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GABAA Receptors (gabaa + receptor)
Kinds of GABAA Receptors Terms modified by GABAA Receptors Selected AbstractsMELATONIN REDUCES BLOOD PRESSURE IN RATS WITH STRESS-INDUCED HYPERTENSION VIA GABAA RECEPTORSCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 4 2009Hua-Li Li SUMMARY 1Several groups have reported that melatonin produces a significant decrease in blood pressure in mammals and that pinealectomy in rats causes hypertension. The purpose of the present study was to investigate the effects of melatonin and bicuculline methiodide on the blood pressure of rats, both in the developing and fully developed stage of stress-induced hypertension (SIH). 2Rats with SIH were generated by mild electric foot shocks for 15 days, after which tail arterial systolic pressure and plasma angiotensin (Ang) II levels were measured. The effects of melatonin injections (i.p. or i.c.v.) on mean arterial pressure (MAP) in rats with SIH were also determined. 3Pretreatment with 1 mg/kg, i.p., melatonin significantly diminished the elevated tail arterial systolic pressure and plasma AngII levels caused by 15 days stress. The suppressive effects of melatonin were blocked by i.p. injection of 1 mg/kg bicuculline methiodide, an antagonist of the GABAA receptor. 4Intraperitoneal (0.2, 0.5 and 1 mg/kg) or i.c.v. (0.15 and 1.5 µg/3 µL) injection of melatonin produced a dose-dependent lowering of MAP in rats with SIH. The antihypertensive response induced by melatonin was blocked by injection of both 1 mg/kg, i.p., and 1.5 × 106 µg/3 µL, i.c.v., bicuculline methiodide. 5In conclusion, melatonin not only prevents increases in blood pressure during the developing stage of SIH, but can also reduce the blood pressure of rats that have already developed SIH. The antihypertensive effect of melatonin may be mediated by GABAA receptors through inhibition of plasma AngII levels. [source] Cardiovascular Regulation through Hypothalamic GABAA Receptors in a Genetic Absence Epilepsy Model in RatEPILEPSIA, Issue 2 2002Rezzan Gülhan Aker Summary: ,Purpose: ,-Aminobutyric acid (GABA) plays a vital role in both central cardiovascular homeostasis and pathogenesis of epilepsy. Epilepsy affects autonomic nervous system functions. In this study, we aimed to clarify the role of GABAA receptors in hypothalamic cardiovascular regulation in a genetically determined animal model of absence epilepsy. Methods: Nonepileptic Wistar rats and genetic absence epilepsy rats from Strasbourg (GAERS) were instrumented with a guide cannula for drug injection and extradural electrodes for EEG recording. After a recovery period, iliac arterial catheters were inserted for direct measurement of mean arterial pressure and heart rate. Bicuculline, a GABAA -receptor antagonist, was injected into the dorsomedial (DMH) or posterior (PH) hypothalamic nuclei of nonepileptic control rats or GAERS. Blood pressure, heart rate, and EEG recordings were performed in conscious unrestrained animals. Results: Bicuculline injections into the hypothalamus produced increases in blood pressure and heart rate of both control rats and GAERS. The DMH group of GAERS showed a twofold increase in the blood pressure and the heart rate compared with those of control rats. Pressor responses to bicuculline, when microinjected into the PH, were similar in the nonepileptic animals and GAERS. Conversely, the amplitude of tachycardic responses to the administration of bicuculline into the PH was significantly higher in GAERS compared with those of control rats. Conclusions: The bicuculline-induced increases in blood pressure and heart rate were more prominent when given in the DMH of GAERS. These results indicate an increased GABAA receptor,mediated cardiovascular response through the DMH in conscious rats with absence epilepsy. [source] Alcohol-Induced Tolerance and Physical Dependence in Mice With Ethanol Insensitive ,1 GABAA ReceptorsALCOHOLISM, Issue 2 2009David F. Werner Background:, Although many people consume alcohol (ethanol), it remains unknown why some become addicted. Elucidating the molecular mechanisms of tolerance and physical dependence (withdrawal) may provide insight into alcohol addiction. While the exact molecular mechanisms of ethanol action are unclear, ,-aminobutyric acid type A receptors (GABAA -Rs) have been extensively implicated in ethanol action. The ,1 GABAA -R subunit is associated with tolerance and physical dependence, but its exact role remains unknown. In this report, we tested the hypothesis that ,1-GABAA -Rs mediate in part these effects of ethanol. Methods:, Ethanol-induced behavioral responses related to tolerance and physical dependence were investigated in knockin (KI) mice that have ethanol-insensitive ,1 GABAA -Rs and wildtype (WT) controls. Acute functional tolerance (AFT) was assessed using the stationary dowel and loss of righting reflex (LORR) assays. Chronic tolerance was assessed on the LORR, fixed speed rotarod, hypothermia, and radiant tail-flick assays following 10 consecutive days of ethanol exposure. Withdrawal-related hyperexcitability was assessed by handling-induced convulsions following 3 cycles of ethanol vapor exposure/withdrawal. Immunoblots were used to assess ,1 protein levels. Results:, Compared with controls, KI mice displayed decreased AFT and chronic tolerance to ethanol-induced motor ataxia, and also displayed heightened ethanol-withdrawal hyperexcitability. No differences between WT and KI mice were seen in other ethanol-induced behavioral measures. Following chronic exposure to ethanol, control mice displayed reductions in ,1 protein levels, but KIs did not. Conclusions:, We conclude that ,1-GABAA -Rs play a role in tolerance to ethanol-induced motor ataxia and withdrawal-related hyperexcitability. However, other aspects of behavioral tolerance and physical dependence do not rely on ,1-containing GABAA -Rs. [source] 3,4-Dihydronaphthalen-1(2H)-ones: Novel Ligands for the Benzodiazepine Site of ,5-Containing GABAA Receptors.CHEMINFORM, Issue 38 2004Helen J. Szekeres Abstract For Abstract see ChemInform Abstract in Full Text. [source] Highly Potent Modulation of GABAA Receptors by Valerenic Acid DerivativesCHEMMEDCHEM, Issue 5 2010Sascha Kopp Dipl.