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Different subtypes of GABA A (gamma-aminobutyric acid A) receptors, through their specific regional and cellular localization, are involved in the manifestation of various functions, both at the central and peripheral levels. We hypothesized that various non-neuronal GABA A receptors are expressed on blood vessels, through which positive allosteric modulators of GABA A receptors exhibit vasodilatory effects. This study involved two parts: one to determine the presence of α1-6 subunit GABA A receptor mRNAs in the rat thoracic aorta, and the other to determine the vasoactivity of the various selective and non-selective positive GABA A receptor modulators: zolpidem (α1-selective), XHe-III-074 (α4-selective), MP-III-022 (α5-selective), DK-I-56-1 (α6-selective), SH-I-048A and diazepam (non-selective). Reverse transcription-polymerase chain reaction (RT-PCR) analysis data demonstrated for the first time the expression of α1, α2, α3, α4 and α5 subunits in the rat thoracic aorta tissue. Tissue bath assays on isolated rat aortic rings revealed significant vasodilatory effects of diazepam, SH-I-048A, XHe-III-074, MP-III-022 and DK-I-56-1, all in terms of achieved relaxations (over 50% of relative tension decrease), as well as in terms of preventive effects on phenylephrine (PE) contraction. Diazepam was the most efficient ligand in the present study, while zolpidem showed the weakest vascular effects. In addition, diazepam-induced relaxations in the presence of antagonists PK11195 or bicuculline were significantly reduced (P < 0.001 and P < 0.05, respectively) at lower concentrations of diazepam (10 -7  M and 3 × 10 -7  M). The present work suggests that the observed vasoactivity is due to modulation of "vascular" GABA A receptors, which after further detailed research may provide a therapeutic target., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Deoxycholic acid (DCA, 3 α , 12 α -dihydroxy-5 β -cholan-24-oic acid) is the major circulating secondary bile acid, which is synthesized by gut flora in the lower gut and selectively oxidized by CYP3A into tertiary metabolites, including 1 β ,3 α ,12 α -trihydroxy-5 β -cholan-24-oic acid (DCA-1 β -ol) and 3 α ,5 β ,12 α -trihydroxy-5 β -cholan-24-oic acid (DCA-5 β -ol) in humans. Since DCA has the similar exogenous nature and disposition mechanisms as xenobiotics, this work aimed to investigate whether the tertiary oxidations of DCA are predictive of in vivo CYP3A activities in beagle dogs. In vitro metabolism of midazolam (MDZ) and DCA in recombinant canine CYP1A1, 1A2, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26 enzymes clarified that CYP3A12 was primarily responsible for either the oxidation elimination of MDZ or the regioselective oxidation metabolism of DCA into DCA-1 β -ol and DCA-5 β -ol in dog liver microsomes. Six male dogs completed the CYP3A intervention studies including phases of baseline, inhibition (ketoconazole treatments), recovery, and induction (rifampicin treatments). The oral MDZ clearance after a single dose was determined on the last day of the baseline, inhibition, and induction phases, and subjected to correlation analysis with the tertiary oxidation ratios of DCA detected in serum and urine samples. The results confirmed that the predosing serum ratios of DCA oxidation, DCA-5 β -ol/DCA, and DCA-1 β -ol/DCA were significantly and positively correlated both intraindividually and interindividually with oral MDZ clearance. It was therefore concluded that the tertiary oxidation of DCA is predictive of CYP3A activity in beagle dogs. Clinical transitional studies following the preclinical evidence are promising to provide novel biomarkers of the enterohepatic CYP3A activities. SIGNIFICANCE STATEMENT: Drug development, clinical pharmacology, and therapeutics are under insistent demands of endogenous CYP3A biomarkers that avoid unnecessary drug exposure and invasive sampling. This work has provided the first proof-of-concept preclinical evidence that the CYP3A catalyzed tertiary oxidation of deoxycholate, the major circulating secondary bile acid synthesized in the lower gut by bacteria, may be developed as novel in vivo biomarkers of the enterohepatic CYP3A activities., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

CYP3A4 is among the most abundant liver and intestinal drug-metabolizing cytochrome P450 enzymes, contributing to the metabolism of more than 30% of clinically used drugs. Therefore, interindividual variability in CYP3A4 activity is a frequent cause of reduced drug efficacy and adverse effects. In this study, we characterized wild-type CYP3A4 and 40 CYP3A4 variants, including 11 new variants, detected among 4773 Japanese individuals by assessing CYP3A4 enzymatic activities for two representative substrates (midazolam and testosterone). The reduced carbon monoxide-difference spectra of wild-type CYP3A4 and 31 CYP3A4 variants produced with our established mammalian cell expression system were determined by measuring the increase in maximum absorption at 450 nm after carbon monoxide treatment. The kinetic parameters of midazolam and testosterone hydroxylation by wild-type CYP3A4 and 29 CYP3A4 variants ( K m , k cat , and catalytic efficiency) were determined, and the causes of their kinetic differences were evaluated by three-dimensional structural modeling. Our findings offer insight into the mechanism underlying interindividual differences in CYP3A4-dependent drug metabolism. Moreover, our results provide guidance for improving drug administration protocols by considering the information on CYP3A4 genetic polymorphisms. SIGNIFICANCE STATEMENT: CYP3A4 metabolizes more than 30% of clinically used drugs. Interindividual differences in drug efficacy and adverse-effect rates have been linked to ethnicity-specific differences in CYP3A4 gene variants in Asian populations, including Japanese individuals, indicating the presence of CYP3A4 polymorphisms resulting in the increased expression of loss-of-function variants. This study detected alterations in CYP3A4 activity due to amino acid substitutions by assessing the enzymatic activities of coding variants for two representative CYP3A4 substrates., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

The insula has emerged as a critical target for electrical stimulation since it influences pathological pain states. We investigated the effects of repetitive electrical stimulation of the insular cortex (ESI) on mechanical nociception, and general locomotor activity in rats subjected to chronic constriction injury (CCI) of the sciatic nerve. We also studied neuroplastic changes in central pain areas and the involvement of GABAergic signaling on ESI effects. CCI rats had electrodes implanted in the left agranular posterior insular cortex (pIC), and mechanical sensitivity was evaluated before and after one or five daily consecutive ESIs (15 min each, 60 Hz, 210 μs, 1 V). Five ESIs (repetitive ESI) induced sustained mechanical antinociception from the first to the last behavioral assessment without interfering with locomotor activity. A marked increase in Fos immunoreactivity in pIC and a decrease in the anterior and mid-cingulate cortex, periaqueductal gray and hippocampus were noticed after five ESIs. The intrathecal administration of the GABA A receptor antagonist bicuculline methiodide reversed the stimulation-induced antinociception after five ESIs. ESI increased GAD65 levels in pIC but did not interfere with GABA, glutamate or glycine levels. No changes in GFAP immunoreactivity were found in this work. Altogether, the results indicate the efficacy of repetitive ESI for the treatment of experimental neuropathic pain and suggest a potential influence of pIC in regulating pain pathways partially through modulating GABAergic signaling., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The activation of GABAA receptors by the neurotransmitter gamma-aminobutyric acid mediates the rapid inhibition response in the central nervous system of mammals. Many neurological and mental health disorders arise from alterations in the structure or function of these pentameric ion channels. GABAA receptors are targets for numerous drugs, including benzodiazepines, which bind to α1β2γ2 GABAA receptors with high affinity to a site in the extracellular domain, between subunits α1 and γ2. It has been established experimentally that the binding of these drugs depends on the presence of one particular amino acid in the α1 subunit: histidine 102. However, the specific role it plays in the intermolecular interaction has not been elucidated. In this work, we applied in silico methods to understand whether certain protonation and rotamer states of α1His102 facilitate the binding of modulators. We analysed diazepam binding, a benzodiazepine, and the antagonist flumazenil to the GABAA receptor using molecular dynamics simulations and adaptive biasing force simulations. The binding free energy follows changes in the protonation state for both ligands, and rotameric states of α1His102 were specific for the different compounds, suggesting distinct preferences for positive allosteric modulators and antagonists. Moreover, in the presence of diazepam and favoured by a neutral tautomer, we identified a water molecule that links loops A, B, and C and may be relevant to the modulation mechanism.

