γ-Hydroxybutyrate inhibits excitatory postsynaptic potentials in rat hippocampal slices

γ-Hydroxybutyrate (GHB) has been shown to mimic different central actions of ethanol, to suppress alcohol withdrawal syndrome, and to reduce alcohol consumption both in rats and in humans. The aim of the present study was to determine if GHB shared with alcohol the ability to inhibit glutamate action at both NMDA and AMPA/kainate receptors. The NMDA or the AMPA/kainate receptors-mediated postsynaptic potentials were evoked in CA1 pyramidal neurons by stimulation of Schaffer-collateral commissural fibers in the presence of CGP 35348, bicuculline to block the GABA_B and GABA_A receptors, and 10 μM 6,7-dinitroquinoxaline-2,3-dione (DNQX) or 30 μM -2-amino-5-phosphonovalerate (d-APV) to block AMPA/kainate or NMDA receptors, respectively. GHB (600 μM) produced a depression of both NMDA and AMPA/kainate receptors-mediated excitatory postsynaptic potentials with recovery on washout. The GHB receptors antagonist, NCS-382, at the concentration of 500 μM had no effect per se on these responses but prevented the depressant effect of GHB (600 μM) on the NMDA and AMPA/kainate-mediated responses. In the paired-pulse experiments, GHB (600 μM) depressed the amplitude of the first and the second evoked AMPA/kainate excitatory postsynaptic potentials, and significantly increased the paired-pulse facilitation (PPF). These results suggest that GHB inhibits excitatory synaptic transmission at Schaffer-collateral commissural–pyramidal neurons synapses by decreasing the probability of release of glutamate.     

Seeking a mechanism of action for the novel anticonvulsant lacosamide

Lacosamide (LCM) is anticonvulsant in animal models and is in phase 3 assessment for epilepsy and neuropathic pain. Here we seek to identify cellular actions for the new drug and effects on recognised target sites for anticonvulsant drugs. Radioligand binding and electrophysiology were used to study the effects of LCM at well-established mammalian targets for clinical anticonvulsants. 10 μM LCM did not bind with high affinity to a plethora of rodent, guinea pig or human receptor sites including: AMPA; Kainate; NMDA (glycine/PCP/MK801); GABA_A (muscimol/benzodiazepine); GABA_B; adenosine A_1,2,3; ₁, ₂; β₁, β₂; M_1,2,3,4,5; H_1,2,3; CB_1,2; D_1,2,3,4,5; 5HT_{1A,1B,2A,2C,3,5A,6,7} and K_ATP. Weak displacement (25%) was evident at batrachotoxin site 2 on voltage gated Na⁺ channels. LCM did not inhibit neurotransmitter transport mechanisms for norepinephrine, dopamine, 5-HT or GABA, nor did it inhibit GABA transaminase. LCM at 100 μM produced a significant reduction in the incidence of excitatory postsynaptic currents (EPSC's) and inhibitory postsynaptic currents (IPSC's) in cultured cortical cells and blocked spontaneous action potentials (EC₅₀ 61 μM). LCM did not alter resting membrane potential or passive membrane properties following application of voltage ramps between −70 to +20 mV. The voltage-gated sodium channel (VGSC) blocker phenytoin potently blocked sustained repetitive firing (SRF) but, in contrast, 100 μM LCM failed to block SRF. No effect was observed on voltage-clamped Ca²⁺ channels (T-, L-, N- or P-type). Delayed-rectifier or A-type potassium currents were not modulated by LCM (100 μM). LCM did not mimic the effects of diazepam as an allosteric modulator of GABA_A receptor currents, nor did it significantly modulate evoked excitatory neurotransmission mediated by NMDA or AMPA receptors (n ≥ 5). Evidently LCM perturbs excitability in primary cortical cultures but does not appear to do so via a high-affinity interaction with an acknowledged recognition site on a target for existing antiepileptic drugs.     

