The linkage between agonist binding and the activation of a GABA(A) receptor ion channel is yet to be resolved. This aspect was examined on human recombinant alpha1beta2gamma2S GABA(A) receptors expressed in human embryonic kidney cells using the following series of receptor agonists: GABA, isoguvacine, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), isonipecotic acid, piperidine-4-sulphonic acid (P4S), imidazole-4-acetic acid (IAA), 5-(4-piperidyl)-3-isothiazolol (thio-4-PIOL) and 5-(4-piperidyl)-3-isoxazolol (4-PIOL). Whole-cell concentration-response curves enabled the agonists to be categorized into four classes based upon their maximum responses. Single channel analyses revealed that the channel conductance of 25-27 pS was unaffected by the agonists. However, two open states were resolved from the open period distributions with mean open times reduced 5-fold by the weakest partial agonists. Using saturating agonist concentrations, estimates of the channel shutting rate, alpha, ranged from 200 to 600 s(-1). The shut period distributions were described by three or four components and for the weakest partial agonists, the interburst shut periods increased whilst the mean burst durations and longest burst lengths were reduced relative to the full agonists. From the burst analyses, the opening rates for channel activation, beta, and the total dissociation rates, k(-1), for the agonists leaving the receptor were estimated. The agonist efficacies were larger for the full agonists (E approximately 7-9) compared to the weak partial agonists ( approximately 0.4-0.6). Overall, changes in agonist efficacy largely determined the different agonist profiles with contributions from the agonist affinities and the degree of receptor desensitization. From this we conclude that GABA(A) receptor activation does not occur in a switch-like manner since the agonist recognition sites are flexible, accommodating diverse agonist structures which differentially influence the opening and shutting rates of the ion channel.

A series of 6-aminonicotinic acid analogues have been synthesized and pharmacologically characterized at native and selected recombinant GABA(A) receptors. 6-Aminonicotinic acid (3) as well as 2- and 4-alkylated analogues (9-11, 14-16) display low to mid-micromolar GABA(A)R binding affinities to native GABA(A) receptors (K(i) 1.1-24 μM). The tetrahydropyridine analogue of 3 (22) shows low-nanomolar affinity (K(i) 0.044 μM) and equipotency as an agonist to GABA itself as well as the standard GABA(A) agonist isoguvacine. Cavities surrounding the core of the GABA binding pocket were predicted by molecular interaction field calculations and docking studies in a α1β2γ2 GABA(A) receptor homology model, and were confirmed by affinities of substituted analogues of 3. The tight steric requirements observed for the remarkably few GABA(A)R agonists reported to date is challenged by our findings. New openings for agonist design are proposed which potentially could facilitate the exploration of different pharmacological profiles within the GABA(A)R area.

Autism spectrum disorder (ASD) is an immensely challenging developmental disorder characterized primarily by two core behavioral symptoms of social communication deficits and restricted/repetitive behaviors. Investigating the etiological process and identifying an appropriate therapeutic target remain as formidable challenges to overcome ASD due to numerous risk factors and complex symptoms associated with the disorder. Among the various mechanisms that contribute to ASD, the maintenance of excitation and inhibition balance emerged as a key factor to regulate proper functioning of neuronal circuitry. In this study, we employed prenatally exposed to valproic acid (VPA) to establish a validated ASD mouse model and found impaired inhibitory gamma-aminobutyric acid (GABAergic) neurotransmission through a presynaptic mechanism in these model mice, which was accompanied with decreased GABA release and GABA-A and GABA-B receptor subunits expression. And acute administration of individual GABA-A or GABA-B receptor agonists partially reversed autistic-like behaviors in the model mice. Furthermore, acute administration of the combined GABA-A and GABA-B receptor agonists palliated sociability deficits, anxiety and repetitive behaviors in the animal model of autistic-like behaviors, demonstrating the therapeutic potential of above cocktail in the treatment of ASD.

