Murray B. Herd

The δ Subunit of γ-Aminobutyric Acid Type A Receptors Does Not Confer Sensitivity to Low Concentrations of Ethanol

GABAA receptors (GABAARs) are usually formed by α, β, and γ or δ subunits. Recently, δ-containing GABAARs expressed in Xenopus oocytes were found to be sensitive to low concentrations of ethanol (1–3 mM). Our objective was to replicate and extend the study of the effect of ethanol on the function of α4β3δ GABAARs. We independently conducted three studies in two systems: rat and human GABAARs expressed in Xenopus oocytes, studied through two-electrode voltage clamp; and human GABAARs stably expressed in the fibroblast L(tk–) cell line, studied through patch-clamp electrophysiology. In all cases, α4β3δ GABAARs were only sensitive to high concentrations of ethanol (100 mM in oocytes, 300 mM in the cell line). Expression of the δ subunit in oocytes was assessed through the magnitude of the maximal GABA currents and sensitivity to zinc. Of the three rat combinations studied, α4β3 was the most sensitive to ethanol, isoflurane, and 5α-pregnan-3α,21-diol-20-one (THDOC); α4β3δ and α4β3γ2S were very similar in most aspects, but α4β3δ was more sensitive to GABA, THDOC, and lanthanum than α4β3γ2S GABAARs. Ethanol at 30 mM did not affect tonic GABA-mediated currents in dentate gyrus reported to be mediated by GABAARs incorporating α4 and δ subunits. We have not been able to replicate the sensitivity of α4β3δ GABAARs to low concentrations of ethanol in four different laboratories in independent studies. This suggests that as yet unidentified factors may play a critical role in the ethanol effects on δ-containing GABAARs.

Neuroactive steroids and inhibitory neurotransmission: mechanisms of action and physiological relevance.

Dysfunction of GABA(A) receptor-mediated inhibition is implicated in a number of neurological and psychiatric conditions including epilepsy and affective disorders. Some of these conditions have been associated with abnormal levels of certain endogenously occurring neurosteroids, which potently and selectively enhance the function of the brains major inhibitory receptor, the GABA(A) receptor. Consistent with their ability to enhance neuronal inhibition, such steroids exhibit in animals and humans anxiolytic, anticonvulsant and anesthetic actions. Neurosteroids, exemplified by the potent progesterone metabolite, 5alpha-pregnan-3alpha-ol-20-one can be synthesized de novo in the CNS both in neurones and glia in levels sufficient to modulate GABA(A) receptor function. Neurosteroid levels are not static, but are subject to dynamic fluctuations, for example during stress, or the later stages of pregnancy. These observations suggest that these endogenous modulators may refine the function of the brains major inhibitory receptor and thus, play an important physiological and pathophysiological role. However, given the ubiquitous expression of GABA(A) receptors throughout the mammalian CNS, changes in neurosteroid levels should be widely experienced, causing a generalized enhancement of neuronal inhibition. Such a non-specific action would seem incompatible with a physiological role. However, neurosteroid action is both brain region and neurone selective. This specificity results from a variety of molecular mechanisms including receptor subunit composition, local steroid metabolism and phosphorylation. This paper will evaluate the relative contribution these mechanisms play in defining the interaction of neurosteroids with synaptic and extra-synaptic GABA(A) receptors.

The Contraceptive Agent Provera Enhances GABAA Receptor-Mediated Inhibitory Neurotransmission in the Rat Hippocampus: Evidence for Endogenous Neurosteroids?

Neurosteroids typified by 5α-pregnan-3α-ol-20-one (5α3α) have emerged as the most potent endogenous positive modulators of the GABAA receptor, the principal mediator of fast inhibitory transmission within the CNS. Neurosteroids can be synthesized de novo in the brain in levels sufficient to modulate GABAA receptor function and, thus, might play an important physiological-pathophysiological role. Indirect support for this proposal comes from the observation that neurosteroid action is region and neuron selective. However, the mechanism(s) that imparts specificity of action remains primarily elusive. Although neurosteroids are relatively promiscuous toward different GABAA receptor isoforms, the contribution of local neurosteroid metabolism has been relatively unexplored. Here, we investigate the role of neurosteroid metabolism by using electrophysiological techniques to compare the actions of 5α3α and its metabolically stable synthetic analog ganaxolone on inhibitory neurotransmission in CA1 and dentate gyrus neurons. Furthermore, we evaluate the contribution of a key enzyme in neurosteroid metabolism [i.e., 3α-hydroxysteroidoxidoreductase (3α-HSOR)] to the inactivation of endogenous, or exogenously applied 5α3α. We show that low concentrations of ganaxolone, but not of 5α3α, enhance inhibitory transmission in dentate gyrus, whereas both steroids are similarly effective in CA1 neurons. Furthermore, inhibition of 3α-HSOR by the contraceptive agent Provera results in enhanced synaptic and extrasynaptic GABAA receptor-mediated inhibition in the dentate gyrus but not in the CA1 region. Collectively, these findings advocate a crucial role for local steroid metabolism in shaping GABAA receptor-mediated inhibition in a regionally dependent manner and suggest a novel action by the contraceptive agent on inhibitory centers in the CNS.

