The GABAB receptor antagonists CGP35348 and CGP52432 inhibit glycine exocytosis: Study with GABAB1- and GABAB2-deficient mice

GABA_B receptors mediate inhibition of neurotransmitter exocytosis from nerve endings. Unexpectedly, the well known GABA_B receptor antagonist CGP35348 and, in part, the compound CGP52432, are now found to inhibit on their own the K⁺-evoked exocytosis of glycine when added at low micromolar concentrations to superfused mouse glycinergic nerve endings prelabelled with [³H]glycine through GLYT2 transporters. CGP35348 inhibited [³H]glycine release both in spinal cord and in hippocampus, but was also able to prevent the inhibitory effect of (?)-baclofen; CGP52432 exhibited intrinsic activity only in the hippocampus; in spinal cord, it behaved exclusively as a silent orthosteric antagonist by blocking the release inhibition brought about by (?)-baclofen. The intrinsic activity of CGP35348 in spinal cord was not prevented by CGP52432, indicating that CGP35348 is not a partial GABA_B agonist in this experimental system. CGP54626, an extremely potent antagonist, exhibited only a minimal intrinsic activity. SCH50911, a GABA_B antagonist belonging to a different chemical class, was devoid of significant activity, while phaclofen was effective only at 100–300 μM. In synaptosomes purified from the spinal cord or the hippocampus of mice lacking either the GABA_B1 (GABA_B1?/? mice) or the GABA_B2 (GABA_B2?/? mice) subunit, the evoked exocytosis of [³H]glycine was no longer inhibited by (?)-baclofen, whereas the intrinsic activity of CGP35348 and CGP52432 was not decreased. Activation of unknown sites on glycinergic terminals is likely to be involved. These unexpected effects should not be ignored when interpreting results obtained with the above GABA_B receptor antagonists.     

Pharmacologically distinct GABAB receptors that mediate inhibition of GABA and glutamate release in human neocortex

1. The release of endogenous γ-aminobutyric acid (GABA) and glutamic acid in the human brain has been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply located tumours.
2. The basal outflows of GABA and glutamate from superfused synaptosomes were largely increased during depolarization with 15 mM KCl. The K⁺-evoked overflows of both amino acids were almost totally dependent on the presence of Ca²⁺ in the superfusion medium.
3. The GABA_B receptor agonist (−)-baclofen (1, 3 or 10 μM) inhibited the overflows of GABA and glutamate in a concentration-dependent manner. The inhibition caused by 10 μM of the agonist ranged from 45–50%.
4. The effect of three selective GABA_B receptor antagonists on the inhibition of the K⁺-evoked GABA and glutamate overflows elicited by 10 μM (−)-baclofen was investigated. Phaclofen antagonized (by about 50% at 100 μM; almost totally at 300 μM) the effect of (−)-baclofen on GABA overflow but did not modify the inhibition of glutamate release. The effect of (−)-baclofen on the K⁺-evoked GABA overflow was unaffected by 3-amino-propyl (diethoxymethyl)phosphinic acid (CGP 35348; 10 or 100 μM); however, CGP 35348 (10 or 100 μM) antagonized (−)-baclofen (complete blockade at 100 μM) at the heteroreceptors on glutamatergic terminals. Finally, [3-[[(3,4-dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic aid (CGP 52432), 1 μM, blocked the GABA_B autoreceptor, but was ineffective at the heteroreceptors. The selectivity of CGP 52432 was lost at 30 μM, as the compound, at this concentration, inhibited completely the (−)-baclofen effect both on GABA and glutamate release.
5. It is concluded that GABA and glutamate release evoked by depolarization of human neocortex nerve terminals can be affected differentially through pharmacologically distinct GABA_B receptors.

