GABAA Receptor Α1 Subunit Research Articles

PSD-95 deficiency alters GABAergic inhibition in the prefrontal cortex

Postsynaptic Density Protein-95 (PSD-95) is a major scaffolding protein in the excitatory synapses in the brain and a critical regulator of synaptic maturation for NMDA and AMPA receptors. PSD-95 deficiency has been linked to cognitive and learning deficits implicated in neurodevelopmental disorders such as autism and schizophrenia. Previous studies have shown that PSD-95 deficiency causes a significant reduction in the excitatory response in the hippocampus. However, little is known about whether PSD-95 deficiency will affect gamma-aminobutyric acid (GABA)ergic inhibitory synapses. Using a PSD-95 transgenic mouse model (PSD-95+/−), we studied how PSD-95 deficiency affects GABAA receptor expression and function in the medial prefrontal cortex (mPFC) during adolescence. Our results showed a significant increase in the GABAA receptor subunit α1. Correspondingly, there are increases in the frequency and amplitude in spontaneous inhibitory postsynaptic currents (sIPSCs) in pyramidal neurons in the mPFC of PSD-95+/− mice, along with a significant increase in evoked IPSCs, leading to a dramatic shift in the excitatory-to-inhibitory balance in PSD-95 deficient mice. Furthermore, PSD-95 deficiency promotes inhibitory synapse function via upregulation and trafficking of NLGN2 and reduced GSK3β activity through tyr-216 phosphorylation. Our study provides novel insights on the effects of GABAergic transmission in the mPFC due to PSD-95 deficiency and its potential link with cognitive and learning deficits associated with neuropsychiatric disorders.

Enriched environment ameliorates memory impairments in rats after postsurgery sleep deprivation

Postsurgery sleep deprivation is a common complication that severely deteriorates the quality of life of patients. Here we aim to investigate the effects and mechanism of enriched environment in ameliorating sleep deprivation and memory impairments. Hernia repair surgery was performed on rats to induce sleep deprivation. Enriched environment (EE) was used to treat rats with sleep deprivation, and open field and Y-maze tests were performed to compare behavioral parameters of sleep deprivation rats with or without EE treatments to those of normal rats. To understand the mechanism, neurotrophic and growth factors including BDNF, NGF, NT-3 and GDNF were analyzed using enzyme-linked immunosorbent assay (ELISA). AMPAR subunits, including GluA1-A3, and GABAA receptor α1 subunit expression in hippocampus tissues were assessed using western blot. EE restored normal levels of anxiety index and freezing behavior in open field test and level of alternation in Y-maze test, suggesting the reduction of anxiolytic effects and spatial memory impairment induced by sleep deprivation. EE increased BDNF levels and reduced NT-3 levels in sleep deprivation rats. GluA1/GluA2 ratio was increased by EE. GABAA receptor α1 subunit expression was decreased by EE. EE is effective in ameliorating the detrimental effects of sleep deprivation in spatial memory impairment, and restoring normal levels of neurotrophic factors, which are potentially mediated by attenuating the changes in AMPAR subunit expression and reducing GABAA receptor α1 subunit expression. These data provide supporting evidences for the use of EE to treat adverse outcomes of sleep deprivation induced by surgery.

Roles of GABAA receptor α5 subunit on locomotion and working memory in transient forebrain ischemia in mice