The family of GABA A receptors is an important drug target group in the treatment of sleep disorders, anxiety, epileptic seizures, and many others. The most frequent GABA A receptor subtype is composed of two α -, two β -, and one γ 2-subunit, whereas the nature of the α -subunit critically determines the properties of the benzodiazepine binding site of those receptors. Nearly all of the clinically relevant drugs target all GABA A receptor subtypes equally. In the past years, however, drug development research has focused on studying α 5-containing GABA A receptors. Beyond the central nervous system, α 5-containing GABA A receptors in airway smooth muscles are considered as an emerging target for bronchial asthma. Here, we investigated a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3 (SH53d-ester). Although SH53d-ester is only moderately selective for α 5-subunit-containing GABA A receptors, the derivative SH53d-acid shows superior (>40-fold) affinity selectivity and is a positive modulator. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes and radioligand displacement assays with human embryonic kidney 293 cells, we demonstrated that an acid group as substituent on the imidazobenzodiazepine scaffold leads to large improvements of functional and binding selectivity for α 5 β 3 γ 2 over other α x β 3 γ 2 GABA A receptors. Atom level structural studies provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABA A receptor α -subunit is the dominant molecular determinant of drug selectivity. Thus, we characterize a promising novel α 5-subunit-selective drug candidate. SIGNIFICANCE STATEMENT: In the current study we present the detailed pharmacological characterization of a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3. We describe its superior (>40-fold) affinity selectivity for α 5-containing GABA A receptors and show atom-level structure predictions to provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABA A receptor α -subunit is the dominant molecular determinant of drug selectivity., (Copyright © 2020 by The Author(s).)

Postmortem studies suggest that schizophrenia is associated with abnormal expression of specific GABA A receptor (GABA A R) α subunits, including α5GABA A R. Positron emission tomography (PET) measures of GABA A R availability in schizophrenia, however, have not revealed consistent alterations in vivo. Animal studies using the GABA A R agonist [ 3 H]-muscimol provide evidence that antipsychotic drugs influence GABA A R availability, in a region-specific manner, suggesting a potential confounding effect of these drugs. No such data, however, are available for more recently developed subunit-selective GABA A R radioligands. To address this, we combined a rat model of clinically relevant antipsychotic drug exposure with quantitative receptor autoradiography. Haloperidol (0.5 and 2 mg/kg/day) or drug vehicle were administered continuously to adult male Sprague-Dawley rats via osmotic mini-pumps for 28 days. Quantitative receptor autoradiography was then performed postmortem using the GABA A R subunit-selective radioligand [ 3 H]-Ro15-4513 and the non-subunit selective radioligand [ 3 H]-flumazenil. Chronic haloperidol exposure increased [ 3 H]-Ro15-4513 binding in the CA1 sub-field of the rat dorsal hippocampus (p<0.01; q<0.01; d=+1.3), which was not dose-dependent. [ 3 H]-flumazenil binding also increased in most rat brain regions (p<0.05; main effect of treatment), irrespective of the haloperidol dose. These data confirm previous findings that chronic haloperidol exposure influences the specific binding of non-subtype selective GABA A R radioligands and is the first to demonstrate a potential effect of haloperidol on the binding of a α1/5GABA A R-selective radioligand. Although caution should be exerted when extrapolating results from animals to patients, our data support a view that exposure to antipsychotics may be a confounding factor in PET studies of GABA A R in the context of schizophrenia., Competing Interests: Conflict of interest ACV discloses receipt of research funding from F. Hoffman La Roche Ltd and UBC Biopharma SPRL as part of a research program on early life immune activation. The views expressed are those of the author and not necessarily those of F. Hoffman La Roche or UCB Biopharma SPRL. All other authors declare that they have no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Positive allosteric modulators (PAMs) of GABA B receptor represent an interesting alternative to receptor agonists such as baclofen, as they act on the receptor in a more physiological way and thus are devoid of the side effects typically exerted by the agonists. Based on our interest in the identification of new GABA B receptor PAMs, we followed a merging approach to design new chemotypes starting from selected active compounds, such as GS39783, rac-BHFF, and BHF177, and we ended up with the synthesis of four different classes of compounds. The new compounds were tested alone or in the presence of 10 µM GABA using [ 35 S]GTPγS binding assay to assess their functionality at the receptor. Unexpectedly, a number of them significantly inhibited GABA-stimulated GTPγS binding thus revealing a functional switch with respect to the prototype molecules. Further studies on selected compounds will clarify if they act as negative modulators of the receptor or, instead, as antagonists at the orthosteric binding site., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Hops (Humulus lupulus L.), a major component of beer, contain potentially neuroactive compounds that made it useful in traditional medicine as a sleeping aid. The present study aims to investigate the individual components in hops acting as allosteric modulators in GABA A receptors and bring further insight into the mode of action behind the sedative properties of hops. GABA-potentiating effects were measured using [ 3 H]ethynylbicycloorthobenzoate (EBOB) radioligand binding assay in native GABA A receptors. Flumazenil sensitivity of GABA-potentiating effects, [ 3 H]Ro 15-4513, and [ 3 H]flunitrazepam binding assays were used to examine the binding to the classical benzodiazepines site. Humulone (alpha acid) and 6-prenylnaringenin (prenylflavonoid) were the most potent compounds displaying a modulatory activity at low micromolar concentrations. Humulone and 6-prenylnaringenin potentiated GABA-induced displacement of [ 3 H]EBOB binding in a concentration-dependent manner where the IC 50 values for this potentiation in native GABA A receptors were 3.2 μM and 3.7 μM, respectively. Flumazenil had no significant effects on humulone- or 6-prenylnaringenin-induced displacement of [ 3 H]EBOB binding. [ 3 H]Ro 15-4513 and [ 3 H]flunitrazepam displacements were only minor with humulone but surprisingly prominent with 6-prenylnaringenin despite its flumazenil-insensitive modulatory activity. Thus, we applied molecular docking methods to investigate putative binding sites and poses of 6-prenylnaringenin at the GABA A receptor α1β2γ2 isoform. Radioligand binding and docking results suggest a dual mode of action by 6-prenylnaringenin on GABA A receptors where it may act as a positive allosteric modulator at α+β- binding interface as well as a null modulator at the flumazenil-sensitive α+γ2- binding interface., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Human CYP3A4 (Cyp3a11 in mice) is one of the most important enzymes for drug metabolism and detoxification. Here, we aimed to investigate a potential role for E4bp4 in regulation of Cyp3a11 expression and activity. The regulatory effects of E4bp4 on Cyp3a11 enzyme were assessed using E4bp4 -/- mice and Hepa-1c1c7 cells. The mRNA and protein levels were quantified using qPCR and Western blotting, respectively. In vitro microsomal Cyp3a11 activity was probed using its specific substrates midazolam and testosterone. Pharmacokinetic studies were performed with wild-type and E4bp4 -/- mice after midazolam administration. Global deletion of E4bp4 led to significant upregulation of Cyp3a11 mRNA and protein in major metabolic organs (i.e., the liver, kidney and small intestine). E4bp4 ablation also caused an increased microsomal Cyp3a11 activity consistent with the enzyme's expression change. Overexpression of E4bp4 in Hepa-1c1c7 cells resulted in reduced levels of Cyp3a11 mRNA and protein, whereas E4bp4 knockdown caused upregulation of Cyp3a11 expression. In addition, the systemic exposure of midazolam was lowered in E4bp4 -/- mice compared with wild-type mice. This was accompanied by enhanced formation of its metabolite 1'-hydroxymidazolam. Furthermore, luciferase reporter and mobility shift assays revealed that E4bp4 repressed Cyp3a11 transcription via direct binding to C-site (-1539/-1529 bp) in the promoter region. In conclusion, E4bp4 negatively regulates Cyp3a11 expression, thereby impacting drug metabolism and pharmacokinetics., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A recent study found that COR659 (methyl 2-[(4-chlorophenyl)carboxamido]-4-ethyl-5-methylthiophene-3-carboxylate) reduced operant alcohol and chocolate self-administration in rats; COR659 also suppressed cue-induced reinstatement of chocolate seeking in rats. COR659 apparently exerts its effects via a composite mechanism, including positive allosteric modulation of the GABA B receptor and an action at the cannabinoid CB 1 receptor. The present study investigated whether the reducing effect of COR659 on alcohol and chocolate self-administration was maintained after repeated treatment and if COR659 affected cue-induced reinstatement of alcohol seeking; additionally, it evaluated the ability of 9 structural analogues of COR659 - designed modifying the substituents on the phenylcarboxamido moiety and replacing the thiophene with the pyridine ring - to affect alcohol and chocolate self-administration. Alcohol self-administration experiments employed Sardinian alcohol-preferring (sP) rats trained to lever-respond for alcohol (15% v/v). Chocolate self-administration experiments employed Wistar rats trained to lever-respond for a chocolate solution (5% w/v Nesquik ® ). In the reinstatement experiment, previously extinguished lever-responding for alcohol in sP rats was reinstated by the non-contingent presentation of an alcohol-associated complex of cues. All drugs were tested at the doses of 0, 2.5, 5, and 10 mg/kg (i.p.). 10-Day treatment with COR659 produced a dose-related reduction of both alcohol and chocolate self-administration, with limited loss of efficacy on continuing treatment. Acute COR659 suppressed reinstatement of alcohol seeking. Among the 9 tested analogues, only COR657 (methyl 2-(benzoylamino)-4-ethyl-5-methylthiophene-3-carboxylate) decreased alcohol self-administration similarly to COR659; all other compounds produced modest, or even no, effect on alcohol self-administration. COR659 excluded, no compound altered chocolate self-administration. These results confirm and extend the ability of COR659 to reduce several behaviors motivated by alcohol and palatable food in rats. Comparison of COR659 to its analogues provided disparate results that do not currently allow any conclusive structure-activity relationship to be hypothesized, as their diverse pharmacological profile apparently does not depend on physicochemical properties., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The extracellular α (+) /γ 2 (-) interface in the α 1,2,3,5 βγ 2 GABA A receptor harbours the allosteric binding site targeted by benzodiazepines and newer generations of subtype-selective modulators. We have probed the molecular determinants for the affinity/potency-based α 1 -preference exhibited by the hypnotic zolpidem (Ambien®, Stilnox®) and the efficacy-based α 3 -over-α 1 selectivity displayed by the analgesic NS11394. Binding affinities and functional properties of the modulators were characterized at wild-type, concatenated, mutant and chimeric α 1,3 β 2 γ 2S receptors expressed in tsA201 cells and Xenopus oocytes by [ 3 H]flumazenil binding and two-electrode voltage clamp electrophysiology. Substitution of Gly 201 in α 1 with the corresponding Glu in α 3 completely eliminated the α 1 -over-α 3 preference exhibited by zolpidem. In contrast, the reverse α 3 -E225G mutation did not yield corresponding increases in the binding affinity or modulatory potency of zolpidem at α 3 β 2 γ 2S , and two additional molecular elements in the extracellular domain of the α-subunit were found also to contribute to its α 1 -preference. Interestingly, the α 1 -Gly 201 /α 3 -Glu 225 residue was also a key determinant of the efficacy-based α 3 -over-α 1 selectivity exhibited by NS11394, and a pronounced correlation existed between the side-chain bulkiness of this residue and the modulatory efficacy of NS11394 at the receptor. The subtype-selectivity determinants identified for zolpidem and NS11394 were found also to apply in different degrees to the α 1 -preferring modulator indiplon and the α 3 -over-α 1 selective modulator L-838,417, respectively. In conclusion, the molecular origins of subtype-selectivity exhibited by benzodiazepine-site modulators at the α 1,2,3,5 βγ 2 GABA A receptor seem more complex than previously appreciated, and the importance of the α 1 -Gly 201 /α 3 -Glu 225 residue for both potency- and efficacy-based subtype-selective modulation through this site is likely to be rooted in different molecular mechanisms., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Odor-guided behaviors, including homing, predator avoidance, or food and mate searching, are ubiquitous in animals. It is only recently that the neural substrate underlying olfactomotor behaviors in vertebrates was uncovered in lampreys. It consists of a neural pathway extending from the medial part of the olfactory bulb (medOB) to locomotor control centers in the brainstem via a single relay in the caudal diencephalon. This hardwired olfactomotor pathway is present throughout life and may be responsible for the olfactory-induced motor behaviors seen at all life stages. We investigated modulatory mechanisms acting on this pathway by conducting anatomical (tract tracing and immunohistochemistry) and physiological (intracellular recordings and calcium imaging) experiments on lamprey brain preparations. We show that the GABAergic circuitry of the olfactory bulb (OB) acts as a gatekeeper of this hardwired sensorimotor pathway. We also demonstrate the presence of a novel olfactomotor pathway that originates in the non-medOB and consists of a projection to the lateral pallium (LPal) that, in turn, projects to the caudal diencephalon and to the mesencephalic locomotor region (MLR). Our results indicate that olfactory inputs can induce behavioral responses by activating brain locomotor centers via two distinct pathways that are strongly modulated by GABA in the OB. The existence of segregated olfactory subsystems in lampreys suggests that the organization of the olfactory system in functional clusters may be a common ancestral trait of vertebrates., Competing Interests: The authors have declared that no competing interests exist.

