GABA Currents Research Articles

β2-nicotinic acetylcholine receptor promotes development of GABAA receptors in mouse hippocampal CA1 and CA3 pyramidal neurons

To explore the role of β2-nicotinic acetylcholine receptor (β2-nAChR) in the development of γ- aminobutyric acid A type receptors (GABAA-Rs) in hippocampal CA1 and CA3 pyramidal neurons of mice. The hippocampal CA1 and CA3 pyramidal neurons were acutely isolated from β2-nAChR gene knockout (β2-KO group) mice. GABA currents in CA1 and CA3 pyramidal neurons were induced with the selective GABAA-R agonist muscimol and recorded using perforated patch-clamp recording technique. The GABA currents of CA1 and CA3 pyramidal neurons were tested for their equilibrium potentials (EMuss) and kinetic parameters and were compared with the measurements in wild-type mice (WT group). The mean EMus of CA1 neurons (n=7) of β2-KO mice (n=4) was -31.7±3.5 mV, showing an obvious depolarizing shift compared with the WT mice (P < 0.05); the mean EMus of CA3 neurons (n=4) was -16.1±4.6 mV, also showing a depolarizing shift (P < 0.01). The difference in the EMuss between CA3 and CA1 neurons in β2-KO mice, but not in WT mice, was significant (P < 0.05). The GABAA-R desensitization was significantly slowed down in both CA1 and CA3 neurons of β2-KO mice, with decay time of 2.2±0.2 s and 3.2±0.1 s, respectively, significantly longer than those in WT mice (1.6±0.1 s and 2.3±0.1 s, respectively; P < 0.05). β2-containing nAChRs may promote the functional maturation of GABAA-R in CA1 and CA3 pyramidal cells in mouse hippocampus.

Differential modulation of human GABAC-\u03c11 receptor by sulfur-containing compounds structurally related to taurine

BackgroundThe amino acid taurine (2-Aminoethanesulfonic acid) modulates inhibitory neurotransmitter receptors. This study aimed to determine if the dual action of taurine on GABAC-ρ1R relates to its structure. To address this, we tested the ability of the structurally related compounds homotaurine, hypotaurine, and isethionic acid to modulate GABAC-ρ1R.ResultsIn Xenopus laevis oocytes, hypotaurine and homotaurine partially activate heterologously expressed GABAC-ρ1R, showing an increment in its deactivation time with no changes in channel permeability, whereas isethionic acid showed no effect. Competitive assays suggest that hypotaurine and homotaurine compete for the GABA-binding site. In addition, their effects were blocked by the ion-channel blockers picrotixin and Methyl(1,2,5,6-tetrahydropyridine-4-yl) phosphinic acid. In contrast to taurine, co-application of GABA with hypotaurine or homotaurine revealed that the dual effect is present separately for each compound: hypotaurine modulates positively the GABA current, while homotaurine shows a negative modulation, both in a dose-dependent manner. Interestingly, homotaurine diminished hypotaurine-induced currents. Thus, these results strongly suggest a competitive interaction between GABA and homotaurine or hypotaurine for the same binding site. “In silico” modeling confirms these observations, but it also shows a second binding site for homotaurine, which could explain the negative effect of this compound on the current generated by GABA or hypotaurine, during co-application protocols.ConclusionsThe sulfur-containing compounds structurally related to taurine are partial agonists of GABAC-ρ1R that occupy the agonist binding site. The dual effect is unique to taurine, whereas in the case of hypotaurine and homotaurine it presents separately; hypotaurine increases and homotaurine decreases the GABA current.

2-(4-methyl-thiazol-5-yl) ethyl nitrate maleate-potentiated GABAA receptor response in hippocampal neurons.

