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Anti γ aminobutyric acid B(GABA B) receptor encephalitis is a rare form of autoimmune encephalitis. Our aim is to study the clinical characteristics and treatment outcomes of anti GABA B receptor encephalitis. This is a retrospective case series from the Neuroimmunology laboratory of Amrita Institute of Medical Sciences, Kochi, Kerala, India from 2016 to 2021. A total of four cases (two males, two females, age range: 13-80 years) tested positive for GABA B antibody during the study period. The clinical manifestations of anti GABA B receptor encephalitis found in our patients consisted of seizures, cognitive function decline (especially recent memory), and behavioral abnormalities. No malignancies were encountered. Patients were treated with steroids and immunoglobulins and showed an improvement in behavioral and cognitive function. However, one patient succumbed to illness while being treated with plasma exchange, and two other patients died at a later date due to other causes. Keywords: Anti-GABA-B receptor encephalitis, autoimmune encephalitis, Author(s): Jeeva P. Jiju [1]; Sneha Santhosh [1]; Chirag S. Lalwani [1]; Smrithi S. Thampi [1]; Sudheeran Kannoth (corresponding author) [1,2]; Vivek Nambiar [1]; Siby Gopinath [1]; Saraf U. Umesh [...]

Author(s): Bipinchandra B Bhimani (corresponding author) [1]; Ajit M Sowani [2]; Himanshu H Zalawadia [3]; Sudheeran Kannoth [4]; Annamma Mathai [4] Sir, Case series have revealed a link between GABA[sub.A] [...]

G-protein-coupled receptors (GPCRs) have central roles in intercellular communication.sup.1,2. Structural studies have revealed how GPCRs can activate G proteins. However, whether this mechanism is conserved among all classes of GPCR remains unknown. Here we report the structure of the class-C heterodimeric GABA.sub.B receptor, which is activated by the inhibitory transmitter GABA, in its active form complexed with G.sub.i1 protein. We found that a single G protein interacts with the GB2 subunit of the GABA.sub.B receptor at a site that mainly involves intracellular loop 2 on the side of the transmembrane domain. This is in contrast to the G protein binding in a central cavity, as has been observed with other classes of GPCR. This binding mode results from the active form of the transmembrane domain of this GABA.sub.B receptor being different from that of other GPCRs, as it shows no outside movement of transmembrane helix 6. Our work also provides details of the inter- and intra-subunit changes that link agonist binding to G-protein activation in this heterodimeric complex. Cryo-electron microscopy structure of heterodimeric GABA.sub.B receptor in complex with G.sub.i1 protein reveals that the mode of G-protein binding in this class-C G-protein-coupled receptor differs from that of other classes., Author(s): Cangsong Shen [sup.1] [sup.2] [sup.3] , Chunyou Mao [sup.2] [sup.3] [sup.4] , Chanjuan Xu [sup.1] [sup.5] , Nan Jin [sup.1] [sup.2] [sup.3] , Huibing Zhang [sup.2] [sup.3] [sup.4] , [...]

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of [gamma]-aminobutyric acid type A (GABA.sub.A) receptors to dampen neuronal activity in the brain.sup.1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABA.sub.A receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABA.sub.A receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABA.sub.A receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain. Cryo-electron microscopy structures of GABA.sub.A receptors bound to intravenous anaesthetics and benzodiazepines reveal both common and distinct transmembrane binding sites, and show that the mechanisms of action of anaesthetics partially overlap with those of benzodiazepines., Author(s): Jeong Joo Kim [sup.1] , Anant Gharpure [sup.1] , Jinfeng Teng [sup.1] , Yuxuan Zhuang [sup.2] , Rebecca J. Howard [sup.2] , Shaotong Zhu [sup.1] , Colleen M. Noviello [...]

The human GABA.sub.B receptor--a member of the class C family of G-protein-coupled receptors (GPCRs)--mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction.sup.1. A unique GPCR that is known to require heterodimerization for function.sup.2-6, the GABA.sub.B receptor has two subunits, GABA.sub.B1 and GABA.sub.B2, that are structurally homologous but perform distinct and complementary functions. GABA.sub.B1 recognizes orthosteric ligands.sup.7,8, while GABA.sub.B2 couples with G proteins.sup.9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail.sup.15. Although the VFT heterodimer structure has been resolved.sup.16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABA.sub.B receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity. The structure of the GABA.sub.B receptor in an inactive state reveals, amongst other features, a latch between the two subunits that locks the transmembrane domain interface, and the presence of large phospholipids that may modulate receptor function., Author(s): Jinseo Park [sup.1] , Ziao Fu [sup.2] , Aurel Frangaj [sup.1] , Jonathan Liu [sup.1] , Lidia Mosyak [sup.1] , Tong Shen [sup.3] , Vesna N. Slavkovich [sup.4] , [...]

Many patients who undergo general anesthesia and surgery experience cognitive dysfunction, particularly memory deficits that can persist for days to months. The mechanisms underlying this postoperative cognitive dysfunction in the adult brain remain poorly understood. Depression of brain function during anesthesia is attributed primarily to increased activity of γ-aminobutyric acid type A receptors ([GABA.sub.A]Rs), and it is assumed that once the anesthetic drug is eliminated, the activity of [GABA.sub.A]Rs rapidly returns to baseline and these receptors no longer impair memory. Here, using a murine model, we found that a single in vivo treatment with the injectable anesthetic etomidate increased a tonic inhibitory current generated by α5 subunitcontaining [GABA.sub.A]Rs (α5[GABA.sub.A]Rs) and cell-surface expression of α5[GABA.sub.A]Rs for at least 1 week. The sustained increase in α5[GABA.sub.A]R activity impaired memory performance and synaptic plasticity in the hippocampus. Inhibition of α5[GABA.sub.A]Rs completely reversed the memory deficits after anesthesia. Similarly, the inhaled anesthetic isoflurane triggered a persistent increase in tonic current and cell-surface expression of α5[GABA.sub.A]Rs. Thus, α5[GABA.sub.A]R function does not return to baseline after the anesthetic is eliminated, suggesting a mechanism to account for persistent memory deficits after general anesthesia., Introduction Each year, more than 234 million surgical procedures are performed worldwide (1). A proportion of patients exhibit cognitive impairment, including memory deficits, after surgery and anesthesia (2). Such postoperative [...]

Type-A γ-aminobutyric acid receptors ([GABA.sub.A]Rs) are the principal mediators of rapid inhibitory synaptic transmission in the human brain. A decline in [GABA.sub.A]R signalling triggers hyperactive neurological disorders such as insomnia, anxiety and epilepsy. Here we present the first three-dimensional structure of a [GABA.sub.A]R, the human β3 homopentamer, at 3 Å resolution. This structure reveals architectural elements unique to eukaryotic Cys-loop receptors, explains the mechanistic consequences of multiple human disease mutations and shows an unexpected structural role for a conserved N-linked glycan. The receptor was crystallized bound to a previously unknown agonist, benzamidine, opening a new avenue for the rational design of [GABA.sub.A]R modulators. The channel region forms a closed gate at the base of the pore, representative of a desensitized state. These results offer new insights into the signalling mechanisms of pentameric ligand-gated ion channels and enhance current understanding of GABAergic neurotransmission., In response to binding the neurotransmitter GABA, released at inhibitory synapses, [GABA.sub.A]R chloride channels open and depress neuronal excitability in the adult central nervous system (1). [GABA.sub.A]Rs belong to a [...]

