Gamma-aminobutyric Acid Type A Receptor Research Articles

Discovering the Intriguing Properties of Extrasynaptic γ-Aminobutyric Acid Type A Receptors.

Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by α5 subunit-containing γ-aminobutyric acid type A receptors. By Caraiscos VB, Elliott EM, You-Ten KE, Cheng VY, Belelli D, Newell JG, Jackson MF, Lambert JJ, Rosahl TW, Wafford KA, MacDonald JF, Orser BA. Proc Natl Acad Sci U S A 2004; 101:3662-7. Reprinted with permission. In this Classic Paper Revisited, the author recounts the scientific journey leading to a report published in the Proceedings of the National Academy of Sciences (PNAS) and shares several personal stories from her formative years and "research truths" that she has learned along the way. Briefly, the principal inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA), was conventionally thought to regulate cognitive processes by activating synaptic GABA type A (GABAA) receptors and generating transient inhibitory synaptic currents. However, the author's laboratory team discovered a novel nonsynaptic form of tonic inhibition in hippocampal pyramidal neurons, mediated by extrasynaptic GABAA receptors that are pharmacologically distinct from synaptic GABAA receptors. This tonic current is highly sensitive to most general anesthetics, including sevoflurane and propofol, and likely contributes to the memory-blocking properties of these drugs. Before the publication in PNAS, the subunit composition of GABAA receptors that generate the tonic current was unknown. The team's research showed that GABAA receptors containing the α5 subunit (α5GABAARs) generated the tonic inhibitory current in hippocampal neurons. α5GABAARs are highly sensitive to GABA, desensitize slowly, and are thus well suited for detecting low, persistent, ambient concentrations of GABA in the extracellular space. Interest in α5GABAARs has surged since the PNAS report, driven by their pivotal roles in cognitive processes and their potential as therapeutic targets for treating various neurologic disorders.

Generation of two patient specific GABRD variants and their isogenic controls for modeling epilepsy

Developmental and epileptic encephalopathies (DEEs) are early-onset conditions that cause intractable seizures and developmental delays. Missense variants in Gamma-aminobutyric acid type A receptor (GABAAR) subunits commonly cause DEEs. Ahring et al. (2022) showed a variant in the gene that encodes the delta subunit (GABRD) is strongly associated with the gain-of-function of extrasynaptic GABAAR. Here, we report the generation of two patient-specific human induced pluripotent stem cells (hiPSC) lines with (i) a de novo variant and (ii) a maternal variant, both for the pathogenic GABRD c.872 C>T, (p.T291I). The variants in the generated cell line were corrected using the CRISPR-Cas9 gene editing technique (respective isogenic control lines).

Extrasynaptic localization is essential for α5GABAA receptor modulation of dopamine system function.

Dopamine system dysfunction, observed in animal models with psychosis-like symptomatology, can be restored by targeting Gamma-Aminobutyric Acid type A receptors (GABAAR) containing the α5, but not α1, subunit in the ventral hippocampus (vHipp). The reason for this discrepancy in efficacy remains elusive; however, one key difference is that α1GABAARs are primarily located in the synapse, whereas α5GABAARs are mostly extrasynaptic. To test whether receptor location is responsible for this difference in efficacy, we injected a small interfering ribonucleic acid (siRNA) into the vHipp to knock down radixin, a scaffolding protein that holds α5GABAARs in the extrasynaptic space. We then administered GL-II-73, a positive allosteric modulator of α5GABAARs (α5-PAM) known to reverse shock-induced deficits in dopamine system function, to determine if shifting α5GABAARs from the extrasynaptic space to the synapse would prevent the effects of α5-PAM on dopamine system function. As expected, knockdown of radixin significantly decreased radixin-associated α5GABAARs and increased the proportion of synaptic α5GABAARs, without changing the overall expression of α5GABAARs. Importantly, GL-II-73 was no longer able to modulate dopamine neuron activity in radixin-knockdown rats, indicating that the extrasynaptic localization of α5GABAARs is critical for hippocampal modulation of the dopamine system. These results may have important implications for clinical use of GL-II-73, as periods of high hippocampal activity appear to favor synaptic α5GABAARs, thus efficacy may be diminished in conditions where aberrant hippocampal activity is present.Significance Statement Currently available treatments for psychosis, a debilitating symptom linked with several brain disorders, are inadequate. While they can help manage symptoms in some patients, they do so imperfectly. They are also associated with severe side effects that can cause discontinuation of medication. This study provides preclinical evidence that the drug, GL-II-73, possesses the ability to modulate dopamine activity, a key player in psychosis symptoms, and further provides some mechanistic details regarding these effects. Overall, this work contributes to the growing body of literature suggesting that GL-II-73 and similar compounds may possess antipsychotic efficacy.

