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Sample records for human gabaa receptors

  1. Antagonistic action of pitrazepin on human and rat GABAA receptors

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    Demuro, Angelo; Martinez-Torres, Ataulfo; Francesconi, Walter; Miledi, Ricardo

    1999-01-01

    Pitrazepin, 3-(piperazinyl-1)-9H-dibenz(c,f) triazolo(4,5-a)azepin is a piperazine antagonist of GABA in a variety of electrophysiological and in vitro binding studies involving GABA and glycine receptors. In the present study we have investigated the effects of pitrazepin, and the GABAA antagonist bicuculline, on membrane currents elicited by GABA in Xenopus oocytes injected with rat cerebral cortex mRNA or cDNAs encoding α1β2 or α1β2γ2S human GABAA receptor subunits.The three types of GABAA receptors expressed were reversibly antagonized by bicuculline and pitrazepin in a concentration-dependent manner. GABA dose-current response curves for the three types of receptors were shifted to the right, in a parallel manner, by increasing concentrations of pitrazepin.Schild analyses gave pA2 values of 6.42±0.62, n=4, 6.41±1.2, n=5 and 6.21±1.24, n=6, in oocytes expressing rat cerebral cortex, α1β2 or α1β2γ2S human GABAA receptors respectively (values are given as means±s.e.mean), and the Hill coefficients were all close to unity. All this is consistent with the notion that pitrazepin acts as a competitive antagonist of these GABAA receptors; and that their antagonism by pitrazepin is not strongly dependent on the subunit composition of the receptors here studied.Since pitrazepin has been reported to act also at the benzodiazepine binding site, we studied the effect of the benzodiazepine antagonist Ro 15-1788 (flumazenil) on the inhibition of α1β2γ2S receptors by pitrazepin. Co-application of Ro 15-1788 did not alter the inhibiting effect of pitrazepin. Moreover, pitrazepin did not antagonize the potentiation of GABA-currents by flunitrazepam. All this suggests that pitrazepin does not affect the GABA receptor-chloride channel by interacting with the benzodiazepine receptor site. PMID:10369456

  2. Adenosine receptor antagonists alter the stability of human epileptic GABAA receptors

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    Roseti, Cristina; Martinello, Katiuscia; Fucile, Sergio; Piccari, Vanessa; Mascia, Addolorata; Di Gennaro, Giancarlo; Quarato, Pier Paolo; Manfredi, Mario; Esposito, Vincenzo; Cantore, Gianpaolo; Arcella, Antonella; Simonato, Michele; Fredholm, Bertil B.; Limatola, Cristina; Miledi, Ricardo; Eusebi, Fabrizio

    2008-01-01

    We examined how the endogenous anticonvulsant adenosine might influence γ-aminobutyric acid type A (GABAA) receptor stability and which adenosine receptors (ARs) were involved. Upon repetitive activation (GABA 500 μM), GABAA receptors, microtransplanted into Xenopus oocytes from neurosurgically resected epileptic human nervous tissues, exhibited an obvious GABAA-current (IGABA) run-down, which was consistently and significantly reduced by treatment with the nonselective adenosine receptor antagonist CGS15943 (100 nM) or with adenosine deaminase (ADA) (1 units/ml), that inactivates adenosine. It was also found that selective antagonists of A2B (MRS1706, 10 nM) or A3 (MRS1334, 30 nM) receptors reduced IGABA run-down, whereas treatment with the specific A1 receptor antagonist DPCPX (10 nM) was ineffective. The selective A2A receptor antagonist SCH58261 (10 nM) reduced or potentiated IGABA run-down in ≈40% and ≈20% of tested oocytes, respectively. The ADA-resistant, AR agonist 2-chloroadenosine (2-CA) (10 μM) potentiated IGABA run-down but only in ≈20% of tested oocytes. CGS15943 administration again decreased IGABA run-down in patch-clamped neurons from either human or rat neocortex slices. IGABA run-down in pyramidal neurons was equivalent in A1 receptor-deficient and wt neurons but much larger in neurons from A2A receptor-deficient mice, indicating that, in mouse cortex, GABAA-receptor stability is tonically influenced by A2A but not by A1 receptors. IGABA run-down from wt mice was not affected by 2-CA, suggesting maximal ARs activity by endogenous adenosine. Our findings strongly suggest that cortical A2–A3 receptors alter the stability of GABAA receptors, which could offer therapeutic opportunities. PMID:18809912

  3. Different subtypes of GABA-A receptors are expressed in human, mouse and rat T lymphocytes.

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    Suresh K Mendu

    Full Text Available γ-Aminobutyric acid (GABA is the most prominent neuroinhibitory transmitter in the brain, where it activates neuronal GABA-A receptors (GABA-A channels located at synapses and outside of synapses. The GABA-A receptors are primary targets of many clinically useful drugs. In recent years, GABA has been shown to act as an immunomodulatory molecule. We have examined in human, mouse and rat CD4(+ and CD8(+ T cells which subunit isoforms of the GABA-A channels are expressed. The channel physiology and drug specificity is dictated by the GABA-A receptor subtype, which in turn is determined by the subunit isoforms that make the channel. There were 5, 8 and 13 different GABA-A subunit isoforms identified in human, mouse and rat CD4(+ and CD8(+ T cells, respectively. Importantly, the γ2 subunit that imposes benzodiazepine sensitivity on the GABA-A receptors, was only detected in the mouse T cells. Immunoblots and immunocytochemistry showed abundant GABA-A channel proteins in the T cells from all three species. GABA-activated whole-cell transient and tonic currents were recorded. The currents were inhibited by picrotoxin, SR95531 and bicuculline, antagonists of GABA-A channels. Clearly, in both humans and rodents T cells, functional GABA-A channels are expressed but the subtypes vary. It is important to bear in mind the interspecies difference when selecting the appropriate animal models to study the physiological role and pharmacological properties of GABA-A channels in CD4(+ and CD8(+ T cells and when selecting drugs aimed at modulating the human T cells function.

  4. Abnormal GABAA receptors from the human epileptic hippocampal subiculum microtransplanted to Xenopus oocytes

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    Palma, Eleonora; Spinelli, Gabriele; Torchia, Gregorio; Martinez-Torres, A.; Ragozzino, Davide; Miledi, Ricardo; Eusebi, Fabrizio

    2005-01-01

    We studied the properties of GABAA receptors microtransplanted from the human temporal lobe epilepsy (TLE)-associated brain regions to Xenopus oocytes. Cell membranes, isolated from surgically resected brain specimens of drug-resistant TLE patients, were injected into frog oocytes, which rapidly incorporated human GABAA receptors, and any associated proteins, into their surface membrane. The receptors originating from different epileptic brain regions had a similar run-down but an affinity for GABA that was ≈60% lower for the subiculum receptors than for receptors issuing from the hippocampus proper or the temporal lobe neocortex. Moreover, GABA currents recorded in oocytes injected with membranes from the subiculum had a more depolarized reversal potential compared with the hippocampus proper or neocortex of the same patients. Quantitative RT-PCR analysis was performed of the GABAA receptor α1- to α5-, β1- to β3-, γ2- to γ3-, and δ-subunit mRNAs. The levels of expression of the α3-, α5-, and β1- to β3- subunit mRNAs are significantly higher, with the exception of γ2-subunit whose expression is lower, in subiculum compared with neocortex specimens. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE subiculum leads to the expression of GABAA receptors with a relatively low affinity. This abnormal behavior of the subiculum GABAA receptors may contribute to epileptogenesis. PMID:15695331

  5. Prefrontal GABA(A) receptor alpha-subunit expression in normal postnatal human development and schizophrenia.

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    Duncan, Carlotta E; Webster, Maree J; Rothmond, Debora A; Bahn, Sabine; Elashoff, Michael; Shannon Weickert, Cynthia

    2010-07-01

    Cortical GABA deficits that are consistently reported in schizophrenia may reflect an etiology of failed normal postnatal neurotransmitter maturation. Previous studies have found prefrontal cortical GABA(A) receptor alpha subunit alterations in schizophrenia, yet their relationship to normal developmental expression profiles in the human cortex has not been determined. The aim of this study was to quantify GABA(A) receptor alpha-subunit mRNA expression patterns in human dorsolateral prefrontal cortex (DLPFC) during normal postnatal development and in schizophrenia cases compared to controls. Transcript levels of GABA(A) receptor alpha subunits were measured using microarray and qPCR analysis of 60 normal individuals aged 6weeks to 49years and in 37 patients with schizophrenia/schizoaffective disorder and 37 matched controls. We detected robust opposing changes in cortical GABA(A) receptor alpha1 and alpha5 subunits during the first few years of postnatal development, with a 60% decrease in alpha5 mRNA expression and a doubling of alpha1 mRNA expression with increasing age. In our Australian schizophrenia cohort we detected decreased GAD67 mRNA expression (p=0.0012) and decreased alpha5 mRNA expression (p=0.038) in the DLPFC with no significant change of other alpha subunits. Our findings confirm that GABA deficits (reduced GAD67) are a consistent feature of schizophrenia postmortem brain studies. Our study does not confirm alterations in cortical alpha1 or alpha2 mRNA levels in the schizophrenic DLPFC, as seen in previous studies, but instead we report a novel down-regulation of alpha5 subunit mRNA suggesting that post-synaptic alterations of inhibitory receptors are an important feature of schizophrenia but may vary between cohorts. Copyright 2009 Elsevier Ltd. All rights reserved.

  6. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

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    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

  7. GABAA receptor subtype involvement in addictive behaviour.

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    Stephens, D N; King, S L; Lambert, J J; Belelli, D; Duka, T

    2017-01-01

    GABAA receptors form the major class of inhibitory neurotransmitter receptors in the mammalian brain. This review sets out to summarize the evidence that variations in genes encoding GABAA receptor isoforms are associated with aspects of addictive behaviour in humans, while animal models of addictive behaviour also implicate certain subtypes of GABAA receptor. In addition to outlining the evidence for the involvement of specific subtypes in addiction, we summarize the particular contributions of these isoforms in control over the functioning of brain circuits, especially the mesolimbic system, and make a first attempt to bring together evidence from several fields to understanding potential involvement of GABAA receptor subtypes in addictive behaviour. While the weight of the published literature is on alcohol dependency, the underlying principles outlined are relevant across a number of different aspects of addictive behaviour. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  8. Role of human GABA(A) receptor beta3 subunit in insecticide toxicity.

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    Ratra, G S; Kamita, S G; Casida, J E

    2001-05-01

    The gamma-aminobutyric acid type A (GABA(A)) receptor is the target for the major insecticides alpha-endosulfan, lindane, and fipronil and for many analogs. Their action as chloride channel blockers is directly measured by binding studies with [(3)H]ethynylbicycloorthobenzoate ([(3)H]EBOB). This study tests the hypothesis that GABA(A) receptor subunit composition determines the sensitivity and selectivity of insecticide toxicity. Human receptor subtypes were expressed individually (alpha1, alpha6, beta1, beta3, and gamma2) and in combination in insect Sf9 cells. Binding parameters were similar for [(3)H]EBOB in the beta3 homooligomer, alpha1beta3gamma2 heterooligomer, and native brain membranes, but toxicological profiles were very different. Surprisingly, alpha-endosulfan, lindane, and fipronil were all remarkably potent on the recombinant beta3 homooligomeric receptor (IC50 values of 0.5-2.4 nM), whereas they were similar in potency on the alpha1beta3gamma2 subtype (IC50 values of 16-33 nM) and highly selective on the native receptor (IC50 values of 7.3, 306, and 2470 nM, respectively). The selectivity order for 29 insecticides and convulsants as IC50 ratios for native/beta3 or alpha1beta3gamma2/beta3 was as follows: fipronil > lindane > 19 other insecticides including alpha-endosulfan and picrotoxinin > 4 trioxabicyclooctanes and dithianes (almost nonselective) > tetramethylenedisulfotetramine, 4-chlorophenylsilatrane, or alpha-thujone. Specificity between mammals and insects at the target site (fipronil > lindane > alpha-endosulfan) paralleled that for toxicity. Potency at the native receptor is more predictive for inhibition of GABA-stimulated chloride uptake than that at the beta3 or alpha1beta3gamma2 receptors. Therefore, the beta3 subunit contains the insecticide target and other subunits differentially modulate the binding to confer compound-dependent specificity and selective toxicity.

  9. Altered GABAA Receptor Subunit Expression and Pharmacology in Human Angelman Syndrome Cortex

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    Roden, William H.; Peugh, Lindsey D.; Jansen, Laura A.

    2011-01-01

    The neurodevelopmental disorder Angelman syndrome is most frequently caused by deletion of the maternally-derived chromosome 15q11-q13 region, which includes not only the causative UBE3A gene, but also the β3-α5-γ3 GABAA receptor subunit gene cluster. GABAergic dysfunction has been hypothesized to contribute to the occurrence of epilepsy and cognitive and behavioral impairments in this condition. In the present study, analysis of GABAA receptor subunit expression and pharmacology was performed in cerebral cortex from four subjects with Angelman syndrome and compared to that from control tissue. The membrane fraction of frozen postmortem neocortical tissue was isolated and subjected to quantitative Western blot analysis. The ratios of β3/β2 and α5/α1 subunit protein expression in Angelman syndrome cortex were significantly decreased when compared with controls. An additional membrane fraction was injected into Xenopus oocytes, resulting in incorporation of the brain membrane vesicles with their associated receptors into the oocyte cellular membrane. Two-electrode voltage clamp analysis of GABAA receptor currents was then performed. Studies of GABAA receptor pharmacology in Angelman syndrome cortex revealed increased current enhancement by the α1-selective benzodiazepine site agonist zolpidem and by the barbiturate phenobarbital, while sensitivity to current inhibition by zinc was decreased. GABAA receptor affinity and modulation by neurosteroids were unchanged. This shift in GABAA receptor subunit expression and pharmacology in Angelman syndrome is consistent with impaired extrasynaptic but intact to augmented synaptic cortical GABAergic inhibition, which could contribute to the epileptic, behavioral, and cognitive phenotypes of the disorder. PMID:20692323

  10. The diversity of GABA(A) receptor subunit distribution in the normal and Huntington's disease human brain.

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    Waldvogel, H J; Faull, R L M

    2015-01-01

    GABA(A) receptors are assembled into pentameric receptor complexes from a total of 19 different subunits derived from a variety of different subunit classes (α1-6, β1-3, γ1-3, δ, ɛ, θ, and π) which surround a central chloride ion channel. GABA(A) receptor complexes are distributed heterogeneously throughout the brain and spinal cord and are activated by the extensive GABAergic inhibitory system. In this chapter, we describe the heterogeneous distribution of six of the most widely distributed subunits (α1, α2, α3, β2,3, and γ2) throughout the human basal ganglia. This review describes the studies we have carried out on the normal and Huntington's disease human basal ganglia using autoradiographic labeling and immunohistochemistry in the human basal ganglia. GABA(A) receptors are known to react to changing conditions in the brain in neurological disorders, especially in Huntington's disease and display a high degree of plasticity which is thought to compensate for loss of function caused by disease. In Huntington's disease, the variable loss of GABAergic medium spiny striatopallidal projection neurons is associated with a loss of GABA(A) receptor subunits in the striosome and/or the matrix compartments of the striatum. By contrast in the globus pallidus, a loss of the GABAergic striatal projection neurons results in a dramatic upregulation of subunits on the large postsynaptic pallidal neurons; this is thought to be a compensatory plastic mechanism resulting from the loss of striatal GABAergic input. Most interestingly, our studies have revealed that the subventricular zone overlying the caudate nucleus contains a variety of proliferating progenitor stem cells that possess a heterogeneity of GABA(A) receptor subunits which may play a role in human brain repair mechanisms. © 2015 Elsevier Inc. All rights reserved.

  11. Modulation of human GABAA receptor function: a novel mode of action of drugs of abuse.

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    Hondebrink, L; Meulenbelt, J; van Kleef, R G D M; van den Berg, M; Westerink, R H S

    2011-12-01

    Drugs of abuse are known to mainly affect the dopaminergic and serotonergic system, although behavioral studies indicated that the GABA-ergic system also plays a role. We therefore investigated the acute effects of several commonly used drugs of abuse (methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA) and meta-chlorophenylpiperazine (mCPP)) on the function of the human α(1)β(2)γ(2) GABA(A) receptor (hGABA(A)-R), expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique. Although none of the tested drugs acted as full agonist on the hGABA(A)-R, some drugs induced differential modulation of hGABA(A)-R function, depending on the degree of receptor occupancy. Methamphetamine did not affect the GABA-evoked current at high receptor occupancy, but induced a minor inhibition at low receptor occupancy. Its metabolite amphetamine slightly potentiated the GABA-evoked current. MDMA and its metabolite MDA both inhibited the current at low receptor occupancy. However, MDMA did not affect the current at high occupancy, whereas MDA induced a potentiation. mCPP induced a strong inhibition (max. ∼ 80%) at low receptor occupancy, but ∼ 25% potentiation at high receptor occupancy. Competitive binding to one of the GABA-binding sites could explain the drug-induced inhibitions observed at low receptor occupancy, whereas an additional interaction with a positive allosteric binding site may play a role in the observed potentiations at high receptor occupancy. This is the first study to identify direct modulation of hGABA(A)-Rs as a novel mode of action for several drugs of abuse. Consequently, hGABA(A)-Rs should be considered as target for psychiatric pharmaceuticals and in developing treatment for drug intoxications.

  12. Axonal GABAA receptors.

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    Trigo, Federico F; Marty, Alain; Stell, Brandon M

    2008-09-01

    Type A GABA receptors (GABA(A)Rs) are well established as the main inhibitory receptors in the mature mammalian forebrain. In recent years, evidence has accumulated showing that GABA(A)Rs are prevalent not only in the somatodendritic compartment of CNS neurons, but also in their axonal compartment. Evidence for axonal GABA(A)Rs includes new immunohistochemical and immunogold data: direct recording from single axonal terminals; and effects of local applications of GABA(A)R modulators on action potential generation, on axonal calcium signalling, and on neurotransmitter release. Strikingly, whereas presynaptic GABA(A)Rs have long been considered inhibitory, the new studies in the mammalian brain mostly indicate an excitatory action. Depending on the neuron that is under study, axonal GABA(A)Rs can be activated by ambient GABA, by GABA spillover, or by an autocrine action, to increase either action potential firing and/or transmitter release. In certain neurons, the excitatory effects of axonal GABA(A)Rs persist into adulthood. Altogether, axonal GABA(A)Rs appear as potent neuronal modulators of the mammalian CNS.

  13. GABAA-current rundown of temporal lobe epilepsy is associated with repetitive activation of GABAA “phasic” receptors

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    Palma, Eleonora; Roseti, Cristina; Maiolino, Francesca; Fucile, Sergio; Martinello, Katiuscia; Mazzuferi, Manuela; Aronica, Eleonora; Manfredi, Mario; Esposito, Vincenzo; Cantore, Gianpaolo; Miledi, Ricardo; Simonato, Michele; Eusebi, Fabrizio

    2007-01-01

    A study was made of the “rundown” of GABAA receptors, microtransplanted to Xenopus oocytes from surgically resected brain tissues of patients afflicted with drug-resistant human mesial temporal lobe epilepsy (mTLE). Cell membranes, isolated from mTLE neocortex specimens, were injected into frog oocytes that rapidly incorporated functional GABAA receptors. Upon repetitive activation with GABA (1 mM), “epileptic” GABAA receptors exhibited a GABAA-current (IGABA) rundown that was significantly enhanced by Zn2+ (≤250 μM), and practically abolished by the high-affinity GABAA receptor inverse agonist SR95531 (gabazine; 2.5–25 μM). Conversely, IGABA generated by “control” GABAA receptors microtransplanted from nonepileptic temporal lobe, lesional TLE, or authoptic disease-free tissues remained stable during repetitive stimulation, even in oocytes treated with Zn2+. We conclude that rundown of mTLE epileptic receptors depends on the presence of “phasic GABAA receptors” that have low sensitivity to antagonism by Zn2+. Additionally, we found that GABAA receptors, microtransplanted from the cerebral cortex of adult rats exhibiting recurrent seizures, caused by pilocarpine-induced status epilepticus, showed greater rundown than control tissue, an event also occurring in patch-clamped rat pyramidal neurons. Rundown of epileptic rat receptors resembled that of human mTLE receptors, being enhanced by Zn2+ (40 μM) and sensitive to the antiepileptic agent levetiracetam, the neurotrophin brain-derived neurotrophic factor, and the phosphatase blocker okadaic acid. Our findings point to the rundown of GABAA receptors as a hallmark of TLE and suggest that modulating tonic and phasic mTLE GABAA receptor activity may represent a useful therapeutic approach to the disease. PMID:18083839

  14. Additive inhibition of human α1β2γ2 GABAA receptors by mixtures of commonly used drugs of abuse.

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    Hondebrink, Laura; Tan, Sijie; Hermans, Elise; van Kleef, Regina G D M; Meulenbelt, Jan; Westerink, Remco H S

    2013-03-01

    Yearly, exposure to drugs of abuse results in ∼1 million emergency department visits in the US. In ∼50% of the visits, stimulant drugs like cocaine and amphetamine-like substances (e.g. 3,4-methylenedioxymethamphetamine (MDMA, the main active ingredient of ecstasy)) are involved, whereas in ∼60% multiple drugs are involved. These drugs induce higher dopamine and serotonin levels resulting in drug-induced toxicity. Since GABA receptors (GABA-R) provide the main inhibitory input on dopaminergic and serotonergic neurons, drug-induced inhibition of GABA-R could contribute to higher neurotransmitter levels and thus toxicity. We therefore investigated the effects of combinations of commonly abused stimulant drugs (cocaine, MDMA, 3,4-methylenedioxyamphetamine (MDA) and meta-chlorophenylpiperazine (mCPP)) on the function of the human α1β2γ2 GABAA receptor (hGABAA-R), expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique. These drugs concentration-dependently inhibited the GABA-evoked current (mCPP>cocaine>MDMA>MDA). Most drug combinations decreased the GABA-evoked current stronger than the single drug. Additivity was observed during combined exposure to low concentrations of cocaine and mCPP as well as during combined exposure to MDA with cocaine or mCPP. However, combinations containing MDMA mainly resulted in sub-additivity or no additivity. At drug concentrations relevant for clinical toxicology, co-exposure to ≥2 drugs can decrease the GABA-evoked current in an additive manner. Thus, in patients exposed to multiple drugs, inhibitory GABA-ergic input is reduced more prominently, likely resulting in higher brain dopamine levels. As this will increase the risk for drug-induced toxicity, treatment of drug-intoxicated patients with drugs that enhance GABA-ergic input should be further optimized.

  15. New GABA amides activating GABAA-receptors.

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    Raster, Peter; Späth, Andreas; Bultakova, Svetlana; Gorostiza, Pau; König, Burkhard; Bregestovski, Piotr

    2013-01-01

    We have prepared a series of new and some literature-reported GABA-amides and determined their effect on the activation of GABAA-receptors expressed in CHO cells. Special attention was paid to the purification of the target compounds to remove even traces of GABA contaminations, which may arise from deprotection steps in the synthesis. GABA-amides were previously reported to be partial, full or superagonists. In our hands these compounds were not able to activate GABAA-receptor channels in whole-cell patch-clamp recordings. New GABA-amides, however, gave moderate activation responses with a clear structure-activity relationship suggesting some of these compounds as promising molecular tools for the functional analysis of GABAA-receptors.

