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Pregnancy is associated with changes in mood and anxiety level as well as with marked hormonal fluctuations. Increases in the brain concentrations of neuroactive steroids during pregnancy in rats are accompanied by changes in expression of subunits of the GABA type A receptor (GABAA-R) in the brain. Granule cells of the dentate gyrus (DGGCs) exhibit two components of inhibitory GABAergic transmission: a phasic component mediated by synaptic GABAA-Rs, and a tonic component mediated by extrasynaptic GABAA-Rs. Recordings of GABAergic currents were obtained from hippocampal slices prepared from rats in estrus, at pregnancy day 15 (P15) or P19, or at 2 d after delivery. Exogenous GABA or 3α,5α-THP induced an increase in tonic current in DGGCs that was significantly greater at P19 than in estrus. Neither tonic nor phasic currents were affected by pregnancy in CA1 pyramidal cells. Immunohistochemical analysis revealed a marked increase in the abundance of the δ subunit of the GABAA-R and a concomitant decrease in that of the γ2 subunit in the hippocampus at P19. Expression of the α4 subunit did not change during pregnancy but was increased 2 d after delivery. Treatment of rats from P12 to P18 with the 5α-reductase inhibitor finasteride prevented the changes in tonic current and in δ and γ2 subunit expression normally apparent at P19. These data suggest that the number of extrasynaptic GABAA-Rs is increased in DGGCs during late pregnancy as a consequence of the associated marked fluctuations in the brain levels of neuroactive steroids. [ABSTRACT FROM AUTHOR]

Alcohol has subjective and behavioral effects at the pharmacological levels typically reached during the consumption of one or two alcoholic drinks. Here we provide evidence that an α4-subunit-containing GABAA receptor contributes to the consumption of low-tomoderate levels of alcohol. Using viral-mediated RNA interference (RNAi), we found that reduced expression of the α4 subunit in the nucleus accumbens (NAc) shell of rats decreased their free consumption of and preference for alcohol. The time course for the reduced alcohol intake paralleled the time course ofα4mRNAreductions achieved after viral-mediated RNAi forα4. Furthermore, the reduction in drinking was region- and alcohol-specific: there was no effect of reductions in α4 expression in the NAc core on alcohol intake, and reductions in α4 expression in the NAc shell did not alter sucrose or water intake. These results indicate that the GABAA receptor α4 subunit in the NAc shell mediates alcohol intake. [ABSTRACT FROM AUTHOR]

Abstract: γ-aminobutyric acid (GABA) is the main neuroinhibitory transmitter in the brain. Here we show that GABA in the extracellular space may affect the fate of pathogenic T lymphocytes entering the brain. We examined in encephalitogenic T cells if they expressed functional GABA channels that could be activated by the low (nM–1 µM), physiological concentrations of GABA present around neurons in the brain. The cells expressed the α1, α4, β2, β3, γ1 and δ GABAA channel subunits and formed functional, extrasynaptic-like GABA channels that were activated by 1 µM GABA. 100 nM and higher GABA concentrations decreased T cell proliferation. The results are consistent with GABA being immunomodulatory. [Copyright &y& Elsevier]

Ethanol alters the distribution and abundance of PKCδ in neural cell lines. Here we investigated whether PKCδ also regulates behavioral responses to ethanol.PKCδ/mice showed reduced intoxication when administered ethanol and reduced ataxia when administered the nonselective GABAA receptor agonists pentobarbital and pregnanolone. However, their response to flunitrazepam was not altered, suggesting that PKCδ regulates benzodiazepine-insensitive GABAA receptors, most of which contain δ subunits and mediate tonic inhibitory currents in neurons. Indeed, the distribution of PKCδ overlapped with GABAA δ subunits in thalamus and hippocampus, and ethanol failed to enhance tonicGABAcurrents inPKCδ-/-thalamic and hippocampal neurons. Moreover, using anATPanalog-sensitive PKCδ mutant in mouse L(tk-) fibroblasts that expressα4β3δ GABAA receptors, we found that ethanol enhancement of GABA currents was PKCδ-dependent. Thus, PKCδ enhances ethanol intoxication partly through regulation of GABAA receptors that contain δ subunits and mediate tonic inhibitory currents. These findings indicate that PKCδ contributes to a high level of behavioral response to ethanol, which is negatively associated with risk of developing an alcohol use disorder in humans. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, we describe GABAA receptor-mediated tonic inhibitory currents in the substantia gelatinosa (SG) region of rat spinal trigeminal nucleus pars caudalis (Vc). The GABAA receptor-mediated tonic currents were identified by bath-application of the GABAA receptor antagonists, picrotoxin (1mM), SR95531 (100μM) and bicuculline (100μM). All three antagonists completely blocked outward spontaneous (phasic) inhibitory postsynaptic currents, but only picrotoxin and bicuculline induced a significant (>5pA) inward shift of holding currents at a holding potential (Vh) of 0mV in 60–70% of SG neurons, revealing the existence of tonic outward currents. The tonic currents were resistant to further the blockades of glycine receptors or those in addition to glutamate receptors and voltage-dependent sodium channels. An acute bath-application of THDOC (0.1μM), the stress-related neurosteroid, did enhance tonic currents, but only in a small population of SG neurons. In addition, slices incubated with THDOC for 30min increased the probability of neurons with significant tonic currents. The GABAergic tonic inhibition demonstrated in this study may play a significant role in the sensory processing system of the Vc. [Copyright &y& Elsevier]

