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Slobodan M Janković,1 Miralem Dješević,2 Snežana V Janković1 1Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia; 2Cardiology Department, Private Policlinic Center Eurofarm, Sarajevo, Bosnia and HercegovinaCorrespondence: Slobodan M JankovićUniversity of Kragujevac, Faculty of Medical Sciences, Svetozara Markovića 69 Street, Kragujevac, 34000, SerbiaEmail slobnera@gmail.comAbstract: GABA A receptors are ubiquitous in the central nervous system and there is a huge diversity of receptor subtypes in almost all regions of the brain. However, the expression of GABA A receptor subtypes is altered in both the gray and white matter of patients with focal epilepsy. Although there is a number of anticonvulsants with marketing authorization for the treatment of focal epilepsy which act through GABA A receptors, potentiating the inhibitory effects of GABA, it is necessary to develop more potent and more specific GABAergic anticonvulsants that are effective in drug-resistant patients with focal epilepsy. There are three orthosteric and at least seven allosteric agonist binding sites at the GABA A receptor. In experimental and clinical studies, full agonists of GABA A receptors showed a tendency to cause desensitization of the receptors, tolerance, and physical dependence; therefore, partial orthosteric agonists and positive allosteric modulators of GABA A receptors were further developed. Preclinical studies demonstrated the anticonvulsant efficacy of positive allosteric modulators with selective action on GABA A receptors with α2/α3 subunits, but only a handful of them were further tested in clinical trials. The best results were obtained for clobazam (already marketed), ganaxolone (in phase III trials), CVL-865 (in phase II trials), and padsevonil (in phase III trials). Several compounds with more selective action on GABA A receptors, perhaps only in certain brain regions, have the potential to become effective drugs against specific subtypes of focal-onset epilepsy. However, their development needs time, and in the near future we can expect only one or two new GABA A agonists to obtain marketing authorization for focal epilepsy, an advance that would be of use for just a fraction of patients with drug-resistant epilepsy.Keywords: GABA A receptors, positive allosteric modulators, focal-onset epilepsy

Background: Clinical and experimental studies support the therapeutic potential of Withania somnifera (WS) (L.) Dunal on anxiety disorders. This potential is attributable to components present in different plant extracts; however, the individual compound(s) endowed with specific anxiolytic effects and potential modulatory activity of the GABAA receptor complex (GABAAR) have remained unidentified until the recent isolation from a WS methanolic root extract of some GABAAR-active compounds, including the long alkyl-chain ferulic acid ester, docosanyl ferulate (DF). Aims: This study was designed to assess whether DF (0.05, 0.25 and 2 mg/kg), similarly to diazepam (2 mg/kg), may exert anxiolytic effects, whether these effects may be significantly blocked by the benzodiazepine antagonist flumazenil (10 mg/kg) and whether DF may lack some of the benzodiazepines' typical motor, cognitive and motivational side effects. Methods: The behavioural paradigms Elevated Plus Maze, Static Rods, Novel Object Recognition, Place Conditioning and potentiation of ethanol-induced Loss of Righting Reflex were applied on male CD-1 mice. Results: Similarly to diazepam, DF exerts anxiolytic effects that are blocked by flumazenil. Moreover, at the full anxiolytic dose of 2 mg/kg, DF lacks typical benzodiazepine-like side effects on motor and cognitive performances and on place conditioning. Moreover, DF fails to potentiate ethanol's (3 g/kg) depressant activity at the ethanol-induced Loss of Righting Reflex paradigm. Conclusions: These data point to DF as an effective benzodiazepine-like anxiolytic compound that, in light of its lack of motor, mnemonic and motivational side effects, could be a suitable candidate for the treatment of anxiety disorders. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric-acid type A (GABA A ) receptors expressing the γ1 or γ3 subunit are only found within a few regions of the brain, some of which are involved in sleep. No known compounds have been reported to selectively target γ1- or γ3-containing GABA A receptors. Pharmacological assessments of this are conflicting, possibly due to differences in experimental models, conditions, and exact protocols when reporting efficacies and potencies. In this study, we evaluated the modulatory properties of five non-benzodiazepine Z-drugs (zaleplon, indiplon, eszopiclone, zolpidem, and alpidem) used in sleep management and the benzodiazepine, diazepam on human α1β2γ receptors using all three γ subtypes. This was accomplished using concatenated GABA A pentamers expressed in Xenopus laevis oocytes and measured via two-electrode voltage clamp. This approach removes the potential for single subunits to form erroneous receptors that could contribute to the pharmacological assessment of these compounds. No compound tested had significant effects on γ1-containing receptors below 10 μM. Interestingly, zaleplon and indiplon were found to modulate γ3-containing receptors equally as efficacious as γ2-containing receptors. Furthermore, zaleplon had a higher potency for γ3- than for γ2-containing receptors, indicating certain therapeutic effects could occur via these γ3-containing receptors. Eszopiclone modulated γ3-containing receptors with reduced efficacy but no reduction in potency. These data demonstrate that the imidazopyridines zaleplon and indiplon are well suited to further investigate potential γ3 effects on sleep in vivo. [ABSTRACT FROM AUTHOR]

The suprachiasmatic nucleus (SCN) contains a pacemaker that generates circadian rhythms and entrains them with the 24-h light-dark cycle (LD). The SCN is composed of 16,000 to 20,000 heterogeneous neurons in bilaterally paired nuclei. γ-amino butyric acid (GABA) is the primary neurochemical signal within the SCN and plays a key role in regulating circadian function. While GABA is the primary inhibitory neurotransmitter in the brain, there is now evidence that GABA can also exert excitatory effects in the adult brain. Cation chloride cotransporters determine the effects of GABA on chloride equilibrium, thereby determining whether GABA produces hyperpolarizing or depolarizing actions following activation of GABAA receptors. The activity of Na-K-2Cl cotransporter1 (NKCC1), the most prevalent chloride influx cotransporter isoform in the brain, plays a critical role in determining whether GABA has depolarizing effects. In the present study, we tested the hypothesis that NKCC1 protein expression in the SCN is regulated by environmental lighting and displays daily and circadian changes in the intact circadian system of the Syrian hamster. In hamsters housed in constant light (LL), the overall NKCC1 immunoreactivity (NKCC1-ir) in the SCN was significantly greater than in hamsters housed in LD or constant darkness (DD), although NKCC1 protein levels in the SCN were not different between hamsters housed in LD and DD. In hamsters housed in LD cycles, no differences in NKCC1-ir within the SCN were observed over the 24-h cycle. NKCC1 protein in the SCN was found to vary significantly over the circadian cycle in hamsters housed in free-running conditions. Overall, NKCC1 protein was greater in the ventral SCN than in the dorsal SCN, although no significant differences were observed across lighting conditions or time of day in either subregion. These data support the hypothesis that NKCC1 protein expression can be regulated by environmental lighting and circadian mechanisms within the SCN. [ABSTRACT FROM AUTHOR]

Abstract—We studied the effect of a 2-week administration of cycloprolylglycine peptide (CPG) (1 and 2 mg/kg/day, i.p.) on the binding characteristics of serotonin 5-HT2A-, NMDA-, metabotropic glutamate mGluII-, GABAA- and GABAB-receptors in the brain of inbred BALB/c mice in vitro and ex vivo. It was found that CPG at doses of 1 and 2 mg/kg/day reduces the density of 5-HT2A receptors in the striatum of mice by 24 and 28%, respectively. A decrease in the density of NMDA receptors in the hippocampus was observed only at a dose of 2 mg/kg/day; it was 22%. At the same time, there was an increase in the density of GABAA receptors in the frontal cortex at a dose of 1 mg/kg/day by 36% compared with the control. The 2-week administration of CPG at both doses did not affect Bmax of mGluII- and GABAB receptors, as well as Kd in all groups. In the in vitro radioligand assay, the CPG did not affect the specific binding of the labeled ligands of the studied receptors. These data indicate the functional involvement of 5-HT2A, NMDA, and GABAA receptors in the formation of the antidepressant-like effect of cycloprolylglycine in BALB/c mice for the first time. [ABSTRACT FROM AUTHOR]

