The initiation and maintenance of cholinergic-induced status epilepticus (SE) are associated with decreased synaptic gamma-aminobutyric acid A receptors (GABAAR) and increased N-methyl-d-aspartate receptors (NMDAR) and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). We hypothesized that trafficking of synaptic GABAAR and glutamate receptors is maladaptive and contributes to the pharmacoresistance to antiseizure drugs; targeting these components should ameliorate the pathophysiological consequences of refractory SE (RSE). We review studies of rodent models of cholinergic-induced SE, in which we used a benzodiazepine allosteric GABAAR modulator to correct loss of inhibition, concurrent with the NMDA antagonist ketamine to reduce excitation caused by increased synaptic localization of NMDAR and AMPAR, which are NMDAR-dependent. Models included lithium/pilocarpine-induced SE in rats and soman-induced SE in rats and in Es1-/- mice, which similar to humans lack plasma carboxylesterase, and may better model soman toxicity. These model human soman toxicity and are refractory to benzodiazepines administered at 40 min after seizure onset, when enough synaptic GABAAR may not be available to restore inhibition. Ketamine-midazolam combination reduces seizure severity, epileptogenesis, performance deficits and neuropathology following cholinergic-induced SE. Supplementing that treatment with valproate, which targets a non-benzodiazepine site, effectively terminates RSE, providing further benefit against cholinergic-induced SE. The therapeutic index of drug combinations is also reviewed and we show the improved efficacy of simultaneous administration of midazolam, ketamine and valproate compared to sequential drug administration. These data suggest that future clinical trials should treat both the lack of sufficient inhibition and the excess excitation that characterize RSE, and include early combination drug therapies. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.

GABA(A) receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABA(A) receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems.

GABA receptors are well known as the inhibitory receptors in the central nervous system and are also found in peripheral tissues. We have previously shown that GABA receptors are involved in lung development and fluid homeostasis. However, the microRNAs that regulate GABA receptors have not yet been identified.

Cultured rat cortical neurons co-expressing VGLUT1 and VGAT (mixed synapses) co-release Glu and GABA. Here, mixed synapses were studied in cultured mouse cortical neurons to verify whether in mice mixed synapses co-release Glu and GABA, and to gain insight into how they may influence excitation/inhibition balance. Results showed the existence of synapses and autapses that co-release Glu and GABA in cultured mouse cortical neurons, and the ability of both neurotransmitters to evoke postsynaptic responses mediated by ionotropic receptors. We studied the short-term plasticity of glutamatergic, GABAergic, and mixed responses and we found that the kinetics of mixPSC amplitude depression was similar to that observed in EPSCs, but it was different from that of IPSCs. We found similar presynaptic release characteristics in glutamatergic and mixed synapses. Analysis of postsynaptic features, obtained by measuring AMPAR- and NMDAR-mediated currents, showed that AMPAR-mediated currents were significantly higher in pure glutamatergic than in mixed synapses, whereas NMDAR-mediated currents were not significantly different from those measured in mixed synapses. Overall, our findings demonstrate that glutamatergic and mixed synapses share similar electrophysiological properties. However, co-release of GABA and Glu influences postsynaptic ionotropic glutamatergic receptor subtypes, thus selectively influencing AMPAR-mediated currents. These findings strengthen the view that mixed neurons can play a key role in CNS development and in maintaining the excitation-inhibition balance.

Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt)/glycogen synthase kinase (GSK)-3 signaling. Here we report that activation of GABA(B) receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABA(B) receptors enhances the phosphorylation of Akt (Thr-308) and enhances the phosphorylation of GSK-3α (Ser-21)/β (Ser-9) in both HEK-293T cells expressing GABA(B) receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABA(B) receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABA(B) receptor agents may exert therapeutic effects in the treatment of schizophrenia.