-Chem. Traditional medicine to potent drug leads: Valerenic acid (1) is a major constituent of common valerian and potentiates the effect of ,-aminobutyric acid on GABAA receptors. Through systematic modification of the carboxyl group of 1 we have discovered a noncarboxylate-containing analogue, tetrazole 10, which exceeds the modulatory activity of 1 at GABAA receptors by one order of magnitude. [source] Cardiovascular Regulation through Hypothalamic GABAA Receptors in a Genetic Absence Epilepsy Model in RatEPILEPSIA, Issue 2 2002Rezzan Gülhan Aker Summary: ,Purpose: ,-Aminobutyric acid (GABA) plays a vital role in both central cardiovascular homeostasis and pathogenesis of epilepsy. Epilepsy affects autonomic nervous system functions. In this study, we aimed to clarify the role of GABAA receptors in hypothalamic cardiovascular regulation in a genetically determined animal model of absence epilepsy. Methods: Nonepileptic Wistar rats and genetic absence epilepsy rats from Strasbourg (GAERS) were instrumented with a guide cannula for drug injection and extradural electrodes for EEG recording. After a recovery period, iliac arterial catheters were inserted for direct measurement of mean arterial pressure and heart rate. Bicuculline, a GABAA -receptor antagonist, was injected into the dorsomedial (DMH) or posterior (PH) hypothalamic nuclei of nonepileptic control rats or GAERS. Blood pressure, heart rate, and EEG recordings were performed in conscious unrestrained animals. Results: Bicuculline injections into the hypothalamus produced increases in blood pressure and heart rate of both control rats and GAERS. The DMH group of GAERS showed a twofold increase in the blood pressure and the heart rate compared with those of control rats. Pressor responses to bicuculline, when microinjected into the PH, were similar in the nonepileptic animals and GAERS. Conversely, the amplitude of tachycardic responses to the administration of bicuculline into the PH was significantly higher in GAERS compared with those of control rats. Conclusions: The bicuculline-induced increases in blood pressure and heart rate were more prominent when given in the DMH of GAERS. These results indicate an increased GABAA receptor,mediated cardiovascular response through the DMH in conscious rats with absence epilepsy. [source] A postnatal switch in GABAergic control of spinal cutaneous reflexesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2006Gareth Hathway Abstract GABAergic signalling exerts powerful inhibitory control over spinal tactile and nociceptive processing, but during development GABA can be depolarizing and the functional consequences of this upon neonatal pain processing is unknown. Here we show a postnatal switch in tonic GABAA receptor (GABAAR) modulation of cutaneous tactile and nociceptive reflexes from excitation to inhibition, but only in the intact spinal cord. Neonatal and 21-day-old (P21) rats were intrathecally treated with one of the GABAAR antagonists bicuculline and gabazine, with both compounds dose-dependently decreasing hindpaw mechanical and thermal withdrawal thresholds in P21 rats but increasing them in P3 neonates. Intrathecal gabazine also produced an increase in the cutaneous evoked electromyography (EMG) response of the biceps femoris in P21 rates but lowering the response in neonates. Injections of 3H-gabazine in the L4,L5 region at P3 confirmed that gabazine binding was restricted to the lumbar spinal cord. Spinalization of P3 neonates at the upper thoracic level prior to drug application reversed the behavioural and EMG responses to GABA antagonists so that they resembled those of P21 rats. The effects of spinalization were consistent with gabazine facilitation of ventral root potentials observed in isolated neonatal spinal cord. These data show a marked postnatal developmental switch in GABAergic control of neonatal nociception that is mediated by supraspinal structures and illustrate the importance of studying developmental circuits in the intact nervous system. [source] Molecular analysis of the A322D mutation in the GABAA receptor ,1 -subunit causing juvenile myoclonic epilepsyEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2005Klaus Krampfl Abstract Juvenile myoclonic epilepsy (JME) belongs to the most common forms of hereditary epilepsy, the idiopathic generalized epilepsies. Although the mode of inheritance is usually complex, mutations in single genes have been shown to cause the disease in some families with autosomal dominant inheritance. The first mutation in a multigeneration JME family has been recently found in the ,1 -subunit of the GABAA receptor (GABRA1), predicting the single amino acid substitution A322D. We further characterized the functional consequences of this mutation by coexpressing ,1 -, ,2 - and ,2 -subunits in human embryonic kidney (HEK293) cells. By using an ultrafast application system, mutant receptors have shown reduced macroscopic current amplitudes at saturating GABA concentrations and a highly reduced affinity to GABA compared to the wild-type (WT). Dose,response curves for current amplitudes, activation kinetics, and GABA-dependent desensitization parameters showed a parallel shift towards 30- to 40-fold higher GABA concentrations. Both deactivation and resensitization kinetics were considerably accelerated in mutant channels. In addition, mutant receptors labelled with enhanced green fluorescent protein (EGFP) were not integrated in the cell membrane, in contrast to WT receptors. Therefore, the A322D mutation leads to a severe loss-of-function of the human GABAA receptor by several mechanisms, including reduced surface expression, reduced GABA-sensitivity, and accelerated deactivation. These molecular defects could decrease and shorten the resulting inhibitory postsynaptic currents (IPSCs) in vivo, which can induce a hyperexcitability of the postsynaptic membrane and explain the occurrence of epileptic seizures. [source] Loss of zolpidem efficacy in the hippocampus of mice with the GABAA receptor ,2 F77I point mutationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2005D. W. Cope Abstract Zolpidem is a hypnotic benzodiazepine site agonist with some ,-aminobutyric acid (GABA)A receptor subtype selectivity. Here, we have tested the effects of zolpidem on the hippocampus of ,2 subunit (,2F77I) point mutant mice. Analysis of forebrain GABAA receptor expression with