This study evaluated the extent to which a reduction in contextual fear contributes to the anxiolytic effect of benzodiazepines in the fear-potentiated startle response. To this end, chlordiazepoxide, an anxiolytic often used as positive control in preclinical drug studies, and zolpidem, known to have sedative properties and to be devoid of anxiolytic effects, were tested in two contexts: the same context as training had taken place and an alternative context. In addition, the level of muscle relaxation was assessed in a grip strength test. Chlordiazepoxide (2.5–10 mg/kg) decreased the fear-potentiated startle response, confirming its anxiolytic activity. In addition, it dose-dependently decreased the overall startle response in the same, but not the alternative context, and did not affect grip strength, indicating that chlordiazepoxide inhibits contextual fear in the absence of non-specific drug effects. Zolpidem (1.0–10 mg/kg) reduced the overall startle response in both contexts equally and decreased grip strength, indicating that its effects on fear-potentiated startle are due to non-specific drug effects, and not anxiolytic effects. The present findings show that chlordiazepoxide reduces contextual conditioned fear in the absence of non-specific drug effects. In addition, they show that training and testing rats in different contexts makes it possible to distinguish between cued, contextual and non-specific drug effects. As exaggerated contextual fear conditioning contributes to the fear generalization processes implicated in pathological anxiety, focus in screening of anxiolytic effects could be directed more towards the suppression of contextual fear and, therefore, this approach would be a valuable addition to standard preclinical screening. [ABSTRACT FROM AUTHOR]

Neurosteroids are the principal endogenous modulators of the γ-Aminobutyric acid receptors (GABA A Rs), pentameric membrane-bound proteins that can be assembled from at least 19 subunits. In the most abundant GABA A R arrangement (α 1 β 2 γ 2 ), neurosteroids can potentiate the GABA action as well as produce a direct activation of the channel. The recent crystal structures of neurosteroids bound to α homopentameric GABA A R reveal binding to five equivalent sites. However, these results have been obtained using receptors that are not physiologically relevant, suggesting a need to investigate neurosteroid binding to heteropentameric receptors that exist in the central nervous system. In a previous work, we predicted the neurosteroid binding site by applying molecular modeling methods on the β 3 homopentamer. Here we construct a homology model of the transmembrane domain of the heteropentameric α 1 β 2 γ 2 receptor and then, by combining docking and molecular dynamics simulations, we analyzed neurosteroid binding. Results show that the five neurosteroid cavities are conserved in the α 1 β 2 γ 2 receptor and all of them are able to bind neurosteroids. Two different binding modes were detected depending on the identity of the residue at position 241 in the transmembrane helix 1. These theoretical findings provide microscopic insights into neurosteroid binding at the heteropentameric GABA A R. The existence of two classes of sites may be associated with how neurosteroids modulate GABA A R. Our finding would represent the essential first step to reach a comprehensive understanding of how these endogenous molecules regulate the central nervous system. [ABSTRACT FROM AUTHOR]

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

GABA A receptors play a dominant role in mediating inhibition in the mature mammalian brain, and defects of GABAergic neurotransmission contribute to the pathogenesis of a variety of neurological and psychiatric disorders. Two types of GABAergic inhibition have been described: αβγ receptors mediate phasic inhibition in response to transient high-concentrations of synaptic GABA release, and αβδ receptors produce tonic inhibitory currents activated by low-concentration extrasynaptic GABA. Both αβδ and αβγ receptors are important targets for general anesthetics, which induce apparently different changes both in GABA-dependent receptor activation and in desensitization in currents mediated by αβγ vs. αβδ receptors. Many of these differences are explained by correcting for the high agonist efficacy of GABA at most αβγ receptors vs. much lower efficacy at αβδ receptors. The stoichiometry and subunit arrangement of recombinant αβγ receptors are well established as β-α-γ-β-α, while those of αβδ receptors remain controversial. Importantly, some potent general anesthetics selectively bind in transmembrane inter-subunit pockets of αβγ receptors: etomidate acts at β + /α − interfaces, and the barbiturate R-5-allyl-1-methyl-5-( m -trifluoromethyl-diazirynylphenyl) barbituric acid (R- m TFD-MPAB) acts at α + /β − and γ + /β − interfaces. Thus, these drugs are useful as structural probes in αβδ receptors formed from free subunits or concatenated subunit assemblies designed to constrain subunit arrangement. Although a definite conclusion cannot be drawn, studies using etomidate and R- m TFD-MPAB support the idea that recombinant α1β3δ receptors may share stoichiometry and subunit arrangement with α1β3γ2 receptors. [ABSTRACT FROM AUTHOR]

β-alanine is a structural analog of glycine and γ-aminobutyric acid (GABA) and is thought to be involved in the modulation of nociceptive information at the spinal cord. However, it is not known whether β-alanine exerts its effect in substantia gelatinosa (SG) neurons of the spinal dorsal horn, where glycine and GABA play an important role in regulating nociceptive transmission from the periphery. Here, we investigated the effects of β-alanine on inhibitory synaptic transmission in adult rat SG neurons using whole-cell patch-clamp. β-alanine dose-dependently induced outward currents in SG neurons. Current-voltage plots revealed a reversal potential at approximately −70 mV, which was close to the equilibrium potential of Cl − . Pharmacological analysis revealed that β-alanine activates glycine receptors, but not GABA A receptors. These results suggest that β-alanine hyperpolarizes the membrane potential of SG neurons by activating Cl − channels through glycine receptors. Our findings raise the possibility that β-alanine may modulate pain sensation through glycine receptors. [ABSTRACT FROM AUTHOR]

This paper describes the novel approach for preparation of [ 18 F]flumazenil ([ 18 F]FMZ), well known radioligand for assessment of GABA A receptors by PET. The optimized reaction conditions allowed us to implement commercially available SPE cartridges for [ 18 F]FMZ purification avoiding HPLC. All procedures were performed with TRACERlab FX N Pro synthesizer in 53 min. Developed approach provided [ 18 F]FMZ with high RCP (> 97%) and low level of chemical impurities (< 5 µg/mL). FMZ was routinely synthesized in 6.4 ± 0.7% RCY at EOS (not decay corrected, n = 8) and the molar radioactivity was > 185 GBq/μmol. [ABSTRACT FROM AUTHOR]

The retigabine analog 2-amino-4-[(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester (AA29504) is a positive allosteric modulator (PAM) of γ-aminobutyric acid A receptors (GABA A Rs), and the modulator has been used in ex vivo / in vivo studies to probe the physiological roles of native δ-containing GABA A Rs. In this study, the functional properties and mode of action of AA29504 were investigated at human GABA A Rs expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology. AA29504 was found to be an allosteric GABA A R agonist displaying low intrinsic activities at 3–30 μM. AA29504 was essentially equipotent as a PAM at the 13 GABA A R subtypes tested (EC 50 : 0.45–5.2 μM), however GABA EC 5 -evoked currents through αβδ subtypes were modulated to substantially higher levels than those through αβγ 2S subtypes (relative to GABA I max ). While the δ/γ 2S -difference clearly was key for this differential GABA efficacy modulation, studies of the AA29504-mediated modulation of different α 4,5,6 -containing αβ, αβγ 2S and αβδ GABA A Rs revealed the α-subunit identity to be another important determinant. Based on its functional properties at numerous mutant GABA A Rs and on in silico analysis of its low-energy conformations, AA29504 is proposed to act through an allosteric site in the transmembrane β (+) /α (−) interface in the GABA A R also targeted by etomidate and several other modulators. In contrast to these modulators, however, AA29504 did not display substantial β 2 /β 3 -over-β 1 GABA A R preference, which challenges the notion of ligands targeting this site always possessing this subtype-selectivity profile. Hence, the detailed pharmacological profiling of AA29504 both highlights the complexity of allosteric GABA A R modulation and provides valuable information about this modulator as a pharmacological tool. [ABSTRACT FROM AUTHOR]

Variations in the early postnatal environment of rodents produce long-term changes in responses to stress that may underlie neuropsychiatric diseases such as anxiety, depression and schizophrenia. GABA A receptors undergo marked changes in their subunit composition during this period, involving a regionally-dependent replacement of α 2 with α 1 subunits, the so-called α-subunit switch. In this study we examined the effects of early-life environment on adulthood GABA A receptor α 1 and α 2 subunit expression and the synaptic clustering of GABA A receptors. Male and female mice were exposed to either 15 min daily handling sessions (EH) or no intervention (NH) over postnatal day (PND) 1–14. Adulthood behavioural differences in anxiety were assessed on the elevated plus-maze. Immunoperoxidase histochemistry was used to examine the density of the α 1 and α 2 subunit proteins. Double-labelling immunofluorescence and confocal microscopy were used to study GABA A receptor synaptic clustering. NH animals showed increased anxiety-type behaviours in the elevated plus maze relative to EH mice. NH males showed a loss of α 2 subunits from the thalamus and lower layers of the somatosensory cortex, whilst NH females showed a reduction of α 2 but increase in α 1 protein in lower layers of the primary somatosensory cortex only. The NH condition also reduced α 1 subunit expression in dentate gyrus (DG) in both males and females. Regardless of sex, NH mice showed reduced colocalisation of GABA A receptor α 2 subunits with the synaptic marker gephyrin relative to the control condition. These findings suggest that early-life environment has long-lasting effects on GABA A receptors, leading to long-term changes in adulthood behaviour, and are of relevance to neurodevelopmental explanations of stress-augmented neuropsychiatric disorders. [ABSTRACT FROM AUTHOR]