Background: The α4 subunit-containing γ-aminobutyric acid (A) receptors (GABAARs) are highly expressed primarily at extrasynaptic sites in the dentate gyrus (DG) and thalamus and are suspected to contribute to tonic inhibition that is sensitive to potentiation by gaboxadol and ethanol (EtOH). Global α4 subunit knockout (KO) mice exhibit greatly reduced tonic currents and insensitivity to ataxic, sedative and analgesic effects of gaboxadol compared to wild type (WT) controls. The α4 KO mice were also significantly more sensitive to pentylenetetrazol-induced seizures. However, no differences were observed between α4 KO and WT mice in other baseline behaviors or in the effects of EtOH on these behaviors. To examine possible functional and pharmacological GABAAR alterations, and search for causes for the lack of differences in EtOH behaviors we studied the effects of acute EtOH application on GABAAR-currents of DG cells from α4 KO and WT control mice complemented by Western blot measurements. Methods: We studied the consequences of α4 subunit deletion using Western immunoblotting and whole cell patch recordings from DG cells in brain slices from α4 KO and WT mice. Results: The magnitude of tonic current and its potentiation by EtOH (10 to 100 mM), alphaxalone (3 μM), and Ro15-4513 (0.3 μM) was greatly attenuated in α4 KO mice. The kinetics of miniature inhibitory postsynaptic currents (mIPSCs) in α4 KO mice were significantly slower compared to WT mice. Potentiation of mIPSCs by alphaxalone was greatly reduced in α4 KO mice. Ro15-4513 had no effect on mIPSCs from WT or KO mice. However, mIPSCs of α4 KO mice were significantly more sensitive to EtOH than those from WT mice. The γ2 subunit protein levels were selectively increased in hippocampus and thalamus, but not cortex of α4 KO mice. Conclusions: These data suggest that the global loss of α4 subunits leads to region- and cell location-specific compensatory increases in γ2 subunits, which in turn alter the pharmacological sensitivity of synaptic and extrasynaptic GABAAR-currents. Our data also suggests that while enhancement of tonic inhibitory currents by gaboxadol, alphaxalone, and EtOH are reduced, and behavioral sensitivity to gaboxadol and alphaxalone may be reduced, compensatory changes in synaptic GABAAR subunits may prevent similar reductions in behavioral sensitivity to EtOH. [ABSTRACT FROM AUTHOR]

Abstract: Certain naturally occurring pregnane steroids act in a nongenomic manner to potently and selectively enhance the interaction of the inhibitory neurotransmitter GABA with the GABAA receptor. Consequently such steroids exhibit anxiolytic, anticonvulsant, analgesic, sedative, hypnotic, and anesthetic properties. In both physiological and pathophysiological scenarios, the pregnane steroids may function as endocrine messengers (e.g., produced in the periphery and cross the blood–brain barrier) to influence behaviour. However, additionally “neurosteroids” can be synthesised in the brain and spinal cord to act in a paracrine or autocrine manner and thereby locally influence neuronal activity. Given the ubiquitous expression of the GABAA receptor throughout the mammalian central nervous system (CNS), physiological, pathophysiological, or drug-induced pertubations of neurosteroid levels may be expected to produce widespread changes in brain excitability. However, the neurosteroid/GABAA receptor interaction is brain region and indeed neuron specific. The molecular basis of this specificity will be reviewed here, including (1) the importance of the subunit composition of the GABAA receptor; (2) how protein phosphorylation may dynamically influence the sensitivity of GABAA receptors to neurosteroids; (3) the impact of local steroid metabolism; and (4) the emergence of extrasynaptic GABAA receptors as a neurosteroid target. [Copyright &y& Elsevier]