immunocytochemistry, quantitative [3H]muscimol and [35S] t-butylbicyclophosphorothionate (TBPS) autoradiography, membrane binding with [3H]flunitrazepam and [3H]muscimol, and comparison of miniature inhibitory postsynaptic current (mIPSC) parameters did not reveal any differences between homozygous ,2I77/I77 and ,2F77/F77 mice. However, quantitative immunoblot analysis of ,2I77/I77 hippocampi showed some increased levels of ,2, ,1, ,4 and , subunits, suggesting that differences between strains may exist in unassembled subunit levels, but not in assembled receptors. Zolpidem (1 µm) enhanced the decay of mIPSCs in CA1 pyramidal cells of control (C57BL/6J, ,2F77/F77) mice by ,,60%, and peak amplitude by ,,20% at 33,34 °C in vitro. The actions of zolpidem (100 nm or 1 µm) were substantially reduced in ,2I77/I77 mice, although residual effects included a 9% increase in decay and 5% decrease in peak amplitude. Similar results were observed in CA1 stratum oriens/alveus interneurons. At network level, the effect of zolpidem (10 µm) on carbachol-induced oscillations in the CA3 area of ,2I77/I77 mice was significantly different compared with controls. Thus, the ,2F77I point mutation virtually abolished the actions of zolpidem on GABAA receptors in the hippocampus. However, some residual effects of zolpidem may involve receptors that do not contain the ,2 subunit. [source] Quantitative effects produced by modifications of neuronal activity on the size of GABAA receptor clusters in hippocampal slice culturesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2004Serge Marty Abstract The number and strength of GABAergic synapses needs to be precisely adjusted for adequate control of excitatory activity. We investigated to what extent the size of GABAA receptor clusters at inhibitory synapses is under the regulation of neuronal activity. Slices from P7 rat hippocampus were cultured for 13 days in the presence of bicuculline or 4-aminopyridine (4-AP) to increase neuronal activity, or DNQX to decrease activity. The changes provoked by these treatments on clusters immunoreactive for the ,1 and ,2 subunits of the GABAA receptor or gephyrin were quantitatively evaluated. While an increase in activity augmented the density of these clusters, a decrease in activity provoked, in contrast, a decrease in their density. An inverse regulation was observed for the size of individual clusters. Bicuculline and 4-AP decreased whilst DNQX increased the mean size of the clusters. When the pharmacological treatments were applied for 2 days instead of 2 weeks, no effects on the size of the clusters were observed. The variations in the mean size of individual clusters were mainly due to changes in the number of small clusters. Finally, a regulation of the size of GABAA receptor clusters occurred during development in vivo, with a decrease of the mean size of the clusters between P7 and P21. This physiological change was also the result of an increase in the number of small clusters. These results indicate that neuronal activity regulates the mean size of GABAA receptor- and gephyrin-immunoreactive clusters by modifying specifically the number of synapses with small clusters of receptors. [source] Organization of GABAA receptor ,-subunit clustering in the developing rat neocortex and hippocampusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2004B. Hutcheon Abstract We compared the expression and co-expression of ,1, ,2, ,3, and ,5-subunit protein clusters of the ,-aminobutyric acid (GABA)A receptor in the neocortex and hippocampus of rat at postnatal days (PND) 5,10 and 30,40 in order to understand how inhibitory receptors reorganize during brain maturation. The size, intensity, density and pattern of co-localization of fluorescently tagged subunit clusters were determined in deconvolved digital images using a novel 2D cross-correlational analysis. The cross-correlation analysis allowed an unbiased identification of GABAA receptor subunit clusters based on staining intensity. Cluster size increased through development; only the ,2 clusters in dentate gyrus (DG) decreased in size. ,5-subunit cluster density either increased or decreased with maturation depending on the brain region. For the other subunits, the cluster density remained rather constant, with noted exceptions (increase in ,2 clusters in cortical layer 5 but a decrease of ,3 clusters in hilus). The co-localization of ,1-subunit with the others was unique and not correlated to overall changes in subunit abundance between developmental époques. So, although ,2-subunit expression went up in the DG, the clusters became less co-localized with ,1. In contrast, ,5-subunit clusters became more co-localized with ,1 as the ,5-subunit expression declined in cortex and CA1. The co-localization of ,3 with ,1 also became greater in layer 6. In the adult brain not all clustering was associated with synapses, as many ,-subunit clusters did not co-localize with synaptophysin. Overall, these data indicate that the regulation of GABAA receptor clustering is both synaptic and extrasynaptic, presumably reflecting complex cellular trafficking mechanisms. [source] Sex differences in anxiety, sensorimotor gating and expression of the ,4 subunit of the GABAA receptor in the amygdala after progesterone withdrawalEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2003M. Gulinello Abstract In a progesterone withdrawal (PWD) model of premenstrual anxiety, we have previously demonstrated that increased hippocampal expression of the ,4 subunit of the GABAA receptor (GABAA -R) is closely associated with higher anxiety levels in the elevated plus maze. However, several studies indicate that sex differences in regulation of the GABAA -R in specific brain regions may be an important factor in the observed gender differences in mood disorders. Thus, we investigated possible sex differences in GABAA -R subunit expression and anxiety during PWD. To this end, we utilized the acoustic startle response (ASR) to assess anxiety levels in male and female rats undergoing PWD as the ASR is also applicable to the assessment of human anxiety responses. We also investigated GABAA -R ,4 subunit expression in the amygdala, as the amygdala directly regulates the primary startle circuit. Female rats exhibited a greater ASR during PWD than controls, indicating