Novel compounds selectively enhance δ subunit containing GABAA receptors and increase tonic currents in thalamus

Inhibition in the brain is dominated by the neurotransmitter gamma-aminobutyric acid (GABA); operating through GABA(A) receptors. This form of neural inhibition was presumed to be mediated by synaptic receptors, however recent evidence has highlighted a previously unappreciated role for extrasynaptic GABA(A) receptors in controlling neuronal activity. Synaptic and extrasynaptic GABA(A) receptors exhibit distinct pharmacological and biophysical properties that differentially influence brain physiology and behavior. Here we used a fluorescence-based assay and cell lines expressing recombinant GABA(A) receptors to identify a novel series of benzamide compounds that selectively enhance, or activate alpha4beta3delta GABA(A) receptors (cf. alpha4beta3gamma2 and alpha1beta3gamma2). Utilising electrophysiological methods, we illustrate that one of these compounds, 4-chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide (DS1) potently (low nM) enhances GABA-evoked currents mediated by alpha4beta3delta receptors. At similar concentrations DS1 directly activates this receptor and is the most potent known agonist of alpha4beta3delta receptors. 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide (DS2) selectively potentiated GABA responses mediated by alpha4beta3delta receptors, but was not an agonist. Recent studies have revealed a tonic form of inhibition in thalamus mediated by the alpha4beta2delta extrasynaptic GABA(A) receptors that may contribute to the regulation of thalamocortical rhythmic activity associated with sleep, wakefulness, vigilance and seizure disorders. In mouse thalamic relay cells DS2 enhanced the tonic current mediated by alpha4beta2delta receptors with no effect on their synaptic GABA(A) receptors. Similarly, in mouse cerebellar granule cells DS2 potentiated the tonic current mediated by alpha6betadelta receptors. DS2 is the first selective positive allosteric modulator of delta-GABA(A) receptors and such compounds potentially offer novel therapeutic opportunities as analgesics and in the treatment of sleep disorders. Furthermore, these drugs may be valuable in elucidating the physiological and pathophysiological roles played by these extrasynaptic GABA(A) receptors.

The expression of GABAAβ subunit isoforms in synaptic and extrasynaptic receptor populations of mouse dentate gyrus granule cells

The subunit composition of GABAA receptors influences their biophysical and pharmacological properties, dictates neuronal location and the interaction with associated proteins, and markedly influences the impact of intracellular biochemistry. The focus has been on α and γ subunits, with little attention given to β subunits. Dentate gyrus granule cells (DGGCs) express all three β subunit isoforms and exhibit both synaptic and extrasynaptic receptors that mediate ‘phasic’ and ‘tonic’ transmission, respectively. To investigate the subcellular distribution of the β subunits we have utilized the patch‐clamp technique to compare the properties of ‘tonic’ and miniature inhibitory postsynaptic currents (mIPSCs) recorded from DGGCs of hippocampal slices of P20–26 wild‐type (WT), β2−/−, β2N265S (etomidate‐insensitive), α1−/− and δ−/− mice. Deletion of either the β2 or the δ subunit produced a significant reduction of the tonic current and attenuated the increase of this current induced by the δ subunit‐preferring agonist 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP). By contrast, mIPSCs were not influenced by deletion of these genes. Enhancement of the tonic current by the β2/3 subunit‐selective agent etomidate was significantly reduced for DGGCs derived from β2N265S mice, whereas this manipulation had no effect on the prolongation of mIPSCs produced by this anaesthetic. Collectively, these observations, together with previous studies on α4−/− mice, identify a population of extrasynaptic α4β2δ receptors, whereas synaptic GABAA receptors appear to primarily incorporate the β3 subunit. A component of the tonic current is diazepam sensitive and is mediated by extrasynaptic receptors incorporating α5 and γ2 subunits. Deletion of the β2 subunit had no effect on the diazepam‐induced current and therefore these extrasynaptic receptors do not contain this subunit. The unambiguous identification of these distinct pools of synaptic and extrasynaptic GABAA receptors should aid our understanding of how they act in harmony, to regulate hippocampal signalling in health and disease.