1. Activation of GABA(B) receptors evokes hypothermia in wildtype (GABA(B(1))+/+) but not in GABA(B) receptor knockout (GABA(B(1))-/-) mice. The aim of the present study was to determine the hypothermic and behavioural effects of the putative GABA(B) receptor agonist gamma-hydroxybutyrate (GHB), and of the GABA(A) receptor agonist muscimol. In addition, basal body temperature was determined in GABA(B(1))+/+, GABA(B(1))+/- and GABA(B(1))-/- mice. 2. GABA(B(1))-/- mice were generated by homologous recombination in embryonic stem cells. Correct gene targeting was assessed by Southern blotting, PCR and Western blotting. GABA(B) receptor-binding sites were quantified with radioligand binding. Measurement of body temperature was done using subcutaneous temperature-sensitive chips, and behavioural changes after drug administration were scored according to a semiquantitative scale. 3. GABA(B(1))-/- mice had a short lifespan, probably caused by generalised seizure activity. No histopathological or blood chemistry changes were seen, but the expression of GABA(B(2)) receptor protein was below the detection limit in brains from GABA(B(1))-/- mice, in the absence of changes in mRNA levels. 4. GABA(B) receptor-binding sites were absent in brain membranes from GABA(B(1))-/- mice. 5. GABA(B(1))-/- mice were hypothermic by approximately 1 degrees C compared to GABA(B(1))+/+ and GABA(B(1))+/- mice. 6. Injection of baclofen (9.6 mg kg-1) produced a large reduction in body temperature and behavioural effects in GABA(B(1))+/+ and in GABA(B(1))+/- mice, but GABA(B(1))-/- mice were unaffected. The same pattern was seen after administration of GHB (400 mg kg-1). The GABA(A) receptor agonist muscimol (2 mg kg-1), on the other hand, produced a more pronounced hypothermia in GABA(B(1))-/-mice. In GABA(B(1))+/+ and GABA(B(1))+/- mice, muscimol induced sedation and reduced locomotor activity. However, when given to GABA(B(1))-/- mice, muscimol triggered periods of intense jumping and wild running. 7. It is concluded that hypothermia should be added to the characteristics of the GABAB(1)-/-phenotype. Using this model, GHB was shown to be a selective GABAB receptor agonist. In addition, GABAB(1)-/- mice are hypersensitive to GABAA receptor stimulation, indicating that GABAB tone normally balances GABAA-mediated effects.

Benzodiazepines (BDZs) are known to increase the amplitude and duration of IPSCs. Moreover, at low [GABA], BDZs strongly enhance GABAergic currents suggesting the up-regulation of agonist binding while their action on gating remains a matter of debate. In the present study we have examined the impact of flurazepam and zolpidem on mIPSCs by investigating their effects on GABA(A)R binding and gating and by considering dynamic conditions of synaptic receptor activation. Flurazepam and zolpidem enhanced the amplitude and prolonged decay of mIPSCs. Both compounds strongly enhanced responses to low [GABA] but, surprisingly, decreased the currents evoked by saturating or half-saturating [GABA]. Analysis of current responses to ultrafast GABA applications indicated that these compounds enhanced binding and desensitization of GABA(A) receptors. Flurazepam and zolpidem markedly prolonged deactivation of responses to low [GABA] but had almost no effect on deactivation at saturating or half-saturating [GABA]. Moreover, at low [GABA], flurazepam enhanced desensitization-deactivation coupling but zolpidem did not. Recordings of responses to half-saturating [GABA] applications revealed that appropriate timing of agonist exposure was sufficient to reproduce either a decrease or enhancement of currents by flurazepam or zolpidem. Recordings of currents mediated by recombinant ('synaptic') alpha1beta2gamma2 receptors reproduced all major findings observed for neuronal GABA(A)Rs. We conclude that an extremely brief agonist transient renders IPSCs particularly sensitive to the up-regulation of agonist binding by BDZs.