Abstract: This review discusses evidence for and apparent controversy about, γ-aminobutyric acid type A (GABAA) receptor (GABAAR) subtypes that mediate alcohol effects experienced during social drinking. GABAARs that contain the β3 and δ subunits were shown to be enhanced by alcohol concentrations that mirror the concentration dependence of alcohol responses in humans. A mutation (α6R100Q) previously found in alcohol nontolerant rats in the cerebellar GABAAR α6 subunit is sufficient for increased alcohol-induced ataxia in rats homozygous for this mutation (α6-100QQ) and further increases alcohol sensitivity of tonic GABA currents (mediated by α6βδ receptors) in cerebellar granule cells of α6-100QQ rats and in recombinant α6R100Qβ3δ receptors. This provided the first direct evidence that these types of receptors mediate behavioral effects of ethanol. Furthermore, the behavioral alcohol antagonist Ro15-4513 specifically reverses ethanol enhancement on α4/6β3δ receptors. Unexpectedly, native and recombinant α4/6β3δ receptors bind the behavioral alcohol antagonist Ro15-4513 with high affinity and this binding is competitive with EtOH, suggesting a specific and mutually exclusive (competitive) ethanol/Ro15-4513 site, which explains the puzzling activity of Ro15-4513 as a behavioral alcohol antagonist. Our conclusion from these findings is that alcohol/Ro15-4513-sensitive GABAAR subtypes are important alcohol targets and that alcohol at relevant concentrations is more specific than previously thought. In this review, we discuss technical difficulties in expressing recombinant δ subunit-containing receptors in oocytes and mammalian cells that may have contributed to negative results and confusion. Not only because we have reproduced detailed positive results numerous times, and we and many others have built extensively on basic findings, but also because we explain and combine many previously puzzling results into a coherent and highly plausible paradigm on how alcohol exerts an important part of its action in the brain, we are confident about our findings and conclusions. However, many important open questions remain to be answered. [Copyright &y& Elsevier]

Abstract: GABA-A receptors mediate both phasic synaptic inhibition and more recently appreciated tonic currents in the vertebrate central nervous system. We addressed discrepancies in the literature regarding the pharmacology of tonic currents by examining tonic currents in a controlled environment of dissociated, solitary glutamatergic neurons. We describe a novel tonically active, bicuculline-sensitive chloride conductance that is insensitive to gabazine and to picrotoxin and thus not mediated by conventional GABA receptors. We exclude a significant contribution from small conductance calcium-gated potassium (SK) channels. We also pharmacologically exclude calcium-gated chloride channels, glycine receptors and the chloride current associated with glutamate transport. Finally, we demonstrate that, although small, this current modulates neuronal excitability. We speculate that this tonic current may provide a complementary mechanism for the regulation of neuronal excitability, particularly in regions with low ambient GABA concentrations. We conclude that this bicuculline-sensitive conductance needs to be accounted for in studies of GABA tonic currents, lest it be confused with currents associated with GABA overflow. [Copyright &y& Elsevier]

Abstract: GABAA receptors (GABAARs) are the main inhibitory neurotransmitter receptors and have long been implicated in mediating at least part of the acute actions of ethanol. For example, ethanol and GABAergic drugs including barbiturates and benzodiazepines share many pharmacological properties. Besides the prototypical synaptic GABAAR subtypes, nonsynaptic GABAARs have recently emerged as important regulators of neuronal excitability. While high doses (≥100 mM) of ethanol have been reported to enhance activity of most GABAAR subtypes, most abundant synaptic GABAARs are essentially insensitive to ethanol concentrations that occur during social ethanol consumption (<30 mM). However, extrasynaptic δ and β3 subunit-containing GABAARs, associated in the brain with α4 or α6 subunits, are sensitive to low millimolar ethanol concentrations, as produced by drinking half a glass of wine. Additionally, we found that a mutation in the cerebellar α6 subunit (α6R100Q), initially reported in rats selectively bred for increased alcohol sensitivity, is sufficient to produce increased alcohol-induced motor impairment and further increases of alcohol sensitivity in recombinant α6β3δ receptors. Furthermore, the behavioral alcohol antagonist Ro15-4513 blocks the low dose alcohol enhancement on α4/6/β3δ receptors, without reducing GABA-induced currents. In binding assays α4β3δ GABAARs bind [3H]Ro15-4513 with high affinity, and this binding is inhibited, in an apparently competitive fashion, by low ethanol concentrations, as well as analogs of Ro15-4513 that are active to antagonize ethanol or Ro15-4513''s block of ethanol. We conclude that most low to moderate dose alcohol effects are mediated by alcohol actions on alcohol/Ro15-4513 binding sites on GABAAR subtypes. [Copyright &y& Elsevier]