Expression of GABAA Receptor Subunits α1 and β2 in Healthy Human Dental Pulp

BACKGROUND: Gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the mammalian central nervous system (CNS), has a well-established role in pain modulation. While numerous studies have delved into the expression of GABA and its receptors in dental pulp, the exact influence of these receptors on dental pain signaling has not been fully elucidated. This study aimed to investigate the gene and protein expression of the two most abundantly expressed GABA type A (GABAA) receptor subunits, GABAA receptor subunit α1 (GABRA1) and β2 (GABRB2), in healthy human dental pulp.METHODS: Six tooth samples were collected from healthy individuals referred for orthodontic treatment. Total RNA was isolated from the pulp tissues of three samples and reverse transcription-polymerase chain reaction (RT-PCR) was performed to assess gene expression of GABRA1 and GABRB2. The other three samples were examined using immunohistochemistry (IHC) for visualization of GABRA1 and GABRB2 proteins within the dental pulp.RESULTS: RT-PCR analysis reported the presence of both GABRA1 and GABRB2 in the dental pulp, and independent t-test analysis revealed that the expression of GABRA1 was significantly higher than GABRB2. The immunohistochemical staining provided compelling visual evidence of the expression of GABRA1 and GABRB2 proteins within the odontoblast layer of dental pulp, clearly indicating their presence in the cell bodies and odontoblastic processes extending into the dentin.CONCLUSION: The presence of α1 and β2 subunits of the GABAA receptor in healthy human dental pulp offers valuable insights for further research into the potential roles of GABAA receptors in dental pain signaling.KEYWORDS: γ-aminobutyric acid, GABAA receptors, GABRA1, GABRB2, dental pain, pain signaling

The Activation of GABAAR Alleviated Cerebral Ischemic Injury via the Suppression of Oxidative Stress, Autophagy, and Apoptosis Pathways.

Ischemic stroke is a devastating disease leading to neurologic impairment. Compounding the issue is the very limited array of available interventions. The activation of a γ-aminobutyric acid (GABA) type A receptor (GABAAR) has been reported to produce neuroprotective properties during cerebral ischemia, but its mechanism of action is not yet fully understood. Here, in a rat model of photochemically induced cerebral ischemia, we found that muscimol, a GABAAR agonist, modulated GABAergic signaling, ameliorated anxiety-like behaviors, and attenuated neuronal damage in rats suffering cerebral ischemia. Moreover, GABAAR activation improved brain antioxidant levels, reducing the accumulation of oxidative products, which was closely associated with the NO/NOS pathway. Notably, the inhibition of autophagy markedly relieved the neuronal insult caused by cerebral ischemia. We further established an oxygen-glucose deprivation (OGD)-induced PC12 cell injury model. Both in vivo and in vitro experiments demonstrated that GABAAR activation obviously suppressed autophagy by regulating the AMPK-mTOR pathway. Additionally, GABAAR activation inhibited apoptosis through inhibiting the Bax/Bcl-2 pathway. These data suggest that GABAAR activation exerts neuroprotective effects during cerebral ischemia through improving oxidative stress and inhibiting autophagy and apoptosis. Our findings indicate that GABAAR serves as a target for treating cerebral ischemia and highlight the GABAAR-mediated autophagy signaling pathway.