The γ-aminobutyric acid A (GABAA) receptor, which contains a chloride channel, is a typical inhibitory neurotransmitter receptor in the central nervous system. Although the GABAergic neurotransmitter system has been discovered to be involved in various psychological behaviors, such as anxiety, convulsions, and cognitive function, its functional changes under conditions of ischemic pathological situation are still uncovered. In the present study, we attempted to elucidate the functional changes in the GABAergic system after transient forebrain ischemia in mice. A bilateral common carotid artery occlusion for 20 min was used to establish a model of transient forebrain ischemia/reperfusion (tI/R). Delayed treatment with diazepam, a positive allosteric modulator of the GABAA receptor, increased locomotor activity in the open field test and spontaneous alternations in the Y-maze test in tI/R mice, but not in shams. Delayed treatment with diazepam did not alter neuronal death or the number of GABAergic neurons in tI/R mice. However, tI/R induced changes in the protein levels of GABAA receptor subunits in the hippocampus. In particular, the most marked increase in the tI/R group was found in the level of α5 subunit of the GABAA receptor. Similar to delayed treatment with diazepam, delayed treatment with imidazenil, an α5-sensitive benzodiazepine, increased spontaneous alternations in the Y-maze in tI/R mice, whereas zolpidem, an α5-insensitive benzodiazepine, failed to show such effects. These results suggest that tI/R-induced changes in the level of the α5 subunit of the GABAA receptor can alter the function of GABAergic drugs in a mouse model of forebrain ischemia.

Neonatal Clonazepam Administration Induced Long-Lasting Changes in GABAA and GABAB Receptors.

Benzodiazepines (BZDs) are widely used in patients of all ages. Unlike adults, neonatal animals treated with BZDs exhibit a variety of behavioral deficits later in life; however, the mechanisms underlying these deficits are poorly understood. This study aims to examine whether administration of clonazepam (CZP; 1 mg/kg/day) in 7–11-day-old rats affects Gama aminobutyric acid (GABA)ergic receptors in both the short and long terms. Using RT-PCR and quantitative autoradiography, we examined the expression of the selected GABAA receptor subunits (α1, α2, α4, γ2, and δ) and the GABAB B2 subunit, and GABAA, benzodiazepine, and GABAB receptor binding 48 h, 1 week, and 2 months after treatment discontinuation. Within one week after CZP cessation, the expression of the α2 subunit was upregulated, whereas that of the δ subunit was downregulated in both the hippocampus and cortex. In the hippocampus, the α4 subunit was downregulated after the 2-month interval. Changes in receptor binding were highly dependent on the receptor type, the interval after treatment cessation, and the brain structure. GABAA receptor binding was increased in almost all of the brain structures after the 48-h interval. BZD-binding was decreased in many brain structures involved in the neuronal networks associated with emotional behavior, anxiety, and cognitive functions after the 2-month interval. Binding of the GABAB receptors changed depending on the interval and brain structure. Overall, the described changes may affect both synaptic development and functioning and may potentially cause behavioral impairment.

Immunohistochemical distribution of 10 GABAA receptor subunits in the forebrain of the rhesus monkey Macaca mulatta.

GABAA receptors are composed of five subunits arranged around a central chloride channel. Their subunits originate from different genes or gene families. The majority of GABAA receptors in the mammalian brain consist of two α‐, two β‐ and one γ‐ or δ‐subunit. This subunit organization crucially determines the physiological and pharmacological properties of the GABAA receptors. Using immunohistochemistry, we investigated the distribution of 10 GABAA receptor subunits (α1, α2, α3, α4, α5, β1, β2, β3, γ2, and δ) in the fore brain of three female rhesus monkeys (Macaca mulatta). Within the cerebral cortex, subunits α1, α5, β2, β3, and γ2 were found in all layers, α2, α3, and β1 were more concentrated in the inner and outer layers. The caudate/putamen was rich in α1, α2, α5, all three β‐subunits, γ2, and δ. Subunits α3 and α5 were more concentrated in the caudate than in the putamen. In contrast, α1, α2, β1, β2, γ2, and δ were highest in the pallidum. Most dorsal thalamic nuclei contained subunits α1, α2, α4, β2, β3, and γ2, whereas α1, α3, β1, and γ2 were most abundant in the reticular nucleus. Within the amygdala, subunits α1, α2, α5, β1, β3, γ2, and δ were concentrated in the cortical nucleus, whereas in the lateral and basolateral amygdala α1, α2, α5, β1, β3, and δ, and in the central amygdala α1, α2, β3, and γ2 were most abundant. Interestingly, subunit α3‐IR outlined the intercalated nuclei of the amygdala. In the hippocampus, subunits α1, α2, α5, β2, β3, γ2, and δ were highly expressed in the dentate molecular layer, whereas α1, α2, α3, α5, β1, β2, β3, and γ2 were concentrated in sector CA1 and the subiculum. The distribution of GABAA receptor subunits in the rhesus monkey was highly heterogeneous indicating a high number of differently assembled receptors. In most areas investigated, notably in the striatum/pallidum, amygdaloid nuclei and in the hippocampus it was more diverse than in the rat and mouse indicating a more heterogeneous and less defined receptor assembly in the monkey than in rodent brain.