Drugs which stimulate or facilitate central GABAergic transmission interact synergistically with delta-9-tetrahydrocannabinol to produce marked catalepsy in mice

In experiments in which mice were placed with their forelegs over a 4 cm high horizontal bar, pretreatment with delta-9-tetrahydrocannabinol (THC; 10 mg/kg i.p.) significantly delayed descent from the bar. This response to THC was markedly enhanced by doses of amino-oxyacetic acid, flurazepam, cis(Z)-flupentixol, muscimol, (−)-baclofen and NO-328 having little or no effect when given alone. No synergism was detected between THC and (+)-baclofen or trans(E)-flupentixol. The interactions between THC and flurazepam, amino-oxyacetic acid and NO-328 were attenuated by (+)-bicuculline and by homotaurine, but not by strychnine. The interaction between THC and (−)-baclofen was prevented by homotaurine but not by (+)-bicuculline whereas only (+)-bicuculline reduced the interactions of THC with muscimol and cis(Z)-flupentixol. Flumazenil prevented the interaction between THC and flurazepam but not that between THC and NO-328. The results suggest that the synergistic interactions observed in this study depended on the activation of GABA_A and/or GABA_B receptors, probably located in extrapyramidal GABAergic pathways.     

A patch clamp study of the effects of ciprofloxacin and biphenyl acetic acid on rat hippocampal neurone GABAA and lonotropic glutamate receptors

The neurotoxic effects of 4-quinolones alone and in combination with certain non-steroidal anti-inflammatory drugs (NSAIDs) may be related to an interaction at GABA_A and/or ionotropic glutamate receptors. In the present study, the effects of the fluoroquinolone, ciprofloxacin, alone and in combination with the NSAID, biphenyl acetic acid (BPAA), were examined on GABA_A-, NMDA-, AMPA-, and kainate-evoked current responses recorded from cultured rat hippocampal neurones, using the whole cell patch clamp technique. GABA-evoked currents were reversibly inhibited by bicuculline (3 μM) and ciprofloxacin (100 μM) to 11 ± 5 and 38 ± 7% of control, respectively. BPAA (100 μM) had little affect on the GABA current (the response was 82 ± 4% of control) but enhanced the inhibitory potency of ciprofloxacin by approx. 3000-fold. The antagonist effects of ciprofloxacin (30 μM) and ciprofloxacin (0.03 μM) together with BPAA (100 μ M) on the GABA-evoked current were not voltage-dependent. Whole cell currents evoked by NMDA, AMPA or kainate were little influenced by ciprofloxacin (100 μM), BPAA (100 μM), or ciprofloxacin plus BPAA (both at 100 μM); the responses being 90% of control in all cases. These data suggest that the proconvulsant effects of quinolones when combined with BPAA may be related to antagonism of central GABA_A receptors but not to an interaction at ionotropic glutamate receptors.     

Direct Activation of GABAA Receptors by Loreclezole, an Anticonvulsant Drug with Selectivity for the β-Subunit

Loreclezole, an anticonvulsant and antiepileptic compound, potentiates γ-aminobutyric acid (GABA) type A receptor function, by interacting with a specific allosteric modulatory site on receptor β-subunits. A similar selectivity for GABA_A receptor β-subunits is apparent for the direct activation of receptor-operated Cl⁻ channels, by the general anesthetics propofol and pentobarbital. The ability of loreclezole to activate GABA_A receptors directly has now been compared, biochemically and electrophysiologically, with that of propofol. In well-washed rat cortical membranes (devoid of endogenous GABA), loreclezole and propofol increased t-[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding by up to 28% (at 5 μM) and 80% (at 10 μM), respectively. Higher concentrations (50–100 μM) of both compounds inhibited [³⁵S]TBPS binding with great efficacy, an effect mimicked by GABA. In contrast, the benzodiazepine diazepam increased [³⁵S]TBPS binding, but failed to inhibit this parameter, even at high concentrations. At concentrations of 50–100 μM, loreclezole induced inward Cl⁻ currents in the absence of GABA, in Xenopus oocytes expressing human recombinant GABA_A receptors, comprised of ₁-, β₂- and γ_2S-subunits. At 100 μM, the current evoked by loreclezole was 26% of that induced by 5 μM GABA. The current evoked by 100 μM propofol was 98% of that induced by 5 μM GABA. Currents induced by loreclezole, like those evoked by propofol, were potentiated by diazepam in a flumazenil-sensitive manner and blocked by either bicuculline or picrotoxin. These data suggest that loreclezole shares, with propofol, an agonistic action at GABA_A receptors containing the β₂-subunit and that the different efficacies of the two compounds in this regard, may underlie the difference in their pharmacological profiles. The failure of loreclezole to activate GABA_A receptors containing the β₁-subunit may be responsible for its lack of hypnotic effect. © 1997 Published by Elsevier Science Ltd. All rights reserved.     

gamma-Hydroxybutyric acid and baclofen decrease extracellular acetylcholine levels in the hippocampus via GABA(B) receptors.

