Peter Wicki

GABA and glutamate release affected by GABAB receptor antagonists with similar potency : no evidence for pharmacologically different presynaptic receptors

1 The effects of a series of nine GABAB receptor antagonists of widely varying potencies on electrically stimulated release from cortical slices of [3H]‐GABA in the absence or presence of 10 μm of the GABABagonist, (−)−baclofen and of endogenous glutamate in the presence of (−)−baclofen were compared. 2 The concentrations of the compounds half maximally increasing [3H]‐GABA release (EC50s) at a stimulation frequency of 2 Hz correlated well with the IC50 values obtained from the inhibition of the binding of the agonist, [3H]‐CGP 27492, to GABAB receptors in rat brain membranes (rank order of potency: CGP 56999 A ≥ CGP 55845 A > CGP 52432 ≥ CGP 56433 A > CGP 57034 A > CGP 57070 A ≥ CGP 57976 > CGP 51176 > CGP 35348). 3 Likewise, the concentrations causing half‐maximal increases of [3H]‐GABA in the absence or presence of (−)−baclofen, and of endogenous glutamate in the presence of (−)−baclofen, correlated well with each other. Reports in the literature suggesting the CGP 35348 exhibits a 70 fold preference for inhibition of (−)−baclofens effects on glutamate over [3H]‐GABA release, and that CGP 52432 shows a 100 fold preference in the opposite sense, could not be confirmed in our model. 4 Therefore, our results suggest that, if there are pharmacological differences between GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings in the rat cortex, they are not revealed by this series of compounds of widely different potencies. 5 In particular, our results with CGP 35348 and CGP 52432 do not support the hypothesis that GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings represent subtypes with different pharmacology.

Potential involvement of a baclofen-sensitive autoreceptor in the modulation of the release of endogenous GABA from rat brain slices in vitro

SummaryThe effects of the GABAA agonist, muscimol, and of the enantiomers of the GABAB agonist, baclofen, on the release of endogenous GABA from slices of the rat cerebral cortex, striatum and hippocampus were measured by means of a HPLC method with electrochemical detection. Moreover, the effect of the GABAA antagonist, bicuculline, and of the frequency of stimulation were studied in cortical slices. The amount of endogenous GABA released per impulse from cortical slices decreased by about 50% when the frequency was increased from 0.25 Hz to 1 Hz. This might indicate that GABA inhibited its own release. (−)-Baclofen at 1 and 10 μM, but not its (+)-enantiomer, markedly inhibited the release of endogenous GABA, to a similar extent in all 3 areas investigated. The effect of (−)-baclofen was dependent on the frequency of stimulation: at lower frequencies (0.25 and 0.5 Hz) it was more marked than at a higher one (4 Hz). This would be expected from the results showing that the release of endogenous GABA decreases with increasing frequency, which suggests that this amino acid inhibits its own release. Muscimol at 10 μM, on the other hand, was ineffective in all 3 areas at a stimulation frequency of 0.5 Hz. Bicuculline (10 μM) at 4 Hz, at which autosuppression of GABA release is maximal did not enhance the release of endogenous GABA from cortical slices. With cerebellar or nigral slices, no adequate stimulation-induced release of endogenous GABA could be obtained under comparable conditions. These data are compatible with, but do not prove the existence of GABAB-type presynaptic autoreceptors modulating the release of this amino acid. More definite conclusions may possibly be drawn when a GABAB antagonist becomes available, which is expected to enhance GABA release under appropriate conditions.

Investigations on GABAB receptor-mediated autoinhibition of GABA release

SummaryIn 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 μmol/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 μmol/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 GABAB 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 μmol/l at 0.125 Hz, it increased to 4.88 + 0.45 μmol/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- GABAB-receptors whose pharmacology is not identical with that of the receptors by which (−)-baclofen exerts its effects on GABA release.

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

SummaryThe 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 μM L-trans-PDC, about 5-fold at 100 μM. 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 μM. Basal as well as evoked release in the presence of dihydrokainate did not exceed ca. 60% of that obtained with 100 μM 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 μM, 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 μM) 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 GABAB receptors was investigated. At 1 Hz, (−)-baclofen reduced evoked glutamate overflow at and above 3 μM by maximally 40% at 30 μM. 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 GABAB antagonist, CGP 55845, induced only a small increase, indicating that tonic inhibition of glutamate by GABA via GABAB receptors was not marked. On the other hand, the GABAB 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.

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

SummaryA 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 μM).

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

SummaryThe 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 μmol/l inhibited release caused by 15 μmol/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 GABAA or GABAB 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.

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

SummaryThe previously reported inhibitory effect of (−)-baclofen on the electrically evoked release of endogenous GABA from rat brain slices indicated the possibility of existence of GABAB 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 μmol/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 μmol/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 μmol/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 GABAB-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.