2-(4-methyl-thiazol-5-yl) ethyl nitrate maleate (NMZM), a derivative of clomethiazole (CMZ), had been investigated for the treatment of Alzheimer's disease (AD). The beneficial effects of NMZM in AD included reversing cognitive deficit, improving learning and memory as well as neuroprotection. The pharmacological effects of NMZM on GABAA receptors were reported previously; however, the mechanisms were unclear and were explored therefore. In this study, we demonstrated that NMZM improved learning and memory by alleviating scopolamine-induced long-term potentiation (LTP) suppression in the dentate gyrus of rats, indicating that NMZM had protective effects against scopolamine-induced depression of LTP. Next, we investigated the action of NMZM on GABAA receptors in hippocampal neurons and the binding site of NMZM on GABAA receptors. NMZM directly activated GABAA receptors in hippocampal neurons in a weak manner. However, NMZM could potentiate the response of GABAA receptors to GABA and NMZM positively modulated GABAA receptors with an EC50 value of 465μmol/L at 3μmol/L GABA while this potentiation at low concentration of GABA (1, 3μmol/L) was more significant than that at high concentration (10, 30μmol/L). In addition, NMZM could enhance GABA currents after using diazepam and pentobarbital, the positive modulators of GABAA receptors. NMZM could not affect the etomidate-potentiated GABAA current. It suggested that the binding site of NMZM on GABAA receptors is the same as etomidate. These results provided support for the neuroprotective effect of NMZM, which was partly dependent on the potentiation of GABAA receptors. The etomidate binding site might be a new target for neuronal protection and for drug development.

Anticonvulsant effect of dipropofol by enhancing native GABA currents in cortical neurons in mice.

Temporal lobe epilepsy (TLE), the most common pharmacoresistant focal epilepsy disorder, remains a major unmet medical need. Propofol is used as a short-acting medication for general anesthesia and refractory status epilepticus with issues of decreased consciousness and memory loss. Dipropofol, a derivative of propofol, has been reported to exert antioxidative and antibacterial activities. Here we report that dipropofol exerted anticonvulsant activity in a mouse model of kainic acid-induced seizures. Whole cell patch-clamp recordings of brain slices from the medial entorhinal cortex (mEC) revealed that dipropofol hyperpolarized the resting membrane potential and reduced the number of action potential firings, resulting in suppression of cortical neuronal excitability. Furthermore, dipropofol activated native tonic GABAA currents of mEC layer II stellate neurons in a dose-dependent manner with an EC50 value of 9.3 ± 1.6 μM (mean ± SE). Taken together, our findings show that dipropofol activated GABAA currents and exerted anticonvulsant activities in mice, thus possessing developmental potential for new anticonvulsant therapy. NEW & NOTEWORTHY The anticonvulsant effect of dipropofol was shown in a mouse model of kainic acid-induced seizures. Whole cell patch-clamp recordings of brain slices showed suppression of cortical neuronal excitability by dipropofol. Dipropofol activated the native tonic GABAA currents in a dose-dependent manner.

Cerebellar α6 -subunit-containing GABAA receptors: a novel therapeutic target for disrupted prepulse inhibition in neuropsychiatric disorders.

The pathophysiological role of α6 -subunit-containing GABAA receptors, which are mainly expressed in cerebellar granule cells, remains unclear. Recently, we demonstrated that hispidulin, a flavonoid isolated from a local herb that remitted a patient's intractable motor tics, attenuated methamphetamine-induced hyperlocomotion in mice as a positive allosteric modulator (PAM) of cerebellar α6 GABAA receptors. Here, using hispidulin and a selective α6 GABAA receptor PAM, the pyrazoloquinolinone Compound 6, we revealed an unprecedented role of cerebellar α6 GABAA receptors in disrupted prepulse inhibition of the startle response (PPI), which reflects sensorimotor gating deficits manifested in several neuropsychiatric disorders. PPI disruptions were induced by methamphetamine and NMDA receptor antagonists in mice. Effects of the tested compounds were measured in Xenopus oocytes expressing recombinant α6 β3 γ2S GABAA receptors. Hispidulin given i.p. or by bilateral intracerebellar (i.cb.) injection rescued PPI disruptions induced by methamphetamine, ketamine, MK-801 and phencyclidine. Intracerebellar effects of hispidulin were mimicked by Ro15-4513 and loreclezole (two α6 GABAA receptor PAMs), but not by diazepam (an α6 GABAA receptor-inactive benzodiazepine) and were antagonized by furosemide (i.cb.), an α6 GABAA receptor antagonist. Importantly, Compound 6 (i.p.) also rescued methamphetamine-induced PPI disruption, an effect prevented by furosemide (i.cb.). Both hispidulin and Compound 6 potentiated α6 β3 γ2S GABAA receptor-mediated GABA currents. Positive allosteric modulation of cerebellar α6 GABAA receptors rescued disrupted PPI by attenuating granule cell activity. α6 GABAA receptor-selective PAMs are potential medicines for treating sensorimotor gating deficits in neuropsychiatric disorders. A mechanistic hypothesis is based on evidence for cerebellar contributions to cognitive functioning including sensorimotor gating.