The activation characteristics of synaptic and extrasynaptic GABA(A) receptors are important for shaping the profile of phasic and tonic inhibition in the central nervous system, which will critically impact on the activity of neuronal networks. Here, we study in isolation the activity of three agonists, GABA, muscimol and 4,5,6,7-tetrahydoisoxazolo[5,4-c]pyridin-3(2H)-one (THIP), to further understand the activation profiles of alpha 1 beta 3 gamma 2, alpha 4 beta 3 gamma 2 and alpha 4 beta 3 delta receptors that typify synaptic- and extrasynaptic-type receptors expressed in the hippocampus and thalamus. The agonists display an order of potency that is invariant between the three receptors, which is reliant mostly on the agonist dissociation constant. At delta subunit-containing extrasynaptic-type GABA(A) receptors, both THIP and muscimol additionally exhibited, to different degrees, superagonist behaviour. By comparing whole-cell and single channel currents induced by the agonists, we provide a molecular explanation for their different activation profiles. For THIP at high concentrations, the unusual superagonist behaviour on alpha 4 beta 3 delta receptors is a consequence of its ability to increase the duration of longer channel openings and their frequency, resulting in longer burst durations. By contrast, for muscimol, moderate superagonist behaviour was caused by reduced desensitisation of the extrasynaptic-type receptors. The ability to specifically increase the efficacy of receptor activation, by selected exogenous agonists over that obtained with the natural transmitter, may prove to be of therapeutic benefit under circumstances when synaptic inhibition is compromised or dysfunctional.

gamma-Hydroxybutyrate (GHB) is believed to function as a neurotransmitter in the mammalian brain by binding to a GHB-specific binding site. In addition, GHB may also indirectly enhance the neuroinhibitory actions of gamma-aminobutyric acid (GABA) by converting to GABA at neuronal synapses. The purpose of the present study was to examine the effects of representative GABA(A) and GABA(B) receptor agonists in rats treated chronically with GHB. Using a rotorod apparatus, the motor-impairing effects of GHB, the indirect GABA(A) receptor agonist, flunitrazepam, and the direct GABA(B) receptor agonist, baclofen, were examined before, during and after chronic treatment with 1000 mg/kg GHB, b.i.d. Prior to chronic treatment, all three drugs produced dose-dependent decreases in motor performance at low (8 rpm) and high (32 rpm) rotational speeds. Chronic treatment with GHB significantly decreased the potency of baclofen at both speeds, but did not alter the potency of either GHB or flunitrazepam. Following termination of chronic treatment, the potency of baclofen increased significantly at both speeds and returned to that observed prior to chronic treatment. These data indicate that chronic treatment with GHB confers tolerance to a GABA(B) receptor agonist under conditions in which tolerance is not conferred to a GABA(A) receptor agonist. These findings are consistent with the in vivo behavioral profile of GHB, which reveals a greater role for GABA(B) receptors than for GABA(A) receptors in its behavioral effects.

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of vertebrate Cys-loop ligand-gated ion channels. It is activated by GABA, and this property, combined with its structural similarity to GABA(A) and other Cys-loop receptors, makes it potentially an excellent model to probe their structure and function. Here we characterise the pharmacological profile of ELIC, examining the effects of compounds that could activate or inhibit the receptor. We confirm that a range of amino acids and classic GABA(A) receptor agonists do not elicit responses in ELIC, and we show the receptor can be at least partially activated by 5-aminovaleric acid and γ-hydroxybutyric acid, which are weak agonists. A range of GABA(A) receptor non-competitive antagonists inhibit GABA-elicited ELIC responses including α-endosulfan (IC₅₀ = 17 μM), dieldrin (IC₅₀ = 66 μM), and picrotoxinin (IC₅₀ = 96 μM) which were the most potent. Docking suggested possible interactions at the 2' and 6' pore-lining residues, and mutagenesis of these residues supports this hypothesis for α-endosulfan. A selection of compounds that act at Cys-loop and other receptors also showed some efficacy at blocking ELIC responses, but most were of low potency (IC₅₀ > 100 μM). Overall our data show that a number of compounds can inhibit ELIC, but it has limited pharmacological similarity to GLIC and to Cys-loop receptors.

Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis) at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P(+)) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2'-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P(+) foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21(Waf1/Cip1), p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P(+) foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P(+) foci by activating GABA(A)R-mediated signaling.

