Presence Of GABA Research Articles

Coronaridine congeners induce anticonvulsant activity in rodents by hippocampal mechanisms involving mainly potentiation of GABAA receptors

The coronaridine congeners catharanthine and 18-methoxycoronaridine (18-MC) display sedative, anxiolytic, and antidepressant properties by acting on mechanisms involving GABAergic and/or monoaminergic transmissions. Here, we expanded their pharmacological properties by studying their anticonvulsant activity in male and female mice using the pentylenetetrazole (PTZ)-induced seizure test. To determine potential neurochemical mechanisms, the effect of congeners on monoamine content and kainic acid (KA)-induced epileptiform discharge was studied in the hippocampus. The behavioral results showed that coronaridine congeners induce acute anticonvulsant activity in a dose-dependent but sex-independent manner. Repeated treatment with a subthreshold dose (20 mg/kg) of each congener produced anticonvulsant activity in a sex-independent manner, but was significantly higher in male mice when compared to its acute effect. Using a behaviourally relevant regimen, we found that PTZ increased dopamine metabolites and serotonin tissue content. Coronaridine congeners, which induced distinct effects on monoamines, blunted the effect of PTZ instead of potentiating it, suggesting the existence of another mechanism in their anticonvulsant activity. The electrophysiological results indicated that both congeners inhibit KA-induced epileptiform discharges in hippocampal slices. A key aspect of this study is that the activity of both congeners was observed only in the presence of GABA, supporting the notion that hippocampal GABAAR potentiation plays an important role. Our study showed that coronaridine congeners induce acute anticonvulsant activity in a sex-independent manner. However, a comparatively higher susceptibility was observed in male mice after repeated treatment. The underlying hippocampal mechanisms mainly involve GABAAR potentiation, whereas monoamines play a minor role in the anticonvulsive action.

Etomidate Depresses Spontaneous Complex Spikes Activity of Cerebellar Purkinje Cells via Cannabinoid 1 Receptor in vivo in Mice

Introduction: Complex spikes (CSs) activity of cerebellar Purkinje cells plays critical roles in motor coordination and motor learning by transferring information to cerebellar cortex, which is an accessible and useful model for neurophysiological investigation. Etomidate is an ultrashort-acting nonbarbiturate intravenous anesthetic, which inhibits the spontaneous activity of cerebellar Purkinje cells through activation of GABA_A and glycine receptors in vivo in mice. However, the effect of etomidate on the spontaneous CSs activity of cerebellar Purkinje cells in living mouse is not clear. Methods: We here investigated the effects of etomidate on spontaneous CSs activity of cerebellar Purkinje cell in urethane-anesthetized mice by electrophysiology recording technique and pharmacological methods. Results: Our results showed that cerebellar surface perfusion of etomidate significantly depressed the activity of spontaneous CSs, which exhibited decreases in the number of spikelets and the area under curve (AUC) of the CSs. The etomidate-produced inhibition of CSs activity was persisted in the presence of GABA_A and glycine receptors antagonists. However, application of cannabinoid 1 (CB1) receptor antagonist, AM-251, completely blocked the etomidate-induced inhibition of CSs. Furthermore, application of the CB1 receptor agonist, WIN55212-2, induced a decrease of CSs. Moreover, in the presence of a specific protein kinase A (PKA) inhibitor, KT5720, etomidate failed to produce decreases in the spikelets number and the AUC of the spontaneous CSs. Conclusion: These results indicate that cerebellar surface application of etomidate facilitates CB1 receptor activity resulting in a depression of spontaneous CSs activity of Purkinje cells via PKA signaling pathway in mouse cerebellar cortex. Our present results suggest that the etomidate administration may impair the function of cerebellar cortical neuronal circuitry by inhibition of the climbing fiber – Purkinje cells synaptic transmission through activation of CB1 receptors in vivo in mice.

GABA has a presynaptic inhibitory effect at Lumbricus terrestris body wall muscle synapses.

