Neuroactive steroids are allosteric modulators of GABAA receptors and are implicated in the etiology and treatment of neuropsychiatric disorders. Existing treatments are helpful but have drawbacks. Neuroactive steroids recently gained attention as rapidly acting antidepressants in postpartum depression and other indications. Unlike other GABAA receptor modulators, neurosteroids may possess anti-inflammatory actions, potentially contributing to therapeutic benefit. Here we seek to understand neuroactive steroid structure-activity relationships relevant to these anti-inflammatory effects. We used murine microglial BV2 cells challenged with lipopolysaccharide (LPS) as an inflammation model. We investigated structure-activity profile of neuroactive steroids and oxysterol-like compounds on cytokine transcription. LPS increased transcripts for cytokines IL-1β, IL-6 and TNF-α. Both allopregnanolone and its enantiomer significantly suppressed these LPS-induced increases, with no effect in the absence of LPS. CONCLUSIONS: Our results suggest that neuroactive steroids exhibit a distinct structure-activity profile compared with their GABAA receptor modulation effects. Certain neuroactive steroids may selectively target neuroinflammation. The enantiomer of AlloP could be a tool compound to differentiate anti-inflammatory effects of neuroactive steroids from GABAergic and other enantioselective effects.

Dysfunction in the GABAergic system has been described in schizophrenia, including decreased expression of α5 subunit-containing GABAA receptors (α5-GABAARs) in patients with schizophrenia. This study explores the therapeutic potential of positive allosteric modulators (PAMs) of the α5-GABAAR to reduce the hyperdopaminergic state produced by the neurodevelopmental methylazoxymethanol acetate (MAM) model of schizophrenia. Male offspring rats generated from pregnant females injected with saline or MAM at gestational day 17 were used for the electrophysiological recordings as adults. In vivo electrophysiological recordings were performed to assess the effects of 10mg/kg of the novel α5-GABAAR-preferring PAM alogabat on dopamine (DA) neuron activity in the ventral tegmental area (VTA); a dose shown to produce sustained, ≥80% α5-GABAAR occupancy over a time period of 0.5-3.5h post-dose. A less extensive confirmatory study was also performed with a second α5-GABAAR PAM, Compound 100. The primary outcome was that at a dose of 10mg/kg, which corresponded to an α5-GABAAR occupancy of ≥80% for alogabat and 70% for Compound 100, reversed the increased number of spontaneously active DA neurons in MAM rats. Alogabat data showed that these effects were driven by a reduction in the central and lateral (but not medial) portions of the VTA; regions that project to the associative striatum. These findings suggest that selective targeting of α5-GABAARs may help normalize aberrant DA activity. The study highlights α5-GABAARs as a promising therapeutic target, potentially addressing positive symptoms by restoring excitatory-inhibitory balance in a key region of the brain implicated in the pathophysiology of schizophrenia.

Neuronal activity drives increases in cerebral blood flow to match metabolic demands, with capillary dilation critical for blood-brain exchange. However, the molecular mechanisms coupling neuronal activity to capillary diameter adjustments remain unclear. We examined the contribution of pericyte pannexin1 channels to capillary responses during increased neuronal excitability and investigated the underlying signaling pathway. To induce neuronal excitability, we administered picrotoxin, a GABAA receptor antagonist, to acute hippocampal slices and invivo, which induces epileptiform activity. Pericyte pannexin1 activity and capillary responses were monitored via dye uptake and capillary diameter measurements in slices from wild-type and pannexin1-deficient mice. Pharmacological blockade of adenosine A1 receptors and exogenous adenosine application were used to identify the signaling pathway. In vivo picrotoxin administration inhibited pericyte pannexin1 channel activity in the hippocampus. Equivalent suppression was observed in picrotoxin-treated acute hippocampal slices, where neuronal excitability led to pericyte pannexin1 inhibition and capillary dilation. Both responses were abolished by tetrodotoxin and absent in pannexin1-deficient mice, confirming dependence on neuronal activity and pannexin1 expression. Pharmacological blockade of adenosine A1 receptors prevented pannexin1 inhibition and the associated vasodilatory response, whereas exogenous adenosine recapitulated these effects, demonstrating that adenosine signaling is required for neuronal activity-dependent modulation of pericyte pannexin1. We identify a previously uncharacterized adenosine-pannexin1 signaling axis in pericytes linking neuronal excitability to capillary relaxation. This mechanism provides a molecular substrate for activity-dependent capillary regulation and supports a role for pericyte pannexin1 in adenosine-mediated neurovascular responses during states of elevated metabolic demand.