In previous work our group described the synthesis and the activity on rat cerebellum granule cell GABAA receptors of new 1,5-benzodiazepine compounds. Here we are describing the synthesis of new triazolobenzodiazepines (mainly 1,5-benzodiazepine derivatives) and the evaluation of their biological activity in terms of effects on those GABAA receptors. Their effects were compared to those of 1,4-benzodiazepine agonists and some known 1,5-benzodiazepines. The activities were evaluated for the two GABAA receptor populations present in cerebellar granule cells, one mediating phasic inhibition and the other one mediating tonic inhibition. Some of the compounds displayed a profile of agonist at the component mediating phasic inhibition. This agonistic activity was prevented by the benzodiazepine site antagonist flumazenil. Interestingly, the active compounds displayed an agonistic activity at these receptors significantly greater than that of "classical" 1,4-benzodiazepine agonists, such as diazepam, flunitrazepam and alprazolam.

Using systematic combination of alpha 1, alpha 3, and alpha 5 with beta 1, beta 2, and beta 3, together with gamma 1, gamma 2, and gamma 3, we have investigated the contributions of the various alpha, beta, and gamma subunits to the pharmacology of gamma-aminobutyric acid (GABA)A agonists. We have characterized GABA, (RS)-dihydromuscimol, piperidine-4-sulfonic acid, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol with recombinant human GABAA receptors expressed in Xenopus oocytes. Our observations indicate that the alpha subunit is the major determinant of efficacy for partial GABAA agonists. When alpha 1 and alpha 3 or alpha 1 and alpha 5 are coexpressed, the alpha 1 subunit determines the maximum efficacy, whereas the affinity is determined by the entire combination of subunits. Thus, the results of the present study demonstrate that the pharmacology of GABAA agonists is dependent on the subunit composition of the GABAA receptor complex. Functional GABAA receptors containing two different alpha subunits show pharmacological profiles distinctly different from those of receptors containing a single alpha subtype, indicating that two different alpha subunits can be coexpressed in one functional GABAA receptor complex.

We present a full-length α(1)β(2)γ(2) GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate-gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode similar to the binding mode of glutamate in the GluCl X-ray structure. Key interactions are predicted with residues α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and the backbone of β(2)S156. Muscimol is predicted to bind similarly, however, with minor differences rationalized with quantum mechanical energy calculations. Muscimol key interactions are predicted to be α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and β(2)F200. Furthermore, we argue that a water molecule could mediate further interactions between muscimol and the backbone of β(2)S156 and β(2)Y157. DZP is predicted to bind with interactions comparable to those of the agonists in the orthosteric site. The carbonyl group of DZP is predicted to interact with two threonines α(1)T206 and γ(2)T142, similar to the acidic moiety of GABA. The chlorine atom of DZP is placed near the important α(1)H101 and the N-methyl group near α(1)Y159, α(1)T206, and α(1)Y209. We present a binding mode of DZP in which the pending phenyl moiety of DZP is buried in the binding pocket and thus shielded from solvent exposure. Our full length GABA(A) receptor is made available as Model S1.

1. The actions of adenosine A(2A) receptor agonists were examined on GABAergic synaptic transmission in the globus pallidus (GP) in rat brain slices using whole-cell patch-clamp recording. GP neurones were characterized into two major groups, type I and type II, according to the degree of time-dependent hyperpolarization-activated inward rectification and the size of input resistance. 2. The A(2A) receptor agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido- adenosine (CGS21680; 0.3-3 microM) enhanced IPSCs evoked by stimulation within the GP. The actions of CGS21680 were blocked by the A(2A) antagonists (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). 3. The CGS21680-induced increase in IPSCs was associated with a reduction in paired-pulse facilitation. CGS21680 (0.3 microM) increased the frequency of miniature IPSCs (mIPSCs) without affecting mIPSC amplitude. These observations demonstrated that the enhancement of IPSCs in the GP was attributable to presynaptic, but not postsynaptic, A(2A) receptors. 4. The results suggest that A(2A) receptors in the GP serve to inhibit GP neuronal activity, thereby disinhibiting subthalamic nucleus neurone activity. Thus, the A(2A) receptor-mediated presynaptic regulation in the GP, together with the A(2A) receptor-mediated intrastriatal presynaptic control of GABAergic neurotransmission described previously, may play a crucial role in controlling the neuronal functions of basal ganglia. This A(2A) receptor-mediated presynaptic dual control in the striatopallidal pathway could also afford the mode of action of A(2A) antagonists for ameliorating the symptoms of Parkinson's disease in an animal model.