Pathogenicity Prediction of GABAA Receptor Missense Variants

AbstractVariants in the genes encoding gamma‐aminobutyric acid type A (GABAA) receptor subunits are associated with epilepsy. To date, over 1000 clinical variants have been identified in these genes. However, the majority of these variants lack functional studies and their clinical significance is uncertain although accumulating evidence indicates that proteostasis deficiency is the major disease‐causing mechanism. Here, we apply two state‐of‐the‐art modeling tools, namely AlphaMissense and Rhapsody to predict the pathogenicity of saturating missense variants in genes that encode the major subunits of GABAA receptors in the central nervous system, including GABRA1, GABRB2, GABRB3, and GABRG2. We demonstrate that the predicted pathogenicity correlates well between AlphaMissense and Rhapsody. In addition, AlphaMissense pathogenicity score correlates modestly with plasma membrane expression, peak current amplitude, and GABA potency of the variants that have available experimental data. Furthermore, almost all annotated pathogenic variants in the ClinVar database are successfully identified from the prediction, whereas uncertain variants from ClinVar partially due to the lack of experimental data are differentiated into different pathogenicity groups. The pathogenicity prediction of GABAA receptor missense variants provides a resource to the community as well as guidance for future experimental and clinical investigations.

Dopaminergic Projections from the Hypothalamic A11 Nucleus to the Spinal Trigeminal Nucleus Are Involved in Bidirectional Migraine Modulation

Clinical imaging studies have revealed that the hypothalamus is activated in migraine patients prior to the onset of and during headache and have also shown that the hypothalamus has increased functional connectivity with the spinal trigeminal nucleus. The dopaminergic system of the hypothalamus plays an important role, and the dopamine-rich A11 nucleus may play an important role in migraine pathogenesis. We used intraperitoneal injections of glyceryl trinitrate to establish a model of acute migraine attack and chronicity in mice, which was verified by photophobia experiments and von Frey experiments. We explored the A11 nucleus and its downstream pathway using immunohistochemical staining and neuronal tracing techniques. During acute migraine attack and chronification, c-fos expression in GABAergic neurons in the A11 nucleus was significantly increased, and inhibition of DA neurons was achieved by binding to GABA A-type receptors on the surface of dopaminergic neurons in the A11 nucleus. However, the expression of tyrosine hydroxylase and glutamic acid decarboxylase proteins in the A11 nucleus of the hypothalamus did not change significantly. Specific destruction of dopaminergic neurons in the A11 nucleus of mice resulted in severe nociceptive sensitization and photophobic behavior. The expression levels of the D1 dopamine receptor and D2 dopamine receptor in the caudal part of the spinal trigeminal nucleus candalis of the chronic migraine model were increased. Skin nociceptive sensitization of mice was slowed by activation of the D2 dopamine receptor in SP5C, and activation of the D1 dopamine receptor reversed this behavioral change. GABAergic neurons in the A11 nucleus were activated and exerted postsynaptic inhibitory effects, which led to a decrease in the amount of DA secreted by the A11 nucleus in the spinal trigeminal nucleus candalis. The reduced DA bound preferentially to the D2 dopamine receptor, thus exerting a defensive effect against headache.

O030 GABAA Receptor Expression in the Suprachiasmatic Nucleus under Different Light Conditions

Abstract Our research group has made inroads investigating the phase shifting effects of general anaesthesia (GA) on the circadian clock. Most clinical anaesthetics function by potentiating the effect of GABA on the GABA type A receptor (GABAARs). While the precise role of GABA in the suprachiasmatic nucleus (SCN) is still not fully understood, it has been implicated in circadian functions including photic entrainment and clock coupling. However, we have discovered that the expression of GABAARs in the central circadian clock remains poorly understood. To address this gap in knowledge, we examined the expression of GABAAR subunits α1, α5, β3, and γ2 in male (n = 80) and female (n = 82) C57Bl/6 mice under light-dark (LD) and constant darkness (DD) conditions (male, n = 95). Analysis of GABAAR subunit expression in the SCN was quantified by immunohistochemical analysis. Additionally, we investigated the time-dependent effects of isoflurane GA and light on GABAAR subunit expression in the SCN (male, n = 60). Our findings revealed rhythmic expression of GABAAR subunits in the mouse SCN under LD conditions which differed by sex, suggesting environmental light conditions may regulate GABAAR expression in the circadian clock in a sex-specific manner. In the SCN, γ2 subunit expression was increased following light and GA treatment compared to light-alone, while α1 subunit expression was increased at times of large behavioural phase delays. We conclude that the expression of GABAAR’s in the SCN is phase-dependent and may contribute to the time-dependent relationship between light and GA on the clock.