Human [GABA.sub.B] (γ-aminobutyric acid class B) receptor is a G-protein-coupled receptor central to inhibitory neurotransmission in the brain. It functions as an obligatory heterodimer of the subunits GBR1 and GBR2. Here we present the crystal structures of a heterodimeric complex between the extracellular domains of GBR1 and GBR2 in the apo, agonist-bound and antagonist-bound forms. The apo and antagonist-bound structures represent the resting state of the receptor; the agonist-bound complex corresponds to the active state. Both subunits adopt an open conformation at rest, and only GBR1 closes on agonist-induced receptor activation. The agonists and antagonists are anchored in the interdomain crevice of GBR1 by an overlapping set of residues. An antagonist confines GBR1 to the open conformation of the inactive state, whereas an agonist induces its domain closure for activation. Our data reveal a unique activation mechanism for [GABA.sub.B] receptor that involves the formation of a novel heterodimer interface between subunits., GABA is the predominant inhibitory neurotransmitter in the central nervous system. Metabotropic [GABA.sub.B] receptor is a G-protein-coupled receptor that mediates slow and prolonged synaptic inhibition through [G.sub.i] or [G.sub.o] protein [...]

Glyoxalase 1 (Glo1) expression has previously been associated with anxiety in mice; however, its role in anxiety is controversial, and the underlying mechanism is unknown. Here, we demonstrate that GLO1 increases anxiety by reducing levels of methylglyoxal (MG), a [GABA.sub.A] receptor agonist. Mice overexpressing Glo1 on a Tg bacterial artificial chromosome displayed increased anxiety-like behavior and reduced brain MG concentrations. Treatment with low doses of MG reduced anxiety-like behavior, while higher doses caused locomotor depression, ataxia, and hypothermia, which are characteristic effects of [GABA.sub.A] receptor activation. Consistent with these data, we found that physiological concentrations of MG selectively activated [GABA.sub.A] receptors in primary neurons. These data indicate that GLO1 increases anxiety by reducing levels of MG, thereby decreasing [GABA.sub.A] receptor activation. More broadly, our findings potentially link metabolic state, neuronal inhibitory tone, and behavior. Finally, we demonstrated that pharmacological inhibition of GLO1 reduced anxiety, suggesting that GLO1 is a possible target for the treatment of anxiety disorders., Introduction Anxiety disorders, such as post-traumatic stress disorder, panic disorder, social phobia, specific phobia, and generalized anxiety disorder, comprise the most common psychiatric diseases in the United States (1). Mouse [...]

Food-borne hemorrhagic Escherichia coli, exemplified by the strains O157:H7 and O104:H4 (refs 1, 2), require elaborate acid-resistance systems (ARs) (3) to survive the extremely acidic environment such as the stomach (pH ≅ 2). AR2 expels intracellular protons through the decarboxylation of L-glutamate (Glu) in the cytoplasm and exchange of the reaction product γ-aminobutyric acid (GABA) with extracellular Glu. The latter process is mediated by the Glu-GABA antiporter GadC (4,5), a representative member of the amino-acid-polyamine-organocation superfamily of membrane transporters. The functional mechanism of GadC remains largely unknown. Here we show, with the use of an in vitro proteoliposome-based assay, that GadC transports GABA/Glu only under acidic conditions, with no detectable activity at pH values higher than 6.5. We determined the crystal structure of E. coli GadC at 3.1 Å resolution under basic conditions. GadC, comprising 12 transmembrane segments (TMs), exists in a closed state, with its carboxy-terminal domain serving as a plug to block an otherwise inward-open conformation. Structural and biochemical analyses reveal the essential transport residues, identify the transport path and suggest a conserved transport mechanism involving the rigid-body rotation of a helical bundle for GadC and other amino acid antiporters., Other homologous amino-acid-polyamine-organocation (APC) family members include the key AR3 component arginine-agmatine (Arg-Agm) antiporter AdiC (6,7) (Fig. 1a), the lysine-cadaverine antiporter CadB, and the putrescine-ornithine antiporter PotE. Structural analysis of [...]

Modification of the number of GABAA receptors ([GABA.sub.A]Rs) clustered at inhibitory synapses can regulate inhibitory synapse strength with important implications for information processing and nervous system plasticity and pathology. Currently, however, the mechanisms that regulate the number of [GABA.sub.A]Rs at synapses remain poorly understood. By imaging superecliptic pHluorin tagged GABAAR subunits we show that synaptic [GABA.sub.A]R clusters are normally stable, but that increased neuronal activity upon glutamate receptor (GluR) activation results in their rapid and reversible dispersal. This dispersal correlates with increases in the mobility of single [GABA.sub.A]RS within the clusters as determined using single-particle tracking of [GABA.sub.A]Rs labeled with quantum dots. GluR-dependent dispersal of [GABA.sub.A]R clusters requires [Ca.sup.2+] influx via NMDA receptors (NMDARs) and activation of the phosphatase calcineurin. Moreover, the dispersal of [GABA.sub.A]R clusters and increased mobility of individual [GABA.sub.A]RS are dependent on serine 327 within the intracellular loop of the [GABA.sub.A]R [gamma]2 subunit. Thus, NMDAR signaling, via calcineurin and a key [GABA.sub.A]R phosphorylation site, controls the stability of synaptic [GABA.sub.A]Rs, with important implications for activity-dependent control of synaptic inhibition and neuronal plasticity. ion channels | plasticity | trafficking | diffusion | calcineurin doi/ 10.1073/pnas.1000589107

[GABA.sub.B] receptors are the G-protein-coupled receptors for γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. They are expressed in almost all neurons of the brain, where they regulate synaptic transmission and signal propagation by controlling the activity of voltage-gated calcium ([Ca.sub.v]) and inward-rectifier potassium ([K.sub.ir]) channels (1). Molecular cloning revealed that functional [GABA.sub.B] receptors are formed by the heteromeric assembly of [GABA.sub.B1] with [GABA.sub.B2] subunits (2-5). However, cloned [GABA.sub.B(12)] receptors failed to reproduce the functional diversity observed with native [GABA.sub.B] receptors (6-8). Here we show by functional proteomics that [GABA.sub.B] receptors in the brain are high-molecular-mass complexes of [GABA.sub.B1], [GABA.sub.B2] and members of a subfamily of the KCTD (potassium channel tetramerization domain-containing) proteins. KCTD proteins 8,12,12b and 16 show distinct expression profiles in the brain and associate tightly with the carboxy terminus of [GABA.sub.B2] as tetramers. This co-assembly changes the properties of the [GABA.sub.B(1,2)] core receptor: the KCTD proteins increase agonist potency and markedly alter the G-protein signalling of the receptors by accelerating onset and promoting desensitization in a KCTD-subtype-specific manner. Taken together, our results establish the KCTD proteins as auxiliary subunits of [GABA.sub.B] receptors that determine the pharmacology and kinetics of the receptor response., To test the hypothesis that functional diversity of native [GABA.sub.B] receptors results from additional as yet unknown subunits, a proteomic analysis was performed with a recently developed approach combining affinity [...]