  16. New GABA amides activating GABAA-receptors

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    Peter Raster

    2013-02-01

    Full Text Available We have prepared a series of new and some literature-reported GABA-amides and determined their effect on the activation of GABAA-receptors expressed in CHO cells. Special attention was paid to the purification of the target compounds to remove even traces of GABA contaminations, which may arise from deprotection steps in the synthesis. GABA-amides were previously reported to be partial, full or superagonists. In our hands these compounds were not able to activate GABAA-receptor channels in whole-cell patch-clamp recordings. New GABA-amides, however, gave moderate activation responses with a clear structure–activity relationship suggesting some of these compounds as promising molecular tools for the functional analysis of GABAA-receptors.

  17. Metabotropic Regulation of Extrasynaptic GABAA Receptors

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    William Martin Connelly

    2013-10-01

    Full Text Available A large body of work now shows the importance of GABAA receptor-mediated tonic inhibition in regulating CNS function. However, outside of pathological conditions, there is relatively little evidence that the magnitude of tonic inhibition is itself under regulation. Here we review the mechanisms by which tonic inhibition is known to be modulated, and outline the potential behavioural consequences of this modulation. Specifically, we address the ability of protein kinase A and C to phosphorylate the extrasynaptic receptors responsible for the tonic GABAA current, and how G-protein coupled receptors can regulate tonic inhibition through these effectors. We then speculate about the possible functional consequences of regulating the magnitude of the tonic GABAA current.

  18. Action of tremorgenic mycotoxins on GABAA receptor.

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    Gant, D B; Cole, R J; Valdes, J J; Eldefrawi, M E; Eldefrawi, A T

    1987-11-09

    The effects of four tremorgenic and one nontremorgenic mycotoxins were studied on gamma-aminobutyric acid (GABAA) receptor binding and function in rat brain and on binding of a voltage-operated Cl- channel in Torpedo electric organ. None of the mycotoxins had significant effect on [3H]muscimol or [3H]flunitrazepam binding to the GABAA receptor. However, only the four tremorgenic mycotoxins inhibited GABA-induced 36Cl- influx and [35S] t-butylbicyclophosphorothionate [( 35S]TBPS) binding in rat brain membranes, while the nontremorgenic verruculotoxin had no effect. Inhibition of [35S]TBPS binding by paspalinine was non-competitive. This suggests that tremorgenic mycotoxins inhibit GABAA receptor function by binding close to the receptor's Cl- channel. On the voltage-operated Cl- channel, only high concentrations of verruculogen and verruculotoxin caused significant inhibition of the channel's binding of [35S]TBPS. The data suggest that the tremorgenic action of these mycotoxins may be due in part to their inhibition of GABAA receptor function.

  19. Positron Emission Tomography (PET Quantification of GABAA Receptors in the Brain of Fragile X Patients.

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    Charlotte D'Hulst

    Full Text Available Over the last several years, evidence has accumulated that the GABAA receptor is compromised in animal models for fragile X syndrome (FXS, a common hereditary form of intellectual disability. In mouse and fly models, agonists of the GABAA receptor were able to rescue specific consequences of the fragile X mutation. Here, we imaged and quantified GABAA receptors in vivo in brain of fragile X patients using Positron Emission Topography (PET and [11C]flumazenil, a known high-affinity and specific ligand for the benzodiazepine site of GABAA receptors. We measured regional GABAA receptor availability in 10 fragile X patients and 10 control subjects. We found a significant reduction of on average 10% in GABAA receptor binding potential throughout the brain in fragile X patients. In the thalamus, the brain region showing the largest difference, the GABAA receptor availability was even reduced with 17%. This is one of the first reports of a PET study of human fragile X brain and directly demonstrates that the GABAA receptor availability is reduced in fragile X patients. The study reinforces previous hypotheses that the GABAA receptor is a potential target for rational pharmacological treatment of fragile X syndrome.

  20. The effects of stiripentol on GABAA receptors

    OpenAIRE

    Fisher, Janet L.

    2011-01-01

    The anti-convulsant Stiripentol (Diacomit™) has been shown to have a positive impact on control of seizures for many patients with Dravet Syndrome. As with most anti-epileptic drugs, stiripentol has multiple mechanisms of action. Its direct anti-convulsant activity is likely due to enhancement of inhibitory, GABAergic neurotransmission. Stiripentol was shown to increase the activity of both neuronal and recombinant GABAA receptors at clinically relevant concentrations. At recombinant receptor...

  1. GABAA receptors modulate cannabinoid-evoked hypothermia.

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    Rawls, S M; Tallarida, R J; Kon, D A; Geller, E B; Adler, Martin W

    2004-05-01

    Cannabinoids evoke hypothermia by stimulating central CB(1) receptors. GABA induces hypothermia via GABA(A) or GABA(B) receptor activation. CB(1) receptor activation increases GABA release in the hypothalamus, a central locus for thermoregulation, suggesting that cannabinoid and GABA systems may be functionally linked in body temperature regulation. We investigated whether GABA receptors modulate the hypothermic actions of [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one] (WIN 55212-2), a selective cannabinoid agonist, in male Sprague-Dawley rats. WIN 55212-2 (2.5 mg/kg im) produced a rapid hypothermia that peaked 45-90 min postinjection. The hypothermia was attenuated by bicuculline (2 mg/kg ip), a GABA(A) antagonist. However, SCH 50911 (1-10 mg/kg ip), a GABA(B) blocker, did not antagonize the hypothermia. Neither bicuculline (2 mg/kg) nor SCH 50911 (10 mg/kg) by itself altered body temperature. We also investigated a possible role for CB(1) receptors in GABA-generated hypothermia. Muscimol (2.5 mg/kg ip), a GABA(A) agonist, or baclofen (5 mg/kg ip), a GABA(B) agonist, evoked a significant hypothermia. Blockade of CB(1) receptors with SR141716A (2.5 mg/kg im) did not antagonize muscimol- or baclofen-induced hypothermia, indicating that GABA-evoked hypothermia does not contain a CB(1)-sensitive component. Our results implicate GABA(A) receptors in the hypothermic actions of cannabinoids and provide further evidence of a functional link between cannabinoid and GABA systems.

  2. GABAA receptor plasticity in Jurkat T cells.

    Science.gov (United States)

    Dionisio, Leonardo; Arias, Verónica; Bouzat, Cecilia; Esandi, María del Carmen

    2013-12-01

    GABAA receptors (GABAAR) mediate inhibitory neurotransmission in the human brain. Neurons modify subunit expression, cellular distribution and function of GABAAR in response to different stimuli, a process named plasticity. Human lymphocytes have a functional neuronal-like GABAergic system with GABAAR acting as inhibitors of proliferation. We here explore if receptor plasticity occurs in lymphocytes. To this end, we analyzed human T lymphocyte Jurkat cells exposed to different physiological stimuli shown to mediate plasticity in neurons: GABA, progesterone and insulin. The exposure to 100 μM GABA differently affected the expression of GABAAR subunits measured at both the mRNA and protein level, showing an increase of α1, β3, and γ2 subunits but no changes in δ subunit. Exposure of Jurkat cells to different stimuli produced different changes in subunit expression: 0.1 μM progesterone decreased δ and 0.5 μM insulin increased β3 subunits. To identify the mechanisms underlying plasticity, we evaluated the Akt pathway, which is involved in the phosphorylation of β subunits and receptor translocation to the membrane. A significant increase of phosphorylated Akt and on the expression of β3 subunit in membrane occurred in cells exposed 15 h to GABA. To determine if plastic changes are translated into functional changes, we performed whole cell recordings. After 15 h GABA-exposure, a significantly higher percentage of cells responded to GABA application when compared to 0 and 40 h exposure, thus indicating that the detected plastic changes may have a role in GABA-modulated lymphocyte function. Our results reveal that lymphocyte GABAAR are modified by different stimuli similarly and by similar mechanisms to those in neurons. This property is of significance for the development of future therapies involving pharmacological modulation of the immune response. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  3. GABA(A) receptor subtype selective cognition enhancers.

    Science.gov (United States)

    Maubach, Karen

    2003-08-01

    Currently the treatment of Alzheimer's disease (AD) and Mild Cognitive Impairment (MCI) is largely unrealised, with no preventive or curative therapies. The marketed acetylcholinesterase inhibitors (eg. donepezil, Aricept) are directed toward temporary symptomatic relief from impaired cognition, but have prominent adverse effects with minimal efficacy. In pursuit of novel cognition enhancers, the observation that classical benzodiazepines (BZ, eg. diazepam) are amnesic, coupled with the preservation of GABA(A) receptors in brain areas most affected by AD, highlighted the GABA(A) receptor as a potential therapeutic target. In contrast to the amnesic BZ agonists, the BZ inverse agonists (eg. DMCM) which attenuate GABA(A) receptor function, have been shown to improve performance in animal models of learning and memory. Unfortunately, such non-selective ligands also induce anxiety and convulsions. More recently, novel ligands have been developed (eg. 6,6-dimethyl-3-(2-hydroxyethyl)thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophen-4(5H)-one) that demonstrate binding selectivity and high inverse agonism for the alpha5 GABA(A) receptor subtype, which is preferentially located in the hippocampus, a region of the brain associated with learning and memory. Pre-clinical results are encouraging, since these alpha5 selective inverse agonists enhance memory in animal models, such as spatial learning in the Morris water-maze, but are devoid of the adverse effects associated with activity at other GABA(A) receptor subtypes in other brain regions. If the efficacy and safety profiles of alpha5 inverse agonists in humans prove to be similar to those seen in pre-clinical studies, these compounds would offer significant benefit to AD and MCI patients.

  4. GABAA receptor partial agonists and antagonists

    DEFF Research Database (Denmark)

    Krall, Jacob; Balle, Thomas; Krogsgaard-Larsen, Niels;

    2015-01-01

    A high degree of structural heterogeneity of the GABAA receptors (GABAARs) has been revealed and is reflected in multiple receptor subtypes. The subunit composition of GABAAR subtypes is believed to determine their localization relative to the synapses and adapt their functional properties...... to the local temporal pattern of GABA impact, enabling phasic or tonic inhibition. Specific GABAAR antagonists are essential tools for physiological and pharmacological elucidation of the different type of GABAAR inhibition. However, distinct selectivity among the receptor subtypes (populations) has been shown...

  5. The role of GABA(A) receptors in the development of alcoholism.

    Science.gov (United States)

    Enoch, Mary-Anne

    2008-07-01

    Alcoholism is a common, heritable, chronic relapsing disorder. GABA(A) receptors undergo allosteric modulation by ethanol, anesthetics, benzodiazepines and neurosteroids and have been implicated in the acute as well as the chronic effects of ethanol including tolerance, dependence and withdrawal. Medications targeting GABA(A) receptors ameliorate the symptoms of acute withdrawal. Ethanol induces plasticity in GABA(A) receptors: tolerance is associated with generally decreased GABA(A) receptor activation and differentially altered subunit expression. The dopamine (DA) mesolimbic reward pathway originating in the ventral tegmental area (VTA), and interacting stress circuitry play an important role in the development of addiction. VTA GABAergic interneurons are the primary inhibitory regulators of DA neurons and a subset of VTA GABA(A) receptors may be implicated in the switch from heavy drinking to dependence. GABA(A) receptors modulate anxiety and response to stress; important elements of sustained drinking and relapse. The GABA(A) receptor subunit genes clustered on chromosome 4 are highly expressed in the reward pathway. Several recent studies have provided strong evidence that one of these genes, GABRA2, is implicated in alcoholism in humans. The influence of the interaction between ethanol and GABA(A) receptors in the reward pathway on the development of alcoholism together with genetic and epigenetic vulnerabilities will be explored in this review.

  6. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site

    Science.gov (United States)

    Snell, Heather D.

    2015-01-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride’s positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound’s potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ. PMID:25829529

  7. Stoichiometry of δ subunit containing GABAA receptors

    Science.gov (United States)

    Patel, B; Mortensen, M; Smart, T G

    2014-01-01

    Background and Purpose Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach. Experimental Approach Using site-directed mutagenesis, we inserted a highly characterized 9′ serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits. Key Results Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose–response curves of cells co-expressing WT subunits with their respective L9′S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits. Conclusions and Implications Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ. PMID:24206220

  8. Metabolic Products of Linalool and Modulation of GABAA Receptors

    Directory of Open Access Journals (Sweden)

    Sinem Milanos

    2017-06-01

    Full Text Available Terpenoids are major subcomponents in aroma substances which harbor sedative physiological potential. We have demonstrated that various monoterpenoids such as the acyclic linalool enhance GABAergic currents in an allosteric manner in vitro upon overexpression of inhibitory α1β2 GABAA receptors in various expression systems. However, in plants or humans, i.e., following intake via inhalation or ingestion, linalool undergoes metabolic modifications including oxygenation and acetylation, which may affect the modulatory efficacy of the generated linalool derivatives. Here, we analyzed the modulatory potential of linalool derivatives at α1β2γ2 GABAA receptors upon transient overexpression. Following receptor expression control, electrophysiological recordings in a whole cell configuration were used to determine the chloride influx upon co-application of GABA EC10−30 together with the modulatory substance. Our results show that only oxygenated linalool metabolites at carbon 8 positively affect GABAergic currents whereas derivatives hydroxylated or carboxylated at carbon 8 were rather ineffective. Acetylated linalool derivatives resulted in non-significant changes of GABAergic currents. We can conclude that metabolism of linalool reduces its positive allosteric potential at GABAA receptors compared to the significant potentiation effects of the parent molecule linalool itself.

  9. Tobacco smoking interferes with GABAA receptor neuroadaptations during prolonged alcohol withdrawal.

    Science.gov (United States)

    Cosgrove, Kelly P; McKay, Reese; Esterlis, Irina; Kloczynski, Tracy; Perkins, Evgenia; Bois, Frederic; Pittman, Brian; Lancaster, Jack; Glahn, David C; O'Malley, Stephanie; Carson, Richard E; Krystal, John H

    2014-12-16

    Understanding the effects of tobacco smoking on neuroadaptations in GABAA receptor levels over alcohol withdrawal will provide critical insights for the treatment of comorbid alcohol and nicotine dependence. We conducted parallel studies in human subjects and nonhuman primates to investigate the differential effects of tobacco smoking and nicotine on changes in GABAA receptor availability during acute and prolonged alcohol withdrawal. We report that alcohol withdrawal with or without concurrent tobacco smoking/nicotine consumption resulted in significant and robust elevations in GABAA receptor levels over the first week of withdrawal. Over prolonged withdrawal, GABAA receptors returned to control levels in alcohol-dependent nonsmokers, but alcohol-dependent smokers had significant and sustained elevations in GABAA receptors that were associated with craving for alcohol and cigarettes. In nonhuman primates, GABAA receptor levels normalized by 1 mo of abstinence in both groups--that is, those that consumed alcohol alone or the combination of alcohol and nicotine. These data suggest that constituents in tobacco smoke other than nicotine block the recovery of GABAA receptor systems during sustained alcohol abstinence, contributing to alcohol relapse and the perpetuation of smoking.

  10. Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

    Directory of Open Access Journals (Sweden)

    Sujatha P Koduvayur

    Full Text Available The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

  11. GABAA Receptors, Anesthetics and Anticonvulsants in Brain Development

    Science.gov (United States)

    Henschel, Oliver; Gipson, Keith E.; Bordey, Angelique

    2008-01-01

    GABA, acting via GABAA receptors, is well-accepted as the main inhibitory neurotransmitter of the mature brain, where it dampens neuronal excitability. The receptor's properties have been studied extensively, yielding important information about its structure, pharmacology, and regulation that are summarized in this review. Several GABAergic drugs have been commonly used as anesthetics, sedatives, and anticonvulsants for decades. However, findings that GABA has critical functions in brain development, in particular during the late embryonic and neonatal period, raise worthwhile questions regarding the side effects of GABAergic drugs that may lead to long-term cognitive deficits. Here, we will review some of these drugs in parallel with the control of CNS development that GABA exerts via activation of GABAA receptors. This review aims to provide a basic science and clinical perspective on the function of GABA and related pharmaceuticals acting at GABAA receptors. PMID:18537647

  12. PGE2 Modulates GABAA Receptors via an EP1 Receptor-Mediated Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Guang Yang

    2015-07-01

    Full Text Available Aims: PGE2 is one of the most abundant prostanoids in mammalian tissues, but its effect on neuronal receptors has not been well investigated. This study examines the effect of PGE2 on GABAA receptor currents in rat cerebellar granule neurons. Methods: GABAA currents were recorded using a patch-clamp technique. Cell surface and total protein of GABAA β1/2/3 subunits was carried out by Western blot analysis. Results: Upon incubation of neurons with PGE2 (1 µM for 60 minutes, GABAA currents were significantly potentiated. This PGE2-driven effect could be blocked by PKC or CaMKII inhibitors as well as EP1 receptor antagonist, and mimicked by PMA or EP1 receptor agonist. Furthermore, Western blot data showed that PGE2 did not increase the total expression level of GABAA receptors, but significantly increased surface levels of GABAA β1/2/3 subunits after 1 h of treatment. Consistently, both PKC and CaMKII inhibitors were able to reduce PGE2-induced increases in cell surface expression of GABAA receptors. Conclusion: Activation of either the PKC or CaMKII pathways by EP1 receptors mediates the PGE2-induced increase in GABAA currents. This suggests that upregulation of postsynaptic GABAA receptors by PGE2 may have profound effects on cerebellar functioning under physiological and pathological conditions.

  13. Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors

    Directory of Open Access Journals (Sweden)

    Milan Fedurco

    2015-01-01

    Full Text Available The California poppy (Eschscholzia californica Cham. contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant α1β2γ2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 μM. However, (S-reticuline behaved as positive allosteric modulator at the α3, α5, and α6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S-reticuline at the α3β2γ2 and α5β2γ2 GABAA receptors. Interestingly, α1, α3, and α5 were not significantly affected by (R-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine—suspected (S-reticuline metabolites in the rodent brain.

  14. In silico comparative genomic analysis of GABAA receptor transcriptional regulation

    Directory of Open Access Journals (Sweden)

    Joyce Christopher J

    2007-06-01

    Full Text Available Abstract Background Subtypes of the GABAA receptor subunit exhibit diverse temporal and spatial expression patterns. In silico comparative analysis was used to predict transcriptional regulatory features in individual mammalian GABAA receptor subunit genes, and to identify potential transcriptional regulatory components involved in the coordinate regulation of the GABAA receptor gene clusters. Results Previously unreported putative promoters were identified for the β2, γ1, γ3, ε, θ and π subunit genes. Putative core elements and proximal transcriptional factors were identified within these predicted promoters, and within the experimentally determined promoters of other subunit genes. Conserved intergenic regions of sequence in the mammalian GABAA receptor gene cluster comprising the α1, β2, γ2 and α6 subunits were identified as potential long range transcriptional regulatory components involved in the coordinate regulation of these genes. A region of predicted DNase I hypersensitive sites within the cluster may contain transcriptional regulatory features coordinating gene expression. A novel model is proposed for the coordinate control of the gene cluster and parallel expression of the α1 and β2 subunits, based upon the selective action of putative Scaffold/Matrix Attachment Regions (S/MARs. Conclusion The putative regulatory features identified by genomic analysis of GABAA receptor genes were substantiated by cross-species comparative analysis and now require experimental verification. The proposed model for the coordinate regulation of genes in the cluster accounts for the head-to-head orientation and parallel expression of the α1 and β2 subunit genes, and for the disruption of transcription caused by insertion of a neomycin gene in the close vicinity of the α6 gene, which is proximal to a putative critical S/MAR.

  15. 5-(N, N-Hexamethylene) amiloride is a GABA-A ρ1 receptor positive allosteric modulator.

    Science.gov (United States)

    Snell, Heather D; Gonzales, Eric B

    2016-11-01

    Guanidine compounds act as ion channel modulators. In the case of Cys-loop receptors, the guanidine compound amiloride antagonized the heteromeric GABA-A, glycine, and nicotinic acetylcholine receptors. However, amiloride exhibits characteristics consistent with a positive allosteric modulator for the human GABA-A (hGABA-A) ρ1 receptor. Site-directed mutagenesis revealed that the positive allosteric modulation was influenced by the GABA-A ρ1 second transmembrane domain 15' position, a site implicated in ligand allosteric modulation of Cys-loop receptors. There are a variety of amiloride derivatives that provide opportunities to assess the significance of amiloride functional groups (e.g., the guanidine group, the pyrazine ring, etc.) in the modulation of the GABA-A ρ1 receptor activity. We utilized 3 amiloride derivatives (benzamil, phenamil, and 5-(N, N-Hexamethylene) amiloride) to assess the contribution of these groups toward the potentiation of the GABA-A ρ1 receptor. Benzamil and phenamil failed to potentiate on the wild type GABA-A ρ1 GABA-mediated current while HMA demonstrated efficacy only at the highest concentration studied. The hGABA-A ρ1 (I15'N) mutant receptor activity was potentiated by lower HMA concentrations compared to the wild type receptor. Our findings suggest that an exposed guanidine group on amiloride and amiloride derivatives is critical for modulating the GABA-A ρ1 receptor. The present study provides a conceptual framework for predicting which amiloride derivatives will demonstrate positive allosteric modulation of the GABA-A ρ1 receptor.

  16. Single-cell genetic expression of mutant GABAA receptors causing Human genetic epilepsy alters dendritic spine and GABAergic bouton formation in a mutation-specific manner

    Directory of Open Access Journals (Sweden)

    Pamela eLachance-Touchette

    2014-10-01

    Full Text Available Mutations in genes encoding for GABAA receptor subunits is a well-established cause of genetic generalized epilepsy. GABA neurotransmission is implicated in several developmental processes including neurite outgrowth and synapse formation. Alteration in excitatory/inhibitory synaptic activities plays a critical role in epilepsy, thus here we investigated whether mutations in α1 subunit of GABAA receptor may affect dendritic spine and GABAergic bouton formation. In particular, we examined the effects of three mutations of the GABRA1 gene (D219N, A322D and K353delins18X that were found in a cohort of families with genetic generalized epilepsy. We used a novel single-cell genetic approach, by preparing cortical organotypic cultures from GABRA1flox/flox mice and simultaneously inactivating endogenous GABRA1 and transfecting mutant α1 subunits in single glutamatergic pyramidal cells and basket GABAergic interneurons by biolistic transfection. We found that GABRA1-/- GABAergic cells showed reduced innervation field, which was rescued by co-expressing α1-A322D and α1-WT but not α1-D219N. We further found that the expression of the most severe GABRA1 missense mutation (α1-A322D induced a striking increase of spine density in pyramidal cells along with an increase in the number of mushroom-like spines. In addition, α1-A322D expression in GABAergic cells slightly increased perisomatic bouton density, whereas other mutations did not alter bouton formation. All together, these results suggest that the effects of different GABAAR mutations on GABAergic bouton and dendritic spine formation are specific to the mutation and cannot be always explained by a simple loss-of-function gene model. The use of single cell genetic manipulation in organotypic cultures may provide a better understanding of the specific and distinct neural circuit alterations caused by different GABAA receptor subunit mutations and will help define the pathophysiology of genetic

  17. The α1, α2, α3, and γ2 subunits of GABAA receptors show characteristic spatial and temporal expression patterns in rhombencephalic structures during normal human brain development.