Developmental Analysis of GABAA Receptor Gene Expression in Gallus gallus Chick Brain

Fetal Alcohol Spectrum Disorders (FASD) are a class of neurological disorders that arise from both ethanol‐related toxicity and alterations in a variety of molecular pathways during fetal development. One result of embryonic ethanol exposure is overactivation of the GABAA receptors within neuronal cells. Due to the varying subunit composition of these pentameric ionotropic receptors in different brain regions and timepoints during development, this experiment characterized the expression levels of nine GABAA receptor subunits (α1–6, β1–3) in an embryonic chick model. RNA was isolated from three brain tissue regions (cerebellum, forebrain, optic tectum). After, the RNA was converted to cDNA by reverse transcription and the gene transcripts were assessed using RT‐qPCR with specifically designed primers for each GABAA subunit type. With the exception of the α6 subunit, expression of the GABAA receptor genes were consistent throughout all brain tissue types and only showed a slight variation in level of expression when it came to the different developmental time points of E7–E17. α6 was absent in forebrain, and not detected until E15 in cerebellum and optic tectum samples. Based on the data collected, it can be concluded that the nine GABAA subunit genes tested are mostly expressed throughout the chicken embryonic timeline. There is currently limited data as to how their gene expression levels change as the embryo develops. Further research is needed to determine the distinct differences between the different subunit types. These subtle differences between the expression of each GABAA receptor subunit gene will point to a better understanding of the influence ethanol has on the developing embryo in the chick model. This evidence may hold meaning to why alcohol exposure during certain time periods in fetal development for humans prove to have more severe effects on the fetus, like FASD, than during other time periods during embryonic growth.

Benzodiazepine exposure induces transcriptional down-regulation of GABAA receptor α1 subunit gene via L-type voltage-gated calcium channel activation in rat cerebrocortical neurons

GABAA receptors are targets of different pharmacologically relevant drugs, such as barbiturates, benzodiazepines, and anesthetics. In particular, benzodiazepines are prescribed for the treatment of anxiety, sleep disorders, and seizure disorders. Benzodiazepines potentiate GABA responses by binding to GABAA receptors, which are mainly composed of α (1–3, 5), β2, and γ2 subunits. Prolonged activation of GABAA receptors by endogenous and exogenous modulators induces adaptive changes that lead to tolerance. For example, chronic administration of benzodiazepines produces tolerance to most of their pharmacological actions, limiting their usefulness. The mechanism of benzodiazepine tolerance is still unknown. To investigate the molecular basis of tolerance, we studied the effect of sustained exposure of rat cerebral cortical neurons to diazepam on the GABAA receptor. Flunitrazepam binding experiments showed that diazepam treatment induced uncoupling between GABA and benzodiazepine sites, which was blocked by co-incubation with flumazenil, picrotoxin, or nifedipine. Diazepam also produced selective transcriptional down-regulation of GABAA receptor α1 subunit gene through a mechanism dependent on the activation of L-type voltage-gated calcium channels. These findings suggest benzodiazepine-induced stimulation of calcium influx through L-type voltage-gated calcium channels triggers the activation of a signaling pathway that leads to uncoupling and an alteration of receptor subunit expression. Insights into the mechanism of benzodiazepine tolerance will contribute to the design of new drugs that can maintain their efficacies after long-term treatments.

MicroRNA-7a overexpression in VMH restores the sympathoadrenal response to hypoglycemia.

