Subunit-containing GABA Research Articles

Alpha2-containing GABA A receptors are involved in mediating stimulant effects of cocaine

α2 subunit-containing GABA A receptors are involved in incentive learning associated with cocaine, and in cocaine addiction. Deletion of α2-containing receptors abolishes cocaine-induced behavioural sensitisation (BS), while selective activation of α2 receptors, achieved using Ro 15-4513's agonist properties in α2(H101R) mice, induced BS. Here, we investigate further the mechanisms underlying Ro 15-4513-induced behavioural sensitisation in α2(H101R) mice. α2(H101R) mice sensitised to Ro 15-4513 (10 mg/kg) showed an enhanced stimulant response to cocaine (10 mg/kg). In contrast, cocaine (10 mg/kg)-sensitised α2(H101R) mice did not show enhanced sensitivity to the stimulant effects of Ro 15-4513 (1, 3 and 10 mg/kg), suggesting that the neural adaptations underlying Ro 15-4513 induced BS are related to, but not identical with those associated with cocaine-induced plasticity. Secondly, we investigated whether α2-containing receptors are involved in mediating the ability of BZs to facilitate cocaine-induced activity. The non-selective (i.e., α1, α2, α3 and α5 subtype) benzodiazepine GABA A receptor agonist midazolam (10 and 30 mg/kg) potentiated cocaine (10 mg/kg) hyperactivity in wildtype mice, but not in α2(H101R) mice, in which α2-containing receptors are insensitive to benzodiazepines. To determine where α2 receptors are localised we compared BZ-insensitive sites between wildtype (α4 and α6) and α2(H101R) (α2, α4 and α6) mice, using quantitative autoradiography to estimate [ 3H]Ro 15-4513 binding in the presence of 10 μM diazepam. α2 receptors were found in projection areas of the mesolimbic dopamine pathway including accumbens, central amygdala, and basolateral amygdala as well as CA1 and CA3 areas of the hippocampus. The involvement of the α2-containing receptor in mediating BZ's potentiating effect on cocaine hyperactivity suggests that the locomotor stimulant effects of BZs and psychostimulants may be mediated by a common neural system, but the lack of cross sensitisation to Ro 15-4513 in cocaine-sensitised α2(H101R) mice, suggests that this form of BS may occur downstream of plastic events underlying cocaine sensitisation.

Cajal–Retzius cells in the mouse neocortex receive two types of pre‐ and postsynaptically distinct GABAergic inputs

Cajal-Retzius (CR) cells are principal cells of layer I in the developing neocortex. They are able to generate action potentials, make synaptic contacts in layer I and receive excitatory GABAergic inputs before birth. Although CR cells participate in neuronal network activity in layer I, the properties of their synaptic inputs are not yet characterized. We recorded miniature (mIPSCs) and evoked (eIPSCs) postsynaptic currents using the whole-cell patch-clamp technique. Most of CR cells displayed two types of mIPSCs, namely those with fast (mIPSC(F)) and slow (mIPSC(S)) rise kinetics. The mIPSC(F) mean amplitude was significantly larger than that of mIPSC(S), while their decay rates were not different. Peak-scaled non-stationary noise analysis revealed that mIPSC(S) and mIPSC(F) differed in their weighted single-channel conductance. In addition, zolpidem (100 nm), a modulator of alpha(1) subunit-containing GABA(A) receptors, selectively affected mIPSC(S) suggesting that different postsynaptic GABA(A) receptors mediate mIPSC(F) and mIPSC(S). eIPSCs also split into two populations with different rise kinetics. Fast eIPSCs (eIPSC(F)) displayed higher paired-pulse ratio (PPR) and lower GABA release probability than slowly rising eIPSCs (eIPSC(S)). As CGP55845, a GABA(B) receptor antagonist, eliminated the observed difference in PPR, the lower release probability at IPSC(F) connections probably reflects a stronger tonic GABA(B) receptor-mediated inhibition of IPSC(F) synapses. At low (0.1 Hz) stimulation frequency both inputs can effectively convert presynaptic action potentials into postsynaptic ones; however, only IPSC(F) connections reliably transfer the presynaptic activity patterns at higher stimulation rates. Thus, CR cells receive two GABAergic inputs, which differ in the quantal amplitude, the probability of GABA release and the frequency dependence of signal transfer.