Alcohol impacts an fMRI marker of neural inhibition in humans and rodents.

Differential sensitivity of RDL1 and RDL2 GABA receptors to Broflanilide, Fluxametamide, Fluralaner, and Fipronil in Heliothis virescens and Helicoverpa zea.

Gain-of-function (GOF) variants in GABAA receptors are increasingly recognised as a cause of severe developmental and epileptic encephalopathies (DEE). However, the mechanisms by which enhanced GABAA receptor activity leads to neuronal network hyperexcitability remains unclear. We engineered a novel mouse model based on the human GOF GABRB3 p.(Glu77Lys) variant (β3E77K), identified in two individuals diagnosed with DEE, to explore the mechanisms underlying GABAA receptor GOF disease. The phenotypes of β3E77K mice included embryonic lethality consistent with a severe early developmental impact of the GOF GABAA receptor variant. These mice display spike-wave-like discharges that were exacerbated by vigabatrin and ameliorated by valproate matching the clinical observations of GOF GABRB3 patients. β3E77K mice also have increased proconvulsant-induced seizure susceptibility and a broad increase in ECoG spectral power amplitude, indicative of cortical hyperexcitability. Additionally, neurological assessments revealed hypoactivity and weakened grip-strength. Ex-vivo electrophysiological recordings demonstrated increased GABAA receptor-mediated current amplitudes at both excitatory and inhibitory synapses in CA1 hippocampus, consistent with the GOF molecular phenotype previously identified in functional studies. In cortical layer 2/3, inhibitory interneurons showed increased synaptic GABAA receptor-mediated current amplitude, while synapses onto pyramidal neurons exhibited reduced inhibitory currents. Enhanced GABAA receptor-mediated synaptic activity among layer 2/3 interneuron populations caused a use-dependent collapse of feed-forward inhibition resulting in increased pyramidal neuron excitability. Computational modelling supported this disinhibition mechanism, showing that enhanced GABAA receptor synaptic strength between interneurons diminishes inhibitory synaptic conductance onto pyramidal cells. Our findings highlight the critical role of interneuron network dysfunction in driving cortical hyperexcitability caused by GOF GABAA receptor variants. They provide a novel pathogenic mechanism in DEE that could have broader implications for disorders involving dysfunction in GABAergic neurons. The β3E77K mouse also provides a unique preclinical model to test therapeutic strategies in GOF GABAA receptor disease.

Polyadic synapses (where each active zone is juxtaposed with multiple postsynaptic targets) are found in many brain regions and are implicated in learning and memory, yet little is known about how they function. To address this question, we analyze Caenorhabditis elegans cholinergic (ACh) motor neurons, which form dyadic synapses with two targets (a body muscle and a GABAergic motor neuron). Decreasing ACh receptors in either target elicits a retrograde decrease in presynaptic type 2 voltage-activated calcium channels (CaV2), thereby decreasing ACh release. By contrast, blocking GABA motor neuron differentiation (thereby eliminating one target) results in ectopic clustering of GABAA receptors at ACh neuromuscular junctions, which elicits a retrograde increase in presynaptic strength. These results suggest that presynaptic CaV2 channels are linked to postsynaptic receptors in both targets, which allows each target to modify transmission to both targets.

Understanding the relationship between lung GABAA receptors and aquaporin-1: a new therapeutic approach for acute lung injury.

