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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] [...]

Systemic inflammatory response syndrome plays an important role in the development of sepsis. GABAergic and cholinergic pathways activation are considered important for inflammatory response regulation. Tumor necrosis factor (TNF)-[alpha], interleukin (IL)-1[beta], IL-12, IL-10, as well as inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) are important inflammatory mediators involved in the pathogenesis of sepsis. Muscimol, an active compound from the mushroom Amanita muscaria (L.) Lam., is a potent [GABA.sub.A] agonist, inhibits inflammatory response via activating [GABA.sub.A] receptor and vagus nerve. However, the effect of muscimol on lipopolysaccharide (LPS)-induced systemic inflammatory response is still unclear. Therefore, we studied the effects of muscimol on systemic inflammatory response and survival rate in endotoxemic mice. Mice endotoxemia was induced by LPS. Muscimol was given to mice or RAW264.7 cells 30 min before LPS (10 mg/kg, i.p., or 10 ng/mL, respectively). Mice received GABAergic and cholinergic receptor antagonists 30 min before muscimol and LPS. Muscimol decreased TNF- [alpha], IL-1[beta], IL-12, iNOS-derived NO, and increased IL-10 levels and survival rate after LPS treatment. Muscimol significantly decreased nuclear factor kappa B (NF-[kappa]B) activity, increased I[kappa]B expression, and decreased pIKK expression in LPS- treated RAW264.7 cells. GABAergic and cholinergic antagonists failed to reverse muscimol's protection in LPS-treated mice. In conclusion, muscimol protected against systemic inflammatory response in endotoxemic mice may be partially independent of GABAergic and cholinergic receptors. Key words: muscimol, systemic inflammatory response, LPS, GABA receptor, cholinergic receptor Le syndrome de reaction inflammatoire generalisee joue un role important dans le developpement de la septicemie. L'activation des voies de signalisation GABAergiques et cholinergiques est consideree comme etant importante dans la regulation de la reaction inflammatoire. Le facteur de necrose tumorale (TNF)-, l'interleukine (IL)-1[beta], l'IL-12, l'IL-10, de meme que l'oxyde nitrique (NO) derive de la NO synthase inductible (iNOS) sont d'importants mediateurs inflammatoires, qui jouent un role dans la pathogenese de la septicemie. Le muscimol, un compose actif issu du champignon Amanita muscaria, puissant agoniste des recepteurs GABAA, permet d'inhiber la reaction inflammatoire par l'intermediaire de l'activation des recepteurs GABAA et du nerf vague. Cependant, l'effet du muscimol sur la reaction inflammatoire generalisee engendree par les lipopolysaccharides (LPS) demeure a clarifier. Par consequent, nous avons etudie les effets du muscimol sur la reaction inflammatoire generalisee et sur le taux de survie chez la souris endotoxemique. Nous avons obtenu une endotoxemie chez des souris a l'aide de LPS. Nous avons administre du muscimol chez des souris ou chez des cellules RAW264.7 30 minutes avant les LPS (10 mg/kg, i.p., ou 10 ng/ml, respectivement). Les souris ont recu des antagonistes GABAergiques et cholinergiques 30 minutes avant le muscimol et les LPS. Apres l'administration des LPS, le muscimol a entraine une diminution des taux de TNF-[alpha], d'IL-1[beta], d'IL-12, de NO derive de l'iNOS, avec une diminution des taux d'IL-10 de meme que du taux de survie. Le muscimol entrainait une diminution marquee de l'activite du NF-[kappa]B, une augmentation de l'activite de I'I[kappa]B, ainsi qu'une diminution de l'expression de pIKK dans les cellules RAW264.7 exposees aux LPS. Les antagonistes GABAergiques et cholinergiques n'ont pas permis de contrer la protection qu'engendrait le muscimol chez les souris auxquelles des LPS etaient administres. En conclusion, la protection contre la reaction inflammatoire generalisee obtenue avec le muscimol chez des souris endotoxemiques pourrait etre partiellement independante des recepteurs GABAergiques et cholinergiques. [Traduit par la Redaction] Mots-cles : muscimol, reaction inflammatoire generalisee, LPS, recepteurs du GABA, recepteur cholinergique, Introduction Systemic inflammatory response syndrome (SIRS), mainly caused by Gram-negative bacteria, plays a vital role in the development of sepsis (Kaukonen et al. 2015; Chakraborty and Burns 2021). It estimates [...]