Pharmacology of the β‐Carboline FG‐7142, a Partial Inverse Agonist at the Benzodiazepine Allosteric Site of the GABAA Receptor: Neurochemical, Neurophysiological, and Behavioral Effects

Given the well-established role of benzodiazepines in treating anxiety disorders, beta-carbolines, spanning a spectrum from full agonists to full inverse agonists at the benzodiazepine allosteric site for the GABA(A) receptor, can provide valuable insight into the neural mechanisms underlying anxiety-related physiology and behavior. FG-7,142 is a partial inverse agonist at the benzodiazepine allosteric site with its highest affinity for the alpha1 subunit-containing GABA(A) receptor, although it is not selective. FG-7,142 also has its highest efficacy for modulation of GABA-induced chloride flux mediated at the alpha1 subunit-containing GABA(A) receptor. FG-7,142 activates a recognized anxiety-related neural network and interacts with serotonergic, dopaminergic, cholinergic, and noradrenergic modulatory systems within that network. FG-7,142 has been shown to induce anxiety-related behavioral and physiological responses in a variety of experimental paradigms across numerous mammalian and non-mammalian species, including humans. FG-7,142 has proconflict actions across anxiety-related behavioral paradigms, modulates attentional processes, and increases cardioacceleratory sympathetic reactivity and neuroendocrine reactivity. Both acute and chronic FG-7,142 treatment are proconvulsive, upregulate cortical adrenoreceptors, decrease subsequent actions of GABA and beta-carboline agonists, and increase the effectiveness of subsequent GABA(A) receptor antagonists and beta-carboline inverse agonists. FG-7,142, as a partial inverse agonist, can help to elucidate individual components of full agonism of benzodiazepine binding sites and may serve to identify the specific GABA(A) receptor subtypes involved in specific behavioral and physiological responses.

Differential Effects of Methylmercury on γ-Aminobutyric Acid Type A Receptor Currents in Rat Cerebellar Granule and Cerebral Cortical Neurons in Culture

Cerebellar granule cells are particularly sensitive to inhibition by methylmercury (MeHg) on GABA(A) receptor function. This is manifested as a more rapid block of inhibitory postsynaptic currents/inhibitory postsynaptic potentials than for Purkinje cells. The underlying mechanism(s) for differential sensitivity of GABAergic transmission to MeHg in cerebellar neurons is unknown. Differential expression of alpha(6) subunit-containing GABA(A) receptors in cerebellar granule and Purkinje neurons could partially explain this. GABA-evoked currents (I(GABA)) were recorded in response to MeHg in alpha(6) subunit-containing cerebellar granule cells and alpha(6) subunit-deficient cerebral cortical cells in culture. Cortical cells were substituted for Purkinje cells, which do not express alpha(6) subunits. They express the same alpha(1)-containing GABA(A) receptor as Purkinje cells but lack characteristics that enhance Purkinje cell resistance to MeHg. I(GABA) were obtained using whole-cell recording and symmetrical [Cl(-)]. MeHg reduced I(GABA) to complete block in both cell types in a time- and concentration-dependent manner. This effect was faster in granule cells than cortical cells. Effects of MeHg on I(GABA) were recorded in granule cells at various developmental stages (days in vitro 4, 6, and 8) to alter the expression level of alpha(6) subunit-containing GABA(A) receptors. Effects of MeHg on I(GABA) were similar in cells at all days. In human embryonic kidney 293 cells expressing either alpha(6) or alpha(1) subunit-containing GABA(A) receptors, time to block of I(GABA) by MeHg was comparable. Thus, the presence of the alpha(6) subunit alone may not underlie the differential effects of MeHg on I(GABA) observed in cerebellar granule and cortical neurons; other factors are likely to be involved as well.