The antihelminthic moxidectin enhances tonic GABA currents in rodent hippocampal pyramidal neurons.

Macrocyclic lactones (MLs) are commonly used treatments for parasitic worm and insect infections in humans, livestock, and companion animals. MLs target the invertebrate glutamate-activated chloride channel that is not present in vertebrates. MLs are not entirely inert in vertebrates, though; they have been reported to have activity in heterologous expression systems consisting of ligand-gated ion channels that are present in the mammalian central nervous system (CNS). However, these compounds are typically not able to reach significant concentrations in the CNS because of the activity of the blood-brain barrier P-glycoprotein extrusion system. Despite this, these compounds are able to reach low levels in the CNS that may be useful in the design of novel "designer" ligand-receptor systems that can be used to directly investigate neuronal control of behavior in mammals and have potential for use in treating human neurological diseases. To determine whether MLs might affect neurons in intact brains, we investigated the activity of the ML moxidectin (MOX) at native GABA receptors. Specifically, we recorded tonic and phasic miniature inhibitory postsynaptic currents (mIPSCs) in ex vivo brain slices. Our data show that MOX potentiated tonic GABA currents in a dose-dependent manner but had no concomitant effects on phasic GABA currents (i.e., MOX had no effect on the amplitude, frequency, or decay kinetics of mIPSCs). These studies indicate that behavioral experiments that implement a ML-based novel ligand-receptor system should take care to control for potential effects of the ML on native tonic GABA receptors. NEW & NOTEWORTHY We have identified a novel mechanism of action in the mammalian central nervous system for the antihelminthic moxidectin, commonly prescribed to animals worldwide and currently being evaluated for use in humans. Specifically, moxidectin applied to rodent brain slices selectively enhanced the tonic GABA conductance of hippocampal pyramidal neurons.

A new approach to detect the coding rule of the cortical spiking model in the information transmission

Investigation of the role of the local field potential (LFP) fluctuations in encoding the received sensory information by the nervous system remains largely unknown. On the other hand, transmission of these translation rules in information transmission between the structure of sensory stimuli and the cortical oscillations to the bio-inspired artificial neural networks operating at the efficiency of the nervous system is still a vague puzzle. In order to move towards this important goal, computational neuroscience tools can be useful so, we simulated a large-scale network of excitatory and inhibitory spiking neurons with synaptic connections consisting of AMPA and GABA currents as a model of cortical populations. Spiking network was equipped with spike-based unsupervised weight optimization based on the dynamical behavior of the excitatory (AMPA) and inhibitory (GABA) synapses using Spike Timing Dependent Plasticity (STDP) on the MNIST benchmark and we specified how the generated LFP by the network contained information about input patterns. The main result of this article is that the calculated coefficients of Prolate spheroidal wave functions (PSWF) from the input pattern with mean square error (MSE) criterion and power spectrum of LFP with maximum correntropy criterion (MCC) are equal. The more important result is that 82.3% of PSWF coefficients are the same as the connecting weights of the cortical neurons to the classifying neurons after the completion of the training process. Higher compliance percentage of coefficients with synaptic weights (82.3%) gives the expectance us that this coding rule will be able to extend to biological systems. Eventually, we introduced the cortical spiking network as an information channel, which transmits the information of the input pattern in the form of PSWF coefficients to the power spectrum of the output generated LFP.