The fall armyworm (FAW), Spodoptera frugiperda, is a highly destructive pest in agriculture. Broflanilide, specifically designed to target the GABA receptors in insects, is effective against various Lepidoptera pests, including FAW. However, the patterns of cross-resistance between broflanilide and other insecticides, especially the underlying molecular mechanisms behind cross-resistance, remain unclear. In this study, a broflanilide-resistant strain of S. frugiperda (F24) was established after 24 generations of continuous selection in the laboratory with a resistance ratio of 4.04-fold. The cross-resistance ratio of broflanilide was found to be 2.95-fold for lambda-cyhalothrin and 2.82-fold for cyproflanilide in the F24 generation. Additionally, an apparent antagonistic effect was observed when broflanilide was mixed with a lambda-cyhalothrin mixture at all volume ratios. Moreover, qPCR combined with CRISPR-Cas9 showed that the gene SfCYP6B42 partially contributed to the cross-resistance between broflanilide and lambda-cyhalothrin, which first elucidated the cross-resistance mechanism between broflanilide and lambda-cyhalothrin.

Rebalancing inhibition: α5 GABAA receptor positive and negative allosteric modulation in Alzheimer's disease.

Gephyrin, the principal scaffolding protein of inhibitory postsynaptic densities, clusters glycine and GABAA receptors via multivalent interactions. It features structured N and C terminal domains connected by an intrinsically disordered linker. Although the structural and functional properties of its terminal domains are well characterized, the mechanism by which full-length gephyrin organizes into higher-order complexes remains unresolved. Here, we combine biochemical reconstitution, cryo-electron microscopy, and mutational analyses to elucidate the structural logic of gephyrin oligomerization. We demonstrate that gephyrin adopts a stable dimeric assembly which constitutes the basic unit for both linear and oblique tetramers as well as linear hexameric arrangements. High resolution structures reveal a critical segment of the flexible linker that adopts two distinct conformations, one of which occludes the receptor-binding site. This segment harbors key phosphorylation sites, suggesting a regulatory control mechanism. Our findings redefine the architecture of inhibitory postsynaptic sites and reconcile gephyrin oligomerization models with published in-situ postsynaptic densities characterized by cryo-electron tomography.

Many antiepileptic drugs act by modulation of ionotropic GABAA receptors or effects on extracellular GABA concentrations. Metabotropic GABAB receptors also affect neuronal excitability but are not recognized as a target of antiepileptic drugs. We investigated effects of GABAB receptors and antiepileptic drug vigabatrin on epileptiform discharges induced by 4-aminopyridine (4AP) in hippocampal brain slices from male mice. The GABAB receptor agonist baclofen caused dose-dependent reduction in frequency of 4AP-induced epileptiform discharges but did not affect amplitude or duration of discharges. The inhibitory effects of baclofen were blocked by GABAB receptor antagonist CGP55845 (CGP) and the K+ channel blocker Ba2+, indicating that baclofen was acting on GABAB receptors and activating GIRK channels. Baclofen effects were independent of GABAA receptors. Vigabatrin inhibits GABA transaminase, thereby increasing GABA concentrations. Pretreatment of brain slices with vigabatrin suppressed 4AP-induced discharges, significantly prolonging the latency to onset of spontaneous activity and reducing frequency of discharges. Similar to baclofen, vigabatrin effects on latency and frequency were reversed by GABAB antagonist CGP and the GIRK channel blocker Ba2+ Vigabatrin had no effect on 4AP-induced activity in GABA transporter type 1 knock-out mice (GAT1KO), suggesting that vigabatrin effects are dependent on GAT1 function. Our results indicate for the first time that a clinically used antiepileptic drug (vigabatrin) suppresses in vitro epileptiform activity via activation of GABAB receptors and GIRK channels. Data are consistent with a model that depicts altered thermodynamic equilibrium of GAT1 after GABA transaminase inhibition that leads to elevated extracellular GABA concentration and activation of GABAB receptors.

