γ-Aminobutyric Acid and Acetylcholine Release in Mouse Brain Synaptosomes

The dependence of gamma-aminobutyric acid (GABA) and acetylcholine (ACh) release on Ca2+ was comparatively studied in synaptosomes from mouse brain, by correlating the influx of 45Ca2+ with the release of the transmitters. It was observed that exposure of synaptosomes to a Na+-free medium notably increases Ca2+ entry, and this condition was used, in addition to K+ depolarization and the Ca2+ ionophore A23187, to stimulate the influx of Ca2+ and the release of labeled GABA and ACh. The effect of ruthenium red (RuR) on these parameters was also investigated. Of the three experimental conditions used, the absence of Na+ in the medium proved to be the most efficient in increasing Ca2+ entry. RuR inhibited by 60-70% the influx of Ca2+ stimulated by K+ depolarization but did not affect its basal influx or its influx stimulated by the absence of Na+ or by A23187. The release of ACh was stimulated by K+ depolarization, absence of Na+ in the medium, and A23187 in a strictly Ca2+-dependent manner, whereas the release of GABA was only partially dependent on the presence of Ca2+ in the medium. The extent of stimulation of ACh release was related to the extent of Ca2+ entry, whereas no such correlation was observed for GABA. In the presence of Na+, RuR did not affect the release of the transmitters induced by A23187. In the absence of Na+, paradoxically RuR notably enhanced the release of both ACh and GABA induced by A23187, in a Ca2+-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Organic and Inorganic Calcium Channel Blockers on γ-Amino-n-Butyiic Acid Release Induced by Monensin and Veratrine in the Absence of External Calcium

The effects of two organic Ca2+ antagonists (verapamil and nitrendipine) and of two inorganic Ca2+ channel blockers (Co2+ and ruthenium red) on the Na+-dependent release of gamma-amino-n-butyric acid (GABA) triggered by veratrine and monensin in the absence of external Ca2+ were studied in mouse brain synaptosomes. Ca2+-independent release of GABA stimulated by the Na+ channel activator veratrine was inhibited with micromolar concentrations of verapamil and nitrendipine. In contrast, GABA release induced by the Na+ ionophore monensin was insensitive to the organic Ca2+ antagonists. Verapamil also failed to modify A23187-stimulated release of GABA in the presence of Ca2+ but inhibited high K+-induced release of the transmitter. Co2+ partially diminished veratrine-induced release but did not change monensin-induced release. Releasing responses to monensin and veratrine were insensitive to ruthenium red, which inhibited the Ca2+-dependent component of GABA release evoked by high K+ depolarization. These data demonstrate that the mechanism of inducing GABA release is different for veratrine and monensin, as evidenced by their differing sensitivities to inhibition by Ca2+ channel antagonists and organic Ca2+ blockers. It is concluded that voltage-sensitive Ca2+ channels of the presynaptic membrane are not involved in the inhibitory action of Ca2+ antagonists on the Na+-dependent, Ca2+-independent mechanism of GABA release.     

γ-Aminobutyric Acid Release from Synaptosomes Prepared from Rats Treated with Isonicotinic Acid Hydrazide and Gabaculine

The potassium-stimulated release of gamma-aminobutyric acid (GABA) from synaptosomes was determined in preparations from control rats and from rats treated with a convulsant agent [isonicotinic acid hydrazide (INH)] and an anticonvulsant agent (gabaculine). INH treatment brought about a significant decrease in Ca2+-dependent release of GABA with no effect on Ca2+-independent release, whereas gabaculine caused an increase in Ca2+-independent release with no effect on Ca2+-dependent release of GABA. Thus, the anticonvulsant action of gabaculine was not a simple reversal of the effects of INH on GABA release. The results indicate that there are at least two pools of GABA in nerve endings and support the hypothesis that exogenous GABA is taken up first into a pool that supplies GABA for Ca2+-independent release and then is transferred to a second pool (Ca2+-dependent releasable), where it mixes with newly synthesized GABA.     

