Characterization of extracellular GABA in the substantia nigra reticulata by means of brain microdialysis

Summary Brain microdialysis was used to characterize extracellular gamma-aminobutyric acid (GABA) in the substantia nigra reticulata (SNR) of freely moving rats. The extracellular GABA in the SNR was characterized using acutely implanted probes (4–8 h after surgery; day 1) and chronically implanted probes (24 h after surgery; day 2).3-Mercaptopropionic acid, a glutamic acid decarboxylase inhibitor, was used to identify GABA. This drug induced an immediate decrease in the extracellular GABA levels to 40% of basal values, suggesting that the detected GABA is, at least in part, newly synthesized.The basal levels of extracellular GABA measured either on day 1 or day 2 were not affected by infusion of micromolar amounts of tetrodotoxin. Therefore, a direct coupling between GABA dialysate concentrations and nerve-impulse flow does not seem to exist. Infusion of the GABA uptake inhibitor nipecotic acid (0.5 mmol/l) resulted in a 4-fold increase in the dialysate levels of GABA lasting at least for 3 h on both days. K⁺ stimulation (60 mmol/l) increased extracellular GABA levels in the SNR to 450% of basal values. This effect again did not differ significantly on day 1 and day 2.The origin of the extracellular GABA in the SNR, as recorded by microdialysis under the two experimental conditions, is discussed.Send offprint requests to W. Timmerman at the above address     

On the importance of GABA-ergic neurons for the AOAA induced accumulation of GABA in the rat brain

Summary The accumulation of GABA induced by an intravenous injection of aminooxyacetic acid (AOAA) was followed for 1 h in five parts of the rat brain, i.e. cerebellum, medulla oblongata-pons, striatum, ventral mesencephalon and hypothalamus. The accumulation was similar in all brain parts studied when expressed as percentual increase from the basal value; an initial rapid accumulation indicating a mean turnovertime of 12–16 min during the first 5 min was gradually decreased to a turnovertime of about 1–3 h during the last 30 min of observation. In other experiments brains were transected unilaterally between the substantia nigra and the striatum. Six days after the hemisection the GABA concentration of the substantia nigra of the transected side had decreased to less than 30% of the control side. The AOAA induced accumulation of GABA in the substantia nigra of the transected side was less pronounced than that of the control side. The initial rapid accumulation seen in all brain parts studies was completely lacking in the substantia nigra of the transected side. In the striatum, the transection did neither alter the GABA concentration nor the AOAA induced accumulation of GABA. As the initial rapid accumulation of GABA disappears after degeneration of GABA-ergic neurons, it is suggested that this initial phase of GABA accumulation produced by an intravenous injection of AOAA probably is the result of GABA accumulating in neurons.A preliminary report of the results was presented at the congress of S.B.P.C., Rio de Janeiro, 1980     

Synaptosomal [3H]GABA uptake and [3H]nipecotic acid binding in audiogenic seizure susceptible (DBA/2) and resistant (C57 B1/6) mice

The kinetics of [3H]GABA uptake into synaptosomes prepared from the cerebral cortex, cerebellum and pons-medulla of DBA/2 (audiogenic seizure susceptible) and C57 B1/6 (audiogenic seizure resistant) mice were determined at various ages. Vmax of uptake decreased with age in all regions of both strains, Km was unchanged. There were no regional differences in the Vmax between strains at any age but Km was lower in DBA/2 mice at 21-23 days in cerebellum and at 40-43 days in pons-medulla. There were no strain differences in the binding of [3H]nipecotic acid to crude cortical membrane fractions at any age. The age related audiogenic seizure susceptibility of DBA/2 mice is not related to an abnormality of neuronal GABA uptake.     

Alteration of GABA metabolism in mammalian brain by l-alpha-amino-beta-chloropropionic acid hydroxamide and related compounds.