A sensitive, accurate and rapid UHPLC-MS/MS method was developed and validated for the simultaneous determination of EVT201 and its two metabolites, Ro46-1927 and Ro18-5528, in human plasma. D 6 -EVT201 was used as the internal standard (IS). Plasma samples were extracted using ethyl acetate after being alkalized with saturated sodium carbonate solution. Chromatographic separation was carried out on an Acquity BEH C18 column (2.1 × 50 mm, 1.7 μm) with a gradient mobile phase at a flow-rate of 0.50 mL/min. The analytical run time was 5.5 min. For mass spectrometric detection, multiple reaction monitoring (MRM) was used. The MRM ion transitions were m/z 373.2 → 58.0 for EVT201, m/z 359.2 → 316.1 for Ro46-1927, m/z 291.2 → 274.1 for Ro18-5528 and m/z 379.3 → 64.0 for the IS. The linear range was 0.2-200 ng/mL for each analyte, with a correlation coefficient (r) over 0.9900. The intra-/inter- precision was within 7.9% and 4.5% for EVT201, 13.2% and 6.3% for Ro46-1927, 3.7% and 4.1% for Ro18-5528. For the accuracy, the relative bias of intra-/inter- run was no more than 6.4% and 4.6% for EVT201, 9.4% and 7.9% for Ro46-1927, -6.9% and -7.5% for Ro18-5528. The validated method was successfully applied to the analysis of more than 1500 samples from a Phase I clinical trial. The incurred sample reanalysis (ISR) of 146 samples from the study was evaluated and the results met the acceptance criteria. It indicated that the method could be used for the pharmacokinetic study of EVT201., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Benzodiazepines facilitate the inhibitory actions of GABA by binding to γ-aminobutyric acid type A receptors (GABA A Rs), GABA-gated chloride/bicarbonate channels, which are the key mediators of transmission at inhibitory synapses in the brain. This activity underpins potent anxiolytic, anticonvulsant and hypnotic effects of benzodiazepines in patients. However, extended benzodiazepine treatments lead to development of tolerance, a process which, despite its important therapeutic implications, remains poorly characterised. Here we report that prolonged exposure to diazepam, the most widely used benzodiazepine in clinic, leads to a gradual disruption of neuronal inhibitory GABAergic synapses. The loss of synapses and the preceding, time- and dose-dependent decrease in surface levels of GABA A Rs, mediated by dynamin-dependent internalisation, were blocked by Ro 15-1788, a competitive benzodiazepine antagonist, and bicuculline, a competitive GABA antagonist, indicating that prolonged enhancement of GABA A R activity by diazepam is integral to the underlying molecular mechanism. Characterisation of this mechanism has revealed a metabotropic-type signalling downstream of GABA A Rs, involving mobilisation of Ca 2+ from the intracellular stores and activation of the Ca 2+ /calmodulin-dependent phosphatase calcineurin, which, in turn, dephosphorylates GABA A Rs and promotes their endocytosis, leading to disassembly of inhibitory synapses. Furthermore, functional coupling between GABA A Rs and Ca 2+ stores was sensitive to phospholipase C (PLC) inhibition by U73122, and regulated by PLCδ, a PLC isoform found in direct association with GABA A Rs. Thus, a PLCδ/Ca 2+ /calcineurin signalling cascade converts the initial enhancement of GABA A Rs by benzodiazepines to a long-term downregulation of GABAergic synapses, this potentially underpinning the development of pharmacological and behavioural tolerance to these widely prescribed drugs.

Fast inhibitory neurotransmission in the brain is principally mediated by the neurotransmitter GABA (γ-aminobutyric acid) and its synaptic target, the type A GABA receptor (GABA A receptor). Dysfunction of this receptor results in neurological disorders and mental illnesses including epilepsy, anxiety and insomnia. The GABA A receptor is also a prolific target for therapeutic, illicit and recreational drugs, including benzodiazepines, barbiturates, anaesthetics and ethanol. Here we present high-resolution cryo-electron microscopy structures of the human α1β2γ2 GABA A receptor, the predominant isoform in the adult brain, in complex with GABA and the benzodiazepine site antagonist flumazenil, the first-line clinical treatment for benzodiazepine overdose. The receptor architecture reveals unique heteromeric interactions for this important class of inhibitory neurotransmitter receptor. This work provides a template for understanding receptor modulation by GABA and benzodiazepines, and will assist rational approaches to therapeutic targeting of this receptor for neurological disorders and mental illness.

The PCDH19 gene (Xp22.1) encodes the cell-adhesion protein protocadherin-19 (PCDH19) and is responsible for a neurodevelopmental pathology characterized by female-limited epilepsy, cognitive impairment and autistic features, the pathogenic mechanisms of which remain to be elucidated. Here, we identified a new interaction between PCDH19 and GABAA receptor (GABAAR) alpha subunits in the rat brain. PCDH19 shRNA-mediated downregulation reduces GABAAR surface expression and affects the frequency and kinetics of miniature inhibitory postsynaptic currents (mIPSCs) in cultured hippocampal neurons. In vivo, PCDH19 downregulation impairs migration, orientation and dendritic arborization of CA1 hippocampal neurons and increases rat seizure susceptibility. In sum, these data indicate a role for PCDH19 in GABAergic transmission as well as migration and morphological maturation of neurons.