Compartments of Labeled and Endogenous γ-Aminobutyric Acid Giving Rise to Release Evoked by Potassium or Veratridine in Rat Cortical Slices

Abstract— To establish compartments involved in depolarization-induced release of γ-aminobutyric acid (GABA) in rat brain slices, the amount of exogenous labeled and endogenous GABA released and retained was followed during 48 min exposure to 50 m m -K⁺ or to 50 μ m -veratridine. Endogenous GABA was measured with high performance liquid chromatography. The presence of 10 μ m -aminooxyacetic acid throughout prevented both the metabolism of GABA and the formation of endogenous GABA due to depolarization. During super-fusion with 50 m m -K⁺ and 2.6 m m -Ca²⁺ the efflux of labeled and endogenous GABA after an initial large increase declined to 10% of the highest value with constant and identical rates. Kinetic analysis of efflux showed that 10% of endogenous and 25% of labeled GABA present is available for release by high K⁺ and Ca²⁺. In the absence of Ca²⁺, release by high K⁺ of both labeled and endogenous GABA was nearly suppressed. Veratridine, unlike high K⁺, caused an efflux which declined with an initial fast and late very slow phase. The slow efflux by veratridine was doubled in the absence of Ca²⁺. Exposure to veratridine in the absence of Ca²⁺ during 120 min released nearly 70% of labeled and endogenous GABA present. Results suggest that only about 0.25 μmol g⁻¹ endogenous GABA is the source of physiological Ca²⁺-dependent release, while much of the remaining GABA present is released only under unphysiological conditions.     

Effect of Nipecotic Acid, a γ-Aminobutyric Acid Transport Inhibitor, on the Turnover and Release of γ-Aminobutyric Acid in Rat Cortical Slices

Abstract: To see the effect of a γ-aminobutyric acid GABA uptake inhibitor on the efflux and content of endogenous and labeled GABA, rat cortical slices were first labeled with [³H]GABA and then superfused in the absence or presence of 1 mM nipecotic acid. Endogenous GABA released or remaining in the slices was measured with high performance liquid chromatography, which was also used to separate [³H]GABA from its metabolites. In the presence of 3 mM K⁺, nipecotic acid released both endogenous and [³H]GABA, with a specific activity four to five times as high as that present in the slices. The release of labeled metabolite(s) of [³H]GABA was also increased by nipecotic acid. The release of endogenous GABA evoked by 50 mM K⁺ was enhanced fourfold by nipecotic acid but that of [³H]GABA was only doubled when expressed as fractional release. In a medium containing no Ca²⁺ and 10 mM Mg²⁺, the release evoked by 50 mMK⁺ was nearly suppressed in either the absence or the presence of nipecotic acid. In the absence of nipecotic acid electrical stimulation (bursts of 64 Hz) was ineffective in evoking release of either endogenous or [³H]GABA, but in the presence of nipecotic acid it increased the efflux of endogenous GABA threefold, while having much less effect on that of [³H]GABA. Tetrodotoxin (TTX) abolished the effect of electrical stimulation. Both high K⁺ and electrical stimulation increased the amount of endogenous GABA remaining in the slices, and this increase was reduced by omission of Ca²⁺ or by TTX. The results suggest that uptake of GABA released through depolarization is of major importance in removing GABA from extracellular spaces, but the enhancement of spontaneous release by nipecotic acid may involve intracellular heteroexchange. Depolarization in the presence of Ca²⁺ leads to an increased synthesis of GABA, in excess of its release, but the role of this excess GABA remains to be established.     

Changes in the Amino Acid Content of Nerve Endings (Synaptosomes) Induced by Drugs that Alter the Metabolism of Glutamate and γ-Aminobutyric Acid

The study was centered on the changes in the amino acid content of nerve endings (synaptosomes) induced by drugs that alter the metabolism of glutamate or gamma-aminobutyric acid (GABA), and that possess convulsant or anticonvulsant properties. The onset of seizures induced by various convulsant agents was associated with a decreased content of GABA and an increased content of glutamate in synaptosomes. The concurrent administration of pyridoxine prevented both the biochemical changes and the convulsions. The administration of gabaculine to mice resulted in large increases in the GABA content of synaptosomes that were counteracted by decreases in glutamate, glutamine, and aspartate levels such that the total content of the four amino acids remained unchanged. The administration of aminooxyacetic acid (0.91 mmol/kg) resulted initially in seizure activity, but subsequently in an anticonvulsant action. No simple relationship existed between the excitable state of the brain induced by aminooxyacetic acid and the changes in the synaptosomal levels of any of the amino acid transmitters. A hypothesis was, however, formulated that explained the convulsant-cum-anticonvulsant action of aminooxyacetic acid on the basis of compartmentation of GABA within the nerve endings.     