Synaptic 5 Subunit-Containing GABAA Receptors Mediate IPSPs Elicited by Dendrite-Preferring Cells in Rat Neocortex

Previous studies indicated that one class of dendrite-preferring hippocampal interneurones inhibits pyramidal cells via alpha 5 gamma-aminobutyric acid (GABA(A)) receptors whereas parvalbumin- and CCK-containing basket cells act via alpha1 and alpha2/3 GABA(A) receptors, respectively. This study asked whether there is selective insertion of different alpha subunit-containing GABA(A) receptors at neocortical inhibitory synapses innervated by specific classes of interneurones. The benzodiazepine site pharmacology of inhibitory postsynaptic potentials (IPSPs) elicited in neocortical pyramidal cells by 3 classes of interneurones was explored with dual whole-cell recordings in neocortical slices from juvenile rats (P18-23). Fast IPSPs activated by multipolar interneurones with narrow spikes and nonadapting firing patterns were powerfully enhanced by the alpha1-preferring agonist zolpidem, suggesting mediation via larger proportion of alpha1 GABA(A) receptors than those activated by multipolar, adapting interneurones, which were less strongly enhanced by zolpidem, but equally insensitive to the alpha 5-selective inverse agonist IA alpha 5 (MSD, Essex, UK) suggesting mediation predominantly via alpha2/3 GABA(A) receptors. In contrast, the IPSPs elicited by bitufted, dendrite-preferring interneurones were reduced by IA alpha 5 and by zinc and insensitive to zolpidem despite enhancement by the broad-spectrum agonist, diazepam. Thus insertion of GABA(A) receptors at synapses on neocortical pyramids is input-specific, with proximal inhibition employing alpha1 and alpha2/3 GABA(A) receptors and dendrite-preferring bitufted interneurones activating alpha 5 GABA(A) receptors.

A 17β‐derivative of allopregnanolone is a neurosteroid antagonist at a cerebellar subpopulation of GABAA receptors with nanomolar affinity

High-affinity, subtype-selective antagonists of the neurosteroid binding sites of GABA(A) receptors are not available. We have characterized an allopregnanolone derivative as an antagonist of cerebellar GABA(A) receptors with nanomolar affinity. Receptor binding and electrophysiological methods were used for the allosteric modulation of cerebellar GABA(A) receptors by an allopregnanolone derivative, (20R)-17beta-(1-hydroxy-2,3-butadienyl)-5alpha-androstane-3alpha-ol (HBAO). GABA(A) receptors of rat cerebellar membranes were labelled with the chloride channel blocker [(3)H]ethynylbicycloorthobenzoate (EBOB). The ionophore function of GABA(A) receptors was studied by whole-cell patch clamp electrophysiology in cultured rat cerebellar granule and cortical cells. Partial displacement of cerebellar [(3)H]EBOB binding by nanomolar HBAO was attenuated by 0.1 mM furosemide, an antagonist of alpha(6) and beta(2-3) subunit-containing GABA(A) receptors. Displacement curves of HBAO were reshaped by 30 nM GABA and shifted to the right. However, the micromolar potency of full displacement by allopregnanolone was not affected by 0.1 mM furosemide or 30 nM GABA. The nanomolar, but not the micromolar phase of displacement of [(3)H]EBOB binding by GABA was attenuated by 100 nM HBAO. Submicromolar HBAO did not affect [(3)H]EBOB binding to cortical and hippocampal GABA(A) receptors. HBAO up to 1 microM did not affect chloride currents elicited by 0.3-10 microM GABA, while it abolished potentiation by 1 microM allopregnanolone with nanomolar potency in cerebellar but not in cortical cells. Furosemide attenuated cerebellar inhibition by 100 nM HBAO. HBAO is a selective antagonist of allopregnanolone, a major endogenous positive modulator via neurosteroid sites of cerebellar (probably alpha(6)beta(2-3)delta) GABA(A) receptors.