Refractory temporal lobe epilepsy (TLE) is associated with a dysfunction of inhibitory signaling mediated by [GABA.sub.A] receptors. In particular, the use-dependent decrease (run-down) of the currents ([I.sub.GABA]) evoked by the repetitive activation of [GABA.sub.A] receptors is markedly enhanced in hippocampal and cortical neurons of TLE patients. Understanding the role of [I.sub.GABA] run-down in the disease, and its mechanisms, may allow development of medical alternatives to surgical resection, but such mechanistic insights are difficult to pursue in surgical human tissue. Therefore, we have used an animal model (pilocarpine-treated rats) to identify when and where the increase in [I.sub.GABA] run-down occurs in the natural history of epilepsy. We found: (i) that the increased run-down occurs in the hippocampus at the time of the first spontaneous seizure (i.e., when the diagnosis of epilepsy is made), and then extends to the neocortex and remains constant in the course of the disease; (ii) that the phenomenon is strictly correlated with the occurrence of spontaneous seizures, because it is not observed in animals that do not become epileptic. Furthermore, initial exploration of the molecular mechanism disclosed a relative increase in [alpha]4-, relative to [alpha]1-containing [GABA.sub.A] receptors, occurring at the same time when the increased run-down appears, suggesting that alterations in the molecular composition of the GABA receptors may be responsible for the occurrence of the increased run-down. These observations disclose research opportunities in the field of epileptogenesis that may lead to a better understanding of the mechanism whereby a previously normal tissue becomes epileptic. [GABA.sub.A] receptor | pilocarpine rat | Xenopus oocytes www.pnas.org/cgi/doi/10.1073/pnas.0914710107

Benzodiazepines are widely used in clinics and for recreational purposes, but will lead to addiction in vulnerable individuals. Addictive drugs increase the levels of dopamine and also trigger long-lasting synaptic adaptations in the mesolimbic reward system that ultimately may induce the pathological behaviour. The neural basis for the addictive nature of benzodiazepines, however, remains elusive. Here we show that benzodiazepines increase firing of dopamine neurons of the ventral tegmental area through the positive modulation of [GABA.sub.A] (γ-aminobutyric acid type A) receptors in nearby interneurons. Such disinhibition, which relies on α1-containing [GABA.sub.A] receptors expressed in these cells, triggers drug- evoked synaptic plasticity in excitatory afferents onto dopamine neurons and underlies drug reinforcement. Taken together, our data provide evidence that benzodiazepines share defining pharmacological features of addictive drugs through cell-type-specific expression of α1-containing [GABA.sub.A] receptors in the ventral tegmental area. The data also indicate that subunit-selective benzodiazepines sparing α1 may be devoid of addiction liability., Addictive drugs can be classified into three groups, according to the cellular mechanism through which they increase mesolimbic dopamine (DA) (1). Opioids, cannabinoids and the club drug γ-hydroxybutyrate reduce release [...]

Because [GABA.sub.A] receptors containing [alpha]2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with [alpha]2 gene deletion showed reduced synaptic [GABA.sub.A] receptor-mediated responses. Behaviorally, the deletion abolished cocaine's ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of [alpha]2-[GABA.sub.A] receptors ([alpha]2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 154513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In [alpha]2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated Iocomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of [alpha]2-[GABA.sub.A] receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction. GABRA2 | behavioral sensitization | nucleus accumbens | mutant mouse | human genetics doi/10.1073/pnas.0910117107

The [[alpha].sub.1][[beta].sub.2][[gamma].sub.2] and [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] two isoforms of [gamma]-aminobutyric acid type A ([GABA.sub.A]) receptor that are widely distributed in the brain. Both are found at synapses, for example in the thalamus, where they mediate distinctly different inhibitory postsynaptic current profiles, particularly with respect to decay time. The two isoforms were expressed in HEK293 cells, and single-channel activity was recorded from outside-out patches. The kinetic characteristics of both isoforms were investigated by analyzing single-channel currents over a wide range of GABA concentrations. [[alpha].sub.1][[beta].sub.2][[gamma].sub.2] channels exhibited briefer active periods than [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] channels over the entire range of agonist concentrations and had lower intraburst open probabilities at subsaturating concentrations. Activation mechanisms were constructed by fitting postulated schemes to data recorded at saturating and subsaturating GABA concentrations simultaneously. Reaction mechanisms were ranked according to log-likelihood values and how accurately they simulated ensemble currents. The highest ranked mechanism for both channels consisted of two sequential binding steps, followed by three conducting and three nonconducting configurations. The equilibrium dissociation constant for GABA at [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] channels was ~2.6 [micro]M compared with ~19 [micro]M for [[alpha].sub.1][[beta].sub.2][[gamma].sub.2] channels, suggesting that GABA binds to the [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] channels with higher affinity. A notable feature of the mechanism was that two consecutive doubly liganded shut states preceded all three open configurations. The lifetime of the third shut state was briefer for the [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] channels. The longer active periods, higher affinity, and preference for conducting states are consistent with the slower decay of inhibitory currents at synapses that contain [[alpha].sub.3][[beta].sub.3][[gamma].sub.2] channels. The reaction mechanism we describe here may also be appropriate for the analysis of other types of [GABA.sub.A] receptors and provides a framework for rational investigation of the kinetic effects of a variety of therapeutic agents that activate or modulate [GABA.sub.A] receptors and hence influence synaptic and extrasynaptic inhibition in the central nervous system. doi/ 10.1085/jgp.200910317

[GABA.sub.A] receptors ([GABA.sub.A]RS), the principal sites of synaptic inhibition in the brain, are dynamic entities on the neuronal cell surface, but the role their membrane trafficking plays in shaping neuronal activity remains obscure. Here, we examined this by using mutant receptor [beta]3 subunits ([beta]3S408/9A), which have reduced binding to the clathrin adaptor protein-2, a critical regulator of [GABA.sub.A]R endocytosis. Neurons expressing [beta]3S408/9A subunits exhibited increases in the number and size of inhibitory synapses, together with enhanced inhibitory synaptic transmission due to reduced [GABA.sub.A]R endocytosis. Furthermore, neurons expressing [beta]3S408/9A subunits had deficits in the number of mature spines and reduced accumulation of postsynaptic density protein-95 at excitatory synapses. This deficit in spine maturity was reversed by pharmacological blockade of [GABA.sub.A]Rs. Therefore, regulating the efficacy of synaptic inhibition by modulating [GABA.sub.A]R membrane trafficking may play a critical role in regulating spine maturity with significant implications for synaptic plasticity together with behavior. endocytosis | inhibition | phosphorylation

Abstract This study aimed to investigate the effects of obovatol isolated from Magnolia obovata on pentobarbital-induced sleeping behaviors and to determine whether these effects were mediated by GABAa receptors/chloride channel [...]

Hypothalamic neurons that contain the neuropeptide orexin (hypocretin) play important roles in the regulation of sleep/wake. Here we analyze the in vivo and in vitro phenotype of mice lacking the [GABA.sub.B1] gene specifically in orexin neurons (oxGKO mice) and demonstrate that [GABA.sub.B] receptors on orexin neurons are essential in stabilizing and consolidating sleep/wake states. In oxGKO brain slices, we show that the absence of [GABA.sub.B] receptors decreases the sensitivity of orexin neurons to both excitatory and inhibitory inputs because of augmented [GABA.sub.A]-mediated inhibition that increases the membrane conductance and shunts postsynaptic currents in these neurons. This increase in [GABA.sub.A]-mediated inhibitory tone is apparently the result of an orexin receptor type 1-mediated activation of local GABAergic interneurons that project back onto orexin neurons, oxGKO mice exhibit severe fragmentation of sleep/wake states during both the light and dark periods, without showing an abnormality in total sleep time or signs of cataplexy. Thus, [GABA.sub.B] receptors on orexin neurons are crucial in the appropriate control of the orexinergic tone through sleep/wake states, thereby stabilizing the state switching mechanisms. hypocretin | interneuron | sleep stability | synaptic input