Developmental maturation of synaptic and extrasynaptic GABAA receptors in mouse thalamic ventrobasal neurones

Thalamic ventrobasal (VB) relay neurones express multiple GABAA receptor subtypes mediating phasic and tonic inhibition. During postnatal development, marked changes in subunit expression occur, presumably reflecting changes in functional properties of neuronal networks. The aims of this study were to characterize the properties of synaptic and extrasynaptic GABAA receptors of developing VB neurones and investigate the role of the α1 subunit during maturation of GABA‐ergic transmission, using electrophysiology and immunohistochemistry in wild type (WT) and α10/0 mice and mice engineered to express diazepam‐insensitive receptors (α1H101R, α2H101R). In immature brain, rapid (P8/9–P10/11) developmental change to mIPSC kinetics and increased expression of extrasynaptic receptors (P8–27) formed by the α4 and δ subunit occurred independently of the α1 subunit. Subsequently (≥ P15), synaptic α2 subunit/gephyrin clusters of WT VB neurones were replaced by those containing the α1 subunit. Surprisingly, in α10/0 VB neurones the frequency of mIPSCs decreased between P12 and P27, because the α2 subunit also disappeared from these cells. The loss of synaptic GABAA receptors led to a delayed disruption of gephyrin clusters. Despite these alterations, GABA‐ergic terminals were preserved, perhaps maintaining tonic inhibition. These results demonstrate that maturation of synaptic and extrasynaptic GABAA receptors in VB follows a developmental programme independent of the α1 subunit. Changes to synaptic GABAA receptor function and the increased expression of extrasynaptic GABAA receptors represent two distinct mechanisms for fine‐tuning GABA‐ergic control of thalamic relay neurone activity during development.

Inhibition of thalamic excitability by 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol: a selective role for delta-GABA(A) receptors.

The sedative and hypnotic agent 4,5,6,7‐tetrahydroisoxazolo[4,5‐c]pyridine‐3‐ol (THIP) is a GABAA receptor (GABAAR) agonist that preferentially activates δ‐subunit‐containing GABAARs (δ‐GABAARs). To clarify the role of δ‐GABAARs in mediating the sedative actions of THIP, we utilized mice lacking the α1‐ or δ‐subunit in a combined electrophysiological and behavioural analysis. Whole‐cell patch‐clamp recordings were obtained from ventrobasal thalamic nucleus (VB) neurones at a holding potential of −60 mV. Application of bicuculline to wild‐type (WT) VB neurones revealed a GABAAR‐mediated tonic current of 92 ± 19 pA, which was greatly reduced (13 ± 5 pA) for VB neurones of δ0/0 mice. Deletion of the δ‐ but not the α1‐subunit dramatically reduced the THIP (1 μm)‐induced inward current in these neurones (WT, −309 ± 23 pA; δ0/0, −18 ± 3 pA; α10/0, −377 ± 45 pA). Furthermore, THIP selectively decreased the excitability of WT and α10/0 but not δ0/0 VB neurones. THIP did not affect the properties of miniature inhibitory post‐synaptic currents in any of the genotypes. No differences in rotarod performance and locomotor activity were observed across the three genotypes. In WT mice, performance of these behaviours was impaired by THIP in a dose‐dependent manner. The effect of THIP on rotarod performance was blunted for δ0/0 but not α10/0 mice. We previously reported that deletion of the α1‐subunit abolished synaptic GABAA responses of VB neurones. Therefore, collectively, these findings suggest that extrasynaptic δ‐GABAARs vs. synaptic α1‐subunit‐containing GABAARs of thalamocortical neurones represent an important molecular target underpinning the sedative actions of THIP.

GABA-independent GABAA receptor openings maintain tonic currents.