The former sedative-hypnotic and recreational drug methaqualone (Quaalude) is a moderately potent, non-selective positive allosteric modulator (PAM) at GABA A receptors (GABA A Rs) (Hammer et al., 2015). In the present study, we have identified a novel methaqualone analog, 2-phenyl-3-( p -tolyl)quinazolin-4(3 H )-one (PPTQ), in a screening of 67 analogs at five αβ 2 γ 2S GABA A R subtypes and delineated its functional properties and mechanism of action at wild-type and mutant GABA A Rs expressed in Xenopus laevis oocytes by two-electrode voltage clamp electrophysiology. PPTQ was found to be an allosteric agonist and a PAM (ago-PAM) at human α 1 β 2 γ 2S and α 4 β 2 δ GABA A Rs, exhibiting intrinsic activity at micromolar concentrations and potentiating the GABA-evoked signaling through the receptors at concentrations down to the low-nanomolar range. Whereas PPTQ exclusively increased the potency of GABA at the α 1 β 2 γ 2S receptor, it increased both GABA potency and efficacy at α 4 β 2 δ and displayed modest potency-based preference for α 4 β 2 δ over α 1 β 2 γ 2S . In elaborate mutagenesis and competition experiments PPTQ was found to act through the same or an overlapping site as etomidate in the transmembrane β (+) /α (−) subunit interfaces, whereas it did not seem to target the other three transmembrane interfaces in the GABA A R. Finally, the PPTQ site was shown to be allosterically linked with sites targeted by neurosteroids and barbiturates but not with the high-affinity benzodiazepine site in the α 1 β 2 γ 2S receptor. In conclusion, the development of a highly potent, bioavailable GABA A R ago-PAM by subtle modifications to the methaqualone scaffold demonstrates that derivatization of this infamous drug from the past can lead to modulators with distinct functional characteristics at the receptors. [ABSTRACT FROM AUTHOR]

Background: In Nepali and Indian system of traditional medicine, Withania somnifera (WS) is considered as a rejuvenative medicine to maintain physical and mental health and has also been shown to improve memory consolidation. Objective: In this study, a methanolic extract of WS (mWS) was applied on mice hippocampal CA1 neurons to identify the receptors activated by the WS. Materials and Methods: The whole cell patch clamp recordings were performed on CA1 pyramidal neurons from immature mice (7-20 postnatal days). The cells were voltage clamped at -60 mV. Extract of WS root were applied to identify the effect of mWS. Results: The application of mWS (400 ng/μl) induced remarkable inward currents (-158.1 ± 28.08 pA, n = 26) on the CA1 pyramidal neurons. These inward currents were not only reproducible but also concentration dependent. mWS-induced inward currents remained persistent in the presence of amino acid receptor blocking cocktail (AARBC) containing blockers for the ionotropic glutamate receptors, glycine receptors and voltage-gated Na + channel (Control: -200.3 ± 55.42 pA, AARBC: -151.5 ± 40.58 pA, P > 0.05) suggesting that most of the responses by mWS are postsynaptic events. Interestingly, these inward currents were almost completely blocked by broad GABA A receptor antagonist, bicuculline- 20 μM (BIC) (BIC: -1.46 ± 1.4 pA, P < 0.001), but only partially by synaptic GABA A receptor blocker gabazine (1 μM) (GBZ: -18.26 ± 4.70 pA, P < 0.01). Conclusion: These results suggest that WS acts on synaptic/extrasynaptic GABA A receptors and may play an important role in the process of memory and neuroprotection via activation of synaptic and extrasynaptic GABA A receptors.

Previous work has shown that S44819 is a novel GABAA receptor (GABA A R) antagonist, which is selective for extrasynaptic GABA A Rs incorporating the α5 subunit (α5-GABA A Rs). The present study reports on the preclinical neuropsychopharmacological profile of S44819. Significantly, no sedative or pro-convulsive side effects of S44819 were found at doses up to 30 mg/kg i.p . Object recognition (OR) memory in intact mice was enhanced by S44819 (0.3 mg/kg p.o. ) given before the acquisition trial. Mice treated with phencyclidine for two weeks and tested six days after the cessation of treatment failed to show OR memory. This deficit was corrected by a single administration of S44819 (0.1, 0.3 or 1 mg/kg p.o. ) prior to the acquisition trial. The amnestic effect of ketamine in rats tested in the eight-arm radial maze (reference and working memory versions) was blocked by S44819 (3 mg/kg p.o .). Extinction of cued fear was preserved during treatment with S44819 (3 mg/kg/ diem i.p. ). Administration of S44819 had no significant effect in the Vogel-conflict test, the elevated plus maze, the forced swim, the marble-burying and the tail-suspension tests. In contrast, anxiolytic/antidepressant-like effects of the compound were found in paradigms that have mnemonic components, such as social interaction, fear-potentiated startle and social avoidance induced by negative life experience. In summary, S44819 enhanced intact recognition memory and ameliorated memory deficits induced by inhibition of NMDA receptors. Anxiolytic/antidepressant efficacy was limited to paradigms involving cognitive function. In conclusion, S44819 is a novel psychoactive pro-cognitive compound with potential as a therapeutic agent in dementia. [ABSTRACT FROM AUTHOR]

This review highlights early efforts to translate pre-clinical and clinical findings regarding the role of neuroactive steroids in stress adaptation and PTSD into new therapeutics for PTSD. Numerous studies have demonstrated PTSD-related alterations in resting levels or the reactivity of neuroactive steroids and their targets. These studies also have demonstrated substantial variability in the dysfunction of specific neuroactive steroid systems among PTSD subpopulations. These variabilities have been related to the developmental timing of trauma, severity and type of trauma, genetic background, sex, reproductive state, lifestyle influences such as substance use and exercise, and the presence of comorbid conditions such as depression and chronic pain. Nevertheless, large naturalistic studies and a small placebo-controlled interventional study have revealed generally positive effects of glucocorticoid administration in preventing PTSD after trauma, possibly mediated by glucocorticoid receptor-mediated effects on other targets that impact PTSD risk, including other neuroactive steroid systems. In addition, clinical and preclinical studies show that administration of glucocorticoids, 17β-estradiol, and GABAergic neuroactive steroids or agents that enhance their synthesis can facilitate extinction and extinction retention, depending on dose and timing of dose in relation to these complex PTSD-relevant recovery processes. This suggests that clinical trials designed to test neuroactive steroid therapeutics in PTSD may benefit from such considerations; typical continuous dosing regimens may not be optimal. In addition, validated and clinically accessible methods for identifying specific neuroactive steroid system abnormalities at the individual level are needed to optimize both clinical trial design and precision medicine based treatment targeting. [ABSTRACT FROM AUTHOR]

Cannabidiol (CBD) is a major non-intoxicating component of cannabis and possesses anti-epileptic, anxiolytic and anti-hyperalgesic properties. The mechanism of action of CBD in producing such effects remains unclear. Despite evidence that some endogenous and synthetic cannabinoids interact with GABA A receptors, no-one has yet investigated the effects of CBD. Here we used two-electrode voltage clamp electrophysiology to compare the actions of CBD with those of the major central endocannabinoid, 2-arachidonoyl glycerol (2-AG) on human recombinant GABA A receptors (synaptic α1-6βγ2 and extrasynaptic α4β2δ) expressed on Xenopus oocytes. CBD and 2-AG were positive allosteric modulators at α1-6βγ2 receptors, with low micromolar potencies. The maximal level of enhancement seen with either CBD or 2-AG were on α2-containing GABA A receptor subtypes, with approximately a 4-fold enhancement of the GABA EC 5 evoked current, more than twice the potentiation seen with other α-subunit receptor combinations. Further we observed β-subunit selectivity, whereby modulatory activity was higher at β2/β3 over β1 subunits. The β1-subunit homologous mutant β2(V436T) substantially diminished the efficacy of both drugs to a third of that obtained with wild-type β2 subunit combinations, but without changing potency. The potency of CBD increased and efficacy preserved in binary α1/α2β2 receptors indicating that their effects do not involve the classic benzodiazepine site. Exploration of extrasynaptic α4β2δ receptors revealed that both compounds enhanced GABA EC 5 evoked currents at concentrations ranging from 0.01–1 μM. Taken together these results reveal a mode of action of CBD on specifically configured GABA A receptors that may be relevant to the anticonvulsant and anxiolytic effects of the compound. [ABSTRACT FROM AUTHOR]