It is proposed that the impaired sympathoadrenal response to hypoglycemia induced by recurrent insulin-induced hypoglycemia (RH) is an adaptive phenomenon induced by specific changes in microRNA expression in the ventromedial hypothalamus (VMH). To test this hypothesis, genome-wide microRNAomic profiling of the VMH by RNA-sequencing was performed in control rats and rats treated for RH. Differential expression analysis identified microRNA-7a-5p and microRNA-665 as potential mediators of this phenomenon. To further test this hypothesis, experiments were conducted consisting of targeted lentiviral-mediated overexpression of microRNA-7a-5p and downregulation of microRNA-665 in the VMH. Hyperinsulinemic hypoglycemic clamp experiments demonstrated that targeted overexpression of microRNA-7a-5p (but not downregulation of microRNA-665) in the VMH of RH rats restored the epinephrine response to hypoglycemia. This restored response to hypoglycemia was associated with a restoration of GABAA receptor gene expression. Finally, a direct interaction of microRNA-7a-5p with the 3'-UTR of GABAA receptor α1-subunit (Gabra1) gene was demonstrated in a luciferase assay. These findings indicate that (a) the impaired sympathoadrenal response RH induces is associated with changes in VMH microRNA expression and (b) microRNA-7a-5p, possibly via direct downregulation of GABA receptor gene expression, may serve as a mediator of the altered sympathoadrenal response to hypoglycemia.

The role of the GABAA receptor Alpha 1 subunit in the ventral hippocampus in stress resilience

Pre-pubertal stress increases post-traumatic stress disorder (PTSD) susceptibility. We have previously demonstrated that enriched environment (EE) intervention immediately after pre-pubertal stress protects from the effects of trauma in adulthood. Here, we examined whether exposure to EE would also be beneficial if applied after exposure to trauma in adulthood. We have recently shown that exposure to juvenile stress and under-water trauma (UWT) is associated with increased expression of GABAA receptor subunit α1 in the ventral hippocampus. However, differentiating between affected and unaffected individuals, this increased expression was confined to stress-exposed, behaviorally unaffected individuals, suggesting upregulation of α1 expression as a potential mechanism of resilience. We now examined whether EE-induced resilience renders increased expression of α1 in the ventral hippocampus redundant when facing a trauma later in life. Adult rats were exposed to UWT, with pre-exposure to juvenile stress, and tested in the open field and elevated plus maze paradigms four weeks later. EE exposure during juvenility prevented pre-pubertal stress-induced vulnerability, but not if performed following UWT in adulthood. Furthermore, juvenile EE exposure prevented the trauma-associated increase in α1 expression levels. Our findings emphasize the importance of early interventions in order to reduce the likelihood of developing psychopathologies in adulthood.

Alterations in GABAA Receptor Subunit Expression in the Amygdala and Entorhinal Cortex in Human Temporal Lobe Epilepsy.

The amygdala has long been implicated in the pathophysiology of human temporal lobe epilepsy (TLE). The different nuclei of this complex structure are interconnected and share reciprocal connections with the hippocampus and other brain structures, partly via the entorhinal cortex. Expression of GABAA receptor subunits α1, α2, α3, α5, β2, β2/3, and γ2 was evaluated by immunohistochemistry in amygdala specimens and the entorhinal cortex of 12 TLE patients and 12 autopsy controls. A substantial decrease in the expression of α1, α2, α3, and β2/3 subunits was found in TLE cases, accompanied by an increase of γ2 subunit expression in many nuclei. In the entorhinal cortex, the expression of all GABAA receptor subunits was decreased except for the α1 subunit, which was increased on cellular somata. The overall reduction in α subunit expression may lead to decreased sensitivity to GABA and its ligands and compromise phasic inhibition, whereas upregulation of the γ2 subunit might influence clustering and kinetics of receptors and impair tonic inhibition. The description of these alterations in the human amygdala is important for the understanding of network changes in TLE as well as the development of subunit-specific therapeutic agents for the treatment of this disease.

