GABAA Receptor Α1 Subunit Research Articles

Hippocampal amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid GluA1 (AMPA GluA1) receptor subunit involves in learning and memory improvement following treatment with Centella asiatica extract in adolescent rats.

Introduction Centella asiatica is an herbal plant that contains phytochemicals that are widely believed to have positive effects on cognitive function. The adolescent stage is a critical development period for the maturation of brain processes that encompass changes in physical and psychological systems. However, the effect of C. asiatica has not been extensively studied in adolescents. The aim of this study was therefore to investigate the effects of a C. asiatica extract on the enhancement of learning and memory in adolescent rats.MethodsThe locomotor activity, learning, and memory were assessed by using open field test and water T‐maze test. This study also examined changes in neuronal cell morphology using cresyl violet and apoptosis staining. We also performed immunohistochemical study to analyse the expression of the glutamate AMPA receptor (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid) GluA1 subunit and the GABA receptor (γ‐Aminobutyric Acid) subtype GABAA α1 subunit in the hippocampus of the same animals.ResultsWe found no significant changes in locomotor activity (p > 0.05). The water T‐maze data showed that 30 mg/kg dose significantly (p < 0.05) improved learning, memory, and the memory consolidation phase but had no effect on reversal learning (p > 0.05). Histological data revealed no neuronal morphological changes. Immunohistochemical analysis revealed increased expression of the AMPA GluA1 receptor subunit but there was no effect on GABAA receptor α1 subunit expression in the CA1 and CA2 subregions of the hippocampus.ConclusionsThe C. asiatica extract therefore improved hippocampus‐dependent spatial learning and memory in a dose‐dependent manner in rats through the GluA1‐containing AMPA receptor in the CA1 and CA2 sub regions of the hippocampus.

Assessment of Cultured Ex Vivo Equine Endometrial Explants as a Model of Uterine Inflammation Using Transcriptome Profiles

Variations in the early postnatal environment of rodents produce long-term changes in responses to stress that may underlie neuropsychiatric diseases such as anxiety, depression and schizophrenia. GABAA receptors undergo marked changes in their subunit composition during this period, involving a regionally-dependent replacement of α2 with α1 subunits, the so-called α-subunit switch. In this study we examined the effects of early-life environment on adulthood GABAA receptor α1 and α2 subunit expression and the synaptic clustering of GABAA receptors. Male and female mice were exposed to either 15 min daily handling sessions (EH) or no intervention (NH) over postnatal day (PND) 1–14. Adulthood behavioural differences in anxiety were assessed on the elevated plus-maze. Immunoperoxidase histochemistry was used to examine the density of the α1 and α2 subunit proteins. Double-labelling immunofluorescence and confocal microscopy were used to study GABAA receptor synaptic clustering. NH animals showed increased anxiety-type behaviours in the elevated plus maze relative to EH mice. NH males showed a loss of α2 subunits from the thalamus and lower layers of the somatosensory cortex, whilst NH females showed a reduction of α2 but increase in α1 protein in lower layers of the primary somatosensory cortex only. The NH condition also reduced α1 subunit expression in dentate gyrus (DG) in both males and females. Regardless of sex, NH mice showed reduced colocalisation of GABAA receptor α2 subunits with the synaptic marker gephyrin relative to the control condition. These findings suggest that early-life environment has long-lasting effects on GABAA receptors, leading to long-term changes in adulthood behaviour, and are of relevance to neurodevelopmental explanations of stress-augmented neuropsychiatric disorders.

The GABAA Receptor α2 Subunit Activates a Neuronal TLR4 Signal in the Ventral Tegmental Area that Regulates Alcohol and Nicotine Abuse.

Alcoholism initiates with episodes of excessive alcohol drinking, known as binge drinking, which is one form of excessive drinking (NIAAA Newsletter, 2004) that is related to impulsivity and anxiety (Ducci et al., 2007; Edenberg et al., 2004) and is also predictive of smoking status. The predisposition of non-alcohol exposed subjects to initiate binge drinking is controlled by neuroimmune signaling that includes an innately activated neuronal Toll-like receptor 4 (TLR4) signal. This signal also regulates cognitive impulsivity, a heritable trait that defines drug abuse initiation. However, the mechanism of signal activation, its function in dopaminergic (TH+) neurons within the reward circuitry implicated in drug-seeking behavior [viz. the ventral tegmental area (VTA)], and its contribution to nicotine co-abuse are still poorly understood. We report that the γ-aminobutyric acidA receptor (GABAAR) α2 subunit activates the TLR4 signal in neurons, culminating in the activation (phosphorylation/nuclear translocation) of cyclic AMP response element binding (CREB) but not NF-kB transcription factors and the upregulation of corticotropin-releasing factor (CRF) and tyrosine hydroxylase (TH). The signal is activated through α2/TLR4 interaction, as evidenced by co-immunoprecipitation, and it is present in the VTA from drug-untreated alcohol-preferring P rats. VTA infusion of neurotropic herpes simplex virus (HSV) vectors for α2 (pHSVsiLA2) or TLR4 (pHSVsiTLR4) but not scrambled (pHSVsiNC) siRNA inhibits signal activation and both binge alcohol drinking and nicotine sensitization, suggesting that the α2-activated TLR4 signal contributes to the regulation of both alcohol and nicotine abuse.

