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     

Release of endogenous GABA from the substantia nigra is not controlled by GABA autoreceptors

Summary The characteristics of the release of GABA from slices of the rat substantia nigra, elicited by electrical stimulation at frequencies of 0.5–48 Hz and by elevated K⁺ concentrations ranging from 15–35 mmol/l, was studied. Comparisons were made with cortical slices where the data were not available from previous studies.No GABA release could be evoked from rat nigral slices by electrical stimulation between 0.5 and 4 Hz, in contrast to cortical slices, in which this pool is sensitive towards inhibition by (–)-baclofen. Also, comparatively less GABA release could be evoked from nigral than from cortical slices by K⁺ concentrations between 15 and 25 mmol/l. While (–)-baclofen at 10 mol/l inhibited release caused by 15 mol/l K⁺ in cortical, it did not in nigral slices. GABA release caused by higher frequencies (8–48 Hz) or 30 mmol/l K⁺ concentrations was Ca²⁺-dependent and in the former case also tetrodotoxin-sensitive. It had similar characteristics as in cortical slices and was insensitive towards (–)-baclofen, muscimol and bicuculline. Even more markedly than in the cortex, 30 mmol/l K⁺ released greater amounts of GABA than electrical stimulation at 24 Hz of a similar duration, suggesting the existence of one or several additional pool(s) of lesser excitability.Since the majority of gabaergic nerve endings in the nigra belong to striato- and pallidonigral projection neurons and those in the cortex probably exclusively to various types of interneurons, it seems that (a) one or several of the latter release GABA at low frequencies in a baclofen-sensitive manner and are absent or rare in the s. nigra, and (b) the striato- and pallidonigral projection neurons are not controlled by presynaptic autoreceptors of the GABA_A or GABA_B type, because neither GABA release elicited by electrical stimulation nor by 30 mmol/l K⁺ was affected by agents interfering with these types of receptors.Send offprint requests to P. C. Waldmeier at the above address     

Systemic administration of baclofen and the GABAB antagonist,CGP 35348, does not affect GABA,glutamate or aspartate in microdialysates of the striatum of conscious rats

Summary Previous in vitro experiments have shown that the GABA_B agonist, baclofen, and the antagonist, CGP 35348, respectively, decrease and increase the autoreceptor-mediated release of GABA in brain slices and synaptosomes. Since it is not clear whether these autoreceptors are operative in vivo, an attempt was made to reproduce these results in brain dialysis experiments, knowing that only positive results would permit a conclusion in view of the doubts expressed in the literature with respect to the origin of extracellular GABA. Because of older reports of an inhibitory action of baclofen on the in vitro release of glutamate, which might be ascribed to the action of presynaptic GABA_B heteroreceptors, extracellular glutamate and aspartate were also measured.Neither (–)-baclofen, administered systemically at a dose of 20 mg/kg i.p., nor the GABA_B antagonist, CGP 35348 (300 mg/kg i.p.) had significant effects on basal overflow of GABA, glutamate, or aspartate nor on that evoked by 100 mmol/l K⁺ in the striatum of the conscious, freely moving rat. To ascertain this result, (–)-baclofen was also administered between two K⁺ stimulations, so that the first stimulation could serve as an intraindividual control of the second. The compound did not significantly affect K⁺ evoked overflow of any of the three transmitter amino acids under these conditions.It must be emphasized that these data do not exclude the operativity of presynaptic GABA_B auto- and hetero-receptors in vivo. They only suggest that this question must, in all probability, be addressed by other techniques than brain dialysis. Send offprint requests to P. Waldmeier at the above address     

Multiple voltage-sensitive calcium channels are probably involved in endogenous GABA release from striatal neurones differentiated in primary culture

