Early-life stress influences acute and sensitized responses of adult mice to cocaine by interacting with GABAA α2 receptor expression.

Early-life stress (ELS) is known to exert long-term effects on brain function, with resulting deleterious consequences for several aspects of mental health, including the development of addiction to drugs of abuse. One potential mechanism in humans is suggested by findings that ELS interacts with polymorphisms of the GABRA2 gene, encoding α2 subunits of GABAA receptors, to increase the risk for both post-traumatic stress disorder and vulnerability to cocaine addiction. We used a mouse model, in which the amount of material for nest building was reduced during early postnatal life, to study interactions between ELS and expression of α2-containing GABAA receptors in influencing cocaine-related behaviour. Breeding of parents heterozygous for a deletion of α2 resulted in litters containing homozygous knockout (α2), heterozygous knockout (α2) and wild-type (α2) offspring. Following the ELS procedure, the mice were allowed to develop to adulthood before being tested for the acute effect of cocaine on locomotor stimulation, behavioural sensitization to repeated cocaine and to cocaine-conditioned activity. Exposure to ELS resulted in increased acute locomotor stimulant effects of cocaine across all genotypes, with the most marked effects in α2 mice (which also showed increased activity following vehicle). Repeated cocaine administration to nonstressed mice resulted in sensitization in α2 and α2 mice, but, in keeping with previous findings, not in α2 mice. Previous exposure to ELS reduced sensitization in α2 mice, albeit not significantly, and abolished sensitization in α2 mice. Conditioned activity was elevated following ELS in all animals, independently of genotype. Thus, while the enhanced acute effects of cocaine following ELS being most marked in α2 mice suggests a function of α2-containing GABAA receptors in protecting against stress, the interaction between ELS and genotype in influencing sensitization may be more in keeping with ELS reducing expression of α2-containing GABAA receptors. The ability of ELS to increase cocaine-conditioned locomotor activity appears to be independent of α2-containing GABAA receptors.

Isotope Dilution-Based Targeted and Nontargeted Carbonyl Neurosteroid/Steroid Profiling.

Neurosteroids are brain-derived steroids, capable of rapidly modulating neuronal excitability in a nongenomic manner. Dysregulation of their synthesis or metabolism has been implicated in many pathological conditions. Here, we describe an isotope dilution based targeted and nontargeted (ID-TNT) profiling of carbonyl neurosteroids/steroids. The method combines stable isotope dilution, hydroxylamine derivatization, high-resolution MS scanning, and data-dependent MS/MS analysis, allowing absolute quantification of pregnenolone, progesterone, 5α-dihydroprogesterone, 3α,5α-tetrahydroprogesterone, and 3ß,5α-tetrahydroprogesterone, and relative quantification of other carbonyl containing steroids. The utility and validity of this approach was tested in an acute stress mouse model and via pharmacological manipulation of the steroid metabolic pathway with finasteride. We report that brain levels of 3α,5α-tetrahydroprogesterone, a potent enhancer of GABAA receptor (GABAAR-mediated inhibitory function, from control mice is in the 5-40 pmol/g range, a value greater than previously reported. The approach allows the use of data from targeted analysis to guide the normalization strategy for nontargeted data. Furthermore, novel findings, including a striking increase of brain pregnenolone following finasteride administration were discovered in this study. Collectively, our results indicate that this approach has distinct advantages for examining targeted and nontargeted neurosteroid/steroid pathways in animal models and could facilitate a better understanding of the physiological and pathological roles of neurosteroids as modulators of brain excitability.

GABAA receptor-acting neurosteroids: a role in the development and regulation of the stress response.