Effects of postnatal handling on adult behavior and brain mRNA expression of serotonin receptor, brain-derived neurotrophic factor and GABA-A receptor subunit

Development of brain and behavior is influenced by the interaction of genetic and environmental factors. Postnatal handling, a manipulation that briefly separates mouse offspring from their mother during the postnatal period, has been reported to yield beneficial effects on the behavior of adult offspring. However, brain mechanisms underlying the effects on the behavior have not been well understood. Here we first examined effects of postnatal handling on the behavior of adult male BALB/c mice. Offspring were separated for 15 min every day between postnatal day 1 (P1) and P14 and then various behaviors were tested in the adulthood. Postnatal handling reduced anxiety-like behavior in elevated plus maze and improved spatial learning and memory in Morris water maze without effects on depression-like behavior in forced swim test. Next, to elucidate mechanisms underlying the behavioral effects, we evaluated mRNA expression of the serotonin 1A (5-HT1A) receptor, brain-derived neurotrophic factor (BDNF), and GABA-A receptor subunits in the medial prefrontal cortex, amygdala, dorsal and ventral hippocampi, and dorsal raphe nucleus by quantitative RT-PCR, since these genes and brain regions have been shown to be involved in cognition and emotion. Postnatal handling up-regulated mRNA expression of the 5-HT1A receptor in the dorsal raphe nucleus and down-regulated 5-HT1A receptor mRNA expression in the amygdala on P15 and P71. In adulthood, mRNA expression of BDNF was up-regulated in the amygdala and dorsal hippocampus and down-regulated in the dorsal raphe nucleus, whereas that of GABA-A receptor α2 subunit was down-regulated in the amygdala. Taken together, the present study suggests that postnatal handling reduced anxiety-like behavior and improved learning and memory, which were accompanied by changes in mRNA expression of 5-HT1A receptor, BDNF and GABA-A receptor α2 subunit in the amygdala, 5-HT1A receptor in the dorsal raphe nucleus and BDNF in the dorsal hippocampus.

SAHA (Vorinostat) Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit.

The GABAA receptor (GABAAR) α1 subunit A295D epilepsy mutation reduces the surface expression of α1A295Dβ2γ2 GABAARs via ER-associated protein degradation. Suberanilohydroxamic acid (SAHA, also known as Vorinostat) was recently shown to correct the misfolding of α1A295D subunits and thereby enhance the functional surface expression of α1A295Dβ2γ2 GABAARs. Here we investigated whether SAHA can also restore the surface expression of γ2 GABAAR subunits that incorporate epilepsy mutations (N40S, R43Q, P44S, R138G) known to reduce surface expression via ER-associated protein degradation. As a control, we also investigated the γ2K289M epilepsy mutation that impairs gating without reducing surface expression. Effects of mutations were evaluated on inhibitory postsynaptic currents (IPSCs) mediated by the major synaptic α1β2γ2 GABAAR isoform. Recordings were performed in neuron-HEK293 cell artificial synapses to minimise contamination by GABAARs of undefined subunit composition. Transfection with α1β2γ2N40S, α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G subunits produced IPSCs with decay times slower than those of unmutated α1β2γ2 GABAARs due to the low expression of mutant γ2 subunits and the correspondingly high expression of slow-decaying α1β2 GABAARs. SAHA pre-treatment significantly accelerated the decay time constants of IPSCs consistent with the upregulation of mutant γ2 subunit expression. This increase in surface expression was confirmed by immunohistochemistry. SAHA had no effect on either the IPSC kinetics or surface expression levels of α1β2γ2K289M GABAARs, confirming its specificity for ER-retained mutant γ2 subunits. We also found that α1β2γ2K289M GABAARs and SAHA-treated α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G GABAARs all mediated IPSCs that decayed at significantly faster rates than wild type receptors as temperature was increased from 22 to 40°C. This may help explain why these mutations cause febrile seizures (FS). Given that SAHA is approved by therapeutic regulatory agencies for human use, we propose that it may be worth investigating as a treatment for epilepsies caused by the N40S, R43Q, P44S and R138G mutations. Although SAHA has already been proposed as a therapeutic for patients harbouring the α1A295D epilepsy mutation, the present study extends its potential utility to a new subunit and four new mutations.