It has been reported that non-subtype-selective GABAA receptor antagonists injected into the nucleus pontis oralis (PnO) of rats induced long-lasting increases in REM sleep. Characteristics of these REM sleep increases were identical to those resulting from injection of muscarinic cholinergic agonists. Both actions were blocked by the muscarinic antagonist, atropine. Microdialysis of GABAA receptor antagonists into the PnO resulted in increased acetylcholine levels. These findings were consistent with GABAA receptor antagonists disinhibiting acetylcholine release in the PnO to result in an acetylcholine-mediated REM sleep induction. Direct evidence has been lacking for localization in the PnO of the specific GABAA receptor-subtypes mediating the REM sleep effects. Here, we demonstrated a dose-related, long-lasting increase in REM sleep following injection (60 nl) in the PnO of the inverse benzodiazepine agonist, methyl-6,7-dimethoxy-4-ethyl-β-carboline (DMCM, 10(-2)M). REM sleep increases were greater and more consistently produced than with the non-selective antagonist gabazine, and both were blocked by atropine. Fluorescence immunohistochemistry and laser scanning confocal microscopy, colocalized in PnO vesicular acetylcholine transporter, a presynaptic marker of cholinergic boutons, with the γ2 subunit of the GABAA receptor. These data provide support for the direct action of GABA on mechanisms of acetylcholine release in the PnO. The presence of the γ2 subunit at this locus and the REM sleep induction by DMCM are consistent with binding of benzodiazepines by a GABAA receptor-subtype in control of REM sleep.

Protein motions in the Cys-loop ligand-gated ion receptors that govern the gating mechanism are still not well understood. The details as to how motions in the ligand-binding domain are translated to the transmembrane domain and how subunit rotations are linked to bring about the cooperative movements involved in gating are under investigation. Homology models of the alpha4beta2 nicotinic acetylcholine (nACh) and beta2alpha1gamma2 GABA receptors were constructed based on the torpedo neuromuscular-like nicotinic receptor structure. The template constructed for the full electron microscopy structure must be considered more reliable for structure-function studies due to the preservation of the E45-R209 salt-link. Many other salt-links are seen to transiently form, including switching off of the E45-R209 link, within a network of potential salt-links at the binding domain to the transmembrane domain interface region. Several potentially important intersubunit salt-links form in both the nAChR and GABAR structures during the simulation and appear conserved across many subunit combinations, such as the salt-link between alpha4.E262 and beta2.K255 in nAChR (beta2.E262 and alpha1.K263 in GABAR), at the top of the pore-lining M2 helices, and the intersubunit link of R210 on the M1-linker to E168 on the beta8-sheet of the adjacent subunit in the GABA receptor (E175-K46 being the structurally equivalent link in the nAChR, with reversed polarity). A network of other salt-links may be vital for transmitting the cooperative gating motions between subunits that become biased upon ligand binding. The changes seen in the simulations suggest that this network of salt-links helps to set limits and specific states for the conformational changes involved in gating of the receptor. We hope that these hypotheses will be tested experimentally in the near future.

Endocytosis is considered as an important mechanism for regulating cell surface numbers and thereby signaling strength of G protein-coupled receptors. Currently, little is known about the endocytotic pathways of GABA(B) receptors in neurons. Here we report that GABA(B) receptors are constitutively internalized presumably via clathrin-dependent endocytosis in cultured cortical neurons. Colocalization of GABA(B) receptors with endosomal marker proteins indicated sorting of GABA(B) receptors from early endosomes to recycling endosomes and to lysosomes. Cell surface biotinylation experiments revealed fast constitutive recycling of GABA(B) receptors as the predominant pathway that was accelerated by the GABA(B) receptor agonist baclofen. Finally, degradation of GABA(B) receptors in lysosomes was demonstrated by their intracellular accumulation upon inhibition of lysosomal proteases and by blocking recycling which resulted in the redirection of receptors to lysosomes for degradation. These data imply rapid constitutive - agonist-accelerated - recycling of GABA(B) receptors presumably via clathrin-coated pits and their final targeting to lysosomes for degradation.

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

Synaptic terminals often contain metabotropic receptors that act as autoreceptors to control neurotransmitter release. Less appreciated is the heterosynaptic crossover of glutamate receptors to control GABA release and vice versa GABA receptors which control glutamate release. In the brainstem, activation of solitary tract (ST) afferents releases glutamate onto second-order neurons within the solitary tract nucleus (NTS). Multiple metabotropic receptors are expressed in NTS for glutamate (mGluRs) and for GABA (GABA(B)). The present report identifies mGluR regulation of glutamate release at second and higher order sensory neurons in NTS slices. We found strong inhibition of glutamate release to group II and III mGluR activation on mechanically isolated NTS neurons. However, the same mGluR-selective antagonists paradoxically decreased glutamate release (miniature, mEPSCs) at identified second-order NTS neurons. Unaltered amplitudes were consistent with selective presynaptic mGluR actions. GABA(B) blockade in slices resolved the paradoxical differences and revealed a group II/III mGluR negative feedback of mEPSC frequency similar to isolated neurons. Thus, the balance of glutamate control is tipped by mGluR receptors on GABA terminals resulting in predominating heterosynaptic GABA(B) inhibition of glutamate release. Regulation by mGluR or GABA(B) was not consistently evident in excitatory postsynaptic currents (EPSCs) in higher-order NTS neurons demonstrating metabotropic receptor distinctions in processing at different NTS pathway stages. These cellular localizations may figure importantly in understanding interventions such as brain-penetrant compounds or microinjections. We conclude that afferent glutamate release in NTS produces a coordinate presynaptic activation of co-localized mGluR and GABA(B) feedback on cranial afferent terminals to regulate glutamate release.