Antagonistic effect of dopamine structural analogues on human GABA\u03c11 receptor

GABAergic and dopaminergic pathways are co-localized in several areas of the central nervous system and recently several reports have shown co-release of both neurotransmitters. The GABA-A receptor (β and ρ1 subunits) is modulated by dopamine (DA) and, interestingly, GABAρ1 can be modulated by several biogenic amines. Here we explored the effects of the metabolites of the dopaminergic pathway and other structural analogues of DA on GABAρ1 and the DA gated ion channel (LGC-53) from Caenorhabditis elegans expressed in Xenopus laevis oocytes. Our findings show an antagonistic effect of the metabolite 3-Methoxytyramine (3-MT, IC50 = 285 ± 30 µM) with similar potency compared to DA on induced GABA currents; however, it was inactive on LGC-53. The structural DA analogues and metabolites, 3, 4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 2-phenylethylamine (β-PEA) and 4-amino-1-butanol (4-AM-1-OH), antagonized GABAρ1 currents, whereas β-PEA acted as partial agonists on LGC-53, indicating that the putative binding sites of both receptors may share structural characteristics. These results suggest that the DA metabolites 3-MT, DOPAC and HVA modulate GABAρ1 and possibly affect the activity of the receptors that include this subunit in vivo.

Gad67 haploinsufficiency reduces amyloid pathology and rescues olfactory memory deficits in a mouse model of Alzheimer\u2019s disease

BackgroundAlzheimer’s disease (AD) is the most common age-related neurodegenerative disorder, affecting millions of people worldwide. Although dysfunction of multiple neurotransmitter systems including cholinergic, glutamatergic and GABAergic systems has been associated with AD progression the underlying mechanisms remain elusive. We and others have recently found that GABA content is elevated in AD brains and linked to cognitive deficits in AD mouse models. The glutamic acid decarboxylase 67 (GAD67) is the major enzyme converting glutamate into GABA and has been implied in a number of neurological disorders such as epilepsy and schizophrenia. However, whether Gad67 is involved in AD pathology has not been well studied. Here, we investigate the functional role of GAD67 in an AD mouse model with Gad67 haploinsufficiency that is caused by replacing one allele of Gad67 with green fluorescent protein (GFP) gene during generation of GAD67-GFP mice.MethodsTo genetically reduce GAD67 in AD mouse brains, we crossed the Gad67 haploinsufficient mice (GAD67-GFP+/−) with 5xFAD mice (harboring 5 human familial AD mutations in APP and PS1 genes) to generate a new line of bigenic mice. Immunostaining, ELISA, electrophysiology and behavior test were applied to compare the difference between groups.ResultsWe found that reduction of GAD67 resulted in a significant decrease of amyloid β production in 5xFAD mice. Concurrently, the abnormal astrocytic GABA and tonic GABA currents, as well as the microglial reactivity were significantly reduced in the 5xFAD mice with Gad67 haploinsufficiency. Importantly, the olfactory memory deficit of 5xFAD mice was rescued by Gad67 haploinsufficiency.ConclusionsOur results demonstrate that GAD67 plays an important role in AD pathology, suggesting that GAD67 may be a potential drug target for modulating the progress of AD.

Orthosteric- versus allosteric-dependent activation of the GABAA receptor requires numerically distinct subunit level rearrangements

Anaesthetic molecules act on synaptic transmission via the allosteric modulation of ligand-gated chloride channels, such as hetero-oligomeric α1β2γ2 GABAA receptors. To elucidate the overall activation paradigm via allosteric versus orthosteric sites, we used highly homologous, but homo-oligomeric, ρ1 receptors that are contrastingly insensitive to anaesthetics and respond partially to several full GABA α1β2γ2 receptor agonists. Here, we coexpressed varying ratios of RNAs encoding the wild-type and the mutated ρ1 subunits, which are anaesthetic-sensitive and respond with full efficacy to partial GABA agonists, to generate distinct ensembles of receptors containing five, four, three, two, one, or zero mutated subunits. Using these experiments, we then demonstrate that, in the pentamer, three anaesthetic-sensitive ρ1 subunits are needed to impart full efficacy to the partial GABA agonists. By contrast, five anaesthetic-sensitive subunits are required for direct activation by anaesthetics alone, and only one anaesthetic-sensitive subunit is sufficient to confer the anaesthetic-dependent potentiation to the GABA current. In conclusion, our data indicate that GABA and anaesthetics holistically activate the GABAA ρ1 receptor through distinct subunit level rearrangements and suggest that in contrast to the global impact of GABA via orthosteric sites, the force of anaesthetics through allosteric sites may not propagate to the neighbouring subunits and, thus, may have only a local and limited effect on the ρ1 GABAA receptor model system.

Recurrently connected and localized neuronal communities initiate coordinated spontaneous activity in neuronal networks.