The administration of L-alpha-amino-beta-chloropropionic acid hydroxamide (L-ACPH) to mice brought about an inhibition in GABA-T activity in the brain of the animals, a significant inhibition occurring with dosage levels as low as 0.25 mmol/kg. Minimum levels of GABA-T activity were reached 3 h after administration of the drug. Brain glutamic acid decarboxylase, DOPA decarboxylase and aspartate aminotransferase activities were not altered by the L-ACPH but alanine aminotransferase activity was totally inhibited. Slight changes in structure caused great changes in the potency of the drugs. For example, the elongation of the L-ACPH structure by one carbon, or a change in the configuration of the amino group from L- to D-, caused a significant decrease in GABA inhibition. The chloro and hydroxamide groups were necessary for inhibitory activity. The administration of L-ACPH to mice delayed the onset of drug induced seizures but had a less noticeable effect against maximal electroshock. The addition of L-ACPH to crude extracts from brain, or to preparations of semipurified GABA-T, also inhibited GABA-T activity. Again the development of the inhibition was time-dependent. Possible mechanisms of action with respect to L-ACPH induced inhibition of GABA-T activity are discussed in the light of the data presented.     

Effect of metabolites of valproic acid on the metabolism of GABA in brain and brain nerve endings

Seven metabolites of valproic acid (VPA), i.e. 2-en-VPA, 3-en-VPA, 4-en-VPA, 3-hydroxy-VPA, 4-hydroxy-VPA, 5-hydroxy-VPA and 3-keto-VPA and valproic acid itself were examined for their effects on the metabolism of γ-aminobutyric acid (GABA) in the brain and brain nerve endings (synaptosomes) in mice. Administered in anticonvulsant doses, valproic acid and its metabolites caused elevations of the synaptosomal GABA content which were correlated with their anticonvulsant potency. No relationship was observed between the relative anticonvulsant activity of the respective compounds and the increase of GABA in the whole brain. The synaptosomal activity of glutamate decarboxylase (GAD) was increased parallel to the elevation of GABA and the activity of GABA aminotransferase (GABA-T) was partly inhibited. The present results emphasise the usefulness of determining the in vivo effects of drugs on GABA metabolism in brain nerve terminals which is thought to be the critical factor controlling the functioning of the amino acid as a neurotransmitter.     

The effect of intracerebroventricularly administered GABA on brain monoamine metabolism

Summary Intracerebroventricular injection of -aminobutyric acid (GABA) was performed in male rats and the brain monoamines, 5-hydroxyindoleacetic acid (5-HIAA), tyrosine and tryptophan levels were measured. GABA induced within 30 min a marked dose-dependent increase in the brain contents of dopamine (DA), serotonin (5-HT), tyrosine and tryptophan, while noradrenaline (NA) was lowered. Large doses of GABA, i.e. 1.5–3 mg/rat, were required for these effects. Aminooxyacetic acid (AOAA), an inhibitor of GABA-transaminase, when given alone in a dose of 25 mg/kg i.p. caused a significant rise of DA, 5-HT and tryptophan. The combination of GABA and AOAA raised these levels more than either agent alone. Picrotoxin (4 mg/kg, i.p.) a claimed GABA receptor antagonist partially counteracted the GABA-induced DA rise. Monoamine synthesis was studied in different parts of the brain by measuring the accumulated dopa and 5-hydroxytryptophan (5-HTP), 30 min after NSD 1015 (3-hydroxybenzylhydrazine HCl, 100 mg/kg) an inhibitor of aromatic L-aminoacid decarboxylase, given i.p. 5 min after GABA. GABA caused a marked rise in dopa formation both in DA-and NA-predominated brain regions. Also 5-HTP formation was enhanced. The effects on both dopa and 5-HTP formation showed marked regional differences. The data suggest that GABA, by activating specific receptors, causes inhibition of firing of dopaminergic neurones and the opposite effect on the noradrenergic neurones. The nature of the effect on 5-HT metabolism needs further investigation.     