Diazepam inhibited lipopolysaccharide (LPS)-induced pyroptotic cell death and alleviated pulmonary fibrosis in mice by specifically activating GABAA receptor α4-subunit

Diazepam could regulate immune system and inflammation, which might be a potential therapeutic agent for pulmonary fibrosis in clinic. This study showed that diazepam reversed LPS-induced inhibition of cell proliferation and promotion of cell apoptosis. Of note, LPS specifically induced Caspase-11 dependent cell pyroptosis, which were significantly attenuated by diazepam or pyroptosis inhibitor necrosulfonamide (NSA) treatment. In addition, α4- and α5-subunits of GABAARs were highly expressed in human bronchial 16HBE cells, human pulmonary epithelial cells (BEAS-2B) and pulmonary epithelial cells isolated from mice (mPECs). Further results showed that only knock-down of α4-GABAARs abrogated the effects of diazepam on LPS induced cell pyroptosis, apoptosis and proliferation. Similiarly, either diazepam or NSA treatment could alleviate development of LPS induced inflammatory reactions and pulmonary fibrosis in mice, which were abrogated by synergistically knocking down α4-GABAARs. Taken together, diazepam alleviated LPS-induced cell pyroptosis and development of pulmonary fibrosis in mice by activating α4-GABAARs.

Increased sensitivity to psychostimulants and GABAergic drugs in Lsamp-deficient mice

Lsamp, in combinations with other members of the IgLON family of cell adhesion molecules, promotes and inhibits neurite outgrowth and synapse formation during development. Mice lacking Lsamp gene display decreased social behaviour, hyperactivity; decreased anxiety level, alongside with altered balance in GABAA receptor α1 and α2 subunits; and decreased sensitivity to amphetamine, alongside with elevated serotonin function. In human studies, Lsamp has been associated with several psychiatric diseases, including schizophrenia, and suicide. Here, we provide a more thorough characterization of the pharmacological phenotype of Lsamp-deficient mice, including testing for sensitivity to morphine, cocaine, MK-801 and ketamine. More thorougly, sensitivity to GABA modulators (diazepam, alprazolam, ethanol, pentobarbital, TP003, and SL651498) was assessed. In brief, Lsamp-deficient mice were more sensitive to the locomotor activating effects of cocaine and morphine, and hypersensitive to the sedative and muscle relaxant effects of GABA modulators, most likely reflecting enhanced function of α1 and α5 subunits of the GABAA receptor. No gross differences in sensitivity to NMDA receptor modulators were observed. Thus, as the lack of Lsamp gene leads to widespread imbalances in major neurotransmitter systems in the brain accompanied by remarkable changes in behavioural phenotype as well, Lsamp-deficient mice are a promising model for mimicking psychiatric disorders.

Potentiating α2 subunit containing perisomatic GABAA receptors protects against seizures in a mouse model of Dravet syndrome.

Dravet syndrome mice (Scn1a+/- ) demonstrate a marked strain dependence for the severity of seizures which is correlated with GABAA receptor α2 subunit expression. The α2 /α3 subunit selective positive allosteric modulator (PAM) AZD7325 potentiates inhibitory postsynaptic currents (IPSCs) specifically in perisomatic synapses. AZD7325 demonstrates stronger effects on IPSCs in the seizure resistant mouse strain, consistent with higher α2 subunit expression. AZD7325 demonstrates seizure protective effects in Scn1a+/- mice without apparent sedative effects in vivo. GABAA receptor potentiators are commonly used for the treatment of epilepsy, but it is not clear whether targeting distinct GABAA receptor subtypes will have disproportionate benefits over adverse effects. Here we demonstrate that the α2 /α3 selective positive allosteric modulator (PAM) AZD7325 preferentially potentiates hippocampal inhibitory responses at synapses proximal to the soma of CA1 neurons. The effect of AZD7325 on synaptic responses was more prominent in mice on the 129S6/SvEvTac background strain, which have been demonstrated to be seizure resistant in the model of Dravet syndrome (Scn1a+/- ), and in which the α2 GABAA receptor subunits are expressed at higher levels relative to in the seizure prone C57BL/6J background strain. Consistent with this, treatment of Scn1a+/- mice with AZD7325 elevated the temperature threshold for hyperthermia-induced seizures without apparent sedative effects. Our results in a model system indicate that selectively targeting α2 is a potential therapeutic option for Dravet syndrome.