Earthworm body wall muscle synapses have been suggested to contain both excitatory and inhibitory inputs, and therefore allow for investigation of excitatory/inhibitory signaling in an easily accessible model system. While previous studies have focused on postsynaptic GABAergic inhibitory mechanisms, this study investigated the hypothesis that GABAergic signaling also has presynaptic inhibitory function. This hypothesis was tested by loading synaptogreen C4 dye (also called FM1-43) into presynaptic vesicles in the presence of GABA at Lumbricus terrestris longitudinal muscle synapses. GABA treatment significantly reduced the fluorescence intensity observed at these synapses, suggesting that GABAergic signaling does indeed have a presynaptic inhibitory mechanism.

Effect of original anticonvulsant ortho-fluoro-benzonal on immune cells functional properties at chronic ethanol intoxication

IntroductionGABAa-receptors proved to be the molecular targets of ethanol on immune and nervous cells, potentiating alcohol influence. ortho-Fluoro-benzonal is known to be a circular urea derivative and an artificial ligand of GABA/BD-receptor and thus a potential candidate drug for alchoholism treatment.ObjectivesWe have shown the alcohol motivation decrease under ortho-fluoro-benzonal influence in experiment. The investigation of molecular mechanisms and functional targets of this substance is an important step in understanding of molecular pathogenesis and approaches to managing alcohol addiction.MethodsSplenocytes from male (CBAxC57Bl/6) F1 mice in a state of alcohol dependence owing to 6-month 10% ethanol exposure were aseptically obtained and cultured in presence of GABA, ortho-fluoro-benzonal and mitogens (LPS or concanavalin A). Proliferative activity of immune cells in vitro was estimated by means of radioactive 3H-thymidine incorporation.Results The intact animals’ splenocytes revealed increased spontaneous proliferation, increased T-mitogen stimulated and decreased B-mitogen stimulated proliferation in the presence of ortho-fluoro-benzonal. The immune cells from alcoholized animals, demonstrating increased spontaneous proliferative activity and weaken susceptibility to the mitogens, showed normal response patterns, except B-mitogen response case, under ortho-fluoro-benzonal inluence. Addition of GABA into the cultures didn’t cancel most positive effects of ortho-fluoro-benzonal inluence, proving existence of their GABAaR-independent pathways, mediated by other barbiturate receptors in addition to GABAaR-dependent ones.ConclusionsImmunomodulating properties of artificial GABA receptor ligand, ortho-fluoro-benzonal, in vitro has been shown. The compound may correct immune cells dysregulation caused by chronic ethanol exposure, so the original anticonvulsant has promise in the treatment of alcoholism.DisclosureThe authors have not supplied a conflict-of-interest statement.

Nipecotic-Acid-Tethered, Naphthalene-Diimide-Based, Orange-Emitting Organic Nanoparticles as Targeted Delivery Vehicle and Diagnostic Probe toward GABAA-Receptor-Enriched Cancer Cells.

This article demonstrates target-specific cellular imaging of GABA (γ-aminobutyric acid) receptor (GABAAR)-enriched cells (SH-SY5Y and A549) with therapeutic efficacy by naphthalene diimide (NDI)-derived fluorescent organic nanoparticles (FONPs). Self-assembly-driven formation of spherical organic particles by nipecotic-acid-tethered l-aspartic acid appended NDI derivative (NDI-nip) took place in DMSO-water through J-type aggregation. NDI-nip having a naphthyl residue and a nipecotic acid unit at both terminals exhibited aggregation-induced emission (AIE) at and above 60% water content in DMSO because of excimer formation at λem = 579 nm. The orange-emitting NDI-nip FONPs (1:99 v/v DMSO-water) having excellent cell viability and high photostability were used for selective bioimaging and killing of GABAAR-overexpressed cancer cells through target-specific delivery of the anticancer drug curcumin. The fluorescence intensity of NDI-nip FONPs were quenched in GABAAR-enriched neuroblastoma cells (SH-SY5Y) and cancerous cells (A549). Notably, in the presence of GABA, the NDI-nip FONPs exhibited their native fluorescence within the same cell lines. Importantly, no such quenching and regaining of NDI-nip FONP emission in the presence of GABA was noted in the case of the noncancerous cell NIH3T3. The killing efficiency of curcumin-loaded NDI-nip FONPs ([curcumin] = 100 μM and [NDI-nip FONPs] = 50 μM) was significantly higher in the cases of SH-SY5Y (88 ± 3%) and A549 (72 ± 2%) than in NIH3T3 (37 ± 2). The presence of a nipecotic acid moiety facilitated the selective cellular internalization of NDI-nip FONPs into GABAAR-overexpressing cells. Hence, these orange-emitting NDI-nip FONPs may be exploited as a targeted diagnostic probe as well as a drug delivery vehicle for GABAAR-enriched cancer cells.