Oysters, the common name for a number of different bivalve molluscs, are the worldwide aquaculture species and also play vital roles in the function of ecosystem. As invertebrate, oysters have evolved an integrated, highly complex innate immune system to recognize and eliminate various invaders via an array of orchestrated immune reactions, such as immune recognition, signal transduction, synthesis of antimicrobial peptides, as well as encapsulation and phagocytosis of the circulating haemocytes. The hematopoietic tissue, hematopoiesis, and the circulating haemocytes have been preliminary characterized, and the detailed annotation of the Pacific oyster Crassostrea gigas genome has revealed massive expansion and functional divergence of innate immune genes in this animal. Moreover, immune priming and maternal immune transfer are reported in oysters, suggesting the adaptability of invertebrate immunity. Apoptosis and autophagy are proved to be important immune mechanisms in oysters. This review will summarize the research progresses of immune system and the immunomodulation mechanisms of the primitive catecholaminergic, cholinergic, neuropeptides, GABAergic and nitric oxidase system, which possibly make oysters ideal model for studying the origin and evolution of immune system and the neuroendocrine-immune regulatory network in lower invertebrates., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Isoflurane and sevoflurane are widely used anesthetics in surgery and administration of these anesthetics could lead to postoperative cognitive dysfunction (POCD). However, the mechanisms remain unclear., Methods: Aged Wistar rats were exposed to isoflurane and sevoflurane for 2 or 4h. Recognition memory and spatial working memory were measured using Novel object recognition (NOR) and Y-maze test, respectively. Apoptotic cells were detected by TUNEL staining. miRNA expression was measured by Real-time PCR while protein expression was measured by Western blot. Dual-Luciferase reporter assay was used to establish the direct relationship between miRNAs and Gabra5 and gephyrin gene expression., Results: Exposure to isoflurane and sevoflurane for 2 or 4h significantly decreased the NOR index in the NOR test and spontaneous alternations in arm entries in the Y-maze test in aged rats. TUNEL staining showed that isoflurane and sevoflurane administration significantly induced apoptosis in the mPFC and hippocampus. The protein level of α5 GABA A receptor (α5GABA A R), gephyrin, and dystrophin were significantly increased, whereas the expression of miR-30a, miR-31, miR-190a, and miR-190b was significantly decreased in the hippocampus and mPFC in aged rats exposed to isoflurane and sevoflurane compared to control rats. The protein levels of α5GABA A R, gephyrin, and dystrophin protein in the hippocampus and the mPFC significantly correlated with NOR index and spontaneous alternations. Dual-Luciferase reporter assay showed that miR-30a and miR-190a/b mimics significantly inhibited Gabra5 and gephyrin gene expression, respectively., Conclusion: There might be a miRNAs-GABAergic transmission pathway which may be involved in the pathophysiological alteration in anesthetics-induced POCD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 GABA A receptor (GABA A R) isoforms (synaptic α 1 β 3 γ 2, extrasynaptic α 4 β 3 δ , and β 3 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]. GABA A R-expressing cells equilibrated with FMP-Red-Dye exhibited depolarized equilibrium membrane potentials compared with GABA A R-null cells. The channel blockers picrotoxin, fipronil, and tetramethylenedisulfotetramine, and the competitive antagonist bicuculline reduced fluorescence near the levels in GABA A R-null cells indicating that FMR-Red-Dye, a barbiturate derivative, activates GABA A R-mediated outward Cl - current in the absence of GABA. GABA caused concentration-dependent increases in fluorescence with rank order of potencies among GABA A R isoforms consistent with results from voltage-clamp experiments (EC 50 values for α 4 β 3 δ , α 1 β 3 γ 2, and β 3 homopentamers were 6 ± 1, 40 ± 11, and >18 mM, respectively), whereas GABA A R-null cells were unresponsive. Neuroactive steroids (NAS) increased fluorescence of GABA A R 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, (3 α ,5 α ,20E)-3-hydroxy-13,24-cyclo-18-norcholan-20-ene-21-carbonitrile, eltanolone, 5 β -pregnan-3 α ,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 GABA A R agonists, antagonists, and PAMs on diverse GABA A R 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., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

278: The Cytochrome P450 (CYP) system is involved in 90% of the human body's interactions with xenobiotics and due to this, it has become an area of avid research including the creation of transgenic mice. This paper proposes a three-compartment model which is used to explain the drug metabolism in the Hepatic Reductase Null (HRN) mouse developed by the University of Dundee (Henderson, C. J., Otto, D. M. E., Carrie, D., Magnuson, M. A., McLaren, A. W., Rosewell, I. and Wolf, C. R. (2003) Inactivation of the hepatic cytochrome p450 system by conditional deletion of hepatic cytochrome p450 reductase. J. Biol. Chem. , 13480-13486). The model is compared with a two-compartment model using experimental data from studies using wild-type and HRN mice. This comparison allowed for metabolic differences between the two types of mice to be isolated. The three sets of drug data (Gefitinib, Midazolam and Thalidomide) showed that the transgenic mouse has a decreased rate of metabolism., (© The authors 2015. Published by Oxford University Press on behalf of the Institute of Mathematics and its Applications. All rights reserved.)

The brain's control of movement is thought to involve coordinated activity between cerebellar Purkinje cells. The results reported here demonstrate that somatic Ca 2+ imaging is a faithful reporter of Na + -dependent "simple spike" pauses and enables us to optically record changes in firing rates in populations of Purkinje cells in brain slices and in vivo. This simultaneous calcium imaging of populations of Purkinje cells reveals a striking spatial organization of pauses in Purkinje cell activity between neighboring cells. The source of this organization is shown to be the presynaptic gamma-Aminobutyric acid producing (GABAergic) network, and blocking ionotropic gamma-Aminobutyric acid receptor (GABA A Rs) abolishes the synchrony. These data suggest that presynaptic interneurons synchronize (in)activity between neighboring Purkinje cells, and thereby maximize their effect on downstream targets in the deep cerebellar nuclei., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Repetitive traumatic brain injury (rTBI) occurs as a result of mild and accumulative brain damage. A prototype of rTBI is chronic traumatic encephalopathy (CTE), which is a degenerative disease that occurs in patients with histories of multiple concussions or head injuries. Boxers have been the most commonly studied patient group because they may experience thousands of subconcussive hits over the course of a career. This study examined the consequences of rTBI with structural brain imaging and biomolecular imaging and investigated whether the neuropsychological features of rTBI were related to the findings of the imaging studies. Five retired professional boxers (mean age, 46.8 ± 3.19 years) and four age-matched controls (mean age, 48.5 ± 3.32 years) were studied. Cognitive-motor related functional impairment was assessed, and all subjects underwent neuropsychological evaluation and behavioral tasks, as well as structural brain imaging and functional-molecular imaging. In neuropsychological tests, boxers showed deficits in delayed retrieval of visuospatial memory and motor coordination, which had a meaningful relationship with biomolecular imaging results indicative of neuronal injury. Morphometric abnormalities were not found in professional boxers by structural magnetic resonance imaging (MRI). Glucose metabolism was impaired in frontal areas associated with cognitive dysfunction, similar to findings in Alzheimer's disease. Low binding potential (BP) of (18)F-flumazenil (FMZ) was found in the angular gyrus and temporal cortical regions, revealing neuronal deficits. These results suggested that cognitive impairment and motor dysfunction reflect chronic damage to neurons in professional boxers with rTBI.