    Science.gov (United States)

    Stojanovic, Tamara; Capo, Ivan; Aronica, Eleonora; Adle-Biassette, Homa; Höger, Harald; Sieghart, Werner; Kovacs, Gabor G; Milenkovic, Ivan

    2016-06-15

    γ-Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in adult mammalian brain, mediating its actions chiefly via a pentameric chloride ion channel, the GABAA receptor. Nineteen different subunits (α1-6, β1-3, γ1-3, δ, ε, π, θ, ρ1-3) can give rise to multiple receptor subtypes that are the site of action of many clinically important drugs. In the developing brain, however, GABAA receptors mediate excitatory actions due to an increased chloride concentration within neurons and seem to control cell proliferation, migration, differentiation, synapse maturation, and cell death. Little is known about the distribution of single subunits in the human brain. Here we describe developmental changes in the immunohistochemical distribution of four subunits (α1, α2, α3, and γ2) in the human rhombencephalon. The γ2 was the most abundant subunit in all rhombencephalic structures during development and in adults, whereas α subunits showed a structure- and age-characteristic distribution. The α1 was expressed prenatally in the molecular and Purkinje cell layer, but only postnatally in the granule cell layer and the dentate nucleus. Expression was completely absent in the inferior olivary nucleus. The α2 gradually increased during development, showing some layer specificity in the cerebellar cortex. The α3-immunoreactivity in the cerebellar cortex was relatively weak, but it was abundantly observed in different cell populations in the subcortical cerebellar structures. Structure- and age-characteristic colocalization between subunits during development suggests differences in GABAA receptor composition. Interestingly, subunit expression in several instances differed between human and rodent brain, underlining the importance of immunohistochemical studies in humans. © 2015 Wiley Periodicals, Inc.

  18. GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster.

    Science.gov (United States)

    Cheung, Samantha K; Scott, Kristin

    2017-01-01

    Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified four GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. Here, we tested this hypothesis by examining GABA receptors and neurons that promote consumption. We find that Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation.

  19. The effects of stiripentol on GABA(A) receptors.

    Science.gov (United States)

    Fisher, Janet L

    2011-04-01

    The anticonvulsant stiripentol (Diacomittm) has been shown to have a positive impact on control of seizures for many patients with Dravet syndrome. As with most antiepileptic drugs, stiripentol has multiple mechanisms of action. Its direct anticonvulsant activity is likely due to enhancement of inhibitory, γ-aminobutyric acid (GABA)ergic neurotransmission. Stiripentol was shown to increase the activity of both neuronal and recombinant GABA(A) receptors at clinically relevant concentrations. At recombinant receptors, stiripentol was found to act through a unique site in a subunit-dependent manner. Positive modulation by stiripentol was most effective at GABA(A) receptors containing an α3 subunit. The expression of the α3 subunit is developmentally regulated, with highest levels in the immature brain. This subunit selectivity may explain the greater clinical efficacy of stiripentol in childhood-onset epilepsies, including Dravet syndrome.

  20. GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster

    Science.gov (United States)

    Cheung, Samantha K.

    2017-01-01

    Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified four GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. Here, we tested this hypothesis by examining GABA receptors and neurons that promote consumption. We find that Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation. PMID:28362856

  1. A propofol binding site on mammalian GABAA receptors identified by photolabeling

    Science.gov (United States)

    Yip, Grace M S; Chen, Zi-Wei; Edge, Christopher J; Smith, Edward H; Dickinson, Robert; Hohenester, Erhard; Townsend, R Reid; Fuchs, Karoline; Sieghart, Werner; Evers, Alex S; Franks, Nicholas P

    2014-01-01

    Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABAA receptors, but where it binds to this receptor is not known and has been a matter of some controversy. We have synthesized a novel propofol analogue photolabeling reagent that has a biological activity very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin which have been identified using X-ray crystallography. Using a combination of the protiated label and a deuterated version, and mammalian receptors labeled in intact membranes, we have identified a novel binding site for propofol in GABAA receptors consisting of both β3 homopentamers and α1β3 heteropentamers. The binding site is located within the β subunit, at the interface between the transmembrane domains and the extracellular domain, and lies close to known determinants of anesthetic sensitivity in transmembrane segments TM1 and TM2. PMID:24056400

  2. The orthosteric GABAA receptor ligand Thio-4-PIOL displays distinctly different functional properties at synaptic and extrasynaptic receptors

    DEFF Research Database (Denmark)

    Hoestgaard-Jensen, K; O'Connor, R M; Dalby, Nils Ole

    2013-01-01

    Explorations into the heterogeneous population of native GABA type A receptors (GABAA Rs) and the physiological functions governed by the multiple GABAA R subtypes have for decades been hampered by the lack of subtype-selective ligands....

  3. Interactions between modulators of the GABAA receptor: Stiripentol and benzodiazepines

    OpenAIRE

    Fisher, Janet L.

    2011-01-01

    Many patients with refractory epilepsy are treated with polytherapy, and nearly 15% of epilepsy patients receive two or more anti-convulsant agents. The anti-convulsant stiripentol is used as an add-on treatment for the childhood epilepsy syndrome known as severe myoclonic epilepsy in infancy (Dravet Syndrome). Stiripentol has multiple mechanisms of action, both enhancing GABAA receptors and reducing activity of metabolic enzymes that break down other drugs. Stiripentol is typically co-admini...

  4. Interneuron- and GABAA receptor-specific inhibitory synaptic plasticity in cerebellar Purkinje cells

    Science.gov (United States)

    He, Qionger; Duguid, Ian; Clark, Beverley; Panzanelli, Patrizia; Patel, Bijal; Thomas, Philip; Fritschy, Jean-Marc; Smart, Trevor G.

    2015-07-01

    Inhibitory synaptic plasticity is important for shaping both neuronal excitability and network activity. Here we investigate the input and GABAA receptor subunit specificity of inhibitory synaptic plasticity by studying cerebellar interneuron-Purkinje cell (PC) synapses. Depolarizing PCs initiated a long-lasting increase in GABA-mediated synaptic currents. By stimulating individual interneurons, this plasticity was observed at somatodendritic basket cell synapses, but not at distal dendritic stellate cell synapses. Basket cell synapses predominantly express β2-subunit-containing GABAA receptors; deletion of the β2-subunit ablates this plasticity, demonstrating its reliance on GABAA receptor subunit composition. The increase in synaptic currents is dependent upon an increase in newly synthesized cell surface synaptic GABAA receptors and is abolished by preventing CaMKII phosphorylation of GABAA receptors. Our results reveal a novel GABAA receptor subunit- and input-specific form of inhibitory synaptic plasticity that regulates the temporal firing pattern of the principal output cells of the cerebellum.

  5. Pharmacological characterisation of murine α4β1δ GABAA receptors expressed in Xenopus oocytes

    DEFF Research Database (Denmark)

    Villumsen, Inge S; Wellendorph, Petrine; Smart, Trevor G

    2015-01-01

    BACKGROUND: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties. The receptor β subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence on extrasyn...

  6. The direct actions of cannabidiol and 2-arachidonoyl glycerol at GABAA receptors.

    Science.gov (United States)

    Bakas, T; van Nieuwenhuijzen, P S; Devenish, S O; McGregor, I S; Arnold, J C; Chebib, M

    2017-02-27

    Cannabidiol (CBD) is a major non-intoxicating component of cannabis and possesses anti-epileptic, anxiolytic and anti-hyperalgesic properties. The mechanism of action of CBD in producing such effects remains unclear. Despite evidence that some endogenous and synthetic cannabinoids interact with GABAA receptors, no-one has yet investigated the effects of CBD. Here we used two-electrode voltage clamp electrophysiology to compare the actions of CBD with those of the major central endocannabinoid, 2-arachidonoyl glycerol (2-AG) on human recombinant GABAA receptors (synaptic α1-6βγ2 and extrasynaptic α4β2δ) expressed on Xenopus oocytes. CBD and 2-AG were positive allosteric modulators at α1-6βγ2 receptors, with low micromolar potencies. The maximal level of enhancement seen with either CBD or 2-AG were on α2-containing GABAA receptor subtypes, with approximately a 4-fold enhancement of the GABA EC5 evoked current, more than twice the potentiation seen with other α-subunit receptor combinations. Further we observed β-subunit selectivity, whereby modulatory activity was higher at β2/β3 over β1 subunits. The β1-subunit homologous mutant β2(V436T) substantially diminished the efficacy of both drugs to a third of that obtained with wild-type β2 subunit combinations, but without changing potency. The potency of CBD increased and efficacy preserved in binary α1/α2β2 receptors indicating that their effects do not involve the classic benzodiazepine site. Exploration of extrasynaptic α4β2δ receptors revealed that both compounds enhanced GABA EC5 evoked currents at concentrations ranging from 0.01-1μM. Taken together these results reveal a mode of action of CBD on specifically configured GABAA receptors that may be relevant to the anticonvulsant and anxiolytic effects of the compound.

  7. Positive allosteric modulation of GABA-A receptors reduces capsaicin-induced primary and secondary hypersensitivity in rats

    DEFF Research Database (Denmark)

    Hansen, Rikke Rie; Erichsen, Helle K; Brown, David T

    2012-01-01

    this concept being tested in humans. Prior to assessing the efficacy of GABA-A receptor PAMs in a human volunteer pain model we have compared compounds capable of variously modulating GABA-A receptor function in comparable rat models of capsaicin-induced acute nocifensive flinching behaviour and secondary...... mechanical hypersensitivity. The subtype-selective PAM NS11394 (0.3-10 mg/kg), and the non-selective PAM diazepam (1-5 mg/kg) variously reduced capsaicin-induced secondary mechanical hypersensitivity (180 min post-injection). However, the low efficacy subtype-selective PAM TPA023 (3-30 mg/kg) was completely......, albeit at doses previously shown to impair locomotor function. Our data indicate that GABA-A receptor PAMs with optimal selectivity and efficacy profiles reduce centrally-mediated mechanical hypersensitivity in capsaicin-injected rats, an observation that we expect can translate directly to human...

  8. Stoichiometry of δ subunit containing GABA(A) receptors.

    Science.gov (United States)

    Patel, B; Mortensen, M; Smart, T G

    2014-02-01

    Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach. Using site-directed mutagenesis, we inserted a highly characterized 9' serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits. Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose-response curves of cells co-expressing WT subunits with their respective L9'S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits. Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ. © 2013 The British Pharmacological Society.

  9. Acutely increasing δGABAA receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus

    OpenAIRE

    2013-01-01

    Extrasynaptic γ-aminobutyric acid type A (GABAA) receptors that contain the δ subunit (δGABAA receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase δGABAA receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABAA receptor–preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enh...

  10. Characterization of the Pharmacology of Recombinant Human GABAA Receptor Complexes Containing A2(α1 isoleucin 148 to valine) or A2(α1 asparatic acid 151 to asparagin) or A2 (α1 threonine 149 to glutamine) in Combination with β2γ2s Subu

    Institute of Scientific and Technical Information of China (English)

    WangXiukun

    2001-01-01

    Recombinant human GABAA receptors were investigated in vitro by coexpression of cDNAs coding for α1,β2 and γ2 subunits in the baculovirus/Sf-9 insect cell system,A single amino acid exchange α1(asparatic acid 151 to asparagin or α1(threonine 149 to glutamine) in the N-terminal,extracellular part of the α1 subunit induced about 10 fold decrease in an antagonist pitrazepine affinity.Other GABAA receptor ligands had little difference in their affinity.It was likely that 151 and 149 amino acid residues were essential for the binding affinity and efficacy of pitrazepine to GABAA receptor combinations containiαααααααααng an α1 subunit.

  11. Heterologous expression of rat testis GABAA receptor βt variant in Chinese hamster ovary cells

    Institute of Scientific and Technical Information of China (English)

    Shi-FengLi; Yu-GuangChen; Yuan-ChangYan; Yi-PingLi

    2004-01-01

    Aim: To study the characteristics and possible retention functionof specific sequence in the 5'-end of rat testis GABAA receptor β 3t variant, Methods: Rat testis GABAA receptor β 3t variant cDNA was cloned and inserted into two eukaryotic expression vectors of pEGFP-N1 and pEGFP-C1 respectively, which have EGFP reporter gene.

  12. GABA_A receptor function is regulated by lipid bilayer elasticity

    DEFF Research Database (Denmark)

    Søgaard, Rikke; Werge, Thomas; Berthelsen, Camilla;

    2006-01-01

    that membrane protein function can be regulated by amphiphile-induced changes in bilayer elasticity and hypothesized that GABAA receptors could be similarly regulated. We therefore studied the effects of four structurally unrelated amphiphiles that decrease bilayer stiffness ( Triton X-100, octyl......-beta-glucoside, capsaicin, and DHA) on GABAA receptor function in mammalian cells. All the compounds promoted GABAA receptor [ (3)H]-muscimol binding by increasing the binding capacity of high- affinity binding without affecting the associated equilibrium binding constant. A semiquantitative analysis found a similar......Docosahexaenoic acid ( DHA) and other polyunsaturated fatty acids ( PUFAs) promote GABA(A) receptor [ (3)H]-muscimol binding, and DHA increases the rate of GABAA receptor desensitization. Triton X-100, a structurally unrelated amphiphile, similarly promotes [ (3)H]-muscimol binding. The mechanism...

  13. GABAA receptor-mediated input change on orexin neurons following sleep deprivation in mice.

    Science.gov (United States)

    Matsuki, T; Takasu, M; Hirose, Y; Murakoshi, N; Sinton, C M; Motoike, T; Yanagisawa, M

    2015-01-22

    Orexins are bioactive peptides, which have been shown to play a pivotal role in vigilance state transitions: the loss of orexin-producing neurons (orexin neurons) leads to narcolepsy with cataplexy in the human. However, the effect of the need for sleep (i.e., sleep pressure) on orexin neurons remains largely unknown. Here, we found that immunostaining intensities of the α1 subunit of the GABAA receptor and neuroligin 2, which is involved in inhibitory synapse specialization, on orexin neurons of mouse brain were significantly increased by 6-h sleep deprivation. In contrast, we noted that immunostaining intensities of the α2, γ2, and β2/3 subunits of the GABAA receptor and Huntingtin-associated protein 1, which is involved in GABAAR trafficking, were not changed by 6-h sleep deprivation. Using a slice patch recording, orexin neurons demonstrated increased sensitivity to a GABAA receptor agonist together with synaptic plasticity changes after sleep deprivation when compared with an ad lib sleep condition. In summary, the GABAergic input property of orexin neurons responds rapidly to sleep deprivation. This molecular response of orexin neurons may thus play a role in the changes that accompany the need for sleep following prolonged wakefulness, in particular the decreased probability of a transition to wakefulness once recovery sleep has begun.

  14. Anaesthetic impairment of immune function is mediated via GABA(A receptors.

    Directory of Open Access Journals (Sweden)

    Daniel W Wheeler

    Full Text Available GABA(A receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients.We demonstrate, using RT-PCR, that monocytes express GABA(A receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin.Our results show that functional GABA(A receptors are present on monocytes with properties similar to CNS GABA(A receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life

  15. [3H]Ethynylbicycloorthobenzoate ([3H]EBOB) binding in recombinant GABAA receptors.

    Science.gov (United States)

    Yagle, Monica A; Martin, Michael W; de Fiebre, Christopher M; de Fiebre, NancyEllen C; Drewe, John A; Dillon, Glenn H

    2003-12-01

    Ethynylbicycloorthobenzoate (EBOB) is a recently developed ligand that binds to the convulsant site of the GABAA receptor. While a few studies have examined the binding of [3H]EBOB in vertebrate brain tissue and insect preparations, none have examined [3H]EBOB binding in preparations that express known configurations of the GABAA receptor. We have thus examined [3H]EBOB binding in HEK293 cells stably expressing human alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and the effects of CNS convulsants on its binding. The ability of the CNS convulsants to displace the prototypical convulsant site ligand, [35S]TBPS, was also assessed. Saturation analysis revealed [3H]EBOB binding at a single site, with a K(d) of approximately 9 nM in alpha1beta2gamma2 and alpha2beta2gamma2 receptors. Binding of both [3H]EBOB and [35S]TBPS was inhibited by dieldrin, lindane, tert-butylbicycloorthobenzoate (TBOB), PTX, TBPS, and pentylenetetrazol (PTZ) at one site in a concentration-dependent fashion. Affinities were in the high nM to low microM range for all compounds except PTZ (low mM range), and the rank order of potency for these convulsants to displace [3H]EBOB and [35S]TBPS was the same. Low [GABA] stimulated [3H]EBOB binding, while higher [GABA] (greater than 10 microM) inhibited [3H]EBOB binding. Overall, our data demonstrate that [3H]EBOB binds to a single, high affinity site in alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and modulation of its binding is similar to that seen with [35S]TBPS. [3H]EBOB has a number of desirable traits that may make it preferable to [35S]TBPS for analysis of the convulsant site of the GABAA receptor.

  16. HZ166, a novel GABAA receptor subtype-selective benzodiazepine site ligand, is antihyperalgesic in mouse models of inflammatory and neuropathic pain

    OpenAIRE

    2011-01-01

    Diminished GABAergic and glycinergic inhibition in the spinal dorsal horn contributes significantly to chronic pain of different origins. Accordingly, pharmacological facilitation of GABAergic inhibition by spinal benzodiazepines (BDZs) has been shown to reverse pathological pain in animals as well as in human patients. Previous studies in GABA(A) receptor point-mutated mice have demonstrated that the spinal anti-hyperalgesic effect of classical BDZs is mainly mediated by GABA(A) receptors co...

  17. Effects of insecticidal ketones present in mint plants on GABAA receptor from mammalian neurons

    Directory of Open Access Journals (Sweden)

    Mariela Eugenia Sánchez-Borzone

    2017-01-01

    Abbreviations used: GABA: gamma aminobutyric acid, GABAA-R: GABAA receptor, MTT: 1-(4,5-dimethylthiazol-2-yl-3,5-diphenylformazam, DMEM: Dulbecco′s modified minimum essential mèdium, [3H]TBOB: [3H] t-Butylbicycloorthobenzoate

  18. Dietary acetylenic oxylipin falcarinol differentially modulates GABAA receptors.

    Science.gov (United States)

    Czyzewska, Marta Magdalena; Chrobok, Lukasz; Kania, Alan; Jatczak, Magdalena; Pollastro, Federica; Appendino, Giovanni; Mozrzymas, Jerzy Wladyslaw

    2014-12-26

    The dietary oxylipins falcarinol (1a) and falcarindiol (1b) trap thiols by direct nucleophilic addition to their diyne system, but despite this, only falcarinol (1a) is a reversible agonist of cannabinoid receptors, providing a rationale for comparing their activity also on other neuronal targets. Because GABAA receptors (GABAARs) are exquisitely sensitive to polyacetylenic oxylipins in terms of either potentiation (falcarindiol, 1b) or inhibition (oenanthotoxin, 2a), the activity of 1a was investigated on synaptic (α1β2γ2L) and extrasynaptic (α1β2δ and α1β2) subtypes of GABAARs. Falcarinol (1a) significantly enhanced the amplitude of currents mediated by α1β2γ2L receptors, but this effect was associated with a use-dependent block. Conversely, α1β2 receptors were inhibited without any sign of use-dependent block for the entire range of concentrations tested (1-10 μM). Interestingly, responses mediated by α1β2δ receptors, showing no or very little macroscopic desensitization, were strongly potentiated by 1a, exhibiting a fading reminiscent of macroscopic desensitization. When compared to the activity of falcarindiol (1b), falcarinol (1a) showed a higher affinity for GABAARs and, overall, a substantially different profile of pharmacological action. Taken together, the present data support the view that modulation of GABAARs might underlie the insecticidal and sedative activity of falcarinol (1a).

  19. GABA potency at GABAA receptors found in synaptic and extrasynaptic zones

    Directory of Open Access Journals (Sweden)

    Martin eMortensen

    2012-01-01

    Full Text Available The potency of GABA is vitally important for its primary role in activating GABAA receptors and acting as an inhibitory neurotransmitter. Although numerous laboratories have presented information, directly or indirectly, on GABA potency, it is often difficult to compare across such studies given the inevitable variations in the methods used, the cell types studied, whether native or recombinant receptors are examined, and their relevance to native synaptic and extrasynaptic GABAA receptors. In this review, we list the most relevant isoforms of synaptic and extrasynaptic GABAA receptors that are thought to assemble in surface membranes of neurons in the central nervous system. Using consistent methodology in one cell type, the potencies of the endogenous neurotransmitter GABA are compared across a spectrum of GABAA receptors. The highest potency for GABA is measured when activating extrasynaptic-type α6 subunit-containing receptors, whereas synaptic-type α2β3γ2 and α3β3γ2 receptors exhibited the lowest potency, and other GABAA receptor subtypes that are found both in synaptic and extrasynaptic compartments, showed intermediate sensitivities to GABA. The relatively simple potency relationship between GABA and its target receptors is important as it serves as one of the major determinants of GABAA receptor activation, with consequences for the development of inhibition, either by tonic or phasic mechanisms.

  20. Souroubea sympetala (Marcgraviaceae): a medicinal plant that exerts anxiolysis through interaction with the GABAA benzodiazepine receptor.

    Science.gov (United States)

    Mullally, Martha; Cayer, Christian; Kramp, Kari; Otárola Rojas, Marco; Sanchez Vindas, Pablo; Garcia, Mario; Poveda Alvarez, Luis; Durst, Tony; Merali, Zul; Trudeau, Vance L; Arnason, John T

    2014-09-01

    The mode of action of the anxiolytic medicinal plant Souroubea sympetala was investigated to test the hypothesis that extracts and the active principle act at the pharmacologically important GABAA-benzodiazepine (GABAA-BZD) receptor. Leaf extracts prepared by ethyl acetate extraction or supercritical extraction, previously determined to have 5.54 mg/g and 6.78 mg/g of the active principle, betulinic acid, respectively, reduced behavioural parameters associated with anxiety in a rat model. When animals were pretreated with the GABAA-BZD receptor antagonist flumazenil, followed by the plant extracts, or a more soluble derivative of the active principle, the methyl ester of betulinic acid (MeBA), flumazenil eliminated the anxiety-reducing effect of plant extracts and MeBA, demonstrating that S. sympetala acts via an agonist action on the GABAA-BZD receptor. An in vitro GABAA-BZD competitive receptor binding assay also demonstrated that S. sympetala extracts have an affinity for the GABAA-BZD receptor, with an EC50 value of 123 μg/mL (EtOAc leaf extract) and 154 μg/mL (supercritical CO2 extract). These experiments indicate that S. sympetala acts at the GABAA-BZD receptor to elicit anxiolysis.

  1. RNA editing of the GABAA receptor α3 subunit alters the functional properties of recombinant receptors

    OpenAIRE

    Nimmich, Mitchell L.; Heidelberg, Laura S.; Fisher, Janet L.

    2009-01-01

    RNA editing provides a post-transcriptional mechanism to increase structural heterogeneity of gene products. Recently, the α3 subunit of the GABAA receptors has been shown to undergo RNA editing. As a result, a highly conserved isoleucine residue in the third transmembrane domain is replaced with a methionine. To determine the effect of this structural change on receptor function, we compared the GABA sensitivity, pharmacological properties and macroscopic kinetics of recombinant receptors co...