Hippocampal α5 subunit-containing GABA A receptors are involved in the development of the latent inhibition effect

Hippocampal GABA A receptors containing the α5 subunit have been implicated in the modulation of hippocampal-dependent learning, presumably via their tonic inhibitory influence on hippocampal glutamatergic activity. Here, we examined the expression of latent inhibition (LI)—a form of selective learning that is sensitive to a number of manipulations targeted at the hippocampal formation, in α5(H105R) mutant mice with reduced levels of hippocampal α5-containing GABA A receptors. A single pre-exposure to the taste conditioned stimulus (CS) prior to the pairing of the same CS with LiCl-induced nausea was effective in reducing the conditioned aversion against the taste CS in wild-type mice—thus constituting the LI effect. LI was however distinctly absent in male α5(H105R) mutant mice. Hence, a partial loss of hippocampal α5 GABA A receptors is sufficient to alter one major form of selective learning, albeit this was not seen in the female. This observed phenotype suggests that specific activation of these extrasynaptic GABA A receptors may confer therapeutic potential against the failure to show selectivity in learning by human psychotic patients.

Phosholipase C-Related Inactive Protein Is Involved in Trafficking of γ2 Subunit-Containing GABAAReceptors to the Cell Surface

The subunit composition of GABA(A) receptors is known to be associated with distinct physiological and pharmacological properties. Previous studies that used phospholipase C-related inactive protein type 1 knock-out (PRIP-1 KO) mice revealed that PRIP-1 is involved in the assembly and/or the trafficking of gamma2 subunit-containing GABA(A) receptors. There are two PRIP genes in mammals; thus the roles of PRIP-1 might be compensated partly by those of PRIP-2 in PRIP-1 KO mice. Here we used PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice and examined the roles for PRIP in regulating the trafficking of GABA(A) receptors. Consistent with previous results, sensitivity to diazepam was reduced in electrophysiological and behavioral analyses of PRIP-DKO mice, suggesting an alteration of gamma2 subunit-containing GABA(A) receptors. The surface numbers of diazepam binding sites (alpha/gamma2 subunits) assessed by [3H]flumazenil binding were reduced in the PRIP-DKO mice as compared with those of wild-type mice, whereas the cell surface GABA binding sites (alpha/beta subunits, assessed by [3H]muscimol binding) were increased in PRIP-DKO mice. The association between GABA(A) receptors and GABA(A) receptor-associated protein (GABARAP) was reduced significantly in PRIP-DKO neurons. Disruption of the direct interaction between PRIP and GABA(A) receptor beta subunits via the use of a peptide corresponding to the PRIP-1 binding site reduced the cell surface expression of gamma2 subunit-containing GABA(A) receptors in cultured cell lines and neurons. These results suggest that PRIP is implicated in the trafficking of gamma2 subunit-containing GABA(A) receptors to the cell surface, probably by acting as a bridging molecule between GABARAP and the receptors.

Synaptic and nonsynaptic localization of GABAA receptors containing the alpha5 subunit in the rat brain.

The alpha5 subunit of the GABA(A) receptors (GABA(A)Rs) has a restricted expression in the brain. Maximum expression of this subunit occurs in the hippocampus, cerebral cortex, and olfactory bulb. Hippocampal pyramidal cells show high expression of alpha5 subunit-containing GABA(A)Rs (alpha5-GABA(A)Rs) both in culture and in the intact brain. A large pool of alpha5-GABA(A)Rs is extrasynaptic and it has been proposed to be involved in the tonic GABAergic inhibition of the hippocampus. Nevertheless, there are no studies on the localization of the alpha5-GABA(A)Rs at the electron microscope (EM) level. By using both immunofluorescence of cultured hippocampal pyramidal cells and EM postembedding immunogold of the intact hippocampus we show that, in addition to the extrasynaptic pool, there is a pool of alpha5-GABA(A)Rs that concentrates at the GABAergic synapses in dendrites of hippocampal pyramidal cells. The results suggest that the synaptic alpha5-GABA(A)Rs might play a role in the phasic GABAergic inhibition of pyramidal neurons in hippocampus and cerebral cortex.