[beta]-elemene (Bel) is a sesquiterpene compound that has shown potential in the antinociceptive treatment. This study focused on the function of Bel in neuropathic pain relief in mice. A murine model with spared nerve injury (SNI) was established and treated with Bel. The paw withdrawal thresholds in response to mechanical and thermal stimulations were examined using von Frey filaments. The L4-L6 spinal dorsal horn tissue samples were collected for histological examination. Bel treatment reduced the sensitivities of model mice to mechanical and thermal stimulations, and it inhibited activation of microglia and the secretion of inflammatory cytokines including tumor necrosis factor-[alpha] (TNF-[alpha]), interleukin-1[beta] (IL-1[beta]), and IL-6 in tissues. Bel treatment reduced the expression of nociceptor excitatory N-methyl-D-aspartate receptor (NMDAR), whereas it enhanced the expression of nociceptor inhibitory gamma-aminobutyric acid A (GABAA) receptor to relieve the nociception of mice. The C-X-C motif chemokine receptor 3 (CXCR3) is a downstream molecule mediated by Bel. Either overexpression of CXCR3 or downregulation of GABAA receptor in the tissues aggravated the neuropathic pain in SNI mice which was initially relieved by Bel. In conclusion, this study suggested that Bel might serve as a drug for nociception management by inhibiting CXCR3 and upregulating GABAA receptor. This study may offer novel insights into the field of neuropathic pain relief. Key words: [beta]-elemene, spared nerve injury, neuropathic pain relief, CXCR3, GABAA receptor. Le [beta]-elemene (Bel) est un compose sesquiterpene dont on a montre le potentiel antinociceptif. Cette etude etait centree sur le fonctionnement du Bel dans le soulagement de la douleur neuropathique chez la souris. Nous avons etabli un modele murin de nerf epargne (ou SNI pour << spared nerve injury >>) et administre du Bel. A l'aide de filaments de von Frey, nous avons examine les seuils de retrait de la patte en reaction a des stimulations mecaniques et thermiques. Nous avons preleve des echantillons tissulaires de corne dorsale de moelle epiniere en L4 a L6 en vue d'un examen histologique. L'administration de Bel permettait de faire diminuer la sensibilite des souris a des stimulations mecaniques et thermiques, ainsi que d'inhiber l'activation de la microglie et la secretion de cytokines inflammatoires dans les tissus, y compris le facteur de necrose tumorale (TNF)-[alpha] et les interleukines (IL)-1[beta] et IL-6. L'administration de Bel entrainait une diminution de l'expression des recepteurs NMDA (nocirecepteurs excitateurs), mais une augmentation de l'expression des recepteurs GABAA (nocirecepteurs inhibiteurs) pour soulager les souris de la nociception. Le CXCR3 etait une molecule en aval de la modulation par le Bel. La surexpression du CXCR3 comme la regulation a la baisse des recepteurs GABAA dans les tissus entrainaient une aggravation de la douleur neuropathique qui etait soulagee initialement par le Bel chez les souris SNI. En conclusion, cette etude laisse entendre que le Bel pourrait servir de medicament dans la prise en charge de la nociception en entrainant une inhibition de CXCR3 et une regulation a la hausse des recepteurs GABAA. Cette etude pourrait permettre d'obtenir de nouvelles connaissances dans le domaine du soulagement de la douleur neuropathique. [Traduit par la Redaction] Mots-cles : [beta]-elemene, nerf epargne, soulagement de la douleur neuropathique, CXCR3, recepteurs GABAA., Introduction Lesions or diseases involving the somatosensory nervous system may not only result in a functional impairment but also in elevated pain sensitivity and spontaneous pain (Scholz et al. 2019). [...]

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 [...]