Altered inhibitory synapses in de novo GABRA5 and GABRA1 mutations associated with early onset epileptic encephalopathies.

We performed next generation sequencing on 1696 patients with epilepsy and intellectual disability using a gene panel with 480 epilepsy-related genes including all GABAA receptor subunit genes (GABRs), and we identified six de novo GABR mutations, two novel GABRA5 mutations (c.880G>T, p.V294F and c.1238C>T, p.S413F), two novel GABRA1 mutations (c.778C>T, p.P260S and c.887T>C, p.L296S/c.944G>T, p.W315L) and two known GABRA1 mutations (c.335G>A, p.R112Q and c.343A>G, p.N115D) in six patients with intractable early onset epileptic encephalopathy. The α5(V294F and S413F) and α1(P260S and L296S/W315L) subunit residue substitutions were all in transmembrane domains, while the α1(R112Q and N115R) subunit residue substitutions were in the N-terminal GABA binding domain. Using multidisciplinary approaches, we compared effects of mutant GABAA receptor α5 and α1 subunits on the properties of recombinant α5β3γ2 and α1β3γ2 GABAA receptors in both neuronal and non-neuronal cells and characterized their effects on receptor clustering, biogenesis and channel function. GABAA receptors containing mutant α5 and α1 subunits all had reduced cell surface and total cell expression with altered endoplasmic reticulum processing, impaired synaptic clustering, reduced GABAA receptor function and decreased GABA binding potency. Our study identified GABRA5 as a causative gene for early onset epileptic encephalopathy and expands the mutant GABRA1 phenotypic spectrum, supporting growing evidence that defects in GABAergic neurotransmission contribute to early onset epileptic encephalopathy phenotypes.

Maternal supplementation with Lactobacillus paracasei DTA 83 alters emotional behavior in Swiss mice offspring

The objective of our study was to evaluate whether maternal supplementation with lactic acid bacteria is able to alter neurobehavioral parameters of Swiss mice offspring. For this purpose, female mice were treated during pregnancy and lactation with milk or milk inoculated with L. paracasei DTA 83 at a concentration of 107 cells/ml (p.o, daily). After the end of the treatment, 6 offspring per group were submitted to euthanasia and the hippocampus was dissected for RNA analysis. After completing 70 days of life, 12 male offspring per group were submitted to a battery of behavioral tests. According to our results, treated offspring had a reduction in the expression of GAD 65, GAD 67 and GABAA receptor α3 subunit in the hippocampus. In the adult phase, the offspring of the treated group had higher time in the central area in the open field test, lower latency to the light side in the light-dark box test and lower number of decision-making behaviors in the elevated plus maze test. These results indicate that maternal supplementation with L. paracasei DTA 83 modulates offspring hippocampal GABAergic system. Such changes could possibly be associated with the reduction of anxiety-like behavior upon reaching adulthood.

Variable colocalisation of GABAA receptor subunits and glycine receptors on neurons in the human hypoglossal nucleus

The hypoglossal nucleus, the nucleus of the twelfth cranial nerve, is located dorsally in the midline of the medulla oblongata. The hypoglossal nucleus contains lower motor neurons which innervate the tongue muscles that control tongue movements involved in speech production, swallowing, mastication and associated respiratory movements. GABAA and glycine receptors are heteropentameric ionotropic receptors that facilitate fast-response, inhibitory neurotransmission in the mammalian brain and spinal cord. We investigated the immunohistochemical distribution of the GABAA receptor α1, α2, β2,3 subunits and glycine receptors as well as their relationship to the vesicular GABA transporter (VGAT) in the human hypoglossal nucleus at the light and confocal laser scanning microscope levels. The results showed that all of the GABAA receptor subunits as well as glycine receptor display punctate labelling indicative of synapses on the soma and dendritic membranes of large neurons within the hypoglossal nucleus. On average, approximately 50% of glycine receptors were co localised with GABAA receptor α1 subunits. Also on average GABAA α2 and β2,3 subunits were colocalised with approximately 30% of glycine receptor subunits. VGAT positive terminals were associated with both GABAA and glycine receptor types. Both glycinergic and GABAergic positive puncta were found adjacent to VGAT terminal-like staining. These results suggest that inhibition of human hypoglossal motor neurons occurs not only through complex interaction of separated GABAAR and glycine receptor regions, but also through synapses containing both inhibitory receptor types co-existing at the same synaptic sites.