  2. Hooked on benzodiazepines: GABAA receptor subtypes and addiction

    Science.gov (United States)

    Tan, Kelly R.; Rudolph, Uwe; Lüscher, Christian

    2011-01-01

    Benzodiazepines are widely used clinically to treat anxiety and insomnia. They also induce muscle relaxation, control epileptic seizures, and can provoke amnesia. Moreover, benzodiazepines are often abused after chronic clinical treatment but also for recreational purposes. Within weeks, tolerance to the pharmacological effects can develop, in addition to dependence and even addiction in vulnerable individuals. Here, we review recent observations from animal models regarding the cellular and molecular basis that may underlie the addictive properties of benzodiazepines. These data reveal how benzodiazepines, acting through specific GABAA receptor subtypes, activate midbrain dopamine neurons and how this may hijack the mesolimbic reward system. Such findings have important implications for the future design of benzodiazepines with reduced or even absent addiction liability. PMID:21353710

  3. α2-containing GABAA receptors: A target for the development of novel treatment strategies for CNS disorders

    OpenAIRE

    2012-01-01

    GABAA receptors have important physiological functions, as revealed by pharmacological studies and experiments involving gene-targeted mouse models, and are the target of widely used drugs such as the benzodiazepines. In this review, we are summarizing current knowledge about the function of α2-containing GABAA receptors, a receptor subtype representing approximately 15–20% of all GABAA receptors. This receptor subtype mediates anxiolytic-like, reward-enhancing, and antihyperalgesic actions o...

  4. The reciprocal regulation of stress hormones and GABAA receptors

    Directory of Open Access Journals (Sweden)

    Istvan eMody

    2012-01-01

    Full Text Available Stress-derived steroid hormones regulate the expression and function of GABAA receptors (GABAARs. Changes in GABAAR subunit expression have been demonstrated under conditions of altered steroid hormone levels, such as stress, as well as following exogenous steroid hormone administration. In addition to the effects of stress-derived steroid hormones on GABAAR subunit expression, stress hormones can also be metabolized to neuroactive derivatives which can alter the function of GABAARs. Neurosteroids allosterically modulate GABAARs at concentrations comparable to those during stress. In addition to the actions of stress-derived steroid hormones on GABAARs, GABAARs reciprocally regulate the production of stress hormones. The stress response is mediated by the hypothalamic-pituitary-adrenal (HPA axis, the activity of which is governed by corticotropin releasing hormone (CRH neurons. The activity of CRH neurons is largely controlled by robust GABAergic inhibition. Recently, it has been demonstrated that CRH neurons are regulated by neurosteroid-sensitive, GABAAR δ subunit-containing receptors representing a novel feedback mechanism onto the HPA axis. Further, it has been demonstrated that neurosteroidogenesis and neurosteroid actions on GABAAR δ subunit-containing receptors on CRH neurons are necessary to mount the physiological response to stress. Here we review the literature describing the effects of steroid hormones on GABAARs as well as the importance of GABAARs in regulating the production of steroid hormones. This review incorporates what we currently know about changes in GABAARs following stress and the role in HPA axis regulation.

  5. The α5 subunit containing GABAA receptors contribute to chronic pain.

    Science.gov (United States)

    Bravo-Hernández, Mariana; Corleto, José A; Barragán-Iglesias, Paulino; González-Ramírez, Ricardo; Pineda-Farias, Jorge B; Felix, Ricardo; Calcutt, Nigel A; Delgado-Lezama, Rodolfo; Marsala, Martin; Granados-Soto, Vinicio

    2016-03-01

    It has been recently proposed that α5-subunit containing GABAA receptors (α5-GABAA receptors) that mediate tonic inhibition might be involved in pain. The purpose of this study was to investigate the contribution of α5-GABAA receptors in the loss of GABAergic inhibition and in formalin-induced, complete Freund's adjuvant (CFA)-induced and L5 and L6 spinal nerve ligation-induced long-lasting hypersensitivity. Formalin or CFA injection and L5 and L6 spinal nerve ligation produced long-lasting allodynia and hyperalgesia. Moreover, formalin injection impaired the rate-dependent depression of the Hofmann reflex. Peripheral and intrathecal pretreatment or post-treatment with the α5-GABAA receptor antagonist, L-655,708 (0.15-15 nmol), prevented and reversed, respectively, these long-lasting behaviors. Formalin injection increased α5-GABAA receptor mRNA expression in the spinal cord and dorsal root ganglia (DRG) mainly at 3 days. The α5-GABAA receptors were localized in the dorsal spinal cord and DRG colabeling with NeuN, CGRP, and IB4 which suggests their presence in peptidergic and nonpeptidergic neurons. These receptors were found mainly in small and medium sized neurons. Formalin injection enhanced α5-GABAA receptor fluorescence intensity in spinal cord and DRG at 3 and 6 days. Intrathecal administration of L-655,708 (15 nmol) prevented and reversed formalin-induced impairment of rate-dependent depression. These results suggest that α5-GABAA receptors play a role in the loss of GABAergic inhibition and contribute to long-lasting secondary allodynia and hyperalgesia.

  6. Regulation of action potential waveforms by axonal GABAA receptors in cortical pyramidal neurons.

    Directory of Open Access Journals (Sweden)

    Yang Xia

    Full Text Available GABAA receptors distributed in somatodendritic compartments play critical roles in regulating neuronal activities, including spike timing and firing pattern; however, the properties and functions of GABAA receptors at the axon are still poorly understood. By recording from the cut end (bleb of the main axon trunk of layer -5 pyramidal neurons in prefrontal cortical slices, we found that currents evoked by GABA iontophoresis could be blocked by picrotoxin, indicating the expression of GABAA receptors in axons. Stationary noise analysis revealed that single-channel properties of axonal GABAA receptors were similar to those of somatic receptors. Perforated patch recording with gramicidin revealed that the reversal potential of the GABA response was more negative than the resting membrane potential at the axon trunk, suggesting that GABA may hyperpolarize the axonal membrane potential. Further experiments demonstrated that the activation of axonal GABAA receptors regulated the amplitude and duration of action potentials (APs and decreased the AP-induced Ca2+ transients at the axon. Together, our results indicate that the waveform of axonal APs and the downstream Ca2+ signals are modulated by axonal GABAA receptors.

  7. Selective Pyramidal Cell Reduction of GABAA Receptor α1 Subunit Messenger RNA Expression in Schizophrenia

    OpenAIRE

    Glausier, Jill R; Lewis, David A.

    2011-01-01

    Levels of messenger RNA (mRNA) for the α1 subunit of the GABAA receptor, which is present in 60% of cortical GABAA receptors, have been reported to be lower in layer 3 of the prefrontal cortex (PFC) in subjects with schizophrenia. This subunit is expressed in both pyramidal cells and interneurons, and thus lower α1 subunit levels in each cell population would have opposite effects on net cortical excitation. We used dual-label in situ hybridization to quantify GABAA α1 subunit mRNA expression...

  8. GABAA receptor signaling in the lateral septum regulates maternal aggression in mice

    OpenAIRE

    2009-01-01

    Maternal aggression (maternal defense) is a fierce aggression produced by lactating females towards intruders that plays an important role in protection of vulnerable offspring. Enhancement of GABAA receptor signaling by benzodiazepines increases maternal aggression and we recently found indirect evidence that lateral septum (LS) could be a key site where benzodiazepines elevate aggression. In this study, we directly tested the hypothesis that activation of GABAA receptors in LS would promote...

  9. GABAA Receptors Implicated in REM Sleep Control Express a Benzodiazepine Binding Site

    OpenAIRE

    Nguyen, Tin Quang; Liang, Chang-Lin; Marks, Gerald A.

    2013-01-01

    It has been reported that non-subtype-selective GABAA receptor antagonists injected into the nucleus pontis oralis (PnO) of rats induced long-lasting increases in REM sleep. Characteristics of these REM sleep increases were identical to those resulting from injection of muscarinic cholinergic agonists. Both actions were blocked by the muscarinic antagonist, atropine. Microdialysis of GABAA receptor antagonists into the PnO resulted in increased acetylcholine levels. These findings were consis...

  10. Propofol suppresses synaptic responsiveness of somatosensory relay neurons to excitatory input by potentiating GABAA receptor chloride channels

    Directory of Open Access Journals (Sweden)

    Goldstein Peter A

    2005-01-01

    Full Text Available Abstract Propofol is a widely used intravenous general anesthetic. Propofol-induced unconsciousness in humans is associated with inhibition of thalamic activity evoked by somatosensory stimuli. However, the cellular mechanisms underlying the effects of propofol in thalamic circuits are largely unknown. We investigated the influence of propofol on synaptic responsiveness of thalamocortical relay neurons in the ventrobasal complex (VB to excitatory input in mouse brain slices, using both current- and voltage-clamp recording techniques. Excitatory responses including EPSP temporal summation and action potential firing were evoked in VB neurons by electrical stimulation of corticothalamic fibers or pharmacological activation of glutamate receptors. Propofol (0.6 – 3 μM suppressed temporal summation and spike firing in a concentration-dependent manner. The thalamocortical suppression was accompanied by a marked decrease in both EPSP amplitude and input resistance, indicating that a shunting mechanism was involved. The propofol-mediated thalamocortical suppression could be blocked by a GABAA receptor antagonist or chloride channel blocker, suggesting that postsynaptic GABAA receptors in VB neurons were involved in the shunting inhibition. GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs were evoked in VB neurons by electrical stimulation of the reticular thalamic nucleus. Propofol markedly increased amplitude, decay time, and charge transfer of GABAA IPSCs. The results demonstrated that shunting inhibition of thalamic somatosensory relay neurons by propofol at clinically relevant concentrations is primarily mediated through the potentiation of the GABAA receptor chloride channel-mediated conductance, and such inhibition may contribute to the impaired thalamic responses to sensory stimuli seen during propofol-induced anesthesia.

  11. Differences in kinetics between GABAC and GABAA receptors on carp retinal bipolar cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The present work was undertaken to characterize kinetics, including activation, desensitization and deactivation, of responses mediated by GABAA and GABAC receptors on carp retinal bipolar cells, using the whole-cell patch-clamp technique. It was revealed that the GABAC response was generally slower in kinetics than the GABAA response. Activation kinetics of both the receptors could be well fit by monoexponential functions with time constants τ, being 44.57 ms (GABAC) and 10.86 ms (GABAA) respectively. Desensitization of the GABAA response was characterized by a fast and a slow exponential component with time constants of τfast = 2.16 s and τslow = 19.78 s respectively, whereas desensitization of the GABAC response was fit by a monoexponential function of the time constant τ = 6.98 s. Deactivation at both the receptors was adequately described by biexponential functions with time constants being much higher for the GABAC response (τfast = 674.8 ms; τslow = 2 090 ms) than those for the GABAA response (τfast = 42.07 ms; τslow = 275.1 ms). These differences in kinetics suggest that GABAC and GABAA receptors may be involved in processing signals in different frequency domains.

  12. Editing modifies the GABA(A) receptor subunit alpha3

    DEFF Research Database (Denmark)

    Ohlson, Johan; Pedersen, Jakob Skou; Haussler, David

    2007-01-01

    Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method...... to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA(A) receptor......, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain...

  13. The possible role of GABAA receptors and gephyrin in epileptogenesis

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    Marco I Gonzalez

    2013-07-01

    Full Text Available The term epileptogenesis refers to a dynamic alteration in neuronal excitability that promotes the appearance of spontaneous seizures. Temporal lobe epilepsy (TLE, the most common type of acquired epilepsy, often develops after an insult to the brain such as trauma, febrile seizures, encephalitis, or status epilepticus (SE. During the pre-epileptic state (also referred as latent or silent period there is a plethora of molecular, biochemical and structural changes that lead to the generation of recurrent spontaneous seizures (or epilepsy. The specific contribution of these alterations to epilepsy development is unclear, but a loss of inhibition has been associated with the increased excitability detected in the latent period. A rapid increase in neuronal hyperexcitability could be due, at least in part, to a decline in the number of physiologically active GABAA receptors (GABAAR. Altered expression of scaffolding proteins involved in the trafficking and anchoring of GABAAR could directly impact the stability of GABAergic synapses and promote a deficiency in inhibitory neurotransmission. Uncovering the molecular mechanisms operating during epileptogenesis and its possible impact on the regulation of GABAAR and scaffolding proteins may offer new targets to prevent the development of epilepsy.

  14. Tonically Active α5GABAA Receptors Reduce Motoneuron Excitability and Decrease the Monosynaptic Reflex

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    Martha Canto-Bustos

    2017-09-01

    Full Text Available Motoneurons, the final common path of the Central Nervous System (CNS, are under a complex control of its excitability in order to precisely translate the interneuronal pattern of activity into skeletal muscle contraction and relaxation. To fulfill this relevant function, motoneurons are provided with a vast repertoire of receptors and channels, including the extrasynaptic GABAA receptors which have been poorly investigated. Here, we confirmed that extrasynaptic α5 subunit-containing GABAA receptors localize with choline acetyltransferase (ChAT positive cells, suggesting that these receptors are expressed in turtle motoneurons as previously reported in rodents. In these cells, α5GABAA receptors are activated by ambient GABA, producing a tonic shunt that reduces motoneurons’ membrane resistance and affects their action potential firing properties. In addition, α5GABAA receptors shunted the synaptic excitatory inputs depressing the monosynaptic reflex (MSR induced by activation of primary afferents. Therefore, our results suggest that α5GABAA receptors may play a relevant physiological role in motor control.

  15. α2-containing GABAA receptors expressed in hippocampal region CA3 control fast network oscillations.

    Science.gov (United States)

    Heistek, Tim S; Ruiperez-Alonso, Marta; Timmerman, A Jaap; Brussaard, Arjen B; Mansvelder, Huibert D

    2013-02-15

    GABA(A) receptors are critically involved in hippocampal oscillations. GABA(A) receptor α1 and α2 subunits are differentially expressed throughout the hippocampal circuitry and thereby may have distinct contributions to oscillations. It is unknown which GABA(A) receptor α subunit controls hippocampal oscillations and where these receptors are expressed. To address these questions we used transgenic mice expressing GABA(A) receptor α1 and/or α2 subunits with point mutations (H101R) that render these receptors insensitive to allosteric modulation at the benzodiazepine binding site, and tested how increased or decreased function of α subunits affects hippocampal oscillations. Positive allosteric modulation by zolpidem prolonged decay kinetics of hippocampal GABAergic synaptic transmission and reduced the frequency of cholinergically induced oscillations. Allosteric modulation of GABAergic receptors in CA3 altered oscillation frequency in CA1, while modulation of GABA receptors in CA1 did not affect oscillations. In mice having a point mutation (H101R) at the GABA(A) receptor α2 subunit, zolpidem effects on cholinergically induced oscillations were strongly reduced compared to wild-type animals, while zolpidem modulation was still present in mice with the H101R mutation at the α1 subunit. Furthermore, genetic knockout of α2 subunits strongly reduced oscillations, whereas knockout of α1 subunits had no effect. Allosteric modulation of GABAergic receptors was strongly reduced in unitary connections between fast spiking interneurons and pyramidal neurons in CA3 of α2H101R mice, but not of α1H101R mice, suggesting that fast spiking interneuron to pyramidal neuron synapses in CA3 contain α2 subunits. These findings suggest that α2-containing GABA(A) receptors expressed in the CA3 region provide the inhibition that controls hippocampal rhythm during cholinergically induced oscillations.

  16. γ-Hydroxybutyric acid (GHB is not an agonist of extrasynaptic GABAA receptors.

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    William M Connelly

    Full Text Available γ-Hydroxybutyric acid (GHB is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABAB receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABAA receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABAA receptors, where GHB acts as an agonist with an EC50 of 140 nM. We investigated three neuronal cell types that express a tonic GABAA receptor current mediated by extrasynaptic receptors: ventrobasal (VB thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM induced any GABAA receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM was able to produce a GABAB receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents.

  17. γ-Hydroxybutyric acid (GHB) is not an agonist of extrasynaptic GABAA receptors.

    Science.gov (United States)

    Connelly, William M; Errington, Adam C; Crunelli, Vincenzo

    2013-01-01

    γ-Hydroxybutyric acid (GHB) is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABAB receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABAA receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABAA receptors, where GHB acts as an agonist with an EC50 of 140 nM. We investigated three neuronal cell types that express a tonic GABAA receptor current mediated by extrasynaptic receptors: ventrobasal (VB) thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM) induced any GABAA receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM) was able to produce a GABAB receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents.

  18. GABA(A) receptor-mediated presynaptic inhibition on glutamatergic transmission.

    Science.gov (United States)

    Yamamoto, Sokatsu; Yoshimura, Megumu; Shin, Min-Chul; Wakita, Masahito; Nonaka, Kiku; Akaike, Norio

    2011-01-15

    We investigated the functional roles of presynaptic GABA(A) receptors on excitatory nerve terminals in contributing to spontaneous and action potential-evoked glutamatergic transmission to rat hippocampal CA3 pyramidal neurons. Single CA3 neurons were mechanically isolated with adherent nerve terminals, namely the 'synaptic bouton preparation', and spontaneous glutamatergic excitatory synaptic potentials (sEPSCs) and EPSCs evoked by focal electrical stimuli of a single presynaptic glutamatergic boutons (eEPSCs) were recorded using conventional whole-cell patch recordings. Selective activation of presynaptic GABA(A) receptors on these excitatory nerve terminals by muscimol, markedly facilitated sEPSCs frequency but inhibited eEPSC amplitude. The facilitation of sEPSC frequency was completely occluded by GABA(A) receptor-Cl⁻ channel blockers bicuculline or penicillin (PN). PN itself concentration-dependently inhibited the GABA(A) receptor response induced by bath application of muscimol, but had no effect on the glutamate receptor response. In addition, pretreatment with a blocker of the Na(+), K(+), 2Cl⁻ co-transporter type 1 (NKCC-1), bumetanide, prevented the muscimol-induced inhibition of eEPSCs. The results indicate that activation of presynaptic GABA(A) receptors directly depolarizes glutamatergic excitatory nerve terminals and thereby differentially modulates sEPSCs and eEPSCs.

  19. The insecticide fipronil and its metabolite fipronil sulphone inhibit the rat α1β2γ2L GABAA receptor

    Science.gov (United States)

    Li, P; Akk, G

    2008-01-01

    Background and purpose: Fipronil is the active ingredient in a number of widely used insecticides. Human exposure to fipronil leads to symptoms (headache, nausea and seizures) typically associated with the antagonism of GABAA receptors in the brain. In this study, we have examined the modulation of the common brain GABAA receptor subtype by fipronil and its major metabolite, fipronil sulphone. Experimental approach: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat α1β2γ2L GABAA receptors. Key results: The major effect of fipronil was to increase the rate of current decay in macroscopic recordings. In single-channel recordings, the presence of fipronil resulted in shorter cluster durations without affecting the intracluster open and closed time distributions or the single-channel conductance. The α1V256S mutation, previously shown alleviate channel inhibition by inhibitory steroids and several insecticides, had a relatively small effect on channel block by fipronil. The mode of action of fipronil sulphone was similar to that of its parent compound but the metabolite was less potent at inhibiting the α1β2γ2L receptor. Conclusions and implications: We conclude that exposure to fipronil induces accumulation of receptors in a novel, long-lived blocked state. This process proceeds in parallel with and independently of, channel desensitization. The lower potency of fipronil sulphone indicates that the conversion serves as a detoxifying process in mammalian brain. PMID:18660823

  20. GABAA receptor drugs and neuronal plasticity in reward and aversion: focus on the ventral tegmental area

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    Elena eVashchinkina

    2014-11-01

    Full Text Available GABAA receptors are the main fast inhibitory neurotransmitter receptors in the mammalian brain, and targets for many clinically important drugs widely used in the treatment of anxiety disorders, insomnia and in anesthesia. Nonetheless, there are significant risks associated with the long-term use of these drugs particularly related to development of tolerance and addiction. Addictive mechanisms of GABAA receptor drugs are poorly known, but recent findings suggest that those drugs may induce aberrant neuroadaptations in the brain reward circuitry. Recently, benzodiazepines, acting on synaptic GABAA receptors, and modulators of extrasynaptic GABAA receptors (THIP and neurosteroids have been found to induce plasticity in the ventral tegmental area (VTA dopamine neurons and their main target projections. Furthermore, depending whether synaptic or extrasynaptic GABAA receptor populations are activated, the behavioral outcome of repeated administration seems to correlate with rewarding or aversive behavioral responses, respectively. The VTA dopamine neurons project to forebrain centers such as the nucleus accumbens and medial prefrontal cortex, and receive afferent projections from these brain regions and especially from the extended amygdala and lateral habenula, forming the major part of the reward and aversion circuitry. Both synaptic and extrasynaptic GABAA drugs inhibit the VTA GABAergic interneurons, thus activating the VTA DA neurons by disinhibition and this way inducing glutamatergic synaptic plasticity. However, the GABAA drugs failed to alter synaptic spine numbers as studied from Golgi-Cox-stained VTA dendrites. Since the GABAergic drugs are known to depress the brain metabolism and gene expression, their likely way of inducing neuroplasticity in mature neurons is by disinhibiting the principal neurons, which remains to be rigorously tested for a number of clinically important anxiolytics, sedatives and anesthetics in different parts of

  1. Α2 GABAA receptor sub-units in the ventral hippocampus and α5 GABAA receptor sub-units in the dorsal hippocampus mediate anxiety and fear memory.

    Science.gov (United States)

    McEown, K; Treit, D

    2013-11-12

    Temporary neuronal inactivation of the ventral hippocampus with the GABAA agonist muscimol suppresses unconditioned fear behavior (anxiety) but inactivation of the dorsal hippocampus does not. On the other hand, inactivating the dorsal hippocampus disrupts fear memory, while inactivating the ventral hippocampus does not. Here we investigate the roles of hippocampal GABAA receptor sub-units in mediating these anxiolytic and amnesic effects of GABAA receptor agonists. We microinfused TPA023 (α2 agonist) or TB-21007 (inverse α5 agonist) into the dorsal or ventral hippocampus prior to testing rats in two animal models of anxiety: the elevated plus-maze and shock-probe burying test. Twenty-four hours later rats were re-tested in the shock-probe chamber with a non-electrified probe to assess their memory of the initial shock-probe experience (i.e., fear memory). We found that TPA023 was anxiolytic in the plus-maze and shock-probe burying tests when microinfused into the ventral hippocampus. However, TPA023 did not affect anxiety-related behavior when infused into the dorsal hippocampus. Conversely, we found that the α5 sub-unit inverse agonist TB-21007 impaired rats' memory of the initial shock-probe experience when infused into the dorsal hippocampus, but not when infused into the ventral hippocampus. This double dissociation suggests that α2 GABAA receptor sub-units in the ventral hippocampus mediate unconditioned fear or anxiety, while α5 GABAA receptor sub-units in the dorsal hippocampus mediate conditioned fear memory.

  2. Synaptically released neurotransmitter fails to desensitize postsynaptic GABA(A) receptors in cerebellar cultures.

    Science.gov (United States)

    Mellor, J R; Randall, A D

    2001-05-01

    GABA concentration jump experiments performed on membrane patches predict that postsynaptic GABA(A) receptors will become desensitized following the release of the contents of a single GABA-containing synaptic vesicle. To examine this we used a single synaptic bouton stimulation technique to directly examine whether postsynaptic GABA(A) receptors in cultured cerebellar granule cells exhibit transmitter-induced desensitization. In a large number of recordings, no evidence was found for desensitization of postsynaptic GABA(A) receptors by vesicularly released transmitter. This was the case even when as many as 40 vesicles were released from a single bouton within 1.5 s. In addition, postsynaptic depolarization and application of the benzodiazepine flunitrazepam, manipulations previously shown to enhance desensitization of GABA(A) receptors, failed to unmask transmitter-induced desensitization. In contrast, a single 2- to 3-s application of a high concentration of exogenous GABA was able to depress synaptic responsiveness for up to 70 s. Furthermore, pharmacological depletion of GABA eliminated inhibitory synaptic communication, suggesting that GABA is the transmitter and the desensitization-resistant inhibitory postsynaptic currents are not mediated by a "nondesensitizing" ligand such as beta-alanine. Overall our data indicate that a specific desensitization-resistant population of GABA(A) receptors are present at postsynaptic sites on cultured cerebellar granule cells.