Purpose The anesthetic urethane is commonly used in physiological experiments. We tested urethane's actions on GABA receptors on the primary afferents in the spinal cord, which are one of the few areas in the adult central nervous system (CNS) that are depolarized by GABA, and on ligand-gated excitatory amino acid (EAA) receptors located on motoneurons. Both receptor types are critically important during anesthetic immobilization. Methods We used the isolated hemisected spinal cord of the frog in a sucrose gap chamber to record glutamate-, N-methyl-D-aspartate (NMDA)-, alpha-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA)-, kainate-, and gamma-aminobutyric acid (GABA)-induced depolarizations of the dorsal root (DR) and ventral root (VR). DR potentials (DRPs) and VR potentials (VRPs) evoked by single supramaximal afferent stimuli were also studied. Urethane (10-80 mM) was applied for 10-30 min. Results Urethane depressed EAA responses on the motoneurons in a dose-dependent manner. At a clinical anesthetic concentration (10 mM), EAA-induced depolarizations were reduced by 8.1 ± 2.2 % (n = 7, P = 0.025), but increasing the concentration to 40 mM revealed a larger, 24.7 [+ or -] 3% (n = 53, P = 0.0001) depressing effect of urethane on all EAA responses in the motoneurons. However, GABA and [K.sup.+] responses recorded in the DR were not altered by the presence of 10 or 40 mM urethane. Evoked DRPs and VRPs were reduced by urethane and spontaneous DR and VR potentials were suppressed by 10 or blocked by 40 mM urethane. Conclusion Urethane appears to be selective for EAA-, sparing GABA responses at a clinical anesthetic concentration. Only a 10 % reduction of EAA activity seems to be necessary to induce anesthesia. Keywords Urethane * Spinal cord * EAA * GABA Introduction, Urethane (ethylcarbamate), an anesthetic thatresults fromthe combination of ethanol and urea, was introduced as an injectable agent more than a century ago [1]. It is considered to have little effect [...]

Autism and epilepsy are common childhood neurological disorders with a great heterogeneity of clinical phenotypes as well as risk factors. There is a high co-morbidity of autism and epilepsy. The neuropathology of autism and epilepsy has similar histology implicating the processes of neurogenesis, neural migration, programmed cell death, and neurite outgrowth. Genetic advances have identified multiple molecules that participate in neural development, brain network connectivity, and synaptic function which are involved in the pathogenesis of autism and epilepsy. Mutations in [GABA.sub.A] receptor subunit have been frequently associated with epilepsy, autism, and other neuropsychiatric disorders. In this paper, we address the hypothesis that functional deficiency of GABAergic signaling is a potential common molecular mechanism underpinning the co-morbidity of autism and epilepsy. Keywords Autism * Epilepsy * Co-morbidity * [GABA.sub.A] receptor * Brain development * Synaptogenesis, Introduction Autism and epilepsy are two common childhood neurological disorders that often occur together. Both autism and epilepsy have heterogeneity of clinical phenotypes as well as molecular underpinnings. Autism is [...]

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 [...]

