The de novo missense mutation F224S in GABRB2, identified in epileptic encephalopathy and developmental delay, impairs GABAAR function.

Genetic variants in genes encoding subunits of the γ-aminobutyric acid-A receptor (GABAAR) have been found to cause neurodevelopmental disorders and epileptic encephalopathy. In a patient with epilepsy and developmental delay, a de novo heterozygous missense mutation c.671 T > C (p.F224S) was discovered in the GABRB2 gene, which encodes the ß2 subunit of GABAAR. Based on previous studies on GABRB2 variants, this new GABRB2 variant (F224S) would be pathogenic. To confirm and investigate the effects of this GABRB2 mutation on GABAAR channel function, we conducted transient expression experiments using GABAAR subunits in HEK293T cells. The GABAARs containing mutant ß2 (F224S) subunit showed poor trafficking to the cell membrane, while the expression and distribution of the normal α1 and γ2 subunits were unaffected. Furthermore, the peak current amplitude of the GABAAR containing the ß2 (F224S) subunit was significantly smaller compared to the wild type GABAAR. We propose that GABRB2 variant F224S is pathogenic and GABAARs containing this ß2 mutant reduce response to GABA under physiological conditions, which could potentially disrupt the excitation/inhibition balance in the brain, leading to epilepsy.

Basmisanil, an α5-GABAAR negative allosteric modulator, produces rapid and sustained antidepressant-like responses in male mice.

There is a critical need for safer and better-tolerated alternatives to address the current limitations of antidepressant treatments for major depressive disorder. Recently, drugs targeting the GABA system via α5-containing GABAA receptors (α5-GABAAR) as negative allosteric modulators (α5-NAMs) have shown promise in alleviating stress-related behaviors in preclinical studies, suggesting that α5-NAMs may have translational relevance as novel antidepressant medications. Here, we evaluated the efficacy of Basmisanil, an α5-NAM that has been evaluated in Phase 2 clinical studies as a cognitive enhancer, in a battery of behavioral tests relevant to coping strategies, motivation, and aversion in male mice, along with plasma and brain pharmacokinetic measurements. Our findings reveal that Basmisanil induces dose-dependent rapid antidepressant-like responses in the forced swim test and sucrose splash test without promoting locomotor stimulating effects. Furthermore, Basmisanil elicits sustained behavioral responses in the female urine sniffing test and sucrose splash test, observed 24 h and 48 h post-treatment, respectively. Bioanalysis of plasma and brain samples confirms effective blood-brain barrier penetration by Basmisanil and extrapolation to previously published data suggest that effects were observed at doses (10 and 30 mg/kg i.p.) corresponding to relatively modest levels of α5-GABAAR occupancy (40-65 %). These results suggest that Basmisanil exhibits a combination of rapid and sustained antidepressant-like effects highlighting the potential of α5-NAMs as a novel therapeutic strategy for depression.

The suppressive effect of the specific KCC2 modulator CLP290 on seizure in mice.

Epilepsy is a chronic neurological disorder characterized by episodic dysfunction of central nervous system. The most basic mechanism of epilepsy falls to the imbalance between excitation and inhibition. In adults, GABAA receptor (GABAAR) is the main inhibitory receptor to prevent neurons from developing hyperexcitability, while its inhibition relies on the low intracellular chloride anion concentration ([Cl-]i). Neuronal-specific electroneutral K+-Cl- cotransporter (KCC2) can mediate chloride efflux to lower [Cl-]i for GABAAR mediated inhibition. Our previous study has revealed that the coordinated downregulation of KCC2 and GABAAR participates in epilepsy. According to a high-throughout screen for compounds that reduce [Cl-]i, CLP290 turns out to be a specific KCC2 functional modulator. In current study, we first confirmed that CLP290 could dose-dependently suppress convulsant-induced seizures in mice in vivo as well as the epileptiform burst activities in cultured hippocampal neurons in vitro. Then, we discovered that CLP290 functioned through preventing the downregulation of the KCC2 phosphorylation at Ser940 and hence the KCC2 membrane expression during convulsant stimulation, and consequently restored the GABA inhibition. In addition, while CLP290 was given in early epileptogenesis period, it also effectively decreased the spontaneous recurrent seizures. Generally, our current results demonstrated that CLP290, as a specific KCC2 modulator by enhancing KCC2 function, not only inhibits the occurrence of the ictal seizures, but also suppresses the epileptogenic process. Therefore, we believe KCC2 may be a suitable target for future anti-epileptic drug development.