Alanine Represses γ-Aminobutyric Acid Utilization and Induces Alanine Transaminase Required for Mitochondrial Function in Saccharomyces cerevisiae.

The γ-aminobutyric acid (GABA) shunt constitutes a conserved metabolic route generating nicotinamide adenine dinucleotide phosphate (NADPH) and regulating stress response in most organisms. Here we show that in the presence of GABA, Saccharomyces cerevisiae produces glutamate and alanine through the irreversible action of Uga1 transaminase. Alanine induces expression of alanine transaminase (ALT1) gene. In an alt1Δ mutant grown on GABA, alanine accumulation leads to repression of the GAD1, UGA1, and UGA2 genes, involved in the GABA shunt, which could result in growth impairment. Induced ALT1 expression and negative modulation of the GABA shunt by alanine constitute a novel regulatory circuit controlling both alanine biosynthesis and catabolism. Consistent with this, the GABA shunt and the production of NADPH are repressed in a wild-type strain grown in alanine, as compared to those detected in the wild-type strain grown on GABA. We also show that heat shock induces alanine biosynthesis and ALT1, UGA1, UGA2, and GAD1 gene expression, whereas an uga1Δ mutant shows heat sensitivity and reduced NADPH pools, as compared with those observed in the wild-type strain. Additionally, an alt1Δ mutant shows an unexpected alanine-independent phenotype, displaying null expression of mitochondrial COX2, COX3, and ATP6 genes and a notable decrease in mitochondrial/nuclear DNA ratio, as compared to a wild-type strain, which results in a petite phenotype. Our results uncover a new negative role of alanine in stress defense, repressing the transcription of the GABA shunt genes, and support a novel Alt1 moonlighting function related to the maintenance of mitochondrial DNA integrity and mitochondrial gene expression.

Alterations of GABA B receptors in the APP/PS1 mouse model of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the progressive decline of memory and cognitive function. The disease is characterized by the presence of amyloid plaques, tau tangles, altered inflammatory signaling, and alterations in numerous neurotransmitter signaling systems, including γ-aminobutyric acid (GABA). Given the extensive role of GABA in regulating neuronal activity, a careful investigation of GABA-related changes is needed. Further, given persistent inflammation has been demonstrated to drive AD pathology, the presence of GABA B receptor expressed on glia that serve a role regulation of the immune response adds to potential implications of altered GABA in AD. There has not previously been a systematic evaluation of GABA-related changes in an amyloid model of AD that specifically focuses on examining changes in GABA B receptors. In the present study, we examined alterations in several GABA-specific targets in the APP/PS1 mouse model at different ages. In the 4-month-old cohort, no significant deficits in spatial learning and memory or alterations in any of the GABAergic targets were observed compared with wild-type controls. However, we identified significant alterations in several GABA-related targets in the 6-month-old cohort that exhibited spatial learning deficits that include changes in glutamic acid decarboxylase 65, GABA transporter type 3, and GABA B receptors protein and mRNA levels. This was the same cohort at which learning and memory deficits and significant amyloid pathology was observed. Overall, our study provides evidence of altered GABAergic signaling in an amyloid model of AD at a time point consistent with AD-related deficits.

Effect of γ-Aminobutyric Acid (GABA) on the Metabolome of Two Strains of Lasiodiplodia theobromae Isolated from Grapevine.