Developing neuronal systems intrinsically generate coordinated spontaneous activity that propagates by involving a large number of synchronously firing neurons. In vivo, waves of spikes transiently characterize the activity of developing brain circuits and are fundamental for activity-dependent circuit formation. In vitro, coordinated spontaneous spiking activity, or network bursts (NBs), interleaved within periods of asynchronous spikes emerge during the development of 2D and 3D neuronal cultures. Several studies have investigated this type of activity and its dynamics, but how a neuronal system generates these coordinated events remains unclear. Here, we investigate at a cellular level the generation of network bursts in spontaneously active neuronal cultures by exploiting high-resolution multielectrode array recordings and computational network modelling. Our analysis reveals that NBs are generated in specialized regions of the network (functional neuronal communities) that feature neuronal links with high cross-correlation peak values, sub-millisecond lags and that share very similar structural connectivity motifs providing recurrent interactions. We show that the particular properties of these local structures enable locally amplifying spontaneous asynchronous spikes and that this mechanism can lead to the initiation of NBs. Through the analysis of simulated and experimental data, we also show that AMPA currents drive the coordinated activity, while NMDA and GABA currents are only involved in shaping the dynamics of NBs. Overall, our results suggest that the presence of functional neuronal communities with recurrent local connections allows a neuronal system to generate spontaneous coordinated spiking activity events. As suggested by the rules used for implementing our computational model, such functional communities might naturally emerge during network development by following simple constraints on distance-based connectivity.

Novel Molecule Exhibiting Selective Affinity for GABAA Receptor Subtypes

Aminoquinoline derivatives were evaluated against a panel of receptors/channels/transporters in radioligand binding experiments. One of these derivatives (DCUK-OEt) displayed micromolar affinity for brain γ-aminobutyric acid type A (GABAA) receptors. DCUK-OEt was shown to be a positive allosteric modulator (PAM) of GABA currents with α1β2γ2, α1β3γ2, α5β3γ2 and α1β3δ GABAA receptors, while having no significant PAM effect on αβ receptors or α1β1γ2, α1β2γ1, α4β3γ2 or α4β3δ receptors. DCUK-OEt modulation of α1β2γ2 GABAA receptors was not blocked by flumazenil. The subunit requirements for DCUK-OEt actions distinguished DCUK-OEt from other currently known modulators of GABA function (e.g., anesthetics, neurosteroids or ethanol). Simulated docking of DCUK-OEt at the GABAA receptor suggested that its binding site may be at the α + β- subunit interface. In slices of the central amygdala, DCUK-OEt acted primarily on extrasynaptic GABAA receptors containing the α1 subunit and generated increases in extrasynaptic “tonic” current with no significant effect on phasic responses to GABA. DCUK-OEt is a novel chemical structure acting as a PAM at particular GABAA receptors. Given that neurons in the central amygdala responding to DCUK-OEt were recently identified as relevant for alcohol dependence, DCUK-OEt should be further evaluated for the treatment of alcoholism.

Modulation of Tonic GABA Currents by Anion Channel and Connexin Hemichannel Antagonists.

Anion channels and connexin hemichannels are permeable to amino acid neurotransmitters. It is hypothesized that these conductive pathways release GABA, thereby influencing ambient GABA levels and tonic GABAergic inhibition. To investigate this, we measured the effects of anion channel/hemichannel antagonists on tonic GABA currents of rat hippocampal neurons. In contrast to predictions, blockade of anion channels and hemichannels with NPPB potentiated tonic GABA currents of neurons in culture and acute hippocampal slices. In contrast, the anion channel/hemichannel antagonist carbenoxolone (CBX) inhibited tonic currents. These findings could result from alterations of ambient GABA concentration or direct effects on GABAA receptors. To test for effects on GABAA receptors, we measured currents evoked by exogenous GABA. Coapplication of NPPB with GABA potentiated GABA-evoked currents. CBX dose-dependently inhibited GABA-evoked currents. These results are consistent with direct effects of NPPB and CBX on GABAA receptors. GABA release from hippocampal cell cultures was directly measured using HPLC. Inhibition of anion channels with NPPB or CBX did not affect GABA release from cultured hippocampal neurons. NPPB reduced GABA release from pure astrocytic cultures by 21%, but the total GABA release from astrocytes was small compared to that of mixed cultures. These data indicate that drugs commonly used to antagonize anion channels and connexin hemichannels may affect tonic currents via direct effects on GABAA receptors and have negligible effects on ambient GABA concentrations. Interpretation of experiments using NPPB or CBX should include consideration of their effects on tonic GABA currents.