Isoliquiritigenin modulates the activity of LTS and non-LTS cells in the ventrolateral preoptic area via GABAA receptors

ObjectiveIsoliquiritigenin (ILTG) is a chalcone compound that exhibits hypnotic effects via gamma-aminobutyric acid type A (GABAA) receptors. The ventrolateral preoptic area (VLPO) is a sleep-promoting center that contains a large number of GABA-releasing cells. There are two cell types in the VLPO: one generates a low-threshold spike (LTS), whereas the other lacks an LTS (non-LTS). MethodWhole-cell patch-clamp technology was used to detect the firing and currents of LTS and non-LTS cells in the VLPO. ResultsBath administration of ILTG (10 μM) increased the firing rate of VLPO LTS cells, reversed by flumazenil (5 μM), a GABAA benzodiazepine site antagonist. However, the firing rate of VLPO non-LTS cells was inhibited by ILTG (10 μM), also reversed by flumazenil (5 μM). No differences were detected regarding resting membrane potential (RMP) amplitude, spike threshold, afterhyperpolarization (AHP) amplitude, or action potential duration (APD50) after ILTG (10 μM) perfusion in VLPO LTS cells. RMP amplitude was more hyperpolarized and spike threshold was higher after ILTG (10 μM) application in VLPO non-LTS cells. In addition, ILTG significantly reduced the frequency of miniature inhibitory postsynaptic currents (mIPSCs) in VLPO LTS cells. ILTG significantly increased the amplitude of mIPSCs in VLPO non-LTS cells. ConclusionsThis study revealed that ILTG suppresses presynaptic GABA release on VLPO LTS cells, thereby increasing their excitability. ILTG enhances postsynaptic GABAA receptor function on VLPO non-LTS cells, thereby decreasing their excitability. These results suggest that ILTG may produce hypnotic effects by modulating the GABAergic synaptic transmission properties of these two cell types.

Cyclin D1—Cdk4 regulates neuronal activity through phosphorylation of GABAA receptors

Nuclear Cyclin D1 (Ccnd1) is a main regulator of cell cycle progression and cell proliferation. Interestingly, Ccnd1 moves to the cytoplasm at the onset of differentiation in neuronal precursors. However, cytoplasmic functions and targets of Ccnd1 in post-mitotic neurons are unknown. Here we identify the α4 subunit of gamma-aminobutyric acid (GABA) type A receptors (GABAARs) as an interactor and target of Ccnd1–Cdk4. Ccnd1 binds to an intracellular loop in α4 and, together with Cdk4, phosphorylates the α4 subunit at threonine 423 and serine 431. These modifications upregulate α4 surface levels, increasing the response of α4-containing GABAARs, measured in whole-cell patch-clamp recordings. In agreement with this role of Ccnd1–Cdk4 in neuronal signalling, inhibition of Cdk4 or expression of the non-phosphorylatable α4 decreases synaptic and extra-synaptic currents in the hippocampus of newborn rats. Moreover, according to α4 functions in synaptic pruning, CCND1 knockout mice display an altered pattern of dendritic spines that is rescued by the phosphomimetic α4. Overall, our findings molecularly link Ccnd1–Cdk4 to GABAARs activity in the central nervous system and highlight a novel role for this G1 cyclin in neuronal signalling.

Implications for Anesthesia and Beyond

AbstractGamma-aminobutyric acid (GABA), a nonpeptide amino acid transmitter, is a major component of modern neuropharmacology and one of the most crucial target sites for general anesthetics and therapeutic drugs. GABA type A receptors (GABAARs) are the most abundant inhibitory neurotransmitter receptors in the central nervous system. They are part of the rapid-acting, ligand-gated ion channel (LGIC) receptor category, a pentameric Cys-loop superfamily member that mediates inhibitory neurotransmission in the mature brain. GABAARs mainly consist of two α subunits, two β subunits, and one additional subunit from either γ or δ arranged around a central chloride (Cl-) selective channel. Multiple GABAAR subunit subtypes and splice variants have been identified. Each variant of GABAAR exhibits distinct biophysical and pharmacologic properties. Several compounds allosterically modulate the GABAAR positively or negatively. The widely used positive GABAAR modulators include benzodiazepines (anxiolytic and anticonvulsant), general anesthetics (volatile agents like isoflurane, and intravenous agents like barbiturates, etomidate, and propofol), long-chain alcohols, some anticonvulsants, and neuroactive steroids. The binding sites for each drug are distinctly different. The anesthetic drugs enhance receptor-mediated synaptic transmission and thus interrupt the thalamocortical transmission, which controls the sleep–wake patterns. Abnormality in the GABAAR function has been implicated in several neurological conditions, such as sleep disorders, seizures, depression, cognitive function, neurological recovery after injury, and neuroplasticity. Understanding the GABAAR lays the foundation for the development of highly specific drugs in the treatment of neurological disorders and general anesthesia.