The effect of γ-aminobutyric acid (GABA) on the metabolome of two strains of Lasiodiplodia theobromae isolated from grapevine that hold a different degree of virulence to the host plant (LA-SOL3 (more virulent), LA-SV1 (less virulent)) was investigated. The culture filtrates and crude extracts from the two strains grown in the presence and absence of 10 mM of GABA were tested for phytotoxicity on tomato plant cuttings and leaves, respectively. Considering the opportunistic nature of this fungus for humans, crude extracts were also tested for cytotoxicity on mammalian cell lines. We found that culture filtrates and crude extracts have a decreased toxicity in the presence of GABA. Metabolomic analysis, conducted on both strains at both growth conditions, revealed the production of several compounds, such as indole-3-carboxylic acid (ICA, which is the main compound produced by L. theobromae), 3-indolecarboxyaldehyde, (3R,4S)-botryodiplodin, (R)-mellein. Finally, data demonstrate that GABA both induces a decrease in the amount of ICA, and a diversification of the metabolites produced by L. theobromae.

Exogenous GABA promotes adaptation and growth by altering the carbon and nitrogen metabolic flux in poplar seedlings under low nitrogen conditions.

Nitrogen (N) deficiency adversely affects tree growth. Additionally, γ-aminobutyric acid (GABA) is closely associated with growth and stress responses because of its effects on carbon (C) and N metabolism. However, little is known about its roles related to plant adaptations to N-deficient conditions. In this study, we analyzed the effects of GABA (0, 2 and 10mM) applications on the growth traits and physiological responses of poplar (Populus alba×P. glandulosa '84K') seedlings under high N (HN) and low N (LN) conditions. We found that the added GABA interacted with N to affect more than half of the studied parameters, with greater effects in LN plants than in HN plants. Under LN conditions, the GABA application tended to increase poplar growth, accompanied by increased xylem fiber cell length and xylem width. In stems, exogenous GABA increased the abundance of non-structural carbohydrates (starch and sugars) and tricarboxylic acid cycle intermediates (succinate, malate and citrate), but had the opposite effect on the structural C contents (hemicellulose and lignin). Meanwhile, exogenous GABA increased the total soluble protein contents in leaves and stems, accompanied by significant increases in nitrate reductase, nitrite reductase and glutamine synthetase activities in leaves, but significant decreases in those (except for the increased glutamate synthetase activity) in stems. A multiple factorial analysis indicated that the nitrate assimilation pathway substantially influences poplar survival and growth in the presence of GABA under LN conditions. Interestingly, GABA applications also considerably attenuated the LN-induced increase in the activities of leaf antioxidant enzymes, including peroxidase and catalase, implying that GABA may regulate the relative allocation of C and N for growth activities by decreasing the energy cost associated with stress defense. Our results suggest that GABA enhances poplar growth and adaptation by regulating the C and N metabolic flux under N-deficient conditions.

New triterpenoids from the roots of Rhododendron molle as positive modulators of GABAA receptors

Phytochemical investigation of the roots of Rhododendron molle led to the isolation of 15 new oleanane- and ursane-type triterpenoid compounds (1–15) along with 2 known triterpenoid compounds (16 and 17). The structures of the new compounds were determined by NMR analysis, ESIMS methods and X-ray diffraction. Notably, compound 1 is the first example of a 23-nor-ursane-type triterpenoid with 5/5/6/6/6/6-fused ring system. Compounds 2–5 are 7/6/6/6/6-fused triterpenoids. Compounds 6–9 are 23-nor-2,3-seco 6/6/6/6-fused triterpenoids. Compounds 10 and 11 are 3, 23, 24-trinor-2,4-seco 6/6/6/6-fused triterpenoids. In a membrane potential FLIPR assay, compounds 2, 14–17 (10 μM) induced the activation of GABAA receptors in the presence of GABA (0.2 μM), with increases in activation of 67.9%, 64.2%, 57.9% and 58%, respectively.

Functional dissection of gamma-aminobutyric acid metabolism in Neurospora crassa.