Human cerebral organoids derived from induced pluripotent stem cells can recapture early developmental processes and reveal changes involving neurodevelopmental disorders. Mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene are associated with Rett syndrome, and disease severity varies depending on the location and type of mutation. Here, we focused on neuronal activity in Rett syndrome patient-derived organoids, analyzing two types of MECP2 mutations-a missense mutation (R306C) and a truncating mutation (V247X)-using calcium imaging with three-photon microscopy. Compared to isogenic controls, we found abnormal neuronal activity in Rett organoids and altered network function based on graph theoretic analyses, with V247X mutations impacting functional responses and connectivity more severely than R306C mutations. These changes paralleled EEG data obtained from patients with comparable mutations. Labeling >/>DLX promoter-driven inhibitory neurons demonstrated differences in activity and functional connectivity of inhibitory and excitatory neurons in the two types of mutations. Transcriptomic analyses revealed HDAC2-associated impairment in R306C organoids and decreased GABAA receptor expression in excitatory neurons in V247X organoids. These findings demonstrate mutation-specific mechanisms of vulnerability in Rett syndrome and suggest targeted strategies for their treatment.

Background. Postpartum depression (PPD) is a common childbirth complication, affecting an estimated 17.2% of women worldwide. Unlike the short-lived “baby blues,” PPD is a unique neurobiological illness defined by an inability to adapt synaptically to reproductive hormone withdrawal. It is the most common cause of maternal suicide, and the offspring of PPD mothers also suffer long-lasting cognitive and emotional impairments. Aim. Our objective was to review the pathophysiology of PPD (with special emphasis on the GABAergic and immuno-inflammatory hypotheses), screening procedures and to outline a “stepped-care” management algorithm, including new neuroactive steroids. Material and Methods. A systematic literature search was conducted using PubMed/MEDLINE, Embase, and Cochrane databases (Jan 2010 – Dec 2024). We prioritized meta-analyses, randomized controlled trials (RCTs), and guidelines from major obstetric societies (ACOG, RCOG). Results. There is evidence for current hypothesis of rapid withdrawal of allopregnanolone as trigger of the development of symptoms that do not initiate the adequate GABA-A receptor reorganization in vulnerable women. Other mechanisms include HPA axis dysregulation and inflammation-induced tryptophan depletion. The gold standard for screening remains the Edinburgh Postnatal Depression Scale (EPDS) Sensitivity: 86%). Pharmacologic treatment has significantly progressed: Sertraline remains the first-line SSRI due to safety in lactation, but the FDA approval of Zuranolone (2023) provides a mechanism-specific, rapid-acting therapeutic option addressing the underlying hormonal dysregulation. Conclusions. Obstetricians are the primary gatekeepers for maternal mental health. Preparation of obstetricians to identify women at higher risk and to treat with pharmacotherapy is an essential part of contemporary obstetric practice.

Alterations of GABAA receptors (GABAARs) following chronic alcohol are thought to be related to deficits in GABAergic signaling in individuals with alcohol use disorder (AUD). Whether those modifications affect the function of synaptic GABAARs is not clear, as the electrophysiological characterization of native synaptic receptors from AUD individuals had not been done. To obtain this information, we microtransplanted synaptic membranes from postmortem dorsolateral prefrontal cortex (DLPFC) samples of AUD and non-AUD subjects to determine functional traits of GABAARs. To follow the path from transcription to function of potential changes of GABAARs in AUD, GABAARs currents and GABA pEC50 values were integrated with RNA-Seq and label-free proteomics datasets of bulk tissue and isolated synaptosomes from the same subjects. Our results outline significant reconfigurations in transcriptomic organization of GABAARs in AUD, higher levels of GABRG1 and significant decrease of mitochondrial transcripts in AUD individuals. Notably, transcriptional differences were gradually lost as the analysis moved from transcription to protein and function within our cohort. This suggests post-translational buffering in AUD resulting in unchanged GABA receptor synaptic activity. Our novel findings establish a proof of concept for reactivating AUD post-synaptic receptors and integrating this information with multiple levels of multi-omic analyses, as well as outline hypothesis-generating insights into this multifaceted disease.