Inhibitory and excitatory synaptic neuroadaptations in the diazepam tolerant brain

Benzodiazepine (BZ) drugs treat seizures, anxiety, insomnia, and alcohol withdrawal by potentiating γ2 subunit containing GABA type A receptors (GABAARs). BZ clinical use is hampered by tolerance and withdrawal symptoms including heightened seizure susceptibility, panic, and sleep disturbances. Here, we investigated inhibitory GABAergic and excitatory glutamatergic plasticity in mice tolerant to benzodiazepine sedation. Repeated diazepam (DZP) treatment diminished sedative effects and decreased DZP potentiation of GABAAR synaptic currents without impacting overall synaptic inhibition. While DZP did not alter γ2-GABAAR subunit composition, there was a redistribution of extrasynaptic GABAARs to synapses, resulting in higher levels of synaptic BZ-insensitive α4-containing GABAARs and a concomitant reduction in tonic inhibition. Conversely, excitatory glutamatergic synaptic transmission was increased, and NMDAR subunits were upregulated at synaptic and total protein levels. Quantitative proteomics further revealed cortex neuroadaptations of key pro-excitatory mediators and synaptic plasticity pathways highlighted by Ca2+/calmodulin-dependent protein kinase II (CAMKII), MAPK, and PKC signaling. Thus, reduced inhibitory GABAergic tone and elevated glutamatergic neurotransmission contribute to disrupted excitation/inhibition balance and reduced BZ therapeutic power with benzodiazepine tolerance.

Flumazenil Pretreatment Reduces Mefenamic Acid-Induced Central Nervous System Toxicity in Mice.

Mefenamic acid (MFA), a common analgesic, causes central nervous system (CNS) toxicity at high doses with a proposed activity on the Gamma-aminobutyric acid (GABA) system. However, it remains unknown whether flumazenil (FMZ), a GABA type A receptor (GABAAR) antagonist, can reverse MFA toxicity. The behavioral and neurophysiological effects of MFA were investigated in mice with and without FMZ pre-treatment. The elevated zero maze (EZM) and marble burying tests were used to assess anxiety-like behaviors and burying activities, respectively. The standard bar test was used to evaluate catalepsy, while the actophotometer test was used to measure locomotor activity. Seizure intensity was scored, and fatalities were counted. Without FMZ pre-treatment, MFA induced behavioral and neurophysiological effects in a dose-dependent manner as follows: At a dose of 20 mg/kg, i.p, MFA-treated mice exhibited anxiety-like behaviors, which was determined by a significant increase in the time spent in the closed areas and a significant decrease in the number of entries to the open areas of the EZM apparatus. These mice also showed a significant decrease in the burying activity, manifested as a significant decrease in the number of buried marbles. At 40 mg/kg, i.p., MFA-treated mice showed catalepsy that was associated with a significant decrease in locomotor activity. At a dose of 80 mg/kg, i.p., mice developed fatal tonic-clonic seizures (seizure score = 4). Pre-treatment with FMZ (5 mg/kg, i.p.) significantly reversed the anxiety-like behaviors and restored marble-burying activity. Additionally, FMZ prevented catalepsy, significantly restored locomotor activity, reduced seizure intensity (seizure score = 0.3) and significantly reduced mortalities. The present study's findings indicate that activation of the GABAAR is involved in the CNS toxicity of MFA, and FMZ reverses MFA toxicity by interfering with this receptor.