The effect of γ-hydroxybutyric acid (GHB) and baclofen, a GABA_B receptor agonist, on extracellular hippocampal acetylcholine levels was studied in freely moving rats by microdialysis. GHB (200 and 500 mg/kg, i.p.) reduced in a dose-dependent manner, extracellular hippocampal acetylcholine concentrations and this effect was prevented by the GABA_B receptor antagonist (2S)(+)-5,5-Dimethyl-2-morpholineacetic acid (SCH 50911), at the dose of 20 mg/kg (i.p.), while the putative GHB receptor antagonist 6,7,8,9-Tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylideneacetic acid (NCS 382) was ineffective. Similar to GHB, the GABA_B agonist baclofen (10 and 20 mg/kg, i.p.) produced a dose-related reduction in extracellular acetylcholine concentrations which was prevented by SCH 50911. These findings indicate that GHB-induced reduction of hippocampal acetylcholine release is mediated by GABA_B receptors and support a possible involvement of hippocampal GABA_B receptors in the control of cognitive processes and in the claimed amnesic effect of GHB intoxication.     

Heterogeneity of functional GABA(A) receptors in rat dentate gyrus neurons revealed by a change in response to drugs during the whole-cell current time-course

We examined if the drug sensitivity of GABA_A receptors in dentate gyrus granule neurons changed during the whole-cell current time-course. Effects of drugs on currents evoked immediately (the peak current) upon drug application and currents remaining about two seconds later (semi-plateau current) were compared. The apparent affinity for GABA (EC₅₀) of the peak and the semi-plateau current were 14 and 4 μM, respectively. Bicuculline inhibited 50% of the peak and the semi-plateau current (IC₅₀) at 7 and 36 μM, respectively, while 100 μM was required for full inhibition of the 100 μM GABA-evoked current. Zinc inhibited about 50% of the peak current with an IC₅₀ value of 94 μM whereas biphasic, but complete inhibition of the semi-plateau current was recorded with IC₅₀ values of 3 and 558 μM. The decay phase of the 100 μM GABA-evoked current was fitted by a fast (τ₁, 100–300 ms) and a slow (τ₂, 1–2 s) time-constants in all cells. The relative current amplitude associated with the fast (A1) and the slow (A2) component varied. The A1 current amplitude appeared more sensitive to bicuculline than the A2 current while the opposite was true for zinc. The results are consistent with heterogenous population of functional GABA_A receptors in the dentate gyrus granule neurons.     

Modulation of GABAA and glycine receptors by chlormethiazole.

The influence of chlormethiazole, on currents evoked by γ-aminobutyric acid (GABA) and glycine, was investigated under voltage-clamp conditions, in bovine chromaffin cells and murine spinal neurones, respectively. Chlormethiazole (30 and 100 μM) dose dependently potentiated currents activated by either inhibitory neurotransmitter. The potentiation of the GABA-evoked response occurred without altering the reversal potential and was not influenced by the benzodiazepine receptor antagonist Ro 15-1788 (300 nM). GABA-gated channels, recorded from outside-out membrane patches, showed increased probability of being in the conducting state in the presence of chlormethiazole. High concentrations of chlormethiazole (3 mM) activated bicuculline (1 μM)-sensitive whole-cell currents with a reversal potential similar to the chloride equilibrium potential. Chlormethiazole potentiates GABA- and glycine-activated currents and at higher doses, directly activates the GABA_A receptor.     