Millimolar concentrations of the barbiturate pentobarbital (PB) activate [gamma]-aminobutyric acid (GABA) type A receptors (GABARs) and cause blockade reported by a paradoxical current increase or 'tail' upon washout. To explore the mechanism of blockade, we investigated PB-triggered currents of recombinant [[alpha].sub.1][[beta].sub.2] [[gamma].sub.2S] GABARs in whole cells and outside-out membrane patches using rapid perfusion. Whole cell currents showed characteristic bell-shaped concentration dependence where high concentrations triggered tail currents with peak amplitudes similar to those during PB application. Tail current time courses could not be described by multi-exponential functions at high concentrations ([greater than or equal to] 3,000 [micro]M). Deactivation time course decayed over seconds and was slowed by increasing PB concentration and application time. In contrast, macropatch tail currents manifested eightfold greater relative amplitude, were described by multi-exponential functions, and had millisecond rise times; deactivation occurred over fractions of seconds and was insensitive to PB concentration and application time. A parsimonious gating model was constructed that accounts for macropatch results ('patch' model). Lipophilic drug molecules migrate slowly through cells due to avid partitioning into lipophilic subcellular compartments. Inclusion of such a pharmacokinetic compartment into the patch model introduced a slow kinetic component in the extracellular exchange time course, thereby providing recapitulation of divergent whole cell results. GABA co-application potentiated PB blockade. Overall, the results indicate that block is produced by PB concentrations sixfold lower than for activation involving at least three inhibitory PB binding sites, suggest a role of blocked channels in GABA-triggered activity at therapeutic PB concentrations, and raise an important technical question regarding the effective rate of exchange during rapid perfusion of whole cells with PB.

Competition among different axons to reach the somatodendritic region of the target neuron is an important event during development to achieve the final architecture typical of the mature brain. Trasmitter-receptor matching is a critical step for the signaling between neurons. In the cerebellar cortex, there is a persistent competition between the two glutamatergic inputs, the parallel fibers and the climbing fibers, for the innervation of the Purkinje cells. The activity of the latter input is necessary to maintain its own synaptic contacts on the proximal dendritic domain and to confine the parallel fibers in the distal one. Here, we show that climbing fiber activity also limits the distribution of the GABAergic input in the proximal domain. In addition, blocking the activity by tetrodotoxin infusion in Wistar rat cerebellum, a synapse made by GABAergic terminals onto the recently formed Purkinje cell spines appear in the proximal dendrites. The density of GABAergic terminals is increased, and unexpected double symmetric/asymmetric postsynaptic densities add to the typical symmetric phenotype of the GABAergic shaft synapses. Moreover, glutamate receptors appear in these ectopic synapses even in the absence of glutamate transmitter inside the presynaptic terminal and close to GABA receptors. These results suggest that the Purkinje cell has an intrinsic tendency to develop postsynaptic assemblies of excitatory types, including glutamate receptors, over the entire dendritic territory. GABA receptors are induced in these assemblies when contacted by GABAergic terminals, thus leading to the formation of hybrid synapses. cerebellum | GABA receptor | spinogenesis | synaptic plasticity

GABAergic synapses are crucial for brain function, but the mechanisms underlying inhibitory synaptogenesis are unclear. Here, we show that postnatal Purkinje cells (PCs) of [GABA.sub.A][alpha]1 knockout (KO) mice express transiently the [alpha]3 subunit, leading to the assembly of functional [GABA.sub.A] receptors and initial normal formation of inhibitory synapses, that are retained until adulthood. Subsequently, down-regulation of the [alpha]3 subunit causes a complete loss of GABAergic postsynaptic currents, resulting in a decreased rate of inhibitory synaptogenesis and formation of mismatched synapses between GABAergic axons and PC spines. Notably, the postsynaptic adhesion molecule neuroligin-2 (NL2) is correctly targeted to inhibitory synapses lacking [GABA.sub.A] receptors and the scaffold molecule gephyrin, but is absent from mismatched synapses, despite innervation by GABAergic axons. Our data indicate that [GABA.sub.A] receptors are dispensable for synapse formation and maintenance and for targeting NL2 to inhibitory synapses. However, GABAergic signaling appears to be crucial for activity-dependent regulation of synapse density during neuronal maturation. gephyrin | Purkinje cell | synaptogenesis

Autism is a complex disorder that arises from the pervasive action of genetic and epigenetic factors that alter synaptic connectivity of the brain. Although GABA and glutamate receptors seem to be two of those factors, very little is known about the functional properties of the autistic receptors. Autistic tissue samples stored in brain banks usually have relatively long postmortem times, and it is highly desirable to know whether neurotransmitter receptors in such tissues are still functional. Here we demonstrate that native receptors microtransplanted from autistic brains, as well as de novo mRNA-expressed receptors, are still functional and susceptible to detailed electrophysiological characterization even after long postmortem intervals. The opportunity to study the properties of human receptors present in diseased brains not only opens new avenues toward understanding autism and other neurological disorders, but it also makes the microtransplantation method a useful translational system to evaluate and develop novel medicinal drugs. autism | human brain | GABA receptors | glutamate receptors

The regulation of the number of [gamma]2-subunit-containing GAB[A.sub.A] receptors (GAB[A.sub.A]RS) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface [gamma]2-subunit-containing GAB[A.sub.A]RS is regulated. Here, we identify a [gamma]2-subunit-specific Yxx[phi]-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for [gamma]2-subunit tyrosine phosphorylation. Blocking GAB[A.sub.A]R-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxx[phi] motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that [gamma]2-subunit-containing heteromeric GAB[A.sub.A]RS may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GAB[A.sub.A]R surface levels and synaptic inhibition. endocytosis | phosphorylation | structure | synaptic transmission | tyrosine kinase

The family of [gamma]-aminobutyric acid type A receptors ([GABA.sub.A]Rs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic [GABA.sub.A]Rs that are mainly comprised of [[alpha].sub.1], [[beta].sub.2], and [[gamma].sub.2] subunits, whereas tonic inhibition is mediated by extrasynaptic [GABA.sub.A]Rs comprised of [[alpha].sub.4/6], [[beta].sub.2], and [delta] subunits. We investigated the activation properties of recombinant [[alpha].sub.4][[beta].sub.2] [delta] and [[alpha].sub.1][[beta].sub.2][gamma].sub.2s] [GABA.sub.A]Rs in response to GABA and 4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridin-3 (2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at [[alpha].sub.4][[beta].sub.2][delta] [GABA.sub.A]Rs ([beta] ~ 1600 [s.sup.-1]) than at [[alpha].sub.1][[beta].sub.2] [gamma].sub.2s] [GABA.sub.A]Rs ([beta] ~ 460 [s.sup.-1]), whereas GABA activated [[alpha].sub.1][[beta].sub.2][gamma].sub.2s] [GABA.sub.A]Rs more rapidly ([beta] ~1800 [s.sup.-1]) than [[alpha].sub.4][[beta].sub.2][delta] [GABA.sub.A]Rs ([beta] < 440 [s.sup.-1). Single channel recordings of [[alpha].sub.1] [[beta].sub.2][gamma].sub.2s] and [[alpha].sub.4][[beta].sub.2] [delta] [GABA.sub.A]Rs showed that both channels open to a main conductance state of ~25 pS at -70 mV when activated by GABA and low concentrations of THIE whereas saturating concentrations of THIP elicited ~36 pS openings at both channels. Saturating concentrations of GABA elicited brief (