Effects of the putative P-type calcium channel blocker, R,R-(−)-daurisoline on neurotransmitter release

The alkaloid and medicinal herb constituent, R,R-(−)-daurisoline, was originally reported to be a N-type Ca2+ channel blocker, but newer evidence indicates that it is a blocker of P-type Ca2+ channels. To clarify its specificity with respect to N- and P-channels, we compared its effects on the electrically induced release of endogenous glutamate, 3H-GABA and 3H-noradrenaline, from brain slices with those of ω-agatoxin IVA and ω-conotoxin GVIA. Like ω-agatoxin IVA (but with about 1000-fold lower potency), and unlike ω-conotoxin GVIA, R,R-(−)-daurisoline inhibited the release of 3H-GABA and glutamate, with IC50 values of 8 and 18 μM. However, inhibition particularly of 3H-GABA release was more complete than by ω-agatoxin IVA, indicating interaction with one or more additional voltage-sensitive Ca2+ channels, possibly the Q-type. Its potency to inhibit glutamate release elicited either electrically, by veratrine or by high concentrations of K+ was similar, in contrast to sodium channel blockkes. The effects of R,R-(−)-daurisoline on the release of 3H-noradrenaline, 3H-dopamine and 3H-acetylcholine were in agreement with previous knowledge from experiments with ω-agatoxin IVA suggesting an involvement of P-channels. A weak inhibition of 3H-noradrenaline release at 10 μM, similar to that by ω-agatoxin IVA at 0.03 μM, was occluded by α2-antagonistic properties and could be unmasked in presence of rauwolscine. At 10 μM, it also inhibited electrically evoked 3H-dopamine and 3H-5-hydroxytryptamine release and caused a marked spontaneous release of all three monoamines in a reserpine-like manner. Spontaneous and evoked release of 3H-acetylcholine was inhibited by about 25% at 10 μM.In radioligand binding studies, R,R-(−)-daurisoline interacted with α1 and α2-adrenoceptors, 5-HT2 and muscarinic cholinergic receptors with IC50 values close to 1 μM, and with μ opiate receptors even with 0.18 μM. Atropine reduced the weak inhibitory effect of R,R-(−)-daurisoline on 3H-acetylcholine release somewhat, suggesting that it was brought about by both P channel blockade and cholinergic agonist activity. The effect on 3H-GABA release was unaffected by naloxone, indicating that the interaction of R,R-(−)-daurisoline with μ opiate receptors is antagonistic.The pattern of effects on neurotransmitter release observed with R,R-(−)-daurisoline resembles that of ω-agatoxin IVA and supports previous electrophysiological data suggesting that the compound blocks P-type voltage-sensitive Ca2+ channels. However, the more complete blockade of amino acid release by R,R-(−)-daurisoline suggests interaction with additional Ca+ channel subtypes. Although it does also possess other pharmacological properties, we think that the compound is suitable to test whether blockade of glutamate release via voltage-sensitive Ca2+ channels is a viable concept to obtain novel neuroprotective and/or anticonvulsant compounds.

Effects of CGP 28014 on the in vivo release and metabolism of dopamine in the rat striatum assessed by brain microdialysis

The effects on rat striatal dopamine (DA) metabolism of systemic and local administration of CGP 28014, an inhibitor of catechol-O-methyl-transferase (COMT), were studied by in vivo microdialysis. CGP 28014 (30 mg/kg i.p.) significantly reduced the levels of homovanillic acid (HVA), but did not modify DA and 3,4-dihydroxyphenylacetic acid (DOPAC). The intrastriatal administration (via the microdialysis probe) of 5, 7.5, 10, and 20 mM of CGP 28014 elicited a concentration-dependent, several-fold increase in extracellular DA but did not alter the levels of HVA and DOPAC. Thus, the effects of CGP 28014 observed after i.p. injection (decrease in HVA levels) are different from those measured after intrastriatal administration (increase in DA release). Therefore, the inhibition of COMT is likely to be due to the action of a metabolite of CGP 28014 formed in the periphery and not in the brain.

GABA RELEASE IN RAT CORTICAL SLICES IS UNABLE TO COPE WITH DEMAND IF THE AUTORECEPTOR IS BLOCKED

Electrically stimulated release of neurotransmitters in brain slices normally displays frequency dependence because of progressive activation of autoreceptors by endogenously released transmitter, which is abolished by blockade of autoreceptors. In consequence, the maximal increase caused by an autoreceptor antagonist in percent of the corresponding controls should be greater at higher than at lower frequencies. In the case of γ-aminobutyric acid (GABA), we have previously found a marked deviation from this expectation. Among several explanations envisaged, computer simulation suggested only one to be compatible with the experimental data: the release mechanism may not be able to cope with high demand.This hypothesis was tested by investigating the frequency dependence of the release of3H-GABA in the presence and absence of a high concentration of the potent GABAB antagonist, CGP 55845, using extremely short stimulation periods. To this end, slices were stimulated with groups of 4 POPS (a POP — pseudo-one-pulse — consists of 4 pulses delivered at 100 Hz). The intervals between the POPs within a group were varied from 60-0.5 s, corresponding to frequencies within the POP group of 0.0167–2 Hz. Under such circumstances, the theoretically expected pattern was indeed observed: the GABAB antagonist abolished the frequency dependence. In a second series of experiments, fractional release per POP was determined when 4–32 POPs were delivered at 2 Hz, with and without CGP 55845. The increase of GABA release elicited by the GABAB antagonist gradually subsided with increasing number of POPS. It was about 50Q7o of that observed during the first 4 POPs in the 4 subsequent ones, and almost nil in the last 16 of a total of 32 POPs.The results of this study support the hypothesis generated with the help of computer simulation, that release may not be able to keep up with high demand. They further suggest that exhaustion of the releasable transmitter pool occurs surprisingly fast. It is not known whether this phenomenon is physiologically relevant or a consequence of metabolic stress to which the slices are subjected during preparation and superfusion, or of their exposure to transaminase and uptake inhibitors, but it provides an explanation for the anomalies observed in the studies of the effects of GABAB antagonists on3H-GABA release.