higher levels of anxiety and arousal. In contrast, male rats undergoing PWD did not demonstrate an increased ASR. The sex differences in the ASR were paralleled by sex differences in the expression of the GABAA -R ,4 subunit in the amygdala such that ,4 subunit expression was up-regulated in females during PWD whereas ,4 levels in males undergoing PWD were not altered relative to controls. These findings might have implications regarding gender differences in human mood disorders and the aetiology of premenstrual anxiety. [source] Differential effects of acute and chronic exercise on plasticity-related genes in the rat hippocampus revealed by microarrayEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2002Raffaella Molteni Abstract Studies were performed to determine the effects of acute and chronic voluntary periods of exercise on the expression of hippocampal genes. RNAs from rodents exposed to a running wheel for 3, 7 and 28 days were examined using a microarray with 1176 cDNAs expressed primarily in the brain. The expression of selected genes was quantified by Taqman RT-PCR or RNase protection assay. The largest up-regulation was observed in genes involved with synaptic trafficking (synapsin I, synaptotagmin and syntaxin); signal transduction pathways (Ca2+/calmodulin-dependent protein kinase II, CaM-KII; mitogen-activated/extracellular signal-regulated protein kinase, MAP-K/ERK I and II; protein kinase C, PKC-,) or transcription regulators (cyclic AMP response element binding protein, CREB). Genes associated with the glutamatergic system were up-regulated (N -methyl- d -aspartate receptor, NMDAR-2A and NMDAR-2B and excitatory amino acid carrier 1, EAAC1), while genes related to the gamma-aminobutyric acid (GABA) system were down-regulated (GABAA receptor, glutamate decarboxylase GAD65). Brain-derived neurotrophic factor (BDNF) was the only trophic factor whose gene was consistently up-regulated at all timepoints. These results, together with the fact that most of the genes up-regulated have a recognized interaction with BDNF, suggest a central role for BDNF on the effects of exercise on brain plasticity. The temporal profile of gene expression seems to delineate a mechanism by which specific molecular pathways are activated after exercise performance. For example, the CaM-K signal system seems to be active during acute and chronic periods of exercise, while the MAP-K/ERK system seems more important during long-term exercise. [source] Synaptic localization of GABAA receptor subunits in the substantia nigra of the rat: effects of quinolinic acid lesions of the striatumEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2002Fumino Fujiyama Abstract The inhibitory amino acid, ,-aminobutyric acid (GABA), plays a critical role in the substantia nigra (SN) in health and disease. GABA transmission is controlled in part by the type(s) of GABA receptor expressed, their subunit composition and their location in relation to GABA release sites. In order to define the subcellular localization of GABAA receptors in the SN in normal and pathological conditions, sections of SN from control rats and rats that had received quinolinic acid lesions of the striatum were immunolabelled using the postembedding immunogold technique with antibodies against subunits of the GABAA receptor. Immunolabelling for ,1, ,2/3 and ,2 subunits was primarily located at symmetrical synapses. Double-labelling revealed that ,2/3 subunit-positive synapses were formed by terminals that were enriched in GABA. Colocalization of ,1, ,2/3 and ,2 subunits occurred at individual symmetrical synapses, some of which were identified as degenerating terminals derived from the striatum. In the SN ipsilateral to the striatal lesion there was a significant elevation of immunolabelling for ,2/3 subunits of the GABAA receptor at symmetrical synapses, but not of GluR2/3 subunits of the AMPA receptor at asymmetrical synapses. It was concluded that fast GABAA -mediated transmission occurs primarily at symmetrical synapses within the SN, that different receptor subunits coexist at individual synapses and that the upregulation of GABAA receptors following striatal lesions is expressed as increased receptor density at synapses. The upregulation of GABAA receptors in Huntington's disease and its models is thus likely to lead to an increased efficiency of transmission at intact GABAergic synapses in the SN and may partly underlie the motor abnormalities of this disorder. [source] Rescue of ,2 subunit-deficient mice by transgenic overexpression of the GABAA receptor ,2S or ,2L subunit isoformsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2000Kristin Baer Abstract The ,2 subunit is an important functional determinant of GABAA receptors and is essential for formation of high-affinity benzodiazepine binding sites and for synaptic clustering of major GABAA receptor subtypes along with gephyrin. There are two splice variants of the ,2 subunit, ,2 short (,2S) and ,2 long (,2L), the latter carrying in the cytoplasmic domain an additional eight amino acids with a putative phosphorylation site. Here, we show that transgenic mice expressing either the ,2S or ,2L subunit on a ,2 subunit-deficient background are phenotypically indistinguishable from wild-type. They express nearly normal levels of ,2 subunit protein and [3H]flumazenil binding sites. Likewise, the distribution, number and size of GABAA receptor clusters colocalized with gephyrin are similar to wild-type in both juvenile and adult mice. Our results indicate that the two ,2 subunit splice variants can substitute for each other and fulfil the basic functions of GABAA receptors, allowing in vivo studies that address isoform-specific roles in phosphorylation-dependent regulatory mechanisms. [source] Fine mapping of a sedative-hypnotic drug withdrawal locus on mouse chromosome 11GENES, BRAIN AND BEHAVIOR, Issue 1 2006H. M. Hood We have established that there is a considerable amount of common genetic influence on physiological dependence and associated withdrawal from sedative-hypnotic drugs including alcohol, benzodiazepines, barbiturates and inhalants. We previously mapped two loci responsible for 12 and 9% of the genetic variance in acute alcohol and pentobarbital withdrawal convulsion liability in mice, respectively, to an approximately 