Effect of aminooxyacetic acid (AOAA) on GABA levels in some parts of the rat brain

Summary The accumulation of GABA in the cerebellum and medulla oblongata-pons of rats has been studied after inhibition of GABA-T (EC 2.6.1.19) by different doses of AOAA. It was found that intraperitoneal (i.p.) injections of AOAA were, at least during the first hour after injection, much less effective than intravenous (i.v.) injections probably due to poor absorption i.p. After i.v. injection, AOAA caused a maximal accumulation of GABA in the cerebellum at a dose of 50 mg/kg. This maximal effect was virtually unchanged up to a dose of 150 mg/kg (the highest dose tested i.v.). If GAD (EC 4.1.1.15) was inhibited by 3-mercaptopropionic acid 30 min after AOAA (90 mg/kg i.v.) the GABA level was stable for at least another 30 min. The rate of GABA accumulation in the cerebellum during the first 15 min after AOAA (50–150 mg/kg i.v.) was 0.086 mol/g/min and thereafter 0.034 mol/g/min. It is concluded that AOAA in vivo in a wide dose range inhibits GABA-T almost 100% without affecting GAD to any great extent, and that the onset of action is rapid after i.v. but not after i.p. injection.     

The γ-aminobutyric acid (GABA) uptake inhibitor, tiagabine, increases extracellular brain levels of GABA in awake rats

The effect of systemic administration of the γ-aminobutyric acid (GABA) uptake inhibitor, R( − )N-(4,4-di(3-methyl-thien-2-yl)-but-3-enyl) nipecotic acid, hydrochloridc (tiagabine) (previously NO-328), on extracellular GABA levels in the globus pallidus, ventral pallidum and substantia nigra of awake Sprague-Dawley rats was investigated using in vivo microdialysis. Tiagabine was administered in doses of 11.5 or 21.0 mg/kg i.p. (ED₅₀, and ED₈₅ doses, respectively, for inhibiting pentylcnctetrazole-induced tonic seizures). Tiagabine increased the extracellular concentrations of GABA in globus pallidus with peak values 310% of basal level (after 21 mg/kg) and 240% of basal level (after 11.5 mg/kg). A significant increase in extracellular GABA levels was also found in the ventral pallidum (280% increase after 11.5 mg/kg and 350% increase after 21 mg/kg) and in the substantia nigra where the ED₈₅ dose of tiagabine 621 mg/kg) produced a peak value of 200% compared to the basal level. Thus, tiagabine acts as a GABA uptake inhibitor in vivo also.     

GABA mimetics increase extracellular DOPAC (as measured by in vivo voltammetry) in the rat locus coeruleus

Summary Previous studies have suggested that GABA exerts an indirect facilitatory influence on cerebral noradrenergic transmission that is likely to be mediated via GABA-ergic synapses involved in polysynaptic neuronal circuits controlling noradrenergic cell activity. In order to confirm these findings, we have studied the effect of GABA mimetics on extracellular DOPAC in the locus coeruleus (a reliable index of noradrenergic neuronal activity) of the rat through the use of in vivo voltammetry with carbon fibre electrodes. Systemic administration of progabide, its active metabolite SL 75.102, of depamide or muscimol increased the height of the DOPAC oxidation peak in the locus coeruleus; the effect of muscimol was antagonized by co-administration of bicuculline. Intra-locus coeruleus infusion of even a high concentration of muscimol (1 g) failed to alter extracellular DOPAC in this area. These results add further weight to the view that GABA exerts a facilitatory influence on noradrenergic neurons (via an increase in their cell firing) which is not effected at the level of their cell bodies or in the surrounding region.     

Pharmacological characterisation of a cell line expressing GABA B1b and GABA B2 receptor subunits

The gamma-aminobutyric acid (GABA(B)) receptor has been shown to be a heterodimer consisting of two receptor subunits, GABA(B1) and GABA(B2). We have stably co-expressed these two subunits in a CHO cell line, characterised its pharmacology and compared it to the native receptor in rat brain membranes. Radioligand binding using [3H]CGP54626A demonstrated a similar rank order of potency between recombinant and native receptors: CGP62349>CGP54626A>SCH 50911>3-aminopropylphosphinicacid(3-APPA)>GABA>baclofen>saclofen>phaclofen. However, differences were observed in the affinity of agonists, which were higher at the native receptor, suggesting that in the recombinant system a large number of the receptors were in the low agonist affinity state. In contrast, [35S]GTPgammaS binding studies did not show any differences between recombinant and native receptors with the full agonists GABA and 3-APPA. Measurement of cAMP accumulation in the cells revealed a degree of endogenous coupling of the receptors to G-proteins. This is most likely to be due to the high expression levels of receptors (B(max)=22.5+/-2.5pmol/mg protein) in this experimental system. There was no evidence of GABA(B2) receptors, when expressed alone, binding [3H]CGP54626A, [3H]GABA, [3H]3-APPA nor of GABA having any effect on basal [35S]GTPgammaS binding or cAMP levels.     