Summary Calcium-dependent release of neurotransmitters is thought to be due to Ca²⁺ entry into nerve terminals, but the identities of the various voltage-sensitive Ca²⁺ channels (VSCC) involved in this process remain obscure. To elucidate the types of VSCCs involved in the release process, we studied the effects of various organic Ca²⁺ channel antagonists and agonists on the release of endogenous -aminobutyric acid (GABA) from mouse striatal neurones differentiated in primary culture. Diltiazem, verapamil and methoxyverapamil (D 600) inhibited K⁺-evoked (30 mM) GABA release at very high concentrations (> 1 M). The dihydropyridine (DHP) nifedipine, at low concentrations (0.01–1.00 M), was able to inhibit part of the K⁺-evoked GABA release (25.6±7.3% inhibition at 1 M). This is in agreement with the high affinity of nifedipine for DHP binding sites. The DHPs, BAY K 8644 (EC₅₀ = 41±15 nM) and CGP 28.392, which possess agonist properties at VSCCs, increased the 15 mM K⁺-evoked GABA release. The release evoked by the combination of K⁺ (15 M) and BAY K 8644 (up to 10 M) remained smaller than the release elicited by 30 mM K⁺. The effect of BAY K 8644 (1 M) was inhibited by nifedipine (IC₅₀ 0.55±0.05 M). When Na⁺ ions were replaced by choline, basal and K⁺-evoked GABA release was significantly increased. Even in the absence of external Na+, nifedipine (1 M) was not able to totally block the K⁺ effect. Moreover amiloride, a drug known to inhibit Na⁺/Ca⁺ exchange, and tetrodotoxin (TTX), did not modify the 30 mM K⁺ response. Therefore, nifedipine-insensitive K⁺-evoked GABA release is not due to Na⁺-dependent Ca²⁺ entry. These results can be explained by the presence of DHP-sensitive and insensitive Ca²⁺ channels on nerve terminals, each involved in the release process.Abbreviations CGP 28.392 4-[2-(difluoromethoxy)phenyl]-1,4,5,7-tetrahydro-2-methyl-5-oxo-furo[3,4-b]pyridine-3-carboxylic acid ethylester - BAY K 8644 (±)-methyl 1,4-dihydro-2,6-dimethyl-3nitro-4-(2 trifluoromethyl) pyridine-5-carboxylate - DHP 1,4-dihydropyridine - DIV days in vitro - D 600 methoxyverapamil - GABA -aminobutyric acid - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - HPLC high performance liquid chromatography - TTX tetrodotoxin - VSCC voltage-sensitive calcium channel Send offprint requests to J.-P. Pin at the above address     

Effect of ethanol on GABA uptake and release from hypothalamic fragments

A study was performed on the effect of ethanol on the basal and K⁺-evoked efflux of endogenous GABA from rat hypothalamic fragments. The amount of GABA present in the medium and in the tissue was measured by radioreceptor assay. In vitro addition of ethanol (50 and 100 mM) enhanced the K⁺-evoked efflux of GABA in a Ca⁺⁺-dependent manner, and increased tissue GABA content. Since K⁺-evoked outflow induced by ethanol was not affected by the presence of nipecotic acid, ethanol appears to alter the uptake of endogenous GABA. An inhibitory effect of ethanol on ³H-GABA uptake was observed under K⁺ depolarization. On the other hand, acute ethanol administration produced a decrease in basal and K⁺-evoked efflux from hypothalamic fragments and in tissue GABA concentration. Changes in GABA efflux may lie behind some of the neuropharmacological effects of ethanol.     

Ca2+-dependent release of endogenous GABA from rat cortical slices from different pools by different stimulation conditions