Benzodiazepines are regularly prescribed for treatment of anxiety though there are side effects. Flavonoids have selective affinity for GABA A receptors implicated in anxiolytic-like activity in rodents, but are devoid of the unwanted side effects of benzodiazepines. In this study, 6-methoxyflavanone (6-MeOF), a positive allosteric modulator of γ-amino butyric acid (GABA) responses at human recombinant GABA A receptors, was evaluated for its behavioral profile in the elevated plus-maze as well as the staircase- plus and open-field tests in mice. In addition, the distribution of 6-MeOF in selected brain areas involved in anxiety (amygdala and cerebral cortex) was also examined using a validated high performance liquid chromatography ultraviolet detection (HPLC/UV) method. 6-MeOF (10, 30 and 50 mg/kg) exerted an anxiolytic-like effect, increasing entries and time spent in the open arm and the central platform, as well as head-dipping frequency in the mouse elevated plus-maze assay. It also decreased rearing incidence without suppressing the number of steps ascended in the staircase test. Whereas, in the open-field anxiety test, 6-MeOF had no effect on locomotor activity at lower doses, a decrease was observed at the highest dose (100 mg/kg). 6-MeOF additionally produced an anxiolytic-like increase in the time spent at the center of the open-field apparatus. These effects were preferentially antagonized by pentylenetetrazole (15 mg/kg). Furthermore, pharmacokinetic studies disclosed a rapid appearance of 6-MeOF in the plasma and discrete brain areas. Taken together, our findings suggest that 6-MeOF readily crosses the blood brain barrier (BBB) generating anxiolytic activity, mediated through the GABAergic system. [ABSTRACT FROM AUTHOR]

GABAergic transmission in the ventral tegmental area (VTA) exerts a tonic inhibitory influence on mesolimbic dopaminergic neurons’ activity. Blockade of VTA GABA A receptors increases dopamine release in the nucleus accumbens (NAcc). Increases in NAcc dopamine levels typically accompany sexual behavior display. Copulation to satiety is characterized by the instatement of a long lasting (72 h) sexual behavior inhibition and the mesolimbic system appears to be involved in this phenomenon. GABAergic transmission in the VTA might play a role in the maintenance of this long lasting sexual inhibitory state. To test this hypothesis, in the present work we investigated the effect of GABA A receptor blockade in sexually exhausted males 24 h after copulation to satiety, once the sexual inhibitory state is established, and compared it with its effect in sexually experienced rats. Results showed that low doses of systemically administered bicuculline induced sexual behavior expression in sexually exhausted rats, but lacked an effect on copulation of sexually experienced animals. Intra-VTA bilateral infusion of bicuculline did not modify sexual behavior of sexually experienced rats, but induced sexual behavior expression in all the sexually exhausted males. Hence, GABA plays a role in the control of sexual behavior expression at the VTA. The role played by GABAergic transmission in male sexual behavior expression of animals with distinct sexual behavior conditions is discussed. [ABSTRACT FROM AUTHOR]

Virtually every neuron within the suprachiasmatic nucleus (SCN) communicates via GABAergic signaling. The extracellular levels of GABA within the SCN are determined by a complex interaction of synthesis and transport, as well as synaptic and non-synaptic release. The response to GABA is mediated by GABA A receptors that respond to both phasic and tonic GABA release and that can produce excitatory as well as inhibitory cellular responses. GABA also influences circadian control through the exclusively inhibitory effects of GABA B receptors. Both GABA and neuropeptide signaling occur within the SCN, although the functional consequences of the interactions of these signals are not well understood. This review considers the role of GABA in the circadian pacemaker, in the mechanisms responsible for the generation of circadian rhythms, in the ability of non-photic stimuli to reset the phase of the pacemaker, and in the ability of the day-night cycle to entrain the pacemaker. [ABSTRACT FROM AUTHOR]

Summary Reduced ocular pigmentation is common in Angelman syndrome (AS) and Prader-Willi syndrome (PWS) and is long thought to be caused by OCA2 deletion. GABRB3 is located in the 15q11-13 region flanked by UBE3A , GABRA5 , GABRG3 , and OCA2 . Mutations in GABRB3 have frequently been associated with epilepsy and autism, consistent with its role in neurodevelopment. We report here a robust phenotype in the mouse in which deletion of Gabrb3 alone causes nearly complete loss of retinal pigmentation due to atrophied melanosomes, as evidenced by electron microscopy. Using exome and RNA sequencing, we confirmed that only the Gabrb3 gene was disrupted while the Oca2 gene was intact. However, mRNA abundance of Oca2 and other genes adjacent to Gabrb3 is substantially reduced in Gabrb3 −/− mice, suggesting complex transcriptional regulation in this region. These results suggest that impairment in GABRB3 downregulates OCA2 and indirectly causes ocular hypopigmentation and visual defects in AS and PWS. [ABSTRACT FROM AUTHOR]

The relatively common view indicates a possible dissociation between the anxiolytic and sedative/hypnotic properties of benzodiazepines (BZs). Indeed, GABA A receptor (GABA A R) subtypes have specific cerebral distribution in distinct neural circuits. Thus, GABA A R subtype-selective drugs may be expected to perform distinct functions. However, standard behavioral test assays provide limited direction towards highlighting new action mechanisms of ligands targeting GABA A Rs. Automated behavioral tests, lack sensitivity as some behavioral characteristics or subtle behavioral changes of drug effects or that are not considered in the overall analysis (Ohl et al., 2001) and observation-based analyses are not always performed. In addition, despite the use of genetically engineered mice, any possible dissociation between the anxiolytic and sedative properties of BZs remains controversial. Moreover, the involvement the different subtypes of GABA A R subtypes in the anxious behavior and the mechanism of action of anxiolytic agents remains unclear since there has been little success in the pharmacological investigations so far. This raises the question of the involvement of the different subunits in anxiolytic-like and/or sedative effects; and the actual implication of these subunits, particularly, α-subunits in the modulation of sedation and/or anxiety-related disorders. This present review was prompted by several conflicting studies on the degree of involvement of these subunits in anxiolytic-like and/or sedative effects. To this end, we explored the GABAergic system, particularly, the role of different subunits containing synaptic GABA A Rs. We report herein the targeting gene encoding the different subunits and their contribution in anxiolytic-like and/or sedative actions, as well as, the mechanism underlying tolerance to BZs. [ABSTRACT FROM AUTHOR]

It is well established that cocaine-induced changes in glutamate receptor expression in the nucleus accumbens (NAc) play a significant role in animal models of cocaine addiction. Far less is known about cocaine-induced changes in GABA transmission, despite its importance in regulating NAc output via local interneurons and medium spiny neuron (MSN) axon collaterals (GABA ‘microcircuit’). Here we investigated whether GABA A receptor surface or total expression is altered following an extended-access cocaine self-administration regimen that produces a time-dependent intensification (incubation) of cue-induced cocaine craving in association with strengthening of AMPA receptor (AMPAR) transmission onto MSN. Rats self-administered cocaine or saline (control condition) 6 h/day for 10 days. NAc tissue was obtained and surface proteins biotinylated on three withdrawal days (WD) chosen to span incubation of craving and associated AMPAR plasticity: WD2, WD25 and WD48. Immunoblotting was used to measure total and surface expression of three GABA A receptor subunits (α1, α2, and α4) that are strongly expressed in the NAc. We found a transient increase in surface, but not total, expression of the α2 subunit on WD2 from cocaine self-administration, an effect that was no longer observed by WD25. The expression of α1 and α4 subunits was not altered at these withdrawal times. On WD48, when AMPAR transmission is significantly potentiated, we did not find any alteration in GABA A receptor surface or total expression. Our findings suggest that the strengthening of AMPAR-mediated glutamate transmission in the NAc is not accompanied by compensatory strengthening of GABAergic transmission through insertion of additional GABA A receptors. [ABSTRACT FROM AUTHOR]