GABA(A) receptor activation by muscimol has sex and age specific effects on substantia nigra reticulata (SNR)-mediated control of generalized seizures. GABA(A) receptor agonists depolarize or hyperpolarize neurons depending upon the level of expression of the neuronal specific potassium chloride contransporter KCC2. We studied KCC2 mRNA expression in the SNR as a function of sex and age and correlated KCC2 expression with the in vivo and in vitro effects of muscimol. Methods included in situ hybridization, gramicidin-perforated patch clamp and fura-2 AM imaging of acute SNR slices. KCC2 mRNA expression increased between postnatal days (PN) 15 and 30 in both sexes, and reached adult levels in males by PN30. Female PN15 and PN30 SNR neurons contained more KCC2 mRNA compared with age-matched males. In male PN14-17 rats, bath application of the GABA(A) receptor agonist muscimol in acute SNR slices depolarized neurons and increased intracellular calcium concentration ([Ca(2+)](i)). Furthermore, acute in vivo administration of muscimol upregulated, whereas blockade of L-type voltage sensitive calcium channels with nifedipine downregulated KCC2 mRNA. In contrast, in female PN14-17 rats, bath application of muscimol hyperpolarized SNR neurons and did not alter [Ca(2+)](i). In vivo muscimol administration acutely downregulated KCC2 mRNA expression whereas nifedipine had no effect. The lower expression of KCC2 mRNA in infantile male SNR neurons may explain why muscimol-induced depolarization and [Ca(2+)](i) increases occur only in males. Consequently, GABA(A) receptor activation selectively upregulates the expression of calcium-regulated genes, such as KCC2, in male SNR, promoting the sexual differentiation of the SNR.

Orexins (ORXs) cross-talking with γ-aminobutyric acid(A) receptor (GABA(A)R) is beginning to constitute a key neuronal signaling feature responsible for the successful promotion of sleep-wake cycle, feeding and motor behaviors plus reward/motivational activities. In this work, ORX-A and the two α GABA(A)R agonists (zolpidem, ZOL; diazepam, DZP) accounted for very great (p<0.001) increases of feeding while only DZP elicited great (p<0.01) levels of food intake in the goldfish (Carassius auratus). It was, however, T-maze and conditioned place preference (CPP) methods that allowed us to specifically establish learning/reward-related events operating in an ORX-A+GABA(A)R-dependent fashion in our experimental model. T-maze data showed that conditioned ORX-A treated-fish were capable of reaching the red/blue chamber and ingesting their food reward in a very greatly reduced latency time with respect to untreated conditioned fish while DZP and ZOL greatly and moderately (p<0.05) reduced their latency time, respectively. Regarding CPP study, conditioned ORX-A- and DZP-treated animals showed comparably greater preferences for the conditioned compartment that became even greater in ORX-A+DZP-treated fish. Surprisingly, ORX receptor expression of the telencephalon was preferentially activated by ORX-A treatments while diencephalic/mesencephalic structures and namely the tuberculum posterioris (TPp) were more sensitive to DZP especially following treatment with ORX-A+DZP. Overall, behavioral performances along with ORX receptor transcriptional properties tend to point to α GABA(A)R agonists as enhancers of palatability while the ORXergic system constitutes a crucial link between satiety-related and cognitive centers through the activation of TPp thus proposing this ascending dopaminergic system as a key target of learning/reward processes in fish.