The Anxioselective Agent 7-(2-Chloropyridin-4-yl)pyrazolo-[1,5-a]-pyrimidin-3-yl](pyridin-2-yl)methanone (DOV 51892) Is More Efficacious Than Diazepam at Enhancing GABA-Gated Currents at α1 Subunit-Containing GABAA Receptors

Studies using mice with point mutations of GABA(A) receptor alpha subunits suggest that the sedative and anxiolytic properties of 1,4-benzodiazepines are mediated, respectively, by GABA(A) receptors bearing the alpha(1) and alpha(2) subunits. This hypothesis predicts that a compound with high efficacy at GABA(A) receptors containing the alpha(1) subunit would produce sedation, whereas an agonist acting at alpha(2) subunit-containing receptors (with low or null efficacy at alpha(1)-containing receptors) would be anxioselective. Electrophysiological studies using recombinant GABA(A) receptors expressed in Xenopus oocytes indicate that maximal potentiation of GABA-stimulated currents by the pyrazolo-[1,5-a]-pyrimidine, DOV 51892, at alpha(1)beta(2)gamma(2S) constructs of the GABA(A) receptor was significantly higher (148%) than diazepam. In contrast, DOV 51892 was considerably less efficacious and/or potent than diazepam in enhancing GABA-stimulated currents mediated by constructs containing alpha(2), alpha(3), or alpha(5) subunits. In vivo, DOV 51892 increased punished responding in the Vogel conflict test, an effect blocked by flumazenil, and increased the percentage of time spent in the open arms of the elevated plus-maze. However, DOV 51892 had no consistent effects on motor function or muscle relaxation at doses more than 1 order of magnitude greater than the minimal effective anxiolytic dose. Although the mutant mouse data predict that the high-efficacy potentiation of GABA(A1a) receptor-mediated currents by DOV 51892 would be sedating, behavioral studies demonstrate that DOV 51892 is anxioselective, indicating that GABA potentiation mediated by alpha(1) subunit-containing GABA(A) receptors may be neither the sole mechanism nor highly predictive of the sedative properties of benzodiazepine recognition site modulators.

Effects of Ethanol on Tonic GABA Currents in Cerebellar Granule Cells and Mammalian Cells Recombinantly Expressing GABAAReceptors

The effects of ethanol on the GABA(A) receptors, which are regarded as one of the most important target sites of ethanol, are very controversial, ranging from potentiation to no effect. The delta subunit-containing GABA(A) receptors expressed in Xenopus oocytes were recently reported to be potently augmented by ethanol. We performed patch-clamp experiments using the cerebellar granule cells and mammalian cells expressing recombinant GABA(A) receptors. In granule cells, the sensitivity to GABA increased from 7 to 11 days in vitro. Furosemide, an antagonist of alpha6-containing GABA(A) receptors, inhibited GABA-induced currents more potently at 11 to 14 days than at 7 days. Ethanol at 30 mM had either no effect or an inhibitory effect on currents induced by low concentrations of GABA in granule cells. On alpha4beta2delta, alpha6beta2delta, or alpha6beta3deltaGABA(A) receptors expressed in Chinese hamster ovary cells, ethanol at 10, 30, and 100 mM had either no effect or an inhibitory effect on GABA currents. Ethanol inhibition of GABA(A) receptor was observed in all of the subunit combinations examined. In contrast, the perforated patch-clamp method to record the GABA currents revealed ethanol effects on the alpha6beta2delta subunits ranging from slight potentiation to slight inhibition. Ethanol seems to exert a dual action on the GABA(A) receptors and the potentiating action may depend on intracellular milieu. Thus, the differences between the GABA(A) receptors expressed in mammalian host cells and those in Xenopus oocytes in the response to ethanol might be due to changes in intracellular components under patch-clamp conditions.