28-cM interval of proximal chromosome 11. Here, we narrow the position of these two loci to a 3-cM interval (8.8 Mb, containing 34 known and predicted genes) using haplotype analysis. These include genes encoding four subunits of the GABAA receptor, which is implicated as a pivotal component in sedative-hypnotic dependence and withdrawal. We report that the DBA/2J mouse strain, which exhibits severe withdrawal from sedative-hypnotic drugs, encodes a unique GABAA receptor ,2 subunit variant compared with other standard inbred strains including the genetically similar DBA/1J strain. We also demonstrate that withdrawal from zolpidem, a benzodiazepine receptor agonist selective for ,1 subunit containing GABAA receptors, is influenced by a chromosome 11 locus, suggesting that the same locus (gene) influences risk of alcohol, benzodiazepine and barbiturate withdrawal. Our results, together with recent knockout studies, point to the GABAA receptor ,2 subunit gene (Gabrg2) as a promising candidate gene to underlie phenotypic differences in sedative-hypnotic physiological dependence and associated withdrawal episodes. [source] Radiosynthesis of novel 18F-labelled derivatives of indiplon as potential GABAA receptor imaging tracers for PETJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 3 2008Steffen Fischer Abstract The involvement of gamma amino butyric acid (GABA) receptors in a variety of neurological and psychiatric diseases has promoted the development and use of radiolabelled benzodiazepines (BZ) for brain imaging by PET. However, these radioligands are unable to distinguish between the various subtypes of GABAA receptors. Novel non-BZ such as the pyrazolo-pyrimidine indiplon proved to be selective for the ,1 -subunit of the GABAA receptor. Here, we describe the syntheses of four novel 18F-labelled indiplon derivatives. Radiosyntheses were performed via n.c.a. 18F-nucleophilic substitution starting from the tosyl, bromo, and 4-nitrobenzoyl precursors to obtain fluorine substituted N -alkylamide side chain derivatives of indiplon, followed by multistep purification using semi-preparative high-performance liquid chromatography and solid phase extraction. Tosyl and bromo precursors were converted into 18F-labelled indiplon derivatives with good and reproducible radiochemical yield (RCY) (35,70%, decay corrected), high radiochemical purity (,98.5%), and high specific activity (,>,150,GBq/µmol). By contrast, a low RCY (5,10%) and specific activity (10,15,GBq/µmol) were achieved for the 4-nitrobenzoyl precursor. Copyright © 2008 John Wiley & Sons, Ltd. [source] Pregnenolone sulfate induces NMDA receptor dependent release of dopamine from synaptic terminals in the striatumJOURNAL OF NEUROCHEMISTRY, Issue 2 2008Matthew T. Whittaker Abstract Neuromodulators that alter the balance between lower-frequency glutamate-mediated excitatory and higher-frequency GABA-mediated inhibitory synaptic transmission are likely to participate in core mechanisms for CNS function and may contribute to the pathophysiology of neurological disorders such as schizophrenia and Alzheimer's disease. Pregnenolone sulfate (PS) modulates both ionotropic glutamate and GABAA receptor mediated synaptic transmission. The enzymes necessary for PS synthesis and degradation are found in brain tissue of several species including human and rat, and up to 5 nM PS has been detected in extracts of postmortem human brain. Here, we ask whether PS could modulate transmitter release from nerve terminals located in the striatum. Superfusion of a preparation of striatal nerve terminals comprised of mixed synaptosomes and synaptoneurosomes with brief-duration (2 min) pulses of 25 nM PS demonstrates that PS increases the release of newly accumulated [3H]dopamine ([3H]DA), but not [14C]glutamate or [3H]GABA, whereas pregnenolone is without effect. PS does not affect dopamine transporter (DAT) mediated uptake of [3H]DA, demonstrating that it specifically affects the transmitter release mechanism. The PS-induced [3H]DA release occurs via an NMDA receptor (NMDAR) dependent mechanism as it is blocked by D-2-amino-5-phosphonovaleric acid. PS modulates DA release with very high potency, significantly increasing [3H]DA release at PS concentrations as low as 25 pM. This first report of a selective direct enhancement of synaptosomal dopamine release by PS at picomolar concentrations via an NMDAR dependent mechanism raises the possibility that dopaminergic axon terminals may be a site of action for this neurosteroid. [source] GABAA receptors in aging and Alzheimer's diseaseJOURNAL OF NEUROCHEMISTRY, Issue 4 2007Robert A. Rissman Abstract In this article we present a comprehensive review of relevant research and reports on the GABAA receptor in the aged and Alzheimer's disease (AD) brain. In comparison to glutamatergic and cholinergic systems, the GABAergic system is relatively spared in AD, but the precise mechanisms underlying differential vulnerability are not well understood. Using several methods, investigations demonstrate that despite resistance of the GABAergic system to neurodegeneration, particular subunits of the GABAA receptor are altered with age and AD, which can induce compensatory increases in GABAA receptor subunits within surrounding cells. We conclude that although aging- and disease-related changes in GABAA receptor subunits may be modest, the mechanisms that compensate for these changes may alter the pharmacokinetic and physiological properties of the receptor. It is therefore crucial to understand the subunit composition of individual GABAA receptors in the diseased brain when developing therapeutics that act at these receptors. [source] Trafficking and potential assembly patterns of ,-containing GABAA receptorsJOURNAL OF NEUROCHEMISTRY, Issue 3 2007Brian L. Jones Abstract Incorporation of the , subunit into the GABAA receptor has been suggested to confer unusual, but variable, biophysical and pharmacological characteristics to both recombinant and native receptors. Due to their structural similarity with the , subunits, , subunits have been assumed to substitute at the single position of the , subunit in assembled receptors. However, prior work suggests that functional variability in ,-containing receptors may reflect alternative sites of incorporation and of not just one, but possibly