Selective inhibition of gamma-aminobutyric acid type A receptors in human IMR-32 cells by low concentrations of toluene

Effects of the neurotoxic organic solvent toluene on human neuronal nicotinic acetylcholine (nACh) and γ-aminobutyric acid type A (GABA_A) neurotransmitter receptors were investigated in whole-cell voltage-clamped IMR-32 neuroblastoma cells. Ion currents evoked by near maximum effective concentrations of 1 mM acetylcholine (ACh) and 1 mM γ-aminobutyric acid (GABA) are inhibited by toluene in a concentration-dependent way. Concentration–effect curves of toluene yield IC₅₀ values of 276±26 and 39±6 μM and slope factors of 1.4±0.2 and 0.8±0.1 for inhibition of the ACh- and GABA-induced ion currents, respectively. The results demonstrate the selective inhibition of human GABA_A receptors by toluene at concentrations comparable with brain concentrations associated with occupational exposure.     

The neuroprotective agent riluzole inhibits release of glutamate and aspartate from slices of hippocampal area CA1

Riluzole is believed to exert its anticonvulsant and neuroprotective actions by reducing glutamate release. This study demonstrated that 10–30 μM riluzole reduces the K⁺-evoked release of glutamate and aspartate from slices of hippocampal area CA1. Only higher concentrations reduced γ-aminobutyrate (GABA) release. These actions of riluzole were not occluded by tetrodotoxin. Riluzole did not diminish the ability of glutamate analogues to depolarize CA1 pyramidal cells, as determined from grease-gap recordings. Therefore the anticonvulsant and neuroprotective actions of riluzole in the hippocampus may be at least partly explained by its ability to inhibit glutamate/aspartate release from synaptic terminals.     

Methionine 286 in transmembrane domain 3 of the GABAA receptor beta subunit controls a binding cavity for propofol and other alkylphenol general anesthetics.

γ-Aminobutyric acid type A (GABA_A) receptors are an important target for general anesthetics in the central nervous system. Site-directed mutagenesis techniques have identified amino acid residues that are important for the positive modulation of GABA_A receptors by general anesthetics. In the present study, we investigate the role of an amino acid residue in transmembrane (TM) domain 3 of the GABA_A receptor β₂ subunit for modulation by the general anesthetic 2,6-diisopropylphenol (propofol). Mutation of methionine 286 to tryptophan (M286W) in the β₂ subunit abolished potentiation of GABA responses by propofol but did not affect direct receptor activation by propofol in the absence of GABA. In contrast, substitution of methionine 286 by alanine, cysteine, glutamate, lysine, phenylalanine, serine, or tyrosine was permissive for potentiation of GABA responses and direct activation by propofol. Using propofol analogs of varying molecular size, we show that the β₂(M286W) mutation resulted in a decrease in the ‘cut-off’ volume for propofol analog molecules to enhance GABA responses at GABA_{A 1}β₂γ_2s receptors. This suggests that mutation of M286 in the GABA_A β₂ subunit alters the dimensions of a ‘binding pocket’ for propofol and related alkylphenol general anesthetics.     

GABAA receptor modulation of 5-HT neuronal firing in the median raphe nucleus: Implications for the action of anxiolytics

5-HT neurones in the median raphe nucleus (MRN) are involved in anxiety and the sleep/wake cycle. Here, using in vitro electrophysiology, we examined if the firing of MRN 5-HT neurones is regulated by GABA_A receptors. The GABA_A receptor agonists THIP and muscimol caused concentration dependent inhibition of MRN 5-HT neurones. The GABA_A receptor antagonist bicuculline blocked the responses to THIP and muscimol. Bicuculline alone increased the basal firing activity. Responses to THIP were enhanced by the Z hypnotic zolpidem at concentrations selective for the ₂/₃ subunits of the GABA_A receptor (0.2 and 1 μM) but not at a concentration selective for the ₁ subunit (0.02 μM). Consistent with these functional data, 5-HT neurones have been shown to express the ₃ (but not ₂) subunit. The anxiolytic effects of GABA_A receptor modulators are reportedly mediated by ₃-containing receptors. Hence the MRN 5-HT system may be a target for anxiolytic drugs.     