Hypocretin/orexin (Hcrt) is a critical neurotransmitter for the maintenance of wakefulness and has been implicated in several other functions, including energy metabolism and reward. Using whole-cell patch-clamp recordings from transgenic mice in which enhanced green fluorescent protein was linked to the Hcrt promoter, we investigated GABAergic control of the Hcrt neurones in hypothalamic slices. Bath application of GABA or muscimol caused an early hyperpolarization mediated by Cl(-) and a late depolarization mediated by the efflux of bicarbonate. These GABA(A) receptor-mediated responses were blocked by picrotoxin and bicuculline. Under the GABA(A) blockade condition, GABA produced consistent hyperpolarization, decreased firing rate and input resistance. The selective GABA(B) agonist (R)-baclofen caused a similar response with an EC(50) of 7.1 mum. The effects of (R)-baclofen were blocked by the GABA(B) antagonist CGP 52432 but persisted in the presence of tetrodotoxin, suggesting direct postsynaptic effects. The existence of GABA(B) modulation was supported by GABA(B(1)) subunit immunoreactivity on Hcrt cells colabelled with antisera to the Hcrt-2 peptide. Furthermore, GABA(B) receptor activation inhibited the presynaptic release of both glutamate and GABA. (R)-Baclofen depressed the amplitude of evoked excitatory postsynaptic currents (EPSCs) and inhibitory synaptic currents (IPSCs), and also decreased the frequency of both spontaneous and miniature EPSCs and IPSCs with a modest effect on their amplitudes. These data suggest that GABA(B) receptors modulate Hcrt neuronal activity via both pre- and postsynaptic mechanisms, which may underlie the promotion of non-rapid eye movement sleep and have implications for the use of GABA(B) agonists in the treatment of substance addiction through direct interaction with the Hcrt system.

Inflammatory processes are known to contribute to tissue damage in the central nervous system (CNS) across a broad range of neurological conditions, including stroke. Gamma amino butyric acid (GABA), the main inhibitory neurotransmitter in the CNS, has been implicated in modulating peripheral immune responses by acting on GABA A receptors on antigen-presenting cells and lymphocytes. Here, we investigated the effects and mechanism of action of the delta-selective compound, DS2, to improve stroke recovery and modulate inflammation. We report a decrease in nuclear factor (NF)-κB activation in innate immune cells over a concentration range in vitro. Following a photochemically induced motor cortex stroke, treatment with DS2 at 0.1 mg/kg from 1 h post-stroke significantly decreased circulating tumor necrosis factor (TNF)-α, interleukin (IL)-17, and IL-6 levels, reduced infarct size and improved motor function in mice. Free brain concentrations of DS2 were found to be lower than needed for robust modulation of central GABA A receptors and were not affected by the presence and absence of elacridar, an inhibitor of both P-glycoprotein and breast cancer resistance protein (BCRP). Finally, as DS2 appears to dampen peripheral immune activation and only shows limited brain exposure, we assessed the role of DS2 to promote functional recovery after stroke when administered from 3-days after the stroke. Treatment with DS2 from 3-days post-stroke improved motor function on the grid-walking, but not on the cylinder task. These data highlight the need to further develop subunit-selective compounds to better understand change in GABA receptor signaling pathways both centrally and peripherally. Importantly, we show that GABA compounds such as DS2 that only shows limited brain exposure can still afford significant protection and promote functional recovery most likely via modulation of peripheral immune cells and could be given as an adjunct treatment.