Dexmedetomidine relieves inflammatory pain by enhancing GABAergic synaptic activity in pyramidal neurons of the anterior cingulate cortex.

Pyramidal neuron (Pyn) hyperactivity in the anterior cingulate cortex (ACC) is involved in the modulation of pain. Previous studies indicate that the activation of α2 adrenoceptors (α2-ARs) by dexmedetomidine (DEX) is a safe and effective means of alleviating multiple types of pain. Here, we showed that systemically administered DEX can ameliorate the inflammatory pain induced by hindpaw injection of formalin (FA) and further examined the molecular and synaptic mechanisms of this DEX-elicited antinociceptive effect. We found that FA caused an increase in c-Fos expression in contralateral layer 2/3 (L2/3) ACC, and that intra-ACC infusion of DEX could also relieve phase 2 inflammatory pain behavior. DEX elicited an increase in the amplitude and frequency of miniature inhibitory post-synaptic currents (mIPSCs) and evoked IPSC amplitude, as well as a reduction in the hyperexcitability and both paired-pulse and excitation/inhibition ratios in contralateral L2/3 ACC Pyns of FA mice. These electrophysiological effects were associated with the upregulation of GABA A receptor (GABAAR) subunits. The interaction of phosphorylated Akt (p-Akt) with GABAAR subunits increased in the ACC following administration of DEX. These results suggest that DEX treatment reduces hyperactivity and enhances GABAergic inhibitory synaptic transmission in ACC Pyns, which produces analgesic effects by increasing GABAAR levels and activating the Akt signaling pathway.

Simulating dynamic interaction between diazepam and ethanol targeting the GABAA receptor via in silico model.

Combined diazepam-ethanol poisoning is common in forensic toxicology. Both diazepam and ethanol can inhibit the central nervous system via the γ-aminobutyric acid (GABA) ligand gated chloride ion channel, GABAA Receptor (GABAAR). As the common target of diazepam and ethanol, whether GABAAR is the key target of their combined action remains unclear. This study aimed to explore their interaction based on the synergistic mechanisms between diazepam and ethanol targeting the GABAAR. Four models were built in silico based on the crystal structure of GABAAR. Molecular dynamic processes of the four models were simulated by the GPU-accelerated pmemd.cuda program in the Amber18 package. Results showed that ethanol inclined to combine the adjacent GABA or diazepam sites, minimized fluctuations of the root-mean-square deviation (RMSD) in the molecular dynamic process of GABA or diazepam binding the GABAAR, and increased the release of binding energy of GABA or diazepam binding the GABAAR. Results also showed that diazepam had less effect on the RMSD fluctuation or the binding energy release of GABA binding GABAAR. The formation of complex of diazepam and GABAAR could minimize the RMSD fluctuation and increase binding energy release of ethanol binding GABAAR. Thus, ethanol, bridging GABA and diazepam, could strengthen the complex of GABA binding the GABAAR, as well as the complex of diazepam binding the GABAAR. However, whether diazepam binds GABAAR or not, it cannot affect GABA binding the GABAAR; and yet the complex of diazepam and GABAAR can stabilize the complex of ethanol and GABAAR.

A role of GABAA receptor α1 subunit in the hippocampus for rapid-acting antidepressant-like effects of ketamine.