Mechanisms for resin acid effects on membrane currents and GABA(A) receptors in mammalian CNS

Resin acids from bleached wood pulp are toxic to fish. 12,14-Dichlorodehydroabietic acid (12,14-Cl₂DHA) raises cytoplasmic Ca²⁺ in synaptosomes and blocks neural GABA_A receptors; however, the underlying mechanism remains unclear in these earlier rodent studies. 12,14-Cl₂DHA (50 μM) almost completely blocked native GABA_A currents (rat cortical cultures) but had no significant effect on picrotoxin-sensitive recombinant human receptors in oocytes (1, β2 and γ2L: the most prevalent isoforms in mammalian brain). In oocytes, 12,14-Cl₂DHA failed to produce a calcium-activated chloride current, in contrast to the calcium ionophore ionomycin (10 μM). However, in cultured cortical pyramidal cells, both ionomycin and 12,14-Cl₂DHA produced chloride-selective currents of similar magnitude (presumably secondary to Ca²⁺ release). 12,14-Cl₂DHA was unable to stimulate phosphate labelling of [³H]-inositol in mouse synaptosomes, indicating that the study compound does not cause Ca²⁺ release via an IP₃ mechanism. Calcium pump ATPase inhibition also seems unlikely since thapsigargin did not elevate free calcium in synaptosomes. 12,14-Cl₂DHA clearly blocks GABA_A currents indirectly: we infer that its toxicity may be secondary to the elevations in cytoplasmic Ca²⁺ via an unidentified recognition site (or receptor) found in neuronal cells.     

Functional evidence for GABA as modulator of the contractility of the longitudinal muscle in mouse duodenum: role of GABA(A) and GABA(C) receptors

We investigated, in vitro, the effects of γ-aminobutyric acid (GABA) on the spontaneous mechanical activity of the longitudinal smooth muscle in mouse duodenum. GABA induced an excitatory effect, consisting in an increase in the basal tone, which was antagonized by the GABA_A-receptor antagonist, bicuculline, potentiated by (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA), a GABA_C-receptor antagonist and it was not affected by phaclofen, a GABA_B-receptor antagonist. Muscimol, GABA_A receptor agonist, induced a contractile effect markedly reduced by bicuculline, tetrodotoxin (TTX), hexamethonium and atropine. Cis-4-aminocrotonic acid (CACA), a specific GABA_C receptor agonist, induced an inhibitory effect, consisting in the reduction of the amplitude of the spontaneous contractions and muscular relaxation, which was antagonised by TPMPA, GABA_C-receptor antagonist, TTX or N^ω-nitro-l-arginine methyl ester (L-NAME), nitric oxide (NO) synthase inhibitor, but not affected by hexamethonium. In conclusion, our study indicates that GABA is a modulator of mechanical activity of longitudinal muscle in mouse duodenum. GABA may act through neuronal presynaptic receptors, namely GABA_A receptors, leading to the release of ACh from excitatory cholinergic neurons, and GABA_C receptors increasing the release of NO from non-adrenergic, non-cholinergic inhibitory neurons.     

Potentiation of inhibitory amino acid receptors-mediated responses by lanthanum in rat sacral dorsal commissural neurons

Lanthanum is one of rare earth cations with extremely active chemical property and has been reported to influence neuronal transmitter systems. To date, little attention has been directed towards the sacral dorsal commissural nucleus (SDCN), which serves as a relay of sensory information from the pelvic viscera in the spinal cord. Therefore, the effect of lanthanum on the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and glycine (Gly) responses in neurons acutely dissociated from the rat SDCN was investigated using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. At a holding potential of − 40 mV, La³⁺ reversibly potentiated GABA (3 μM)-activated currents (I_GABA) in a concentration-dependent manner over the concentration range of 10 μM to 30 mM, with the EC₅₀ value of 67.3 ± 16.4 μM. Similarly, La³⁺ reversibly potentiated glycine (10 μM)-activated currents (I_Gly) in a concentration-dependent manner over the concentration range of 1 μM to 1 mM, with the EC₅₀ value of 52.3 ± 10.9 μM. The effects of La³⁺ on I_GABA and I_Gly were voltage-independent. Moreover, both of the potentiations were not use-dependent and were overcome by increasing the concentration of agonist. Our results indicate that La³⁺ potentiates the inhibitory amino acid receptors-mediated responses in SDCN, which may reduce the transmission of the pelvic visceral information. The information provided by this work may help to elucidate the mechanisms and effects of lanthanum on brain functions.     