Summary The previously reported inhibitory effect of (–)-baclofen on the electrically evoked release of endogenous GABA from rat brain slices indicated the possibility of existence of GABA_B autoreceptors. In this study, we have tested an alternative explanation, i. e. the possibility that (–)-baclofen reduced an excitatory glutamatergic input to GABAergic neurons by inhibiting glutamate release, by investigating the interaction of 10 mmol/1 l-glutamate with the inhibitory effect of 10 mol/1(–)-baclofen. l-Glutamate did not affect the electrically evoked release of GABA on its own and did not abolish the effect of (–)-baclofen, suggesting that the latter was not secondary to a reduction of glutamate release. On the other hand, it greatly increased the basal release of GABA and more than doubled the GABA content of the slices at the end of the perfusion, indicating a marked enhancement of GABA synthesis. This additional GABA, apparently formed from exogenous l-glutamate, was not releasable by electrical stimulation at 0.5 or 24 Hz, but at least in part by stimulation with 30 mmol/l K⁺. The previously reported increase of GABA release at 12 Hz as compared to 4 Hz was studied in more detail. GABA released by electrical stimulation at 8–48 Hz was Ca²⁺-dependent and tetrodotoxin-sensitive. No evidence was obtained for a decrease of the amount of GABA released per impulse with increasing frequency in this range. Moreover, neither (–)-baclofen nor muscimol at 10 mol/l altered the release of the amino acid at 24 Hz; the former was also tested at a low Ca²⁺ concentration (0.3 mmol/l) and found to be inactive under these conditions. Thirty mmol/l K⁺ released about 30% higher amounts of GABA than electrical stimulation at 24 Hz under comparable conditions, in a Ca²⁺-dependent manner. K⁺-induced release was not modified by 10 mol/l (–)-baclofen or muscimol. Our results suggest the existence of at least 2 different, presumably neuronally located, releasable pools of GABA. One is sensitive to electrical stimulation at 0.25–4 Hz and responds to (–)-baclofen, suggesting control by GABA_B-type autoreceptors. The existence of a 2nd pool is indicated by the fact that K⁺ releases substantially more GABA than electrical stimulation and by the exclusive sensitivity of K⁺-evoked GABA release to exogenous l-glutamate. GABA released by electrical stimulation at frequencies above 4 Hz may come from a 3rd pool. Both the 2nd and the 3rd pool seem to be insensitive to (–)-baclofen and muscimol. Send offprint requests to P. C. Waldmeier at the above address     

Release of endogenous glutamate from rat cortical slices in presence of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid

Summary The effect of the new glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), on the electrically evoked release or, rather, overflow of endogenous glutamate in superfusates from rat cortical slices was compared with that of dihydrokainate. In the absence of these presumed uptake inhibitors, electrical stimulation for 4 min at 1 Hz did not elicit a measurable glutamate overflow over baseline at all. Basal overflow increased concentration-dependently in the presence of 10–100 M L-trans-PDC, about 5-fold at 100 M. Also, electrical stimulation caused increases of glutamate overflow over basal levels progressive with increasing concentrations of trans-PDC; a stimulated overflow corresponding to about 50% of basal overflow was obtained at 100 M. Basal as well as evoked release in the presence of dihydrokainate did not exceed ca. 60% of that obtained with 100 M L-trans-PDC. In synaptosomes, L-trans-PDC much more than dihydrokainate caused a transient increase of spontaneous glutamate release which was diminished in the absence of Na⁺, indicating that it is transported into the cytoplasm by the glutamate carrier and induces some efflux of the amino acid from this compartment. Moreover, trans-PDC caused a weak to moderate inhibition of K⁺-evoked glutamate release from synaptosomes at 10–300 M, without obvious concentration-dependence.Glutamate overflow elicited from rat cortical slices by electrical field stimulation at 1 Hz was Ca²⁺-dependent to about 80%. Tetrodotoxin (0.3 M) reduced it by about 90%. Lowering the temperature from 37°C to 22°C increased the ratio between evoked and basal overflow.As an application for L-trans-PDC as a glutamate uptake inhibitor in release studies, the regulation of glutamate release by GABA_B receptors was investigated. At 1 Hz, (–)-baclofen reduced evoked glutamate overflow at and above 3 M by maximally 40% at 30 M. This maximal effect was not increased when higher or lower stimulation frequencies were used nor when the Ca²⁺ concentration in the medium was increased or lowered, nor when the slices were prepared from other brain areas (hippocampus or striatum). The GABA uptake inhibitor, SK&F 89976, had no significant effect on evoked glutamate overflow, and the potent GABA_B antagonist, CGP 55845, induced only a small increase, indicating that tonic inhibition of glutamate by GABA via GABA_B receptors was not marked. On the other hand, the GABA_B antagonist was able to prevent the inhibitory effect of (–)-baclofen when applied before it and to abolish it when applied afterwards. The conclusion is that L-trans-PDC is a useful tool in glutamate release studies in brain slices for many purposes, with the reservation that its inhibitory effect on evoked glutamate release in synaptosomes is not yet understood. Correspondence to: P. C. Waldmeier at the above address     