Cys-loop receptor neurotransmitter-gated ion channels are pentameric assemblies of subunits that contain three domains: extracellular, transmembrane, and intracellular. The extracellular domain forms the agonist binding site. The transmembrane domain forms the ion channel. The cytoplasmic domain is involved in trafficking, localization, and modulation by cytoplasmic second messenger systems but its role in channel assembly and function is poorly understood and little is known about its structure. The intracellular domain is formed by the large (>100 residues) loop between the [alpha]-helical M3 and M4 transmembrane segments. Putative prokaryotic Cys-loop homologues lack a large M3M4 loop. We replaced the complete M3M4 loop (115 amino acids) in the 5-hydroxytryptamine type 3A (5-[HT.sub.3A]) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-[HT.sub.3A]-glvM3M4 receptors were comparable to 5-[HT.sub.3A] wild type. The channels remained cation-selective but the 5-[HT.sub.3A]-glvM3M4 single channel conductance was 43.5 pS as compared with the subpicosiemens wild-type conductance. Coexpression of hRIC-3, a protein that modulates expression of 5-[HT.sub.3] and acetylcholine receptors, significantly attenuated 5-HT-induced currents with wild-type 5-[HT.sub.3A] but not 5-[HT.sub.3A]-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-[rho]1 receptor yielded functional, GABA-activated, anion-selective channels. These results imply that the M3M4 loop is not essential for receptor assembly and function and suggest that the cytoplasmic domain may fold as an independent module from the transmembrane and extracellular domains.

Author(s): Julia Knabl [1]; Robert Witschi [2]; Katharina Hösl [1]; Heiko Reinold [1]; Ulrike B. Zeilhofer [1]; Seifollah Ahmadi [1, 6]; Johannes Brockhaus [2, 6]; Marina Sergejeva [1]; Andreas Hess [...]

Selective permeability is a fundamental property of ion channels. The Cys-loop receptor superfamily is composed of both excitatory (ACh, 5-HT) and inhibitory (GABA, glycine) neurotransmitter-operated ion channels. In the GABA receptor, it has been previously shown that the charge selectivity of the integral pore can be altered by a single mutation near the intracellular end of the second transmembrane-spanning domain (TM2). We have extended these findings and now show that charge selectivity of the anionic [rho]1 GABA receptor can be influenced by the introduction of glutamates, one at a time, over an 8-amino acid stretch (-2' to 5') in the proposed intracellular end of TM2 and the TM1-TM2 intracellular linker. Depending on the position, glutamate substitutions in this region produced sodium to chloride permeability ratios ([P.sub.Na]+/[sub.Ck]-) varying from 0.64 to 3.4 (wild type [P.sub.Na] + [sub./Cl]- = 0). In addition to providing insight into the mechanism of ion selectivity, this functional evidence supports a model proposed for the homologous nicotinic acetylcholine receptor in which regions of the protein, in addition to TM2, form the ion pathway.

A study was made of the 'rundown' of [GABA.sub.A] receptors, micro-transplanted to Xenopus oocytes from surgically resected brain tissues of patients afflicted with drug-resistant human mesial temporal lobe epilepsy (mTLE). Cell membranes, isolated from mTLE neocortex specimens, were injected into frog oocytes that rapidly incorporated functional [GABA.sub.A] receptors. Upon repetitive activation with GABA (1 mM), 'epileptic' [GABA.sub.A] receptors exhibited a [GABA.sub.A]-current ([I.sub.GABA]) rundown that was significantly enhanced by [Zn.sup.2+] ([less than or equal to]250 [micro]M), and practically abolished by the high-affinity [GABA.sub.A] receptor inverse agonist SR95531 (gabazine; 2.5-25 [micro]M). Conversely, [IGAB.sub.A] generated by 'control' [GABA.sub.A] receptors microtransplanted from nonepileptic temporal lobe, lesional TLE, or authoptic disease-free tissues remained stable during repetitive stimulation, even in oocytes treated with [Zn.sup.2+]. We conclude that rundown of mTLE epileptic receptors depends on the presence of 'phasic [GABA.sub.A] receptors' that have low sensitivity to antagonism by [Zn.sup.2+]. Additionally, we found that [GABA.sub.A] receptors, microtransplanted from the cerebral cortex of adult rats exhibiting recurrent seizures, caused by pilocarpine-induced status epilepticus, showed greater rundown than control tissue, an event also occurring in patch-clamped rat pyramidal neurons. Rundown of epileptic rat receptors resembled that of human mTLE receptors, being enhanced by [Zn.sup.2+] (40 [micro]M) and sensitive to the antiepileptic agent levetiracetam, the neurotrophin brain-derived neurotrophic factor, and the phosphatase blocker okadaic acid. Our findings point to the rundown of [GABA.sub.A] receptors as a hallmark of TLE and suggest that modulating tonic and phasic mTLE [GABA.sub.A] receptor activity may represent a useful therapeutic approach to the disease. Xenopus oocytes | tonic | epileptic rat

Phasic (synaptic) and tonic (extrasynaptic) inhibition represent the two most fundamental forms of GAB[A.sub.A] receptor-mediated transmission. Inhibitory postsynaptic currents (IPSCs) generated by GAB[A.sub.A] receptors are typically extremely rapid synaptic events that do not last beyond a few milliseconds. Although unusually slow GAB[A.sub.A] IPSCs, lasting for tens of milliseconds, have been observed in recordings of spontaneous events, their origin and mechanisms are not known. We show that neocortical GAB[A.sub.A,slow] IPSCs originate from a specialized interneuron called neurogliaform cells. Compared with classical GAB[A.sub.A, fast] IPSCS evoked by basket cells, single spikes in neurogliaform cells evoke extraordinarily prolonged GAB[A.sub.A] responses that display tight regulation by transporters, low peak GABA concentration, unusual benzodiazepine modulation, and spillover. These results reveal a form of GAB[A.sub.A] receptor mediated communication by a dedicated cell type that produces slow ionotropic responses with properties intermediate between phasic and tonic inhibition. inhibition | neocortex | neurogliaform cell

A form of autosomal dominant juvenile myoclonic epilepsy is caused by a nonconservative missense mutation, A322D, in the GABA[A.sub.A] receptor [alpha]1 subunit M3 transmembrane helix. We reported previously that the A322D mutation reduced total and surface [alpha]1(A322D) subunit protein and that residual [alpha]1(A322D) subunit resided in the endoplasmic reticulum. Here, we demonstrate that the reduction in [alpha]1(A322D) expression results from rapid endoplasmic reticulum-associated degradation of the [alpha]1(A322D) subunit through the ubiquitin-proteasome system. We provide direct evidence that the [alpha]1(A322D) subunit misfolds and show that in at least 33% of [alpha]1(A322D) subunits, M3 failed to insert into the lipid bilayer. We constructed a series of mutations in the M3 domain and empirically determined the apparent free energy cost ([DELTA][G.Sub.app]) of membrane insertion failure, and we show that the [DELTA][G.sub.app] correlated directly with the recently elucidated transmembrane sequence code ([DELTA][G.sub.Lep]). These data provide a biochemical mechanism for the pathogenesis of this epilepsy mutation and demonstrate that [DELTA][G.sub.Lep] predicts the efficiency of lipid partitioning of a naturally occurring protein's transmembrane domain expressed in vivo. Finally, we calculated the [DELTA][DELTA][G.sub.Lep] for 277 known transmembrane missense mutations associated with Charcot--Marie--Tooth disease, diabetes insipidus, retinitis pigmentosa, cystic fibrosis, and severe myoclonic epilepsy of infancy and showed that the majority of these mutations also are likely to destabilize transmembrane domain membrane insertion, but that only a minority of the mutations would be predicted to be as destabilizing as the A322D mutation. ion channel | peripheral myelin protein | protein folding | sodium channel | translocon