TRPV1 antagonist, SB-366791, inhibits glutamatergic synaptic transmission in rat spinal dorsal horn following peripheral inflammation

The anti-hyperalgesic effects of TRPV1 receptor antagonists are well documented in animal models of pain, however, the precise site of their action is not known. Here we have examined the effects of the selective TRPV1 antagonist SB-366791 on glutamatergic synaptic transmission in substantia gelatinosa using spinal cord slices from either control rats or animals that had undergone a peripheral inflammation induced by intraplantar injection of Freund's complete adjuvant (FCA). In control animals, SB-366791 (30 μM) had no effect on spontaneous excitatory post-synaptic currents (sEPSC) or evoked EPSCs. In slices from FCA-inflamed animals, SB-366791 decreased sEPSC frequency to 66 ± 8% of control in 5/10 neurones, and decreased miniature glutamatergic EPSCs (mEPSC) frequency to 63 ± 4% of control, in 6/7 neurones; with no significant effect on sEPSC or mEPSC amplitude. Dorsal root evoked EPSCs at C-fibre intensity were reduced to 72 ± 6% of control by SB-366791 (30 μM) in 3/4 neurones from FCA-treated animals. In conclusion, SB-366791 inhibited glutamatergic transmission in a subset of neurones via a pre-synaptic mechanism following peripheral inflammation. We hypothesise that during peripheral inflammation spinal TRPV1 becomes tonically active, promoting the synaptic release of glutamate. These results provide evidence for a mechanism by which TRPV1 contributes to inflammatory pain and provides a basis for the understanding of the efficacy of TRPV1 antagonists.     

Local modulation of the 5-HT release in the dorsal striatum of the rat: an in vivo microdialysis study

Using in vivo microdialysis in freely moving rats, we examined the involvement of major striatal transmitters on the local modulation of the 5-HT release. Tetrodotoxin reduced the striatal 5-HT output to 15–20% of baseline. The selective 5-HT_1B receptor agonist CP 93129 (50 μM) reduced (50%) and the 5-HT_2A/2C receptor agonist DOI (1–100 μM) increased (220%) the 5-HT output. Neither GABA nor baclofen (100 nM–100 μM) altered the 5-HT output. The glutamate reuptake inhibitor -trans-PDC (1–4 mM) raised 5-HT to 280% of baseline. This effect was not antagonized by the NMDA receptor antagonist MK-801 (0.5 mg/kg i.p.). Local MK-801 (10–100 μM) did not significantly alter the 5-HT output. Finally, neither carbachol (10–100 μM) nor quipirole (10 μM–1 mM) affected 5-HT. These data suggest that the striatal 5-HT release is influenced by local serotonergic and glutamatergic (but not GABAergic) inputs.     

Stimulus-evoked glutamate release is diminished by acute exposure to uranium in vitro

Uranium is used in civilian applications, in the manufacture of nuclear fuel, and by the military for munitions and armament, but little information is available on its neurotoxicity. Neurological dysfunctions have been observed after chronic exposure in both animals and humans, but the actions of acute exposure on amino acid neurotransmission have not been investigated. The following study was performed to examine the effects of uranyl ion (UO₂^+ 2) on hippocampal glutamatergic and GABAergic function as possible bases for the neurotoxicity and to assess the direct effects on the exocytotic process. Nominal UO₂^+ 2 concentrations were applied to superfused hippocampal synaptosomes to permit estimation of the metal's potency on endogenous transmitter release in the presence and absence of Ca^+ 2. K⁺-evoked glutamate release was diminished in the range of 10 nM–316 μM UO₂^+ 2, resulting in an IC₅₀ of 1.92 μM. In contrast, the potency of UO₂^+ 2 to decrease stimulated GABA release was reduced, producing an IC₅₀ ≈ 2.6 mM. In the absence of Ca^+ 2 in the superfusion medium there was no systematic change in the magnitude of glutamate or GABA release, suggesting that UO₂^+ 2 does not possess Ca^+ 2-mimetic properties. The inhibitory potency of UO₂^+ 2 on glutamate release is similar to the potencies of other multivalent metal ions, suggesting by inference an action exerted on voltage-sensitive Ca^+ 2 channels. The bases for the reduced potency to inhibit GABA release is not known, but differential sensitivity to other heavy metals has been reported for glutamate and GABA neurotransmission. These findings indicate a profile of neurotoxicity not unlike that of other metal ions, and indicate the importance of extending subsequent studies to chronic exposure models.     