Regulation of hypothalamic-pituitary-adrenocortical (HPA) axis activity by stress is a fundamental survival mechanism and HPA-dysfunction is implicated in psychiatric disorders. Adverse early life experiences, e.g. poor maternal care, negatively influence brain development and programs an abnormal stress response by encoding long-lasting molecular changes, which may extend to the next generation. How HPA-dysfunction leads to the development of affective disorders is complex, but may involve GABAA receptors (GABAARs), as they curtail stress-induced HPA axis activation. Of particular interest are endogenous neurosteroids that potently modulate the function of GABAARs and exhibit stress-protective properties. Importantly, neurosteroid levels rise rapidly during acute stress, are perturbed in chronic stress and are implicated in the behavioural changes associated with early-life adversity. We will appraise how GABAAR-active neurosteroids may impact on HPA axis development and the orchestration of the stress-evoked response. The significance of these actions will be discussed in the context of stress-associated mood disorders.

Control of Inhibition by the Direct Action of Cannabinoids on GABAA Receptors.

Cannabinoids are known to regulate inhibitory synaptic transmission via activation of presynaptic G protein-coupled cannabinoid CB1 receptors (CB1Rs). Additionally, recent studies suggest that cannabinoids can also directly interact with recombinant GABAA receptors (GABAARs), potentiating currents activated by micromolar concentrations of γ-aminobutyric acid (GABA). However, the impact of this direct interaction on GABAergic inhibition in central nervous system is unknown. Here we report that currents mediated by recombinant GABAARs activated by high (synaptic) concentrations of GABA as well as GABAergic inhibitory postsynaptic currents (IPSCs) at neocortical fast spiking (FS) interneuron to pyramidal neuron synapses are suppressed by exogenous and endogenous cannabinoids in a CB1R-independent manner. This IPSC suppression may account for disruption of inhibitory control of pyramidal neurons by FS interneurons. At FS interneuron to pyramidal neuron synapses, endocannabinoids induce synaptic low-pass filtering of GABAAR-mediated currents evoked by high-frequency stimulation. The CB1R-independent suppression of inhibition is synapse specific. It does not occur in CB1R containing hippocampal cholecystokinin-positive interneuron to pyramidal neuron synapses. Furthermore, in contrast to synaptic receptors, the activity of extrasynaptic GABAARs in neocortical pyramidal neurons is enhanced by cannabinoids in a CB1R-independent manner. Thus, cannabinoids directly interact differentially with synaptic and extrasynaptic GABAARs, providing a potent novel context-dependent mechanism for regulation of inhibition.

Molecular and functional diversity of GABA-A receptors in the enteric nervous system of the mouse colon.

The enteric nervous system (ENS) provides the intrinsic neural control of the gastrointestinal tract (GIT) and regulates virtually all GI functions. Altered neuronal activity within the ENS underlies various GI disorders with stress being a key contributing factor. Thus, elucidating the expression and function of the neurotransmitter systems, which determine neuronal excitability within the ENS, such as the GABA-GABAA receptor (GABAAR) system, could reveal novel therapeutic targets for such GI disorders. Molecular and functionally diverse GABAARs modulate rapid GABAergic-mediated regulation of neuronal excitability throughout the nervous system. However, the cellular and subcellular GABAAR subunit expression patterns within neurochemically defined cellular circuits of the mouse ENS, together with the functional contribution of GABAAR subtypes to GI contractility remains to be determined. Immunohistochemical analyses revealed that immunoreactivity for the GABAAR gamma (γ) 2 and alphas (α) 1, 2, 3 subunits was located on somatodendritic surfaces of neurochemically distinct myenteric plexus neurons, while being on axonal compartments of submucosal plexus neurons. In contrast, immunoreactivity for the α4-5 subunits was only detected in myenteric plexus neurons. Furthermore, α-γ2 subunit immunoreactivity was located on non-neuronal interstitial cells of Cajal. In organ bath studies, GABAAR subtype-specific ligands had contrasting effects on the force and frequency of spontaneous colonic longitudinal smooth muscle contractions. Finally, enhancement of γ2-GABAAR function with alprazolam reversed the stress-induced increase in the force of spontaneous colonic contractions. The study demonstrates the molecular and functional diversity of the GABAAR system within the mouse colon providing a framework for developing GABAAR-based therapeutics in GI disorders.