Role of GABA(A) receptors in the ethanol-mediated inhibition of extracellular signal-regulated kinase

In the present study, we demonstrate the involvement of GABA(A) receptors in the ethanol-mediated modulation of extracellular signal-regulated kinases (ERK). Intraperitoneal (i.p.) administration of ethanol (3.5 g), flurazepam (75 mg) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cycloheptane-5,10-iminemaleate (MK-801) (0.4 mg/kg body weight) decreased, while picrotoxin (10 mg/kg body weight) increased, the phosphorylation of ERK following 10 min of their injection. However, the picrotoxin-induced phosphorylation of ERK was inhibited by ethanol, but was not affected by MK-801. These results indicate that ethanol's inhibitory effect on ERK phosphorylation may involve the modulation of GABA(A) receptor function.     

Weak effects of local and systemic administration of the GABA uptake inhibitor,SK&F 89976, on extracellular GABA in the rat striatum

Summary The effects of local and systemic administration of the potent GABA uptake inhibitor, SK&F 89976, on GABA overflow from the striatum of conscious rats were investigated in brain dialysis experiments. Administration of the compound via the dialysis probe at concentrations of 25 or 100 gmol/l significantly increased basal GABA overflow about 2-fold. Overflow evoked by 104 mmol/l K⁺ remained unaltered at the lower and was almost doubled at the higher concentration; this increase did, however, not reach statistical significance.Given systemically at 50 mg/kg i.p., a dose which is severalfold higher than those which exhibit anticonvulsant effects, SK&F 89976 caused a significant enhancement of K⁺-stimulated GABA overflow by about a factor of 2; the lower dose of 20 mg/kg i.p. was not effective. Basal GABA overflow was not significantly increased by either dose. These results suggest that the marked effects of nipecotic acid on basal GABA overflow reported by several authors seem to be related to GABA displacement rather than uptake inhibition, and that uptake inhibition does not improve the interpretability of measurements of GABA release by brain dialysis. They neither support the idea that the relative insensitivity of extracellular GABA to low Ca²⁺ and tetrodotoxin is indirectly due to very efficient removal of GABA by neuronal and/or glial uptake, leaving only residual amounts to be measured. Send offprint requests to P. Waldmeier at the above address     

Anxiolytic activity of a brain delivery system for GABA

We evaluated the anxiolytic property of a brain-specific gamma-aminobutyric acid delivery system (GABA-CDS) in male rats by means of a drink-foot shock conflict procedure. Brain-specific delivery of the active compound was achieved by combination of GABA benzyl ester with an interconvertible dihydropyridinepyridinium salt carrier, which is locked in to the brain upon its oxidation. Pharmacokinetic studies revealed that the hydrophilic pyridinium salt form (G-Q⁺) of the GABA-CDS formed in situ remained in the brain for 12 h but was cleared from the blood and other peripheral tissues by 0.5–4 h. While the lipophilic form (G-DH) of the GABA-CDS caused a marked and sustained anxiolytic response when administered systemically, GABA and the charged pyridinium salt (G-Q⁺ form) of the GABA-CDS were ineffective. G-DH was injected at either 0, 4, 10 or 25 mg/kg IV in DMSO after rats were water and food deprived. After either 0.5, 2, 4, 8 or 24 h, rats were permitted 10 s of shock-free drinking of 10% sucrose, then given a 35 mA (DC) current through the drinking tube. Drinking time was recorded for 3 min. All doses of G-DH caused a significant increase in anxiolysis over control levels through 8 h. An increase (4 to 7-fold) in anxiolytic activity was observed through the 10 mg/kg dose with the 25 mg/kg dose causing no additional increase. No sedation or analgesia was observed at 2 h with any anxiolytic-producing dose of G-DH. These results suggest that G-DH elicits anxiolysis with minimal sedation, through the local brain action the G-Q⁺ or subsequent to the release of GABA.This work was presented at the Joint Meeting of the American Society for Pharmacology and Experiment Therapeutics and The Society of Toxicology in Baltimore, MD (August 17–21, 1986)     