Sex differences in the synergistic effect of prior binge drinking and traumatic stress on subsequent ethanol intake and neurochemical responses in adult C57BL/6J mice.

Alcohol-use disorders (AUDs) are characterized by repeated episodes of binge drinking. Based on reports that exposure to predator odor stress (PS) consistently increases ethanol intake, the present studies examined whether prior binge drinking differentially altered responsivity to PS and subsequent ethanol intake in male and female mice, when compared to mice without prior binge exposure. Initial studies in naïve male and female C57BL/6J mice confirmed that 30-min exposure to dirty rat bedding significantly increased plasma corticosterone (CORT) levels and anxiety-related behavior, justifying the use of dirty rat bedding as PS in the subsequent drinking studies. Next, separate groups of male and female C57BL/6J mice received seven binge ethanol sessions (binge) or drank water (controls), followed by a 1-month period of abstinence. Then, 2-bottle choice ethanol intake (10% or 10E vs. water, 23h/day) was measured in lickometer chambers for 4 weeks. After baseline intake stabilized, exposure to intermittent PS (2×/week×2 weeks) significantly enhanced ethanol intake after the 2nd PS in male, but not female, binge mice vs. baseline and vs. the increase in controls. However, in a subgroup of females (with low baselines), PS produced a similar increase in 10E intake in control and binge mice vs. baseline. Analysis of lick behavior determined that the enhanced 10E intake in binge male mice and in the female low baseline subgroup was associated with a significant increase in 10E bout frequency and 10E licks throughout the circadian dark phase. Thus, PS significantly increased 10E intake and had a synergistic interaction with prior binge drinking in males, whereas PS produced a similar significant increase in 10E intake in the low baseline subgroup of binge and control females. Plasma CORT levels were increased significantly in both binge and control animals after PS. CORT levels at 24-h withdrawal from daily 10E intake were highest in the groups with elevated 10E licks (i.e., binge males and control females). At 24-h withdrawal, protein levels of GABAA receptor α1 subunit, corticotropin releasing factor receptor 1, and glucocorticoid receptor in prefrontal cortex (PFC) and hippocampus (HC) were differentially altered in the male and female mice vs. levels in separate groups of age-matched naïve mice, with more changes in HC than in PFC and in females than in males. Importantly, the sexually divergent changes in protein levels in PFC and HC add to evidence for sex differences in the neurochemical systems influenced by stress and binge drinking, and argue for sex-specific pharmacological strategies to treat AUD.

Histone deacetylases mediate GABAA receptor expression, physiology, and behavioral maladaptations in rat models of alcohol dependence.

Alcohol use disorders are chronic debilitating diseases characterized by severe withdrawal symptoms that contribute to morbidity and relapse. GABAA receptor (GABAAR) adaptations have long been implicated in the chronic effects of alcohol and contribute to many withdrawal symptoms associated with alcohol dependence. In rodents, GABAAR hypofunction results from decreases in Gabra1 expression, although the underlying mechanism controlling Gabra1 expression after chronic ethanol exposure is still unknown. We found that chronic ethanol exposure using either ethanol gavage or two-bottle choice voluntary access paradigms decreased Gabra1 expression and increased Hdac2 and Hdac3 expression. Administration of the HDAC inhibitor trichostatin A (TSA) after chronic ethanol exposure prevents the decrease in Gabra1 expression and function as well as the increase in Hdac2 and Hdac3 expression in both the cortex and the medial prefrontal cortex (mPFC). Chronic ethanol exposure and withdrawal, but not acute ethanol exposure or acute withdrawal, cause a selective upregulation of HDAC2 and HDAC3 associated with the Gabra1 promoter that accompanies a decrease in H3 acetylation of the Gabra1 promoter and the reduction in GABAAR α1 subunit expression. TSA administration prevented each of these molecular events as well as behavioral manifestations of ethanol dependence, including tolerance to zolpidem-inducedloss of righting reflex, reduced open-arm time in the elevated plus maze, reduced center-time and locomotor activity in the open-field assay, and TSA reduced voluntary ethanol consumption. The results show how chronic ethanol exposure regulates the highly prominent GABAAR α1 subunit by an epigenetic mechanism that represents a potential treatment modality for alcohol dependence.