γ-Aminobutyric acid (GABA) receptors are important targets of parasiticides/insecticides. Several 4-substituted analogs of the partial GABAA receptor agonist 5-(4-piperidyl)-3-isothiazolol (Thio-4-PIOL) were synthesized and examined for their antagonism of insect GABA receptors expressed in Drosophila S2 cells or Xenopus oocytes. Thio-4-PIOL showed weak antagonism of three insect GABA receptors. The antagonistic activity of Thio-4-PIOL was enhanced by introducing bicyclic aromatic substituents into the 4-position of the isothiazole ring. The 2-naphthyl and the 3-biphenylyl analogs displayed antagonist potencies with half maximal inhibitory concentrations in the low micromolar range. The 2-naphthyl analog induced a parallel rightward shift of the GABA concentration-response curve, suggesting competitive antagonism by these analogs. Both compounds exhibited weak insecticidal activities against houseflies. Thus, the orthosteric site of insect GABA receptors might be a potential target site of insecticides.

Resilience means "the ability to withstand or recover quickly in the face of adversity". Elucidating the neural and molecular mechanisms underlying stress resilience will facilitate the development of more effective treatments for stress-induced psychiatric disorders such as depression. The habenular nuclei, which consist of the medial and lateral sub-regions (MHb and LHb, respectively), have been described as a critical node in emotional regulations. GABA(B) receptors play an important regulatory role in habenular activity. In this study, we assessed the functional role of GABA(B) receptors within the habenula in stress resilience and vulnerability by using chronic social defeat stress (CSDS) model in C57BL/6 male mice. Approximately 47.1% of mice exhibited depression- or anxiety-like behaviors after exposure to CSDS. The vulnerable mice presented elevated c-Fos expression in the LHb when confronted with an attacker. On the other hand, the expression of GABA(B) receptors, including both GABA(B1) and GABA(B2) subunits, was significantly down-regulated in the LHb of the susceptible mice. Finally, we found the stress-induced social withdrawal symptoms could be rapidly relieved by intra-LHb injection of both baclofen and CGP36216 (a GABA(B) receptor agonist and antagonist respectively). The above results indicated that GABA(B) receptors in the LHb may play an important role in stress resilience and vulnerability, and thus, may be an important therapeutic target for treatments of stress-induced psychiatric disorders.

Using patch-clamp techniques we studied several aspects of intracellular GABA(A) and glycine Cl- current regulation in cortical and spinal cord neurons, respectively. Activation of PKA with a permeable analog of cyclic AMP (cAMP) produced a potentiation of the Cl- current activated with glycine, but not of the current induced with GABA. The inactive analog was without effect. Activation of PKC with 1 microM PMA reduced the amplitude of the GABA(A) and glycine currents. Internal application of 1 mM cGMP, on the other hand, had no effect on the amplitude of either current. The amplitude of these inhibitory currents changed slightly during 20 min of patch-clamp recording. Internal perfusion of the neurons with 1 microM okadaic acid, a phosphatase inhibitor, induced potentiation in both currents. The amplitude of GABA(A) and glycine currents recorded with 1 mM internal CaCl2 and 10 mM EGTA (10 nM free Ca2+) decayed by less than 30% of control. Increasing the CaCl2 concentration to 10 mM (34 microM free Ca2+) induced a transient potentiation of the GABA(A) current. A strong depression of current amplitude was found with longer times of dialysis. The glycine current, on the contrary, was unchanged by increasing the intracellular Ca2+ concentration. Activation of G proteins with internal FAl4- induced an inhibition of the GABA(A) current, but potentiated the amplitude of the strychnine-sensitive Cl- current. These results indicate that GABA(A) and glycine receptors are differentially regulated by activation of protein kinases, G proteins and Ca2+. This conclusion supports the existence of selectivity in the intracellular regulation of these two receptor types.