The GABA shunt is one of the metabolic pathways that is ubiquitous in prokaryotes and eukaryotes. γ-aminobutyric acid (GABA) in fungi is required in the stress responses, virulence and development. The number of genes encoding glutamate decarboxylase (gad), GABA transaminase (gta) and succinic semialdehyde dehydrogenase (ssadh) varies between fungal species. The genome-wide analysis in Neurospora crassa resulted in the identification of a gta and a ssadh. Disruption of either gta or ssadh decreased respiration rate and biomass accumulation, reduced growth on GABA and beta-alanine. The gta and ssadh mutants exhibited aberrant hyphal morphology and displayed differential transcription of the GABA shunt genes. In the gta mutant, protoperithecia and perithecia formation was almost completely suppressed in the presence of GABA and beta-alanine, indicating GTA requirement for the turnover of these amino acids. The strains displayed differential metabolic dysregulations in response to different nitrogen sources. The phenotypic differences between the gta and ssadh mutants could be contributed to accumulation of intermediates of the GABA shunt and/or GABA shunt-independent functions. Together, our data suggest that the GABA shunt could function as a moderate modulator of multiple biological events, including respiration, energy metabolism, carbon and nitrogen metabolism, growth, as well as sexual development in N. crassa.

Steady-state activation of the high-affinity isoform of the \u03b14\u03b22\u03b4 GABAA receptor

Activation of GABAA receptors consisting of α4, β2 (or β3), and δ subunits is a major contributor to tonic inhibition in several brain regions. The goal of this study was to analyze the function of the α4β2δ receptor in the presence of GABA and other endogenous and clinical activators and modulators under steady-state conditions. We show that the receptor has a high constitutive open probability (~0.1), but is only weakly activated by GABA that has a maximal peak open probability (POpen,peak) of 0.4, taurine (maximal POpen,peak = 0.4), or the endogenous steroid allopregnanolone (maximal POpen,peak = 0.2). The intravenous anesthetic propofol is a full agonist (maximal POpen,peak = 0.99). Analysis of currents using a cyclic three-state Resting-Active-Desensitized model indicates that the maximal steady-state open probability of the α4β2δ receptor is ~0.45. Steady-state open probability in the presence of combinations of GABA, taurine, propofol, allopregnanolone and/or the inhibitory steroid pregnenolone sulfate closely matched predicted open probability calculated assuming energetic additivity. The results suggest that the receptor is active in the presence of physiological concentrations of GABA and taurine, but, surprisingly, that receptor activity is only weakly potentiated by propofol.

Abstract 4379: Prostate cancer cells utilize the GABA shunt to enhance oxidative metabolism

Abstract Although an upregulation of aerobic glycolysis (the Warburg effect) is a canonical hallmark of cancer, it is now recognized that most cancerous cells continue to rely on the TCA cycle as a source of both biosynthetic precursors (DeBarardinis & Chandel, 2016) and the majority of ATP production (Zu & Guppy, 2004). Drug-resistant cancers have been associated with metabolic reprogramming (Rahman & Hasan, 2015) involving an upregulation in oxidative phosphorylation (Wolf, 2014). Two advanced, treatment-resistant variants of prostate cancer are castration-resistant adenocarcinoma (CRPC-adeno) and neuroendocrine prostate cancer (NEPC). Emerging evidence suggests that the γ-aminobutyric acid (GABA) shunt—an evolutionarily conserved pathway for energy production, biosynthetic precursors, and ROS management—may be used by both CRPC-adeno and NEPC. The GABA shunt is a bypass for two steps of the TCA cycle, wherein GABA is synthesized from glutamate by a GAD1-encoded decarboxylase, transaminated into succinic semialdehyde, and metabolized into the TCA cycle intermediate succinate. Analysis of publicly available transcript datasets (via cBioPortal) indicates that GAD1 mRNA expression increases with increasing Gleason score (p < 0.00005, prostate adenocarcinoma TCGA provisional dataset), with higher expression in both CRPC-adeno compared to primary adenocarcinoma (Ippolito and Piwnica-Worms, 2014), and NEPC compared to CRPC-adeno (p < 0.005, Beltran, et al., 2016). Expression of GAD1 transcripts appears to be contingent on low levels of AR transcripts (Trento/Cornell/Broad data set), consistent with the AR independence of the NEPC phenotype. Similarly, GABA levels increase as AR expression is lost during transdifferentiation to NEPC (Solorzano, et al., 2018). GAD67 protein levels in prostate tumors are a more accurate predictor of patient risk for metastasis than Gleason score (Azuma, et al., 2003), and flux through the GABA shunt is critical for the growth of brain tumors in mouse xenograft models (Schnepp, et al., 2017; Neman, et al., 2014). Our experiments show that treatment of an early-stage prostate cancer cell line model (LNCaP) with GABA (100 µM, 24h) induces a shift from aerobic glycolysis to oxidative metabolism. This metabolic reprogramming toward increased oxidative phosphorylation requires at least 6 hours to emerge, and is maximal by 18 hours. During this time period, the presence of GABA in the mitochondrial matrix steadily increases, as shown by our newly-developed GABA-sensing fluorescent receptor, mitoGABASnFR. The observed increases in oxygen consumption are accompanied by GABA-induced decreases in extracellular acidification and lactate levels. In light of these findings, we propose that the GABA shunt represents an understudied pathway for inducing a metabolic shift towards oxidative phosphorylation in advanced prostate cancer, and therefore may be a target for future anticancer therapies. Citation Format: Erika L. Knott, Sumitra Miriyala, Manikandan Panchatcharam, Nancy Leidenheimer. Prostate cancer cells utilize the GABA shunt to enhance oxidative metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4379.

Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in the α1β3γ2 GABAAR Transmembrane Domain.

GABAA receptors (GABAARs) are targets for important classes of clinical agents (e.g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs). Previously, using photoreactive analogs of etomidate ([3H]azietomidate) and mephobarbital [[3H]1-methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid ([3H]R-mTFD-MPAB)], we identified two homologous but pharmacologically distinct classes of general anesthetic binding sites in the α1β3γ2 GABAAR transmembrane domain at β +-α - (β + sites) and α +-β -/γ +-β - (β - sites) subunit interfaces. We now use competition photolabeling with [3H]azietomidate and [3H]R-mTFD-MPAB to identify para-substituted propofol analogs and other drugs that bind selectively to intersubunit anesthetic sites. Propofol and 4-chloro-propofol bind with 5-fold selectivity to β +, while derivatives with bulkier lipophilic substitutions [4-(tert-butyl)-propofol and 4-(hydroxyl(phenyl)methyl)-propofol] bind with ∼10-fold higher affinity to β - sites. Similar to R-mTFD-MPAB and propofol, these drugs bind in the presence of GABA with similar affinity to the α +-β - and γ +-β - sites. However, we discovered four compounds that bind with different affinities to the two β - interface sites. Two of these bind with higher affinity to one of the β - sites than to the β + sites. We deduce that 4-benzoyl-propofol binds with >100-fold higher affinity to the γ +-β - site than to the α +-β - or β +-α - sites, whereas loreclezole, an anticonvulsant, binds with 5- and 100-fold higher affinity to the α +-β - site than to the β + and γ +-β - sites. These studies provide a first identification of PAMs that bind selectively to a single intersubunit site in the GABAAR transmembrane domain, a property that may facilitate the development of subtype selective GABAAR PAMs.

A Novel Orally Available Asthma Drug Candidate That Reduces Smooth Muscle Constriction and Inflammation by Targeting GABAA Receptors in the Lung.

We describe lead compound MIDD0301 for the oral treatment of asthma based on previously developed positive allosteric α5β3γ2 selective GABAA receptor (GABAAR) ligands. MIDD0301 relaxed airway smooth muscle at single micromolar concentrations as demonstrated with ex vivo guinea pig tracheal rings. MIDD0301 also attenuated airway hyperresponsiveness (AHR) in an ovalbumin murine model of asthma by oral administration. Reduced numbers of eosinophils and macrophages were observed in mouse bronchoalveolar lavage fluid without changing mucous metaplasia. Importantly, lung cytokine expression of IL-17A, IL-4, and TNF-α were reduced for MIDD0301-treated mice without changing antiinflammatory cytokine IL-10 levels. Automated patch clamp confirmed amplification of GABA induced current mediated by α1-3,5β3γ2 GABAARs in the presence of MIDD0301. Pharmacodynamically, transmembrane currents of ex vivo CD4+ T cells from asthmatic mice were potentiated by MIDD0301 in the presence of GABA. The number of CD4+ T cells observed in the lung of MIDD0301-treated mice were reduced by an oral treatment of 20 mg/kg b.i.d. for 5 days. A half-life of almost 14 h was demonstrated by pharmacokinetic studies (PK) with no adverse CNS effects when treated mice were subjected to sensorimotor studies using the rotarod. PK studies also confirmed very low brain distribution. In conclusion, MIDD0301 represents a safe and improved oral asthma drug candidate that relaxes airway smooth muscle and attenuates inflammation in the lung leading to a reduction of AHR at a dosage lower than earlier reported GABAAR ligands.