Hepatocellular carcinoma (HCC) accompanied by severe liver dysfunction is a serious disease, which results in altered hepatic clearance. Generally, maintenance doses depend upon drug clearance, so individual dosage regimens should be customized for HCC patients based on the condition of patients. Based on clearance of CYP isoform-specific substrates at the microsomal level (CLM), microsomal protein per gram of liver (MPPGL), liver weight, hepatic blood flow, hepatic clearance values (CLH) for 10 CYPs in HCC patients (n=102) were extrapolated using a predictive bottom-up pharmacokinetic model. Compared with controls, the CLM values for CYP2C9, 2D6, 2E1 were significantly increased in HCC patients. Additionally, CYP1A2, 2C8, 2C19 CLM values decreased while the values for CYP2A6, 2B6, 3A4/5 were unchanged. The MPPGL values in HCC tissues were significantly reduced. CLH values of HCC patients for CYP1A2, 2A6, 2B6, 2C8, 2C19, and 3A4/5 were significantly reduced, while this for CYP2E1 were markedly increased and those for CYP2C9 and 2D6 did not change. Moreover, disease (fibrosis and cirrhosis) and polymorphisms of the CYP genes have influenced the CLH for some CYPs. Prediction of the effects of HCC on drug clearance may be helpful for the design of clinical studies and the clinical management of drugs in HCC patients., Competing Interests: The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Recently, the detection of pharmaceuticals in surface waters has increased worldwide. Pharmaceuticals are typically found in the environment at concentrations well below therapeutic levels in humans; however, their mechanisms of action may be largely unknown in non-target organisms, such as teleost species. Thus, chronic exposure to these types of compounds warrants further investigation. The goal of this study was to examine the potential for diazepam, a model benzodiazepine drug, to bioconcentrate in tissues of channel catfish and to examine its ability to interact with the endocrine system through modulation of steroid hormones and/or steroidogenic genes. To investigate the bioconcentration potential of diazepam, channel catfish (Ictalurus punctatus) were exposed to 1 ng/mL diazepam for seven days, followed by clean water for another seven days, using an abbreviated OECD 305 Fish Bioconcentration Test study design. This concentration of diazepam is well below environmentally relevant concentrations of diazepam (ng/L). To evaluate steroidogenic effects, fish were exposed to 1 ng/mL diazepam for seven days only. Steroid hormone concentrations were analyzed for various tissues, as well as expression of selected steroidogenic genes. Calculated bioconcentration factors for diazepam were well below regulatory threshold values in all tissues analyzed. No changes in steroid hormone concentration were detected in any tissue analyzed; however, the steroidogenic gene cytochrome P450 side chain cleavage (P450scc) was significantly down-regulated at day 5 and 3β-hydroxy steroid dehydrogenase (3β-HSD) was significantly down-regulated at day 7 in the gonad. These results indicate that although diazepam does not significantly bioconcentrate, low-level chronic exposure to diazepam may have the potential to interact with endocrine function by altering gene expression., (Copyright © 2016. Published by Elsevier Inc.)

In certain women, increased negative mood relates to the progesterone metabolite, allopregnanolone (allo), during the luteal phase of ovulatory menstrual cycles, the premenstrual dysphoric disorder (PMDD). In anovulatory cycles, no symptom or sex steroid increase occurs but symptoms return during progesterone/allo treatment. Allo is a potent GABAA receptor-modulating steroid and as such is expected to be calming and anxiolytic. A relation to negative mood is unexpected. However, this paradoxical effect can be induced by all GABAA receptor modulators in low concentrations whereas higher concentrations are calming. The severity of the mood symptoms relate to allo in an inverted U-shaped curve at endogenous luteal-phase serum concentrations. Allo's effects on the GABAA receptor can be antagonized by isoallopregnanolone (ISO), an antagonist to allo. ISO has also been used in a preliminary clinical trial on PMDD ameliorating symptoms with good effect in PMDD patients.

Neuregulin1 (NRG1) is a single transmembrane protein that plays a critical role in neural development and synaptic plasticity. Both NRG1 and its receptor, ErbB4, are well-established risk genes of schizophrenia. The NRG1 ecto-domain (ED) binds and activates ErbB4 following proteolytic cleavage of pro-NRG1 precursor protein. Although several studies have addressed the function of NRG1 in brain, very little is known about the cleavage and shedding mechanism. Here we show that the neuronal vesicular protein calcyon is a potent activator and key determinant of NRG1 ED cleavage and shedding. Calcyon stimulates clathrin-mediated endocytosis and endosomal targeting; and its levels are elevated in postmortem brains of schizophrenics. Overexpression of calcyon stimulates NRG1 cleavage and signaling in vivo, and as a result, GABA transmission is enhanced in calcyon overexpressing mice. Conversely, NRG1 cleavage, ErbB4 activity and GABA transmission are decreased in calcyon null mice. Moreover, stimulation of NRG1 cleavage by calcyon was recapitulated in HEK 293 cells suggesting the mechanism involved is cell-autonomous. Finally, studies with site-specific mutants in calcyon and inhibitors for the major sheddases indicate that the stimulatory effects of calcyon on NRG1 cleavage and shedding depend on clathrin-mediated endocytosis, β-secretase 1, and interaction with clathrin adaptor proteins. Together these results identify a novel mechanism for NRG1 cleavage and shedding.

Flavonoids that are found in nutraceuticals have many and varied effects on the activation of ionotropic receptors for GABA, the major inhibitory neurotransmitter in our brains. They can act as positive or negative modulators enhancing or reducing the effect of GABA. They can act as allosteric agonists. They can act to modulate the action of other modulators. There is considerable evidence that these flavonoids are able to enter the brain to influence brain function. They may have a range of effects including relief of anxiety, improvement in cognition, acting as neuroprotectants and as sedatives. All of these effects are sought after in nutraceuticals. A number of studies have likened flavonoids to the widely prescribed benzodiazepines as 'a new family of benzodiazepine receptor ligands'. They are much more than that with many flavonoid actions on ionotropic GABA receptors being insensitive to the classic benzodiazepine antagonist flumazenil and thus independent of the classic benzodiazepine actions. It is time to consider flavonoids in their own right as important modulators of these vital receptors in brain function. Flavonoids are rarely consumed as a single flavonoid except as dietary supplements. The effects of mixtures of flavonoids and other modulators on GABAA receptors need to be more thoroughly investigated., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Intellectual disability, autism spectrum disorder, and epilepsy are prime examples of neurodevelopmental disorders that collectively affect a significant percentage of the world population. Recent technological breakthroughs allowed the elucidation of the genetic causes of many of these disorders. As neurodevelopmental disorders are genetically heterogeneous, the development of rational therapy is extremely challenging. Fortunately, many causative genes are interconnected and cluster in specific cellular pathways. Targeting a common node in such a network would allow us to interfere with a series of related neurodevelopmental disorders at once. Here, we argue that the GABAergic system is disturbed in many neurodevelopmental disorders, including fragile X syndrome, Rett syndrome, and Dravet syndrome, and is a key candidate target for therapeutic intervention. Many drugs that modulate the GABAergic system have already been tested in animal models with encouraging outcomes and are readily available for clinical trials., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: AZD6280 is a novel γ-aminobutyric acid A receptor modulator with higher in vitro efficacy at the α2,3 subtypes as compared to the α1 and α5 subtypes. This study compared the pharmacodynamic effects of single oral dose AZD6280 10 mg and 40 mg on the central nervous system with 2 mg of lorazepam., Methods: Sixteen healthy males were enrolled into the double-blind, randomized, 4-way crossover study. Two validated central nervous system test batteries, Neurocart and CogState, were administered to measure drug effects on cognition, neurophysiologic function, and psychomotor and subjective feelings. Statistical analysis was performed using mixed model analysis of variance, with fixed factors of treatment, period, time and treatment by time, and random factors of subject, subject by treatment and subject by time, and the average prevalue as covariate., Results: Most pharmacodynamic parameters were affected by lorazepam. AZD6280 induced dose-dependent smaller-than-lorazepam effects on saccadic peak velocity (SPV) (AZD6280, 10 mg vs. AZD6280, 40 mg vs. lorazepam [deg/s]: -22.6 vs. -50.0 vs. -62.9, P < 0.001), whereas the impacts on adaptive-tracking, body-sway, smooth-pursuit, and the one-card-learning tests were significant but much smaller than lorazepam. Thus, the slopes of regression lines for the ΔLog(Sway)-ΔSPV, ΔTracking-ΔSPV, and ΔSmooth-ΔSPV relations were flatter with AZD6280 than with lorazepam. AZD6280 caused a distinct electroencephalography signature from that of lorazepam., Conclusions: The SPV responses to AZD6280 suggest potential concentration-related anxiolytic effects, whereas the smaller SPV-normalized effects of AZD6280 on various non-SPV pharmacodynamic parameters suggest a more favorable side effect profile compared to lorazepam. Overall, the pharmacodynamic profile of AZD6280 matches the pharmacological specificity and selectivity of this compound at the α2,3 γ-aminobutyric acid A receptor subtypes.