On the origin of extracellular GABA collected by brain microdialysis and assayed by a simplified on-line method

Summary The present study describes a simplified on-line system for determination of GABA in brain dialysates. GABA was determined with an isocratic HPLC method after derivatization with o-phtaldialdehyde. One peristaltic pump was sufficient to transport both the perfusion fluid and the derivatizing reagent.The basal release of GABA was stimulated by infusion with either elevated K⁺ or the GABA uptake inhibitor (–)-nipecotic acid. Basal as well as stimulated GABA release were investigated for possible calcium-dependency by infusing submmolar amounts of the potent calcium antagonist cadmium. Infusion of cadmium did not modify the dialysate concentrations of GABA. In addition basal as well as nipecotic acid enhanced release of GABA dialysate concentrations were investigated for nerve-impulse dependency by infusing molar amounts of tetrodotoxin. No change in the GABA output was observed during infusion of TTX. From these results it is concluded that the basal as well as the nipecotic acid induced release of GABA did not fulfill the criteria for classic exocytotic release. Possible explanations for these unexpected findings are discussed. Send offprint requests to B. H. C. Westerink at the above address     

A novel type of GABA receptor in rat spinal cord?

Summary The depolarization-evoked release of -aminobutyric acid (GABA) and its possible modulation mediated by autoreceptors were studied in nerve endings isolated from rat spinal cord and prelabeled with the radioactive aminoacid. In the presence of the GABA uptake inhibitor SK&F 89976A [N-(4,4-diphenyl-3-butenyl)-nipecotic acid], used to minimize carrier-mediated homoexchange, exogenous GABA (1–10 µmol/l) decreased in a concentration-dependent way the release of³H-GABA evoked by 15 mmol/l KCl. The GABA_A receptor agonist muscimol (10–100 µmol/l) did not affect the K⁺(15 mmol/l)-evoked³H-GABA release. Similarly ineffective was the GABA_B receptor agonist (–)-baclofen (3–100 µmol/l). The effect of GABA was not counteracted by the GABA_A receptor antagonists bicuculline,picrotoxinorSR95531 [2-(3-carbethoxy-2-propenyl)-3-amino-6-paramethoxy-phenyl-pyridazinium bromide]. In a set of experiments performed in parallel with spinal cord or cerebral cortex synaptosomes depolarized with 9 mmol/l KCI it was found that: (a) GABA (1–10 µmol/l) decreased in a concentration-dependent way the release of³H-GABA in both brain regions and its effects were practically doubled when compared with those in synaptosomes depolarized with 15 mmol/l KCI; - (b) muscimol (1–100 µmol/l) was ineffective both in spinal cord and in cerebral cortex; - (c) (–)-baclofen (1-100 µmol/l) concentration-dependently inhibited³H-GABA release in cerebral cortex synaptosomes but was almost inactive in the spinal cord; the (+)-enantiomer of baclofen (100 µmol/l) did not affect significantly the release of³H-GABA; - (d) the GABA_B receptor antagonist phaclofen [3-amino-2-(4chlorophenyl)-propyl phosphonic acid] antagonized the effect of GABA in cerebral cortex but not in spinal cord synaptosomes. It is concluded that the release of GABA in rat spinal cord may be modulated by an autoreceptor which does not belong to the known GABA receptor subtypes. Send offprint requests to M. Raiteri at the above address     

Inhibition of Na+, K+-ATPase in rat renal proximal tubules by dopamine involved DA-1 receptor activation