Methylene blue inhibits GABAA receptors by interaction with GABA binding site

Methylene blue (MB) is commonly used in diagnostic procedures and is also used to treat various medical conditions. Neurological effects of MB have been reported in clinical observations and experimental studies. Thus the modulation of GABAA receptor function by MB was investigated. Whole-cell GABA-activated currents were recorded from HEK293 cells expressing various GABAA receptor subunit configurations. MB inhibition of GABA currents was apparent at 3 μM, and it had an IC50 of 31 μM in human α1β2γ2 receptors. The MB action was rapid and reversible. MB inhibition was not mediated via the picrotoxin site, as a mutation (T6′F of the β2 subunit) known to confer resistance to picrotoxin had no effect on MB-induced inhibition. Blockade of GABAA receptors by MB was demonstrated across a range of receptors expressing varying subunits, including those expressed at extrasynaptic sites. The sensitivity of α1β2 receptors to MB was similar to that observed in α1β2γ2 receptors, indicating that MB's action via the benzodiazepine or Zn2+ site is unlikely. MB-induced inhibition of GABA response was competitive with respect to GABA. Furthermore, mutation of α1 F64 to A and β2 Y205 to F in the extracellular N-terminus, both residues which are known to comprise GABA binding pocket, remarkably diminished MB inhibition of GABA currents. These data suggest that MB inhibits GABAA receptor function by direct or allosteric interaction with the GABA binding site. Finally, in mouse hippocampal CA1 pyramidal neurons, MB inhibited GABA-activated currents as well as GABAergic IPSCs. We demonstrate that MB directly inhibits GABAA receptor function, which may underlie some of the effects of MB on the CNS.

Loss of Plasticity in the D2-Accumbens Pallidal Pathway Promotes Cocaine Seeking

Distinct populations of D1- and D2-dopamine receptor-expressing medium spiny neurons (D1-/D2-MSNs) comprise the nucleus accumbens, and activity in D1-MSNs promotes, whereas activity in D2-MSNs inhibits, motivated behaviors. We used chemogenetics to extend D1-/D2-MSN cell specific regulation to cue-reinstated cocaine seeking in a mouse model of self-administration and relapse, and found that either increasing activity in D1-MSNs or decreasing activity in D2-MSNs augmented cue-induced reinstatement. Both D1- and D2-MSNs provide substantial GABAergic innervation to the ventral pallidum, and chemogenetic inhibition of ventral pallidal neurons blocked the augmented reinstatement elicited by chemogenetic regulation of either D1- or D2-MSNs. Because D1- and D2-MSNs innervate overlapping populations of ventral pallidal neurons, we next used optogenetics to examine whether changes in synaptic plasticity in D1- versus D2-MSN GABAergic synapses in the ventral pallidum could explain the differential regulation of VP activity. In mice trained to self-administer cocaine, GABAergic LTD was abolished in D2-, but not in D1-MSN synapses. A μ opioid receptor antagonist restored GABA currents in D2-, but not D1-MSN synapses of cocaine-trained mice, indicating that increased enkephalin tone on presynaptic μ opioid receptors was responsible for occluding the LTD. These results identify a behavioral function for D1-MSN innervation of the ventral pallidum, and suggest that losing LTD_GABA in D2-MSN, but not D1-MSN input to ventral pallidum may promote cue-induced reinstatement of cocaine-seeking. <b>SIGNIFICANCE STATEMENT</b> More than 90% of ventral striatum is composed of two cell types, those expressing dopamine D1 or D2 receptors, which exert opposing roles on motivated behavior. Both cell types send GABAergic projections to the ventral pallidum and were found to differentially promote cue-induced reinstatement of cocaine seeking via the ventral pallidum. Furthermore, after cocaine self-administration, synaptic plasticity was selectively lost in D2, but not D1 inputs to the ventral pallidum. The selective impairment in D2 afferents may promote the influence of D1 inputs to drive relapse to cocaine seeking.