The flavonoid myricetin is found in several sedative herbs, for example, the St. John's Wort, but its influence on sedation and its possible mechanism of action are unknown. Using patch-clamp technique on a brain slice preparation, the present study found that myricetin promoted GABAergic activity in the neurons of hypothalamic paraventricular nucleus (PVN) by increasing the decay time and frequency of the inhibitory currents mediated by GABA(A) receptor. This effect of myricetin was not blocked by the GABA(A) receptor benzodiazepine- (BZ-) binding site antagonist flumazenil, but by KN-62, a specific inhibitor of the Ca(2+)/calmodulin-stimulated protein kinase II (CaMK-II). Patch clamp and live Ca(2+) imaging studies found that myricetin could increase Ca(2+) current and intracellular Ca(2+) concentration, respectively, via T- and L-type Ca(2+) channels in rat PVN neurons and hypothalamic primary culture neurons. Immunofluorescence staining showed increased phosphorylation of CaMK-II after myricetin incubation in primary culture of rat hypothalamic neurons, and the myricetin-induced CaMK-II phosphorylation was further confirmed by Western blotting in PC-12 cells. The present results suggest that myricetin enhances GABA(A) receptor activity via calcium channel/CaMK-II dependent mechanism, which is distinctively different from that of most existing BZ-binding site agonists of GABA(A) receptor.

It is unclear whether GABA(A) receptor-mediated hyperpolarizing and depolarizing synaptic potentials (IPSP(A)s and DPSP(A)s, respectively) are evoked by (a) the same populations of GABAergic interneurones and (b) exhibit similar regulation by allosteric modulators of GABA(A) receptor function. We have attempted to address these questions by investigating the effects of (a) known agonists for presynaptic receptors on GABAergic terminals, and (b) a range of GABA(A) receptor ligands, on each response. The GABA uptake inhibitor NNC 05-711 (10 microM) enhanced whereas bicuculline (10 microM) inhibited both IPSP(A)s and DPSP(A)s. (-)-Baclofen (5 microM), [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO; 0.5 microM), and carbachol (10 microM) caused substantial depressions (up to 99%) of DPSP(A)s that were reversed by CGP 55845A (1 microM), naloxone (10 microM) and atropine (5 microM), respectively. In contrast, 2-chloroadenosine (CADO; 10 microM) only slightly depressed DPSP(A)s. Quantitatively, the effect of each agonist was similar to that reported for IPSP(A)s. The neurosteroid ORG 21465 (1 - 10 microM), the anaesthetic propofol (50-500 microM), the barbiturate pentobarbitone (100-300 microM) and zinc (50 microM) all enhanced DPSP(A)s and IPSP(A)s. The benzodiazepine (BZ) agonist flunitrazepam (10-50 microM) and inverse agonist DMCM (1 microM) caused a respective enhancement and inhibition of both IPSP(A)s and DPSP(A)s. The BZomega1 site agonist zolpidem (10-30 microM) produced similar effects to flunitrazepam. The anticonvulsant loreclezole (1-100 microM) did not affect either response. These data demonstrate that similar populations of inhibitory interneurones can generate both IPSP(A)s and DPSP(A)s by activating GABA(A) receptors that are subject to similar allosteric modulation.

Emerging evidence has implicated a potential role for 5-HT(4) receptors in cognition and anxiolysis. One of the main target structures of 5-HT(4) receptors on 'cognitive and emotional' pathways is the prefrontal cortex (PFC). As GABAergic signalling plays a key role in regulating PFC functions, we examined the effect of 5-HT(4) receptors on GABA(A) receptor channels in PFC pyramidal neurons. Application of 5-HT(4) receptor agonists produced either an enhancement or a reduction of GABA-evoked currents in PFC neurons, which are both mediated by anchored protein kinase A (PKA). Although PKA phosphorylation of GABA(A) receptor beta3 or beta1 subunits leads to current enhancement or reduction respectively in heterologous expression systems, we found that beta3 and beta1 subunits are co-expressed in PFC pyramidal neurons. Interestingly, altering PKA activation levels can change the direction of the dual effect, switching enhancement to reduction and vice versa. In addition, increased neuronal activity in PFC slices elevated the PKA activation level, changing the enhancing effect of 5-HT(4) receptors on the amplitude of GABAergic inhibitory postsynaptic currents (IPSCs) to a reduction. These results suggest that 5-HT(4) receptors can modulate GABAergic signalling bidirectionally, depending on the basal PKA activation levels that are determined by neuronal activity. This modulation provides a unique and flexible mechanism for 5-HT(4) receptors to dynamically regulate synaptic transmission and neuronal excitability in the PFC network.