Filial imprinting of domestic chicks has a well-defined sensitive (critical) period lasting in the laboratory from hatching to day 3. It is a typical model to investigate the molecular mechanisms underlying memory formation in early learning. We recently found that thyroid hormone 3,5,3'-triiodothyronine (T3) is a determinant of the sensitive period. Rapid increases in cerebral T3 levels are induced by imprinting training, rendering chicks imprintable. Furthermore, the administration of exogenous T3 makes chicks imprintable on days 4 or 6 even after the sensitive period has ended. However, how T3 affects neural transmission to enable imprinting remains mostly unknown. In this study, we demonstrate opposing roles for gamma-aminobutyric acid (GABA)-A and GABA-B receptors in imprinting downstream of T3. Quantitative reverse transcription polymerase chain reaction and immunoblotting showed that the GABA-A receptor expression increases gradually from days 1 to 5, whereas the GABA-B receptor expression gradually decreases. We examined whether neurons in the intermediate medial mesopallium (IMM), the brain region responsible for imprinting, express both types of GABA receptors. Immunostaining showed that morphologically identified putative projection neurons express both GABA-A and GABA-B receptors, suggesting that those GABA receptors interact with each other in these cells to modulate the IMM outputs. The roles of GABA-A and GABA-B receptors were investigated using various agonists and antagonists. Our results show that GABA-B receptor antagonists suppressed imprinting on day 1, while its agonists made day 4 chicks imprintable without administration of exogenous T3. By contrast, GABA-A receptor agonists suppressed imprinting on day 1, while its antagonists induced imprintability on day 4 without exogenous T3. Furthermore, both GABA-A receptor agonists and GABA-B receptor antagonists suppressed T3-induced imprintability on day 4 after the sensitive period has ended. Our data from these pharmacological experiments indicate that GABA-B receptors facilitate imprinting downstream of T3 by initiating the sensitive period, while the GABA-A receptor contributes to the termination of the sensitive period. In conclusion, we propose that opposing roles of GABA-A and GABA-B receptors in the brain during development determine the induction and termination of the sensitive period.

The external globus pallidus (GP) firing rate synchronizes the basal ganglia-thalamus-cortex network controlling GABAergic output to different nuclei. In this context, two findings are significant: the activity and GABAergic transmission of the GP modulated by GABA B receptors and the presence of the GP-thalamic reticular nucleus (RTn) pathway, the functionality of which is unknown. The functional participation of GABA B receptors through this network in cortical dynamics is feasible because the RTn controls transmission between the thalamus and cortex. To analyze this hypothesis, we used single-unit recordings of RTn neurons and electroencephalograms of the motor cortex (MCx) before and after GP injection of the GABA B agonist baclofen and the antagonist saclofen in anesthetized rats. We found that GABA B agonists increase the spiking rate of the RTn and that this response decreases the spectral density of beta frequency bands in the MCx. Additionally, injections of GABA B antagonists decreased the firing activity of the RTn and reversed the effects in the power spectra of beta frequency bands in the MCx. Our results proved that the GP modulates cortical oscillation dynamics through the GP-RTn network via tonic modulation of RTn activity.

The nucleus tractus solitarii (NTS) is essential for coordinating arterial baroreflex control of blood pressure. The primary baroreceptor afferent fibres make their first excitatory synaptic contact at second-order NTS neurones with glutamate as the major neurotransmitter. Glutamate regulates its own release by activating presynaptic metabotropic glutamate autoreceptors (mGluRs) on the baroreceptor central terminals to suppress its further release in frequency-dependent manner. Gamma-aminobutyric acid (GABA) interneurones provide the major inhibitory synaptic input. It is the integration of excitatory and inhibitory inputs that shapes the NTS output of baroreceptor signals. We hypothesized that glutamate released from the primary central afferent terminals can spill over to presynaptic mGluRs on GABA interneurones to suppress GABA release at the second-order baroreceptor neurones. We assessed GABA transmission in second-order baroreceptor neurones identified by attached aortic depressor nerve (ADN) boutons. The medial NTS was stimulated to evoke GABA inhibitory postsynaptic currents (eIPSCs). Glutamate spillover, generated by brief 2 s, 25 Hz trains of stimuli applied to the tractus solitarius (TS), induced a small (10%) but significant reduction in the eIPSC amplitudes. The depression was enhanced to a 25% decrease by increasing glutamate in the cleft with a glutamate-uptake inhibitor (M-trans-pyrrolidine-2,4-dicarboxylic acid, 1 mum), blocked by a Group II mGluR antagonist (LY341495, 200 nm) and mimicked by a Group II agonist ((2S,3S,4S)-CCG/(2S,1'S,2'S)-2-carboxycyclopropyl; L-CCG-I). A presynaptic mGluR locus was established by the mGluR agonist-mediated increase in the paired-pulse ratio of two consecutive eIPSCs in conjunction with the decrease in the first eIPSC, and a decrease in the frequency (39-46% reduction at EC(50) concentration), but not amplitude, of spontaneous and miniature GABA IPSCs. The data indicate that endogenous glutamate activation of Group II presynaptic mGluRs can decrease GABA release at the first central synapses, suggesting a heterosynaptic role for the Group II mGluRs in shaping baroreceptor signal transmission.