     

Effects of Post-Training Administration of (−)-Baclofen and Chlordiazepoxide on Memory Retention in ICRC Swiss Mice: Interactions with GABAA and GABAB Receptor Antagonists

Abstract: The effects of post-training administration of chlordiazepoxide and (?)-badofen on memory retention was studied in ICRC Swiss mice by measuring the retest stepdown latency 24 hr after foot-shock in a passive avoidance task. Chlordiazepoxide 20 mg/kg impaired memory retention and a similar effect was produced by 10 mg/kg of diazepam. The effect of chlordiazepoxide was antagonised when combined with picrotoxin but not by the addition of a specific GABA_B antagonist CGP 35348. The effect of chlordiazepoxide on memory retention seems to be mediated by action at the GABA_A-benzodiazepine receptor complex. (?) Baclofen, the active isomer of the GABA_B agonist enhanced memory in ICRC mice and this effect was antagonised by CGP 35348 at a dose of 10 mg/kg. The inactive isomer of baclofen, (+)-baclofen did not produce any effect. This indicates that GABA_B receptors contribute to the effects of (?)-baclofen on memory.     

The role of GABAB receptors in mediating the stimulatory effects of ethanol in mice

Recently, the GABA_B receptor antagonist phaclofen has been shown to attenuate the stimulation of locomotor activity induced by ethanol (Allan and Harris 1989). In the present study, the effects of a range of recently developed GABA_B receptor antagonists (phaclofen, 2-hydroxysaclofen, beta-phenyl-beta-alanine, CGP 35348) and the GABA_B receptor agonist baclofen, were studied for their ability to block the locomotor stimulation induced by a low dose of ethanol administered IP to mice (1.75 g/kg). Results showed that phaclofen, 2-hydroxysaclofen, BPBA and baclofen all dose-dependently decreased ethanol-induced locomotor activity, and, of these, baclofen and BPBA did so at doses which did not attenuate locomotor activity when administered alone. CGP 35348 had no effect on the activity produced by ethanol. The action of baclofen on ethanol-induced activity appeared to be GABA_B receptor mediated, as the effects were stereospecific and were reversed by the antagonist, CGP 35348. However phaclofen, 2-hydroxysaclofen and BPBA failed to reverse the effects of baclofen. These results suggest that the GABA_B receptor may modulate locomotor stimulation induced by low doses of ethanol, and furthermore, that agonist, rather than antagonist activity at the GABA_B receptor is responsible for this reduction. The GABA_B receptor subtype responsible for modulating the effects of ethanol may have properties different from those GABA_B receptors characterised to date.     

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     

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     

Do GABAB receptors have a role in causing behavioural hyperexcitability,both during ethanol withdrawal and in naive mice?

The effects of the GABA_B agonist baclofen and the GABA_B antagonist CGP35348 were examined on the behavioural hyperexcitability which is seen on cessation of chronic ethanol treatment. When baclofen was given to mice of the TO strain after withdrawal from ethanol inhalation, there was evidence of increased hyperexcitability with one dose, 2.5 mg/kg, but no significant change was seen with other doses, 1.25 and 10 mg/kg. When given after withdrawal from a liquid diet containing ethanol, baclofen, 10 mg/kg, produced a large, but short lasting, increase in the ratings of hyperexcitability during the withdrawal period. This effect was significantly decreased when the antagonist CGP35348, 300 mg/kg, was given with baclofen 10 mg/kg. When the antagonist was given alone at 300 mg/kg it significantly decreased the hyperexcitability during ethanol withdrawal. Increases in the ratings of hyperexcitability were seen when baclofen was given to control mice, which had not received ethanol, and these effects were significant, so the effects during ethanol withdrawal were not confined to that syndrome. CGP35348 decreased the behavioural ratings in control animals, and blocked the effects of baclofen 10 mg/kg. When the effects of the compounds on spontaneous locomotor activity in control mice were measured, this parameter was decreased both by baclofen and by CGP35348, at does which were effective in altering the handling-induced behaviour.     