Summary Endogenous kidney dopamine (DA) causes natriuresis and diuresis, at least partly, via inhibition of proximal tubular Na⁺,K⁺-ATPase. The present study was done to identify the dopamine receptor subtype(s) involved in dopamine-induced inhibition of Na⁺,K⁺-ATPase activity. Suspensions of renal proximal tubules from Sprague-Dawley rats were incubated with dopamine, the DA-1 receptor agonist fenoldopam or the DA-2 receptor agonist SK&F 89124 in the presence or absence of either the DA-1 receptor antagonist SCH 23390 or the DA-2 receptor antagonist domperidone. Dopamine and fenoldopam (10^–5 to 10^–8 mol/1) produced a concentration-dependent inhibition of Na⁺,K⁺-ATPase activity. However, SK&F 89124 failed to produce any significant effect over the same concentration range. Incubation with fenoldopam (10^–5 to 10^–8 mol/1) in the presence of SK&F 89124 (10^–6 mol/l) inhibited Na+,K+-ATPase activity to a degree similar to that with fenoldopam alone. Furthermore, DA-induced inhibition of Na⁺,K⁺-ATPase activity was attenuated by SCH 23390, but not by domperidone. Since -adrenoceptor activation is reported to stimulate Na⁺,K⁺-ATPase activity and, at higher concentrations, dopamine also acts as an a-adrenoceptor agonist, the potential opposing effect from -adrenoceptor activation on DA-induced inhibition of Na⁺,K⁺-ATPase activity was investigated by using the -adrenoceptor blocker phentolamine. We found that, in the lower concentration range (10^–5 to 10^–7 mol/1), dopamine-induced inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine was similar in magnitude to that observed with dopamine alone. However, at the highest concentration used (10^–4 mol/1), dopamine produced a significantly larger degree of inhibition of Na⁺,K⁺-ATPase activity in the presence of phentolamine. These results indicate that the DA-1 dopamine receptor subtype, but not the DA-2 receptor subtype, is involved in dopamine-mediated inhibition of Na⁺,K⁺-ATPase. At higher concentrations of dopamine, the DA-1 receptor-mediated inhibitory effect on Na⁺,K⁺-ATPase activity may be partly opposed by a simultaneous -adrenoceptor-mediated stimulation of the activity of this enzyme.     

Age-dependent changes in the susceptibility to thiopental anesthesia in mice: Analysis of the relationship to the functional expression of GABA transporter

The potency of anesthetics changes during development, probably due not only to pharmacokinetic factors such as differential distribution and/or metabolism, but also to pharmacodynamic factors such as changes to the GABAergic system in the brain. To explore the latter mechanism, we focused on the GABA transporter (GAT), the uptake system for GABA, which participates in the synaptic clearance of GABA. Thiopental-induced anesthesia, as assessed by the onset and duration of loss of the righting reflex, was more pronounced in 3-week-old mice than in 7-week-old mice. Both NO-711 and SKF89976A, selective GAT-1 inhibitors, significantly enhanced the anesthesia in the 7-week-old but not in the 3-week-old mice. In synaptosomes prepared from the cerebral cortex, the kinetics of GABA transport was similar between the two age groups, as assessed by [³H]GABA uptake assay. In addition, expression of GAT mRNA was similar between the two age groups, as assessed by quantitative RT-PCR. Thiopental reduced [³H]GABA uptake only at high concentrations in a similar manner at both ages. Conversely, the ability of SKF89976A to inhibit [³H]GABA uptake was greater in the 7-week-old mice than in the 3-week-old mice. Based on these results, GAT seems unlikely to contribute to the greater susceptibility to thiopental anesthesia in 3-week-old mice, while the increased ability of GABA uptake inhibitors to enhance thiopental-induced anesthesia in 7-week-old mice is at least partly due to higher sensitivity of GAT to the inhibitors.     

Facilitation by GABA of the potassium-evoked release of 3H-noradrenaline from the rat occipital cortex