Ketamine can produce rapid-acting antidepressant effects in treatment-resistant patients with depression. Although alterations in glutamatergic and GABAergic neurotransmission in the brain play a role in depression, the precise molecular mechanisms in these neurotransmission underlying ketamine's antidepressant actions remain largely unknown. Mice exposed to FSS (forced swimming stress) showed depression-like behavior and decreased levels of GABA (γ-aminobutyric acid), but not glutamate, in the hippocampus. Ketamine increased GABA levels and decreased glutamate levels in the hippocampus of mice exposed to FSS. There was a correlation between GABA levels and depression-like behavior. Furthermore, ketamine increased the levels of enzymes and transporters on the GABAergic neurons (SAT1, GAD67, GAD65, VGAT and GAT1) and astrocytes (EAAT2 and GAT3), without affecting the levels of enzymes and transporters (SAT2, VGluT1 and GABAAR γ2) on glutamatergic neurons. Moreover, ketamine caused a decreased expression of GABAAR α1 subunit, which was specifically expressed on GABAergic neurons and astrocytes, an increased GABA synthesis and metabolism in GABAergic neurons, a plasticity change in astrocytes, and an increase in ATP (adenosine triphosphate) contents. Finally, GABAAR antagonist bicuculline or ATP exerted a rapid antidepressant-like effect whereas pretreatment with GABAAR agonist muscimol blocked the antidepressant-like effects of ketamine. In addition, pharmacological activation and inhibition of GABAAR modulated the synthesis and metabolism of GABA, and the plasticity of astrocytes in the hippocampus. The present data suggest that ketamine could increase GABA synthesis and astrocyte plasticity through downregulation of GABAAR α1, increases in GABA, and conversion of GABA into ATP, resulting in a rapid-acting antidepressant-like action. This article is part of the Special Issue on 'Ketamine and its Metabolites'.

The m6A mRNA demethylase FTO regulates GnRH secretion in Mn-induced precocious puberty.

The GABAA receptor (GABAAR) plays important roles in the regulation of Mn-induced GnRH secretion in immature female rats. However, the underlying molecular mechanisms remain unknown. Here, we assessed whether FTO and its substrate m6A are correlated with GABAAR expression in GnRH neurons after treatment with Mn in vitro and in vivo. Our study indicated that Mn treatment increased the expression of GnRH mRNA and decreased the levels of GABAAR protein but had no effect on GABAAR mRNA. Moreover, Mn upregulated the levels of FTO and inhibited global cellular m6A levels and GABAAα2 mRNA m6A levels. Knockdown of FTO increased the expression of GABAAR protein and GABAAα2 mRNA m6A levels. Data from rat models further demonstrate that inhibition of FTO suppressed GABAAR protein expression in the hypothalamus, causing delayed puberty onset. Collectively, our findings suggest that FTO-dependent m6A demethylation plays a critical role in regulating GABAAR mRNA processing in GnRH neurons.

The efficacy of γ-aminobutyric acid type A receptor (GABA AR) subtype-selective positive allosteric modulators in blocking tetramethylenedisulfotetramine (TETS)-induced seizure-like behavior in larval zebrafish with minimal sedation.

The chemical threat agent tetramethylenedisulfotetramine (TETS) is a γ-aminobutyric acid type A receptor (GABA AR) antagonist that causes life threatening seizures. Currently, there is no specific antidote for TETS intoxication. TETS-induced seizures are typically treated with benzodiazepines, which function as nonselective positive allosteric modulators (PAMs) of synaptic GABAARs. The major target of TETS was recently identified as the GABAAR α2ß3γ2 subtype in electrophysiological studies using recombinantly expressed receptor combinations. Here, we tested whether these in vitro findings translate in vivo by comparing the efficacy of GABAAR subunit-selective PAMs in reducing TETS-induced seizure behavior in larval zebrafish. We tested PAMs targeting α1, α2, α2/3/5, α6, ß2/3, ß1/2/3, and δ subunits and compared their efficacy to the benzodiazepine midazolam (MDZ). The data demonstrate that α2- and α6-selective PAMs (SL-651,498 and SB-205384, respectively) were effective at mitigating TETS-induced seizure-like behavior. Combinations of SB-205384 and MDZ or SL-651,498 and 2-261 (ß2/3-selective) mitigated TETS-induced seizure-like behavior at concentrations that did not elicit sedating effects in a photomotor behavioral assay, whereas MDZ alone caused sedation at the concentration required to stop seizure behavior. Isobologram analyses suggested that SB-205384 and MDZ interacted in an antagonistic fashion, while the effects of SL-651,498 and 2-261 were additive. These results further elucidate the molecular mechanism by which TETS induces seizures and provide mechanistic insight regarding specific countermeasures against this chemical convulsant.