Structure-Function Evaluation of Imidazopyridine Derivatives Selective for δ-Subunit-Containing γ-Aminobutyric Acid Type A (GABAA) Receptors.

δ-Selective compounds 1 and 2 (DS1, compound 22; DS2, compound 16) were introduced as functionally selective modulators of δ-containing GABA type A receptors (GABAAR). In our hands, [3H]EBOB-binding experiments with recombinant GABAAR and compound 22 showed no proof of δ-selectivity, although there was a minimally higher preference for the α4β3δ and α6β2/3δ receptors with respect to potency. In order to delineate the structural determinants of δ preferences, we synthesized 25 derivatives of DS1 and DS2, and investigated their structure-activity relationships (SAR). Four of our derivatives showed selectivity for α6β3δ receptors (29, 38, 39, and 41). For all of them, the major factors that distinguished them from compound 22 were variations at the para-positions of their benzamide groups. However, two compounds (29 and 39), when tested in the presence of GABA, revealed effects at several additional GABAAR. The newly synthesized compounds will still serve as useful tools to investigate α6β3δ receptors.

Rapid Throughput Analysis of GABAA Receptor Subtype Modulators and Blockers Using DiSBAC1(3) Membrane Potential Red Dye.

Fluorometric imaging plate reader membrane potential dye (FMP-Red-Dye) is a proprietary tool for basic discovery and high-throughput drug screening for G-protein-coupled receptors and ion channels. We optimized and validated this potentiometric probe to assay functional modulators of heterologous expressed GABAA receptor (GABAAR) isoforms (synaptic α1β3γ2, extrasynaptic α4β3δ, and β3 homopentomers). High-resolution mass spectrometry identified FMP-Red-Dye as 5,5'-(1-propen-1-yl-3-ylidene)bis[1,3-dimethyl-2-thio-barbituric acid]. GABAAR-expressing cells equilibrated with FMP-Red-Dye exhibited depolarized equilibrium membrane potentials compared with GABAAR-null cells. The channel blockers picrotoxin, fipronil, and tetramethylenedisulfotetramine, and the competitive antagonist bicuculline reduced fluorescence near the levels in GABAAR-null cells indicating that FMR-Red-Dye, a barbiturate derivative, activates GABAAR-mediated outward Cl- current in the absence of GABA. GABA caused concentration-dependent increases in fluorescence with rank order of potencies among GABAAR isoforms consistent with results from voltage-clamp experiments (EC50 values for α4β3δ, α1β3γ2, and β3 homopentamers were 6 ± 1, 40 ± 11, and >18 mM, respectively), whereas GABAAR-null cells were unresponsive. Neuroactive steroids (NAS) increased fluorescence of GABAAR expressing cells in the absence of GABA and demonstrated positive allosteric modulation in the presence of GABA, whereas benzodiazepines only exhibited positive allosteric modulator (PAM) activity. Of 20 NAS tested, allopregnanolone, (3α,5α,20E)-3-hydroxy-13,24-cyclo-18-norcholan-20-ene-21-carbonitrile, eltanolone, 5β-pregnan-3α,21-diol-20-one, and ganaxolone showed the highest potency. The FMP-Red-Dye-based assay described here provides a sensitive and quantitative method of assessing the activity of GABAAR agonists, antagonists, and PAMs on diverse GABAAR isoforms. The assay has a wide range of applications, including screening for antiseizure agents and identifying channel blockers of interest to insecticide discovery or biosecurity.