  3. Abnormal benzodiazepine and zinc modulation of GABAA receptors in an acquired absence epilepsy model.

    Science.gov (United States)

    Wu, Jie; Ellsworth, Kevin; Ellsworth, Marc; Schroeder, Katherine M; Smith, Kris; Fisher, Robert S

    2004-07-01

    Brain cholesterol synthesis inhibition (CSI) at a young age in rats has been shown to be a faithful model of acquired absence epilepsy, a devastating condition for which few therapies or models exist. We employed the CSI model to study cellular mechanisms of acquired absence epilepsy in Long-Evans Hooded rats. Patch-clamp, whole-cell recordings were compared from neurons acutely dissociated from the nucleus reticularis of thalamus (nRt) treated and untreated with a cholesterol synthesis inhibitor, U18666A. In U18666A-treated animals, 91% of rats developed EEG spike-waves (SWs). Patchclamp results revealed that although there was no remarkable change in GABAA receptor affinity, both a loss of ability of benzodiazepines to enhance GABAA-receptor responses and an increase of Zn2+ inhibition of GABAA-receptor responses of nRt neurons occurred in Long-Evans Hooded rats previously administered U18666A. This change was specific, since no significant changes were found in neurons exposed to the GABA allosteric modulator, pentobarbital. Taken collectively, these findings provide evidence for abnormalities in benzodiazepine and Zn2+ modulation of GABAA receptors in the CSI model, and suggest that decreased gamma2 subunit expression may underlie important aspects of generation of thalamocortical SWs in atypical absence seizures. The present results are also consistent with recent findings that mutation of the gamma2 subunit of the GABAA receptor changes benzodiazepine modulation in families with generalized epilepsy syndromes.

  4. Memory Deficits Induced by Inflammation Are Regulated by α5-Subunit-Containing GABAA Receptors

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    Dian-Shi Wang

    2012-09-01

    Full Text Available Systemic inflammation causes learning and memory deficits through mechanisms that remain poorly understood. Here, we studied the pathogenesis of memory loss associated with inflammation and found that we could reverse memory deficits by pharmacologically inhibiting α5-subunit-containing γ-aminobutyric acid type A (α5GABAA receptors and deleting the gene associated with the α5 subunit. Acute inflammation reduces long-term potentiation, a synaptic correlate of memory, in hippocampal slices from wild-type mice, and this reduction was reversed by inhibition of α5GABAA receptor function. A tonic inhibitory current generated by α5GABAA receptors in hippocampal neurons was increased by the key proinflammatory cytokine interleukin-1β through a p38 mitogen-activated protein kinase signaling pathway. Interleukin-1β also increased the surface expression of α5GABAA receptors in the hippocampus. Collectively, these results show that α5GABAA receptor activity increases during inflammation and that this increase is critical for inflammation-induced memory deficits.

  5. Effects of common anesthetic agents on [(18)F]flumazenil binding to the GABAA receptor

    DEFF Research Database (Denmark)

    Palner, Mikael; Beinat, Corinne; Banister, Sam

    2016-01-01

    mice. CONCLUSIONS: Anesthesia has pronounced effects on the binding and blood-brain distribution of [(18)F]flumazenil. Consequently, considerable caution must be exercised in the interpretation of preclinical and clinical PET studies of GABAA receptors involving the use of anesthesia....... in preclinical imaging studies and clinical imaging studies involving patient populations that do not tolerate relatively longer scan times. The objective of this study was to examine the effects of anesthesia on the binding of [(18)F]flumazenil to GABAA receptors in mice. METHODS: Brain and whole blood......BACKGROUND: The availability of GABAA receptor binding sites in the brain can be assessed by positron emission tomography (PET) using the radioligand, [(18)F]flumazenil. However, the brain uptake and binding of this PET radioligand are influenced by anesthetic drugs, which are typically needed...

  6. Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABA(A) receptors expressed in Xenopus laevis oocytes.

    Science.gov (United States)

    Hammer, Harriet; Ebert, Bjarke; Jensen, Henrik Sindal; Jensen, Anders A

    2015-01-01

    The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences. The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABA(A)R) subtypes α1β2γ(2S), α2β2γ(2S), α3β2γ(2S), α5β2γ(2S) and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited by the 1,4-benzodiazepine clonazepam. All three compounds potentiated GABA EC20-evoked responses through the α(1,2,3,5)β2γ(2S) GABA(A)Rs in a reversible and concentration-dependent manner, with each displaying similar EC50 values at the four subtypes. Furthermore, the degrees of potentiation of the GABA EC20 currents through the four receptors mediated by saturating modulator concentrations did not differ substantially for any of the three benzodiazepines. The three compounds were substantially less potent (200-3900 fold) as positive allosteric modulators at the α6β2δ GABA(A)R than at the α(1,2,3,5)β2γ(2S) receptors. Interestingly, however, clobazam and especially N-desmethylclobazam were highly efficacious potentiators of α6β2δ receptor signaling. Although this activity component is unlikely to contribute to the in vivo effects of clobazam/N-desmethylclobazam, the 1,5-benzodiazepine could constitute an interesting lead for novel modulators targeting this low-affinity binding site in GABAARs. In conclusion, the non-selective modulation

  7. Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABA(A receptors expressed in Xenopus laevis oocytes.

    Directory of Open Access Journals (Sweden)

    Harriet Hammer

    Full Text Available The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences. The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABA(AR subtypes α1β2γ(2S, α2β2γ(2S, α3β2γ(2S, α5β2γ(2S and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited by the 1,4-benzodiazepine clonazepam. All three compounds potentiated GABA EC20-evoked responses through the α(1,2,3,5β2γ(2S GABA(ARs in a reversible and concentration-dependent manner, with each displaying similar EC50 values at the four subtypes. Furthermore, the degrees of potentiation of the GABA EC20 currents through the four receptors mediated by saturating modulator concentrations did not differ substantially for any of the three benzodiazepines. The three compounds were substantially less potent (200-3900 fold as positive allosteric modulators at the α6β2δ GABA(AR than at the α(1,2,3,5β2γ(2S receptors. Interestingly, however, clobazam and especially N-desmethylclobazam were highly efficacious potentiators of α6β2δ receptor signaling. Although this activity component is unlikely to contribute to the in vivo effects of clobazam/N-desmethylclobazam, the 1,5-benzodiazepine could constitute an interesting lead for novel modulators targeting this low-affinity binding site in GABAARs. In conclusion, the non

  8. GABA(A) receptors implicated in REM sleep control express a benzodiazepine binding site.

    Science.gov (United States)

    Nguyen, Tin Quang; Liang, Chang-Lin; Marks, Gerald A

    2013-08-21

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

  9. GABA(A) receptor subtype-selectivity of novel bicuculline derivatives.

    Science.gov (United States)

    Ramerstorfer, Joachim; Foppa, Verena; Thiery, Hanna; Hermange, Philippe; Janody, Simon; Berger, Michael L; Dodd, Robert H; Sieghart, Werner

    2015-01-01

    GABA(A) receptors are the major inhibitory neurotransmitter receptors in the central nervous system and are targets of clinically important drugs modulating GABA induced ion flux by interacting with distinct allosteric binding sites. ROD 185 is a previously investigated structural analogue of the GABA site antagonist bicuculline, and a positive allosteric modulator acting via the benzodiazepine binding site. Here, we investigated 13 newly synthesized structural analogues of ROD 185 for their interaction with rat GABA(A) receptors. Using [(3)H]flunitrazepam binding assays, we identified four compounds exhibiting a higher affinity for the benzodiazepine binding site than ROD 185. Two electrode voltage clamp electrophysiology at recombinant GABA(A) receptors indicated that most of these compounds positively modulated GABA-induced currents at these receptors. Additionally, these experiments revealed that this compound class not only interacts with the benzodiazepine binding site at αβγ receptors but also with a novel, so far unidentified binding site present in αβ receptors. Compounds with a high affinity for the benzodiazepine binding site stimulated GABA-induced currents stronger at αβγ than at αβ receptors and preferred α3β3γ2 receptors. Compounds showing equal or smaller effects at αβγ compared to αβ receptors differentially interacted with various αβ or αβγ receptor subtypes. Surprisingly, five of these compounds interacting with αβ receptors showed a strong stimulation at α6β3γ2 receptors. The absence of any direct effects at GABA(A) receptors, as well as their potential selectivity for receptor subtypes not being addressed by benzodiazepines, make this compound class to a starting point for the development of drugs with a possible clinical importance.

  10. Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABAA receptors expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Hammer, Harriet; Ebert, Bjarke; Jensen, Henrik S.

    2015-01-01

    The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit...... different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences....... The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABAAR) subtypes α1β2γ2S, α2β2γ2S, α3β2γ2S, α5β2γ2S and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited...

  11. Early expression of GABA(A) receptor delta subunit in the neonatal rat hippocampus.

    Science.gov (United States)

    Didelon, F; Mladinic', M; Cherubini, E; Bradbury, A

    2000-12-01

    The cDNA library screening strategy was used to identify the genes encoding for GABA(A) receptor subunits in the rat hippocampus during development. With this technique, genes encoding eleven GABA(A) receptor subunits were identified. The alpha5 subunit was by far the most highly expressed, followed by the gamma2, alpha2 and alpha4 subunits respectively. The expression of the beta2, alpha1, gamma1, beta1 and beta3 subunits was moderate, although that of the alpha3 and delta subunits was weak. In situ hybridization experiments, using digoxigenin-labeled cRNA probes, confirmed that the delta subunit was expressed in the neonatal as well as in the adult hippocampus, and is likely to form functional receptors in association with other subunits of the GABA(A) receptor. When the more sensitive RT-PCR approach was used, the gamma3 subunit was also detected, suggesting that this subunit is present in the hippocampus during development but at low levels of expression. The insertion of the delta subunit into functional GABA(A) receptors may enhance the efficacy of GABA in the immediate postnatal period when this amino acid is still exerting a depolarizing and excitatory action.

  12. Loss of functional GABAA receptors in the Alzheimer diseased brain

    Science.gov (United States)

    Limon, Agenor; Reyes-Ruiz, Jorge Mauricio; Miledi, Ricardo

    2012-01-01

    The cholinergic and glutamatergic neurotransmission systems are known to be severely disrupted in Alzheimer's disease (AD). GABAergic neurotransmission, in contrast, is generally thought to be well preserved. Evidence from animal models and human postmortem tissue suggest GABAergic remodeling in the AD brain. Nevertheless, there is no information on changes, if any, in the electrophysiological properties of human native GABA receptors as a consequence of AD. To gain such information, we have microtransplanted cell membranes, isolated from temporal cortices of control and AD brains, into Xenopus oocytes, and recorded the electrophysiological activity of the transplanted GABA receptors. We found an age-dependent reduction of GABA currents in the AD brain. This reduction was larger when the AD membranes were obtained from younger subjects. We also found that GABA currents from AD brains have a faster rate of desensitization than those from non-AD brains. Furthermore, GABA receptors from AD brains were slightly, but significantly, less sensitive to GABA than receptors from non-AD brains. The reduction of GABA currents in AD was associated with reductions of mRNA and protein of the principal GABA receptor subunits normally present in the temporal cortex. Pairwise analysis of the transcripts within control and AD groups and analyses of the proportion of GABA receptor subunits revealed down-regulation of α1 and γ2 subunits in AD. In contrast, the proportions of α2, β1, and γ1 transcripts were up-regulated in the AD brains. Our data support a functional remodeling of GABAergic neurotransmission in the human AD brain. PMID:22691495

  13. Role of spinal GABAA receptor reduction induced by stress in rat thermal hyperalgesia.

    Science.gov (United States)

    Ma, Xuelian; Bao, Weiying; Wang, Xiujun; Wang, Zhilong; Liu, Qiaoran; Yao, Zhenyu; Zhang, Di; Jiang, Hong; Cui, Shuang

    2014-11-01

    The mechanisms underlying stress-induced hyperalgesia (SIH) remain poorly understood. Recent findings have provided strong evidence indicating that SIH could be related, at least in part, to alterations in spinal cord GABA activity. In the present study, we first investigated how acute restraint stress impacted pain responses as assessed using the tail flick immersion test. These results showed that rats developed hyperalgesia at 6 h after being subjected to 1-h acute restraint stress. Second, we measured the activation of spinal neurons and alterations in expression of GABAA receptor β2 and β3 subunits as related to stress-induced hyperalgesia. Results from Western blot and immunofluorescence assays showed that c-fos protein increased in the dorsal horn of the lumbar spinal cord and GABAA receptor β2 and β3 subunit proteins decreased significantly at 6 h after exposure to 1 h of acute restraint stress. Finally, the effects of spinal GABAA receptor alteration on SIH were evaluated. These results showed that intrathecal administration of muscimol inhibited hyperalgesia induced by stress while bicuculline enhanced hyperalgesia in the control groups. Taken together, the present data reveal that GABAA receptor β2 and β3 decrease following 1 h of acute restraint stress and may play a critical role in SIH.

  14. Flavylium salts as in vitro precursors of potent ligands to brain GABA-A receptors

    DEFF Research Database (Denmark)

    Kueny-Stotz, Marie; Chassaing, Stefan; Brouillard, Raymond

    2008-01-01

    The synthesis of a series of derivatized flavylium cations was undertaken and the affinity to the benzodiazepine binding site of the GABA-A receptor evaluated. The observed high affinity for some derivatives (sub-muM range) was explained by an in vitro transformation of the flavylium cations into...

  15. In vitro blood-brain barrier permeability predictions for GABAA receptor modulating piperine analogs

    DEFF Research Database (Denmark)

    Eigenmann, Daniela Elisabeth; Dürig, Carmen; Jähne, Evelyn Andrea

    2016-01-01

    The alkaloid piperine from black pepper (Piper nigrum L.) and several synthetic piperine analogs were recently identified as positive allosteric modulators of γ-aminobutyric acid type A (GABAA) receptors. In order to reach their target sites of action, these compounds need to enter the brain...

  16. GABA(A) receptor- and GABA transporter polymorphisms and risk for essential tremor

    DEFF Research Database (Denmark)

    Thier, S; Kuhlenbäumer, G; Lorenz, D

    2011-01-01

    Background:  Clinical features and animal models of essential tremor (ET) suggest gamma-aminobutyric acid A receptor (GABA(A) R) subunits and GABA transporters as putative candidate genes. Methods:  A total of 503 ET cases and 818 controls were investigated for an association between polymorphisms...

  17. Azaflavones compared to flavones as ligands to the benzodiazepine binding site of brain GABAA receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Nielsen, Elsebet Østergaard; Liljefors, Tommy

    2008-01-01

    A series of azaflavone derivatives and analogues were prepared and evaluated for their affinity to the benzodiazepine binding site of the GABA(A) receptor, and compared to their flavone counterparts. Three of the compounds, the azaflavones 9 and 12 as well as the new flavone 13, were also assayed...

  18. Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABA(A) receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Gidlöf, Ritha; Nielsen, Elsebet Østergaard

    2011-01-01

    Based on a pharmacophore model of the benzodiazepine-binding site of GABA(A) receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity ligands of the binding site. For several compounds, K...

  19. GABAA receptors in visual and auditory cortex and neural activity changes during basic visual stimulation

    Directory of Open Access Journals (Sweden)

    Pengmin eQin

    2012-12-01

    Full Text Available Recent imaging studies have demonstrated that levels of resting GABA in the visual cortex predict the degree of stimulus-induced activity in the same region. These studies have used the presentation of discrete visual stimulus; the change from closed eyes to open also represents a simple visual stimulus, however, and has been shown to induce changes in local brain activity and in functional connectivity between regions. We thus aimed to investigate the role of the GABA system, specifically GABAA receptors, in the changes in brain activity between the eyes closed (EC and eyes open (EO state in order to provide detail at the receptor level to complement previous studies of GABA concentrations. We conducted an fMRI study involving two different modes of the change from EC to EO: An EO and EC block design, allowing the modelling of the haemodynamic response, followed by longer periods of EC and EO to allow the measuring of functional connectivity. The same subjects also underwent [18F]Flumazenil PET measure GABAA receptor binding potentials. It was demonstrated that the local-to-global ratio of GABAA receptor binding potential in the visual cortex predicted the degree of changes in neural activity from EC to EO. This same relationship was also shown in the auditory cortex. Furthermore, the local-to-global ratio of GABAA receptor binding potential in the visual cortex also predicts the change of functional connectivity between visual and auditory cortex from EC to EO. These findings contribute to our understanding of the role of GABAA receptors in stimulus-induced neural activity in local regions and in inter-regional functional connectivity.

  20. GABAA receptors are located in cholinergic terminals in the nucleus pontis oralis of the rat: implications for REM sleep control.

    Science.gov (United States)

    Liang, Chang-Lin; Marks, Gerald A

    2014-01-16

    The oral pontine reticular formation (PnO) of rat is one region identified in the brainstem as a rapid eye movement (REM) sleep induction zone. Microinjection of GABA(A) receptor antagonists into PnO induces a long lasting increase in REM sleep, which is similar to that produced by cholinergic agonists. We previously showed that this REM sleep-induction can be completely blocked by a muscarinic antagonist, indicating that the REM sleep-inducing effect of GABA(A) receptor antagonism is dependent upon the local cholinergic system. Consistent with these findings, it has been reported that GABA(A) receptor antagonists microdialyzed into PnO resulted in increased levels of acetylcholine. We hypothesize that GABA(A) receptors located on cholinergic boutons in the PnO are responsible for the REM sleep induction by GABA(A) receptor antagonists through blocking GABA inhibition of acetylcholine release. Cholinergic, varicose axon fibers were studied in the PnO by immunofluorescence and confocal, laser scanning microscopy. Immunoreactive cholinergic boutons were found to be colocalized with GABA(A) receptor subunit protein γ2. This finding implicates a specific subtype and location of GABA(A) receptors in PnO of rat in the control of REM sleep.

  1. Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

    DEFF Research Database (Denmark)

    Hansen, G H; Hösli, E; Belhage, B;

    1991-01-01

    GABAA-receptors were localized in explant cultures of rat cerebellum and in dissociated primary cultures of rat cerebellar granule cells and rat cerebellar astrocytes using the monoclonal antibody bd-17 directed against the beta-subunit of the GABAA/benzodiazepine/chloride channel complex. At the...

  2. Presynaptic α2-GABAA Receptors in Primary Afferent Depolarization and Spinal Pain Control

    OpenAIRE

    2011-01-01

    Spinal dorsal horn GABAA receptors are found both postsynaptically on central neurons and presynaptically on axons and/or terminals of primary sensory neurons, where they mediate primary afferent depolarization (PAD) and presynaptic inhibition. Both phenomena have been studied extensively on a cellular level, but their role in sensory processing in vivo has remained elusive, due to inherent difficulties to selectively interfere with presynaptic receptors. Here, we address the contribution of ...

  3. Presynaptic {alpha}2-GABAA receptors in primary afferent depolarization and spinal pain control

    OpenAIRE

    2011-01-01

    Spinal dorsal horn GABA(A) receptors are found both postsynaptically on central neurons and presynaptically on axons and/or terminals of primary sensory neurons, where they mediate primary afferent depolarization (PAD) and presynaptic inhibition. Both phenomena have been studied extensively on a cellular level, but their role in sensory processing in vivo has remained elusive, due to inherent difficulties to selectively interfere with presynaptic receptors. Here, we address the contribution o...

  4. Distinct roles of synaptic and extrasynaptic GABAA receptors in striatal inhibition dynamics

    Directory of Open Access Journals (Sweden)

    Ruixi eLuo

    2013-11-01

    Full Text Available Striatonigral and striatopallidal projecting medium spiny neurons (MSNs express dopamine D1 (D1+ and D2 receptors (D2+, respectively. Both classes receive extensive GABAergic input via expression of synaptic, perisynaptic and extrasynaptic GABAA receptors. The activation patterns of different presynaptic GABAergic neurons produce transient and sustained GABAA receptor-mediated conductance that fulfill distinct physiological roles. We performed single and dual whole cell recordings from striatal neurons in mice expressing fluorescent proteins in interneurons and MSNs. We report specific inhibitory dynamics produced by distinct activation patterns of presynaptic GABAergic neurons as source of synaptic, perisynaptic and extrasynaptic inhibition. Synaptic GABAA receptors in MSNs contain the α2, γ2 and a β subunit. In addition, there is evidence for the developmental increase of the α1 subunit that contributes to faster inhibitory postsynaptic current (IPSC. Tonic GABAergic currents in MSNs from adult mice are carried by extrasynaptic receptors containing the α4 and δ subunit, while in younger mice this current is mediated by receptors that contain the α5 subunit. Both forms of tonic currents are differentially expressed in D1+ and D2+ MSNs. This study extends these findings by relating presynaptic activation with pharmacological analysis of inhibitory conductance in mice where the β3 subunit is conditionally removed in fluorescently labeled D2+ MSNs and in mice with global deletion of the δ subunit. Our results show that responses to low doses of gaboxadol (2μM, a GABAA receptor agonist with preference to δ subunit, are abolished in the δ but not the β3 subunit knock out mice. This suggests that the β3 subunit is not a component of the adult extrasynaptic receptor pool, in contrast to what has been shown for tonic current in young mice. Deletion of the β3 subunit from D2+ MSNs however, removed slow spontaneous IPSCs, implicating its

  5. First direct electron microscopic visualization of a tight spatial coupling between GABAA-receptors and voltage-sensitive calcium channels

    DEFF Research Database (Denmark)

    Hansen, G H; Belhage, B; Schousboe, A

    1992-01-01

    Using cerebellar granule neurons in culture it was demonstrated that exposure of the cells to the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) leads to an increase in the number of voltage-gated calcium channels as revealed by quantitative preembedding indirect...... immunogold labelling using a monoclonal antibody specific for phenylalkylamine and dihydropyridine sensitive Ca2+ channels. Using the same technique and a monoclonal antibody (bd-17) to the beta 2/beta 3-subunit of the GABAA-receptor, double labelling of Ca2+ channels and GABAA-receptors with gold particles...... of THIP-treated cultures. This suggests that primarily low affinity GABAA-receptors are closely associated with Ca2+ channels and this may be important for the ability of these receptors to mediate an inhibitory action on transmitter release even under extreme depolarizing conditions....