'Proximity frequencies' a new parameter to evaluate the profile of GABAAR modulators.

We reported the synthesis of new 8-methoxypyrazolo[1,5-a]quinazolines bearing an amide fragment at the 3-position. The final compounds, as aromatic (2a-i) and 4,5-dihydro derivatives (3a-i), have been evaluated in vitrofor their ability to modulate the chlorine current on recombinant GABAA receptors of the α1ß2γ2L type (expressed in frog oocytes of the Xenopus laevis species). From electrophysiological test two groups of compounds emerged: positive modulators agonist (2e, h, i and 3e, h) and null modulators antagonist (2a, b, d, f, g and 3a-d, f, g) of GABAA subtype receptor. Using a set of compounds (new derivatives, known products and GABAA subtype receptor ligands from our library) we identify the amino acids at the α+/γ- interface, which could be involved in the agonist or antagonist profile, using the 'Proximity Frequencies', namely the frequencies with which a ligand intercepts two or more binding-site amino acids during the molecular dynamic simulation. The linear discriminant analysis (LDA) evidences that the combination of amino acids αVAL203- γTHR142 and αTYR 160- γTYR 58 allowed to collocate 70.6% of agonists and 72.7% of antagonists in their respective class.

Baicalin attenuates fibrogenic process in human renal proximal tubular cells (HK-2) exposed to diabetic milieu.

AIMS: Baicalin, a flavonoid glycoside substance extracted from Scutellaria baicalensis Georgi, has been shown to exhibit multiple therapeutic properties owing to its anti-inflammatory effect. Diabetes is characterized by chronic hyperglycemia, inflammation and oxidative stress, which promote renal fibrosis and kidney failure. Although anti-fibrogenic effects of baicalin in lung and liver have been reported previously, no study has investigated its roles in renal fibrosis. Here, we demonstrated protective effects of baicalin against fibrogenic process in human kidney proximal tubular epithelial cells (HK-2) exposed to diabetic milieu. MAIN METHODS: To investigate the effects of baicalin on oxidative stress- and inflammation-induced fibrosis in HK-2 cells, protein and gene expressions of NF-κB- and STAT3-associated inflammatory molecules and TGFß-associated extracellular matrix proteins were examined by western blotting, immunocytochemistry and qRT-PCR. To determine physiological changes of HK-2 exposed to diabetic milieu in response to baicalin, production of cAMP and cGMP and Ca2+ influx were measured. KEY FINDINGS: Baicalin attenuated oxidative stress- and inflammation-inudced IκB and JAK2 phosphorylations and, subsequent, NF-κB nuclear translocation and STAT3 phosphorylation. Consequently, it markedly reduced transactivation of NF-κB- and STAT3-associated inflammatory genes such as ICAM1, VCAM1, TGFß, IL1ß and MCP1, and protein expression of TGFß-associated extracellular matrix proteins, such as fibronectin and collagen IV. These effects are, partially, attributed to its regulatory function of intracellular concentration of Ca2+ via interaction with type A γ-aminobutyric acid receptor. SIGNIFICANCE: This is the first study which investigated anti-fibrogenic effect of baicalin in human kidney cells, and our results highlight a potential therapeutic application of baicalin for diabetic nephropathy.

Recurrent seizures cause immature brain injury and changes in GABA a receptor α1 and γ2 subunits.