Interneuron synaptopathy in developing rat cortex induced by the pro-inflammatory cytokine LIF

Pro-inflammatory cytokines of the IL-6 family have been linked to the etiology of epilepsy and mental disorders. After infusion of the IL-6 family member leukemia inhibitory factor (LIF) into postnatal day 12–20 rat visual cortex (period of synaptogenesis; equals early childhood in human) and equivalent ages in organotypic cultures (OTC), somatic growth of cortical and subcortical neurons is delayed, expression of interneuron markers reduced and expression of neurotrophins transiently reduced. Further, LIF prevents NT4 from promoting soma growth of GABA-ergic interneurons. We now show that LIF-treatment from DIV 12–20 in OTC compromises the differentiation of fast-spiking GABA-ergic basket neurons: GAD-65/67, Kv3.2, and synaptotagmin-2 proteins are reduced, dendrites are shorter, and axonal varicosities are smaller. Bitufted and Martinotti interneurons are barely affected. Pyramidal cells display lower dendritic spine densities, more filopodia, and shorter axon initial segments. βIV-spectrin, Cav3.1, GABAA receptor subunits α1 and α2, and the phosphorylation of GluN2B at Y1472 are reduced. The frequency of calcium events in pyramidal cells and interneurons is increased, and the antiepileptic neuropeptide Y is upregulated, suggesting a hyperexcitability of the network. In the presence of LIF, neurotrophins fail to activate MAP kinase phosphorylation and c-fos expression, and exogenous NT4 fails to promote the maturation of pyramidal cells and interneurons as it normally would. After discontinuing LIF treatment, bouton size and expression levels of affected proteins normalize except for synaptotagmin-2; moreover, hyperexcitability persists. The results suggest that elevated levels of inflammatory cytokines during this developmental period cause a lasting maldevelopment in particular of basket cells.

Identification and expression of a unique neonatal variant of the GABAA receptor α3 subunit.

The GABAA receptor provides the majority of inhibitory neurotransmission in the adult central nervous system but in immature brain is responsible for much of the excitatory drive, a requirement for normal brain development. It is well established that GABAA receptor subunit expression changes across the course of brain development. In the present study, we have identified a splice variant of the GABAA receptor α3 subunit which appears unique to the developing brain, referred to here as the GABAA receptor α3 subunit neonatal variant (GABAA receptor α3N). RT-PCR and sequence analysis revealed splicing of exon 8 of the α3 subunit. Western blot analysis showed expression of GABAA receptor α3N in the cortex of several neonatal species and significantly reduced expression of this splice variant in the corresponding adult brains. Expression was evident in multiple brain regions and decreased across development in the pig. Fractionation revealed differential cellular localisation in the parietal cortex, hippocampus and thalamus of the full-length GABAA receptor α3 and GABAA receptor α3N. Immunoprecipitation showed direct interaction with the GABAA receptor subunits α1 and γ2 but not with gephyrin.

GABAA and GABAB receptor subunit localization on neurochemically identified neurons of the human subthalamic nucleus.

The subthalamic nucleus (STN) is a critical excitatory signaling center within the basal ganglia circuitry. The activity of subthalamic neurons is tightly controlled by upstream inhibitory signaling centers in the basal ganglia. In this study, we used immunohistochemical techniques to firstly, visualize and quantify the STN neurochemical organization based on neuronal markers including parvalbumin (PV), calretinin (CR), SMI-32, and GAD65/67 . Secondly, we characterized the detailed regional, cellular and subcellular expression of GABAA (α1 , α2 , α3 , β2/3 , and γ2 ) and GABAB (R1 and R2) receptor subunits within the normal human STN. Overall, we found seven neurochemically distinct populations of principal neurons in the human STN. The three main populations detected were: (a) triple-labeled PV+ /CR+ /SMI32+ ; (b) double-labeled PV+ /CR+ ; and (c) single-labeled CR+ neurons. Subthalamic principal neurons were found to express GABAA receptor subunits α1 , α3 , β2/3 , γ2 , and GABAB receptor subunits R1 and R2. However, no expression of GABAA receptor α2 subunit was detected. We also found a trend of increasing regional staining intensity for all positive GABAA receptor subunits from the dorsolateral pole to ventromedial extremities. The GAD+ interneurons showed relatively low expression of GABAA receptor subunits. These results provide the morphological basis of GABAergic transmission within the normal human subthalamic nucleus and evidence of GABA innervation through both GABAA and GABAB receptors on subthalamic principal neurons.