Sudden unexpected death in epilepsy (SUDEP) is the leading cause for death in individuals with epilepsy. The frequency of SUDEP correlates with the severity of epilepsies and lack of response to antiepileptic drug treatment, but the underlying mechanisms of SUDEP have not been elucidated fully. GABRG2(Q390X) is a mutation associated with the epileptic encephalopathy Dravet syndrome (DS) and with genetic epilepsy with febrile seizures plus (GEFS+) in patients. The Gabrg2 +/Q390X knockin (KI) mouse phenocopies the major features of DS and GEFS+ and has SUDEP throughout life. The Gabrg2 +/− knockout (KO) mouse is associated with infrequent absence seizures and represents a model of mild absence epilepsy syndrome without increased mortality. To explore the basis for SUDEP in DS and GEFS+, we compared mutant γ2 subunit and wild-type α1 and β2/3 subunit expression in mice in brainstem nuclei associated with respiratory function including the solitary tract, pre-Botzinger complex and Kolliker-Fuse nuclei. We found that synaptic GABA A receptors were reduced while intracellular nonfunctional γ2(Q390X) subunits were increased in the heterozygous DS and GEFS+ KI mice, but not in the heterozygous absence epilepsy KO mice. Given the critical role of these nuclei in cardiorespiratory function, it is likely the impaired GABAergic transmission and neuronal dysfunction in these brainstem nuclei are involved in the cardiorespiratory collapse in SUDEP. The study provides novel mechanistic insights into cardiorespiratory failure of SUDEP. [ABSTRACT FROM AUTHOR]

GABA A receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs. Among the many modulatory compounds are also the intravenous anesthetics propofol and etomidate, and barbiturates. The mechanism of receptor modulation by these compounds is of mayor relevance. The site of action of these compounds has been located to subunit interfaces in the intra-membrane region of the receptor. In α 1 β 2 γ 2 GABA A receptors there are five such interfaces, two β+/α− and one each of α+/β−, α+/γ− and γ+/β− subunit interfaces. We have used reporter mutations located in the second trans-membrane region in different subunits to probe the effects of changes at these subunit interfaces on modulation by propofol, etomidate and pentobarbital. We provide evidence for the fact that each of these compounds either modulates through a different set of subunit interfaces or through the same set of subunit interfaces to a different degree. As a GABA A receptor pentamer harbors two β+/α− subunit interfaces, we used concatenated receptors to dissect the contribution of individual interfaces and show that only one of these interfaces is important for receptor modulation by etomidate. [ABSTRACT FROM AUTHOR]

The hypothalamo–pituitary–adrenal (HPA) axis plays a critical role in regulating responses to stress and long term dysregulation of the HPA axis is associated with higher rates of mood disorders. There are circumstances where the HPA axis is more or less responsive to stress. For example, during late pregnancy ACTH and corticosterone responses to stress are markedly suppressed, whereas in offspring born to mothers that experienced repeated stress during pregnancy, the HPA axis is hyper-responsive to stress. Neuroactive steroids such as allopregnanolone, tetrahydrodeoxycorticosterone (THDOC) and androstanediol can modulate HPA axis activity and concentrations of some neuroactive steroids in the brain are altered during pregnancy and following stress. Thus, here altered neurosteroidogenesis is proposed as a mechanism that could underpin the dynamic changes in HPA axis regulation typically observed in late pregnant and in prenatally stressed individuals. In support of this hypothesis, evidence in rats demonstrates that elevated levels of allopregnanolone in pregnancy induce a central inhibitory opioid mechanism that serves to minimize stress-induced HPA axis activity. Conversely, in prenatally stressed rodents, where HPA axis stress responses are enhanced, evidence indicates the capacity of the brain for neurosteroidogenesis is reduced. Understanding the mechanisms involved in adaptations in HPA axis regulation may provide insights for manipulating stress sensitivity and for developing therapies for stress-related disorders in humans. [ABSTRACT FROM AUTHOR]

The hypothalamic–pituitary–adrenal (HPA) and hypothalamic–pituitary–gonadal (HPG) axes are regulated by GABAergic signaling at the level of corticotropin-releasing hormone (CRH) and gonadotropin-releasing hormone (GnRH) neurons, respectively. Under basal conditions, activity of CRH and GnRH neurons are controlled in part by both phasic and tonic GABAergic inhibition, mediated by synaptic and extrasynaptic GABA A receptors (GABA A Rs), respectively. For CRH neurons, this tonic GABAergic inhibition is mediated by extrasynaptic, δ subunit-containing GABA A Rs. Similarly, a THIP-sensitive tonic GABAergic current has been shown to regulate GnRH neurons, suggesting a role for δ subunit-containing GABA A Rs; however, this remains to be explicitly demonstrated. GABA A Rs incorporating the δ subunit confer neurosteroid sensitivity, suggesting a potential role for neurosteroid modulation in the regulation of the HPA and HPG axes. Thus, stress-derived neurosteroids may contribute to the impact of stress on reproductive function. Interestingly, excitatory actions of GABA have been demonstrated in both CRH neurons at the apex of control of the HPA axis and in GnRH neurons which mediate the HPG axis, adding to the complexity for the role of GABAergic signaling in the regulation of these systems. Here we review the effects that stress has on GnRH neurons and HPG axis function alongside evidence supporting GABA A Rs as a major interface between the stress and reproductive axes. [ABSTRACT FROM AUTHOR]

The premammillary nucleus (PMM) of the turkey mediobasal hypothalamus, where dopamine-melatonin (DA-Mel) neurons are localized, is a site for photoreception and photoperiodic time measurement, which is essential for the initiation of avian reproductive seasonality. In addition, this area could also be responsible for the onset and maintenance of photorefractoriness at the end of the breeding season due to the enhanced inhibitory effect of γ-aminobutyric acid (GABA). GABA is an inhibitory neurotransmitter in the central nervous system which interferes with the photosexual response in the turkey, a seasonally breeding bird. Here, we further characterized the GABA A receptor subunits in the PMM DA-Mel neurons related to reproductive seasonality and the onset of photorefractoriness. GABA A receptor subunits and GABA synthesis enzymes in the PMM of photosensitive and photorefractory turkey hens were identified using real-time qRT-PCR. The upregulation of GABA A receptor α1-3, β2-3, γ1-3, ρ1-3, δ, and θ mRNA expression were observed in the PMM of photorefractory birds when compared to those of photosensitive ones while there is no change observed in the GABA synthesis enzymes, glutamate decarboxylase 1 and 2. Those upregulated GABA A receptor subunits were further examined using immunohistochemical staining and they appeared to be co-localized within the PMM DA-Mel neurons. The upregulation of GABA A receptor subunits observed in the PMM of photorefractory birds coincides with a lack of responsiveness to a light stimulus provided during the photosensitive phase. This is supported by the absence of c-fos induction and TH upregulation in the PMM and a subsequence inhibition of c-fos and GnRH-I expression in the nucleus commissurae pallii. The augmented GABA A receptor subunits expression may mediate an enhancement of inhibitory GABAergic neurotransmission and the subsequent interference with the photosexual response. This could contribute to the state of photorefractoriness and the termination of breeding activities in the turkey, a temperate zone bird. [ABSTRACT FROM AUTHOR]

Temporal lobe epilepsy (TLE), the most prevalent form of epilepsy, is often associated with drug-resistant seizures. In TLE, altered function of γ-aminobutyric acid (GABA) A receptors (GABA A Rs) results in potentiation of excitatory and/or failure of inhibitory neurotransmission, which contributes to seizure induction and propagation. Our previous study suggested that chloride channel-2 (Clc-2) contributed to chronically elevated tonic inhibition mediated by GABA A Rs in a rat model of TLE. In the present study, we used Clc-2 knockout mice to investigate further the role of Clc-2 and its interaction with tonic GABAergic inhibition in a model of TLE. The results revealed that knockout of Clc-2 decreased tonic seizure protection, latency of clonic seizure, seizure threshold and mortality protection in mice. Clc-2 knockout decreased the action potential (AP) peak and AP threshold , Clc-2 currents and GABA A R-mediated tonic inhibition in CA1 pyramidal neurons. Thus, the voltage-gated chloride channel Clc-2, which was functionally upregulated in CA1 pyramidal cells after seizures, may provide protection against TLE by its regulation of action potentials, Clc-2 currents and GABA A Rs in the CA1 region of the hippocampus. [ABSTRACT FROM AUTHOR]