Uptake of γ-Aminobutyric Acid and Glycine by Synaptosomes from Postmortem Human Brain

Synaptosomes prepared from frozen postmortem human brain accumulated the neurotransmitter gamma-aminobutyric acid (GABA) and the conformationally restricted GABA analogue cis-3-aminocyclohexanecarboxylic acid (ACHC) by a sodium-dependent, temperature-sensitive, high-affinity transport process into an osmotically sensitive compartment. This transport process could be inhibited by GABA analogues (ACHC, 2,4-diaminobutyric acid, nipecotic acid, arecaidine, guvacine) that have been shown in studies on other species to be relatively selective for neuronal rather than glial uptake systems, whereas the glial uptake inhibitor beta-alanine was ineffective. Synaptosomes prepared from frozen post-mortem human medulla and spinal cord, but not cerebral cortex, took up the neurotransmitter glycine by a sodium-dependent high-affinity transport process. The kinetic parameters for the high-affinity uptake of GABA, ACHC, and glycine were Km = 10 +/- 3, 49 +/- 19, and 35 +/- 19 microM; and Vmax = 98 +/- 15, 84 +/- 25, and 5.5 +/- 2.5 nmol/min/100 mg protein, respectively. These results demonstrate the feasibility of using human CNS preparations for studying GABA and glycine uptake, and suggest that such studies may be useful neurochemical markers for transmitter-specific presynaptic terminals in health and disease.     

Distribution of Glycine, γ-Aminobutyric Acid, Glutamate Decarboxylase, and γ-Aminobutyric Acid Transaminase in Rabbit and Mudpuppy Retinas

Abstract: The distributions of glycine, γ-aminobutyric acid (GABA), glutamate decarboxylase (EC 4.1.1.15), and GABA transaminase (EC 2.6.1.19) were determined in rabbit and mudpuppy retinas. In both species, peak levels of the amino acids and the enzymes occurred in the inner plexiform layer. Glutamate decarboxylase was almost entirely confined to the inner plexiform layer. Determinations were also made of the GABA content of 107 individual putative amacrine cell somas from mudpuppy retina. About 30% of those somas were found to have high endogenous GABA levels.     

Radiation Inactivation Studies of the Benzodiazepine/γ-Aminobutyric Acid/Chloride Ionophore Receptor Complex

Radiation inactivation was used to estimate the molecular weight of the benzodiazepine (BZ), gamma-aminobutyric acid (GABA), and associated chloride ionophore (picrotoxinin/barbiturate) binding sites in frozen membranes prepared from rat forebrain. The target size of the BZ recognition site (as defined by the binding of the agonists [3H]diazepam and [3H]flunitrazepam, the antagonists [3H]Ro 15-1788 and [3H]CGS 8216, and the inverse agonist [3H]ethyl-beta-carboline-3-carboxylate) averaged 51,000 +/- 2,000 daltons. The presence or absence of GABA during irradiation had no effect on the target size of the BZ recognition site. The apparent molecular weight of the GABA binding site labelled with [3H]muscimol was identical to the BZ receptor when determined under identical assay conditions. However the target size of the picrotoxinin/barbiturate binding site labelled with the cage convulsant [35S]t-butylbicyclophosphorothionate was about threefold larger (138,000 daltons). The effects of lyophilization on BZ receptor binding activity and target size analysis were also determined. A decrease in the number of BZ binding sites (Bmax) was observed in the nonirradiated, lyophilized membranes compared with frozen membranes. Lyophilization of membranes prior to irradiation at -135 degrees C or 30 degrees C resulted in a 53 and 151% increase, respectively, in the molecular weight (target size) estimates of the BZ recognition site when compared with frozen membrane preparations. Two enzymes were also added to the membrane preparations for subsequent target size analysis. In lyophilized preparations irradiated at 30 degrees C, the target size for beta-galactosidase was also increased 71% when compared with frozen membrane preparations. In contrast, the target size for glucose-6-phosphate dehydrogenase was not altered by lyophilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the Effect of Monensin on γ-Amino-n-Butyric Acid Release from Isolated Nerve Terminals