Tonic inhibition of accumbal spiny neurons by extrasynaptic α4ßδ 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, ß, 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 (α4(D1-/-)) showed increased CPP. Furthermore, THIP administered systemically or directly into the NAc of WT, but not α4(-/-) or α4(D1-/-) mice, blocked cocaine enhancement of CPP. In comparison, α4(D2-/-) 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, α4ßδ GABAARs may represent a viable target for the development of novel therapeutics to better understand and influence addictive behaviors.

Developmentally regulated neurosteroid synthesis enhances GABAergic neurotransmission in mouse thalamocortical neurones.

KEY POINTS: During neuronal development synaptic events mediated by GABAA receptors are progressively reduced in their duration, allowing for rapid and precise network function. Here we focused on ventrobasal thalamocortical neurones, which contribute to behaviourally relevant oscillations between thalamus and cortex. We demonstrate that the developmental decrease in the duration of inhibitory phasic events results predominantly from a precisely timed loss of locally produced neurosteroids, which act as positive allosteric modulators of the GABAA receptor. The mature thalamus retains the ability to synthesise neurosteroids, thus preserving the capacity to enhance both phasic and tonic inhibition, mediated by synaptic and extrasynaptic GABAA receptors, respectively, in physiological and pathophysiological scenarios associated with perturbed neurosteroid levels. Our data establish a potent, endogenous mechanism to locally regulate the GABAA receptor function and thereby influence thalamocortical activity. During brain development the duration of miniature inhibitory postsynaptic currents (mIPSCs) mediated by GABAA receptors (GABAA Rs) progressively reduces, to accommodate the temporal demands required for precise network activity. Conventionally, this synaptic plasticity results from GABAA R subunit reorganisation. In particular, in certain developing neurones synaptic α2-GABAA Rs are replaced by α1-GABAA Rs. However, in thalamocortical neurones of the mouse ventrobasal (VB) thalamus, the major alteration to mIPSC kinetics occurs on postnatal (P) day 10, some days prior to the GABAA R isoform change. Here, whole-cell voltage-clamp recordings from VB neurones of mouse thalamic slices revealed that early in postnatal development (P7-P8), the mIPSC duration is prolonged by local neurosteroids acting in a paracrine or autocrine manner to enhance GABAA R function. However, by P10, this neurosteroid 'tone' rapidly dissipates, thereby producing brief mIPSCs. This plasticity results from a lack of steroid substrate as pre-treatment of mature thalamic slices (P20-24) with the GABAA R-inactive precursor 5α-dihydroprogesterone (5α-DHP) resulted in markedly prolonged mIPSCs and a greatly enhanced tonic conductance, mediated by synaptic and extrasynaptic GABAA Rs, respectively. In summary, endogenous neurosteroids profoundly influence GABAergic neurotransmission in developing VB neurones and govern a transition from slow to fast phasic synaptic events. Furthermore, the retained capacity for steroidogenesis in the mature thalamus raises the prospect that certain physiological or pathophysiological conditions may trigger neurosteroid neosynthesis, thereby providing a local mechanism for fine-tuning neuronal excitability.

Extrasynaptic GABA(A) 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 α1ß2γ2 GABA(A) receptors (GABA(A)Rs). Importantly, TC neurons also express extrasynaptic α4ß2δ GABA(A)Rs, although how they cooperate with synaptic GABA(A)Rs 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 GABA(A)R-lacking, α4 "knock-out" (α4(0/0)) mice. We demonstrate that the duration of VB phasic inhibition generated in response to nRT burst firing is greatly reduced in α4(0/0) pairs, suggesting that action potential-dependent phasic inhibition is prolonged by recruitment of extrasynaptic GABA(A)Rs. Furthermore, the influence of nRT tonic firing frequency on VB holding current is also greatly reduced in α4(0/0) pairs, implying that the α4-GABA(A)R-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 GABA(A)Rs 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.