A Functional Switch in Tonic GABA Currents Alters the Output of Central Amygdala Corticotropin Releasing Factor Receptor-1 Neurons Following Chronic Ethanol Exposure.

The corticotropin releasing factor (CRF) system in the central amygdala (CeA) has been implicated in the effects of acute and chronic ethanol. We showed previously that CRF receptor 1-expressing (CRF1+) neurons in the CeA are under tonic inhibitory control and are differentially regulated by acute ethanol (Herman et al., 2013). Here we show that the inhibitory control of CRF1+ CeA neurons is lost with chronic ethanol exposure, likely by a functional switch in local tonic signaling. The loss of tonic inhibition is seen in CRF1+ projection neurons, suggesting that a critical consequence of chronic ethanol exposure is an increase in the output of the CeA CRF1 system, a neuroadaptation that may contribute to the behavioral consequences of alcohol dependence.

Acute Stress Suppresses Synaptic Inhibition and Increases Anxiety via Endocannabinoid Release in the Basolateral Amygdala

Stress and glucocorticoids stimulate the rapid mobilization of endocannabinoids in the basolateral amygdala (BLA). Cannabinoid receptors in the BLA contribute to anxiogenesis and fear-memory formation. We tested for rapid glucocorticoid-induced endocannabinoid regulation of synaptic inhibition in the rat BLA. Glucocorticoid application to amygdala slices elicited a rapid, nonreversible suppression of spontaneous, but not evoked, GABAergic synaptic currents in BLA principal neurons; the effect was also seen with a membrane-impermeant glucocorticoid, but not with intracellular glucocorticoid application, implicating a membrane-associated glucocorticoid receptor. The glucocorticoid suppression of GABA currents was not blocked by antagonists of nuclear corticosteroid receptors, or by inhibitors of gene transcription or protein synthesis, but was blocked by inhibiting postsynaptic G-protein activity, suggesting a postsynaptic nongenomic steroid signaling mechanism that stimulates the release of a retrograde messenger. The rapid glucocorticoid-induced suppression of inhibition was prevented by blocking CB1 receptors and 2-arachidonoylglycerol (2-AG) synthesis, and it was mimicked and occluded by CB1 receptor agonists, indicating it was mediated by the retrograde release of the endocannabinoid 2-AG. The rapid glucocorticoid effect in BLA neurons in vitro was occluded by prior in vivo acute stress-induced, or prior in vitro glucocorticoid-induced, release of endocannabinoid. Acute stress also caused an increase in anxiety-like behavior that was attenuated by blocking CB1 receptor activation and inhibiting 2-AG synthesis in the BLA. Together, these findings suggest that acute stress causes a long-lasting suppression of synaptic inhibition in BLA neurons via a membrane glucocorticoid receptor-induced release of 2-AG at GABA synapses, which contributes to stress-induced anxiogenesis. We provide a cellular mechanism in the basolateral amygdala (BLA) for the rapid stress regulation of anxiogenesis in rats. We demonstrate a nongenomic glucocorticoid induction of long-lasting suppression of synaptic inhibition that is mediated by retrograde endocannabinoid release at GABA synapses. The rapid glucocorticoid-induced endocannabinoid suppression of synaptic inhibition is initiated by a membrane-associated glucocorticoid receptor in BLA principal neurons. We show that acute stress increases anxiety-like behavior via an endocannabinoid-dependent mechanism centered in the BLA. The stress-induced endocannabinoid modulation of synaptic transmission in the BLA contributes, therefore, to the stress regulation of anxiety, and may play a role in anxiety disorders of the amygdala.

Zolpidem is a potent stoichiometry-selective modulator of α1β3 GABAA receptors: evidence of a novel benzodiazepine site in the α1-α1 interface.