Postnatal expression of thalamic GABAA receptor subunits in the stargazer mouse model of absence epilepsy.

Absence seizures are known to originate from disruptions within the corticothalamocortical network; however, the precise underlying cellular and molecular mechanisms that induce hypersynchronicity and hyperexcitability are debated and likely to be complex and multifactorial. Recent studies implicate impaired thalamic GABAergic inhibition as a common feature in multiple animal models of absence epilepsy, including the well-established stargazer mouse model. Recently, we demonstrated region-specific increases in the whole tissue and synaptic levels of GABAA receptor (GABAAR) subunits α1 and β2, within the ventral posterior region of the thalamus in adult epileptic stargazer mice compared with nonepileptic control littermates. The objective of this study was to investigate whether such changes in GABAAR subunits α1 and β2 can be observed before the initiation of seizures, which occur around postnatal (PN) days 16-18 in stargazers. Semiquantitative western blotting was used to analyze the relative tissue level expression of GABAAR α1 and β2 subunits in the thalamus of juvenile stargazer mice compared with their nonepileptic control littermates at three different time points before the initiation of seizures. We show that there is a statistically significant increase in the expression of α1 and β2 subunits in the thalamus of stargazer mice, at the PN7-9 stage, compared with the control littermates, but not at PN10-12 and PN13-15 stages. These results suggest that an aberrant expression of GABAAR subunits α1 and β2 in the stargazers does not occur immediately before seizure onset and therefore is unlikely to directly contribute to the initiation of absence seizures.

PKC/CREB pathway mediates the expressions of GABAA receptor subunits in cultured hippocampal neurons after low-Mg2+ solution treatment

ObjectiveTo investigate the potential effects of the PKC/CREB pathway on the expressions of GABAA receptor subunits α1, γ2, and δ in cultured hippocampal neurons using a model of epilepsy that employed conditions of low magnesium (Mg2+). MethodsA total of 108 embryonic rats at the age of 18 embryonic days (E18)prepared from adult female SD rats were used as experimental subjects. Primary rat hippocampal cultures were prepared from the embryonic 18 days rats. The cultured hippocampal neurons were then treated with artificial cerebrospinal fluid containing low Mg2+ solutions to generate a low Mg2+ model of epilepsy. The low Mg2+ stimulation lasted for 3 h and then returned to in maintenance medium for 20 h. The changes of the GABAA receptor subunit α1, γ2, δ were observed by blocking or activating the function of the CREB. The quantification of the GABAA receptor subunit α1, γ2, δ and the CREB were determined by a qRT-PCR and a Western blot method. ResultsAfter the neurons were exposed to a low-Mg2+ solution for 3 h, GABAA receptor mRNA expression markedly increased compared to the control, and then gradually decreased. In contrast, CREB mRNA levels exhibited a dramatic down-regulation 3 h after terminating low-Mg2+ treatment, and then peaked at 9 h. Western blot analyses verified that staurosporine suppressed CREB phosphorylation (p-CREB). The mRNA expression of GABAA receptor subunit α1 increased only in the presence of staurosporine, whereas the expressions of subunits γ2 and δ significantly increased in the presence of either KG-501 or staurosporine. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the expressions of GABAA subunits α1, γ2, and δ when administered alone. However, the administration of either KG-501 or staurosporine reversed the inhibitory effects of PMA. ConclusionsThe PKC/CREB pathway may negatively regulate the expressions of GABAA receptor subunits α1, γ2, and δ in cultured hippocampal neurons in low Mg2+ model of epilepsy.

Effects of different ratios of medicine dosage for isoflurane and propofol on GABAA receptor α1 subunit proteostasis during hypoxia injury to hippocampal neurons of rats