Baclofen and antidepressant – induced antinociception in formalin test: possible GABAB mechanism involvement

In this study, the influences of GABA_B agents on antidepressant-induced antinociception in the mouse formalin test have been investigated. The GABA_B receptor agonist baclofen (2.5, 5 and 10 mg/kg) induced a dose-dependent antinociception in the second phase of the formalin test. This response was inhibited by the GABA_B receptor antagonist, CGP35348 [P-(3-aminopropyl)-p-diethoxymethyl-phosphinic acid], in a dose-dependent manner. The antagonist by itself also induced antinociception. Single administration of the antidepressants citalopram (10, 20, 40 and 80 mg/kg), desipramine (20, 40, 80 mg/kg) or imipramine (10, 20 and 40 mg/kg) also induced antinociception in both phases of the formalin test. CGP35348 (100 and 200 mg/kg) pretreatment reduced the response induced by the tricyclic antidepressants. A combination of baclofen with the tricyclic antidepressant did not potentiate antinociception induced by antidepressants, but a decrease in the response induced by higher dose of baclofen was shown. It is concluded that GABA mechanism(s) may modulate the antidepressant-induced antinociception. Received: 3 August 1998/Final version: 28 September 1998     

Muscarinic M1 receptors stimulated by intracerebroventricular administration of McN-A-343 reduces the nerve injury-induced mechanical hypersensitivity via GABAB receptors rather than GABAA receptors in mice

Cholinergic neurons play an important role in the higher functions of the brain, such as the memory, cognition, and nociception. However, the exact mechanism behind how the stimulation of all the muscarinic M₁ receptors in the entire brain results in the alleviation of partial sciatic nerve ligation (PSNL)-induced mechanical hypersensitivity has not been investigated. Thus, we examined which subtype of GABA receptor was involved in the alleviation of PSNL-induce mechanical hypersensitivity produced by an intracerebroventricular administration of a muscarinic M₁ receptor agonist, McN-A-343. Administering a GABA_A receptor antagonist, bicuculline, resulted in no changes to the McN-A-343-induced anti-hypersensitivity in PSNL mice whereas a GABA_B receptor antagonist, CGP35348, dose-dependently inhibited the anti-hypersensitivity. Furthermore, CGP35348 increased mechanical hypersensitivity in naïve mice, and the hypersensitivity was blocked by NMDA receptor antagonists, MK-801 and D-AP5. Additionally, muscarinic M₁ receptors colocalized with GABA_B1 receptors and an NMDA receptor subunit, GluN2A, in a large region of the brain. Consequently, these results suggest that the activation of muscarinic M₁ receptors in the entire brain reduces nerve injury-induced mechanical hypersensitivity via the GABA_B receptors, and the activation of the GABA_B receptors regulates glutamatergic transmission via NMDA receptors.     

The GABAB antagonist,CGP 35348, antagonizes the effects of baclofen, γ-butyrolactone and HA 966 on rat striatal dopamine synthesis

Summary The effects of the new GABA_B antagonist, CGP 35348 (3-aminopropane-diethoxymethylphosphinic acid), on rat striatal dopamine synthesis and the increases thereof, caused by (–)-baclofen, -butyrolactone (GBL), and HA 966 (3-amino-l-hydroxypyrrolid-2-one), were investigated. CGP 35348 did not alter dopamine synthesis on its own up to the highest dose tested (500 mg/kg i.p.). However, it antagonized the increase elicited by 50 mg/kg s. c. (–)-baclofen at doses above 100 mg/kg i. p.; at 500 mg/kg i. p. this antagonism disappeared within about 6 h of interval between the administration of the compound and (–)-baclofen. CGP 35348 also clearly and significantly attenuated the effects of graded doses of GBL and HA 966 at 500 mg/kg i. p., but was unable to alter the responses elicited by 0.3 mg/kg i. p. haloperidol or 10 mg/kg i. p. tetrabenazine. This indicates that the compound did not generally attenuate increases of dopamine synthesis. It is likely that its GABA_B antagonistic properties are responsible for the attenuation of the effect of (–)-baclofen, and our results suggest that this compound is useful for the characterization of the role of GABAB receptors in vivo, e.g. in behaviour. On the other hand, they also suggest the possibility that GBL and HA 966 elicit their effects on dopamine synthesis by means of an interaction with GABA_B receptors; a weak in vitro interaction with the latter in radioligand binding experiments has been found for GBL, but not for HA 966.     

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     

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.     