Activation of GABAA receptors (GABAARs) produces two forms of inhibition: phasic inhibition generated by the rapid, transient activation of synaptic GABAARs by presynaptic GABA release, and tonic inhibition generated by the persistent activation of perisynaptic or extrasynaptic GABAARs, which can detect extracellular GABA. Such tonic GABAAR-mediated currents are particularly evident in dentate granule cells in which they play a major role in regulating cell excitability. Here we show that in rat dentate granule cells in ex vivo hippocampal slices, tonic currents are predominantly generated by GABA-independent GABAA receptor openings. This tonic GABAAR conductance is resistant to the competitive GABAAR antagonist SR95531 (gabazine), which at high concentrations acts as a partial agonist, but can be blocked by an open channel blocker, picrotoxin. When slices are perfused with 200 nm GABA, a concentration that is comparable to CSF concentrations but is twice that measured by us in the hippocampus in vivo using zero-net-flux microdialysis, negligible GABA is detected by dentate granule cells. Spontaneously opening GABAARs, therefore, maintain dentate granule cell tonic currents in the face of low extracellular GABA concentrations.

Extrasynaptic GABAA Receptors Couple Presynaptic Activity to Postsynaptic Inhibition in the Somatosensory Thalamus

Thalamocortical circuits govern cognitive, sensorimotor, and sleep-related network processes, and generate pathological activities during absence epilepsy. Inhibitory control of thalamocortical (TC) relay neurons is partially mediated by GABA released from neurons of the thalamic reticular nucleus (nRT), acting predominantly via synaptic α1β2γ2 GABAA receptors (GABAARs). Importantly, TC neurons also express extrasynaptic α4β2δ GABAARs, although how they cooperate with synaptic GABAARs to influence relay cell inhibition, particularly during physiologically relevant nRT output, is unknown. To address this question, we performed paired whole-cell recordings from synaptically coupled nRT and TC neurons of the ventrobasal (VB) complex in brain slices derived from wild-type and extrasynaptic GABAAR-lacking, α4 “knock-out” (α40/0) mice. We demonstrate that the duration of VB phasic inhibition generated in response to nRT burst firing is greatly reduced in α40/0 pairs, suggesting that action potential-dependent phasic inhibition is prolonged by recruitment of extrasynaptic GABAARs. Furthermore, the influence of nRT tonic firing frequency on VB holding current is also greatly reduced in α40/0 pairs, implying that the α4-GABAAR-mediated tonic conductance of relay neurons is dynamically influenced, in an activity-dependent manner, by nRT tonic firing intensity. Collectively, our data reveal that extrasynaptic GABAARs of the somatosensory thalamus do not merely provide static tonic inhibition but can also be dynamically engaged to couple presynaptic activity to postsynaptic excitability. Moreover, these processes are highly sensitive to the δ-selective allosteric modulator, DS2 and manipulation of GABA transport systems, revealing novel opportunities for therapeutic intervention in thalamocortical network disorders.

Tonic Inhibition of Accumbal Spiny Neurons by Extrasynaptic α4βδ GABAA Receptors Modulates the Actions of Psychostimulants

Within the nucleus accumbens (NAc), synaptic GABAA receptors (GABAARs) mediate phasic inhibition of medium spiny neurons (MSNs) and influence behavioral responses to cocaine. We demonstrate that both dopamine D1- and D2-receptor-expressing MSNs (D-MSNs) additionally harbor extrasynaptic GABAARs incorporating α4, β, and δ subunits that mediate tonic inhibition, thereby influencing neuronal excitability. Both the selective δ-GABAAR agonist THIP and DS2, a selective positive allosteric modulator, greatly increased the tonic current of all MSNs from wild-type (WT), but not from δ−/− or α4−/− mice. Coupling dopamine and tonic inhibition, the acute activation of D1 receptors (by a selective agonist or indirectly by amphetamine) greatly enhanced tonic inhibition in D1-MSNs but not D2-MSNs. In contrast, prolonged D2 receptor activation modestly reduced the tonic conductance of D2-MSNs. Behaviorally, WT and constitutive α4−/− mice did not differ in their expression of cocaine-conditioned place preference (CPP). Importantly, however, mice with the α4 deletion specific to D1-expressing neurons (α4D1−/−) showed increased CPP. Furthermore, THIP administered systemically or directly into the NAc of WT, but not α4−/− or α4D1−/− mice, blocked cocaine enhancement of CPP. In comparison, α4D2−/− mice exhibited normal CPP, but no cocaine enhancement. In conclusion, dopamine modulation of GABAergic tonic inhibition of D1- and D2-MSNs provides an intrinsic mechanism to differentially affect their excitability in response to psychostimulants and thereby influence their ability to potentiate conditioned reward. Therefore, α4βδ GABAARs may represent a viable target for the development of novel therapeutics to better understand and influence addictive behaviors.