Changes in functional GABA A R signaling in hippocampus have previously been evaluated using pre-clinical animal models of either diffuse brain injury or extreme focal brain injury that precludes measurement of cells located ipsilateral to injury. As a result, there is little information about the status of functional GABA A R signaling in dentate granule cells (DGCs) located ipsilateral to focal brain injury, where significant cellular changes have been documented. We used whole-cell patch-clamp recordings from hippocampal slices to measure changes in GABA A Rs in dentate granule cells (DGCs) at 1–2, 3–5, and 8–13 weeks after controlled cortical impact (CCI) brain injury. Synaptic and tonic GABA A R currents (I TonicGABA ) were measured in DGCs at baseline conditions and during application of the GABA A R agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride (THIP) to assess in the function of δ subunit-containing GABA A Rs. DGCs ipsilateral to CCI exhibited no changes in the amplitude of resting I TonicGABA relative to DGCs after sham-injury or contralateral to CCI. In contrast, there was a significant reduction in the THIP-evoked I TonicGABA in DGCs ipsilateral to CCI at both time-points. Tonic GABAergic inhibition of DGCs ipsilateral to injury also exhibited reduced responsiveness to the neurosteroid THDOC. I TonicGABA in DGCs ipsilateral to CCI did not exhibit a change in sensitivity to L655,708, an inverse agonist with selectivity for α 5 subunit-containing GABA A Rs, suggesting a lack of functional change in GABA A Rs containing this subunit. At the 8–13 week time-point, gene expression of GABA A R subunits expected to contribute to I TonicGABA (i.e., α 4 , α 5 and δ) was not significantly altered by CCI injury in isolated dentate gyrus. Collectively, these results demonstrate enduring functional changes in I TonicGABA in DGCs ipsilateral to focal brain injury that occur independent of altered gene expression. [ABSTRACT FROM AUTHOR]

Aside from their use in the treatment of anxiety disorders and insomnia, benzodiazepines and other GABA A receptor positive modulators are widely used as add-on treatments in schizophrenic and non-schizophrenic psychoses. However, there is relatively little direct clinical or pre-clinical evidence for antipsychotic effects of GABAergic medications. Previous studies have indicated that zolpidem, a GABAergic drug acting preferentially at α1-containing GABA A receptors, may produce catalepsy through interactions with dopaminergic neurotransmission. The aim of the present study was to investigate effects of zolpidem in experimental models of antipsychotic activity and extrapyramidal side effects in Wistar rats. Effects of zolpidem were compared with that of a classic benzodiazepine drug, diazepam and a second-generation antipsychotic medication, risperidone. High doses of risperidone (10.0 mg/kg, i.p.) and zolpidem (10.0 mg/kg, i.p.), but not diazepam, induced relatively short-lasting cataleptic responses in the bar test. Zolpidem and risperidone, but not diazepam, produced some antipsychotic-like effects at doses, which produced no catalepsy and did not inhibit spontaneous locomotor activity and apomorphine-induced stereotypies. The present results tend to indicate that zolpidem exerts some neuroleptic-like effects at doses, which do not produce motor side effects. Our findings may provide further rationale for the development of new subtype-selective GABA A receptor modulators for the treatment of psychotic symptoms. [ABSTRACT FROM AUTHOR]

Previous studies in our laboratory have shown that the neuron-specific specificity protein 4 (Sp4) transcriptionally regulates many excitatory neurotransmitter receptor subunit genes, such as those for GluN1, GluN2A, and GluN2B of N-methyl- d -aspartate (NMDA) receptors and Gria2 of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. It also regulates Atp1a1 and Atp1b1 subunit genes of Na + /K + -ATPase, a major energy-consuming enzyme, as well as all 13 subunits of cytochrome c oxidase (COX), an important energy-generating enzyme. Thus, there is a tight coupling between energy consumption, energy production, and excitatory neuronal activity at the transcriptional level in neurons. The question is whether inhibitory neurotransmitter receptors are also regulated by Sp4. In the present study, we tested our hypothesis that Sp4 regulates receptor subunit genes of a major inhibitory neurotransmitter, GABA, specifically GABA A receptors. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, real-time quantitative PCR, chromatin immunoprecipitation, promoter mutational analysis, over-expression and shRNA of Sp4, functional assays, and western blots, we found that Sp4 functionally regulates the transcription of Gabra1 (GABA A α1) and Gabra2 (GABA A α2), but not Gabra3 (GABA A α3) subunit genes. The binding sites of Sp4 are conserved among rats, humans, and mice. Thus, our results substantiate our hypothesis that Sp4 plays a key role in regulating the transcription of GABA A receptor subunit genes. They also indicate that Sp4 is in a position to transcriptionally regulate the balance between excitatory and inhibitory neurochemical expressions in neurons. [ABSTRACT FROM AUTHOR]

The goal of this study was to determine whether there are region-specific or time-dependent changes in GABA-mediated synaptic inhibition of principal neurons in the hippocampus during in vivo status epilepticus. Standard whole cell patch clamp electrophysiological techniques were used to characterize miniature inhibitory postsynaptic currents (mIPSCs) in recordings from the principal neurons (PNs) of the dentate gyrus, CA1, and CA3 in acutely-obtained hippocampal slices from control and lithium/pilocarpine-induced status epilepticus(SE)-treated animals. The reduction in mIPSC amplitude was pervasive across the 3 regions examined in hippocampal slices obtained after 60 min (late) or just 15 min after the onset of SE. The mIPSC frequency was reduced in all 3 regions after 60 min and 2 regions (dentate, CA1) after 15 min. These findings lend further support to the hypothesis that a rapid modification of the postsynaptic GABA A receptor population leads to a widespread decline in GABA-mediated inhibition that, in part, contributes to both the self-sustaining nature of SE and to the decrease in the efficacy of benzodiazepines. [ABSTRACT FROM AUTHOR]

Introduction The purpose of these studies was to describe a novel application of an automated data acquisition/data reduction system, DanioVision™ by Noldus. DanioVision™ has the ability to detect changes in locomotor activity in third instar Drosophila melanogaster larvae. The noncompetitive GABA A receptor antagonist picrotoxin (PTX), was used as a pharmacologic agent to decrease locomotor activity. Methods Two strains of Drosophila were used in these studies; wild-type flies and flies with a mutation in the Rdl gene ( Rdl MD-RR ). Rdl MD-RR Drosophila are naturally occurring mutants that express an aberrant form of the GABA A receptor, which has a lower affinity for PTX, but not GABA itself. Larvae, extracted from food in 20% sucrose, were randomly placed into vials containing vehicle or PTX (0.03–3 mM). After incubation of 2–24 h, individual larvae were put in each well of a 6-well culture plate previously coated with 2% agar, the plate was then placed in the DanioVision™ apparatus. The activity of individual larva was recorded for 5 min, digitized and analyzed using Ethovision® XT software. Results Incubation of third instar wild-type larvae in 1 mM PTX for 4 or 24 h decreased activity; whereas, a 2 h incubation in PTX was without effect. PTX caused a concentration-dependent decrease in activity as demonstrated by consistently reduced locomotor activity with 1.0 and 3.0 mM: 0.3 mM resulted in variable decreases in locomotor activity and 0.03 mM yielded no effect. By contrast, PTX did not affect activity in Rdl MD-RR larvae even at the highest concentration, 3.0 mM. Discussion Using an automated data acquisition system, it was found that PTX decreases activity in third instar Drosophila larvae due to a selective blockade of the GABA A receptor. The method will reduce the likelihood of human error and bias, as well as increase the speed and ease of data collection and analysis. [ABSTRACT FROM AUTHOR]