Topical borneol relieves nonhistaminergic pruritus via targeting TRPA1 and TRPM8 channels in peripheral nerve terminals of mice

Borneol has been used successfully for the treatment of itchy skin in traditional Chinese medicine. However, the antipruritic effect of borneol has rarely been studied, and the mechanism is unclear. Here, we showed that topical application of borneol on skin substantially suppressed pruritogen chloroquine- and compound 48/80-induced itching in mice. The potential targets of borneol, including transient receptor potential cation channel subfamily V member 3 (TRPV3), transient receptor potential cation channel subfamily A member 1 (TRPA1), transient receptor potential cation channel subfamily M member 8 (TRPM8), and gamma-aminobutyric acid type A (GABAA) receptor were pharmacologically inhibited or genetically knocked out one by one in mouse. Itching behavior studies demonstrated that the antipruritic effect of borneol is largely independent of TRPV3 and GABAA receptor, and TRPA1 and TRPM8 channels are responsible for a major portion of the effect of borneol on chloroquine-induced nonhistaminergic itching. Borneol activates TRPM8 and inhibits TRPA1 in sensory neurons of mice. Topical co-application of TRPA1 antagonist and TRPM8 agonist mimicked the effect of borneol on chloroquine-induced itching. Intrathecal injection of a group II metabotropic glutamate receptor antagonist partially attenuated the effect of borneol and completely abolished the effect of TRPM8 agonist on chloroquine-induced itching, suggesting that a spinal glutamatergic mechanism is involved. In contrast, the effect of borneol on compound 48/80-induced histaminergic itching occurs through TRPA1-and TRPM8-independent mechanisms. Our work demonstrates that borneol is an effective topical itch reliever, and TRPA1 inhibition and TRPM8 activation in peripheral nerve terminals account for its antipruritic effect.

Long-term α5 GABA A receptor negative allosteric modulator treatment reduces NMDAR-mediated neuronal excitation and maintains basal neuronal inhibition

α5 subunit-containing GABA type-A receptors (α5 GABAARs) are enriched in the hippocampus and play critical roles in neurodevelopment, synaptic plasticity, and cognition. α5 GABAAR preferring negative allosteric modulators (α5 NAMs) show promise mitigating cognitive impairment in preclinical studies of conditions characterized by excess GABAergic inhibition, including Down syndrome and memory deficits post-anesthesia. However, previous studies have primarily focused on acute application or single-dose α5 NAM treatment. Here, we measured the effects of chronic (7-day) in vitro treatment with L-655,708 (L6), a highly selective α5 NAM, on glutamatergic and GABAergic synapses in rat hippocampal neurons. We previously showed that 2-day in vitro treatment with L6 enhanced synaptic levels of the glutamate NMDA receptor (NMDAR) GluN2A subunit without modifying surface α5 GABAAR expression, inhibitory synapse function, or L6 sensitivity. We hypothesized that chronic L6 treatment would further increase synaptic GluN2A subunit levels while maintaining GABAergic inhibition and L6 efficacy, thus increasing neuronal excitation and glutamate-evoked intracellular calcium responses. Immunofluorescence experiments revealed that 7-day L6 treatment slightly increased the synaptic levels of gephyrin and surface α5 GABAARs. Functional studies showed that chronic α5 NAM treatment did not alter inhibition or α5 NAM sensitivity. Surprisingly, chronic L6 exposure decreased surface levels of GluN2A and GluN2B subunits, concurrent with reduced NMDAR-mediated neuronal excitation as seen by faster synaptic decay rates and reduced glutamate-evoked calcium responses. Together, these results show that chronic in vitro treatment with an α5 NAM leads to subtle homeostatic changes in inhibitory and excitatory synapses that suggest an overall dampening of excitability.

Altered GABAA receptor function in women with endometriosis: a possible pain-related mechanism.