Differential composition of GABAA receptor (GABAAR) subunits underlies the variability of fast inhibitory synaptic transmission; alteration of specific GABAAR subunits in localized brain regions may contribute to abnormal brain states such as absence epilepsy. We combined immunocytochemistry and high-resolution ImmunoGold electron microscopy to study cellular and subcellular localization of [GABA.sub.A]R [alpha]l, [alpha]3, and [beta]2/[beta]3 subunits in ventral posterior nucleus (VP) and reticular nucleus (RTN) of control rats and WAG/Rij rats, a genetic model of absence epilepsy. In control rats, [alpha]1 subunits were prominent at inhibitory synapses in VP and much less prominent in RTN; in contrast, the [alpha]3 subunit was highly evident at inhibitory synapses in RTN. [beta]2/[beta]3 subunits were evenly distributed at inhibitory synapses in both VP and RTN. ImmunoGold particles representing all subunits were concentrated at postsynaptic densities with no extrasynaptic localization. Calculated mean number of particles for al subunit per postsynaptic density in nonepileptic VP was 6.1 [+ or -] 3.7, for a3 subunit in RTN it was 6.6 [+ or -] 3.4, and for [beta]2/[beta]3 subunits in VP and RTN the mean numbers were 3.7 [+ or -] 1.3 and 3.5 [+ or -] 1.2, respectively. In WAG/Rij rats, there was a specific loss of [alpha]3 subunit immunoreactivity at inhibitory synapses in RTN, without reduction in [alpha]3 subunit mRNA or significant change in immunostaining for other markers of RTN cell identity such as GABA or parvalbumin, [alpha]3 immunostaining in cortex was unchanged. Subtle, localized changes in GABAAR expression acting at highly specific points in the interconnected thalamocortical network lie at the heart of idiopathic generalized epilepsy. absence epilepsy | inhibition | quantitative electron microscopy

During development of the nervous system, the tip of a growing axon, the growth cone (GC), must respond accurately to stimuli that direct its growth. This axonal navigation depends on extracellular concentration gradients of numerous guidance cues, including GABA. GCs can detect even weak directional signals, yet the mechanisms underlying this sensitivity remain unclear. Past studies in other eukaryotic chemotactic systems have pointed to the role of the spatial reorganization of the transduction pathway in their sensitive response. Here we have developed a single-molecule assay to observe individual [GABA.sub.A] receptors ([GABA.sub.A]Rs) in the plasma membrane of nerve GCs subjected to directional stimuli. We report that in the presence of an external GABA gradient [GABA.sub.A]Rs redistribute asymmetrically across the GC toward the gradient source. Single-particle tracking of [GABA.sub.A]RS shows that the redistribution results from transient interactions between the receptors and the microtubules. Moreover, the relocalization is accompanied by an enhancement in the asymmetry of intracellular calcium concentration. Altogether, our results reveal a microtubule-dependent polarized reorganization of chemoreceptors at the cell surface and suggest that this polarization serves as an amplification step in GABA gradient sensing by nerve GCs. axonal guidance | chemotaxis | single molecule | polarity

In the olfactory bulb (OB), odorants induce oscillations in the [gamma], range (20-80 Hz) that play an important role in the processing of sensory information. Synaptic transmission between dendrites is a major contributor to this processing. Glutamate released from mitral cell dendrites excites the dendrites of granule cells, which in turn mediate GABAergic inhibition back onto mitral cells. Although this reciprocal synapse is thought to be a key element supporting oscillatory activity, the mechanisms by which dendrodendritic inhibition induces and maintains [gamma] oscillations remain unknown. Here, we assessed the role of the dendrodendritic inhibition, using mice lacking the [GABA.sub.A] receptor [alpha]1-subunit, which is specifically expressed in mitral cells but not in granule cells. The spontaneous inhibitory postsynaptic current frequency in these mutants was low and was consistent with the reduction of [GABA.sub.A] receptor clusters detected by immunohistochemistry. The remaining [GABA.sub.A] receptors in mitral cells contained the [alpha]3-subunit and supported slower decaying currents of unchanged amplitude. Overall, inhibitory-mediated interactions between mitral cells were smaller and slower in mutant than in WT mice, although the strength of sensory afferent inputs remained unchanged. Consequently, both experimental and theoretical approaches revealed slower [gamma] oscillations in the OB network of mutant mice. We conclude, therefore, that fast oscillations in the OB circuit are strongly constrained by the precise location, subunit composition and kinetics of [GABA.sub.A] receptors expressed in mitral cells. [alpha]1 knockout | [GABA.sub.A] receptor | olfaction | reciprocal synapses

Our previous studies with estrogen receptor [beta] knockout (ER[[beta].sub.-/-]) mice demonstrated that ER[beta] is necessary for embryonic development of the brain as early as embryonic day 14.5 (E14.5) and is involved in neuronal migration. Such early effects of ER were unexpected because estradiol synthesis and action in the brain occur at E18.5. In the present study, we examined the distribution of ER[beta] in the developing brain and identified a population of ER[beta]-regulated interneurons. ER[beta] appears in the brain at E12.5, mainly localized in the wall of the midbrain, neuromere, hypothalamus, thalamus, and basal plate of pons. At E15.5 and E16.5, ER[beta] expression increased in the hypothalamus, thalamus, and midbrain and appeared in the limbic forebrain. At E18.5, ER[beta] expression was strongly expressed throughout the brain, including the cerebellum and striatum, whereas there were very few positive cells in the ventricular region. In the paraventricular thalamic nucleus and parafascicular nucleus, most of the calretinin-immunopositive interneurons expressed ER[beta]. In ER[[beta].sup.-/-] mice, calretinin expression was markedly lower than in WT mice in the hippocampus, thalamus, and amygdala both at E16.5 and at E18.5. Epidermal growth factor receptor expression was lower in the cortex of ER[[beta].sup.-/-]] than in WT mice at E15.5 and, unlike WT mice, was absent from the superficial marginal zone. These findings suggest that ER[beta] in the embryonic brain is necessary for the development of calretininimmunoreactive GABAergic interneurons and for neuronal migration in the cortex through modulating epidermal growth factor receptor expression at middle and later embryonic stages. cortex | EGF receptor | striatum | thalamus

Selected mutations in the human [alpha]4 or [beta]2 neuronal nicotinic acetylcholine receptor subunit genes cosegregate with a partial epilepsy syndrome known as autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To examine possible mechanisms underlying this inherited epilepsy, we engineered two ADNFLE mutations (Chrna[4.sup.S252F] and Chrna[4.sup.+L264]) in mice. Heterozygous ADNFLE mutant mice show persistent, abnormal cortical electroencephalograms with prominent delta and theta frequencies, exhibit frequent spontaneous seizures, and show an increased sensitivity to the proconvulsant action of nicotine. Relative to WT, electrophysiological recordings from ADNFLE mouse layer II/III cortical pyramidal cells reveal a >20-fold increase in nicotine-evoked inhibitory postsynaptic currents with no effect on excitatory postsynaptic currents, i.p. injection of a subthreshold dose of picrotoxin, a use-dependent [gamma]-aminobutyric acid receptor antagonist, reduces cortical electroencephalogram delta power and transiently inhibits spontaneous seizure activity in ADNFLE mutant mice. Our studies suggest that the mechanism underlying ADNFLE seizures may involve inhibitory synchronization of cortical networks via activation of mutant [alpha]4-containing nicotinic acetylcholine receptors located on the presynaptic terminals and somatodendritic compartments of cortical GABAergic interneurons. cortex | GABAegic interneuron | nicotinic acetylcholine receptor