Summary The effects of GABA were studied on the release of ³H-noradrenaline (³H-NA) evoked by potassium or by tyramine from slices of the rat occipital cortex and on the release of ³H-NA elicited by nerve stimulation from the cat nictitating membrane.GABA (30–1000M) facilitated the potassium-evoked release of ³H-NA in a concentration dependent manner. This facilitatory effect was not antagonized by bicuculline (1–100 M) or by picrotoxin (1–100 M). Exposure to the GABA agonist muscimol (1–100 M) did not affect either the spontaneous or the potassium-evoked release of ³H-NA.The facilitatory effect of GABA on the release of ³H-NA elicited by potassium was observed when the occipital cortex slices were exposed to 20 mM K⁺ during 1 min. When depolarization was induced by 35 mM K⁺ exposure to GABA failed to enhance the release of the neurotransmitter. GABA 300 M did not affect the release of the labelled neurotransmitter evoked by exposure to tyramine 0.6 M.In the cat nictitating membrane prelabelled with ³H-NA, the stimulation evoked release of the labelled neurotransmitter was not affected by GABA (10–300 M).In conclusion, GABA has a facilitatory effect on the calcium-dependent, potassium evoked release of ³H-NA when the depolarization is of moderate degree. This effect of GABA appears to be selective for the central nervous system.Some of the findings described in this paper have been presented at the Meeting of the British Pharmacological Society (March, 1978, Guildford, U.K.)     

Enhancement by GABA of the stimulation-evoked catecholamine release from cultured bovine adrenal chromaffin cells

Summary The possible involvement of GABAergic mechanisms in the catecholamine (CA) release from adrenal medulla was investigated in a primary culture of bovine adrenal chromaffin cells. GABA elicited CA release and enhanced acetylcholine (ACh)-, excess K⁺-and veratridine-evoked CA release. Muscimol, a selective GABA_A receptor agonist, mimicked the action of GABA on CA release. On the other hand, baclofen, a GABA_B receptor agonist, failed to affect basal or evoked CA release. Furthermore, bicuculline and picrotoxin blocked the enhancement by GABA of veratridine-evoked CA release without affecting basal CA release and CA release evoked by veratridine. In Ca²⁺-free medium, GABA failed to affect basal and caffeine-evoked CA release. ACh-evoked CA release was slightly reduced by bicuculline, whereas excess K⁺-evoked CA release was not, suggesting the involvement of endogenous GABA in CA release evoked by ACh. These results suggest a facilitatory modulation by GABA of basal and evoked release of CA from bovine adrenal medulla through GABA_A receptor-mediated mechanisms. Send offprint requests to A. Tsujimoto at the above address     

GABA induced changes in acetylcholine release from slices of guinea-pig brain

Summary The effect of GABA on acetylcholine (ACh) release was investigated on superfused slices of guinea-pig cerebral cortex (CC), caudate nucleus (CN), tuberculum olfactorium and brain stem.GABA (1–6×10^–3 mol/l) increased the spontaneous and KCl-evoked ACh overflow in CC and CN, reduced the electrically-evoked release in all areas tested (most evidently in CC and CN) and lowered the threshold of electric stimulation-induced ACh release in CC. These effects were also caused by 3-amino-1-propane sulphonic acid (1×10^–3mol/l) and ethanolamine-O-sulphate (2×10^–4mol/l), were reduced by bicuculline (1×10^–4mol/l) and fully antagonized by picrotoxin (8×10^–5mol/l), but they were not influenced by phentolamine, methysergide, spiroperidol or strychnine.Tetrodotoxin (TTX) (5×10^–7mol/l) blocked the facilitation of spontaneous ACh release by GABA only when the slices were perfused with normal Krebs solution, but not when perfused with a KCl-enriched medium. These results suggest that GABA affects the cholinergic transmitter release through bicuculline- and picrotoxin-sensitive receptors, showing low affinity toward the agonist. Moreover GABA modulation of resting ACh release requires action potentials only in normal [K⁺]₀, but not in high [K⁺]₀, suggesting that GABA-receptive sites are located at cholinergic terminals.     