Absence seizures are known to result from disturbances within the cortico-thalamocortical network, which remains partially synchronous under normal conditions but switches to a state of hypersynchronicity and hyperexcitability during absence seizures. There is evidence to suggest that impaired GABAergic inhibitory function within the thalamus could contribute to the generation of hypersynchronous oscillations in some animal models of absence epilepsy. Recently, we demonstrated region-specific alterations in the tissue expression level of GABA A receptors (GABA A Rs) α1 and β2 subunits within the thalamus of the stargazer mouse model of absence epilepsy. In the present study we investigated whether changes in these subunits also occur at synapses in the ventral posterior (VP) complex where they are components of phasic GABA A R receptors. Postembedding immunogold cytochemistry and electron microscopy were used to analyze the relative synaptic expression of α1 and β2 subunits in the VP thalamic region in epileptic stargazer mice compared to their non-epileptic littermates. We show that there is a significant increase in expression of α1 and β2 subunits (53.6% and 45.8%, respectively) at synapses in the VP region of stargazers, indicative of an increase in phasic GABA A Rs at thalamocortical (TC) relay neurons. Furthermore, we investigated whether tissue expression of GABA A R subunits α4 and δ, which constitute part of tonic GABA A Rs in the VP region, is altered in the stargazer mouse. Semi-quantitative Western blotting showed a significant increase in GABA A R α4 and δ subunits in the VP region of stargazer thalamus, which would indicate an increase in tonic GABA A R expression. Our findings show that there are changes in the levels of both phasic and tonic GABA A Rs in the VP thalamus; altered GABAergic inhibition within the VP could be one of many mechanisms contributing to the generation of absence seizures in this model. [ABSTRACT FROM AUTHOR]

In clinical medicine, morphine is widely used to relieve many types of pain, but it has several side effects such as the development of tolerance and dependence. In order to decrease the side effects of morphine administration for the treatment of pain, the combination of minocycline as a glial inhibitor and morphine has been suggested in previous studies. It is important to understand which synaptic mechanisms are involved in the potentiative effect of minocycline on morphine antinociception. To this aim, male Wistar rats were bilaterally cannulated in the basolateral amygdala by srereotaxic instrument. A tail-flick apparatus was used to measure the pain threshold. The results revealed that intraperitoneal injection of morphine (2.5–7.5 mg/kg) induced antinociception. Intra-basolateral amygdala microinjection of minocycline (5–10 µg/rat) by itself had no effect on tail-flick latency, while the microinjection of the same doses of minocycline with an ineffective dose of morphine (5 mg/kg) induced antinociception. Intra-basolateral amygdala microinjection of different doses of muscimol (0.001–0.005 µg/rat) increased the minocycline-induced potentioation on morphine response in the tail-flick test. Intra-basolateral amygdala microinjection of muscimol by itself had no effect on tail-flick latency. On the other hand, intra-basolateral amygdala microinjection of bicuculline (0.01–0.1 µg/rat) inhibited minocycline-induced potentiation of morphine antinociception. It should be noted that intra-basolateral amygdala bicucculine by itself had no effect on tail-flick latency. It can thus be concluded that intra-basolateral amygdala minocycline potentiates morphine response in the tail-flick test. Moreover, basolateral amygdala GABAergic system may be involved in the minocycline-induced potentiation of morphine response via GABA A receptors. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Tilia tomentosa Moench bud extracts (TTBEs) is used in traditional medicine for centuries as sedative compound. Different plants belonging to the Tilia genus have shown their efficacy in the treatment of anxiety but still little is known about the mechanism of action of their bud extracts. Aim of the study To evaluate the action of TTBEs as anxiolytic and sedative compound on in vitro hippocampal neurons. Material and methods The anxiolytic effect of TTBEs was assayed by testing the effects of these compounds on GABA A receptor-activated chloride current of hippocampal neurons by means of the patch-clamp technique and microelectrode-arrays (MEAs). Results TTBEs acutely administered on mouse hippocampal neurons, activated a chloride current comparable to that measured in the presence of GABA (100 µM). Bicuculline (100 µM) and picrotoxin (100 µM) blocked about 90% of this current, while the remaining 10% was blocked by adding the benzodiazepine (BDZ) antagonist flumazenil (30 µM). Flumazenil alone blocked nearly 60% of the TTBEs activated current, suggesting that TTBEs binds to both GABA A and BDZ receptor sites. Application of high-doses of TTBEs on spontaneous active hippocampal neurons grown for 3 weeks on MEAs blocked the synchronous activity of these neurons. The effects were mimicked by GABA and prevented by picrotoxin (100 µM) and flumazenil (30 µM). At minimal doses, TTBEs reduced the frequency of synchronized bursts and increased the cross-correlation index of synchronized neuronal firing. Conclusions Our data suggest that TTBEs mimics GABA and BDZ agonists by targeting hippocampal GABAergic synapses and inhibiting network excitability by increasing the strength of inhibitory synaptic outputs. Our results contribute toward the validation of TTBEs as effective sedative and anxiolytic compound. [ABSTRACT FROM AUTHOR]

Central GABA A receptors mediate GABAergic phasic and tonic inhibition. While synaptic αβγ GABA A receptors primarily mediate phasic inhibition, extrasynaptic αβδ receptors play an important role in mediating tonic inhibition. Etomidate is a general anesthetic that produces its effects by enhancing GABA A receptor activity. We previously showed that etomidate modulates the gating of oocyte-expressed αβγ and αβδ receptors with similar overall allosteric impact, but different pharmacological patterns. In αβγ receptors, etomidate enhances apparent GABA sensitivity (reduces GABA EC 50 ), modestly increases maximal GABA efficacy, and slows current deactivation without affecting desensitization (Zhong et al., 2008). In αβδ receptors characterized by low GABA efficacy, etomidate dramatically increases responses to both low and maximal GABA. The effects of etomidate on desensitization and deactivation of αβδ receptors are unknown. To investigate the kinetic effects of etomidate on α1β3δ receptors of defined subunit arrangement, we expressed concatenated trimer (β3-α1-δ) and dimer (β3-α1) GABA A receptor subunit assemblies in human embryonic kidney (HEK)293T cells and recorded whole-cell voltage-clamp currents during rapid external solution exchanges. As expected, etomidate substantially increased maximal GABA-induced currents and prolonged deactivation. Moreover, desensitization was significantly decreased by etomidate. During prolonged GABA applications, etomidate enhanced steady-state currents more than peak currents. Thus, etomidate enhances tonic GABAergic inhibition through extrasynaptic αβδ receptors by both augmenting gating and reducing desensitization. [ABSTRACT FROM AUTHOR]

Tonic conductance mediated by extrasynaptic GABA A receptors has been implicated in the modulation of network oscillatory activity. Using an in vitro brain slice to produce oscillatory activity and a kinetic model of GABA A receptor dynamics, we show that changes in tonic inhibitory input to fast spiking interneurons underlie benzodiazepine-site mediated modulation of neuronal network synchrony in rat primary motor cortex. We found that low concentrations (10 nM) of the benzodiazepine site agonist, zolpidem, reduced the power of pharmacologically-induced beta-frequency (15–30 Hz) oscillatory activity. By contrast, higher doses augmented beta power. Application of the antagonist, flumazenil, also increased beta power suggesting endogenous modulation of the benzodiazepine binding site. Voltage-clamp experiments revealed that pharmacologically-induced rhythmic inhibitory postsynaptic currents were reduced by 10 nM zolpidem, suggesting an action on inhibitory interneurons. Further voltage-clamp studies of fast spiking cells showed that 10 nM zolpidem augmented a tonic inhibitory GABA A receptor mediated current in fast spiking cells whilst higher concentrations of zolpidem reduced the tonic current. A kinetic model of zolpidem-sensitive GABA A receptors suggested that incubation with 10 nM zolpidem resulted in a high proportion of GABA A receptors locked in a kinetically slow desensitized state whilst 30 nM zolpidem favoured rapid transition into and out of desensitized states. This was confirmed experimentally using a challenge with saturating concentrations of GABA. Selective modulation of an interneuron-specific tonic current may underlie the reversal of cognitive and motor deficits afforded by low-dose zolpidem in neuropathological states. [ABSTRACT FROM AUTHOR]

GABA A receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ− subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABA A receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ− site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs. [ABSTRACT FROM AUTHOR]