The action of the polyether antibiotic monensin on the release of gamma-[3H]amino-n-butyric acid [( 3H]GABA) from mouse brain synaptosomes is characterized. Monensin enhances the release of this amino acid transmitter in a dose-dependent manner and does not modify the efflux of the nontransmitter amino acid alpha-[3H]aminoisobutyrate. The absence of external Ca2+ fails to prevent the stimulatory effect of monensin on [3H]GABA release. Furthermore, monensin is less effective in stimulating [3H]GABA release in the presence of Ca2+. The releasing response to monensin is absolutely dependent on external Na+. The blockade of voltage-sensitive Na+ or Ca2+ channels does not modify monensin-induced release of the transmitter. Also, the blockade of the GABA uptake pathway fails to prevent the stimulatory effect of monensin on [3H]GABA release. Although monensin markedly increases Na+ permeability in synaptosomes, these data indicate that the Ca2+-independent monensin-stimulated transmitter release is not mediated by the Na+-dependent uptake pathway. It is concluded that the entrance of Na+ through monensin molecules inserted in the presynaptic membrane might be sufficient to initiate the intraterminal molecular events underlying transmitter release.     

Lipid Peroxidation-Induced Inhibition of γ-Aminobutyric Acid Uptake in Rat Brain Synaptosomes: Protection by Glucocorticoids

Incubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) resulted in an inhibition of gamma-aminobutyric acid (GABA) uptake. The inhibitory effects of X/XO were temperature- and time-dependent, and were characterized by an increased Km for GABA and a decreased Vmax. Inhibition of GABA uptake by X/XO was associated with both the formation of malonyldialdehyde (MDA) and conjugated dienes, indicating that lipid peroxidation was involved. Studies with catalase, superoxide dismutase (SOD), mannitol, and chelated iron suggested that hydroxyl radical (OH X) was probably responsible for the initiation of lipid peroxidation. Both the peroxidation of synaptosomal membranes and the inhibition of GABA uptake by X/XO were enhanced by the addition of ADP and FeCl2. The X/XO-induced inhibition of GABA uptake by synaptosomes could be prevented by preincubation of synaptosomes with certain glucocorticoids prior to X/XO exposure. Methylprednisolone sodium succinate (MPSS), dexamethasone sodium phosphate (DMSP), and prednisolone sodium succinate (PSS) all prevented the inhibition of GABA uptake by X/XO. MPSS was most effective at concentrations around 100 microM, DMSP was slightly more potent, and PSS was optimal at around 300 microM. On the other hand, hydrocortisone sodium succinate (HCSS) was ineffective at preventing X/XO-induced inhibition of GABA uptake at concentrations up to 3 mM. The steroids are presumed to work through a mechanism that blocked the formation of lipid peroxides, as MPSS inhibited the formation of conjugated dienes in synaptosomes exposed to X/XO at a concentration that also protected GABA uptake.     

The Differential Effect of Ischemia on the Active Uptake of Dopamine, γ-Aminobutyric Acid, and Glutamate by Brain Synap to somes

Abstract: Ischemic stroke was induced in the Mongolian gerbil by left common carotid ligation. No change in uptake of [³H]dopamine, [³H]γ-aminobutyric acid ([³H]GABA), or [¹⁴C]glutamate in synaptosomes obtained from the ischemic hemisphere was observed for up to 8 h. At 16 h after ligation, marked decrements in uptake were observed in animals showing hemiparesis: Uptake values expressed as a percent of the corresponding control hemisphere were 15.2% for dopamine, 28.0% for GABA, and 47.5% for glutamate. The differential sensitivity of dopamine terminals compared with glutamate terminals was highly significant. Separate experiments performed with synaptosomes isolated from the corpus striatum showed that the greater sensitivity to damage was intrinsic to the dopamine nerve terminal and not the result of regional variations in ischemic damage in brain. No bilateral effect of ischemia on dopamine uptake was evident. In animals exhibiting milder behavioral deficits (circling), a smaller and comparable decrement in uptake of dopamine, GABA, and glutamate was evident at 16 h, whereas animals not affected behaviorally showed no decrement at 16 h. Following uptake, the subsequent fractional release of neurotransmitter stimulated by 60 mM-potassium ions was not affected at any time point studied. Therefore, the loss in uptake at 16 h probably represents overt destruction of nerve terminals. Experiments with urethane used in place of pentobarbital for anesthesia during carotid occlusion showed that protection by pentobarbital was not a factor in the delayed response to ischemia. These results show that damage or destruction of nerve terminals is a delayed event following ischemia and that dopamine terminals are intrinsically more sensitive than glutamate terminals.     