Abstract: The potassium-chloride cotransporter 2 (KCC2)-dependent intracellular chloride level determines whether neurons respond to GABA and/or glycine by depolarization or hyperpolarization. However, still unknown is the role of KCC2-dependent chloride homeostasis in regulating the spontaneous activity of neuronal circuits via GABAA receptor (GABAAR) and the glycine receptor (GlyR). In this study, patch-clamp recordings were performed to measure the change of spontaneous neuronal activity in cultured hippocampal neurons. Our results showed that inhibition of KCC2 with furosemide, as well as blockade of GABAAR with bicuculline, significantly enhanced circuit activity. Perfusion with bicuculline further enhanced the effects of furosemide on spontaneous circuit activity, while furosemide did not alter the effects of bicuculline. Surprisingly, blockade of GlyR not only induced obvious tonic currents, but also significantly decreased spontaneous synaptic activity. Moreover, inhibition of KCC2 did not change the depressive effect of strychnine on neuronal circuits. Our findings suggest that KCC2-dependent chloride homeostasis is mainly involved in GABAAR-mediated synaptic inhibition whereas GlyR-mediated tonic action plays a totally different role in regulating hippocampal circuit activity. [Copyright &y& Elsevier]

Abstract: Thiocolchicoside (TCC) is used clinically for its muscle relaxant, anti-inflammatory, and analgesic properties, and it has been shown to interact with γ-aminobutyric acid (GABA) type A receptors (GABAARs) and strychnine-sensitive glycine receptors in the rat central nervous system. In contrast to a proposed agonistic action at these two types of inhibitory receptors, pharmacological evidence has shown that, under certain conditions, TCC manifests convulsant activity in animals and humans. We now show that the phasic and tonic GABAAR-mediated currents recorded from Purkinje cells and granule neurons, respectively, in parasagittal cerebellar slices from adult male rats were inhibited by TCC in a concentration-dependent manner. The median inhibitory concentrations of TCC for these effects were ∼0.15 and ∼0.9μM, respectively. TCC did not potentiate GABABR-mediated currents in hippocampal slices, suggesting that its muscle relaxant action is not mediated by GABABRs. Intraperitoneal injection of TCC in rats either alone or in combination with negative modulators of GABAergic transmission revealed convulsant and proconvulsant actions of this drug. Our data, consistent with clinical observations of the epileptogenic effect of this compound, suggest that TCC is a potent competitive antagonist of GABAAR function. [Copyright &y& Elsevier]

Abstract: In this study, 48h administration of 3α-OH-5β-pregnan-20-one (3α,5β-THP) or 17β-estradiol (E2)+progesterone (P) to female rats increased expression of the δ subunit of the GABAA receptor (GABAR) in CA1 hippocampus. Coexpression of α4 and δ subunits was suggested by an increased response of isolated pyramidal cells to the GABA agonist 4,5,6,7- tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), following 48h steroid treatment, and nearly complete blockade by 300μM lanthanum (La3+). Because α4βδ GABAR are extrasynaptic, we also recorded pharmacologically isolated GABAergic holding current from CA1 hippocampal pyramidal cells in the slice. The La3+-sensitive THIP current, representative of current gated by α4βδ GABAR, was measurable only following 48h steroid treatment. In contrast, the bicuculline-sensitive current was not altered by steroid treatment, assessed with or without 200nM gabazine to block synaptic current. However, 48h steroid treatment resulted in a tonic current insensitive to the benzodiazepine agonists lorazepam (10μM) and zolpidem (100nM). These results suggest that 48h steroid treatment increases expression of α4βδ GABAR which replace the ambient receptor population. Increased anxiolytic effects of THIP were also observed following 48h steroid treatment. The findings from the present study may be relevant for alterations in mood and benzodiazepine sensitivity reported across the menstrual cycle. [Copyright &y& Elsevier]