Zolpidem is not a typical GABAA receptor hypnotic. Unlike benzodiazepines, zolpidem modulates tonic GABA currents in the rat dorsal motor nucleus of the vagus, exhibits residual effects in mice lacking the benzodiazepine binding site, and improves speech, cognitive and motor function in human patients with severe brain injury. The receptor by which zolpidem mediates these effects is not known. In this study we evaluated binary α1β3 GABAA receptors in either the 3α1:2β3 or 2α1:3β3 subunit stoichiometry, which differ by the existence of either an α1-α1 interface, or a β3-β3 interface, respectively. Both receptor stoichiometries are readily expressed in Xenopus oocytes, distinguished from each other by using GABA, zolpidem, diazepam and Zn2+. At the 3α1:2β3 receptor, clinically relevant concentrations of zolpidem enhanced GABA in a flumazenil-sensitive manner. The efficacy of diazepam was significantly lower compared to zolpidem. No modulation by either zolpidem or diazepam was detected at the 2α1:3β3 receptor, indicating that the binding site for zolpidem is at the α1-α1 interface, a site mimicking the classical α1-γ2 benzodiazepine site. Activating α1β3 (3α1:2β3) receptors may, in part, mediate the physiological effects of zolpidem observed under distinct physiological and clinical conditions, constituting a potentially attractive drug target.

The terpenoids Myrtenol and Verbenol act on δ subunit-containing GABAA receptors and enhance tonic inhibition in dentate gyrus granule cells

Sideritis plants and their extracts have been used in traditional medicine as sedatives, anxiolytics and anticonvulsant agents. Pinenes are the most prevalent of the volatile aroma components in Siderites extracts and the pinene metabolites myrtenol and verbenol have been identified as the most potent positive allosteric modulators of synaptic GABAA receptors composed of α1β2 and α1β2γ2 subunits. In view of their therapeutic spectrum, we wondered whether these two terpenoids would also augment tonic GABA currents mediated by extrasynaptic GABAA receptors containing the δ subunit. When we expressed α4β2δ receptors in HEK293 cells, we found that co-application of myrtenol or verbenol enhanced whole-cell current responses to GABA by up to 100%. Consistent with their effects on heterologous α1β2γ2 receptors, we found that myrtenol and verbenol, when co-applied with GABA via local perfusion, increased the amplitude and area of miniature inhibitory postsynaptic potentials (mIPSCs) recorded in whole-cell voltage-clamp recordings from granule cells in the dentate gyrus of mouse hippocampal brain slices. In addition, co-application of terpenoids with GABA was also able to enhance tonic GABA current, measured from the change in baseline current and current noise, compared to GABA perfusion alone. Our results suggest that myrtenol and verbenol act as positive allosteric modulators at synaptic and extrasynaptic GABAA receptors, thereby augmenting phasic and tonic GABAergic inhibition. Thus, our study reveals an important pharmacological and therapeutic target of bicyclic monoterpenoids.

Differential Potency of 2,6-Dimethylcyclohexanol Isomers for Positive Modulation of GABAA Receptor Currents.

GABAA receptors meet all of the pharmacological requirements necessary to be considered important targets for the action of general anesthetic agents in the mammalian brain. In the following patch-clamp study, the relative modulatory effects of 2,6-dimethylcyclohexanol diastereomers were investigated on human GABAA (α1β3γ2s) receptor currents stably expressed in human embryonic kidney cells. Cis,cis-, trans,trans-, and cis,trans-isomers were isolated from commercially available 2,6-dimethylcyclohexanol and were tested for positive modulation of submaximal GABA responses. For example, the addition of 30 μM cis,cis-isomer resulted in an approximately 2- to 3-fold enhancement of the EC20 GABA current. Coapplications of 30 μM 2,6-dimethylcyclohexanol isomers produced a range of positive enhancements of control GABA responses with a rank order for positive modulation: cis,cis > trans,trans ≥ mixture of isomers > > cis,trans-isomer. In molecular modeling studies, the three cyclohexanol isomers bound with the highest binding energies to a pocket within transmembrane helices M1 and M2 of the β3 subunit through hydrogen-bonding interactions with a glutamine at the 224 position and a tyrosine at the 220 position. The energies for binding to and hydrogen-bond lengths within this pocket corresponded with the relative potencies of the agents for positive modulation of GABAA receptor currents (cis,cis > trans,trans > cis,trans-2,6-dimethylcyclohexanol). In conclusion, the stereochemical configuration within the dimethylcyclohexanols is an important molecular feature in conferring positive modulation of GABAA receptor activity and for binding to the receptor, a consideration that needs to be taken into account when designing novel anesthetics with enhanced therapeutic indices.