We present the synthesis of new derivatives of natural products magnolol (1) and honokiol (2) and their evaluation as allosteric ligands for modulation of GABAA receptor activity. New derivatives were prepared via metal assisted cross-coupling reactions in two consecutive steps. Compounds were tested by means of two-electrode voltage clamp electrophysiology at the α1β2γ2 receptor subtype at low GABA concentrations. We have identified several compounds enhancing GABA induced current (IGABA) in the range similar or even higher than the lead structures. At 3μM, compound 8g enhanced IGABA by factor of 443, compared to 162 and 338 of honokiol and magnolol, respectively. Furthermore, 8g at EC10-20 features a much bigger window of separation between the α1β2γ2 and the α1β1γ2 subtypes compared to honokiol, and thus improved subtype selectivity., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: γ-Aminobutyric acid (GABA) plasticity plays an important role in stroke motor recovery in a mouse model. However, little is known about changes over time in neuronal excitability mediated by GABA receptors in human stroke patients., Objectives: To establish the mechanism of neuroplasticity during the recovery phase following ischemic stroke by assessing the changes in cerebral GABA activity using [(18)F]flumazenil ([(18)F]FMZ) positron emission tomography (PET)., Methods: A total of 10 patients with unilateral ischemic stroke were studied at 1 month (T0) and 3 months (T1) postonset using [(18)F]FMZ PET. Changes in regional GABAergic activity were assessed longitudinally, and values were also compared with those in 15 age-matched controls. Upper-extremity motor function was evaluated using the Fugl-Meyer score (FMS)., Results: During the follow-up period, statistical parametric mapping analysis demonstrated a decrease in GABAA receptor availability throughout the cerebral cortex and cerebellum, especially the contralateral hemisphere. GABAA availability in the bilateral primary motor cortex, contralateral supplemental motor cortex, and globus pallidus at T0 was positively correlated with the FMS score at T1 CONCLUSIONS: This is the first prospective, controlled longitudinal study showing that the change in GABA receptor availability over time is significantly related to motor recovery after stroke in humans. This work supports the rationale for a novel strategy to promote motor recovery after stroke., (© The Author(s) 2014.)

A series of cyclopropane-based conformationally restricted γ-aminobutyric acid (GABA) analogs with stereochemical diversity, that is, the trans- and cis-2,3-methano analogs Ia and Ib and their enantiomers ent-Ia and ent-Ib, and also the trans- and cis-3,4-methano analogs IIa and IIb and their enantiomers ent-IIa and ent-Iib, were synthesized from the chiral cyclopropane units Type-a and Type-b that we developed. These analogs were systematically evaluated with four GABA transporter (GAT) subtypes. The trans-3,4-methano analog IIa had inhibitory effects on GAT3 (IC50=23.9μM) and betaine-GABA transporter1 (5.48μM), indicating its potential as an effective lead compound for the development of potent GAT inhibitors due to its hydrophilic and low molecular weight properties and excellent ligand efficiency., (Copyright © 2013. Published by Elsevier Ltd.)

The decades-long quest for the phenobarbital (PhB) receptor that mediates activation of Cyp2B would appear fulfilled with the discovery by Mutoh et al., who found that PhB binds with pharmacological affinity to the epidermal growth factor receptor (EGFR). This finding provides a molecular basis for the suppression of hepatocyte EGFR signaling observed with PhB treatment, as previously noted in the context of tumor promotion. Although the PhB-mediated induction of Cyp2B expression through the association of a canonical nuclear receptor with the 5'-enhancer PBREM of Cyp2B is well known, direct binding of PhB to constitutive active androstane receptor (CAR, also known as NR1I3) typical of other xenobiotic-activated nuclear receptors has eluded detection. One EGF-activated pathway affected by the PhB-EGFR interaction is the loss of tyrosine phosphorylation of the scaffold protein RACK1. Dephosphorylated RACK1 provides the mechanistic link between the binding of PhB to EGFR and its effects on CAR by facilitating the interaction of serine/threonine phosphatase PP2A with inactive phosphorylated CAR. The dephosphorylation of CAR enables its translocation to the nucleus and activation of Cyp2B expression. Because EGFR and transducers RACK1, PP2A, and other partners are highly networked in numerous cellular pathways, this newly discovered partnership will surely reveal new fundamental roles for PhB beyond the regulation of drug metabolism.

Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

Background: GABA(A) receptors (GABA(A)R) are composed of several subunits that determine sensitivity to drugs, synaptic localisation and function. Recent studies suggest that agonists targeting selective GABA(A)R subunits may have therapeutic value against the cognitive impairments observed in schizophrenia. In this study, we determined whether GABA(A)R binding deficits exist in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia and tested if changes in GABA(A)R binding are related to the changes in subunit mRNAs. The GABA orthosteric and the benzodiazepine allosteric binding sites were assessed autoradiographically using [(3)H]Muscimol and [(3)H]Flumazenil, respectively, in a large cohort of individuals with schizophrenia (n = 37) and their matched controls (n = 37). We measured, using qPCR, mRNA of β (β1, β2, β3), γ (γ1, γ2, γ2S for short and γ2L for long isoform, γ3) and δ subunits and used our previous measurements of GABA(A)R α subunit mRNAs in order to relate mRNAs and binding through correlation and regression analysis., Results: Significant increases in both [(3)H]Muscimol (p = 0.016) and [(3)H]Flumazenil (p = 0.012) binding were found in the DLPFC of schizophrenia patients. Expression levels of mRNA subunits measured did not show any significant difference in schizophrenia compared to controls. Regression analysis revealed that in schizophrenia, the [(3)H]Muscimol binding variance was most related to α4 mRNA levels and the [(3)H]Flumazenil binding variance was most related to γ2S subunit mRNA levels. [(3)H]Muscimol and [(3)H]Flumazenil binding were not affected by the lifetime anti-psychotics dose (chlorpromazine equivalent)., Conclusions: We report parallel increases in orthosteric and allosteric GABA(A)R binding sites in the DLPFC in schizophrenia that may be related to a "shift" in subunit composition towards α4 and γ2S respectively, which may compromise normal GABAergic modulation and function. Our results may have implications for the development of treatment strategies that target specific GABA(A)R receptor subunits.

Neurosteroids are synthesized in the brain and modulate brain excitability. There is increasing evidence of their sedative, anesthetic and antiseizure properties, as well as their influence on mood. Currently neurosteroids are classified as pregnane neurosteroids (allopregnanolone and allotetrahydrodeoxycorticosterone), androstane neurosteroids (androstanediol and etiocholanone) or sulfated neurosteroids (pregnenolone sulfate and dehydroepiandrosterone sulfate). Both preclinical and clinical findings indicate that progesterone derivative neurosteroids such as allopregnanolone and allotetrahydrodeoxycorticosterone play a role in mood disorders. Clozapine and olanzapine, which were shown to be effective in stabilizing bipolar disorder, elevate pregnenolone levels in rat hippocampus, cerebral cortex, and serum. In lithium-treated mice, the blood levels of allopregnanolone and pregnenolone were elevated compared to control levels. Women diagnosed with bipolar disorder typically show symptomatic exacerbation in relation to the menstrual cycle, and show vulnerability to the onset or recurrence of mood disorders immediately after giving birth, when the levels of neurosteroid derivatives of progesterone drop. Whereas in women who had recovered from bipolar disorder, the plasma concentration of allopregnanolone was elevated compared to either healthy controls or women with major depressive disorder during the premenstrual period. During depressive episodes, blood level of allopregnanolone is low. Treatment with fluoxetine tends to stabilize the levels of neurosteroids in depression. These findings converge to suggest that these steroids have significant mood-stabilizing effect. This hypothesis is consistent with the observation that a number of anticonvulsants are effective therapies for bipolar disorder, a finding also consistent with the antiseizure properties of neurosteroids. Further exploration of action of neuroactive steroids is likely to open new frontiers in the investigation of the etiology and treatment of mood disorders, particularly bipolar disorders.

Epilepsies consist of a spectrum of neurologic disorders typically characterized by unpredictable and dysfunctional network behaviors in the central nervous system (CNS), which lead to discrete episodes of large bouts of uncontrolled neuronal synchrony that interfere with the normal functioning of the brain. Temporal lobe epilepsy (TLE) is accompanied by changes in interneuronal innervation and modifications in different γ-aminobutyric acid (GABA)(A) receptor subunits. Hormones play an important role in modulating the overall excitability of neurons, and at the same time hormonal pathways are frequently modified during epilepsy. This review focuses on TLE-correlated modifications of GABAergic transmission, and in particular on the implications of some of our own findings related to GABA(A) Rs containing the δ subunits (δ-GABA(A) Rs). These are extra- or perisynaptic GABA(A) Rs that mediate tonic inhibition, a major component of the inhibitory mechanism in the brain. The most potent endogenous modulators of δ-GABA(A) Rs are neurosteroids, which act as positive allosteric modulators. Plasticity of δ-GABA(A) Rs during TLE consists of down-regulation of the subunit in the dentate gyrus granule cells (DGGCs), while being up-regulated in interneurons. Surprisingly, the level of tonic inhibition in DGGCs remains unchanged, consistent with the idea that it becomes mediated by GABA(A) Rs containing other subunits. In parallel, tonic inhibition in a TLE model ceases to be sensitive to neurosteroid potentiation. In contrast, as predicted by the anatomic plasticity, interneuronal tonic current is increased, and remains sensitive to neurosteroids. These findings have important pharmacologic implications. Where neurosteroids normally have sedative and anticonvulsant effects, bimodal and cell-type specific modulations in their natural targets might weaken the inhibitory control on the dentate gate, under circumstances of altered neurosteroids levels (stress, ovarian cycle, or the postpartum period)., (Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.)