Changing receptor abundance at synapses is an important mechanism for regulating synaptic strength. Synapses contain two pools of receptors, immobilized and diffusing receptors, both of which are confined to post-synaptic elements. Here we show that immobile and diffusing GABA(A) receptors are stabilized by distinct synaptic scaffolds at C. elegans neuromuscular junctions. Immobilized GABA(A) receptors are stabilized by binding to FRM-3/EPB4.1 and LIN-2A/CASK. Diffusing GABA(A) receptors are stabilized by the synaptic adhesion molecules Neurexin and Neuroligin. Inhibitory post-synaptic currents are eliminated in double mutants lacking both scaffolds. Neurexin, Neuroligin, and CASK mutations are all linked to Autism Spectrum Disorders (ASD). Our results suggest that these mutations may directly alter inhibitory transmission, which could contribute to the developmental and cognitive deficits observed in ASD.

The regulation of pre-synaptic glutamate release is important in the maintenance and fidelity of excitatory transmission in the nervous system. In this study, we report a novel interaction between a ligand-gated ion channel and a G-protein coupled receptor which regulates glutamate release from parallel fiber axon terminals. Immunocytochemical analysis revealed that GABA(A) receptors and the high affinity group III metabotropic glutamate receptor subtype 4 (mGlu4) are co-localized on glutamatergic parallel fiber axon terminals in the cerebellum. GABA(A) and mGlu4 receptors were also found to co-immunoprecipitate from cerebellar membranes. Independently, these two receptors have opposing roles on glutamate release: pre-synaptic GABA(A) receptors promote, while mGlu4 receptors inhibit, glutamate release. However, coincident activation of GABA(A) receptors with muscimol and mGlu4 with the agonist (2S)-S-2-amino-4-phosphonobutanoic acid , increased glutamate release from [(3) H]glutamate-loaded cerebellar synaptosomes above that observed with muscimol alone. Further support for an interaction between GABA(A) and mGlu4 receptors was obtained in the mGlu4 knockout mouse which displayed reduced binding of the GABA(A) ligand [(35) S]tert-butylbicyclophosphorothionate, and decreased expression of the α1, α6, β2 GABA(A) receptor subunits in the cerebellum. Taken together, our data suggest a new role for mGlu4 whereby simultaneous activation with GABA(A) receptors acts to amplify glutamate release at parallel fiber-Purkinje cell synapses.

Selectively activating (by optogenetics) parvalbumin-expressing (PV) interneurons induces GABA release onto CA1 pyramidal cells. Here we report that this release was attenuated by presynaptic mu opioid receptors (MORs) activation. On the other hand, conventional electric shock, presumably activating non-selectively presynaptic GABAergic terminals, also induced GABA release; however, this release showed relatively limited depression by MORs activation. The data suggest that MORs specifically inhibit GABA release from PV terminals and therefore, further support the idea that MORs contribute to homeostasis in CA1 neuro-circuit.

Flavonoids, important plant pigments, have been shown to allosterically modulate brain GABA(A) receptors (GABA(A)Rs). We previously reported that trans-6,4'-dimethoxyretrochalcone (Rc-OMe), a hydrolytic derivative of the corresponding flavylium salt, displayed nanomolar affinity for the benzodiazepine binding site of GABA(A)Rs. Here, we evaluate the functional modulations of Rc-OMe, along with two other synthetic derivatives trans-6-bromo-4'-methoxyretrochalcone (Rc-Br) and 4,3'-dimethoxychalcone (Ch-OMe) on GABA(A)Rs.