Purpose: Remimazolam is a novel, ultra–short-acting benzodiazepine. Developed as a “soft drug,” it is rapidly metabolized by tissue esterases into an inactive metabolite. This metabolism enables fast onset, predictable offset, and straightforward reversibility with flumazenil, distinguishing it from non-benzodiazepine hypnotics.Current concepts: Pharmacokinetically, remimazolam exhibits high clearance, a small steady-state volume of distribution, and a short context-sensitive half-time, which help minimize accumulation during prolonged infusions. Pharmacodynamically, it produces dose-dependent sedation by modulating the GABAA receptor and demonstrates consistent concentration–response relationships. Clinically, randomized and observational studies support its efficacy in procedural sedation and general anesthesia. Remimazolam is associated with stable hemodynamics, reduced vasopressor requirements, and minimal respiratory compromise. Older adults and high-risk patients often experience favorable recovery profiles, and remimazolam shows promise in neurosurgery, where rapid neurological assessment is essential.Discussion and conclusion: Several challenges remain, presenting opportunities for innovation. Sporadic cases of reported anaphylaxis may involve remimazolam itself or the dextran-40 excipient, necessitate careful administration and warrant research into optimized infusion strategies. Reports of resedation after flumazenil antagonism underscore the need for extended recovery monitoring and highlight areas for clinical refinement. Limited pediatric data emphasize the importance of establishing standardized dosing regimens. Discrepancies between bispectral index values and actual hypnotic depth indicate the need to identify drug-specific electroencephalography (EEG) markers. Overall, remimazolam represents a promising alternative to conventional sedative-hypnotics, especially when hemodynamic stability and rapid, reversible hypnosis are priorities. Future studies should prioritize refining dosing strategies for special populations, validating EEG markers, and evaluating long-term cognitive outcomes and cost-effectiveness.

Noise pollution, particularly by aircraft, is a significant risk factor for cardiovascular disease. Aircraft noise activates stress response pathways in the brain, via the amygdala, the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. Male C57BL/6J mice were treated with citalopram (a selective serotonin (5-HT)-reuptake inhibitor) or diazepam (a benzodiazepine) 1 day before aircraft noise exposure for 4 days. Aortic vascular function was measured by isometric tension method, microvascular function by video microscopy in pressurized cerebral arterioles, blood pressure by tail cuff, reactive oxygen species formation by dihydrothidium staining of vascular tissue and markers of inflammation and oxidative stress by western blotting. In support of the stress response concept, we report here that noise exposure of mice implicates an increase in activity primarily in the left amygdala, envisaged by [18F]fluorodeoxyglucose positron emission tomography (PET) scan. Both neuro-active drugs, diazepam and citalopram, ameliorated the adverse cardiovascular and neurobiological effects of noise exposure, partially preventing blood pressure increases and endothelial dysfunction in both large (aorta) and small vessels (cerebral arterioles). Diazepam showed slightly greater efficacy. Noise exposure also increased markers of oxidative stress and inflammation in the heart and brain (cortex and hippocampus), and both drugs mostly prevented these pathophysiological changes. The study provides indirect evidence that modulating the stress response pathway may represent a pharmacological approach to mitigate the negative effects of noise exposure. This may have implications for patients with neuropsychiatric disease suffering from aircraft noise exposure.

With the goal of identifying new therapeutic leads to treat pain, we screened a library of microbial chemical extracts using the Embryonic Zebrafish Irritant-evoked Hyperlocomotion (EZIH) assay, an in vivo phenotypic assay for analgesic/antinociceptive activity. We found that extracts from the bacterium Croceibacter atlanticus substantially altered behavioral responses. Using behavior-guided fractionation to identify the active partitions, we determined that the known 1,4-benzothiazin-3-one BPSS2111-2113:C and the new benzothiazole croceithiazole A suppress behavioral responses to painful and/or nociceptive stimuli without apparent acute toxicity. Further investigation with radioligand binding assays revealed that BPSS2111-2113:C shows selective binding to both the serotonin receptor 5-HT2B and the benzodiazepine site of GABAA receptors, while croceithiazole A appears to bind the peripheral benzodiazepine receptor. These results suggest that BPSS2111-2113:C and croceithiazole A are interesting leads as analgesics, sedatives, anesthetics, or molecular probes.