Objective To evaluate the effects of different ratios of medicine dosage for isoflurane and propofol on GABAA receptor(GABAAR)α1 subunit proteostasis during hypoxia injury to hippocampal neurons of rats. Methods The hippocampal neurons isolated from fetal rats obtained from Wistar rats were primarily cultured and divided into 6 groups(n=60 each)using a random number table: control group(group C), hypoxia group(group H), isoflurane group(group I), propofol group(group P)and different ratios of medicine dosage for isoflurane and propofol groups(group IP1 and group IP2). The cells were subjected to hypoxia for 6 h in group H. Cells were incubated for 3 h with 1.9 % isoflurane and with 22.4 μmol/L propofol after being subjected to hypoxia for 6 h in I and P groups, respectively.Cells were incubated for 3 h with 1.0% isoflurane and 6.7 μmol/L propofol and with 1.4 % isoflurane and 3.4 μmol/L propofol after being subjected to hypoxia for 6 h in IP1 and IP2 groups, respectively.Then the culture medium was replaced with plain culture medium.At 24 h of incubation, the cells were collected for measurement of cell viability by CCK-8 assay, GABAAR α1 mRNA expression(by quantitative polymerase chain reaction), GABAAR α1 expression in the cytomembrane(by Western blot), level of GABAAR α1 subunit endoplasmic reticulum-associated degradation(ERAD) (by immunoprecipitation and Western blot)and CCAAT/enhancer-binding protein homologous protein(CHOP)expression (by immunofluorescence). Results Compared with group C, the cell viability was significantly decreased, the expression of GABAAR α1 mRNA and GABAAR α1 in cytomembrane was down-regulated, the expression of CHOP was up-regulated, and the level of GABAAR α1 subunit ERAD was increased in the other five groups(P<0.05). Compared with group H, the cell viability was significantly decreased, the expression of GABAAR α1 mRNA and GABAAR α1 in cytomembrane was down-regulated, the expression of CHOP was up-regulated, and the level of GABAAR α1 subunit ERAD was increased in I, P and IP2 groups(P <0.05), and no significant change was found in the parameters mentioned above in group IP1(P<0.05). Compared with group I or group P, the cell viability was significantly increased, the expression of GABAAR α1 mRNA and GABAAR α1 in cytomembrane was up-regulated, the expression of CHOP was down-regulated, and the level of GABAAR α1 subunit ERAD was decreased in IP1 and IP2 groups(P<0.05). Compared with group IP1, the cell viability was significantly decreased, the expression of GABAAR α1 mRNA and GABAAR α1 in cytomembrane was down-regulated, the expression of CHOP was up-regulated, and the level of GABAAR α1 subunit ERAD was increased in group IP2(P<0.05). Conclusion Combination of 1.0% isoflurane and 6.7 μmol/L propofol does not aggravate hypoxia-induced destruction of GABAAR α1 subunit proteostasis in hippocampal neurons of rats. Key words: Propofol; Isoflurane; Receptors, GABA-A; Hypoxia-ischemia, brain

GABAA receptor subunits in the human amygdala and hippocampus: Immunohistochemical distribution of 7 subunits.

GABAergic neurotransmission in the amygdala contributes to the regulation of emotional processes in anxiety, stress, reward, mnestic functions, addiction, and epilepsy. Species-specific differences in the distribution and composition of GABAA receptors may account for distinct effects and side-effects of GABAergic agents. However, data on the distribution and composition of GABAA receptors in the human amygdala are lacking. Here, the expression of GABAA receptor subunits α1, α2, α3, α5, β2, β2/3, and γ2 was studied in the human amygdala using immunohistochemistry. Hippocampi were evaluated as a reference structure. Neuronal counts and field fraction analyses were performed, and subcellular expression of GABAA receptor subunits was analyzed semiquantitatively. In the amygdala, field fraction analyses showed the highest α1 expression in the lateral nucleus (La), whereas α3 was prominent in intercalated nuclei (IC), and α5 and γ2 in the cortical nuclei, and amygdalo-hippocampal/parahippocampal-amygdala transition areas. In the hippocampus, α1 and α3 were accentuated in the dentate gyrus, CA1 region, and subiculum, whereas α5 expression was rather uniform. In both regions, α2 was homogenously distributed, and the two β subunits and γ2 showed faint immunostaining. The intensity of subunit expression also varied in the neuropil, neuronal somata, and/or cellular processes in the subregions. GABAA receptors containing subunit α1, showing the strongest expression in the La, and α3, with the strongest expression in the IC and subiculum, could be targets for treating amygdala-related disorders. Differences in GABAA receptor subunit expression between the human and rodent amygdala should be taken into consideration when developing subunit-selective drugs.

Thalamic inhibition regulates critical-period plasticity in visual cortex and thalamus

During critical periods of development, experience shapes cortical circuits, resulting in the acquisition of functions used throughout life. The classic example of critical-period plasticity is ocular dominance (OD) plasticity, which optimizes binocular vision but can reduce the responsiveness of the primary visual cortex (V1) to an eye providing low-grade visual input. The onset of the critical period of OD plasticity involves the maturation of inhibitory synapses withinV1, specifically those containing the GABAA receptor α1 subunit. Here we show that thalamic relay neurons in mouse dorsolateral geniculate nucleus (dLGN) also undergo OD plasticity. This process depends on thalamic α1-containing synapses and is required for consolidation of the OD shift in V1 during long-term deprivation. Our findings demonstrate that thalamic inhibitory circuits play a central role in the regulation of the critical period. This has far-reaching consequences for the interpretation of studies investigating the molecular and cellular mechanisms regulating critical periods of brain development.