The mechanism underlying endometriosis-related pain remains poorly understood. Previous studies have indicated that γ-aminobutyric acid (GABA) type A (GABAA ) receptors and GABAergic substances (eg endogenous neurosteroids) play important mechanistic roles in various pain conditions. Our primary objective was to compare GABAA receptor function between women with endometriosis and healthy controls by performing a challenge test with diazepam, a GABAA receptor agonist, using the saccadic eye velocity as the main outcome. The secondary objective was to investigate the relation between GABAA receptor function and serum levels of allopregnanolone, an endogenous positive modulator of the GABAA receptor, in the participating women. 15 women with pelvic pain and laparoscopically confirmed endometriosis and 10 healthy, symptom-free, control women, aged 18-40 years, underwent the diazepam challenge test during the follicular phase of the menstrual cycle. Basal serum allopregnanolone levels were measured prior to diazepam injection. Compared with healthy controls, women with pelvic pain and confirmed endometriosis had a significantly smaller change in saccadic eye velocity after GABAA receptor stimulation with diazepam, indicating lower sensitivity to diazepam. The saccadic eye velocity response was not correlated with the serum allopregnanolone levels. Women with painful endometriosis show altered GABAA receptor function, depicted as a muted response to an exogenous GABAA receptor agonist.

Gamma-aminobutyric acid type A receptor alpha 4 coordinates autophagy, inflammation, and immunometabolism to promote innate immune activation

Gamma-aminobutyric acid type A receptor (GABAAR), the ionotropic receptor of GABA, is expressed in macrophages and in the nervous system; however, its role in innate immunity is unknown. Herein, we identified myeloid GABAAR subunit α4 (Gabra4) as a critical regulator of autophagy and a promoter of host innate defense during infection and inflammation. Myeloid Gabra4 deficiency led to defective mycobacterial clearance during infection and increased susceptibility to septic shock. Gabra4 deletion exaggerated inflammatory responses and suppressed the activation of autophagy in macrophages upon infectious and inflammatory stimuli. Mechanistically, Gabra4-mediated signaling led to upregulation of autophagy in macrophages via intracellular calcium release and AMP-activated protein kinase (AMPK) signaling activation, which was required for linking autophagy and antimicrobial responses. Additionally, Gabra4 was required to generate mitochondrial reactive oxygen species, thereby triggering autophagy and antimicrobial responses to mycobacteria. Metabolomics analysis showed that Gabra4 was critical for glucose metabolism and aerobic glycolysis in macrophages. Our findings demonstrate that myeloid Gabra4 coordinates autophagy, inflammation, and immunometabolism to promote innate host defense against pathogenic and dangerous stimuli.Abbreviation AM: Alveolar macrophage; AMPK: AMP-activated protein kinase; ASC: Apoptosis-associated speck-like protein containing a CARD; ATP: Adenosine 5’-triphosphate; BAL: Bronchoalveolar lavage; BCG: Mycobacterium bovis Bacillus Calmette–Guérin; BMDM: Bone marrow-derived macrophage; CCL: CC motif chemokine ligand; CFU: Colony forming unit; CKO: Conditional knock out; CXCL: C-X-C motif ligand; Dpi: Days post-infection; ECAR: Extracellular acidification rate; EGFP: Enhanced green fluorescent protein; FOXO3: Forkhead box O3; GABA: Gamma-aminobutyric acid; GABAAR: GABA type A receptor; Gabarap: GABA type A receptor-associated protein; Gabarapl1: GABA type A receptor-associated protein like 1; GABRA4: Gamma-aminobutyric acid type A receptor subunit alpha4; HIF-1α: Hypoxia-inducible factor-1 alpha; IL: Interleukin; i.n.: Intranasal; i.p.: Intraperitoneal; LDHA: Lactate dehydrogenase A; LPS: Lipopolysaccharide; Mabc: Mycobacteroides abscessus subsp. abscessus; MOI: Multiplicities of infection; Mtb: Mycobacterium tuberculosis; mtROS: Mitochondrial reactive oxygen species; OCR: Oxygen consumption rate; OXPHOS: Oxidative phosphorylation; PM: Peritoneal macrophage; TNF: Tumor necrosis factor; WT: Wild type

Electroacupuncture-induced activation of GABAergic system alleviates airway inflammation in asthma model by suppressing TLR4/MyD88/NF-κB signaling pathway.