Interaction between 5-HT uptake inhibition and activation of 5-HT autoreceptors by exogenous agonists in rat cerebral cortex slices and synaptosomes

Summary Rat cerebral cortex slices or synaptosomes were labelled with ³H-5-hydroxytryptamine (³H-5-HT) and subsequently superfused. They were depolarized by electrical stimulation (slices) or with high K⁺ (slices and synaptosomes). Continuous electrical stimulation (2 Hz, 24 mA, 2 ms) and continuous or discontinuous K⁺ depolarization (15–25 mM) were used. 1. Continuous electrical stimulation or continuous K⁺-depolarization of slices evoked a steady overflow of tritium that slowly decayed with time. 2. Exposure to increasing concentrations of 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole succinate (RU 24969) (0.001–0.1 M) during continuous electrical stimulation produced a concentration-dependent decrease in tritium overflow. Citalopram (1 M) counteracted the effect of RU 24969. 3. RU 24969 inhibited the evoked ³H-overflow and citalopram reduced the effect of RU 24969 also during continuous depolarization of slices with 20 mM K⁺. Similar results were obtained by using 5-methoxytryptamine or LSD. 4. In slices 1 M citalopram increased significantly the tritium overflow evoked by electrical stimulation or by 20 mM K⁺-depolarization. 5. Increasing the K⁺ concentration from 20 mM to 25 mM mimicked the effects of 1 M citalopram both on the RU 24969 activity and on the evoked tritium overflow. 6. RU 24969 (0.001–0.1 M) decreased in a concentration-dependent way the release of tritium from cortical synaptosomes depolarized with K⁺ (15–20 mM). The presence of 1 M citalopram did not modify significantly the effect of the agonist. Citalopram was ineffective also when the serotonin uptake carrier in superfused synaptosomes was activated by tryptamine. In conclusion, in slices of rat cerebral cortex, the action of exogenous 5-HT autoreceptor agonists is inhibited by 5-HT uptake blockers independently of the depolarizing agent (electrical stimulation or high-K⁺) used to elicit ³H-5-HT release. Increasing K⁺-concentration, which probably increases serotonin in the biophase, mimics the presence of the reuptake inhibitor. These data together with the finding that, in superfused synaptosomes, 5-HT uptake inhibition did not affect the potency of autoreceptor agonists, favours the idea that, in cerebral cortex slices, inhibitors of 5-HT reuptake prevent activation of autoreceptors by exogenous agonists by increasing the concentration of 5-HT in the autoreceptor biophase. Send offprint requests to M. Raiteri at the above address     

GABA terminal autoreceptors in the pars compacta and in the pars reticulata of the rat substantia nigra are GABAB

The depolarization-evoked release of gamma-aminobutyric acid (GABA) and its modulation mediated by autoreceptors were studied in superfused synaptosomes prepared from the pars compacta and from the pars reticulata of the rat substantia nigra. The release of [3H]GABA evoked by 9 mM KCl was almost totally calcium-dependent in both nigral subregions. In the presence of SK&F 89976A (N-(4,4-diphenyl-3-butenyl)nipecotic acid), a GABA uptake inhibitor added to minimize carrier-mediated homoexchange, GABA (0.3-10 microM) inhibited, in a concentration-dependent way, the K(+)-evoked overflow of [3H]GABA from both pars compacta and pars reticulata synaptosomes. Similarly to GABA, (-)-baclofen (0.3-10 microM) reduced the [3H]GABA overflow, being roughly equipotent to GABA in both nigral subregions. The (+) enantiomer of baclofen was ineffective. The overflow of [3H]GABA was not consistently affected by muscimol in either the pars compacta or the pars reticulata. The effects of GABA were bicuculline- and picrotoxin-insensitive. However, the inhibition by GABA of the [3H]GABA overflow was antagonized by phaclofen. It is concluded that (a) GABA autoreceptors are sited on GABAergic nerve endings in both the pars compacta and pars reticulata of the rat substantia nigra; (b) these autoreceptors belong to the GABAB type.     