Long-lasting effects of early-life intervention in mice on adulthood behaviour, GABAA receptor subunit expression and synaptic clustering

Variations in the early postnatal environment of rodents produce long-term changes in responses to stress that may underlie neuropsychiatric diseases such as anxiety, depression and schizophrenia. GABAA receptors undergo marked changes in their subunit composition during this period, involving a regionally-dependent replacement of α2 with α1 subunits, the so-called α-subunit switch. In this study we examined the effects of early-life environment on adulthood GABAA receptor α1 and α2 subunit expression and the synaptic clustering of GABAA receptors. Male and female mice were exposed to either 15min daily handling sessions (EH) or no intervention (NH) over postnatal day (PND) 1–14. Adulthood behavioural differences in anxiety were assessed on the elevated plus-maze. Immunoperoxidase histochemistry was used to examine the density of the α1 and α2 subunit proteins. Double-labelling immunofluorescence and confocal microscopy were used to study GABAA receptor synaptic clustering. NH animals showed increased anxiety-type behaviours in the elevated plus maze relative to EH mice. NH males showed a loss of α2 subunits from the thalamus and lower layers of the somatosensory cortex, whilst NH females showed a reduction of α2 but increase in α1 protein in lower layers of the primary somatosensory cortex only. The NH condition also reduced α1 subunit expression in dentate gyrus (DG) in both males and females. Regardless of sex, NH mice showed reduced colocalisation of GABAA receptor α2 subunits with the synaptic marker gephyrin relative to the control condition. These findings suggest that early-life environment has long-lasting effects on GABAA receptors, leading to long-term changes in adulthood behaviour, and are of relevance to neurodevelopmental explanations of stress-augmented neuropsychiatric disorders.

Parvalbumin, but not calretinin, neurons express high levels of α1-containing GABAA receptors, α7-containing nicotinic acetylcholine receptors and D2-dopamine receptors in the basolateral amygdala of the rat

The generation of emotional responses by the basolateral amygdala is largely determined by the balance of excitatory and inhibitory inputs to its principal neurons − the pyramidal cells. The activity of these neurons is tightly controlled by g-aminobutyric acid (GABA)ergic interneurons, especially by those expressing parvalbumin (PV) and calretinin (CR). Although it is known that GABAergic, cholinergic and dopaminergic fibres make synapses on PV and CR cells, knowledge of the various receptors which are used by these cells is still incomplete. Thus, the present study investigates whether neurons expressing PV or CR co-express specific GABA, acetylcholine and/or dopamine receptors in the basolateral amygdala of the rat. The results show that almost two-thirds of PV neurons co-express high concentrations of α1 subunit of GABAA receptor, and more than half of them co-express high levels of α7 subunit of nicotinic acetylcholine receptor and/or D2-subtype of dopamine receptor. In contrast, a smaller percentage of CR neurons had detectable amounts of these receptors and at lower levels of abundance in most cases. In conclusion, the present results indicate that not only principal neurons but also GABAergic interneurons have specific receptors, which allow these cells to respond to the GABAergic, cholinergic and dopaminergic inputs coming to the basolateral amygdala of the rat. Since these cells receive intrinsic GABAergic inputs, they are strongly interconnected. Since they also receive extrinsic cholinergic and dopaminergic inputs, such stimulation may result in stimulus-driven feed-forward control of the principal neurons. The effects of such control may be either feed-forward inhibition of the principal neurons via α7 nicotinic acetylcholine receptors or disinhibition of these cells via D2-dopamine receptors.

Erbb4 Deletion from Medium Spiny Neurons of the Nucleus Accumbens Core Induces Schizophrenia-Like Behaviors via Elevated GABAA Receptor α1 Subunit Expression.