  6. Hyperalgesic effect induced by barbiturates, midazolam and ethanol: pharmacological evidence for GABA-A receptor involvement

    Directory of Open Access Journals (Sweden)

    M.A.K.F. Tatsuo

    1997-02-01

    Full Text Available The involvement of GABA-A receptors in the control of nociception was studied using the tail-flick test in rats. Non-hypnotic doses of the barbiturates phenobarbital (5-50 mg/kg, pentobarbital (17-33 mg/kg, and thiopental (7.5-30 mg/kg, of the benzodiazepine midazolam (10 mg/kg or of ethanol (0.4-1.6 g/kg administered by the systemic route reduced the latency for the tail-flick response, thus inducing a 'hyperalgesic' state in the animals. In contrast, non-convulsant doses of the GABA-A antagonist picrotoxin (0.12-1.0 mg/kg administered systemically induced an increase in the latency for the tail-flick response, therefore characterizing an 'antinociceptive' state. Previous picrotoxin (0.12 mg/kg treatment abolished the hyperalgesic state induced by effective doses of the barbiturates, midazolam or ethanol. Since phenobarbital, midazolam and ethanol reproduced the described hyperalgesic effect of GABA-A-specific agonists (muscimol, THIP, which is specifically antagonized by the GABA-A antagonist picrotoxin, our results suggest that GABA-A receptors are tonically involved in the modulation of nociception in the rat central nervous system

  7. Potentiating Action of Propofol at GABAA Receptors of Retinal Bipolar Cells

    Science.gov (United States)

    Yue, Lan; Xie, An; Bruzik, Karol S.; Frølund, Bente; Qian, Haohua

    2011-01-01

    Purpose. Propofol (2,6-diisopropyl phenol), a widely used systemic anesthetic, is known to potentiate GABAA receptor activity in a number of CNS neurons and to produce changes in electroretinographically recorded responses of the retina. However, little is known about propofol's effects on specific retinal neurons. The authors investigated the action of propofol on GABA-elicited membrane current responses of retinal bipolar cells, which have both GABAA and GABAC receptors. Methods. Single, enzymatically dissociated bipolar cells obtained from rat retina were treated with propofol delivered by brief application in combination with GABA or other pharmacologic agents or as a component of the superfusing medium. Results. When applied with GABA at subsaturating concentrations and with TPMPA (a known GABAC antagonist), propofol markedly increased the peak amplitude and altered the kinetics of the response. Propofol increased the response elicited by THIP (a GABAA-selective agonist), and the response was reduced by bicuculline (a GABAA antagonist). The response to 5-methyl I4AA, a GABAC-selective agonist, was not enhanced by propofol. Serial brief applications of (GABA + TPMPA + propofol) led to a progressive increase in peak response amplitude and, at higher propofol concentrations, additional changes that included a prolonged time course of response recovery. Pre-exposure of the cell to perfusing propofol typically enhanced the rate of development of potentiation produced by (GABA + TPMPA + propofol) applications. Conclusions. Propofol exerts a marked and selective potentiation on GABAA receptors of retinal bipolar cells. The data encourage the use of propofol in future studies of bipolar cell function. PMID:21071744

  8. Immunochemical Localization of GABAA Receptor Subunits in the Freshwater Polyp Hydra vulgaris (Cnidaria, Hydrozoa).

    Science.gov (United States)

    Concas, A; Imperatore, R; Santoru, F; Locci, A; Porcu, P; Cristino, L; Pierobon, P

    2016-11-01

    γ-aminobutyric acid (GABA) receptors, responding to GABA positive allosteric modulators, are present in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa), one of the most primitive metazoans to develop a nervous system. We examined the occurrence and distribution of GABAA receptor subunits in Hydra tissues by western blot and immunohistochemistry. Antibodies against different GABAA receptor subunits were used in Hydra membrane preparations. Unique protein bands, inhibited by the specific peptide, appeared at 35, 60, ∼50 and ∼52 kDa in membranes incubated with α3, β1, γ3 or δ antibodies, respectively. Immunohistochemical screening of whole mount Hydra preparations revealed diffuse immunoreactivity to α3, β1 or γ3 antibodies in tentacles, hypostome, and upper part of the gastric region; immunoreactive fibers were also present in the lower peduncle. By contrast, δ antibodies revealed a strong labeling in the lower gastric region and peduncle, as well as in tentacles. Double labeling showed colocalization of α3/β1, α3/γ3 and α3/δ immunoreactivity in granules or cells in tentacles and gastric region. In the peduncle, colocalization of both α3/β1 and α3/γ3 immunoreactivity was found in fibers running horizontally above the foot. These data indicate that specific GABAA receptor subunits are present and differentially distributed in Hydra body regions. Subunit colocalization suggests that Hydra GABA receptors are heterologous multimers, possibly sub-serving different physiological activities.

  9. Fibrous and protoplasmic astrocytes express GABAA receptors that differ in benzodiazepine pharmacology.

    Science.gov (United States)

    Rosewater, K; Sontheimer, H

    1994-02-04

    Astrocytes cultured from spinal cord contain two morphologically distinguishable types of astrocytes: fibrous and protoplasmic cells. Both astrocyte subtypes, in culture, are able to express GABAA receptors, and their activation results in inward currents at the resting potential. Using patch-clamp electrophysiology we characterized their basic receptor pharmacology and compared it to spinal cord neurons that were also present in small numbers in these cultures. As in neuronal GABAA receptors, the local anesthetic pentobarbital effectively potentiated GABA-induced currents in both astrocyte subtypes. Similarly, the benzodiazepine diazepam, on average doubled GABA-induced currents in both astrocytes subtypes. In contrast to these effects that were similar in both astrocytes types and similar to spinal cord neurons, the response to the convulsant methyl-4-ethyl-6,7-dimethoxy-beta-carboline-3-carboxylate (DMCM), which is an inverse benzodiazepine agonist differs between astrocyte subtypes. DMCM reduced GABA-induced currents by about 50% in fibrous astrocytes as we also observed with spinal cord neurons. In contrast, DMCM increased GABA currents in protoplasmic astrocytes by up to 150%, an effect never observed in neurons. DMCM potentiations of GABA currents have recently been attributed to differences in receptor subunit composition. Our results thus indicate that subtypes of astrocytes express GABAA receptors that differ pharmacologically and likely differ also in subunit composition.

  10. Antiseizure Activity of Midazolam in Mice Lacking δ-Subunit Extrasynaptic GABA(A) Receptors.

    Science.gov (United States)

    Reddy, Sandesh D; Younus, Iyan; Clossen, Bryan L; Reddy, Doodipala Samba

    2015-06-01

    Midazolam is a benzodiazepine anticonvulsant with rapid onset and short duration of action. Midazolam is the current drug of choice for acute seizures and status epilepticus, including those caused by organophosphate nerve agents. The antiseizure activity of midazolam is thought to result from its allosteric potentiation of synaptic GABA(A) receptors in the brain. However, there are indications that benzodiazepines promote neurosteroid synthesis via the 18-kDa cholesterol transporter protein (TSPO). Therefore, we investigated the role of neurosteroids and their extrasynaptic GABA(A) receptor targets in the antiseizure activity of midazolam. Here, we used δ-subunit knockout (DKO) mice bearing a targeted deletion of the extrasynaptic receptors to investigate the contribution of the extrasynaptic receptors to the antiseizure activity of midazolam using the 6-Hz and hippocampus kindling seizure models. In both models, midazolam produced rapid and dose-dependent protection against seizures (ED50, 0.4 mg/kg). Moreover, the antiseizure potency of midazolam was undiminished in DKO mice compared with control mice. Pretreatment with PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide], a TSPO blocker, or finasteride, a 5α-reductase neurosteroid inhibitor, did not affect the antiseizure effect of midazolam. The antiseizure activity of midazolam was significantly reversed by pretreatment with flumazenil, a benzodiazepine antagonist. Plasma and brain levels of the neurosteroid allopregnanolone were not significantly greater in midazolam-treated animals. These studies therefore provide strong evidence that neurosteroids and extrasynaptic GABA(A) receptors are not involved in the antiseizure activity of midazolam, which mainly occurs through synaptic GABA(A) receptors via direct binding to benzodiazepine sites. This study reaffirms midazolam's use for controlling acute seizures and status epilepticus.

  11. Searching for perfect sleep: the continuing evolution of GABAA receptor modulators as hypnotics.

    Science.gov (United States)

    Nutt, David J; Stahl, S M

    2010-11-01

    The non-benzodiazepine GABA(A) receptor modulators ('Z-drugs') - zaleplon, zolpidem, zopiclone and eszopiclone - have become the accepted treatments for insomnia where they are available. However, recent randomized, placebo-controlled trials suggest that, for these drugs, there may be particular efficacy and tolerability profiles and distinct clinical outcomes in specific patient populations. This is particularly apparent when hypnotic/ selective serotonin reuptake inhibitor co-therapy is used to treat patients with co-morbid insomnia and psychiatric disorders, as patient recovery appears to be accelerated and enhanced by some drugs but not others. Emerging evidence of why this should be the case is that these hypnotic drugs may differ significantly from each other in their pharmacodynamic and pharmacokinetic profiles. Functional selectivity for specific GABA(A) receptor subtypes may determine each drug's clinical attributes, while the pharmacokinetic characteristics of Z-drugs also determine to a large extent how they perform in the clinic. For example, activity at GABA(A) alpha 1 receptor subtypes may be associated with sedative effects, whereas activity at alpha 2 and alpha 3 receptor subtypes may be associated with anxiolytic and antidepressant effects. In summary, the distinct clinical outcomes of zaleplon, zolpidem, zopiclone and eszopiclone may be explained by each drug's unique GABA(A) receptor subunit selectivity and pharmacokinetic profile. Further investigation of GABA( A) receptor subtype effects would help to increase understanding of current hypnotic drug effects, while knowledge of each drug's specific binding profile should enable clinicians to tailor treatment to individual patient's needs.

  12. Antiseizure Activity of Midazolam in Mice Lacking δ-Subunit Extrasynaptic GABAA Receptors

    Science.gov (United States)

    Reddy, Sandesh D.; Younus, Iyan; Clossen, Bryan L.

    2015-01-01

    Midazolam is a benzodiazepine anticonvulsant with rapid onset and short duration of action. Midazolam is the current drug of choice for acute seizures and status epilepticus, including those caused by organophosphate nerve agents. The antiseizure activity of midazolam is thought to result from its allosteric potentiation of synaptic GABAA receptors in the brain. However, there are indications that benzodiazepines promote neurosteroid synthesis via the 18-kDa cholesterol transporter protein (TSPO). Therefore, we investigated the role of neurosteroids and their extrasynaptic GABAA receptor targets in the antiseizure activity of midazolam. Here, we used δ-subunit knockout (DKO) mice bearing a targeted deletion of the extrasynaptic receptors to investigate the contribution of the extrasynaptic receptors to the antiseizure activity of midazolam using the 6-Hz and hippocampus kindling seizure models. In both models, midazolam produced rapid and dose-dependent protection against seizures (ED50, 0.4 mg/kg). Moreover, the antiseizure potency of midazolam was undiminished in DKO mice compared with control mice. Pretreatment with PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide], a TSPO blocker, or finasteride, a 5α-reductase neurosteroid inhibitor, did not affect the antiseizure effect of midazolam. The antiseizure activity of midazolam was significantly reversed by pretreatment with flumazenil, a benzodiazepine antagonist. Plasma and brain levels of the neurosteroid allopregnanolone were not significantly greater in midazolam-treated animals. These studies therefore provide strong evidence that neurosteroids and extrasynaptic GABAA receptors are not involved in the antiseizure activity of midazolam, which mainly occurs through synaptic GABAA receptors via direct binding to benzodiazepine sites. This study reaffirms midazolam’s use for controlling acute seizures and status epilepticus. PMID:25784648

  13. GABA-A and NMDA receptor expression is altered in the caudate but not the putamen of the postmortem brains of alcoholics.

    Directory of Open Access Journals (Sweden)

    Amol K Bhandage

    2014-12-01

    Full Text Available Chronic consumption of alcohol by humans has been shown to lead to impairment of executive and cognitive functions. Here we have studied the changes that take place in the dorsal striatum in post-mortem brains of alcoholics and normal controls. The results show a significant change in the expression of both the excitatory ionotropic glutamate receptor and the inhibitory GABA-A receptor subunit genes in the caudate but not the putamen of the striatum. The mRNA levels in the caudate encoding the glutamate receptor subunit GluN2A and the GABA-A receptor subunits δ, ε and ρ2 were significantly decreased whereas the GluD1, GluD2 and the GABA-A γ1 mRNA levels were significantly increased in the alcoholics as compared to controls. Interestingly in controls, 11 glutamate and 5 GABA-A receptor genes were more prominently (fold-increase varied from 1.24 to 2.91 expressed in the caudate than the putamen. We have previously shown in post-mortem samples from alcoholics that the expression level of glutamate and GABA-A receptor genes in the dorsal-lateral prefrontal cortex is similar to that of normal controls (Jin et al., 2011a;Jin et al., 2014b. This is in contrast to the present study. As the caudate is vital for automatic thinking, the results indicate that the balance between voluntary and automatic control of behaviours is altered in alcoholics. Our results suggest that there may be diminished executive control on goal-directed alcohol-seeking behaviour and, rather, a shift to greater striatal control over behaviours that may be critical in the progress of becoming an alcoholic.

  14. Interactions between modulators of the GABA(A) receptor: Stiripentol and benzodiazepines.

    Science.gov (United States)

    Fisher, Janet L

    2011-03-05

    Many patients with refractory epilepsy are treated with polytherapy, and nearly 15% of epilepsy patients receive two or more anti-convulsant agents. The anti-convulsant stiripentol is used as an add-on treatment for the childhood epilepsy syndrome known as severe myoclonic epilepsy in infancy (Dravet syndrome). Stiripentol has multiple mechanisms of action, both enhancing GABA(A) receptors and reducing activity of metabolic enzymes that break down other drugs. Stiripentol is typically co-administered with other anti-convulsants such as benzodiazepines which also act through GABA(A) receptor modulation. Stiripentol slows the metabolism of some of these drugs through inhibition of a variety of cytochrome P450 enzymes, but could also influence their effects on GABAergic neurotransmission. Is it rational to co-administer drugs which can act through the same target? To examine the potential interaction between these modulators, we transiently transfected HEK-293T cells to produce α3β3γ2L or α3β3δ recombinant GABA(A) receptors. Using whole-cell patch clamp recordings, we measured the response to each benzodiazepine alone and in combination with a maximally effective concentration of stiripentol. We compared the responses to four different benzodiazepines: diazepam, clonazepam, clobazam and norclobazam. In all cases we found that these modulators were equally effective in the presence and absence of stiripentol. The δ-containing receptors were insensitive to modulation by the benzodiazepines, which did not affect potentiation by stiripentol. These data suggest that stiripentol and the benzodiazepines act independently at GABA(A) receptors and that polytherapy could be expected to increase the maximum effect beyond either drug alone, even without consideration of changes in metabolism.

  15. Antidepressant-like effects of ascorbic acid and ketamine involve modulation of GABAA and GABAB receptors.

    Science.gov (United States)

    Rosa, Priscila B; Neis, Vivian B; Ribeiro, Camille M; Moretti, Morgana; Rodrigues, Ana Lúcia S

    2016-10-01

    It has been suggested that dysregulation of γ-aminobutyric acid (GABA)-mediated neurotransmission is involved in the etiology of major depressive disorder and in the action of the fast-acting antidepressant ketamine. Considering that recent evidence has suggested that ascorbic acid may exert an antidepressant-like effect through mechanisms similar to ketamine, this study evaluated the involvement of GABAA and GABAB receptors in the antidepressant-like effect of ascorbic acid, comparing the results with those obtained with ketamine. To investigate the involvement of GABAA in the antidepressant-like effect of ascorbic acid and ketamine in the tail suspension test (TST), mice were treated with a sub-effective dose of ascorbic acid (0.1mg/kg, po), ketamine (0.1mg/kg, ip) or vehicle and 30minutes later, a sub-effective dose of muscimol (0.1mg/kg, ip, GABAA receptor agonist) or vehicle was administered. In another set of experiments, mice were treated with ascorbic acid (1mg/kg, po, active dose in the TST) or vehicle and 30minutes later, baclofen (1mg/kg, ip, GABAB receptor agonist) was administered. A similar experimental protocol was performed with ketamine (1mg/kg, ip). The administration of muscimol combined with ascorbic acid or ketamine produced a synergistic antidepressant-like effect in the TST. Moreover, the antidepressant-like effects of ascorbic acid and ketamine were abolished by baclofen. There was no alteration in spontaneous locomotion in any experimental group. Results indicate that the anti-immobility effect of ascorbic acid and ketamine in TST may involve an activation of GABAA receptors and a possible inhibition of GABAB receptors. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  16. GABA-A receptor inhibition of local calcium signaling in spines and dendrites.

    Science.gov (United States)

    Marlin, Joseph J; Carter, Adam G

    2014-11-26

    Cortical interneurons activate GABA-A receptors to rapidly control electrical and biochemical signaling at pyramidal neurons. Different populations of interneurons are known to uniquely target the soma and dendrites of pyramidal neurons. However, the ability of these interneurons to inhibit Ca(2+) signaling at spines and dendrites is largely unexplored. Here we use whole-cell recordings, two-photon microscopy, GABA uncaging and optogenetics to study dendritic inhibition at layer 5 (L5) pyramidal neurons in slices of mouse PFC. We first show that GABA-A receptors strongly inhibit action potential (AP)-evoked Ca(2+) signals at both spines and dendrites. We find robust inhibition over tens of milliseconds that spreads along the dendritic branch. However, we observe no difference in the amount of inhibition at neighboring spines and dendrites. We then examine the influence of interneurons expressing parvalbumin (PV), somatostatin (SOM), or 5HT3a receptors. We determine that these populations of interneurons make unique contacts onto the apical and basal dendrites of L5 pyramidal neurons. We also show that SOM and 5HT3a but not PV interneurons potently inhibit AP Ca(2+) signals via GABA-A receptors at both spines and dendrites. These findings reveal how multiple interneurons regulate local Ca(2+) signaling in pyramidal neurons, with implications for cortical function and disease.

  17. Differential involvement of GABAA and GABAB receptors in propofol self-administration in rats

    Institute of Scientific and Technical Information of China (English)

    Bo YANG; Ben-fu WANG; Miao-jun LAI; Fu-qiang ZHANG; Xiao-wei YANG; Wen-hua ZHOU; Qing-quan LIAN

    2011-01-01

    Propofol has shown abuse potential.The aim of the present study is to investigate the effects of GABAA antagonist and GABAB agonist on propofol reinforcement.Methods:Sprague-Dawley rats were trained to self-administer propofol at a dose of 1.7 mg/kg per infusion under a fixed ratio (FR1) schedule of reinforcement for 14 d.In a separate set of experiments,food-maintained self-administration under a fixed ratio (FR5) schedule and locomotor activities of Sprague-Dawley rats were examined.Results:GABAA receptor antagonist bicuculline (0.25 mg/kg,ip) significantly increased the number of injections and active responses.Pretreatment with GABAB receptor agonist baclofen (3 mg/kg,ip) significantly decreased the number of active responses and total infusions of propofol during the training session.Moreover,microinjection of baclofen (50 and 100 ng/side) into the ventral tegmental area (VTA) significantly decreased the number of active responses and total infusions of propofol.Neither baclofen (1-3 mg/kg,ip) nor bicuculline (0.25-1 mg/kg,ip) affected food-maintained responses or motor activities.Conclusion:Propofol maintains its reward properties partially through GABAA receptor activation.Stimulation of GABA~ receptors in VTA may counteract the reinforcing properties of propofol.

  18. Waking action of ursodeoxycholic acid (UDCA involves histamine and GABAA receptor block.

    Directory of Open Access Journals (Sweden)

    Yevgenij Yanovsky

    Full Text Available Since ancient times ursodeoxycholic acid (UDCA, a constituent of bile, is used against gallstone formation and cholestasis. A neuroprotective action of UDCA was demonstrated recently in models of Alzheimer's disease and retinal degeneration. The mechanisms of UDCA action in the nervous system are poorly understood. We show now that UDCA promotes wakefulness during the active period of the day, lacking this activity in histamine-deficient mice. In cultured hypothalamic neurons UDCA did not affect firing rate but synchronized the firing, an effect abolished by the GABA(AR antagonist gabazine. In histaminergic neurons recorded in slices UDCA reduced amplitude and duration of spontaneous and evoked IPSCs. In acutely isolated histaminergic neurons UDCA inhibited GABA-evoked currents and sIPSCs starting at 10 µM (IC(50 = 70 µM and did not affect NMDA- and AMPA-receptor mediated currents at 100 µM. Recombinant GABA(A receptors composed of α1, β1-3 and γ2L subunits expressed in HEK293 cells displayed a sensitivity to UDCA similar to that of native GABA(A receptors. The mutation α1V256S, known to reduce the inhibitory action of pregnenolone sulphate, reduced the potency of UDCA. The mutation α1Q241L, which abolishes GABA(AR potentiation by several neurosteroids, had no effect on GABA(AR inhibition by UDCA. In conclusion, UDCA enhances alertness through disinhibition, at least partially of the histaminergic system via GABA(A receptors.

  19. Imidazenil: A Low Efficacy Agonist at α1- but High Efficacy at α5-GABAA Receptors Fail to Show Anticonvulsant Cross Tolerance to Diazepam or Zolpidem

    OpenAIRE

    2008-01-01

    Whereas advances in the molecular biology of GABAA receptor complex using knock-out and knock-in mice have been valuable in unveiling the structure, composition, receptor assembly, and several functions of different GABAA receptor subtypes, the mechanism(s) underlying benzodiazepine (BZ) tolerance and withdrawal remain poorly understood. Studies using specific GABAA receptor subunit knock-in mice suggest that tolerance to sedative action of diazepam requires long-term activation of α1 and α5 ...

  20. Moderate alcohol exposure during the rat equivalent to the third trimester of human pregnancy alters dopamine regulation of GABAA receptor-mediated transmission in the basolateral amygdala

    Directory of Open Access Journals (Sweden)

    Marvin Rafael Diaz

    2014-05-01

    Full Text Available Fetal ethanol (EtOH exposure leads to a range of neurobehavioral alterations, including deficits in emotional processing. The basolateral amygdala (BLA plays a critical role in modulating emotional processing, in part, via dopamine (DA regulation of GABA transmission. This BLA modulatory system is acquired during the first two weeks of postnatal life in rodents (equivalent to the 3rd trimester of human pregnancy and we hypothesized that it could be altered by EtOH exposure during this period. We found that exposure of rats to moderate levels of EtOH vapor during the 3rd trimester-equivalent (postnatal days (P 2-12 alters DA modulation of GABAergic transmission in BLA pyramidal neurons during periadolescence. Specifically, D1R-mediated potentiation of spontaneous inhibitory postsynaptic currents (IPSCs was significantly attenuated in EtOH-exposed animals. However, this was associated with a compensatory decrease in D3R-mediated suppression of miniature IPSCs. Western blot analysis revealed that these effects were not a result of altered D1R or D3R levels. BLA samples from EtOH-exposed animals also had significantly lower levels of the DA precursor (L-3,4-dihydroxyphenylalanine but DA levels were not affected. This is likely a consequence of reduced catabolism of DA, as indicated by reduced levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid in the BLA samples. Anxiety-like behavior was not altered in EtOH-exposed animals. This is the first study to demonstrate that the modulatory actions of DA in the BLA are altered by developmental EtOH exposure. Although compensatory adaptations were engaged in our moderate EtOH exposure paradigm, it is possible that these are not able to restore homeostasis and correct anxiety-like behaviors under conditions of heavier EtOH exposure. Therefore, future studies should investigate the potential role of alterations in the modulatory actions of DA in the pathophysiology of fetal alcohol spectrum

  1. Potentiating action of propofol at GABAA receptors of retinal bipolar cells

    DEFF Research Database (Denmark)

    Yue, Lan; Xie, An; Bruzik, Karol S

    2011-01-01

    Purpose. Propofol (2,6-diisopropyl phenol), a widely used systemic anesthetic, is known to potentiate GABA(A) receptor activity in a number of CNS neurons and to produce changes in electroretinographically recorded responses of the retina. However, little is known about propofol's effects...... on specific retinal neurons. The authors investigated the action of propofol on GABA-elicited membrane current responses of retinal bipolar cells, which have both GABA(A) and GABA(C) receptors. Methods. Single, enzymatically dissociated bipolar cells obtained from rat retina were treated with propofol...... delivered by brief application in combination with GABA or other pharmacologic agents or as a component of the superfusing medium. Results. When applied with GABA at subsaturating concentrations and with TPMPA (a known GABA(C) antagonist), propofol markedly increased the peak amplitude and altered...