multiple , subunits in the pentameric receptor complex. Here we present data indicating that increased expression of ,, in conjunction with ,2 and ,3 subunits, results in expression of GABAA receptors with correspondingly altered rectification, deactivation and levels of spontaneous openings, but not increased total current density. We also provide data that the , subunit, like the ,3 subunit, can self-export and data from chimeric receptors suggesting that similarities between the assembly domains of the ,3 and the , subunits may allow the , subunit to replace the ,, as well as the ,, subunit. The substitution of an , for a ,, as well as the , subunit and formation of receptors with alternative patterns of assembly with respect to , incorporation may underlie the observed variability in both biophysical and pharmacological properties noted not only in recombinant, but also in native receptors. [source] GABAA receptor associated proteins: a key factor regulating GABAA receptor functionJOURNAL OF NEUROCHEMISTRY, Issue 2 2007Zi-Wei Chen Abstract ,-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in both vertebrates and invertebrates, acts on GABA receptors that are ubiquitously expressed in the CNS. GABAA receptors also represent a major site of action of clinically relevant drugs, such as benzodiazepines, barbiturates, ethanol, and general anesthetics. It has been shown that the intracellular M3-M4 loop of GABAA receptors plays an important role in regulating GABAA receptor function. Therefore, studies of the function of receptor intracellular loop associated proteins become important for understanding mechanisms of regulating receptor activity. Recently, several labs have used the yeast two-hybrid assay to identify proteins interacting with GABAA receptors, for example, the interaction of GABAA receptor associated protein (GABARAP) and Golgi-specific DHHC zinc finger protein (GODZ) with , subunits, PRIP, phospholipase C-related, catalytically inactive proteins (PRIP-1) and (PRIP-2) with GABARAP and receptor ,2 and , subunits, Plic-1 with some , and , subunits, radixin with the ,5 subunit, HAP1 with the ,1 subunit, GABAA receptor interacting factor-1 (GRIF-1) with the ,2 subunit, and brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) with the ,3 subunit. These proteins have been shown to play important roles in modulating the activities of GABAA receptors ranging from enhancing trafficking, to stabilizing surface and internalized receptors, to regulating modification of GABAA receptors. This article reviews the current studies of GABAA receptor intracellular loop-associated proteins. [source] Consequence of the presence of two different , subunit isoforms in a GABAA receptorJOURNAL OF NEUROCHEMISTRY, Issue 6 2005Nathalie Boulineau Abstract The major isoforms of GABAA receptors are thought to be composed of two ,, two , and one , subunit(s). GABAA receptors containing two ,1 subunits respond differently to the anticonvulsive compound loreclezole and the general anaesthetic etomidate than receptors containing two ,2 subunits. Receptors containing ,2 subunits show a much larger allosteric stimulation by these agents than those containing ,1 subunits. We were interested to know how receptors containing both ,1 and ,2 subunits, in different positions respond to loreclezole and etomidate. To answer this question, subunits were fused at the DNA level to form dimeric and trimeric subunits. Concatenated receptors (,1 -,1 -,1/,2 -,1, ,1 -,2 -,1/,2 -,1, ,1 -,1 -,1/,2 -,2 and ,1 -,2 -,1/,2 -,2) were expressed in Xenopus ooctyes and functionally compared in their response to the agonist GABA and to the positive allosteric modulators, loreclezole and etomidate. We have shown that (I) in the presence of both ,1 and ,2 subunits in the same pentamer (mixed receptors) direct gating by etomidate is similar to exclusively ,1 containing receptors; (II) In mixed receptors, stimulation by etomidate assumed characteristics intermediate to exclusively ,1 or ,2 containing receptors, but the values for the concentrations < 10 µm were always much closer to those observed in ,1 -,1 -,1/,2 -,1 receptors; and (III) mixed receptors show no positional effects. [source] Insulin exerts neuroprotection by counteracting the decrease in cell-surface GABAA receptors following oxygen,glucose deprivation in cultured cortical neuronsJOURNAL OF NEUROCHEMISTRY, Issue 1 2005John G. Mielke Abstract A loss of balance between excitatory and inhibitory signaling leads to excitoxicity, and contributes to ischemic cell death. Reduced synaptic inhibition as a result of dysfunction of the ionotropic GABAA receptor has been suggested as one of the major causes for this imbalance, although the underlying mechanisms remain poorly understood. In the present study, we investigated whether oxygen,glucose deprivation (OGD), an ischemia-like challenge, alters cell-surface expression of GABAA receptors in cultured hippocampal neurons, and thereby leads to excitotoxic cell death. Using cell culture ELISA as a cell surface receptor assay, we found that OGD produced a marked decrease in cell surface GABAA receptors, without altering the total amount of receptors. Furthermore, the reduction could be prevented by inhibition of receptor endocytosis with hypertonic sucrose treatment. Notably, insulin significantly limited OGD-induced changes in cell-surface GABAA receptors. In parallel, insulin protected cultured neurons against both glutamate toxicity and OGD, as assayed by mitochondrial reduction of Alamar Blue. Importantly, insulin-mediated neuroprotection was eliminated when bicuculline, a GABAA receptor antagonist, was co-applied with insulin during OGD. Together, our results strongly suggest that ischemia-like insults decrease cell surface GABAA receptors in neurons via accelerated internalization, and that insulin provides neuroprotection by counteracting this reduction. [source] Diazepam Promotes ATP Recovery and Prevents Cytochrome c Release in Hippocampal Slices After In Vitro IschemiaJOURNAL OF NEUROCHEMISTRY, Issue 3 2000Francesca Galeffi Abstract: Benzodiazepines protect hippocampal neurons when administered within the first few hours after transient cerebral ischemia. Here, we examined the ability of diazepam to prevent early signals of cell injury (before cell death) after in vitro