Dysfunctional astrocytic and synaptic regulation of hypothalamic glutamatergic transmission in a mouse model of early-life adversity: relevance to neurosteroids and programming of the stress response.

Adverse early-life experiences, such as poor maternal care, program an abnormal stress response that may involve an altered balance between excitatory and inhibitory signals. Here, we explored how early-life stress (ELS) affects excitatory and inhibitory transmission in corticotrophin-releasing factor (CRF)-expressing dorsal-medial (mpd) neurons of the neonatal mouse hypothalamus. We report that ELS associates with enhanced excitatory glutamatergic transmission that is manifested as an increased frequency of synaptic events and increased extrasynaptic conductance, with the latter associated with dysfunctional astrocytic regulation of glutamate levels. The neurosteroid 5α-pregnan-3α-ol-20-one (5α3α-THPROG) is an endogenous, positive modulator of GABAA receptors (GABAARs) that is abundant during brain development and rises rapidly during acute stress, thereby enhancing inhibition to curtail stress-induced activation of the hypothalamic-pituitary-adrenocortical axis. In control mpd neurons, 5α3α-THPROG potently suppressed neuronal discharge, but this action was greatly compromised by prior ELS exposure. This neurosteroid insensitivity did not primarily result from perturbations of GABAergic inhibition, but rather arose functionally from the increased excitatory drive onto mpd neurons. Previous reports indicated that mice (dams) lacking the GABAAR δ subunit (δ(0/0)) exhibit altered maternal behavior. Intriguingly, δ(0/0) offspring showed some hallmarks of abnormal maternal care that were further exacerbated by ELS. Moreover, in common with ELS, mpd neurons of δ(0/0) pups exhibited increased synaptic and extrasynaptic glutamatergic transmission and consequently a blunted neurosteroid suppression of neuronal firing. This study reveals that increased synaptic and tonic glutamatergic transmission may be a common maladaptation to ELS, leading to enhanced excitation of CRF-releasing neurons, and identifies neurosteroids as putative early regulators of the stress neurocircuitry.

GABA-independent GABAA receptor openings maintain tonic currents.

Activation of GABA(A) receptors (GABA(A)Rs) produces two forms of inhibition: phasic inhibition generated by the rapid, transient activation of synaptic GABA(A)Rs by presynaptic GABA release, and tonic inhibition generated by the persistent activation of perisynaptic or extrasynaptic GABA(A)Rs, which can detect extracellular GABA. Such tonic GABA(A)R-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 GABA(A) receptor openings. This tonic GABA(A)R conductance is resistant to the competitive GABA(A)R 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 GABA(A)Rs, therefore, maintain dentate granule cell tonic currents in the face of low extracellular GABA concentrations.

The general anaesthetic etomidate inhibits the excitability of mouse thalamocortical relay neurons by modulating multiple modes of GABAA receptor-mediated inhibition.