Changes in the function of the inhibitory neurotransmitter system in the rat brain following subchronic inhalation exposure to 1-bromopropane

1-Bromopropane (1-BP) has been widely used as a cleaning agent and a solvent in industries, but the central neurotoxicity of 1-BP remains to be clarified. In the present study, we investigated the effects of subchronic inhalation exposure to 1-BP vapor on the function of the inhibitory neurotransmitter system mediated by γ-aminobutyric acid (GABA) in the rat brain. Male Wistar rats were exposed to 1-BP vapor for 12 weeks (6 h/day, 5 days/week) at a concentration of 400 ppm, and, in order to investigate the expression and function of brain GABA type A (GABA A) receptors, total/messenger RNA was prepared from the neocortex, hippocampus, and cerebellum of the control and 1-BP-exposed rats. Moreover, hippocampal slices were prepared, and the population spike (PS) amplitude and the slope of the field excitatory postsynaptic potential (fEPSP) were investigated in the paired-pulse configuration of the extracellular recording technique. Using the Xenopus oocyte expression system, we compared GABA concentration–response curves obtained from oocytes injected with brain subregional mRNAs of control and 1-BP exposed rats, and observed no significant differences in apparent GABA affinity. On the other hand, paired-pulse inhibition of PS amplitude was significantly decreased in the hippocampal dentate gyrus (DG) by exposure to 1-BP, without any effect on the paired-pulse ratio of the fEPSP slopes, suggesting neuronal disinhibition in the DG. Moreover, RT-PCR analysis indicated decreased levels of GABA A receptor β3 and δ subunit mRNAs in the hippocampus of 1-BP-exposed rats. These results demonstrate that subchronic inhalation exposure to 1-BP vapor reduces the function of the hippocampal GABAergic system, which could be due to changes in the expression and function of GABA A receptors, especially the δ subunit-containing GABA A receptors.

Update on the neurobiology of alcohol withdrawal seizures.

Abrupt cessation of alcohol intake after prolonged heavy drinking may trigger alcohol withdrawal seizures. Generalized tonic-clonic seizures are the most characteristic and severe type of seizure that occur in this setting. Generalized seizures also occur in rodent models of alcohol withdrawal. In these models, the withdrawal seizures are triggered by neuronal networks in the brainstem, including the inferior colliculus; similar brainstem mechanisms may contribute to alcohol withdrawal seizures in humans. Alcohol causes intoxication through effects on diverse ion channels and neurotransmitter receptors, including GABA(A) receptors--particularly those containing delta subunits that are localized extrasynaptically and mediate tonic inhibition--and N-methyl-D-aspartate (NMDA) receptors. Alcohol dependence results from compensatory changes during prolonged alcohol exposure, including internalization of GABA(A) receptors, which allows adaptation to these effects. Withdrawal seizures are believed to reflect unmasking of these changes and may also involve specific withdrawal-induced cellular events, such as rapid increases in alpha4 subunit-containing GABA(A) receptors that confer reduced inhibitory function. Optimizing approaches to the prevention of alcohol withdrawal seizures requires an understanding of the distinct neurobiologic mechanisms that underlie these seizures.

Clustered and non-clustered GABA A receptors in cultured hippocampal neurons

In cultured hippocampal neurons, γ 2 subunit-containing GABA ARs form large postsynaptic clusters at GABAergic synapses and small clusters outside GABAergic synapses. We now show that a pool of non-clustered γ 2 subunit-containing GABA ARs are also present at the cell surface. We also demonstrate that myc- or EGFP-tagged γ 2, α 2, β 3 or α 1 subunits expressed in these neurons assemble with endogenous subunits, forming GABA ARs that target large postsynaptic clusters, small clusters outside GABAergic synapses or a pool of non-clustered surface GABA ARs. In contrast, myc- or EGFP-tagged δ subunits only form non-clustered GABA ARs, which can be induced to form clusters by antibody capping. A myc-tagged chimeric γ 2 subunit possessing the large intracellular loop (IL) of the δ-subunit IL ( myc γ 2S/δ-IL) assembled into GABA ARs, but it did not form clusters, therefore behaving like the δ subunit. Thus, the large intracellular loops of γ 2 and δ play an important role in determining the synaptic clustering/non-clustering capacity of the GABA ARs.