Electroacupuncture (EA) has been shown to attenuate airway inflammation in asthmatic mice; however, the underlying mechanism is not fully understood. Studies have shown that EA can significantly increase the inhibitory neurotransmitter γ-aminobutyric acid (GABA) content in mice, and can also increase the expression level of GABA type A receptor (GABAAR). Furthermore, activating GABAAR may relieve inflammation in asthma by suppressing toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB) signaling pathway. Therefore, this study aimed to investigate the role of GABAergic system and TLR4/MyD88/NF-κB signaling pathway in asthmatic mice treated with EA. A mouse model of asthma was established, and a series of methods including Western blot and histological staining assessment were employed to detect the level of GABA, and expressions of GABAAR and TLR4/MyD88/NF-κB in lung tissue. In addition, GABAAR antagonist was used to further validate the role and mechanism of GABAergic system in mediating the therapeutic effect of EA in asthma. The mouse model of asthma was established successfully, and EA was verified to alleviate airway inflammation in asthmatic mice. The release of GABA and the expression of GABAAR were significantly increased in asthmatic mice treated with EA compared with untreated asthmatic mice (P < 0.01), and the TLR4/MyD88/NF-κB signaling pathway was down-regulated. Moreover, inhibition of GABAAR attenuated the beneficial effects of EA in asthma, including the regulation of airway resistance and inflammation, as well as the inhibitory effects on TLR4/MyD88/NF-κB signaling pathway. Our findings suggest that GABAergic system may be involved in mediating the therapeutic effect of EA in asthma, possibly by suppressing the TLR4/MyD88/NF-κB signaling pathway.

Translational patterns of ionotropic glutamate and GABA receptors during brain development and behavioral stimuli revealed by polysome profiling.

mRNA translation is critical for regulation of various aspects of the nervous system. Ionotropic glutamate and gamma-aminobutyric acid type A (GABAA ) receptors are fundamental synaptic ion channels that control excitatory and inhibitory synaptic transmission, respectively. However, little is known about the translation of these receptors during brain development and function. By utilizing polysome profiling, a powerful tool for investigating translational machinery and mRNA translational states, we characterized the translational patterns of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-d-aspartate (NMDA), and GABAA receptor subunits, and compared them with total mRNA and protein levels during mouse brain development, in different brain regions, and in response to behavioral stimuli. Most of the receptor subunits exhibited developmental changes at total mRNA, translation, and protein levels, among which translation of Gria1, Gria2, Grin1, Grin2a, Gabra1, and Gabrg2 contributed greatly to their protein levels. Most of the receptor subunits also displayed differentiated levels of total mRNA, translation, and protein in the prefrontal cortex and hippocampus, among which translation of Gria1, Gria2, Gabrb2, and Gabrg2 contributed to their protein levels. Finally, we showed that acute foot shock stress had a rapid influence in both the prefrontal cortex and hippocampus, with the prefrontal cortex displaying more changes at translational and protein levels. Notably, Grin2a is translationally repressed by stress which was followed by a decrease of GluN2A protein in both brain regions. Together, this study provides a new understanding of the translational patterns of critical ionotropic synaptic receptors during brain development and behavioral stress.

Lateral Diffusion of NKCC1 Contributes to Chloride Homeostasis in Neurons and Is Rapidly Regulated by the WNK Signaling Pathway.

An upregulation of the Na+-K+-2Cl- cotransporter NKCC1, the main chloride importer in mature neurons, can lead to depolarizing/excitatory responses mediated by GABA type A receptors (GABAARs) and, thus, to hyperactivity. Understanding the regulatory mechanisms of NKCC1 would help prevent intra-neuronal chloride accumulation that occurs in pathologies with defective inhibition. The cell mechanisms regulating NKCC1 are poorly understood. Here, we report in mature hippocampal neurons that GABAergic activity controls the membrane diffusion and clustering of NKCC1 via the chloride-sensitive WNK lysine deficient protein kinase 1 (WNK1) and the downstream Ste20 Pro-line Asparagine Rich Kinase (SPAK) kinase that directly phosphorylates NKCC1 on key threonine residues. At rest, this signaling pathway has little effect on intracellular Cl- concentration, but it participates in the elevation of intraneuronal Cl- concentration in hyperactivity conditions associated with an up-regulation of NKCC1. The fact that the main chloride exporter, the K+-Cl- cotransporter KCC2, is also regulated in mature neurons by the WNK1 pathway indicates that this pathway will be a target of choice in the pathology.