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     

GABAA and GABAB receptor-mediated effects in guinea-pig ileum

1 The effects of γ-aminobutyric acid (GABA) and related substances were examined in guinea-pig ileum longitudinal muscle.

2 GABA at doses ranging from 10^-7 M to 3 × 10^-6 M elicited a relaxation while at higher doses (3 × 10^-6 M — 10^-4 M), as previously described, it caused a contraction followed by relaxation.

3 GABA-induced relaxation was bicuculline-insensitive, was mimicked by (-)-baclofen but not by homotaurine and muscimol. The effect of baclofen was stereospecific. GABA- and (-)-baclofen-induced relaxations were dose-dependent and their ED₅₀ values were similar. A specific cross-desensitization occurred between GABA and (-)-baclofen.

4 The bicuculline-insensitive relaxation induced by GABA and (-)-baclofen was prevented by tetrodotoxin and hyoscine but not by phentolamine plus propranolol, naloxone or theophylline.

5 In preparations in which the muscle tone was raised by histamine or prostaglandin F_2α, GABA and (-)-baclofen induced relaxation to the same extent as before increasing the tone. If the tone was raised by DMPP, a greater bicuculline-insensitive relaxation occurred.

6 Contraction caused by GABA was bicuculline-sensitive and was mimicked by homotaurine and muscimol. Contraction was dose-dependent and muscimol was about three times more potent than GABA or homotaurine. A specific cross-desensitization occurred between the contractile effects of GABA and those of homotaurine or muscimol.

7 Bicuculline competitively antagonized the contractile effects of GABA, homotaurine and muscimol and gave closely similar pA₂ values. The slope of the Schild plot for the above drugs was near 1, confirming the competitive nature of the antagonism.

8 The bicuculline-sensitive contraction induced by GABA, homotaurine and muscimol was abolished by tetrodotoxin and was non-competitively antagonized by hyoscine, while it was unaffected by hexamethonium, mepyramine and methysergide.

9 It is concluded that two receptors mediate the GABA effects in guinea-pig ileum: a bicuculline-sensitive GABA_A receptor, which elicits contraction through an excitatory action on cholinergic post-ganglionic neurones; and a bicuculline-insensitive GABA_B receptor which causes relaxation through an inhibitory presynaptic action on cholinergic post-ganglionic neurones. We confirm that GABA, homotaurine and muscimol are GABA_A agonists, while GABA and (-)-baclofen are GABA_B agonists.

     

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     

Effects of selective h5-HT1B (SB-216641) and h5-HT1D (BRL-15572) receptor ligands on guinea-pig and human 5-HT auto- and heteroreceptors

Human cerebral cortical slices and synaptosomes, guinea-pig cerebral cortical slices and human right atrial appendages were used to study the effects of SB-216641, a preferential h5-HT_1B receptor ligand, and of BRL-15572, a preferential h5-HT_1D receptor ligand, on the presynaptic h5-HT_1B and h5-HT_1B-like autoreceptors in the human and guinea-pig brain preparations, respectively, and on the presynaptic h5-HT_1D heteroreceptors in the human atrium. The brain preparations, preincubated with [³H]serotonin ([³H]5-HT), and the segments of atrial appendages, preincubated with [³H]noradrenaline, were superfused with modified Krebs’ solution and tritium overflow was evoked electrically (human and guinea-pig cerebral cortex slices and human atrial appendages) or by high K⁺ (human cerebral cortex synaptosomes). The electrically evoked tritium overflow from guinea-pig cerebral cortex slices was reduced by the 5-HT receptor agonist 5-carboxamidotryptamine (5-CT). This effect was not modified by BRL-15572 (2μM; concentration 154 times higher than its K_i at h5-HT_1D receptors) but was antagonized by SB-216641 (0.1μM; concentration 100 times higher than its K_i at h5-HT_1B receptors; apparent pA₂ 8.45). SB-216641 (0.1μM) by itself facilitated, whereas BRL-15572 (2μM) did not affect, the evoked overflow. In human cerebral cortex slices SB-216641 (0.1μM) also facilitated, and BRL-15572 (2μM) again failed to affect, the electrically evoked tritium overflow. In human cerebral cortical synaptosomes, 5-CT reduced the K⁺-evoked tritium overflow. This response was unaffected by BRL-15572 (300nM) but antagonized by SB-216641 (15nM; drug concentrations 23 and 15 times higher than their K_i at h5-HT_1D and h5-HT_1B receptors, respectively). Both drugs, given alone, did not modify the K⁺-evoked tritium overflow. In human atrial appendages, the electrically evoked tritium overflow was inhibited by 5-HT in a manner susceptible to antagonism by BRL-15572 (300nM; 23 times K_i at h5-HT_1D receptors) but not by SB-216641 (30nM; 30 times K_i at h5-HT_1B receptors). Both drugs by themselves did not change the electrically evoked tritium overflow. In conclusion, SB-216641 behaves as a preferential antagonist at native human 5-HT_1B receptors and BRL-15572 as a preferential antagonist at native human 5-HT_1D receptors. These compounds are clearly useful tools for the differentiation between human 5-HT_1B and 5-HT_1D receptors in functional studies. Received: 14 March 1997 / Accepted: 18 May 1997     