ischemia. Ischemia in vitro or in vivo causes a rapid depletion of ATP and the generation of cell death signals, such as the release of cytochrome c from mitochondria. Hippocampal slices from adult rats were subjected to 7 min of oxygen-glucose deprivation (OGD) and assessed histologically 3 h after reoxygenation. At this time, area CA1 neurons appeared viable, although slight abnormalities in structure were evident. Immediately following OGD, ATP levels in hippocampus were decreased by 70%, and they recovered partially over the next 3 h of reoxygenation. When diazepam was included in the reoxygenation buffer, ATP levels recovered completely by 3 h after OGD. The effects of diazepam were blocked by picrotoxin, indicating that the protection was mediated by an influx of Cl - through the GABAA receptor. It is interesting that the benzodiazepine antagonist flumazenil did not prevent the action of diazepam, as has been shown in other studies using the hippocampus. Two hours after OGD, the partial recovery of ATP levels occurred simultaneously with an increase of cytochrome c (,400%) in the cytosol. When diazepam was included in the reoxygenation buffer, it completely prevented the increase in cytosolic cytochrome c. Thus, complete recovery of ATP and prevention of cytochrome c release from mitochondria can be achieved when diazepam is given after the loss of ATP induced by OGD. [source] GABAergic Modulation of the Expression of Genes Involved in GABA Synaptic Transmission and Stress in the Hypothalamus and Telencephalon of the Female Goldfish (Carassius auratus)JOURNAL OF NEUROENDOCRINOLOGY, Issue 5 2005C. J. Martyniuk Abstract GABA is one of the most abundant neurotransmitters in the vertebrate central nervous system and is involved in neuroendocrine processes such as development, reproduction, feeding and stress. To examine the effect of GABA on gene expression in the brain, we used a cDNA macroarray containing 26 genes involved in GABA synaptic transmission (GABA receptor subunits, GABA transporters), reproduction (gonadotrophin-releasing hormone isoforms and oestrogen receptor ,), feeding (neuropeptide Y and cholecystokinin), and stress [corticotrophin-releasing factor (CRF)]. To elevate GABA levels in the brain, we injected female goldfish with gamma-vinyl GABA (300 µg/g of body weight) (24 h), an irreversible inhibitor of the enzyme GABA transaminase (GABA-T). We found that increased levels of GABA in the hypothalamus resulted in a 2.2-fold down-regulation of GABAA receptor ,4 subunit mRNA. In the telencephalon, we found that increased GABA levels resulted in a 1.5-fold increase of CRF mRNA and a 1.8-fold decrease of GABAA receptor ,2 subunit mRNA. Increasing GABA in the hypothalamus and telencephalon of the goldfish did not significantly affect the mRNA abundance of genes involved in GABA synthesis (glutamic acid decarboxylase isoforms) and degradation (GABA-T), feeding, or reproduction. Our preliminary study suggests that the regulation of GABA receptor subunit mRNA expression by GABA may be a conserved evolutionary mechanism in vertebrates to modulate GABAergic synaptic transmission. [source] Altering the Relative Abundance of GABAA Receptor Subunits Changes GABA- and Ethanol-Responses in Xenopus OocytesALCOHOLISM, Issue 6 2009Joyce H. Hurley Background:, Variations in GABRA2 and GABRG3, genes encoding the ,2 and ,3 subunits of the pentameric GABAA receptor, are associated with the risk of developing alcoholism in adults, conduct disorder at younger ages, and with differences in electroencephalographic power in the , frequency range. The SNPs associated with alcoholism did not alter the coding of these genes, and extensive DNA sequencing of GABRA2 did not find coding changes in the high-risk haplotypes. Therefore, we hypothesize that the associations arise from differences in gene expression. Methods:, Here we report studies in Xenopus oocytes to examine the functional effects of altering the relative abundance of these 2 receptor subunits on GABA current and response to ethanol, as a model of potential effects of regulatory differences. Results:, When human ,2,2,3 subunits are co-expressed, increasing the amount of the ,2 subunit mRNA increased GABA current; in contrast, increasing the amount of the ,3 subunit decreased GABA currents. Acute ethanol treatment of oocytes injected with a 1:1:1 or 2:2:1 ratio of ,2:,2:,3 subunit mRNAs resulted in significant potentiation of GABA currents, whereas ethanol inhibited GABA currents in cells injected with a 6:2:1 ratio. Overnight treatment with ethanol significantly reduced GABA currents in a manner dependent on the ratio of subunits. Conclusions:, These studies demonstrate that changes in relative expression of GABAA receptor subunits alter the response of the resulting channels to GABA and to ethanol. [source] Chronic Ethanol Administration Alters Immunoreactivity for GABAA Receptor Subunits in Rat Cortex in a Region-Specific MannerALCOHOLISM, Issue 8 2000A. Chistina Grobin Background: Chronic ethanol administration has a plethora of physiological effects. Among the most consistently observed findings is a change in the expression pattern of ,-aminobutyric acid type A (GABAA) receptor subunits in the rat brain cortex. These findings led to the hypothesis of "subunit substitution" to account for changes in receptor function without changes in receptor number. Methods: We used subunit (,1 and ,4) specific antibodies and a combination of immunohistochemistry and immunoblotting to examine subregions of cortex (prefrontal, cingulate, motor, parietal, and piriform) for their response to 2 weeks of forced ethanol administration. Results: Overall, cortical immunoreactivity for the ,1 subunit was decreased and for the ,4 subunit increased whether measured immunohistochemically or by immunoblotting. Piriform cortex exhibited a bidirectional change in GABAA receptor ,1 and ,4 immunoreactivity, similar to that previously observed in preparations of whole cortex. However, in parietal cortex, declines in ,1 immunoreactivity (55 ± 12% control value [CV] and 88.3 ± 4.3% CV; immunohistochemistry and immunoblotting, respectively) were not accompanied by concomitant increases in ,4 immunoreactivity (104 ± 8% CV and 116 ± 9.3% CV; immunohistochemistry and immunoblotting, respectively). Conversely, ,4 immunoreactivity