Alterations in benzodiazepine and GABA receptor binding in rat brain following systemic injection of kainic acid

The binding of gamma-aminobutyric acid (GABA) and benzodiazepine to receptors was examined in regions of rat brain at various times after subcutaneous injection of kainic acid (KA, 15 mg/kg). The animals exhibited pronounced convulsions 90 min-4 hr after this treatment. During this period (2 hr after the injection of kainic acid) no alterations in the binding of [3H]-GABA or [3H]flunitrazepam to receptors were detected in the frontal cortex, the hippocampus or the amygdala-pyriform cortex. After recovery from the acute convulsive phase, the rats appeared to be hyperexcitable, hyperactive, and displayed marked aggression and occasional clonic convulsions one to 80 days later. During this period a marked increase (80-200%) in the number of binding sites for GABA in the amygdala-pyriform cortex occurred but this was associated with a slow decrease in the number of binding sites for [3H]flunitrazepam to 70% control value at 3 weeks. Binding of the "peripheral"-type of benzodiazepine ligand, [3H]-Ro5-4864, was increased to 450% of control 3 weeks after injection. In addition, the ability of GABA to stimulate the binding of [3H]flunitrazepam was reduced when measured 3 days after the injection of kainic acid. It is suggested that the long-term behavioural syndrome observed in kainic acid-treated rats, as well as the reduced effectiveness of diazepam in preventing seizures in animals treated with kainic acid, (Czuczwar, Turski, Turski and Kleinrock, 1981) may be explained in part by a reduction in the number of neuronal benzodiazepine receptors and a "desensitization" of the GABA receptors which are coupled to benzodiazepine receptors.     

Cadmium exposure disrupts GABA and taurine regulation of prolactin secretion in adult male rats

This work was undertaken to evaluate the possible effects of cadmium exposure on 24 h changes of gamma-aminobutyric acid (GABA) and taurine median eminence and pituitary contents. Also the possible alterations of the regulatory mechanisms of GABA and taurine on prolactin secretion were evaluated. Adult male rats were given cadmium at a dose of 25 mg/l of cadmium chloride in the drinking water for 30 days. Control age-matched rats received cadmium free water. Metal exposure induced the appearance of a maximal value of prolactin at 08:00 h. In median eminence, cadmium abolished the GABA and taurine maximal values and decreased GABA and taurine mean levels. In the anterior pituitary, cadmium treatment phase advanced 12 h the peak observed in controls at 00:00 h for both amino acids. There was a positive correlation between GABA and taurine contents in median eminence and the anterior pituitary in both control and cadmium-exposed animals. However, the correlation between GABA or/and taurine with prolactin levels disappeared in cadmium-exposed animals. These results suggest that cadmium exposure affects GABA and taurine daily pattern in the median eminence and anterior pituitary, and those changes explain, at least in part, the modification in the regulatory pattern of prolactin secretion.     

The measurement of the release of endogenous GABA from rat brain slices by liquid chromatography with electrochemical detection

Summary A method for the determination of GABA by derivatization with 2,4,6-trinitrobenzenesulphonic acid and subsequent separation and quantitation by HPLC with electrochemical detection was characterized with respect to specificity, reproducibility and sensitivity. No other amino acid occurring in significant amounts in the brain was found to interfere; however, adequate separation of the derivatives of GABA and tryptophan must be carefully checked in each experiment. The sensitivity of the method is essentially determined by baseline noise, which mainly depends on the quality of the HPLC pump; under our conditions, it was about 2 ng/ml analyte. The coefficients of variation determined at two different concentrations relevant for the subsequent experiments were well below 10%. The method proved useful for the assessment of endogenous release of GABA from superfused rat cortical slices by electrical stimulation, which, in contrast to the basal release, was found to be completely calcium-dependent at stimulation frequencies of 5 and 12 Hz, under our conditions. Both stimulated and basal release of GABA was enhanced 4–5-fold by the inhibitor of GABA uptake, SK&F 89976 (10 M).Send offprint requests to P. C. Waldmeier     

Uptake of GABA by bovine adrenal medulla slices

Summary A sodium dependent GABA uptake system has been found in bovine adrenal medulla slices. This uptake has a K _m of 83.19±38.45 M and a V _max of 9.20±1.36 pmol/min×mg of tissue. It was inhibited by nipecotic acid and 2,4-diaminobutyric acid (IC50 67 and 38.5 M, respectively) but not by -alanine at concentrations up to 5 mM, a result which is similar to those found for the neuronal GABA uptake rather than the glial uptake. It is suggested that GABA uptake together with catabolic action of GABA-transaminase, also found in this tissue, could be regulating the GABA levels disposable for the proposed modulator role on catecholamine secretion of this amino acid in adrenal medulla.     