Effect of Pressure on the Release of Radioactive Glycine and γ-Aminobutyric Acid from Spinal Cord Synaptosomes

Exposure to high hydrostatic pressure produces neurological changes referred to as the high-pressure nervous syndrome (HPNS). Manifestations of HPNS include tremor, EEG changes, and convulsions. These symptoms suggest an alteration in synaptic transmission, particularly with inhibitory neural pathways. Because spinal cord transmission has been implicated in HPNS, this study investigated inhibitory neurotransmitter function in the cord at high pressure. Guinea pig spinal cord synaptosome preparations were used to study the effect of compression to 67.7 atmospheres absolute on [3H]glycine and [3H]gamma-aminobutyric acid ([3H]GABA) release. Pressure was found to exert a significant suppressive effect on the depolarization-induced calcium-dependent release of glycine and GABA by these spinal cord presynaptic nerve terminals. This study suggests that decreased tonic inhibitory regulation at the level of the spinal cord contributes to the hyperexcitability observed in animals with compression to high pressure.     

Developmental Changes in the Calcium Dependency of γ-Aminobutyric Acid Release from Isolated Growth Cones: Correlation with Growth Cone Morphology

We have investigated the development of Ca2+-dependent gamma-[3H]aminobutyric acid [( 3H]GABA) release in superfused growth cone fractions isolated from rats between the postnatal ages of 1 and 11 days. We have compared this release with the overall morphology of the subcellular fractions, and identified those structures taking up [3H]GABA by electron microscopical autoradiography. In fractions isolated from rats between 1 and 5 days, K+-evoked [3H]GABA release was completely independent of extracellular Ca2+. After 5 days a Ca2+ dependency appeared, which increased with age, such that by 10 days approximately 50% of the K+-evoked release was Ca2+ dependent. Electron microscopical analysis showed that, at all ages, large numbers of GABAergic growth cones were present in the subcellular fractions. Up to postnatal day 5, the growth cones were synaptic vesicle sparse but, after this age, increasing numbers of synaptic vesicle-containing growth cones were seen. These results suggest that during maturation of GABAergic growth cones into synapses there is, initially, a mechanism for release that is independent of extracellular Ca2+ and that the appearance of a Ca2+-dependent [3H]GABA release from growth cones correlates with the appearance of synaptic vesicles.     

The Release of γ-Aminobutyric Acid, Glutamate, and Acetylcholine from Striatal Slices: A Mass Fragmentographic Study

Abstract: The release processes of endogenous Acetylcholine (ACh), γ-aminobutyric acid (GABA), glutamate (Glu) and glutamine (GLN) were studied in superfused guinea-pig caudatal slices. Basal ACh release remained constant for up to 2 h, while the basal release of GABA, Glu and GLN declined to half or less of its initial values after 1 h of superfusion. Electrical stimulation increased the ACh release by 700–800% and that of GABA by 80% whereas it decreased the output of Glu by 50% and failed to modify the GLN efflux. KCl (25 mM) increased the output of ACh by 400%, that of GABA by approximately 500% and decreased that of Glu by 40%. Substituting of CaCl₂ by MgCl₂ in the superfusion medium reduced the basal ACh release by 70% whereas no differences were observed in the basal efflux of GABA, Glu and GLN. Under these conditions, no evoked release of ACh or of GABA was detected, following electrical or KCl stimulation. Tetrodotoxin 5 × 10^-7 M decreased the basal ACh release by 60% and increased the GABA efflux by 40%. The toxin abolished the stimulus-evoked ACh efflux but scarcely affected that of GABA. These results are consistent with a possible neurotransmitter role of ACh and GABA in the striatum and show some differences in the ionic mechanisms underlying GABA and ACh release.     