We used the open-channel blocker, penicillin (10 mM), as a tool to investigate if the human α1β1 or α1β1γ2S γ-aminobutyric acid type A (GABAA) receptor channels opened in the absence of GABA. Application of penicillin to cells expressing the receptors resulted in a transient inward whole-cell current, the off-current, upon penicillin removal. The amplitude of the off-current was dependent on the duration of the penicillin application, it reversed in polarity at depolarized potentials and exhibited “run-down” similar to the GABA-activated currents. Bicuculline (100 μM) blocked the off-current response. Pentobarbital (50 μM) enhanced the peak off-current amplitude by 2.8 and 3.4 in α1β1 and α1β1γ2S receptors, respectively. Diazepam (1 μM) only enhanced the off-current peak response in α1β1γ2S receptors (1.6) and induced the development of an inward current when applied alone. The results are consistent with that the α1β1 or α1β1γ2S GABAA receptors can open in the absence of GABA and raise the question of what role spontaneous channel openings have in the function of GABAA receptors. [Copyright &y& Elsevier]

Background: ATP-sensitive K+ (KATP) channels couple metabolic state to cellular excitability. Activation of neuronal and astrocytic mitochondrial KATP (mitoKATP) channels regulates a variety of neuronal functions. However, less is known about the impact of mitoKATP on tonic γ-aminobutyric acid (GABA) inhibition. Tonic GABA inhibition is mediated by the binding of ambient GABA on extrasynaptic GABA A-type receptors (GABAARs) and is involved in regulating neuronal excitability., Methods: We determined the impact of activation of KATP channels with diazoxide (DIZ) on tonic inhibition and recorded tonic current from rat cortical layer 5 pyramidal cells by patch-clamp recordings., Results: We found that neonatal tonic current increased with an increase in GABA concentration, which was partially mediated by the GABA A-type receptor (GABAAR) α5, and likely the δ subunits. Activation of KATP channels resulted in decreased tonic current in newborns, but there was increased tonic current during the second postnatal week., Conclusions: These findings suggest that activation of KATP channels with DIZ regulates GABAergic transmission in neocortical pyramidal cells during development.

Summary: GABAA receptors (GABAR) mediate most inhibition in the CNS and are also a target for neuroactive steroids such as 3α,5[α]β-THP (3αOH-5[α]β-OH-pregnan-20-one or [allo]pregnanolone). Although these steroids robustly enhance current gated by α1β2δ GABAR, we have shown that 3α,5[α]β-THP effects at recombinant α4β2δ GABAR depend on the direction of Cl− flux, where the steroid increases outward flux, but decreases inward flux through the receptor. This polarity-dependent inhibition of α4β2δ GABAR resulted from an increase in the rate and extent of rapid desensitization of the receptor, recorded from recombinant receptors expressed in HEK-293 cells with whole cell voltage clamp techniques. This inhibitory effect of 3α,5[α]β-THP was not observed at other receptor subtypes, suggesting it was selective for α4β2δ GABAR. Furthermore, it was prevented by a selective mutation of basic residue arginine 353 in the intracellular loop of the receptor, suggesting that this might be a putative chloride modulatory site. Expression of α4βδ GABAR increases markedly at extrasynaptic sites at the onset of puberty in female mice. At this time, 3α,5[α]β-THP decreased the inhibitory tonic current, recorded with perforated patch techniques to maintain the physiological Cl− gradient. By decreasing this shunting inhibition, 3α,5[α]β-THP increased the excitability of CA1 hippocampal pyramidal cells at puberty. These effects of the steroid were opposite to those observed before puberty when 3α,5[α]β-THP reduced neuronal excitability as a pre-synaptic effect. Behaviorally, the excitatory effect of 3α,5[α]β-THP was reflected as an increase in anxiety at the onset of puberty in female mice. Taken together, these findings suggest that the emergence of α4β2δ GABAR at the onset of puberty reverses the effect of a stress steroid. These findings may be relevant for the mood swings and increased response to stressful events reported in adolescence. [Copyright &y& Elsevier]