Drug-refractory status epilepticus (RSE) is a major medical emergency with a mortality of up to 40% and the risk of severe long-term consequences. The mechanisms involved in RSE are incompletely understood. Animal models are important in developing treatment strategies for more effective termination of SE and prevention of its long-term outcomes. The pilocarpine and lithium-pilocarpine rat models are widely used in this respect. In these models, resistance to diazepam and other antiseizure drugs (ASDs) develops during SE so that an SE that is longer than 30 min is difficult to suppress. Furthermore, because all ASDs used in SE treatment are much more rapidly eliminated by rodents than by humans, SE recurs several hours after ASD treatment. Long-term consequences include hippocampal damage, behavioral alterations, and epilepsy with spontaneous recurrent seizures. In this review, different rational polytherapies for SE, which are more effective than monotherapies, are discussed, including a novel polytherapy recently developed by our group. Based on data from diverse seizure models, we hypothesized that cholinergic mechanisms are involved in the mechanisms underlying ASD resistance of SE. We, therefore, developed an intravenous drug cocktail, consisting of diazepam, phenobarbital, and the anticholinergic scopolamine. This drug combination irreversibly terminated SE when administered 60, 90, or 120 min after SE onset. The efficacy of this cocktail in terminating SE was comparable with the previously reported efficacy of polytherapies with the glutamate receptor antagonist ketamine. Furthermore, when injected 60 min after SE onset, the scopolamine-containing cocktail prevented development of epilepsy and hippocampal neurodegeneration, which was not observed with high doses of diazepam or a combination of phenobarbital and diazepam. Our data add to the existing preclinical evidence that rational polytherapy can be more effective than monotherapy in the treatment of SE and that combinatorial therapy may offer a clinically useful option for the treatment of RSE. This article is part of a Special Issue entitled “Status Epilepticus”. [ABSTRACT FROM AUTHOR]

Abtract Chronic pain is a major public health problem categorized as inflammatory or neuropathic, each involving impaired GABAergic control in the spinal cord of mammals. (+)-Borneol, a bicyclic monoterpene present in the essential oil of plants, is used for analgesia and anesthesia in traditional Chinese medicine. It has been reported that (+)-borneol directly potentiates GABA activity at recombinant human GABA A receptors. Although borneol has antinociceptive effect on acute pain models, little is known about its effect on chronic pain and its mechanism. Here we report that (+)-borneol has remarkable anti-hyperalgesic effects on neuropathic and inflammatory pain in animal models. Neuropathic hypersensitivity was induced by segmental spinal nerve ligation (SNL), and inflammatory hypersensitivity was induced by intraplantar (i.pl.) injection of complete Freund׳s adjuvant (CFA). Both oral administration (125, 250 or 500 mg/kg) and intrathecal injection (i.t.) (15, 30 and 60 μg) of (+)-borneol reduced mechanical hypersensitivity dose-dependently in SNL and CFA models. The anti-hyperalgesic effects of (+)-borneol were abolished by a selective GABA A receptor (GABA A R) antagonist bicuculline (i.t., at 30 min after (+)-borneol injection). Furthermore, (+)-borneol (500 mg/kg, p.o. or 60 μg, i.t.) did not influence motor function. These findings suggest that (+)-borneol may ameliorate mechanical hyperalgesia by enhancing GABA A R-mediated GABAergic transmission in the spinal cord, and could serve as a therapeutic for chronic pain. [ABSTRACT FROM AUTHOR]

Etifoxine (EFX) is a non-benzodiazepine anxiolytic which potentiate GABA A receptor (GABA A R) function directly or indirectly via the production of 3α-reduced neurosteroids. The later effect is now recognized to account for the long-term reduction of pain symptoms in various neuropathic and inflammatory pain models. In the present study, we characterized the acute antinociceptive properties of EFX during spinal pain processing in naive and monoarthritic rats using in vivo electrophysiology. The topical application of EFX on lumbar spinal cord segment, at concentrations higher than 30 μM, reduced the excitability of wide dynamic range neurons receiving non-nociceptive and nociceptive inputs. Windup discharge resulting from the repetitive stimulation of the peripheral receptive field, and recognized as a short-term plastic process seen in central nociceptive sensitization, was significantly inhibited by EFX at these concentrations. In good agreement, mechanical nociceptive thresholds were also significantly increased following an acute intrathecal injection of EFX. The acute modulatory properties of EFX on spinal pain processing were never seen in the simultaneous presence of bicuculline. This result further confirmed EFX antinociception to result from the potentiation of spinal GABA A receptor function. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is involved in synaptic plasticity in the hippocampus through different presynaptic and postsynaptic mechanisms that include the modulation of the GABAergic neurotransmission. Inhibitory synapses on hippocampal pyramidal neurons are known to possess the molecular machinery for retrograde NO-signaling, but the modulation of GABA A Rs function by NO in these neurons and the mechanisms of action involved have not been fully characterized. Here we show that suppression of the endogenous NO generation by the nitric oxide synthase (NOS) inhibitor L-NAME produces significant and reversible increases in the magnitude of both tonic and phasic GABAergic currents in CA1 hippocampal pyramidal neurons. GABA-evoked chloride currents were measured in the presence or absence of L-NAME using whole-cell patch-clamp recordings in acute hippocampal slices from young adult mice. Enhancement of the tonic GABA responses induced by L-NAME was insensitive to TTX and decreased by co-incubation with the NO donor DEA/NO. Applications of DEA/NO alone did not produce significant effects on tonic GABA responses. L-NAME treatment also increased the amplitude of phasic GABAergic currents evoked by GABA-puffs. Our results indicate that the extent of tonic and phasic inhibition mediated by GABA A receptors in CA1 hippocampal pyramidal neurons is affected by endogenous NO production. [ABSTRACT FROM AUTHOR]

Although an extensive body of literature confirmed γ-aminobutyric acid (GABA) as mediator within the enteric nervous system (ENS) controlling gastrointestinal (GI) function, the true significance of GABAergic signalling in the gut is still a matter of debate. GABAergic cells in the bowel include neuronal and endocrine-like cells, suggesting GABA as modulator of both motor and secretory GI activity. GABA effects in the GI tract depend on the activation of ionotropic GABA A and GABA C receptors and metabotropic GABA B receptors, resulting in a potential noteworthy regulation of both the excitatory and inhibitory signalling in the ENS. However, the preservation of GABAergic signalling in the gut could not be limited to the maintenance of physiologic intestinal activity. Indeed, a series of interesting studies have suggested a potential key role of GABA in the promising field of neuroimmune interaction, being involved in the modulation of immune cell activity associated with different systemic and enteric inflammatory conditions. Given the urgency of novel therapeutic strategies against chronic immunity-related pathologies, i.e. multiple sclerosis and Inflammatory Bowel Disease, an in-depth comprehension of the enteric GABAergic system in health and disease could provide the basis for new clinical application of nerve-driven immunity. Hence, in the attempt to drive novel researches addressing both the physiological and pathological importance of the GABAergic signalling in the gut, we summarized current evidence on GABA and GABA receptor function in the different parts of the GI tract, with particular focus on the potential involvement in the modulation of GI motility and inflammation. [ABSTRACT FROM AUTHOR]

Based on the diverse array of anaesthetic structures, a single anaesthetic target site seems unlikely. With the knowledge that anaesthesia likely results from central nervous system depression, it can be hypothesized that anaesthesia results from either enhanced inhibitory transmission or reduced excitatory transmission. Two main targets have been extensively described: GABA A receptors and N-methyl- d -aspartate (NMDA) glutamate receptors. On γ-aminobutyric acid (GABA) binding to GABA A receptors, an influx of Cl − results to produce hyperpolarization. With the exception of ketamine, xenon and nitrous oxide, all anaesthetic agents potentiate GABA-mediated conductance. On binding of the main excitatory transmitter glutamate, NMDA receptors gate an influx of Ca 2+ and Na + . Ketamine, xenon and nitrous oxide inhibit this ion movement to depress excitatory transmission. [ABSTRACT FROM AUTHOR]

GABA A receptors are pentameric chloride ion channels that are opened by GABA. We have screened a cell line derived from human glioblastoma, U3047MG, for expression of GABA A receptor subunit isoforms and formation of functional ion channels. We identified GABA A receptors subunit α2, α3, α5, β1, β2, β3, δ, γ3, π, and θ mRNAs in the U3047MG cell line. Whole-cell GABA-activated currents were recorded and the half-maximal concentration (EC 50 ) for the GABA-activated current was 36 μM. The currents were activated by THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) and enhanced by the benzodiazepine diazepam (1 μM) and the general anesthetics etomidate and propofol (50 μM). In line with the expressed GABA A receptors containing at least the α3β3θ subunits, the receptors were highly sensitive to etomidate (EC 50 =55 nM). Immunocytochemistry identified expression of the α3 and β3 subunit proteins. Our results show that the GABA A receptors in the glial cell line are functional and are modulated by classical GABA A receptor drugs. We propose that the U3047MG cell line may be used as a model system to study GABA A receptors function and pharmacology in glial cells. [ABSTRACT FROM AUTHOR]