Benzodiazepines modify the agonist responses at a presynaptic GABA receptor

The stimulus-induced release of [3H]glutamic acid from striatal tissue was actively modulated by a presynaptic GABA receptor. The agonist responses of this GABA receptor were modified stereoselectively by benzodiazepines at low concentrations. The results correlate well with effects seen in [3H]GABA binding experiments and as they demonstrate benzodiazepine-induced changes in GABA receptor properties in a tissue prism preparation, these results suggest that the mechanisms described for extensively disrupted preparations may also apply to whole tissue.     

Effect of GABA Analogues on Blood Pressure and Central GABA Metabolism in the Rat

Abstract: Gamma aminobutyric acid (GABA) and different GABA analogues were examined for their cardiovascular actions and their influence on striatal dopamine (DA) levels and GABA accumulation after aminooxyacetic acid (AOAA). Gamma hydroxybutyric acid (GHBA) and baclofen caused hypertension and tachycardia after systemic as well as intracerebroventricular administration, while the opposite was true for GABA and muscimol. The hypertension after GHBA and baclofen was not reduced by picrotoxin or bicuculline and was not influenced by varying GABA levels by 3-mercaptopropionic acid (3-MPA)or AOAA. GHBA and muscimol but not baclofen reduced GABA accumulation induced by AOAA. Picrotoxin in a subconvulsive dose increased GABA accumulation and antagonized the inhibition after GHBA or muscimol. Bicuculline and a moderate dose of picrotoxin tended to decrease GABA accumulation by themselves and if anything augmented the effects of GHBA and muscimol. GHBA and baclofen but not muscimol in combination with AOAA increased DA levels, which was not prevented by picrotoxin or bicuculline. We conclude that the cardiovascular actions of GHBA and baclofen are probably not mediated by mechanisms identical to those of muscimol or exogenous GABA. In view of the biochemical results their actions would however be compatible with a concept of different GABA receptors.     

Effects of phenelzine and imipramine on the steady-state levels of mRNAs that encode glutamic acid decarboxylase (GAD67 and GAD65), the GABA transporter GAT-1 and GABA transaminase in rat cortex

There is an increasing body of evidence suggesting that GABA plays an important role in the therapeutic effects of antidepressant/antipanic drugs. Phenelzine and imipramine are efficacious in the treatment of depression and panic disorder and phenelzine has been reported to elevate GABA levels while imipramine enhances GABA release in rat brains. In the present study, using a multiprobe quantitative solution hybridization assay, we measured the steady-state levels of mRNAs that encode glutamic acid decarboxylase (GAD₆₇ and GAD₆₅), the GABA transporter GAT-1 and GABA transaminase (GABA-T) in rat cortex after treatment with constant infusion (via osmotic minipumps) of phenelzine or imipramine for a short-term (3 days) or long-term (21 days) period. We found that none of the treatments gave rise to significant changes in the steady-state levels of mRNAs encoding GAD₆₇, GAD₆₅ or GABA-T at any time point. The steady-state levels of GAT-1 mRNA were increased significantly (23%) after long-term, but not by short-term, treatment with phenelzine. Imipramine treatment, short- or long-term, did not alter the steady-state levels of GAT-1 mRNA. These results suggest that the GABA enhancing effects of phenelzine or imipramine in rat cortex do not affect the steady-state levels of mRNAs that encode GAD₆₇, GAD₆₅ and GABA-T. Further, the previously observed increases in GABA levels or GABA release induced by these drugs are probably not a consequence of changes in the expression of these genes. Received: 20 June 1997 / Accepted: 23 September 1997