Modulation of thalamocortical (TC) relay neuron function has been implicated in the sedative and hypnotic effects of general anaesthetics. Inhibition of TC neurons is mediated predominantly by a combination of phasic and tonic inhibition, together with a recently described 'spillover' mode of inhibition, generated by the dynamic recruitment of extrasynaptic γ-aminobutyric acid (GABA)A receptors (GABAA Rs). Previous studies demonstrated that the intravenous anaesthetic etomidate enhances tonic and phasic inhibition in TC relay neurons, but it is not known how etomidate may influence spillover inhibition. Moreover, it is unclear how etomidate influences the excitability of TC neurons. Thus, to investigate the relative contribution of synaptic (α1ß2γ2) and extrasynaptic (α4ß2δ) GABAA Rs to the thalamic effects of etomidate, we performed whole-cell recordings from mouse TC neurons lacking synaptic (α1(0/0) ) or extrasynaptic (δ(0/0) ) GABAA Rs. Etomidate (3 µm) significantly inhibited action-potential discharge in a manner that was dependent on facilitation of both synaptic and extrasynaptic GABAA Rs, although enhanced tonic inhibition was dominant in this respect. Additionally, phasic inhibition evoked by stimulation of the nucleus reticularis exhibited a spillover component mediated by δ-GABAA Rs, which was significantly prolonged in the presence of etomidate. Thus, etomidate greatly enhanced the transient suppression of TC spike trains by evoked inhibitory postsynaptic potentials. Collectively, these results suggest that the deactivation of thalamus observed during etomidate-induced anaesthesia involves potentiation of tonic and phasic inhibition, and implicate amplification of spillover inhibition as a novel mechanism to regulate the gating of sensory information through the thalamus during anaesthetic states.

Reducing the harms of alcohol: nutritional interventions and functional alcohol alternatives.

The health risks and harm associated with regular alcohol consumption are well documented. In a recent WHO statement published in The Lancet Public Health alcohol consumption has been estimated to contribute worldwide to 3 million deaths in 2016 while also being responsible for 5·1% of the global burden of disease and injury. The total elimination of alcohol consumption, which has been long imbedded in human culture and society, is not practical and prohibition policies have proved historically ineffective. However, valuable strategies to reduce alcohol harms are already available and improved alternative approaches are currently being developed. Here, we will review and discuss recent advances on two main types of approaches, that is nutritional interventions and functional alcohol alternatives.

Neurosteroid Modulation of Synaptic and Extrasynaptic GABAA Receptors of the Mouse Nucleus Accumbens.

The recent approval of formulations of the endogenous neurosteroid allopregnanolone (brexanolone) and the synthetic neuroactive steroid SAGE-217 (zuranolone) to treat postpartum depression (PPD) has encouraged further research to elucidate why these potent enhancers of GABAAR function are clinically effective in this condition. Dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens are associated with reward/motivation and brain imaging studies report that individuals with PPD show reduced activity of this pathway in response to reward and infant engagement. However, the influence of neurosteroids on GABA-ergic transmission in the nucleus accumbens has received limited attention. Here, we investigate, in the medium spiny neurons (MSNs) of the mouse nucleus accumbens core, the effect of allopregnanolone, SAGE-217 and other endogenous and synthetic steroids of interest on fast phasic and tonic inhibition mediated by synaptic (α1/2ßγ2) and extrasynaptic (α4ßδ) GABAARs, respectively. We present evidence suggesting the resident tonic current results from the spontaneous opening of δ-GABAARs, where the steroid-enhanced tonic current is GABA-dependent. Furthermore, we demonstrate local neurosteroid synthesis in the accumbal slice preparation and reveal that GABA-ergic neurotransmission of MSNs is influenced by an endogenous neurosteroid tone. Given the dramatic fluctuations in allopregnanolone levels during pregnancy and postpartum, this neurosteroid-mediated local fine-tuning of GABAergic transmission in the MSNs will probably be perturbed.

Cocaine effects on mouse incentive-learning and human addiction are linked to alpha2 subunit-containing GABAA receptors.

Because GABA(A) receptors containing alpha2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with alpha2 gene deletion showed reduced synaptic GABA(A) receptor-mediated responses. Behaviorally, the deletion abolished cocaine's ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of alpha2-GABA(A) receptors (alpha2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In alpha2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of alpha2-GABA(A) receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction.

α4-Containing GABAA Receptors on DRD2 Neurons of the Nucleus Accumbens Mediate Instrumental Responding for Conditioned Reinforcers and Its Potentiation by Cocaine.