The neurotransmitter GABA mediates the majority of rapid inhibition in the CNS. Inhibition can occur via the conventional mechanism, the transient activation of subsynaptic [GABA.sub.A] receptors ([GABA.sub.A]-RS), or via continuous activation of high-affinity receptors by low concentrations of ambient GABA, leading to 'tonic' inhibition that can control levels of excitability and network activity. The [GABA.sub.A]-R [alpha]4 subunit is expressed at high levels in the dentate gyrus and thalamus and is suspected to contribute to extrasynaptic [GABA.sub.A]-R-mediated tonic inhibition. Mice were engineered to lack the [alpha]4 subunit by targeted disruption of the Gabra4 gene. [alpha]4 Subunit knockout mice are viable, breed normally, and are superficially indistinguishable from WT mice. In electrophysiological recordings, these mice show a lack of tonic inhibition in dentate granule cells and thalamic relay neurons. Behaviorally, knockout mice are insensitive to the ataxic, sedative, and analgesic effects of the novel hypnotic drug, gaboxadol. These data demonstrate that tonic inhibition in dentate granule cells and thalamic relay neurons is mediated by extrasynaptic [GABA.sub.A]-RS containing the [alpha]4 subunit and that gaboxadol achieves its effects via the activation of this [GABA.sub.A]-R subtype. tonic inhibition | THIP | analgesia | sedative/hypnotic

Allopregnanolone (ALLO) and tetrahydrodeoxycorticosterone (THDOC) are potent positive allosteric modulators of GABA action at GABAA receptors. ALLO and THDOC are synthesized in the brain from progesterone or deoxycorticosterone, respectively, by the sequential action of two enzymes: 5[alpha]-reductase (5[alpha]-R) type I and 3[alpha]-hydroxysteroid dehydrogenase (3[alpha]-HSD). This study evaluates 5[alpha]-R type I and 3[alpha]-HSD mRNA expression level in mouse brain by using in situ hybridization combined with glutamic acid decarboxylase 67/65, vesicular glutamate transporter 2, glial fibrillary acidic protein, and $100[beta] immunohistochemistry. We demonstrate that 5[alpha]-R type I and 3[alpha]-HSD colocalize in cortical, hippocampal, and olfactory bulb glutamatergic principal neurons and in some output neurons of the amygdala and thalamus. Neither 5[alpha]-R type I nor 3[alpha]-HSD mRNAs are expressed in $100[beta]- or glial fibrillary acidic protein-positive glial cells. Using glutamic acid decarboxylase 67/65 antibodies to mark GABAergic neurons, we failed to detect 5[alpha]-R type I and 3[alpha]-HSD in cortical and hippocampal GABAergic interneurons. However, 5[alpha]-R type I and 3[alpha]-HSD are significantly expressed in principal GABAergic output neurons, such as striatal medium spiny, reticular thalamic nucleus, and cerebellar Purkinje neurons. A similar distribution and cellular location of neurosteroidogenic enzymes was observed in rat brain. Taken together, these data suggest that ALLO and THDOC, which can be synthesized in principal output neurons, modulate GABA action at [GABA.sub.A] receptors, either with an autocrine or a paracrine mechanism or by reaching [GABA.sub.A] receptor intracellular sites through lateral membrane diffusion. 3[alpha]-hydroxysteroid dehydrogenase | 5[alpha]-reductase (type I) | GABAergic neurons | glutamatergic neurons

Several major insecticides, including [alpha]-endosulfan, lindane, and fipronil, and the botanical picrotoxinin are noncompetitive antagonists (NCAs) for the GABA receptor. We showed earlier that human [[beta].sub.3] homopentameric GAB[A.sub.A] receptor recognizes all of the important GABAergic insecticides and reproduces the high insecticide sensitivity and structure-activity relationships of the native insect receptor. Despite large structural diversity, the NCAs are proposed to fit a single binding site in the chloride channel lumen lined by five transmembrane 2 segments. This hypothesis is examined with the [[beta].sub.3] homopentamer by mutagenesis, pore structure studies, NCA binding, and molecular modeling. The 15 amino acids in the cytoplasmic half of the pore were mutated to cysteine, serine, or other residue for 22 mutants overall. Localization of A-1'C, A2'C, T6'C, and L9'C (index numbers for the transmembrane 2 region) in the channel lumen was established by disulfide cross-linking. Binding of two NCA radioligands [[sup.3]H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane and [[sup.3]H] 3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile was dramatically reduced with 8 of the 15 mutated positions, focusing attention on A2', T6', and L9' as proposed binding sites, consistent with earlier mutagenesis studies. The cytoplasmic half of the [beta]3 homopentamer pore was modeled as an [alpha]-helix. The six NCAs listed above plus t-butylbicyclophosphorothionate fit the 2' to 9' pore region forming hydrogen bonds with the T6' hydroxyl and hydrophobic interactions with A2', T6', and L9' alkyl substituents, thereby blocking the channel. Thus, widely diverse NCA structures fit the same GABA receptor [beta] subunit site with important implications for insecticide cross-resistance and selective toxicity between insects and mammals. [beta]3 homopentamer | transmembrane 2 | insecticide | disulfide trapping | receptor model

In cortex and hippocampus, protracted (>4 weeks) social isolation of adult male mice alters the subunit expression of GABA type A receptors (GABAA-Rs) as follows: (i) the mRNAs encoding GABAA-R [alpha]1, [alpha]2, and [gamma]2 subunits are decreased by [approximately equal to] 50%, whereas those encoding [alpha]4 and [alpha]5 subunits are increased by [approximately equal to] 100%; (ii) similarly, the synaptic membrane expression of the [alpha]1 subunit protein is down-regulated, and that of the [alpha]5 subunit protein is up-regulated; and (ii) the binding of [[sup.3]H]flumazenil to hippocampal synaptic membranes is decreased. Behaviorally, socially isolated (Sl) mice are resistant to the sedative effects of the positive allosteric GAB[A.sub.A]-R modulators diazepam (DZP) and zolpidem. This resistance seems to be attributable to the decrease of [alpha]1-containing GAB[A.sub.A]Rs. Paradoxically, DZP, which, unlike zolpidem, acts at [alpha]5-containing GAB[A.sub.A]-Rs, increases the locomotor activity of Sl mice. Imidazenil, which fails to modulate [alpha]1-, [alpha]4-, and [alpha]6-containing GAB[A.sub.A]-RS but is a selective positive allosteric modulator of [alpha]5containing GAB[A.sub.A]-Rs, also increases locomotor activity in SI mice. Importantly, SI mice responded to muscimol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one, and allopregnanolone similar to group-housed mice. These data suggest that a switch (a decrease in [alpha]1/[alpha]2 and [gamma]2 and an increase in [alpha]4 and [alpha]5 subunits) in the composition of the heteropentameric GAB[A.sub.A]-R subunit assembly without a change in total GAB[A.sub.A]-R number occurs during social isolation. Thus, the repertoire of DZP and imidazenil actions in SI mice appears to be elicited by the allosteric modulation of GAB[A.sub.A]-Rs overexpressing [alpha]5 subunits. Benzodiazepine response mediated by [alpha]1-containing GAB[A.sub.A]-Rs is expected to be silent or reduced. GABAA receptors | nested RT-PCR

Human brain oscillations represent important features of information processing and are highly heritable. A common feature of beta oscillations (13-28 Hz) is the critical involvement of networks of inhibitory interneurons as pacemakers, gated by [gamma]-aminobutyric acid type A (GABAA) action. Advances in molecular and statistical genetics permit examination of quantitative traits such as the beta frequency of the human electroencephalogram in conjunction with DNA markers. We report a significant linkage and linkage disequilibrium between beta frequency and a set of GABAA receptor genes. Uncovering the genes influencing brain oscillations provides a better understanding of the neural function involved in information processing.