Medium spiny neurons (MSNs), the major GABAergic projection neurons in the striatum, are implicated in many neuropsychiatric diseases such as schizophrenia, but the underlying mechanisms remain unclear. We found that a deficiency in Erbb4, a schizophrenia risk gene, in MSNs of the nucleus accumbens (NAc) core, but not the dorsomedial striatum, markedly induced schizophrenia-like behaviors such as hyperactivity, abnormal marble-burying behavior, damaged social novelty recognition, and impaired sensorimotor gating function in male mice. Using immunohistochemistry, Western blot, RNA interference, electrophysiology, and behavior test studies, we found that these phenomena were mediated by increased GABAA receptor α1 subunit (GABAAR α1) expression, which enhanced inhibitory synaptic transmission on MSNs. These results suggest that Erbb4 in MSNs of the NAc core may contribute to the pathogenesis of schizophrenia by regulating GABAergic transmission and raise the possibility that GABAAR α1 may therefore serve as a new therapeutic target for schizophrenia.SIGNIFICANCE STATEMENT Although ErbB4 is highly expressed in striatal medium spiny neurons (MSNs), its role in this type of neuron has not been reported previously. The present study demonstrates that Erbb4 deletion in nucleus accumbens (NAc) core MSNs can induce schizophrenia-like behaviors via elevated GABAA receptor α1 subunit (GABAAR α1) expression. To our knowledge, this is the first evidence that ErbB4 signaling in the MSNs is involved in the pathology of schizophrenia. Furthermore, restoration of GABAAR α1 in the NAc core, but not the dorsal medium striatum, alleviated the abnormal behaviors. Here, we highlight the role of the NAc core in the pathogenesis of schizophrenia and suggest that GABAAR α1 may be a potential pharmacological target for its treatment.

Huntingtin-Interacting Protein 1-Related Protein Plays a Critical Role in Dendritic Development and Excitatory Synapse Formation in Hippocampal Neurons.

Huntingtin-interacting protein 1-related (HIP1R) protein is considered to be an endocytic adaptor protein like the other two members of the Sla2 family, Sla2p and HIP1. They all contain homology domains responsible for the binding of clathrin, inositol lipids and F-actin. Previous studies have revealed that HIP1R is highly expressed in different regions of the mouse brain and localizes at synaptic structures. However, the function of HIP1R in the nervous system remains unknown. In this study, we investigated HIP1R function in cultured rat hippocampal neurons using an shRNA knockdown approach. We found that, after HIP1R knockdown, the dynamics and density of dendritic filopodia, and dendritic branching and complexity were significantly reduced in developing neurons, as well as the densities of dendritic spines and PSD95 clusters in mature neurons. Moreover, HIP1R deficiency led to significantly reduced expression of the ionotropic glutamate receptor GluA1, GluN2A and GluN2B subunits, but not the GABAA receptor α1 subunit. Similarly, HIP1R knockdown reduced the amplitude and frequency of the miniature excitatory postsynaptic current, but not of the miniature inhibitory postsynaptic current. In addition, the C-terminal proline-rich region of HIP1R responsible for cortactin binding was found to confer a dominant-negative effect on dendritic branching in cultured developing neurons, implying a critical role of cortactin binding in HIP1R function. Taken together, the results of our study suggest that HIP1R plays important roles in dendritic development and excitatory synapse formation and function.

The postnatal 5-HT1A receptor regulates adult anxiety and depression differently via multiple molecules

Serotonin (5-HT) and the 5-HT1A receptor during development are known to modulate anxiety and depression in later life. However, the brain mechanisms linking the postnatal 5-HT system and adult behavior remain unknown. Here, we examined the effects of pharmacological 5-HT1A receptor activation during the postnatal period on anxiety and depression-like behavior in adult BALB/c male mice. To elucidate the underlying mechanisms, we measured mRNA expression of the 5-HT1A receptor, brain-derived neurotrophic factor (BDNF), GABAA receptor subunits, and AMPA receptor subunits in the medial prefrontal cortex (mPFC), amygdala, and hippocampus. Treatment with the selective 5-HT reuptake inhibitor (fluoxetine) and 5-HT1A receptor agonist (8-OH-DPAT) during the postnatal period decreased anxiety-like behavior in adulthood, whereas only 8-OH-DPAT treatment increased depression-like behavior. Concomitantly with the behavioral effects, postnatal treatment with fluoxetine and 8-OH-DPAT decreased the mRNA expression of the GABAA receptor α3 subunit in the mPFC and ventral hippocampus in adulthood, while 8-OH-DPAT, but not fluoxetine, decreased the mRNA expression of the 5-HT1A receptor and BDNF in the mPFC and the GABAA receptor α2 subunit in the mPFC and ventral hippocampus. On the basis of the correlative changes between behavior and mRNA expression, these results suggest that the GABAA receptor α3 subunit in the mPFC and ventral hippocampus may regulate anxiety-like behavior. In contrast, depression-like behavior may be regulated by the 5-HT1A receptor and BDNF in the mPFC and by the GABAA receptor α2 subunit in the mPFC and ventral hippocampus. In summary, activation of the 5-HT1A receptor during the postnatal period may reduce anxiety levels, but increase depression levels during adulthood via different multiple molecules in the mPFC and ventral hippocampus.