increased in cingulate cortex (210 ± 30% CV and 134 ± 9.5% CV; immunohistochemistry and immunoblotting, respectively) without a decline in ,1 immunoreactivity (90 ± 4% CV and 91.3 ± 3.9% CV; immunohistochemistry and immunoblotting, respectively). Prefrontal and motor cortex exhibited GABAA receptor subunit peptide alterations, but these changes varied with the method of analysis. Conclusions: These findings demonstrate that ethanol dependence results in nonuniform changes in GABAA receptor subunit peptide levels across the rat brain cortex and suggest that mechanisms which subserve functional changes in receptor activity may vary in accordance with anatomic or cellular differences within the cortex. [source] GABAmimetic intravenous anaesthetics inhibit spontaneous Ca2+ -oscillations in cultured hippocampal neuronsACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 6 2006B. Sinner Background:, Spontaneous Ca2+ -oscillations are a possible mechanism of Ca2+ -mediated signal transduction in neurons. They develop by a periodical interplay of Ca2+, which enters the neuron from the extracellular medium and triggers Ca2+ release from the endoplasmic reticulum (ER). Ca2+ -oscillations are terminated by reuptake into the ER or plasmalemmal extrusion. Spontaneous Ca2+ -oscillations are glutamate dependent and appear to be responsible for neuronal plasticity and integration of information. Here, we examined the role of the gamma-aminobutyric acid (GABAA) receptor on spontaneous Ca2+ -oscillations and studied the effects of the anaesthetics midazolam, thiopental and the non-anesthetic barbituric acid on spontaneous Ca2+ -oscillations. Methods:, Hippocampal neuronal cell cultures of 19-day-old embryonic Wistar rats 17,18 days in culture were loaded with the Ca2+ -sensitive dye Fura-2AM. Experiments were performed using dual wave-length excitation fluorescence microscopy and calibration constants were obtained from in situ calibration. Results:, Spontaneous Ca2+ -oscillations are influenced by the GABAA receptor. The intravenous anaesthetics midazolam and thiopental suppressed the amplitude and frequency reversibly in a dose-dependent manner with EC50 in clinically relevant concentrations. This effect was mediated via the GABAA receptor as it could be reversed by the GABAA receptor antagonist bicuculline. In contrast, the application of barbituric acid had no effects on the spontaneous Ca2+ -oscillations. Conclusion:, Spontaneous Ca2+ -oscillations are influenced by the GABAA receptor. Spontaneous Ca2+ -oscillations might represent an interesting model system to study anaesthetic mechanisms on neuronal information processing. [source] Comparative cellular distribution of GABAA and GABAB receptors in the human basal ganglia: Immunohistochemical colocalization of the ,1 subunit of the GABAA receptor, and the GABABR1 and GABABR2 receptor subunitsTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 4 2004Henry J. Waldvogel Abstract The GABAB receptor is a G-protein linked metabotropic receptor that is comprised of two major subunits, GABABR1 and GABABR2. In this study, the cellular distribution of the GABABR1 and GABABR2 subunits was investigated in the normal human basal ganglia using single and double immunohistochemical labeling techniques on fixed human brain tissue. The results showed that the GABAB receptor subunits GABABR1 and GABABR2 were both found on the same neurons and followed the same distribution patterns. In the striatum, these subunits were found on the five major types of interneurons based on morphology and neurochemical labeling (types 1, 2, 3, 5, 6) and showed weak labeling on the projection neurons (type 4). In the globus pallidus, intense GABABR1 and GABABR2 subunit labeling was found in large pallidal neurons, and in the substantia nigra, both pars compacta and pars reticulata neurons were labeled for both receptor subunits. Studies investigating the colocalization of the GABAA ,1 subunit and GABAB receptor subunits showed that the GABAA receptor ,1 subunit and the GABABR1 subunit were found together on GABAergic striatal interneurons (type 1 parvalbumin, type 2 calretinin, and type 3 GAD neurons) and on neurons in the globus pallidus and substantia nigra pars reticulata. GABABR1 and GABABR2 were found on substantia nigra pars compacta neurons but the GABAA receptor ,1 subunit was absent from these neurons. The results of this study provide the morphological basis for GABAergic transmission within the human basal ganglia and provides evidence that GABA acts through both GABAA and GABAB receptors. That is, GABA acts through GABAB receptors, which are located on most of the cell types of the striatum, globus pallidus, and substantia nigra. GABA also acts through GABAA receptors containing the ,1 subunit on specific striatal GABAergic interneurons and on output neurons of the globus pallidus and substantia nigra pars reticulata. J. Comp. Neurol. 470:339,356, 2004. © 2004 Wiley-Liss, Inc. [source] Stoichiometry of a pore mutation that abolishes picrotoxin-mediated antagonism of the GABAA receptorTHE JOURNAL OF PHYSIOLOGY, Issue 2 2006Anna Sedelnikova Picrotoxin, a potent antagonist of the inhibitory central nervous system GABAA and glycine receptors, is believed to interact with residues that line the central ion pore. These pore-lining residues are in the second transmembrane domain (TM2) of each of the five constituent subunits. One of these amino acids, a threonine at the 6, location, when mutated to phenylalanine, abolishes picrotoxin sensitivity. It has been suggested that this threonine, via hydrogen bonding, directly interacts with the picrotoxin molecule. We previously demonstrated that this mutation, in the ,, , or , subunit, can impart picrotoxin resistance to the GABA receptor. Since the functional pentameric GABA receptor contains two , subunits, two , subunits and one , subunit, it is not clear how many , and , subunits must carry this mutation to impart the resistant phenotype. In this study, by coexpression of mutant , or , subunits with their wild-type counterparts in various defined ratios, we demonstrate that any single subunit carrying the 6, mutation imparts picrotoxin resistance. Implications of this finding in terms of the mechanism of antagonism are considered. 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