Extrasynaptic GABAA receptors (GABAARs) composed of α4, ß, and δ subunits mediate GABAergic tonic inhibition and are potential molecular targets in the modulation of behavioral responses to natural and drug rewards. These GABAARs are highly expressed within the nucleus accumbens (NAc), where they influence the excitability of the medium spiny neurons. Here, we explore their role in modulating behavioral responses to food-conditioned cues and the behavior-potentiating effects of cocaine. α4-Subunit constitutive knock-out mice (α4-/-) showed higher rates of instrumental responding for reward-paired stimuli in a test of conditioned reinforcement (CRf). A similar effect was seen following viral knockdown of GABAAR α4 subunits within the NAc. Local infusion of the α4ßδ-GABAAR-preferring agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; Gaboxadol) into the NAc had no effect on responding when given alone but reduced cocaine potentiation of responding for conditioned reinforcers in wild-type, but not α4-/- mice. Finally, specific deletion of α4-subunits from dopamine D2, but not D1, receptor-expressing neurons (DRD2 and DRD1 neurons), mimicked the phenotype of the constitutive knockout, potentiating CRf responding, and blocking intra-accumbal THIP attenuation of cocaine-potentiated CRf responding. These data demonstrate that α4-GABAAR-mediated inhibition of DRD2 neurons reduces instrumental responding for a conditioned reinforcer and its potentiation by cocaine and emphasize the importance of GABAergic signaling within the NAc in mediating the effects of cocaine.

Relating neurosteroid modulation of inhibitory neurotransmission to behaviour.

Studies in the 1980s revealed endogenous metabolites of progesterone and deoxycorticosterone to be potent, efficacious, positive allosteric modulators (PAMs) of the GABAA receptor (GABAA R). The discovery that such steroids are locally synthesised in the central nervous system (CNS) promoted the thesis that neural inhibition in the CNS may be "fine-tuned" by these neurosteroids to influence behaviour. In preclinical studies, these neurosteroids exhibited anxiolytic, anticonvulsant, analgesic and sedative properties and, at relatively high doses, induced a state of general anaesthesia, a profile consistent with their interaction with GABAA Rs. However, realising the therapeutic potential of either endogenous neurosteroids or synthetic "neuroactive" steroids has proven challenging. Recent approval by the Food and Drug Administration of the use of allopregnanolone (brexanolone) to treat postpartum depression has rekindled enthusiasm for exploring their potential as new medicines. Although neurosteroids are selective for GABAA Rs, they exhibit little or no selectivity across the many GABAA R subtypes. Nevertheless, a relatively minor population of receptors incorporating the δ-subunit (δ-GABAA Rs) appears to be an important contributor to their behavioural effects. Here, we consider how neurosteroids acting upon GABAA Rs influence neuronal signalling, as well as how such effects may acutely and persistently influence behaviour, and explore the case for developing selective PAMs of δ-GABAA R subtypes for the treatment of psychiatric disorders.

Functional Alternatives to Alcohol.

The consumption of alcohol is associated with well-known health harms and many governments worldwide are actively engaged in devising approaches to reduce them. To this end, a common proposed strategy aims at reducing alcohol consumption. This approach has led to the development of non-alcoholic drinks, which have been especially welcome by younger, wealthier, health-conscious consumers, who have been turning away from alcohol to look toward alternatives. However, a drawback of non-alcoholic drinks is that they do not facilitate social interaction in the way alcohol does, which is the main reason behind social drinking. Therefore, an alternative approach is to develop functional drinks that do not use alcohol yet mimic the positive, pro-social effects of alcohol without the associated harms. This article will discuss (1) current knowledge of how alcohol mediates its effects in the brain, both the desirable, e.g., antistress to facilitate social interactions, and the harmful ones, with a specific focus on the pivotal role played by the gamma-aminobutyric acid (GABA) neurotransmitter system and (2) how this knowledge can be exploited to develop functional safe alternatives to alcohol using either molecules already existing in nature or synthetic ones. This discussion will be complemented by an analysis of the regulatory challenges associated with the novel endeavour of bringing safe, functional alternatives to alcohol from the bench to bars.