OBJECTIVE: Recurrent seizures can cause brain damage and affect the cognitive outcome, particularly in developing children. We aimed to determine the effects of recurrent seizures on the expression of gamma-aminobutyric acid A receptor (GABAAR) α1 and γ2 subunit and neurodevelopment in immature rats. The role of the GABAAR agonist clonazepam and antagonist/partial agonist flumazenil in seizure-induced brain injury was also studied. METHODS: Recurrent seizures (RS) were induced by flurothyl inhalation in immature rats. Clonazepam (CZP) and flumazenil (FMZ) were administered to modulate GABAAR subunit expression in different experimental groups. Neurobehavioral changes and GABAAR α1 and γ2 subunit expression were studied. RESULTS: Inhalation of flurothyl for five days triggered RS and caused reflex delay, inability to adapt to new environments in adulthood, and deficits in long-term learning and memory ability in rats. Down-regulation of GABAAR α1 and γ2 subunits occurred after seizure onset and persisted for a long time. CZP treatment decreased the expression of GABAAR α1 and γ2 subunits and delayed neurodevelopment of the immature rats, whereas FMZ did not show any significant effects. CONCLUSIONS: Changes in GABAAR α1 and γ2 subunit expression and neurodevelopment were related to recurrent seizures and administration of CZP. Thus, GABAAR α1 and γ2 subunits likely play a significant role in the development of immature rats with RS and provide a novel target for therapeutic intervention.

Prepubertal overexposure to manganese induce precocious puberty through GABAA receptor/nitric oxide pathway in immature female rats.

Gamma-aminobutyric acid (GABA) plays a critical role in regulation of gonadotropin-releasing hormone (GnRH) through GABAA receptor (GABAAR). Nitric oxide (NO) production has correlation with GABA and regulates GnRH secretion. This study was performed to examine the mechanisms by which manganese (Mn) accelerate puberty onset involves GABAAR/NO pathway in the preoptic area-anterior hypothalamus (POA-AH) in immature female rats. First, female rats received daily dose of MnCl2 0 (saline), 2.5, 5 and 10 mg/kg b.w by oral gavage during postnatal day (PND) 21-32. Animals administered with 10 mg/kg MnCl2 exhibited earlier puberty onset age and advanced ovary and uterus development than these in saline-treatment group. Furthermore, we found that decrease of GABAAR result in elevated production of nitric oxide synthase1 (NOS1), NO and GnRH in the POA-AH. Second, we recorded the neuronal spikes alternation after perfusion with GABAAR inhibitor bicuculline (BIC), GABAAR agonist isoguvacine (isog), and MnCl2 from the POA-AH in acute brain slices of PND21 rats. Spontaneous firing revealed a powerful GABAAR-mediated action on immature POA-AH and confirm that MnCl2 has a significant effect on GABAAR. Third, we revealed that decrease in NOS1 and NO production by treatment with isog-alone or isog+MnCl2 contribute to the decrease of GnRH in the POA-AH and a delayed puberty onset age compared to treatment with MnCl2-alone. Together, these results suggested that excessive exposure to MnCl2 stimulates NO production through decreased GABAAR in the POA-AH to advance puberty onset in immature female rats.

Fractional occupancy of synaptic binding sites and the molecular plasticity of inhibitory synapses.

The postsynaptic density (PSD) at inhibitory synapses is a complex molecular assembly that serves as a platform for the interaction of neurotransmitter receptors, scaffold and adapter proteins, cytoskeletal elements and signalling molecules. The stability of the PSD depends on a multiplicity of interactions linking individual components. At the same time the PSD retains a substantial degree of flexibility. The continuous exchange of synaptic molecules and the preferential addition or removal of certain components induce plastic changes in the synaptic structure. This property necessarily implies that interactors are in dynamic equilibrium and that not all synaptic binding sites are occupied simultaneously. This review discusses the molecular plasticity of inhibitory synapses in terms of the connectivity of their components. Whereas stable protein complexes are marked by stoichiometric relationships between subunits, the majority of synaptic interactions have fractional occupancy, which is here defined as the non-saturation of synaptic binding sites. Fractional occupancy can have several causes: reduced kinetic or thermodynamic stability of the interactions, an imbalance in the concentrations or limited spatio-temporal overlap of interacting proteins, negative cooperativity or mutually exclusive binding. The role of fractional occupancy in the regulation of synaptic structure and function is explored based on recent data about the connectivity of inhibitory receptors and scaffold proteins. I propose that the absolute quantification of interactors and their stoichiometry at identified synapses can provide new mechanistic insights into the dynamic properties of inhibitory PSDs at the molecular level. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