Modification of cardiovascular responses to spinal GABAB receptor stimulation by cAMP and by KATP channel blockade in anaesthetized rats

1 Intrathecal (i.t.) injection of baclofen (30, 60 and 100 nmol), a GABA_B receptor agonist, produced a dose-dependent decrease in blood pressure (BP) and heart rate (HR). 2 Pretreatment with 5-aminovaleric acid (50 nmol), a GABA_B receptor antagonist, blocked the depressor and bradycardic effects of baclofen (100 nmol). 3 Pretreatment with 8-bromo-cAMP (10 nmol), a cAMP analogue, attenuated the depressor and bradycardic effects of baclofen (100 nmol), but not with 8-bromo-cGMP (10 nmol), a cGMP analogue. 4 In addition, pretreatment with glipizide (20 nmol), an ATP-sensitive K⁺ channel (K_ATP) blocker, attenuated the depressor and bradycardic effects of baclofen (100 nmol). 5 These results suggest that GABA_B receptors in the spinal cord have an inhibitory role in the central cardiovascular regulation and that these depressive and bradycardic actions are modified by cAMP and by K_ATP channel blockade.     

GABA<Subscript>A</Subscript> autoreceptors enhance GABA release from human neocortex: towards a mechanism for high-frequency stimulation (HFS) in brain?

High-frequency stimulation (HFS) in human neocortical slices induces γ-aminobutyric acid (GABA) release via GABA_A receptor (GABA_AR) activation. The mechanism of this effect and the localization of these GABA_ARs were now studied. Fresh human neocortical slices were subjected to HFS (130 Hz) in the presence of veratridine (3 μM). As measured by high-performance liquid chromatography, only GABA but not glutamate outflow was affected by HFS/veratridine stimulation. The evoked GABA overflow was abolished by tetrodotoxin and furosemide, suggesting an involvement of action potentials and plasmalemmal chloride gradients. Double immunolabeling showed that GABA_ARs are localized on soma and dendrites of GABAergic neurons in the human neocortex. Moreover, in support of a terminal localization of GABA_ARs, the K⁺-evoked [³H]-GABA release from synaptosomes was enhanced by the GABA_AR agonist muscimol (antagonized by GABA_AR blockers). We conclude that HFS in human brain neocortex leads to a specific increase of GABA release, which is mediated by facilitatory GABA_A autoreceptors located on soma, dendrites, and axon terminals of GABAergic neurons. A preliminary report of this work was presented at the German Society for Experimental and Clinical Pharmacology and Toxicology, Mainz, Germany, March 13–15, 2007 and at the 37th annual meeting of the Society for Neuroscience, San Diego, CA, November 3–7, 2007.     