Ovarian cycle–linked changes in GABAA receptors mediating tonic inhibition alter seizure susceptibility and anxiety

Disturbances of neuronal excitability changes during the ovarian cycle may elevate seizure frequency in women with catamenial epilepsy and enhance anxiety in premenstrual dysphoric disorder (PMDD). The mechanisms underlying these changes are unknown, but they could result from the effects of fluctuations in progesterone-derived neurosteroids on the brain. Neurosteroids and some anxiolytics share an important site of action: tonic inhibition mediated by delta subunit-containing GABA(A) receptors (deltaGABA(A)Rs). Here we demonstrate periodic alterations in specific GABA(A)R subunits during the estrous cycle in mice, causing cyclic changes of tonic inhibition in hippocampal neurons. In late diestrus (high-progesterone phase), enhanced expression of deltaGABA(A)Rs increases tonic inhibition, and a reduced neuronal excitability is reflected by diminished seizure susceptibility and anxiety. Eliminating cycling of deltaGABA(A)Rs by antisense RNA treatment or gene knockout prevents the lowering of excitability during diestrus. Our findings are consistent with possible deficiencies in regulatory mechanisms controlling normal cycling of deltaGABA(A)Rs in individuals with catamenial epilepsy or PMDD.

In vivo labelling of α5 subunit-containing GABA A receptors using the selective radioligand [ 3H]L-655,708

L-655,708 is an imidazobenzodiazepine possessing 30–70-fold selectivity for the benzodiazepine binding site of GABA A receptors containing an α5 rather than α1, α2 or α3 subunit. In the present study, [ 3H]L-655,708 was used to label mouse brain benzodiazepine binding sites in vivo. When compared to inhibition of in vivo binding of the non-selective ligand [ 3H]Ro 15-1788, the pharmacology of mouse in vivo [ 3H]L-655,708 binding was consistent with selective in vivo labelling of α5 subunit-containing GABA A receptors. Thus, diazepam was equipotent at inhibiting in vivo [ 3H]L-655,708 and [ 3H]Ro 15-1788 binding; zolpidem, which has very low affinity for α5-containing GABA A receptors, gave no inhibition of in vivo [ 3H]L-655,708 binding despite inhibiting in vivo [ 3H]Ro 15-1788 binding; and L-655,708 was more potent at inhibiting the in vivo binding of [ 3H]L-655,708 compared to [ 3H]Ro 15-1788. This pharmacological specificity of in vivo [ 3H]L-655,708 binding was confirmed autoradiographically. Hence, the anatomical distribution of in vivo [ 3H]L-655,708 binding was comparable to the distribution of α5-containing GABA A receptors identified in vitro. Moreover, this distribution was distinct from that identified using [ 3H]Ro 15-1788. These data therefore suggest that [ 3H]L-655,708 can be used to identify α5-containing GABA A receptors in vivo and that this ligand can be used to measure receptor occupancy of α5-selective ligands.

α1 Subunit-Containing GABA Type A Receptors in Forebrain Contribute to the Effect of Inhaled Anesthetics on Conditioned Fear

Inhaled anesthetics are believed to produce anesthesia by their actions on ion channels. Because inhaled anesthetics robustly enhance GABA A receptor (GABA(A)-R) responses to GABA, these receptors are considered prime targets of anesthetic action. However, the importance of GABA(A)-Rs and individual GABA(A)-R subunits to specific anesthetic-induced behavioral effects in the intact animal is unknown. We hypothesized that inhaled anesthetics produce amnesia, as assessed by loss of fear conditioning, by acting on the forebrain GABA(A)-Rs that harbor the alpha1 subunit. To test this, we used global knockout mice that completely lack the alpha1 subunit and forebrain-specific, conditional knockout mice that lack the alpha1 subunit only in the hippocampus, cortex, and amygdala. Both knockout mice were 75 to 145% less sensitive to the amnestic effects of the inhaled anesthetic isoflurane. These results indicate that alpha1-containing GABA(A)-Rs in the hippocampus, amygdala, and/or cortex influence the amnestic effects of inhaled anesthetics and may be an important molecular target of the drug isoflurane.