Gabapentin is not a GABAB receptor agonist.

Recent experiments have demonstrated that formation of functional type B gamma-aminobutyric acid (GABA_B) receptors requires co-expression of two receptor subunits, GABA_B1 and GABA_B2. Despite the identification of these subunits and a number of associated splice variants, there has been little convincing evidence of pharmacological diversity between GABA_B receptors comprising different subunit combinations. However, Ng et al. [Mol. Pharmacol., 59 (2000) 144] have recently suggested a novel and important pharmacological difference between GABA_B receptor heterodimers expressing the GABA_B1a and GABA_B1b receptor subunits. This study suggested that the antiepileptic GABA analogue gabapentin (Neurontin) is an agonist at GABA_B receptors expressing the GABA_B1a but not the GABA_B1b receptor subunit. The importance of this finding with respect to identifying novel GABA_B receptor subunit specific agonists prompted us to repeat these experiments in our own [³⁵S]−GTPγS binding and second messenger assay systems. Here we report that gabapentin was completely inactive at recombinant GABA_B heterodimers expressing either GABA_B1a or GABA_B1b receptor subunits in combination with GABA_B2 receptor subunits. In addition, in both CA1 and CA3 pyramidal neurones from rodent hippocampal slices we were unable to demonstrate any agonist-like effects of gabapentin at either pre- or post-synaptic GABA_B receptors. In contrast, gabapentin activated a GABA_A receptor mediated chloride conductance. Our data suggest that gabapentin is not a GABA_B-receptor agonist let alone a GABA_B receptor subunit selective agonist.     

Activity of 2,3-benzodiazepines at Native Rat and Recombinant Human Glutamate Receptors In Vitro: Stereospecificity and Selectivity Profiles

The activity and selectivity of the glutamate receptor antagonists belonging to the 2,3-benzodiazepine class of compounds have been examined at recombinant human non-NMDA glutamate receptors expressed in HEK293 cells and on native rat NMDA and non-NMDA receptors in vitro. The racemic 2,3-benzodiazepines GYK152466, LY293606 (GYKI53405) and LY300168 (GYKI53655) inhibited AMPA (10 μM)-mediated responses in recombinant human GluR1 receptors expressed in HEK293 cells with approximate ₅₀ values of 18 μM, 24 μM and 6 μM, respectively and AMPA (10 μM) responses in recombinant human GluR4 expressing HEK293 cells with approximate ₅₀ values of 22 μM, 28 μM and 5 μM, respectively. GYKI 52466, LY293606 and LY300168 were non-competitive antagonists of AMPA receptor-mediated responses in acutely isolated rat cerebellar Purkinje neurons with approximate ₅₀ values of 10 μM, 8 μM and 1.5 μM, respectively. The activity of racemic compounds LY293606 and LY300168 was established to reside in the (−) isomer of each compound. At a concentration of 100 μM, GYKI52466, LY293606 and LY300168 produced <30% inhibition of kainate-activated currents evoked in HEK293 cells expressing either human homomeric GluR5 or GluR6 receptors or heteromeric GluR6+KA2 kainate receptors. The activity of the 2,3-benzodiazepines at 100 μM was weak at kainate receptors, but was stereoselective. Similar levels of inhibition were observed for kainate-induced currents in dorsal root ganglion neurons. Intact tissue preparations were also used to examine the stereoselective actions of the 2,3-benzodiazepines. In the cortical wedge preparation, the active isomer of LY300168, LY303070, produced a non-competitive antagonism of AMPA-evoked depolarizations with smaller changes in depolarizations induced by kainate and no effect on NMDA-dependent depolarizations. LY303070 was also effective in preventing 30 μM AMPA-induced depolarizations in isolated spinal cord dorsal roots with an approximate ₅₀ value of 1 μM. Synaptic transmission in the hemisected spinal cord preparation was stereoselectively antagonized by the active isomers of LY300168 and LY293606. In summary, these results indicate that 2,3-benzodiazepines are potent, selective and stereospecific antagonists of the AMPA subtype of the non-NMDA glutamate receptor. © 1997 Elsevier Science Ltd. All rights reserved.