Endogenous Neurosteroid (3\u03b1,5\u03b1)3-Hydroxypregnan-20-one Inhibits Toll-like-4 Receptor Activation and Pro-inflammatory Signaling in Macrophages and Brain

The endogenous neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone) has protective activity in animal models of alcoholism, depression, traumatic brain injury, schizophrenia, multiple sclerosis, and Alzheimer’s disease that is poorly understood. Because these conditions involve proinflammatory signaling through toll-like receptors (TLRs), we examined the effects of 3α,5α-THP, and pregnenolone on TLR4 activation in both the periphery and the central nervous system (CNS). We used monocytes/macrophages (RAW264.7) as a model of peripheral immune signaling and studied innately activated TLR4 in the ventral tegmental area (VTA) of selectively bred alcohol-preferring (P) rats. LPS activated the TLR4 pathway in RAW264.7 cells as evidenced by increased levels of p-TAK1, TRAF6, NF-κB p50, phospho-NF-κB- p65, pCREB, HMGB1, and inflammatory mediators, including MCP-1 and TNFα. Both 3α,5α-THP and pregnenolone (0.5–1.0μM) substantially (~80%) inhibited these effects, indicating pronounced inhibition of TLR4 signaling. The mechanism of inhibition appears to involve blockade of TLR4/MD-2 protein interactions in RAW246.7 cells. In VTA, 3α,5α-THP (15 mg/kg, IP) administration reduced TRAF6 (~20%), CRF (~30%), and MCP-1 (~20%) levels, as well as TLR4 binding to GABAA receptor α2 subunits (~60%) and MyD88 (~40%). The data suggest that inhibition of proinflammatory neuroimmune signaling underlies protective effects of 3α,5α-THP in immune cells and brain, apparently involving blocking of protein-protein interactions that initiate TLR4-dependent signaling. Inhibition of pro-inflammatory TLR4 activation represents a new mechanism of 3α,5α-THP action in the periphery and the brain.

Sexually divergent changes in select brain proteins and neurosteroid levels after a history of ethanol drinking and intermittent PTSD-like stress exposure in adult C57BL/6J mice.

Human studies reported that the number of past-year stressors was positively related to current drinking patterns, including binge drinking. In animal models, exposure to predator odor stress (PS), considered a model of traumatic stress, consistently increased ethanol intake. Recently, we reported that repeated PS significantly increased ethanol intake and had a synergistic interaction with prior binge drinking (binge group) in male but not in female C57BL/6J mice, when compared to mice without prior binge exposure (control group). The current studies utilized plasma and dissected prefrontal cortex (PFC) and hippocampal tissue from these animals and from age-matched naïve mice (naïve group). Western blots assessed relative protein levels of P450scc (an enzyme involved in the first step of steroidogenesis), of GABAA receptor α2 and α4 subunits, and of two proteins involved in synaptic plasticity - ARC (activity-regulated cytoskeletal protein) and synaptophysin. Gas chromatography-mass spectrometry simultaneously quantified 10 neurosteroid levels in plasma. A history of ethanol drinking and PS exposure produced brain regional and sex differences in the changes in proteins examined as well as in the pattern of neurosteroid levels versus (vs.) values in naïve mice. For instance, P450scc levels were significantly increased only in binge and control female PFC and hippocampus vs. naïve mice. Some neurosteroid levels were significantly altered by binge treatment in both males and females, whereas others were only significantly altered in males. These sexually divergent changes in neurosteroid and protein levels add to evidence for sex differences in the neurochemical systems influenced by traumatic stress and a history of ethanol drinking.