Clustering of Extrasynaptic GABAA Receptors Modulates Tonic Inhibition in Cultured Hippocampal Neurons

Tonic inhibition plays a crucial role in regulating neuronal excitability because it sets the threshold for action potential generation and integrates excitatory signals. Tonic currents are known to be largely mediated by extrasynaptic gamma-aminobutyric acid type A (GABA(A)) receptors that are persistently activated by submicromolar concentrations of ambient GABA. We recently reported that, in cultured hippocampal neurons, the clustering of synaptic GABA(A) receptors significantly affects synaptic transmission. In this work, we demonstrated that the clustering of extrasynaptic GABA(A) receptors modulated tonic inhibition. Depolymerization of the cytoskeleton with nocodazole promoted the disassembly of extrasynaptic clusters of delta and gamma(2) subunit-containing GABA(A) receptors. This effect was associated with a reduction in the amplitude of tonic currents and diminished shunting inhibition. Moreover, diffuse GABA(A) receptors were less sensitive to the GAT-1 inhibitor NO-711 and to flurazepam. Quantitative analysis of GABA-evoked currents after prolonged exposure to submicromolar concentrations of GABA and model simulations suggest that clustering affects the gating properties of extrasynaptic GABA(A) receptors. In particular, a larger occupancy of the singly and doubly bound desensitized states can account for the modulation of tonic inhibition recorded after nocodazole treatment. Moreover, comparison of tonic currents recorded during spontaneous activity and those elicited by exogenously applied low agonist concentrations allows estimation of the concentration of ambient GABA. In conclusion, receptor clustering appears to be an additional regulating factor for tonic inhibition.

Altered expression of the delta subunit of the GABAA receptor in a mouse model of temporal lobe epilepsy.

delta Subunit-containing GABA(A) receptors are located predominantly at nonsynaptic sites in the dentate gyrus where they may play important roles in controlling neuronal excitability through tonic inhibition and responses to GABA spillover. Immunohistochemical methods were used to determine whether delta subunit expression was altered after pilocarpine-induced status epilepticus in C57BL/6 mice in ways that could increase excitability of the dentate gyrus. In pilocarpine-treated animals, the normal diffuse labeling of the delta subunit in the dentate molecular layer was decreased by 4 d after status epilepticus (latent period) and remained low throughout the period of chronic seizures. In contrast, diffuse labeling of alpha4 and gamma2 subunits, potentially interrelated GABA(A) receptor subunits, was increased during the chronic period. Interestingly, delta subunit labeling of many interneurons progressively increased after pilocarpine treatment. Consistent with the observed changes in delta subunit labeling, physiological studies revealed increased excitability in the dentate gyrus of slices obtained from the pilocarpine-treated mice and demonstrated that physiological concentrations of the neurosteroid tetrahydrodeoxycorticosterone were less effective in reducing excitability in the pilocarpine-treated animals than in controls. The findings support the idea that alterations in nonsynaptic delta subunit-containing GABA(A) receptors in both principal cells and interneurons could contribute to increased seizure susceptibility in the hippocampal formation in a temporal lobe epilepsy model.

Low Ethanol Concentrations Selectively Augment the Tonic Inhibition Mediated by δ Subunit-Containing GABAAReceptors in Hippocampal Neurons

In central neurons, a tonic conductance is activated by ambient levels of the inhibitory transmitter GABA. Here, we show that in dentate gyrus granule cells, where tonic inhibition is mediated by delta subunit-containing GABA(A) receptors, this conductance is augmented by low concentrations (30 mM) of ethanol. In contrast, the tonic inhibition mediated by alpha5 subunit-containing receptors of CA1 pyramidal cells is not affected. The effect of ethanol on tonic inhibition specifically reduces the excitability of the dentate gyrus and identifies the delta subunit-dependent tonic inhibition as a likely site of ethanol action in the brain.