Postnatal Lipopolysaccharide Exposure Impairs Adult Neurogenesis and Causes Depression-like Behaviors Through Astrocytes Activation Triggering GABAA Receptor Downregulation.

Growing evidence indicates that early-life inflammation has adverse effects on adult hippocampal neurogenesis and GABA system. Based the report that hippocampal GABA system is a key modulator in adult hippocampal neurogenesis, the aim of this study was to investigate whether and how early inflammation affects GABAergic system resulting in the alterations of adult hippocampal neurogenesis and related behaviors. Neonatal mice received a daily subcutaneous injection of lipopolysaccharide (LPS, 50 µg/kg) or saline on postnatal days (PND) 3-5. Behavioral tests were used to explore LPS-induced depression-like behaviors of adult mice. Immunohistochemistry staining and western blot were employed to detect adult neurogenesis, GABAergic system, glia activation and BDNF-TrkB pathway in the hippocampus. LPS-treated mice developed a depression phenotype with the inhibited maturation of hippocampal newborn neurons in adulthood. Compared with controls, LPS mice showed a decreased expression of GABAA receptor (GABAAR) protein. GABAAR agonist phenobarbital could rectify the decrease of BrdU+/NeuN+ cells in LPS mice. Additionally, postnatal LPS treatment resulted in the activation of astrocytes and the increase expression of toll-like receptor 4 (TLR4) in the second postnatal week and the downregulation of BDNF-TrkB pathway in adulthood. The treatment with TLR4 inhibitor TAK-242 restored the decrease of BrdU+/NeuN+ cells and depression-like behaviors in LPS mice via improving GABAAR. The results indicate that postnatal LPS exposure impairs adult hippocampal neurogenesis and causes depression-like behaviors through early astrocytes activation triggering the later GABAAR downregulation.

Propofol reduces acute lung injury by up-regulating gamma-aminobutyric acid type a receptors.

BACKGROUND: We used a two-hit lung injury rat model that involves mechanical ventilation (MV) following lipopolysaccharide exposure to investigate the effects of propofol on the expression of GABAA receptors (GABAAR) and cytokine responses, and we then determined the specific effects of GABA on cytokine responses in vitro in alveolar epithelial cells (AECs). METHODS: Forty-eight adult male Wister rats were equally and randomly divided into the following 4 groups (n = 12) using a random number table: sham group, sham+propofol group, lipopolysaccharide (LPS) + VILI group, and LPS + VILI + propofol group. All animals were anesthetized, and the animals received a 3.75 mg/kg intratracheal instillation of endotoxins or phosphate-buffered saline (PBS) as the control, as described previously. After 30 min, rats were ventilated for 5 h in a volume-controlled ventilation mode. In the LPS + VILI group, animals were ventilated with a tidal volume (Vt) of 22 ml/kg and zero positive end-expiratory pressure (PEEP) at a respiratory rate of 16-18 breaths/min, whereas control (sham) rats were ventilated with a Vt of 6 ml/kg and PEEP of 5 cmH2O at a rate of 45-55 breaths/min. The FiO2 remained constant as 0.4, propofol was administered intravenously in the LPS + VILI + propofol and sham + propofol groups at a rate of 10 mg·kg-1·h-1 while normal saline at the same rate was intravenously administered in the LPS + VILI and sham groups during the entire mechanical ventilation period. Five hours after mechanical ventilation, the rats were killed. Survival rates, histopathology, concentrations of inflammatory mediators in bronchoalveolar lavage fluid (BALF), wet weight/dry weight (W/D) ratio of the lung, myeloperoxidase (MPO) activity in lung tissues, and expression of GAD and GABAAR by immunohistochemical detection and Western blotting were assessed. Then, human type II-like alveolar epithelial cells (A549 cells) were cultured to full confluence and incubated with GABA (100 nM) alone, picrotoxin alone, a GABAAR antagonist (PTX, 50 nM), or GABA + PTX for 10 min, followed by stimulation with LPS (control) at 100 ng/ml for 4 h. The concentrations of IL-1ß, IL-2, IL-8, and IL-10 were then measured. RESULTS: Administration of propofol in a two-hit lung injury rat model can increase survival rates and the expression of GAD and GABAAR (P < .05). The administration of propofol can attenuate the release of pro-inflammatory cytokines both in vivo and in vitro, and the administration of propofol can attenuate histopathological changes, the W/D ratio, and MPO activity (P < .05). CONCLUSIONS: In this study, we found that the administration of propofol improved lung function, alleviated lung injury, and up-regulated the GAD and GABAAR expressions in a two-hit model of acute lung injury (ALI) characterized by intratracheal instillation of an endotoxin and prolonged MV. Therefore, the protective effects of propofol may be associated with the up-regulation of GABAA receptors in AECs.