Brain iron loading impairs DNA methylation and alters GABAergic function in mice.

Iron deficiency is closely associated with altered GABA metabolism and affective behavior. While mutation in the hemochromatosis ( HFE) gene disrupts iron homeostasis and promotes oxidative stress that increases the risk of neurodegeneration, it is largely unknown whether HFE mutation modifies GABAergic homeostasis and emotional behavior. The goal of our study was to investigate the impact of HFE on GABAergic neurochemistry and redox-epigenetic regulation in the brain using H67D HFE-mutant mice that recapitulates the H63D-HFE mutation in humans. H67D mice displayed elevated redox-active iron levels in the brain by 32% compared to age-matched wild-type mice. Moreover, the H67D brain had increased isoprostane and decreased glutathione, indicating elevated oxidative stress. Additionally, the H67D brain had decreased global methylation and attenuated DNA methyltransferase (DNMT) activity. Direct addition of iron to purified DNMT in vitro decreased enzyme activity in a concentration-dependent manner. Last, H67D mice exhibited decreased anxiety-like behavior, which was associated with increased expression of the GABAA receptor α2 subunits by 93%, and these changes were also observed in H67D mice fed a low-iron diet. Taken together, our results suggest a putative role of HFE in regulating labile iron status in the brain, and mutation in H67D perturbs redox-methylation status, contributing to GABAergic dysfunction.-Ye, Q., Trivedi, M., Zhang, Y., Böhlke, M., Alsulimani, H., Chang, J., Maher, T., Deth, R., Kim, J. Brain iron loading impairs DNA methylation and alters GABAergic function in mice.

Histone Deacetylase Inhibitor Suberanilohydroxamic Acid Treatment Reverses Hyposensitivity to γ-Aminobutyric Acid in the Ventral Tegmental Area During Ethanol Withdrawal.

BackgroundThe ventral tegmental area (VTA) is important for alcohol‐related reward and reinforcement. Mouse VTA neurons are hyposensitive to γ‐aminobutyric acid (GABA) during ethanol (EtOH) withdrawal, and GABA responsiveness is normalized by in vitro treatment with histone deacetylase inhibitors (HDACi). The present study examined the effect of a systemically administered HDACi, suberanilohydroxamic acid (SAHA) on GABA sensitivity, and related molecular changes in VTA neurons during withdrawal after chronic EtOH intake in rats.MethodsSprague Dawley male adult rats were fed with Lieber‐DeCarli diet (9% EtOH or control diet) for 16 days. Experimental groups included control diet‐fed and EtOH diet‐fed (0‐ or 24‐hour withdrawal) rats treated with either SAHA or vehicle injection. Single‐unit recordings were used to measure the response of VTA neurons to GABA. Immunohistochemistry was performed to examine levels of HDAC2, acetylated histone H3 lysine 9 (acH3K9), and GABAA receptor α1 and α5 subunits in the VTA; quantitative polymerase chain reaction was performed to examine the mRNA levels of HDAC2 and GABAA receptor subunits.ResultsVTA neurons from the withdrawal group exhibited GABA hyposensitivity. In vivo SAHA treatment 2 hours before sacrifice normalized the sensitivity of VTA neurons to GABA. EtOH withdrawal was associated with increased HDAC2 and decreased acH3K9 protein levels; SAHA treatment normalized acH3K9 levels. Interestingly, no significant change was observed in the mRNA levels of HDAC2. The mRNA levels, but not protein levels, of GABAA receptor α1 and α5 subunits were increased during withdrawal.ConclusionsWithdrawal from chronic EtOH exposure results in a decrease in GABA‐mediated inhibition, and this GABA hyposensitivity is normalized by in vivo SAHA treatment. Disruption of signaling in the VTA produced by alteration of GABA neurotransmission could be 1 neuroadaptive physiological process leading to craving and relapse. These results suggest that HDACi pharmacotherapy with agents like SAHA might be an effective treatment for alcoholism.