Exogenous application of agonists at the kainate subtype of glutamate receptors has been shown to depress evoked monosynaptic inhibition by [Gamma]-aminobutyric acid (GABA)ergic interneurons in the hippocampus. This observation has led to the hypothesis that synaptic release of endogenous glutamate might have a disinhibitory effect on neuronal circuits, in addition to depolarizing neurons via postsynaptic [Alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, and N-methyl-D-aspartic acid (NMDA) receptors. It is not known, however, if glutamate released from excitatory neurons has the same kainate receptor-mediated effect on monosynaptic inhibitory transmission as exogenous agonist application. Indeed, the recent demonstration that excitatory synaptic signals elicited in interneurons are partly mediated by kainate receptors suggests that these receptors may have a pro- rather than disinhibitory role. Here, we examine the effect of synaptically released glutamate on monosynaptic inhibitory signaling. In the presence of antagonists to AMPA and NMDA receptors, brief bursts of activity in glutamatergic afferent fibers reduce GABAergic transmission. This depression of inhibition is reversibly abolished by blocking kainate receptors. It persists when [GABA.sub.B] receptors are blocked and is enhanced by blocking metabotropic glutamate receptors, possibly explained by presynaptic regulation of glutamate release from excitatory afferents by metabotropic autoreceptors. We conclude that the net kainate receptor-mediated effect of synaptically released glutamate is to reduce monosynaptic inhibition. Since this form of disinhibition may contribute to seizure initiation, kainate receptors may constitute an important target for anticonvulsant drug development.

[Gamma]-Aminobutyric acid type A (GABA-A) receptors are a major mediator of inhibitory neurotransmission in the mammalian central nervous system, and the site of action of a number of clinically important drugs. These receptors exist as a family of subtypes with distinct temporal and spatial patterns of expression and distinct properties that presumably underlie a precise role for each subtype. The newest member of this gene family is the [Theta] subunit. The deduced polypeptide sequence is 627 amino acids long and has highest sequence identity (50.5%) with the [Beta]1 subunit. Within the rat striatum, this subunit coassembles with [Alpha]2, [Beta]1, and [Gamma]1, suggesting that [Gamma]-aminobutyric acid type A receptors consisting of arrangements other than [Alpha][Beta] + [Gamma], [Delta], or [Epsilon] do exist. Expression of [Alpha]2[Beta]1[Gamma]1[Theta] in transfected mammalian cells leads to the formation of receptors with a 4-fold decrease in the affinity for [Gamma]-aminobutyric acid compared with [Alpha]2[Beta]1[Gamma]1. This subunit has a unique distribution, with studies so far suggesting significant expression within monoaminergic neurons of both human and monkey brain.

Postsynaptic responses to stereotyped activation of single axons are known to fluctuate, but the origin of synaptic variability in the vertebrate central nervous system is still unclear. To test the hypothesis that fluctuations of inhibitory postsynaptic currents reflect variations in presynaptic [Ca.sup.2+] concentration, we examined single GABAergic axodendritic contacts in low-density cultures. Collicular neurons from rat embryos were loaded with the [Ca.sup.2+] indicator Oregon Green 488 BAPTA-1. Presynaptic axon terminals were visualized by staining with the styryl dye RH414. Under the condition of action potential block, RH414-labeled boutons were activated selectively by current pulses applied through a fine-tipped glass pipette. Short (1- to 3-ms) depolarization of isolated boutons resulted in stimulus-locked changes of presynaptic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.pre]) and in evoked inhibitory postsynaptic currents (eIPSCs). Varying the strength of the stimulating currents produced a wide amplitude range of both presynaptic fluorescence transients (up to 220% of the resting value) and postsynaptic conductance changes (up to 2-3 nS). It was found that average eIPSCs displayed an approximately third-power dependency on [[[Ca.sup.2+]].sub.pre]. Transmitter release retained its probabilistic character throughout the range of observed [[[Ca.sup.2+]].sub.pre] values. In any tested single bouton, maximal eIPSCs occurred in association with the largest [[[Ca.sup.2+]].sub.pre] transients, but failures were present at any [[[Ca.sup.2+]].sub.pre]. The increase of maximal eIPSC amplitudes in connection with higher [[[Ca.sup.2+]].sub.pre] supports the hypothesis that GABAergic boutons have the capacity to regulate synaptic strength by changing the number of simultaneously released vesicles.

The [Gamma]-aminobutyric acid type A ([GABA.sub.A]) receptor is the predominant [CI.sup.-] channel protein mediating inhibition in the olfactory bulb and elsewhere in the mammalian brain. The olfactory bulb is rich in neurons containing both GABA and dopamine. Dopamine D1 and D2 receptors are also highly expressed in this brain region with a distinct and complementary distribution pattern. This distribution suggests that dopamine may control the GABAergic inhibitory processing of odor signals, possibly via different signal-transduction mechanisms. We have observed that [GABA.sub.A] receptors in the rat olfactory bulb are differentially modulated by dopamine in a cell-specific manner. Dopamine reduced the currents through GABA-gated [CI.sup.-] channels in the interneurons, presumably granule cells. This action was mediated via D1 receptors and involved phosphorylation of [GABA.sub.A] receptors by protein kinase A. Enhancement of GABA responses via activation of D2 dopamine receptors and phosphorylation of [GABA.sub.n] receptors by protein kinase C was observed in mitral/tufted cells. Decreasing or increasing the binding affinity for GABA appears to underlie the modulatory effects of dopamine via distinct receptor subtypes. This dual action of dopamine on inhibitory [GABA.sub.A] receptor function in the rat olfactory bulb could be instrumental in odor detection and discrimination, olfactory learning, and ultimately odotopic memory formation.

Neurotransmitter GABA B-type receptors restrict neuronal activity. Drug efficiencies are shown to be different at different GABAB receptors, and it is likely that subtypes exists. GABAB-receptor splice variants were cloned and native GABAB receptors showed differences in agonist-binding potencies. A new GABAB receptor subtype, GABABR2 is described, which does not bind GABAB antagonists. Evidence is provided for the assembly of heteromeric GABAB receptors in vivo and GABABR2 and GABABR1a/b proteins are shown to immunoprecipitate and localize at dendritic spines.

The main inhibitory neurotransmitter GABA operates via two ligand-gated channels GABAA and GABAC receptors, and GABAB, a receptor. GABABR2 is a new member of the GABAB polypeptide group, and has a sequence homology to GABABR1. GIRK-type potassium channels are not activated when GABABR1 or GABAR2 are expressed individually. Both are co-expressed in individual neurons and they co-localize in transfected cells. Their co-expression is required for signal transduction by GABAB receptors.

Changes in synaptic efficacy are required for neuronal development, states of neuronal excitability and learning and memory formation. A quantal analysis of evoked inhibitory postsynaptic currents was combined with quantitative immunogold localization of synaptic GABAA receptors in hippocampal granule cells. The aim was to determine the basis of inhibitory synaptic plasticity caused by an experiment temporal-lobe epilepsy model.

The influence of extracellular calcium ions and other divalent metal ions on the activity of the gamma amino butyric acid receptor c (GABAc) of the horizontal cells of the catfish retina was evaluated using patch clamp analysis. Results indicate that calcium has an excitatory effect while zinc has an inhibitory effect on GABAc responses. The implications of these findings on the regulation of events in the horizontal cells are discussed.