Cenobamate is a novel alkyl-carbamate antiseizure medication (ASM) that represents a major breakthrough in the treatment of drug-resistant epilepsy, particularly focal seizures. Unlike other ASMs, cenobamate achieves seizure freedom in up to one third of patients with focal drug-resistant epilepsy-a response not observed with other therapies. Herein, we describe the chemocentric and phenotypic screening strategy employed by one of us (Yong Moon Choi) while at SK Biopharmaceuticals and how cenobamate was developed by optimizing earlier alkyl-carbamates such as carisbamate and felbamate. We discuss how the incorporation of a tetrazole moiety likely played a significant role in its successful development. Cenobamate was found to display potent broad-spectrum activity in diverse disease-relevant rodent models, including maximal electroshock, pentylenetetrazole, kindling, and lithium-pilocarpine-induced status epilepticus. When compared to 12 other ASMs, it ranked highest across four models of difficult-to-treat focal seizures. We discuss how the two known molecular activities of cenobamate, i.e., positive allosteric modulation of GABAA receptors via a non-benzodiazepine site and inhibition of persistent sodium currents (INaP), may contribute to its clinical efficacy and how pharmacokinetic/pharmacodynamic modeling confirmed that the effective brain concentrations of cenobamate align with clinically relevant plasma concentrations observed in seizure-free patients. The discovery of cenobamate has significant implications for future ASM development. It validates phenotypic screening as a powerful tool for identifying first-in-class central nervous system therapeutics and highlights the value of chemocentric optimization using known scaffolds. The predictive utility of specific preclinical models-especially kindling and lithium-pilocarpine-suggests that future ASM discovery should prioritize models that better mimic pharmacoresistant epilepsy. The dual mechanisms of cenobamate may serve as a blueprint for designing ASMs with synergistic actions. Approval of cenobamate represents a paradigm shift in epilepsy treatment and offers a promising framework for discovering more effective therapies for drug-resistant epilepsy.

Background: We investigated whether a water extract of Lilii bulbus (Lilium lancifolium Thunberg; WELB) could modulate inhibitory synaptic organization in a mouse model of pentylenetetrazol (PTZ)-induced kindling. Methods: Starting 14 days prior to the initial PTZ challenge, WELB (500 mg/kg) was delivered via oral gavage once daily. This treatment regimen was maintained for a total of 40 days, spanning the entire period until the animals reached the fully kindled state. Results: Behavioral assessments revealed that WELB treatment significantly reduced seizure severity and Racine scores, prolonged the latency to clonic seizures, and shortened seizure duration, demonstrating potent anticonvulsant activity. Two-photon calcium imaging further showed that WELB markedly suppressed PTZ-induced neuronal hyperexcitability in the posterior parietal cortex, accompanied by decreased expression of neuronal activation markers, including c-fos, phosphorylated-calcium/calmodulin-dependent protein kinase IIα (p-CaMKIIα), and N-methyl-D-aspartate receptor 1 (NR1). In the hippocampus, WELB modulated the expression of GABAergic interneuron markers [glutamate decarboxylase 67 (GAD67), vesicular GABA transporter (VGAT), parvalbumin (PV), somatostatin (SOM)] and upregulated GABAergic gene transcripts [GABA-A receptor α1 subunit (Gabra1), GABA-A receptor α2 subunit (Gabra2), GABA transporter 1 (Gat1), GABA transporter 3 (Gat3), PV, SOM, cholecystokinin (CCK)] that were downregulated by PTZ kindling. Moreover, WELB enhanced the expression of GABAergic synaptic organization-related proteins (gephyrin, collybistin, neurexin-1β, neuroligin-2, and neuropilin-2), indicating its regulatory effect on inhibitory synaptic integrity. Conclusions: Collectively, these findings suggest that WELB may exert its anticonvulsant effects by functionally remodeling GABAergic synaptic organization-related factors, thereby restoring inhibitory circuit integrity and providing a mechanism-based therapeutic strategy for epilepsy and seizure-related neurological disorders.