Differential effects of GABAB autoreceptor activation on ethanol potentiation of local and lateral paracapsular GABAergic synapses in the rat basolateral amygdala

Many studies have demonstrated that GABAergic inhibition within the basolateral amygdala (BLA) plays an integral role in the regulation of anxiety, an important behavioral component in the etiology of alcoholism. Although ethanol has recently been shown to enhance BLA GABAergic inhibition via two distinct populations of inhibitory cells, local and lateral paracapsular (lpcs) interneurons, little is known about the mechanisms underlying ethanol potentiation of these two inhibitory pathways. Ethanol is known to enhance GABAergic inhibition in many brain regions via a complex array of pre- and postsynaptic mechanisms. In addition, ethanol's presynaptic effects are often subject to GABA_B autoreceptor (GABA_B-R) modulation. Therefore, in this study, we characterized GABA_B-R function and modulation of ethanol actions at local and lpcs GABAergic synapses. At local synapses, we found significant paired-pulse depression (PPD, 250 ms inter-pulse interval) which was abated by SCH-50911 (GABA_B-R antagonist). No significant PPD was detected at lpcs synapses, but SCH-50911 significantly potentiated lpcs-evoked IPSCs. Baclofen (GABA_B-R agonist) had similar depressant effects on local- and lpcs-evoked IPSCs, however baclofen pretreatment only reduced ethanol potentiation at local synapses. Ethanol also significantly enhanced the frequency of spontaneous and miniature IPSCs, and these effects were also sensitive to GABA_B-R modulators. Collectively, these data suggest that stimulus-independent inhibitory responses recorded from BLA principal neurons primarily reflect the activity of local GABAergic interneurons and provide additional evidence that ethanol potentiates local BLA inhibitory synapses primarily via a presynaptic enhancement of GABA release that is tightly regulated by GABA_B-Rs. In contrast, ethanol potentiation of lpcs GABAergic synapses is not sensitive to GABA_B-R activation and does not appear to involve increased presynaptic GABA release.     

Autoreceptor-mediated regulation of GABA release: role of uptake inhibition and effects of novel GABAB antagonists

Summary While the role of GABA_B autoreceptors in the regulation of GABA release in synaptosomes and brain slices is well established, little is known about their role in vivo. Doubts have arisen because there is an apparent discrepancy between the frequencies at which GABA neurons fire and the frequency range within which autoreceptor regulation is observed in vitro. To see whether this apparent mismatch could be due to the use of a GABA uptake inhibitor in the release experiments in slices, we have compared the frequency dependencies of GABA release in the presence and absence of uptake inhibition. Beforehand, the previously incomplete frequency curve in the presence of uptake inhibition was extended at the lower end. To achieve this, stimulation was performed by means of groups of 4 pseudo-one-pulses (POP's) at inter-POP intervals corresponding to frequencies of 0.015625-0.5 Hz. It could be shown that activation of the GABA_B autoreceptor by endogenously released GABA begins at a stimulation frequency as low as 0.0625 Hz. Experiments with the antagonist, CGP 35348, at inter-POP intervals of 1 min, at which the preceding POP has no longer an effect on GABA release during the next one, showed that basal release alone already substantially activated the autoreceptor. The frequency dependence in the absence as compared to the presence of uptake inhibition was shifted towards higher frequencies by a factor of 4. We do not consider this enough to remove our doubts about the in vivo operativity of GABA_B autoreceptors.Further experiments in the presence of uptake inhibition were made to see whether the expectation was met that autoreceptor-mediated regulation of GABA release could be blocked out by sufficiently high concentrations of potent antagonists. As judged from the frequency dependence in the presence of 1 mol/l of the potent compound CGP 55845 as well as from the similarity of the maximal increases of GABA release caused at 0.125 and 2 Hz by various other potent, novel GABA_B antagonists, this was not the case. Possible explanations are the occurrence of an agonist and an antagonist state of the autoreceptor prevailing at low GABA concentrations or, less likely, the occurrence of two autoreceptor subtypes with different relative sensitivities towards GABA and baclofen on the one hand and towards aminophosphonous acid antagonists on the other.Finally, it was shown that also in the absence of uptake inhibition, regulation of GABA release was mediated entirely by GABA_B autoreceptors. Both muscimol and bicuculline at 1 and 10 mol/1 were without effect. Correspondence to P. Waldmeier at the above address