Early stress-induced impaired microglial pruning of excitatory synapses on immature CRH-expressing neurons provokes aberrant adult stress responses.

Several mental illnesses, characterized by aberrant stress reactivity, often arise after early-life adversity (ELA). However, it is unclear how ELA affects stress-related brain circuit maturation, provoking these enduring vulnerabilities. We find that ELA increases functional excitatory synapses onto stress-sensitive hypothalamic corticotropin-releasing hormone (CRH)-expressing neurons, resulting from disrupted developmental synapse pruning by adjacent microglia. Microglial process dynamics and synaptic element engulfment were attenuated in ELA mice, associated with deficient signaling of the microglial phagocytic receptor MerTK. Accordingly, selective chronic chemogenetic activation of ELA microglia increased microglial process dynamics and reduced excitatory synapse density to control levels. Notably, selective early-life activation of ELA microglia normalized adult acute and chronic stress responses, including stress-induced hormone secretion and behavioral threat responses, as well as chronic adrenal hypertrophy of ELA mice. Thus, microglial actions during development are powerful contributors to mechanisms by which ELA sculpts the connectivity of stress-regulating neurons, promoting vulnerability to stress and stress-related mental illnesses.

Distinct mechanisms regulate GABAA receptor and gephyrin clustering at perisomatic and axo-axonic synapses on CA1 pyramidal cells.

Pyramidal cells express various GABA(A) receptor (GABA(A)R) subtypes, possibly to match inputs from functionally distinct interneurons targeting specific subcellular domains. Postsynaptic anchoring of GABA(A)Rs is ensured by a complex interplay between the scaffolding protein gephyrin, neuroligin-2 and collybistin. Direct interactions between these proteins and GABA(A)R subunits might contribute to synapse-specific distribution of GABA(A)R subtypes. In addition, the dystrophin-glycoprotein complex, mainly localized at perisomatic synapses, regulates GABA(A)R postsynaptic clustering at these sites. Here, we investigated how the functional and molecular organization of GABAergic synapses in CA1 pyramidal neurons is altered in mice lacking the GABA(A)R α2 subunit (α2-KO). We report a marked, layer-specific loss of postsynaptic gephyrin and neuroligin-2 clusters, without changes in GABAergic presynaptic terminals. Whole-cell voltage-clamp recordings in slices from α2-KO mice show a 40% decrease in GABAergic mIPSC frequency, with unchanged amplitude and kinetics. Applying low/high concentrations of zolpidem to discriminate between α1- and α2/α3-GABA(A)Rs demonstrates that residual mIPSCs in α2-KO mice are mediated by α1-GABA(A)Rs. Immunofluorescence analysis reveals maintenance of α1-GABA(A)R and neuroligin-2 clusters, but not gephyrin clusters, in perisomatic synapses of mutant mice, along with a complete loss of these three markers on the axon initial segment. This striking subcellular difference correlates with the preservation of dystrophin clusters, colocalized with neuroligin-2 and α1-GABA(A)Rs on pyramidal cell bodies of mutant mice. Dystrophin was not detected on the axon initial segment in either genotype. Collectively, these findings reveal synapse-specific anchoring of GABA(A)Rs at postsynaptic sites and suggest that the dystrophin-glycoprotein complex contributes to stabilize α1-GABA(A)R and neuroligin-2, but not gephyrin, in perisomatic postsynaptic densities.

GABAA receptors in GtoPdb v.2021.3.