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)     

Investigations on GABAB receptor-mediated autoinhibition of GABA release

Summary In this study, we have investigated the effects of phaclofen on the [³H] overflow from [³H]GABA prelabelled rat cortical slices and its interaction with the effects of (–)-baclofen in dependence of the stimulation frequency. (–)-Baclofen strongly depressed the [³H] overflow in the frequency range of 0.125 to 4 Hz to a constant residual level (ICIn50 = 0.37 mol/l at 0.125 Hz), but became inactive above. The potency of the (+)-enantiomer was considerably weaker by a factor of nearly 1000. The GABAB antagonist, phaclofen, increased [³H] overflow at 300 mol/l and, moremarkedly, at 3 and 1 mmol/l, respectively. However, the increase was virtually independent of the frequency between 0.125 and 16 Hz. If the compound interacted only with the putative GABA_B autoreceptor involved in the regulation of GABA release, the extent of the enhancing effect should increase with increasing frequency because of the concomitant rise in synaptic GABA concentration. In order to further investigate this phenomenon, the IC₅₀ of (–)-baclofen and antagonism of phaclofen against (–)-baclofen were determined at 0.125 Hz and 2 Hz, respectively. Whereas the IC₅₀ of (–)-baclofen was 0.63 ± 0.04 mol/l at 0.125 Hz, it increased to 4.88 + 0.45 mol/l at 2 Hz. The pA₁₀-values of phaclofen were about the same at both frequencies, whereas the pA₂-values differed by a factor of 2.3. Therefore, the possibility should be considered that (–)-baclofen does not only interact with presynaptic GABA autoreceptors, but also may interact with other - presumably somatodendritic- GABA_B-receptors whose pharmacology is not identical with that of the receptors by which (–)-baclofen exerts its effects on GABA release. Send offprint requests to P. A. Baumann at the above address     

Potassium ion is indispensable to the catecholamine releasing response of dog adrenals to γ-aminobutyric acid

Summary The effect of potassium ion on the GABA-evoked catecholamine (CA) release from isolated perfused adrenal glands of the dog was investigated. When omitting the external potassium ion, the basal release of CA was increased. During this period GABA no longer caused the increase in CA release and moreover the increased basal release was diminished reversibly by GABA. 3-Amino-1-propane-sulfonic acid, a GABA_A agonist, mimicked the action of GABA in K⁺-free solution, while baclofen, a GABA_B agonist, did not cause CA release in normal solution and did not alter the basal release in K⁺-free solution. The inhibition by GABA of the basal CA release in K⁺-free solution was blocked by bicuculline. The potency of the CA releasing action of GABA was dependent on the concentration of external K⁺ between 1–10 mM. Reintroduction of K⁺ to glands which had been perfused with K⁺-free solution immediately reduced the basal release of CA whereas it recovered the CA releasing action of GABA. These results suggest that GABA-evoked CA release is dependent on potassium ion. The possible mechanisms by which GABA evoked CA release are discussed.     

Coexistence of carriers for dopamine and GABA uptake on a same nerve terminal in the rat brain.

The ability of gamma-aminobutyric acid (GABA) to affect the release of [3H]-dopamine in rat brain synaptosomes prepared from corpus striatum, frontal cortex and hypothalamus and prelabelled with the radioactive catecholamine in the presence of desipramine was examined. GABA (10-300 microM) increased in a concentration-dependent way the basal release of [3H]-dopamine from striatum and cortical synaptosomes; however, its effect was much less pronounced in hypothalamic nerve terminals. 2,4-Diaminobutyric acid (DABA) mimicked GABA although less potently. Neutral amino acids such as leucine, valine or alpha-aminoisobutyric acid (100-300 microM) did not affect or increased minimally the release of [3H]-dopamine. The GABA-induced [3H]-dopamine release was not prevented by the GABAA-receptor antagonists, bicuculline or picrotoxin. The GABAA-receptor agonist, muscimol (10-300 microM), displayed only a very weak, not significant, enhancing effect on [3H]-dopamine release. The GABAB-receptor agonist (-)-baclofen (100 or 300 microM) had no effect. Three novel and selective inhibitors of GABA uptake, N-(4,4-diphenyl-3-butenyl)-nipecotic acid (SK&F 89976A), N-(4,4-diphenyl-3-butenyl)-guvacine (SK&F 100330A) and N-(4,4-diphenyl-3-butenyl)-homo-beta-proline (SK&F 100561) potently counteracted the enhancing effect of GABA on [3H]-dopamine release. Nipecotic acid also reduced the effect of GABA. It is concluded that carriers for the uptake of dopamine and GABA may coexist on the same nerve terminal in the rat brain.