  2. Dual Modulators of GABA-A and Alpha7 Nicotinic Receptors for Treating Autism

    Science.gov (United States)

    2014-08-01

    figure 3). The effect was dose- dependent with no significant effect observed at a dose of 0.3 mg/kg i.p. (data not shown). 8...Interestingly recent studies with the benzodiazepine agonist clonazepam, a non-selective GABAA receptor PAM, resulted in a bell-shaped dose response...These dose- dependence studies are underway and will not be complete until August 2014 because of changes in baseline BTBR behavior described below

  3. THIP, a hypnotic and antinociceptive drug, enhances a tonic GABAA receptor mediated conductance in mouse neocortex

    DEFF Research Database (Denmark)

    Drasbek, Kim Ryun; Jensen, Kimmo

    2006-01-01

    its cellular actions in the neocortex are uncertain, we studied the effects of THIP on neurons in slices of frontoparietal neocortex of 13- to 19-day-old (P13-19) mice. Using whole-cell patch-clamp recordings, we found that the clinically relevant THIP concentration of 1 μM induced a robust tonic GABA...... suggest that THIP activates an extrasynaptic GABA(A) receptor-mediated conductance in the neocortex, which may alter the cortical network activity....

  4. Dual Modulators of GABA-A and Alpha 7 Nicotinic Receptors for Treating Autism

    Science.gov (United States)

    2015-10-01

    AWARD NUMBER: W81XWH-13-1-0144 TITLE: Dual Modulators of GABA-A and Alpha 7 Nicotinic Receptors for Treating Autism PRINCIPAL INVESTIGATOR...Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for public release; distribution...searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send

  5. Implication for treatment: GABAA receptors in aging, Down syndrome and Alzheimer's disease

    Science.gov (United States)

    Rissman, Robert A.; Mobley, William C.

    2011-01-01

    In addition to progressive dementia, Alzheimer's disease (AD) is characterized by increased incidence of seizure activity. Although originally discounted as a secondary process occurring as a result of neurodegeneration, more recent data suggest that alterations in excitatory-inhibitory (E/I) balance occur in AD and may be a primary mechanism contributing AD cognitive decline. In this study, we discuss relevant research and reports on the GABAA receptor in developmental disorders, such as Down syndrome, in healthy aging, and highlight documented aberrations in the GABAergic system in AD. Stressing the importance of understanding the subunit composition of individual GABAA receptors, investigations demonstrate alterations of particular GABAA receptor subunits in AD, but overall sparing of the GABAergic system. In this study, we review experimental data on the GABAergic system in the pathobiology of AD and discuss relevant therapeutic implications. When developing AD therapeutics that modulate GABA it is important to consider how E/I balance impacts AD pathogenesis and the relationship between seizure activity and cognitive decline. PMID:21388375

  6. Implications for treatment: GABAA receptors in aging, Down syndrome and Alzheimer's disease.

    Science.gov (United States)

    Rissman, Robert A; Mobley, William C

    2011-05-01

    In addition to progressive dementia, Alzheimer's disease (AD) is characterized by increased incidence of seizure activity. Although originally discounted as a secondary process occurring as a result of neurodegeneration, more recent data suggest that alterations in excitatory-inhibitory (E/I) balance occur in AD and may be a primary mechanism contributing AD cognitive decline. In this study, we discuss relevant research and reports on the GABA(A) receptor in developmental disorders, such as Down syndrome, in healthy aging, and highlight documented aberrations in the GABAergic system in AD. Stressing the importance of understanding the subunit composition of individual GABA(A) receptors, investigations demonstrate alterations of particular GABA(A) receptor subunits in AD, but overall sparing of the GABAergic system. In this study, we review experimental data on the GABAergic system in the pathobiology of AD and discuss relevant therapeutic implications. When developing AD therapeutics that modulate GABA it is important to consider how E/I balance impacts AD pathogenesis and the relationship between seizure activity and cognitive decline.

  7. CO2-induced ocean acidification increases anxiety in rockfish via alteration of GABAA receptor functioning.

    Science.gov (United States)

    Hamilton, Trevor James; Holcombe, Adam; Tresguerres, Martin

    2014-01-22

    The average surface pH of the ocean is dropping at a rapid rate due to the dissolution of anthropogenic CO2, raising concerns for marine life. Additionally, some coastal areas periodically experience upwelling of CO2-enriched water with reduced pH. Previous research has demonstrated ocean acidification (OA)-induced changes in behavioural and sensory systems including olfaction, which is due to altered function of neural gamma-aminobutyric acid type A (GABAA) receptors. Here, we used a camera-based tracking software system to examine whether OA-dependent changes in GABAA receptors affect anxiety in juvenile Californian rockfish (Sebastes diploproa). Anxiety was estimated using behavioural tests that measure light/dark preference (scototaxis) and proximity to an object. After one week in OA conditions projected for the next century in the California shore (1125 ± 100 µatm, pH 7.75), anxiety was significantly increased relative to controls (483 ± 40 µatm CO2, pH 8.1). The GABAA-receptor agonist muscimol, but not the antagonist gabazine, caused a significant increase in anxiety consistent with altered Cl(-) flux in OA-exposed fish. OA-exposed fish remained more anxious even after 7 days back in control seawater; however, they resumed their normal behaviour by day 12. These results show that OA could severely alter rockfish behaviour; however, this effect is reversible.

  8. Modulation of synaptic GABAA receptor function by zolpidem in substantia nigra pars reticulata

    Institute of Scientific and Technical Information of China (English)

    Li-li ZHANG; Lei CHEN; Yan XUE; Wing-ho YUNG

    2008-01-01

    Aim: The substantia nigra pars reticulata (SNr) constitutes one of the output centers of the basal ganglia, and its abnormal activity is believed to contribute to some basal ganglia motor disorders. Different lines of evidence revealed a major contribution of GABAA receptor-mediated synaptic inhibition in controlling the activity of SNr. The benzodiazepine binding site within the GABAA receptor is a modulation site of significant clinical interest. A high density of benzodiazepine binding sites has been reported in the rat SNr. In the present study, we investi-gate the effects of activating benzodiazepine binding sites in the SNr. Methods: Whole-cell patch-clamp recordings and motor behavior were applied. Results: Superfusion of zolpidem, a benzodiazepine binding agonist, at 100 nmol/L signifi-cantly prolonged the decay time of GABAA receptor-mediated postsynaptic currents. The prolongation on decay time induced by zolpidem was sensitive to the benzodiazepine antagonist flumazenil, confirming the specificity on the ben-zodiazepine site. Zolpidem at 1 μmol/L exerted a stronger prolongation on the decay time. A further experiment was performed on behaving rats. A unilateral microinjection of zolpidem into the rat SNr caused a robust contralateral rotation, which was significantly different from that of control animals receiving the vehicle injection. Conclusion: The present in vitro and in vivo findings that zolpidem significantly potentiated GABA currents and thus inhibited the activity of the SNr provide a rationale for further investigations into its potential in the treatment of basal ganglia disorders.

  9. Partial Agonism of Taurine at Gamma-Containing Native and Recombinant GABAA Receptors

    Science.gov (United States)

    Kletke, Olaf; Gisselmann, Guenter; May, Andrea; Hatt, Hanns; A. Sergeeva, Olga

    2013-01-01

    Taurine is a semi-essential sulfonic acid found at high concentrations in plasma and mammalian tissues which regulates osmolarity, ion channel activity and glucose homeostasis. The structural requirements of GABAA-receptors (GABAAR) gated by taurine are not yet known. We determined taurine potency and efficacy relative to GABA at different types of recombinant GABAAR occurring in central histaminergic neurons of the mouse hypothalamic tuberomamillary nucleus (TMN) which controls arousal. At binary α1/2β1/3 receptors taurine was as efficient as GABA, whereas incorporation of the γ1/2 subunit reduced taurine efficacy to 60–90% of GABA. The mutation γ2F77I, which abolishes zolpidem potentiation, significantly reduced taurine efficacy at recombinant and native receptors compared to the wild type controls. As taurine was a full- or super- agonist at recombinant αxβ1δ-GABAAR, we generated a chimeric γ2 subunit carrying the δ subunit motif around F77 (MTVFLH). At α1/2β1γ2(MTVFLH) receptors taurine became a super-agonist, similar to δ-containing ternary receptors, but remained a partial agonist at β3-containing receptors. In conclusion, using site-directed mutagenesis we found structural determinants of taurine’s partial agonism at γ-containing GABAA receptors. Our study sheds new light on the β1 subunit conferring the widest range of taurine-efficacies modifying GABAAR function under (patho)physiological conditions. PMID:23637894

  10. Nootropic alpha7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators.

    Science.gov (United States)

    Ng, Herman J; Whittemore, Edward R; Tran, Minhtam B; Hogenkamp, Derk J; Broide, Ron S; Johnstone, Timothy B; Zheng, Lijun; Stevens, Karen E; Gee, Kelvin W

    2007-05-08

    Activation of brain alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of alpha7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective alpha7 nAChR-positive allosteric modulator (PAM) from a library of GABAA receptor PAMs. Compound 6 (N-(4-chlorophenyl)-alpha-[[(4-chloro-phenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at alpha7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of alpha7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction.

  11. Benzodiazepine-induced anxiolysis and reduction of conditioned fear are mediated by distinct GABAA receptor subtypes in mice.

    Science.gov (United States)

    Smith, Kiersten S; Engin, Elif; Meloni, Edward G; Rudolph, Uwe

    2012-08-01

    GABA(A) receptor modulating drugs such as benzodiazepines (BZs) have been used to treat anxiety disorders for over five decades. In order to determine whether the same or different GABA(A) receptor subtypes are necessary for the anxiolytic-like action of BZs in unconditioned anxiety and conditioned fear models, we investigated the role of different GABA(A) receptor subtypes by challenging wild type, α1(H101R), α2(H101R) and α3(H126R) mice bred on the C57BL/6J background with diazepam or chlordiazepoxide in the elevated plus maze and the fear-potentiated startle paradigms. Both drugs significantly increased open arm exploration in the elevated plus maze in wild type, α1(H101R) and α3(H126R), but this effect was abolished in α2(H101R) mice; these were expected results based on previous published results. In contrast, while administration of diazepam and chlordiazepoxide significantly attenuated fear-potentiated startle (FPS) in wild type mice and α3(H126R) mice, the fear-reducing effects of these drugs were absent in both α1(H101R) and α2(H101R) point mutants, indicating that both α1- and α2-containing GABA(A) receptors are necessary for BZs to exert their effects on conditioned fear responses. Our findings illustrate both an overlap and a divergence between the GABA(A) receptor subtype requirements for the impact of BZs, specifically that both α1- and α2-containing GABA(A) receptors are necessary for BZs to reduce conditioned fear whereas only α2-containing GABA(A) receptors are needed for BZ-induced anxiolysis in unconditioned tests of anxiety. This raises the possibility that GABAergic pharmacological interventions for specific anxiety disorders can be differentially tailored.

  12. Probing α4βδ GABAA Receptor Heterogeneity

    DEFF Research Database (Denmark)

    Hoestgaard-Jensen, Kirsten; Dalby, Nils Ole; Krall, Jacob

    2014-01-01

    in cerebellar granule cells. In contrast, the compound did not elicit significant currents in dentate gyrus granule cells or in striatal medium spiny neurons (MSNs), indicating predominant expression of extrasynaptic α4β2δ receptors in these cells. Interestingly, Thio-THIP evoked differential degrees...... recorded from dentate gyrus granule cells, most likely by targeting perisynaptic α4βδ receptors expressed at distal dendrites of these cells. Being the first published ligand capable of discriminating between β2- and β3-containing receptor subtypes, Thio-THIP could be a valuable tool in explorations...

  13. Imidazenil: a low efficacy agonist at alpha1- but high efficacy at alpha5-GABAA receptors fail to show anticonvulsant cross tolerance to diazepam or zolpidem.

    Science.gov (United States)

    Auta, James; Impagnatiello, Francesco; Kadriu, Bashkim; Guidotti, Alessandro; Costa, Erminio

    2008-08-01

    Whereas advances in the molecular biology of GABA(A) receptor complex using knock-out and knock-in mice have been valuable in unveiling the structure, composition, receptor assembly, and several functions of different GABA(A) receptor subtypes, the mechanism(s) underlying benzodiazepine (BZ) tolerance and withdrawal remain poorly understood. Studies using specific GABA(A) receptor subunit knock-in mice suggest that tolerance to sedative action of diazepam requires long-term activation of alpha1 and alpha5 GABA(A) receptor subunits. We investigated the role of long-term activation of these GABA(A) receptor subunits during anticonvulsant tolerance using high affinity and high intrinsic efficacy ligands for GABA(A) receptors expressing the alpha5 subunit (imidazenil) or alpha1 subunit (zolpidem), and a non-selective BZ recognition site ligand (diazepam). We report here that long-term activation of GABA(A) receptors by zolpidem and diazepam but not by imidazenil elicits anticonvulsant tolerance. Although anticonvulsant cross-tolerance occurs between diazepam and zolpidem, there is no cross-tolerance between imidazenil and diazepam or zolpidem. Furthermore, diazepam or zolpidem long-term treatment decreased the expression of mRNA encoding the alpha1 GABA(A) receptor subunit in prefrontal cortex by 43% and 20% respectively. In addition, diazepam but not zolpidem long-term treatment produced a 30% increase in the expression of the alpha5 GABA(A) receptor subunit mRNA in prefrontal cortex. In contrast, imidazenil which is devoid of anticonvulsant tolerance does not elicit significant changes in the expression of alpha1 or alpha5 GABA(A) receptor subunit. These findings suggest that long-term activation of GABA(A) receptors containing the alpha1 or other subunits but not the alpha5 receptor subunit is essential for the induction of anticonvulsant tolerance.

  14. Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 β1/3 δ GABAA Receptor Subtypes

    DEFF Research Database (Denmark)

    Falk-Petersen, Christina B; Søgaard, Rikke; Madsen, Kenneth L

    2017-01-01

    format cell-based assay for extrasynaptic α4 β1/3 δ receptors, we have engineered and validated a HEK293 Flp-In™ cell line stably expressing the human GABAA δ-subunit. Upon co-transfection of α4 and β1/3 subunits, at optimized ratios, we have established a well-defined system for expressing α4 β1/3 δ...

  15. Identification of amino acids involved in histamine potentiation of GABA(A receptors

    Directory of Open Access Journals (Sweden)

    Ulrike eThiel

    2015-05-01

    Full Text Available Histamine is a neurotransmitter involved in a number of physiological and neuronal functions. In mammals, such as humans and rodents, the histaminergic neurons found in the tuberomamillary nucleus (TMN project widely throughout the central nervous system (CNS. Histamine acts as positive modulator of GABA(A receptors (GABA(ARs and, in high concentrations (10 mM, as negative modulator of the strychnine-sensitive glycine receptor. However, the exact molecular mechanisms by which histamine acts on GABA(ARs are unknown. In our study, we aimed to identify amino acids potentially involved in the modulatory effect of histamine on GABA(ARs. We expressed GABA(ARs with 12 different point mutations in Xenopus laevis oocytes and characterized the effect of histamine on GABA-induced currents using the two-electrode voltage clamp technique. Our data demonstrate that the amino acid residues ß2(N265 and ß2(M286, which are important for modulation by propofol, are not involved in the action of histamine. However, we found that histamine modulation is dependent on the amino acid residues alpha1(R120, ß2(Y157, ß3(D163, ß3(V175 and ß3(Q185. We showed that the amino acid residues ß2(Y157 and ß3(Q185 mediate the positive modulatory effect of histamine on GABA-induced currents, whereas alpha1(R120 and ß2(D163 form a potential histamine interaction site in GABA(ARs.

  16. Axon-to-Glia Interaction Regulates GABAA Receptor Expression in Oligodendrocytes.

    Science.gov (United States)

    Arellano, Rogelio O; Sánchez-Gómez, María Victoria; Alberdi, Elena; Canedo-Antelo, Manuel; Chara, Juan Carlos; Palomino, Aitor; Pérez-Samartín, Alberto; Matute, Carlos

    2016-01-01

    Myelination requires oligodendrocyte-neuron communication, and both neurotransmitters and contact interactions are essential for this process. Oligodendrocytes are endowed with neurotransmitter receptors whose expression levels and properties may change during myelination. However, only scant information is available about the extent and timing of these changes or how they are regulated by oligodendrocyte-neuron interactions. Here, we used electrophysiology to study the expression of ionotropic GABA, glutamate, and ATP receptors in oligodendrocytes derived from the optic nerve and forebrain cultured either alone or in the presence of dorsal root ganglion neurons. We observed that oligodendrocytes from both regions responded to these transmitters at 1 day in culture. After the first day in culture, however, GABA sensitivity diminished drastically to less than 10%, while that of glutamate and ATP remained constant. In contrast, the GABA response amplitude was sustained and remained stable in oligodendrocytes cocultured with dorsal root ganglion neurons. Immunochemistry and pharmacological properties of the responses indicated that they were mediated by distinctive GABAA receptors and that in coculture with neurons, the oligodendrocytes bearing the receptors were those in direct contact with axons. These results reveal that GABAA receptor regulation in oligodendrocytes is driven by axonal cues and that GABA signaling may play a role in myelination and/or during axon-glia recognition.

  17. Association between alcoholism and the genetic polymorphisms of the GABAA receptor genes on chromosome 5q33-34 in Korean population.

    Science.gov (United States)

    Park, Chul-Soo; Park, So-Young; Lee, Chul-Soon; Sohn, Jin-Wook; Hahn, Gyu-Hee; Kim, Bong-Jo

    2006-06-01

    Family, twin, and adoption studies have demonstrated that genes play an important role in the development of alcoholism. We investigated the association between alcoholism and the genetic polymorphisms of the GABAA receptor genes on chromosome 5q33-34 in Korean population. The genotype of the GABAA receptor gene polymorphisms were determined by performing polymerase chain reaction genotyping for 172 normal controls and 162 male alcoholics who are hospitalized in alcoholism treatment institute. We found a significant association between the genetic polymorphisms of the GABAA alpha1 and GABAA alpha6 receptor gene and alcoholism. The GG genotype of the GABAA alpha1 receptor gene was associated with the onset age of alcoholism and alcohol withdrawal symptoms, and a high score on the Korean version of the ADS. However, there was no association between the genetic polymorphisms of the GABAA beta2 and gamma2 receptor gene and alcoholisms. Our finding suggest that genetic polymorphisms of the GABAA alpha1 and GABAA alpha6 receptor gene may be associated with the development of alcoholism and that the GG genotype of the GABAA alpha1 receptor gene play an important role in the development of the early onset and the severe type of alcoholism.

  18. Bicuculline, a GABAA-receptor antagonist, blocked HPA axis activation induced by ghrelin under an acute stress.

    Science.gov (United States)

    Gastón, M S; Cid, M P; Salvatierra, N A

    2017-03-01

    Ghrelin is a peptide of 28 amino acids with a homology between species, which acts on the central nervous system to regulate different actions, including the control of growth hormone secretion and metabolic regulation. It has been suggested that central ghrelin is a mediator of behavior linked to stress responses and induces anxiety in rodents and birds. Previously, we observed that the anxiogenic-like behavior induced by ghrelin injected into the intermediate medial mesopallium (IMM) of the forebrain was blocked by bicuculline (a GABAA receptor competitive antagonist) but not by diazepam (a GABAA receptor allosteric agonist) in neonatal meat-type chicks (Cobb). Numerous studies have indicated that hypothalamic-pituitary-adrenal (HPA) axis activation mediates the response to stress in mammals and birds. However, it is still unclear whether this effect of ghrelin is associated with HPA activation. Therefore, we investigated whether anxiety behavior induced by intra-IMM ghrelin and mediated through GABAA receptors could be associated with HPA axis activation in the neonatal chick. In the present study, in an Open Field test, intraperitoneal bicuculline methiodide blocked anxiogenic-like behavior as well as the increase in plasma ACTH and corticosterone levels induced by ghrelin (30pmol) in neonatal chicks. Moreover, we showed for the first time that a competitive antagonist of GABAA receptor suppressed the HPA axis activation induced by an anxiogenic dose of ghrelin. These results show that the anxiogenic ghrelin action involves the activation of the HPA axis, with a complex functional interaction with the GABAA receptor.

  19. First direct electron microscopic visualization of a tight spatial coupling between GABAA-receptors and voltage-sensitive calcium channels

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Belhage, B; Schousboe, A

    1992-01-01

    Using cerebellar granule neurons in culture it was demonstrated that exposure of the cells to the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) leads to an increase in the number of voltage-gated calcium channels as revealed by quantitative preembedding indirect imm...... of THIP-treated cultures. This suggests that primarily low affinity GABAA-receptors are closely associated with Ca2+ channels and this may be important for the ability of these receptors to mediate an inhibitory action on transmitter release even under extreme depolarizing conditions....

  20. A Review of the Updated Pharmacophore for the Alpha 5 GABA(A Benzodiazepine Receptor Model

    Directory of Open Access Journals (Sweden)

    Terry Clayton

    2015-01-01

    Full Text Available An updated model of the GABA(A benzodiazepine receptor pharmacophore of the α5-BzR/GABA(A subtype has been constructed prompted by the synthesis of subtype selective ligands in light of the recent developments in both ligand synthesis, behavioral studies, and molecular modeling studies of the binding site itself. A number of BzR/GABA(A α5 subtype selective compounds were synthesized, notably α5-subtype selective inverse agonist PWZ-029 (1 which is active in enhancing cognition in both rodents and primates. In addition, a chiral positive allosteric modulator (PAM, SH-053-2′F-R-CH3 (2, has been shown to reverse the deleterious effects in the MAM-model of schizophrenia as well as alleviate constriction in airway smooth muscle. Presented here is an updated model of the pharmacophore for α5β2γ2 Bz/GABA(A receptors, including a rendering of PWZ-029 docked within the α5-binding pocket showing specific interactions of the molecule with the receptor. Differences in the included volume as compared to α1β2γ2, α2β2γ2, and α3β2γ2 will be illustrated for clarity. These new models enhance the ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists at the Bz BS of GABA(A receptors.