Does kisspeptin participate in GABA-mediated modulation of GnRH and GnRH receptor biosynthesis in the hypothalamic-pituitary unit of follicular-phase ewes?

BACKGROUND: The inverse relationship between GnRH transcript level and GABA neurons activity has suggested that GABA at the hypothalamic level may exert a suppressive effect on subsequent steps of the GnRH biosynthesis. In the present study, we analyzed the effects of GABA type A receptor agonist (muscimol) or antagonist (bicuculline) on molecular mechanisms governing GnRH/LH secretion in follicular-phase sheep. METHODS: ELISA technique was used to investigate the effects of muscimol and/or bicuculline on levels of post-translational products of genes encoding GnRH ligand and GnRH receptor (GnRHR) in the preoptic area (POA), anterior (AH) and ventromedial (VMH) hypothalamus, stalk/median eminence (SME), and GnRHR in the anterior pituitary (AP). Real-time PCR was chosen for determination of the effect of drugs on kisspeptin (Kiss 1) mRNA level in POA and VMH including arcuate nucleus (VMH/ARC), and on Kiss1 receptor (Kiss1r) mRNA abundance in POA-hypothalamic structures. These analyses were supplemented by RIA method for measurement of plasma LH concentration. RESULTS: The study demonstrated that muscimol and bicuculline significantly decreased or increased GnRH biosynthesis in all analyzed structures, respectively, and led to analogous changes in plasma LH concentration. Similar muscimol- and bicuculline-related alterations were observed in levels of GnRHR. However, the expression of Kiss 1 and Kiss1r mRNAs in selected POA-hypothalamic areas of either muscimol- and bicuculline-treated animals remained unaltered. CONCLUSIONS: Our data suggest that GABAergic neurotransmission is involved in the regulatory pathways of GnRH/GnRHR biosynthesis and then GnRH/LH release in follicular-phase sheep conceivably via indirect mechanisms that exclude involvement of Kiss 1 neurons.

GABAergic and glutamatergic cells in the inferior colliculus dynamically express the GABAAR γ1 subunit during aging.

Age-related hearing loss may result, in part, from declining levels of γ-amino butyric acid (GABA) in the aging inferior colliculus (IC). An upregulation of the GABAAR γ1 subunit, which has been shown to increase sensitivity to GABA, occurs in the aging IC. We sought to determine whether the upregulation of the GABAAR γ1 subunit was specific to GABAergic or glutamatergic IC cells. We used immunohistochemistry for glutamic acid decarboxylase and the GABAAR γ1 subunit at 4 age groups in the IC of Fisher Brown Norway rats. The percentage of somas that expressed the γ1 subunit and the number of subunits on each soma were quantified. Our results show that GABAergic and glutamatergic IC cells increasingly expressed the γ1 subunit from young age until expression peaked during middle age. At old age (∼77% of life span), the number of GABAAR γ1 subunits per cell sharply decreased for both cell types. These results, along with previous studies, suggest inhibitory and excitatory IC circuits may express the GABAAR γ1 subunit in response to the age-related decline of available GABA.