Drugs of abuse are known to mainly affect the dopaminergic and serotonergic system, although behavioral studies indicated that the GABA-ergic system also plays a role. We therefore investigated the acute effects of several commonly used drugs of abuse (methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA) and meta-chlorophenylpiperazine (mCPP)) on the function of the human α(1)β(2)γ(2) GABA(A) receptor (hGABA(A)-R), expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique. Although none of the tested drugs acted as full agonist on the hGABA(A)-R, some drugs induced differential modulation of hGABA(A)-R function, depending on the degree of receptor occupancy. Methamphetamine did not affect the GABA-evoked current at high receptor occupancy, but induced a minor inhibition at low receptor occupancy. Its metabolite amphetamine slightly potentiated the GABA-evoked current. MDMA and its metabolite MDA both inhibited the current at low receptor occupancy. However, MDMA did not affect the current at high occupancy, whereas MDA induced a potentiation. mCPP induced a strong inhibition (max. ∼ 80%) at low receptor occupancy, but ∼ 25% potentiation at high receptor occupancy. Competitive binding to one of the GABA-binding sites could explain the drug-induced inhibitions observed at low receptor occupancy, whereas an additional interaction with a positive allosteric binding site may play a role in the observed potentiations at high receptor occupancy. This is the first study to identify direct modulation of hGABA(A)-Rs as a novel mode of action for several drugs of abuse. Consequently, hGABA(A)-Rs should be considered as target for psychiatric pharmaceuticals and in developing treatment for drug intoxications., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A conjugable analogue of the benzodiazepine 5-(2-hydroxyphenyl)-7-nitro-benzo[e][1,4]diazepin-2(3H)-one containing a bromide C(12)-aliphatic chain (BDC) at nitrogen N1 was synthesized. One-pot preparation of this benzodiazepine derivative was achieved using microwave irradiation giving 49% yield of the desired product. BDC inhibited FNZ binding to GABA(A)-R with an inhibition binding constant K(i) = 0.89 μM and expanded a model membrane packed up to 35 mN m(-1) when penetrating in it from the aqueous phase. BDC exhibited surface activity, with a collapse pressure π = 9.8 mN m(-1) and minimal molecular area A(min) = 52 Å(2)/molecule at the closest molecular packing, resulted fully and non-ideally mixed with a phospholipid in a monolayer up to a molar fraction x≅ 0.1. A geometrical-thermodynamic analysis along the π-A phase diagram predicted that at low x(BDC) (<0.1) and at all π, including the equilibrium surface pressures of bilayers, dpPC-BDC mixtures dispersed in water were compatible with the formation of planar-like structures. These findings suggest that, in a potential surface grafted BDC, this compound could be stabilize though London-type interactions within a phospholipidic coating layer and/or through halogen bonding with an electron-donor surface via its terminal bromine atom while GABA(A)-R might be recognized through the CNZ moiety.

Benzodiazepines are widely used clinically to treat anxiety and insomnia. They also induce muscle relaxation, control epileptic seizures, and can produce amnesia. Moreover, benzodiazepines are often abused after chronic clinical treatment and also for recreational purposes. Within weeks, tolerance to the pharmacological effects can develop as a sign of dependence. In vulnerable individuals with compulsive drug use, addiction will be diagnosed. Here we review recent observations from animal models regarding the cellular and molecular basis that might underlie the addictive properties of benzodiazepines. These data reveal how benzodiazepines, acting through specific GABA(A) receptor subtypes, activate midbrain dopamine neurons, and how this could hijack the mesolimbic reward system. Such findings have important implications for the future design of benzodiazepines with reduced or even absent addiction liability., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A small library of anilino enaminones was analyzed for potential anticonvulsant agents. We examined the effects of three anilino enaminones on neuronal activity of output neurons, mitral cells (MC), in an olfactory bulb brain slice preparation using whole-cell patch-clamp recording. These compounds are known to be effective in attenuating pentylenetetrazol-induced convulsions. Among the three compounds tested, 5-methyl-3-(4-trifluoromethoxy-phenylamino)-cyclohex-2-enone (KRS-5Me-4-OCF₃) showed potent inhibition of MC activity with an EC₅₀ of 24.5 μM. It hyperpolarized the membrane potential of MCs accompanied by suppression of spontaneous firing. Neither ionotropic glutamate receptor blockers nor a GABA(B) receptor blocker prevented the KRS-5Me-4-OCF(3)-evoked inhibitory effects. In the presence of GABA(A) receptor antagonists, KRS-5Me-4-OCF(3) completely failed to evoke inhibition of MC spiking activity, suggesting that KRS-5Me-4-OCF₃-induced inhibition may be mediated by direct action on GABA(A) receptors or indirect action through the elevation of tissue GABA levels. Neither vigabatrin (a selective GABA-T inhibitor) nor 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid hydrochloride (NNC-711) (a selective inhibitor of GABA uptake by GABA transporter 1) eliminated the effect of KRS-5ME-4-OCF₃ on neuronal excitability, indicating that the inhibitory effect of the enaminone resulted from direct activation of GABA(A) receptors. The concentration-response curves for GABA are left-shifted by KRS-5Me-4-OCF₃, demonstrating that KRS-5Me-4-OCF₃ enhanced GABA affinity and acted as a positive allosteric modulator of GABA(A) receptors. The effect of KRS-5Me-4-OCF₃ was blocked by applying a benzodiazepine site antagonist, suggesting that KRS-5Me-4-OCF₃ binds at the classic benzodiazepine site to exert its pharmacological action. The results suggest clinical use of enaminones as anticonvulsants in seizures and as a potential anxiolytic in mental disorders.

Benzodiazepines (BZDs) and barbiturates exert their CNS actions by binding to GABA-A receptors (GABARs). The structural mechanisms by which these drugs allosterically modulate GABAR function, to either enhance or inhibit GABA-gated current, are poorly understood. Here, we used the substituted cysteine accessibility method to examine and compare structural movements in the GABA-binding site interface triggered by a BZD positive (flurazepam), zero (flumazenil) and negative (3-carbomethoxy-4-ethyl-6,7-dimethoxy-β-carboline, DMCM) modulator as well as the barbiturate pentobarbital. Ten residues located throughout the GABA-binding site interface were individually mutated to cysteine. Wild-type and mutant α(1)β(2)γ(2) GABARs were expressed in Xenopus laevis oocytes and functionally characterized using two-electrode voltage clamp. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive methanethiosulfonate (MTS) reagents in the absence and presence of BZD-site ligands and pentobarbital. Flurazepam and DMCM each accelerated the rate of reaction at α(1)R131C and slowed the rate of reaction at α(1)E122C, whereas flumazenil had no effect indicating that simple occupation of the BZD binding site is not sufficient to cause movements near these positions. Therefore, BZD-induced movements at these residues are likely associated with the ability of the BZD to modulate GABAR function (BZD efficacy). Low, modulating concentrations of pentobarbital accelerated the rate of reaction at α(1)S68C and β(2)P206C, slowed the rate of reaction at α(1)E122C and had no effect at α(1)R131C. These findings indicate that pentobarbital and BZDs induce different movements in the receptor, providing evidence that the structural mechanisms underlying their allosteric modulation of GABAR function are distinct., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objectives: To clarify whether alterations in midazolam pharmacokinetics resulting from changes in cytochrome P450 3A (CYP3A) activity lead to changes in its pharmacodynamic effects, benzodiazepine receptor occupancy was measured in the brain of rats after oral administration of midazolam., Methods: Receptor occupancy was measured by radioligand binding assay in rats pretreated with ursodeoxycholic acid (UDCA), ketoconazole and dexamethasone, and the plasma concentration of midazolam was simultaneously determined., Key Findings: There was a significant increase in the apparent dissociation constant and decrease in the maximum number of binding sites for specific [(3) H]flunitrazepam binding after oral administration of midazolam at pharmacologically relevant doses, suggesting that midazolam binds significantly to brain benzodiazepine receptors. Pretreatment with UDCA significantly enhanced the binding. This correlated well with significant enhancement by UDCA of the plasma midazolam concentration. The brain benzodiazepine receptor binding of oral midazolam was significantly enhanced by pretreatment with ketoconazole, a potent inhibitor of CYP3A, whereas it was significantly reduced by treatment with dexamethasone, an inducer of this enzyme. These effects paralleled changes in the plasma concentration of midazolam., Conclusions: The results indicate that pharmacokinetic changes such as altered CYP3A activity significantly influence the pharmacodynamic effect of midazolam by affecting occupancy of benzodiazepine receptors in the brain. They also suggest in-vivo or ex-vivo time-dependent measurements of receptor occupancy by radioligand binding assay to be a tool for elucidating the pharmacokinetic interaction of benzodiazepines with other agents in pre-clinical and clinical evaluations., (© 2010 The Authors. JPP © 2010 Royal Pharmaceutical Society.)