The GABAA receptor is a ligand-gated ion channel of the Cys-loop family that includes the nicotinic acetylcholine, 5-HT3 and strychnine-sensitive glycine receptors. GABAA receptor-mediated inhibition within the CNS occurs by fast synaptic transmission, sustained tonic inhibition and temporally intermediate events that have been termed 'GABAA, slow' [45]. GABAA receptors exist as pentamers of 4TM subunits that form an intrinsic anion selective channel. Sequences of six α, three ß, three γ, one δ, three ρ, one ε, one π and one θ GABAA receptor subunits have been reported in mammals [278, 235, 236, 283]. The π-subunit is restricted to reproductive tissue. Alternatively spliced versions of many subunits exist (e.g. α4- and α6- (both not functional) α5-, ß2-, ß3- and γ2), along with RNA editing of the α3 subunit [71]. The three ρ-subunits, (ρ1-3) function as either homo- or hetero-oligomeric assemblies [359, 50]. Receptors formed from ρ-subunits, because of their distinctive pharmacology that includes insensitivity to bicuculline, benzodiazepines and barbiturates, have sometimes been termed GABAC receptors [359], but they are classified as GABA A receptors by NC-IUPHAR on the basis of structural and functional criteria [16, 235, 236]. Many GABAA receptor subtypes contain α-, ß- and γ-subunits with the likely stoichiometry 2α.2ß.1γ [168, 235]. It is thought that the majority of GABAA receptors harbour a single type of α- and ß - subunit variant. The α1ß2γ2 hetero-oligomer constitutes the largest population of GABAA receptors in the CNS, followed by the α2ß3γ2 and α3ß3γ2 isoforms. Receptors that incorporate the α4- α5-or α 6-subunit, or the ß1-, γ1-, γ3-, δ-, ε- and θ-subunits, are less numerous, but they may nonetheless serve important functions. For example, extrasynaptically located receptors that contain α6- and δ-subunits in cerebellar granule cells, or an α4- and δ-subunit in dentate gyrus granule cells and thalamic neurones, mediate a tonic current that is important for neuronal excitability in response to ambient concentrations of GABA [209, 272, 83, 19, 288]. GABA binding occurs at the ß+/α- subunit interface and the homologous γ+/α- subunits interface creates the benzodiazepine site. A second site for benzodiazepine binding has recently been postulated to occur at the α+/ß- interface ([254]; reviewed by [282]). The particular α-and γ-subunit isoforms exhibit marked effects on recognition and/or efficacy at the benzodiazepine site. Thus, receptors incorporating either α4- or α6-subunits are not recognised by 'classical' benzodiazepines, such as flunitrazepam (but see [356]). The trafficking, cell surface expression, internalisation and function of GABAA receptors and their subunits are discussed in detail in several recent reviews [52, 140, 188, 316] but one point worthy of note is that receptors incorporating the γ2 subunit (except when associated with α5) cluster at the postsynaptic membrane (but may distribute dynamically between synaptic and extrasynaptic locations), whereas as those incorporating the δ subunit appear to be exclusively extrasynaptic. NC-IUPHAR [16, 235, 3, 2] class the GABAA receptors according to their subunit structure, pharmacology and receptor function. Currently, eleven native GABAA receptors are classed as conclusively identified (i.e., α1ß2γ2, α1ßγ2, α3ßγ2, α4ßγ2, α4ß2δ, α4ß3δ, α5ßγ2, α6ßγ2, α6ß2δ, α6ß3δ and ρ) with further receptor isoforms occurring with high probability, or only tentatively [235, 236]. It is beyond the scope of this Guide to discuss the pharmacology of individual GABAA receptor isoforms in detail; such information can be gleaned in the reviews [16, 95, 168, 173, 143, 278, 216, 235, 236] and [9, 10]. Agents that discriminate between α-subunit isoforms are noted in the table and additional agents that demonstrate selectivity between receptor isoforms, for example via ß-subunit selectivity, are indicated in the text below. The distinctive agonist and antagonist pharmacology of ρ receptors is summarised in the table and additional aspects are reviewed in [359, 50, 145, 223]. Several high-resolution cryo-electron microscopy structures have been described in which the full-length human α1ß3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam [198].