Endogenous Release of γ-Aminobutyric Acid from the Medial Preoptic Area Measured by Microdialysis in the Anaesthetised Rat

The characteristics of gamma-aminobutyric acid (GABA) release as monitored by microdialysis have been investigated in the chloral hydrate anaesthetised rat. The high outflow of GABA following insertion of the microdialysis probe (membrane 2 mm in length, 0.5 mm in diameter) into the medial preoptic area was found to decline to a stable baseline level after 2 h. After this time, perfusion with a medium containing 100 mM potassium ions evoked a 56-fold increase in GABA outflow. The addition of the calcium channel blocker verapamil (100 microM) to the perfusion medium induced significant 25 and 50% reductions in basal and potassium-stimulated GABA outflow, respectively. In the same animals, verapamil caused an 80% decrease in potassium-stimulated noradrenaline outflow. The glutamic acid decarboxylase inhibitors 3-mercaptopropionic acid and L-allylglycine added to the perfusion medium at a concentration of 10 mM reduced basal GABA release by approximately 50% with different time-courses of action. Ethanolamine-O-sulfate, a GABA-transaminase inhibitor, induced significant increases in basal GABA outflow 90 min after inclusion in the perfusion medium. These results demonstrate that microdialysis is a suitable technique with which to monitor extracellular levels of GABA and provide in vivo data on GABA release and degradation mechanisms.     

Influence of Sodium-Independent γ-Aminobutyric Acid Binding on the Assay of Sodium-Dependent γ-Aminobutyric Acid Binding in a Membrane Preparation of Rat Brain

A large amount of [³H]GABA was bound to crude synaptic membrane fractions of rat. by sodium-independent process in a medium that contained 100 μM [³H]GABA used for assaying GABA uptake site. This [³H]-GABA binding was different from receptor binding of GABA. It was confirmed that this sodium-independent [²H]GABA binding scarcely occurred in the presence of a physiological concentration of sodium chloride, and that sodium-independent GABA binding had a negligible influence on sodium-dependent GABA binding.     

Regional Variation in γ-Aminobutyric Acid Turnover: Effect of Castration on γ-Aminobutyric Acid Turnover in Microdissected Brain Regions of the Male Rat

Abstract: This study compared the turnover of GABA neurons in different brain areas of the male rat and examined the effect of castration on GABA turnover in regions of the brain associated with the control of gonadotropin secretion. To estimate GABA turnover, GABA was quantified by HPLC in microdissected brain regions 0,30,60,90, and 120 min after inhibition of GABA degradation by aminooxyacetic acid (100 mg/kg, i.p.). GABA accumulation was linear in all areas for 90 min ( p < 0.01), and GABA turnover was estimated as the slope of the line formed by increased GABA concentration versus time, determined by linear regression. There was considerable regional variation both in the initial steady-state concentrations of GABA and in the rates of GABA turnover. Of 10 discrete brain structures, GABA turnover was highest in the medial preoptic nucleus and lowest in the caudate nucleus. Turnover times in the terminal fields of known GABAergic projection neurons ranged sevenfold, from 2.6 h in the substantia nigra to 0.4 h in the lateral vestibular nucleus. The effect of castration on GABA turnover in 13 microdissected brain regions was investigated by measuring regional GABA concentrations before and 30 min after injection of aminooxyacetic acid in intact rats or 2 or 6 days postcastration. Following castration, steady-state GABA concentrations were increased, and GABA turnover decreased in the diagonal band of Broca, the medial preoptic area, and the median eminence. GABA turnover increased in the medial septal nucleus and was unaffected in the cortex, striatum, and hindbrain. These results are consistent with the hypothesis that testosterone negative-feedback control of luteinizing hormone-releasing hormone involves steroid-sensitive GABAergic neurons in the rostral and medial basal hypothalamus.     

Inhibition by Neurotoxic Phospholipases A2 of Synaptosomal Uptake of γ-Aminobutyric Acid

A comparison has been made of the abilities of several neurotoxic and nontoxic phospholipases A₂ from snake venoms to inhibit the intake of γ-aminobutyric acid into synaptosomes from rat cerebral cortex. The neurotoxic phospholipases A₂ inhibited GABA uptake more than the nontoxic enzymes did. However, there was a poor correlation between the measured specific enzyme activity of a phospholipase A₂ and its ability to inhibit the uptake of GABA.