HIV gp120 upregulates tonic inhibition through α5-containing GABAARs.

HIV-Associated Neurocognitive disorder (HAND) affects nearly half of infected patients. The HIV envelope protein gp120 is shed by infected cells and is a potent neurotoxin in vitro that reproduces many aspects of HAND when expressed in vivo. Here, we show that HIV gp120 increases the amplitude of a tonic current mediated by γ-aminobutyric acid type-A receptors (GABAARs). Treating rat hippocampal cultures with 600 pM gp120IIIB for 4 h increased a tonic bicuculline-sensitive current, which remained elevated for 24 h. The increased current resulted from upregulation of extrasynaptic α5-containing GABAARs, as indicated by inhibition with the selective inverse agonist basmisanil. Treatment with gp120 increased α5-GABAAR immunoreactivity on the cell surface without new protein synthesis. The increase in tonic inhibition was prevented by a C-X-C chemokine receptor type 4 (CXCR4) antagonist or elimination of microglia from the culture. Treatment with interleukin-1ß (IL-1ß) increased the tonic current and an IL-1 receptor antagonist blocked the gp120-evoked response. Pharmacological or genetic inhibition of p38 mitogen-activated protein kinase (MAPK) prevented the gp120-evoked increase in tonic current and direct activation of a mutant form of p38 MAPK expressed in neurons increased the current. Collectively, these data show that gp120 activates CXCR4 to stimulate microglia to release IL-1ß. Subsequent stimulation of IL-1 receptors activates p38 MAPK in neurons leading to the upregulation of α5-containing GABAARs. Increased tonic inhibition impairs neuroplasticity and inhibition of α5-containing GABAARs improves cognitive function in disease models. Thus, gp120-induced upregulation of α5-containing GABAARs presents a novel therapeutic target for HAND.

Gephyrin Palmitoylation in Basolateral Amygdala Mediates the Anxiolytic Action of Benzodiazepine.

BACKGROUND: Benzodiazepines (BZDs) have been used to treat anxiety disorders for more than five decades as the allosteric modulator of the gamma-aminobutyric acid A receptor (GABAAR). Little is known about other mechanisms of BZDs. Here, we describe how the rapid stabilization of postsynaptic GABAAR is essential and sufficient for the anxiolytic effect of BZDs via a palmitoylation-dependent mechanism. METHODS: Palmitoylated proteins in the basolateral amygdala (BLA) of rats with different anxious states were assessed by a biotin exchange protocol. Both pharmacological and genetic approaches were used to investigate the role of palmitoylation in anxiety behavior. Electrophysiological recording, reverse transcription polymerase chain reaction, Western blotting, and coimmunoprecipitation were used to investigate the mechanisms. RESULTS: Highly anxious rats were accompanied by the deficiency of gephyrin palmitoylation and decreased the synaptic function of GABAAR in the BLA. We then identified that the dysfunction of DHHC12, a palmitoyl acyltransferase that specifically palmitoylates gephyrin, contributed to the high-anxious state. Furthermore, diazepam, as an anxiolytic drug targeting GABAARs, was found to increase gephyrin palmitoylation in the BLA via a GABAAR-dependent manner to activate DHHC12. The anxiolytic effect of diazepam was nearly abolished by the DHHC12 knockdown. Specifically, similar to the effect of BZD, the overexpression of DHHC12 in the BLA exerted a significant anxiolytic action, which was prevented by flumazenil. CONCLUSIONS: Our results support the view that the strength of inhibitory synapse was controlled by gephyrin palmitoylation in vivo and proposes a previously unknown palmitoylation-centered mode of BZD's action.