There are conflicting data regarding whether activation of γ-aminobutyric acid-B (GABA-B) receptors results in inhibition of tumor growth and invasion. The objectives of this study were to document the effects of the GABA-B receptor agonist baclofen on malignant hepatocyte proliferation and migration. We also sought to determine whether any effects on cell migration were mediated by changes in cyclic adenosine monophosphate (cAMP) signaling or matrix metalloproteinase (MMP) expression. Finally, GABA-[B.sub.1] and -[B.sub.2] receptor expression was documented in 2 malignant hepatocyte cell lines (PLC/PRF/5 a h-7) and 12 sets of human hepatocellular carcinoma and adjacent nontumor tissues. Cell proliferative activity was documented by WST-1 absorbance, migration by wound healing assays, cAMP levels by enzyme-linked immunoassay (ELISA), MMP by immunohistochemistry and ELISA, and GABA-B receptor expression by flow cytometry and reverse transcriptase--polymerase chain reaction. Although baclofen had no effect on cell proliferation, wound healing was delayed, an effect that was reversed by the GABA-B receptor antagonist CGP. cAMP levels were decreased in Huh-7 but not PLC cells exposed to baclofen. MMP expression remained unaltered in both cell lines. Finally, GABA-[B.sub.1] receptor expression was present and consistently expressed, but GABA-[B.sub.2] expressi on was limited and varied with the number of cell passages and (or) duration of culture. In conclusion, activation of GABA-B receptors has no effect on malignant hepatocyte proliferation but does decrease cell migration. This inhibitory effect may involve cAMP signaling but not MMP expression. GABA-[B.sub.2] receptor expression is limited and variable, which may help to explain discrepancies with previously published results. Key words: hepatocellular carcinoma, metastases, GABA, baclofen, neurotransmitters, proliferation, migration. Il existe des donnees contradictoires quant a savoir si l'activation des recepteurs de l'acide g-aminobutyrique B (GABA-B) provoque l'inhibition de la croissance des tumeurs et de leur invasion. La presente etude a eu comme objectif, d'une part, de documenter les effets de l'agoniste des recepteurs GABA-B, baclofene, sur la proliferation et la migration des hepatocytes malins; d'autre part, de determiner si les effets sur la migration cellulaire sont vehicules par des modifications dans la signalisation de l'adenosine monophosphate cyclique (AMPc) ou dans l'expression de la metalloproteinase matricielle (MMP); finalement, d'examiner l'expression des recepteurs GABA-[B.sub.1] et GABA-[B.sub.2] dans deux lignees cellulaires d'hepatocytes malins (PLC/PRF/5 et Huh-7) et 12 ensembles de carcinomes hepatocellulaires humains et tissus sains adjacents. On a examine l'activite proliferative des cellules par l'absorbance de WST-1, la migration par des essais de cicatrisation, les taux d'AMPc par la methode ELISA, la MMP par immunohistochimie et ELISA, et l'expression des recepteurs GABA-B par cytometrie de flux et RT-PCR. Bien que le baclofene n'ait pas eu d'effet sur la proliferation des cellules, la cicatrisation a ete retardee, un effet qui a ete renverse par l'antagoniste des recepteurs GABA-B, CGP. Les taux d'AMPc ont diminue dans les cellules Huh-7 mais pas dans les cellules PLC exposees au baclofene. L'expression de la MMP est demeuree inchangee dans les deux lignees de cellules. En fin, l'expression des recepteurs GABA-[B.sub.1] a ete constante, mais celle des recepteurs GABA-[B.sub.2] a ete limitee et a varie avec le nombre de passages cellulaires et (ou) la duree de la culture. Ainsi, l'activation des recepteurs GABA-B n'a pas d'effet sur la proliferation des hepatocytes malins, mais elle ne diminue pas la migration des cellules. Cet effet inhibiteur pourrait impliquer la signalisation de l'AMPc, mais pas l'expression de la MMP. L'expression des recepteurs GABA-[B.sub.2] est limitee et variable, ce qui pourrait expliquer les divergences entre ces resultats et ceux publies anterieurement. Mots-cles : carcinome hepatocellulaire, metastases, GABA, baclofene, neurotransmetteurs, proliferation, migration., Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most common cause of cancer death in the world today (El-Serag and Rudolph 2007). Although most prevalent [...]

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 [...]

GABA, the main inhibitory neurotransmitter, acts through [GABA.sub.A/C] and [GABA.sub.B] receptors ([GABA.sub.B]Rs); it is critical for gonadotropin regulation. We studied whether the lack of functional [GABA.sub.B]Rs in [GABA.sub.B1] knockout ([GABA.sub.B]KO) mice affected the gonadotropin axis physiology. Adult male and female [GABA.sub.B1]KO and wild-type (WT) mice were killed to collect blood and tissue samples. Gonadotropin-releasing hormone (GnRH) content in whole hypothalami (HT), olfactory bulbs (OB), and frontoparietal cortexes (CT) were determined (RIA). GnRH expression by quantitative real-time PCR (qRT-PCR) was evaluated in preoptic area-anterior hypothalamus (POA-AH), medial basal-posterior hypothalamus (MBH-PH), OB, and CT. Pulsatile GnRH secretion from hypothalamic explants was measured by RIA. GABA, glutamate, and taurine contents in HT and CT were determined by HPLC. Glutamic acid decarboxylase-67 (GAD-67) mRNA was measured by qRT-PCR in POA-AH, MBH-PH, and CT. Gonadotropin content, serum levels, and secretion from adenohypophyseal cell cultures (ACC) were measured by RIA. GnRH mRNA expression was increased in POA-AH of WT males compared with females; this pattern of expression was inversed in [GABA.sub.B1]KO mice. MBH-PH, OB, and CT did not follow this pattern. In [GABA.sub.B1]KO females, GnRH pulse frequency was increased and GABA and glutamate contents were augmented. POA-AH GAD-67 mRNA showed the same expression pattern as GnRH mRNA in this area. Gonadotropin pituitary contents and serum levels showed no differences between genotypes. Increased basal LH secretion and decreased GnRH-stimulated gonadotropin response were observed in [GABA.sub.B1]KO female ACCs. These results support the hypothesis that the absence of functional [GABA.sub.B]Rs alters GnRH physiology and critically affects sexual dimorphic expression of GnRH and GAD-67 in POA-AH. gonadotropin-releasing hormone pulsatility; gonadotropins; amino acid neurotransmitters doi:10.1152/ajpendo.00532.2009.

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