Homeostatic regulation through GABA and acetylcholine muscarinic receptors of motor trigeminal neurons following sleep deprivation

Muscle tone is regulated across sleep-wake states, being maximal in waking, reduced in slow wave sleep (SWS) and absent in paradoxical or REM sleep (PS or REMS). Such changes in tone have been recorded in the masseter muscles and shown to correspond to changes in activity and polarization of the trigeminal motor 5 (Mo5) neurons. The muscle hypotonia and atonia during sleep depend in part on GABA acting upon both GABAA and GABAB receptors (Rs) and acetylcholine (ACh) acting upon muscarinic 2 (AChM2) Rs. Here, we examined whether Mo5 neurons undergo homeostatic regulation through changes in these inhibitory receptors following prolonged activity with enforced waking. By immunofluorescence, we assessed that the proportion of Mo5 neurons positively stained for GABAARs was significantly higher after sleep deprivation (SD, ~65%) than sleep control (SC, ~32%) and that the luminance of the GABAAR fluorescence was significantly higher after SD than SC and sleep recovery (SR). Although, all Mo5 neurons were positively stained for GABABRs and AChM2Rs (100%) in all groups, the luminance of these receptors was significantly higher following SD as compared to SC and SR. We conclude that the density of GABAA, GABAB and AChM2 receptors increases on Mo5 neurons during SD. The increase in these receptors would be associated with increased inhibition in the presence of GABA and ACh and thus a homeostatic down-scaling in the excitability of the Mo5 neurons after prolonged waking and resulting increased susceptibility to muscle hypotonia or atonia along with sleep.

Putative Role of Taurine as Neurotransmitter During Perinatal Cortical Development.

Neurotransmitters and neuronal activity affect neurodevelopmental events like neurogenesis, neuronal migration, apoptosis and differentiation. Beside glutamate and gamma-amino butyric acid, the aminosulfonic acid taurine has been considered as possible neurotransmitter that influences early neuronal development. In this article I review recent studies of our group which demonstrate that taurine can affect a variety of identified neuronal populations in the immature neocortex and directly modulates neuronal activity. These experiments revealed that taurine evoke dose-dependent membrane responses in a variety of neocortical neuron populations, including Cajal-Retzius cells, subplate neurons and GABAergic interneurons. Taurine responses persist in the presence of GABA(A) receptor antagonists and are reduced by the addition of strychnine, suggesting that glycine receptors are involved in taurine-mediated membrane responses. Gramicidin-perforated patch-clamp and cell-attached recordings demonstrated that taurine evokes depolarizing and mainly excitatory membrane responses, in accordance with the high intracellular Cl- concentration in immature neurons. In addition, taurine increases the frequency of postsynaptic GABAergic currents (PSCs) in a considerable fraction of immature pyramidal neurons, indicating a specific activation of presynaptic GABAergic networks projecting toward and exciting pyramidal neurons. In summary, these results suggest that taurine may be critically involved in the regulation of network excitability in the immature neocortex and hippocampus via interactions with glycine receptors.

Pax2 is persistently expressed by GABAergic neurons throughout the adult rat dorsal horn

The transcription factor Pax2 is required for the differentiation of GABAergic neurons in the mouse dorsal horn. Pax2 continues to be expressed in the adult murine spinal cord and has been used as a presumed marker of GABAergic neurons in the superficial dorsal horn of the adult mouse, although a strict association between adult Pax2 expression and presence of GABA throughout the dorsal horn has not been firmly established. Moreover, whether Pax2 is selectively expressed in GABAergic dorsal horn neurons also in the rat is unknown. Here, immunofluorescent labeling of Pax2 and GABA in the lumbar spinal cord of adult rats was used to investigate this issue. Indeed, essentially all GABA immunoreactive neurons in laminae I–V were immunolabeled for Pax2. Conversely, essentially all Pax2 immunopositive neurons in these laminae exhibited somatic GABA immunolabeling. These results indicate persistent Pax2 expression in GABAergic neurons in the adult rat dorsal horn, supporting the hypothesis that Pax2 may be required for the maintenance of a GABAergic phenotype in mature inhibitory dorsal horn neurons in the rat. Furthermore, Pax2 may be used as a selective and specific general somatic marker of such neurons.