Background: The structural arrangement of the γ-aminobutyric acid type A receptor (GABAAR) is known to be crucial for the maintenance of cerebral-dependent homeostatic mechanisms during the promotion of highly adaptive neurophysiological events of the permissive hibernating rodent, i.e the Syrian golden hamster. In this study, in vitro quantitative autoradiography and in situ hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR) tests were performed for specific GABAAR receptor subunit gene primers synthases of non-hibernating (NHIB) and hibernating (HIB) hamsters. Attempts were made to identify the type of αβγ subunit combinations operating during the switching ON/OFF of neuronal activities in some hypothalamic nuclei of hibernators., Results: Both autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p < 0.01) prevalence of α1 ratio (over total α subunits considered in the present study) in the medial preoptic area (MPOA) and arcuate nucleus (Arc) of NHIBs with respect to HIBs. At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe) and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%). Regarding the other two subunits (β and γ), elevated β3 and γ3 mRNAs levels mostly characterized MPOA of HIBs, while prevalently elevated expression concentrations of the same subunits were also typical of Sch, even though this time during the awakening state. In the case of Arc, notably elevated levels were obtained for β3 and γ2 during hibernating conditions., Conclusion: We conclude that different αβγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster. The identification of a brain regional distribution pattern of distinct GABAAR subunit combinations may prove to be very useful for highlighting GABAergic mechanisms functioning at least during the different physiological states of hibernators and this may have interesting therapeutic bearings on neurological sleeping disorders.

The CXC chemokine receptor 4 (CXCR4) for the chemokine (C-X-C motif) ligand 12/stromal cell-derived factor-1 alpha (CXCL12/SDF-1 alpha) is highly expressed in the postnatal CA1 stratum lacunosum-moleculare. However, both the network events triggered by SDF-1 alpha in this microcircuit and the cellular targets of this chemokine remain virtually unexplored. Here, we have studied SDF-1 alpha-mediated neuromodulation of the stratum lacunosum-moleculare by directly comparing the properties of CXCR4-expressing Cajal-Retzius cells vs. CXCR4-non-expressing interneurons, and by recording the electrophysiological effects caused by application of SDF-1 alpha on either cell type. We demonstrate that SDF-1 alpha dramatically reduces spontaneous firing in Cajal-Retzius cells via hyerpolarization, and that cessation of firing is prevented by the CXCR4-specific antagonist AMD3100. In contrast, no effects on the excitability of interneurons of the same layer were observed following exposure to the chemokine. We also provide evidence that, despite the expression of functional glutamate receptors, Cajal-Retzius cells are integrated in the synaptic network of the stratum lacunosum-moleculare via excitatory GABAergic input. Furthermore, we show that the axons of Cajal-Retzius cells target specifically the stratum lacunosum-moleculare and the dentate gyrus, but lack postsynaptic specializations opposite to their axonal varicosities. These results, taken together with our observation that SDF-1 alpha reduces evoked field responses at the entorhinal cortex-CA1 synapse, suggest that Cajal-Retzius cells produce a diffuse output that may impact information processing of stratum lacunosum-moleculare. We propose that pathological alterations of local levels of SDF-1 alpha or CXCR4 expression may affect the functions of an important hippocampal microcircuit.

Interactions between coral reef topography, tide cycles, and photoperiod provided selection pressure for adaptive physiological changes in sheltered hypoxic niches to be exploited by specialized tropical reef fish. The epaulette shark Hemiscyllium ocellatum withstands cyclic hypoxia in its natural environment, many hours of experimental hypoxia, and anoxia for up to 5h. It shows neuronal hypometabolism in response to 5% oxygen saturation. Northern-hemisphere hypoxia- and anoxia-tolerant vertebrates that over-winter under ice alter their inhibitory to excitatory neurotransmitter balance to forestall brain ATP depletion in the absence of oxidative phosphorylation. GABA immunochemistry, HPLC analysis and receptor binding studies in H. ocellatum cerebellum revealed a heterogeneous regional accumulation of neuronal GABA despite no change in its overall concentration, and a significant increase in GABA(A) receptor density without altered binding affinity. Increased GABA(A) receptor density would protect the cerebellum during reoxygenation when transmitter release resumes. While all hypoxia- and anoxia-tolerant teleosts examined to date respond to low oxygen levels by elevating brain GABA, the phylogenetically older epaulette shark did not, suggesting that it uses an alternative neuroprotective mechanism for energy conservation. This may reflect an inherent phylogenetic difference, or represent a novel ecophysiological adaptation to cyclic variations in the availability of oxygen.

Coadministration of grapefruit juice (GFJ) has been proposed to enhance the systemic availability and decrease the required dose of drugs such as cyclosporine that are extensively metabolized in the intestine and liver. Although GFJ inhibits human cytochrome P450 (CYP) 3A, effects on dog CYP have not yet been reported. Consequently, we determined whether GFJ inhibits triazolam hydroxylation by Beagle dog liver microsomes (DLM) using human liver microsomes (HLM) as positive control. Results were compared with the effects of lyophilized GFJ and commercially-available powdered grapefruit capsules, which may be more convenient dosage forms. GFJ inhibited alpha-hydroxytriazolam formation in both DLM and HLM with similar IC(50) (inhibitor concentration producing a 50% decrease in reaction velocity) values of 0.56% and 0.52% (v/v), respectively. Lyophilized GFJ and powdered grapefruit also inhibited DLM alpha-hydroxytriazolam formation with IC(50) values of 0.76 and 1.2 mg/mL, respectively. Consistent with mechanism-based enzyme inhibition, preincubation of DLM with any of the grapefruit products for 20 min resulted in significant enhancement of inhibition of triazolam alpha-hydroxylation by 8-20%. The results indicate that 16 g of lyophilized GFJ or 23 g of powdered grapefruit would be equivalent to dosing 100 mL of GFJ. In vivo pharmacokinetic interaction studies are needed to confirm these in vitro findings.

A higher throughput method of screening for the metabolism dependent inhibition of 56 marketed drugs was evaluated and compared data from the PHLM assay (using midazolam as probe) with Cypex assay (using diethoxyfluoresin (DEF) as probe) for CYP3A4 by using 96 well plate. Also 27 marketed drugs were selected to evaluate the reproducibility of Cypex assay using 7-benzyloxyquinoline (7-BQ) as second probe substrate for CYP3A4. Furthermore Cypex CYP2D6 was used to evaluate the reproducibility of this system using 4-methylaminomethyl-7-methoxycoumarin (MMC) as probe substrate with 15 marketed drugs. The fold change was estimated using the fold change obtained from triplicates experiments in same day or different days. All replicates in agreement (i.e. all positive or all negative) for >80% of compounds. The IC50 values for the two assays closely matched only for 13 compounds (23%). Only 5 of the variant 56 compounds had higher IC50 values with the recombinant enzymes, whereas 38 had lower IC50 values with the recombinant cypex CYP3A4 enzyme. The Cypex assay is comparable to PHLM assay in terms of predictivity and reproducibility. The Cypex assay therefore offers a higher throughput, reproducible alternative to PHLM for placement earlier in the lead optimisation process. In conclusion, the results obtained from a fluorescence-based method using Cypex CYP3A4 reflect mostly those obtained from conventional assay using human liver microsomes. This method provides more rapid and reliable detection of MDI inhibitors and may be useful in drug discovery.

Upon activation by therapeutics, the nuclear xenobiotic/ constitutive active/androstane receptor (CAR) regulates various liver functions ranging from drug metabolism and excretion to energy metabolism. CAR can also be a risk factor for developing liver diseases such as hepatocellular carcinoma. Here we have characterized the conserved threonine 38 of human CAR as the primary residue that regulates nuclear translocation and activation of CAR. Protein kinase C phosphorylates threonine 38 located on the alpha-helix spanning from residues 29-42 that constitutes a part of the first zinc finger and continues into the region between the zinc fingers. Molecular dynamics study has revealed that this phosphorylation may destabilize this helix, thereby inactivating CAR binding to DNA as well as sequestering it in the cytoplasm. We have found, in fact, that helix-stabilizing mutations reversed the effects of phosphorylation. Immunohistochemical study using an anti-phospho-threonine 38 peptide antibody has, in fact, demonstrated that the classic CAR activator phenobarbital dephosphorylates the corresponding threonine 48 of mouse CAR in the cytoplasm of mouse liver and translocates CAR into the nucleus. These results define CAR as a cell signal-regulated constitutive active nuclear receptor. These results also provide phosphorylation/dephosphorylation of the threonine as the primary drug target for CAR activation.