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Prolonged exposure to volatile anesthetics may raise the risk of developing cognitive impairment by acting on gamma-a Aminobutyric acid A receptors (GABAAR). The dentate gyrus plays an important role in the hippocampus and has a high potential for neural plasticity. However, it is unknown whether prolonged anesthesia induces a change in acute phasic or tonic inhibition in dentate gyrus granule cells (DGGCs) by acting on GABAAR. In order to verify the effects of volatile anesthetics on the current in DGGCs, a whole-cell patch was applied to record acute brain slices, and this study indicated that 4 h but not 2 h of isoflurane (ISO) exposure induced significantly larger tonic currents in DGGCs other than hippocampal CA1 pyramidal and thalamic relay neurons. Furthermore, this study demonstrated that the increased tonic current in DGGCs was dependent on the δ subunit-containing GABAARs by using transgenic δ subunit knockout mice. In conclusion, the δ subunit specific GABAAR is the key element that increased acute tonic inhibition in DGGCs of mice after prolonged ISO exposure, which may be one of the mechanisms of ISO neurotoxicity to the developing brain.

Aim: We examined if tonic γ‐aminobutyric acid (GABA)‐activated currents in primary hippocampal neurons were modulated by insulin in wild‐type and tg‐APPSwe mice, an Alzheimer's disease (AD) model. Methods: GABA‐activated currents were recorded in dentate gyrus (DG) granule cells and CA3 pyramidal neurons in hippocampal brain slices, from 8 to 10 weeks old (young) wild‐type mice and in dorsal DG granule cells in adult, 5‐6 and 10‐12 (aged) months old wild‐type and tg‐APPSwe mice, in the absence or presence of insulin, by whole‐cell patch‐clamp electrophysiology. Results: In young mice, insulin (1 nmol/L) enhanced the total spontaneous inhibitory postsynaptic current (sIPSCT) density in both dorsal and ventral DG granule cells. The extrasynaptic current density was only increased by insulin in dorsal CA3 pyramidal neurons. In absence of action potentials, insulin enhanced DG granule cells and dorsal CA3 pyramidal neurons miniature IPSC (mIPSC) frequency, consistent with insulin regulation of presynaptic GABA release. sIPSCT densities in DG granule cells were similar in wild‐type and tg‐APPSwe mice at 5‐6 months but significantly decreased in aged tg‐APPSwe mice where insulin normalized currents to wild‐type levels. The extrasynaptic current density was increased in tg‐APPSwe mice relative to wild‐type littermates but, only in aged tg‐APPSwe mice did insulin decrease and normalize the current. Conclusion: Insulin effects on GABA signalling in hippocampal neurons are selective while multifaceted and context‐based. Not only is the response to insulin related to cell‐type, hippocampal axis‐location, age of animals and disease but also to the subtype of neuronal inhibition involved, synaptic or extrasynaptic GABAA receptors‐activated currents. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid (GABA) depolarizes embryonic cerebrocortical neurons and continuous activation of the GABAA receptor (GABAAR) contributes to their tonic depolarization. Although multiple reports have demonstrated a role of GABAAR activation in neocortical development, including in migration, most of these studies have used pharmacological blockers. Herein, we performed in utero electroporation in GABA synthesis-lacking homozygous GAD67-GFP knock-in mice (GAD67GFP/GFP) to label neurons born in the ventricular zone. Three days after electroporation, there were no differences in the distribution of labeled cells between the genotypes. The dose-response properties of cells labeled to detect GABA were equivalent among genotypes. However, continuous blockade of GABAAR with the GABAAR antagonist SR95531 accelerated radial migration. This effect of GABAAR blockade in GAD67GFP/GFP mice suggested a role for alternative endogenous GABAAR agonists. Thus, we tested the role of taurine, which is derived from maternal blood but is abundant in the fetal brain. The taurine-evoked currents in labeled cells were mediated by GABAAR. Taurine uptake was blocked by a taurine transporter inhibitor, 2-(guanidino)ethanesulfonic acid (GES), and taurine release was blocked by a volume-sensitive anion channel blocker, 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB), as examined through high-performance liquid chromatography (HPLC). GES increased the extracellular taurine concentration and induced an inward shift of the holding current, which was reversed by SR95531. In a taurine-deficient mouse model, the GABAAR-mediated tonic currents were greatly reduced, and radial migration was accelerated. As the tonic currents were equivalent among the genotypes of GAD67-GFP knock-in mice, taurine, rather than GABA, might play a major role as an endogenous agonist of embryonic tonic GABAAR conductance, regulating the radial migration of neurons in the developing neocortex.

Hydrogen peroxide (H2O2), a key reactive oxygen species, is produced at low levels during normal cellular metabolism and at higher concentrations under pathological conditions such as ischemia-reperfusion injury. The mechanisms by which H2O2 contributes to physiological and pathological processes in the brain remain poorly understood. Inhibitory GABA type A (GABAA) receptors critically regulate brain function by generating tonic and synaptic currents; however, it remains unknown whether H2O2 directly modulates GABAA receptor function. Here, we performed patch-clamp recordings, together with pharmacological and genetic approaches, to investigate the effects of H2O2 on GABAA receptor-mediated tonic and synaptic currents recorded in cultured mouse hippocampal neurons and CA1 pyramidal neurons in hippocampal slices. We found that H2O2 caused a dramatic increase in tonic current, whereas synaptic currents were unaffected. This increase in tonic current resulted from an extracellular oxidative reaction, which increased the potency of GABA, but only when GABAA receptors were activated by low concentrations of GABA. Oxygen-glucose deprivation, which produces high endogenous levels of H2O2, similarly increased the tonic current. These results suggest that GABAA receptor-mediated tonic current, which is potentiated by H2O2, might contribute to H2O2-induced brain dysfunction. [ABSTRACT FROM AUTHOR]

γ -Aminobutyric acid (GABA) depolarizes embryonic cerebrocortical neurons and continuous activation of the GABAA receptor (GABAAR) contributes to their tonic depolarization. Although multiple reports have demonstrated a role of GABAAR activation in neocortical development, including in migration, most of these studies have used pharmacological blockers. Herein, we performed in utero electroporation in GABA synthesis-lacking homozygous GAD67-GFP knock-in mice (GAD67GFP/GFP) to label neurons born in the ventricular zone. Three days after electroporation, there were no differences in the distribution of labeled cells between the genotypes. The dose-response properties of cells labeled to detect GABA were equivalent among genotypes. However, continuous blockade of GABAAR with the GABAAR antagonist SR95531 accelerated radial migration. This effect of GABAAR blockade in GAD67GFP/GFP mice suggested a role for alternative endogenous GABAAR agonists. Thus, we tested the role of taurine, which is derived from maternal blood but is abundant in the fetal brain. The taurine-evoked currents in labeled cells were mediated by GABAAR. Taurine uptake was blocked by a taurine transporter inhibitor, 2-(guanidino)ethanesulfonic acid (GES), and taurine release was blocked by volume-sensitive anion channel blocker, 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB), as examined through high-performance liquid chromatography (HPLC). GES increased the extracellular taurine concentration and induced an inward shift of the holding current, which was reversed by SR95531. In a taurine-deficient mouse model, the GABAAR-mediated tonic currents were greatly reduced, and radial migration was accelerated. As the tonic currents were equivalent among the genotypes of GAD67-GFP knock-in mice, taurine, rather than GABA, might play a major role as an endogenous agonist of embryonic tonic GABAAR conductance, regulating the radial migration of neurons in the developing neocortex. [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]

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: 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: 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]

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]