  1. GABA(A) receptor mediated inhibition contributes to corticostriatal frequency filtering.

    Science.gov (United States)

    Jelinek, Devin A; Partridge, L Donald

    2012-11-21

    The striatum plays an important role in the initiation and learning of skilled motor behavior [6] and receives topographic input from most areas of the cortex. Cortical afferents make divergent contact with many striatal medium spiny neurons while individual medium spiny neurons receive tens of thousands of these glutamatergic synapses [13]. Temporal filtering of frequency information within synaptic fields plays an important role in the processing of neuronal signals. We have previously shown differential filtering characteristics within CA1, CA3, and the dentate gyrus of the hippocampus [26] and have now extended these studies to the cortical input to the dorsal striatum in order to address the network filtering characteristics in this important synaptic field. We measured field potentials of striatal medium spiny neurons in response to layer V cortical input over a range of stimulus frequencies from 2Hz to 100Hz. The average population spike amplitude in response to these stimulus trains exhibited a non-linear relationship to frequency, with characteristics of a low pass filter. In order to assess potential modulation of these filter properties, we examined the frequency response in the presence of antagonists to CB1, D2, nACh, and GABA(A) receptors, which are all known to be expressed at these synapses [13]. Of these, only GABA(A) receptor antagonists significantly modulated the frequency filtering characteristics over the examined frequency range. High frequency stimulation induces long term plasticity at corticostriatal synapses [4] and this process is strengthened when GABA(A) receptors are blocked [7,20,29]. Our results suggest a model whereby a temporary decrease in GABA level would modulate the filtering parameters of the corticostriatal circuit, allowing a more robust induction of high frequency-dependent plasticity.

  2. Studying cerebellar circuits by remote control of selected neuronal types with GABA-A receptors

    Directory of Open Access Journals (Sweden)

    William Wisden

    2009-12-01

    Full Text Available Although GABA-A receptor-mediated inhibition of cerebellar Purkinje cells by molecular layer interneurons (MLIs has been studied intensely on the cellular level, it has remained unclear how this inhibition regulates cerebellum-dependent behaviour. We have implemented two complementary approaches to investigate the function of the MLI-Purkinje cell synapse on the behavioral level. In the first approach we permanently disrupted inhibitory fast synaptic transmission at the synapse by genetically removing the postsynaptic GABA-A receptors from Purkinje cells (PC-Δγ2 mice. We found that chronic disruption of the MLI-Purkinje cell synapse strongly impaired cerebellar learning of the vestibular occular reflex (VOR, presumably by disrupting the temporal patterns of Purkinje cell activity. However, in PC-Δγ2 mice the baseline VOR reflex was only mildly affected; indeed PC-Δγ2 mice showed no ataxia or gait abnormalities suggesting that MLI control of Purkinje cell activity is either not involved in ongoing motor tasks or that the system has found a way to compensate for its loss. To investigate the latter possibility we have developed an alternative genetic technique; we made the MLI-Purkinje cell synapse selectively sensitive to rapid manipulation with the GABAA receptor modulator zolpidem (PC-γ2-swap mice. Minutes after intraperitoneal zolpidem injection, these PC-γ2-swap mice developed severe motor abnormalities, revealing a substantial contribution of the MLI-Purkinje cell synapse to real time motor control. The cell-type selective permanent knockout of synaptic GABAergic input, and the fast reversible modulation of GABAergic input at the same synapse illustrate how pursuing both strategies gives a fuller view.

  3. Role of spinal GABAA receptors in pudendal inhibition of nociceptive and nonnociceptive bladder reflexes in cats.

    Science.gov (United States)

    Xiao, Zhiying; Reese, Jeremy; Schwen, Zeyad; Shen, Bing; Wang, Jicheng; Roppolo, James R; de Groat, William C; Tai, Changfeng

    2014-04-01

    Picrotoxin, an antagonist for γ-aminobutyric acid receptor subtype A (GABAA), was used to investigate the role of GABAA receptors in nociceptive and nonnociceptive reflex bladder activities and pudendal inhibition of these activities in cats under α-chloralose anesthesia. Acetic acid (AA; 0.25%) was used to irritate the bladder and induce nociceptive bladder overactivity, while saline was used to distend the bladder and induce nonnociceptive bladder activity. To modulate the bladder reflex, pudendal nerve stimulation (PNS) was applied at multiple threshold (T) intensities for inducing anal sphincter twitching. AA irritation significantly (P < 0.01) reduced bladder capacity to 34.3 ± 7.1% of the saline control capacity, while PNS at 2T and 4T significantly (P < 0.01) increased AA bladder capacity to 84.0 ± 7.8 and 93.2 ± 15.0%, respectively, of the saline control. Picrotoxin (0.4 mg it) did not change AA bladder capacity but completely removed PNS inhibition of AA-induced bladder overactivity. Picrotoxin (iv) only increased AA bladder capacity at a high dose (0.3 mg/kg) but significantly (P < 0.05) reduced 2T PNS inhibition at low doses (0.01-0.1 mg/kg). During saline cystometry, PNS significantly (P < 0.01) increased bladder capacity to 147.0 ± 7.6% at 2T and 172.7 ± 8.9% at 4T of control capacity, and picrotoxin (0.4 mg it or 0.03-0.3 mg/kg iv) also significantly (P < 0.05) increased bladder capacity. However, picrotoxin treatment did not alter PNS inhibition during saline infusion. These results indicate that spinal GABAA receptors have different roles in controlling nociceptive and nonnociceptive reflex bladder activities and in PNS inhibition of these activities.

  4. GABA-A and NMDA receptor subunit mRNA expression is altered in the caudate but not the putamen of the postmortem brains of alcoholics.

    Science.gov (United States)

    Bhandage, Amol K; Jin, Zhe; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R; Birnir, Bryndis

    2014-01-01

    Chronic consumption of alcohol by humans has been shown to lead to impairment of executive and cognitive functions. Here, we have studied the mRNA expression of ion channel receptors for glutamate and GABA in the dorsal striatum of post-mortem brains from alcoholics (n = 29) and normal controls (n = 29), with the focus on the caudate nucleus that is associated with the frontal cortex executive functions and automatic thinking and on the putamen area that is linked to motor cortices and automatic movements. The results obtained by qPCR assay revealed significant changes in the expression of specific excitatory ionotropic glutamate and inhibitory GABA-A receptor subunit genes in the caudate but not the putamen. Thus, in the caudate we found reduced levels of mRNAs encoding the GluN2A glutamate receptor and the δ, ε, and ρ2 GABA-A receptor subunits, and increased levels of the mRNAs encoding GluD1, GluD2, and GABA-A γ1 subunits in the alcoholics as compared to controls. Interestingly in the controls, 11 glutamate and 5 GABA-A receptor genes were more prominently expressed in the caudate than the putamen (fold-increase varied from 1.24 to 2.91). Differences in gene expression patterns between the striatal regions may underlie differences in associated behavioral outputs. Our results suggest an altered balance between caudate-mediated voluntarily controlled and automatic behaviors in alcoholics, including diminished executive control on goal-directed alcohol-seeking behavior.

  5. Abuse and dependence liability of benzodiazepine-type drugs: GABA(A) receptor modulation and beyond.

    Science.gov (United States)

    Licata, Stephanie C; Rowlett, James K

    2008-07-01

    Over the past several decades, benzodiazepines and the newer non-benzodiazepines have become the anxiolytic/hypnotics of choice over the more readily abused barbiturates. While all drugs from this class act at the GABA(A) receptor, benzodiazepine-type drugs offer the clear advantage of being safer and better tolerated. However, there is still potential for these drugs to be abused, and significant evidence exists to suggest that this is a growing problem. This review examines the behavioral determinants of the abuse and dependence liability of benzodiazepine-type drugs. Moreover, the pharmacological and putative biochemical basis of the abuse-related behavior is discussed.

  6. GABA(A) receptors on calbindin-immunoreactive myenteric neurons of guinea pig intestine.

    Science.gov (United States)

    Zhou, X; Galligan, J J

    2000-01-14

    These studies were carried out to characterize the properties of gamma-aminobutyric acidA (GABA(A)) receptors on guinea pig intestinal myenteric neurons maintained in primary culture. In addition, the type of neuron expressing GABA(A) receptors was identified using immunohistochemical methods. Whole-cell patch clamp recordings of currents elicited by GABA and acetylcholine (ACh) were obtained using pipettes containing Neurobiotin. After electrophysiological studies, neurons were processed for localization of calbindin-D28K-immunoreactivity (calbindin-ir). GABA (1 mM) and ACh (3 mM) caused inward currents in most cells tested. GABA currents were mimicked by muscimol (1-300 microM) and were blocked by bicuculline (10 microM) indicating that GABA was acting at GABA(A) receptors. GABA currents were associated with a conductance increase and a linear current/voltage relationship with a reversal potential of 1 +/- 1 mV (n = 5). Pentobarbital (PB, 3-1000 microM) and diazepam (DZP, 0.01-10 microM) potentiated GABA-induced currents. A maximum concentration of DZP (1 microM) increased GABA-induced currents 3.1 +/- 0.3 times while PB (1000 microM) increased GABA currents by 11 +/- 2 times. In outside-out patches, the amplitude of GABA-activated single-channel currents was linearly related to membrane potential with a single-channel conductance of 28.5 + 0.5 pS (n = 10). PB and DZP increased the open probability of GABA-induced single-channel currents. Neurons containing calbindin-ir were large, were isolated from other neurons and had GABA current amplitudes of -3.4 +/- 0.3 nA (n = 48). Neurons with weak or absent calbindin-ir were smaller, were localized in clusters of cells and had GABA-induced current amplitudes of -0.6 +/- 0.1 nA (n = 20). ACh-induced currents were smaller in calbindin-ir neurons (-0.7 +/- 0.1 nA) compared to weakly calbindin-ir neurons (-1.4 +/- 0.1 nA). These results indicate that myenteric calbindin-ir neurons express a high density of GABA(A

  7. Modulation of GABAA receptor-mediated synaptic transmission by Zn2+ at a dentate gyrus circuit

    OpenAIRE

    Grauert, A.

    2013-01-01

    Zinc (ionic form Zn2+) is a common trace element in the forebrain, and is especially enriched in the hippocampus, a brain structure important for learning and memory. A large amount of vesicular Zn2+ which is thought to be released upon presynaptic depolarisation is found at synapses formed by the axons of dentate granule cells (GCs), known as mossy fibres (MFs). Zn2+ inhibits NMDA and GABAA receptors (NMDAR and GABAAR) at mono-synaptic inputs between MFs and CA3 pyramidal neurons but its rol...

  8. Affinity of 3-acyl substituted 4-quinolones at the benzodiazepine site of GABAA receptors

    DEFF Research Database (Denmark)

    Lager, Erik; Nilsson, Jakob; Nielsen, Elsebet Østergaard

    2008-01-01

    The finding that alkyl 1,4-dihydro-4-oxoquinoline-3-carboxylate and N-alkyl-1,4-dihydro-4-oxoquinoline-3-carboxamide derivatives may be high-affinity ligands at the benzodiazepine binding site of the GABA(A) receptor, prompted a study of 3-acyl-1,4-dihydro-4-oxoquinoline (3-acyl-4-quinolones......). In general, the affinity of the 3-acyl derivatives was found to be comparable with the 3-carboxylate and the 3-carboxamide derivatives, and certain substituents (e.g., benzyl) in position 6 were again shown to be important. As it is believed that the benzodiazepine binding site is situated between an alpha...

  9. Participation of central GABAA receptors in the trigeminal processing of mechanical allodynia in rats

    Science.gov (United States)

    Kim, Min Ji; Park, Young Hong; Yang, Kui Ye; Ju, Jin Sook; Bae, Yong Chul

    2017-01-01

    Here we investigated the central processing mechanisms of mechanical allodynia and found a direct excitatory link with low-threshold input to nociceptive neurons. Experiments were performed on male Sprague-Dawley rats weighing 230-280 g. Subcutaneous injection of interleukin 1 beta (IL-1β) (1 ng/10 µL) was used to produce mechanical allodynia and thermal hyperalgesia. Intracisternal administration of bicuculline, a gamma aminobutyric acid A (GABAA) receptor antagonist, produced mechanical allodynia in the orofacial area under normal conditions. However, intracisternal administration of bicuculline (50 ng) produced a paradoxical anti-allodynic effect under inflammatory pain conditions. Pretreatment with resiniferatoxin (RTX), which depletes capsaicin receptor protein in primary afferent fibers, did not alter the paradoxical anti-allodynic effects produced by the intracisternal injection of bicuculline. Intracisternal injection of bumetanide, an Na-K-Cl cotransporter (NKCC 1) inhibitor, reversed the IL-1β-induced mechanical allodynia. In the control group, application of GABA (100 µM) or muscimol (3 µM) led to membrane hyperpolarization in gramicidin perforated current clamp mode. However, in some neurons, application of GABA or muscimol led to membrane depolarization in the IL-1β-treated rats. These results suggest that some large myelinated Aβ fibers gain access to the nociceptive system and elicit pain sensation via GABAA receptors under inflammatory pain conditions. PMID:28066142

  10. Differences in kinetics between GABA_C and GABA_A receptors on carp retinal bipolar cells

    Institute of Scientific and Technical Information of China (English)

    韩明虎; 沈颖; 杨雄里

    2000-01-01

    The present work was undertaken to characterize kinetics, including activation, desensitization and deactivation, of responses mediated by GABAA and GABAc receptors on carp retinal bipolar cells, using the whole-cell patch-clamp technique. It was revealed that the GABAC response was generally slower in kinetics than the GABAA response. Activation kinetics of both the receptors could be well fit by monoexponential functions with time constants r, being 44.57 ms (GABAC) and 10.86 ms (GABAA) respectively. Desensitization of the GABAA response was characterized by a fast and a slow exponential component with time constants of τfast = 2.16 s and τslow = 19.78 s respectively, whereas desensitization of the GABAc response was fit by a monoexponential function of the time constant T = 6.98 s. Deactivation at both the receptors was adequately described by biexponential functions with time constants being much higher for the GABAC response (τfast = 674.8 ms; τslow = 2 090 ms) than those for the GABAA response

  11. Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Hösli, E; Belhage, B

    1991-01-01

    . At the light microscope level specific staining of GABAA-receptors was localized in various types of neurones in explant cultures of rat cerebellum using the indirect peroxidase-antiperoxidase (PAP) technique, whereas no specific staining was found in astrocytes. At the electron microscope level labeling...... of GABAA-receptors was observed in the plasma membrane of both the cell bodies and processes in dissociated primary cultures of cerebellar granule cells using an indirect preembedding immunogold staining technique which in contrast to the classical PAP technique allows quantitative estimations...

  12. 3-Alkyl- and 3-amido-isothiazoloquinolin-4-ones as ligands for the benzodiazepine site of GABAA receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Nielsen, Elsebet Østergaard; Liljefors, Tommy

    2012-01-01

    Based on a pharmacophore model of the benzodiazepine binding site of the GABA(A) receptors, developed with synthetic flavones and potent 3-carbonylquinolin-4-ones, 3-alkyl- and 3-amido-6-methylisothiazoloquinolin-4-ones were designed, prepared and assayed. The suggestion that the interaction...... interaction with the lipophilic pockets of the pharmacophore model. The most potent 3-alkyl derivative, 3-pentyl-6-methylisothiazoloquinolin-4-one, has an affinity (K(i) value) for the benzodiazepine binding site of the GABA(A) receptors of 13nM. However, by replacing the 3-pentyl with a 3-butyramido group...

  13. Effect of a GABA agonist on the expression and distribution of GABAA receptors in the plasma membrane of cultured cerebellar granule cells: an immunocytochemical study

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Belhage, B; Schousboe, A

    1991-01-01

    , the density of the GABAA receptors was significantly increased in the THIP-treated cultures as compared to the control cultures and this effect of THIP was particularly pronounced in the processes. GABAA receptors were occasionally observed to form 'hot spots' in process-like structures and again......The effect of the gamma-aminobutyric acid (GABA) agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 microM) on the localization and density of GABAA receptors in the plasma membrane of rat cerebellar granule cells in primary cultures was studied at the electron microscope (EM) level...... by preembedding immunogold staining using the monoclonal antibody bd-17 directed against the beta-subunit of the GABAA receptor complex. In THIP-treated as well as untreated control cultures, GABAA receptors were found to be evenly distributed in the plasma membrane of cell bodies as well as processes. However...

  14. Parvalbumin immunoreactivity and expression of GABAA receptor subunits in the thalamus after experimental TBI.

    Science.gov (United States)

    Huusko, N; Pitkänen, A

    2014-05-16

    Traumatic brain injury (TBI) causes 10-20% of acquired epilepsy in humans, resulting in an ictogenic region that is often located in the cerebral cortex. The thalamus provides heavy projections to the cortex and the activity of thalamocortical pathways is controlled by GABAergic afferents from the reticular nucleus of the thalamus (RT). As rats with TBI induced by lateral fluid-percussion injury (FPI) undergo epileptogenesis, we hypothesized that damage to the parvalbumin (PARV)-immunoreactive (ir) neurons in the RT is associated with seizure susceptibility after lateral FPI. To address this hypothesis, adult Sprague-Dawley rats (n=13) were injured with lateral FPI. At 6months post-TBI, each animal underwent a pentylenetetrazol (PTZ) seizure susceptibility test and 2weeks of continuous video-electroencephalography (EEG) monitoring for detection of the occurrence of spontaneous seizures. Thereafter, the brain was processed for PARV immunohistochemistry. We (a) estimated the total number of PARV-ir neurons in the RT using unbiased stereology, (b) measured the volume of the ventroposteromedial (VPM) and ventroposterolateral (VPL) nuclei of the thalamus, which receive PARV-ir inputs from the RT and project to the perilesional cortex, (c) quantified the density of PARV-ir terminals in the VPM-VPL, and (d) studied the expression of GABAA receptor subunits in a separate group of rats using laser-dissection of the thalamus followed by Real-Time polymerase chain reaction (RT-PCR) array studies. At 6months post-TBI, only 64% of PARV-ir neurons were remaining in the RT ipsilaterally (p0.05). Also, the volume of the VPM-VPL was only 51% of that in controls ipsilaterally (p<0.001) and 91% contralaterally (p<0.05). The density of PARV-ir axonal labeling was remarkably increased in the lateral aspects of the VPM and VPL (both p<0.001). Expression of the ε- and θ-subunits of the GABAA receptor was down-regulated (0.152, p<0.01 and 0.302, p<0.05, respectively), which could relate

  15. βCCT, an antagonist selective for α(1)GABA(A) receptors, reverses diazepam withdrawal-induced anxiety in rats.

    Science.gov (United States)

    Divljaković, Jovana; Milić, Marija; Namjoshi, Ojas A; Tiruveedhula, Veera V; Timić, Tamara; Cook, James M; Savić, Miroslav M

    2013-02-01

    The abrupt discontinuation of prolonged benzodiazepine treatment elicits a withdrawal syndrome with increased anxiety as a major symptom. The neural mechanisms underlying benzodiazepine physical dependence are still insufficiently understood. Flumazenil, the non-selective antagonist of the benzodiazepine binding site of GABA(A) receptors was capable of preventing and reversing the increased anxiety during benzodiazepine withdrawal in animals and humans in some, but not all studies. On the other hand, a number of data suggest that GABA(A) receptors containing α(1) subunits are critically involved in processes developing during prolonged use of benzodiazepines, such are tolerance to sedative effects, liability to physical dependence and addiction. Hence, we investigated in the elevated plus maze the level of anxiety 24 h following 21 days of diazepam treatment and the influence of flumazenil or a preferential α(1)-subunit selective antagonist βCCt on diazepam withdrawal syndrome in rats. Abrupt cessation of protracted once-daily intraperitoneal administration of 2 mg/kg diazepam induced a withdrawal syndrome, measured by increased anxiety-like behavior in the elevated plus maze 24 h after treatment cessation. Acute challenge with either flumazenil (10mg/kg) or βCCt (1.25, 5 and 20 mg/kg) alleviated the diazepam withdrawal-induced anxiety. Moreover, both antagonists induced an anxiolytic-like response close, though not identical, to that seen with acute administration of diazepam. These findings imply that the mechanism by which antagonism at GABA(A) receptors may reverse the withdrawal-induced anxiety involves the α(1) subunit and prompt further studies aimed at linking the changes in behavior with possible adaptive changes in subunit expression and function of GABA(A) receptors.

  16. Differential effects of short-and long-term zolpidem treatment on recombinant α1β2γ2s subtype of GABAA receptors in vitro

    Institute of Scientific and Technical Information of China (English)

    Josipa VLAINI(C); Maja Jazvin(s)(c)ak JEMBREK; Toni VLAINI(C); Dubravka (S)vob (S)TRAC; Danka PERI(C)I(C)

    2012-01-01

    Aim: Zolpidem is a non-benzodiazepine agonist at benzodiazepine binding site in GABAA receptors,which is increasingly prescribed.Recent studies suggest that prolonged zolpidem treatment induces tolerance.The aim of this study was to explore the adaptive changes in GABAA receptors following short and long-term exposure to zolpidem in vitro.Methods: Human embryonic kidney (HEK) 293 cells stably expressing recombinant α1β2γ2s GABAA receptors were exposed to zolpidem (1 and 10 μmol/L) for short-term (2 h daily for 1,2,or 3 consecutive days) or long-term (continuously for 48 h).Radioligand binding studies were used to determine the parameters of [3H]flunitrazepam binding sites.Results: A single (2 h) or repeated (2 h daily for 2 or 3 d) short-term exposure to zolpidem affected neither the maximum number of [3H]flunitrazepam binding sites nor the affinity.In both control and short-term zolpidem treated groups,addition of GABA (1 nmol/L-1 mmol/L) enhanced [3H]flunitrazepam binding in a concentration-dependent manner.The maximum enhancement of [3H]flunitrazepam binding in short-term zolpidem treated group was not significantly different from that in the control group.In contrast,long-term exposure to zolpidem resulted in significantly increase in the maximum number of [3H]flunitrazepam binding sites without changing the affinity.Furthermore,long-term exposure to zolpidem significantly decreased the ability of GABA to stimulate [3H]flunitrazepam binding.Conclusion: The results suggest that continuous,but not intermittent and short-term,zolpidem-exposure is able to induce adaptive changes in GABAA receptors that could be related to the development of tolerance and dependence.

  17. Allosteric modulation by benzodiazepine receptor ligands of the GABAA receptor channel expressed in Xenopus oocytes.

    Science.gov (United States)

    Sigel, E; Baur, R

    1988-01-01

    Chick brain mRNA was isolated and injected into Xenopus oocytes. This led to the expression in the surface membrane of functional GABA-activated channels with properties reminiscent of vertebrate GABAA channels. The GABA-induced current was analyzed quantitatively under voltage-clamp conditions. Picrotoxin inhibited this current in a concentration-dependent manner with IC50 = 0.6 microM. The allosteric modulation of GABA currents by a number of drugs acting at the benzodiazepine binding site was characterized quantitatively. In the presence of the benzodiazepine receptor ligands diazepam and clorazepate, GABA responses were enhanced, and in the presence of the convulsant beta-carboline compound methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), they were depressed. Maximal stimulation of the response elicited by 10 microM GABA was 160% with diazepam and 90% with clorazepate, and maximal inhibition was 42% with DMCM, 30% with methyl beta-carboline-3-carboxylate (beta-CCM), 15% with ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a][1,4]benzodiazepine-3-carboxylate (Ro 15-1788), and 12% with ethyl beta-carboline-3-carboxylate (beta-CCE). Half-maximal stimulation was observed with 20 nM diazepam and 390 nM clorazepate, respectively, and half-maximal inhibition with 6 nM DMCM. beta-CCM had a similar effect to DMCM, whereas beta-CCE and Ro 15-1788 showed only small inhibition at low concentrations (less than 1 microM). All the tested carboline compounds and Ro 15-1788 showed a biphasic action and stimulated GABA current at concentrations higher than 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. GABAA receptor β3 subunit expression regulates tonic current in developing striatopallidal medium spiny neurons

    Directory of Open Access Journals (Sweden)

    Megan eJanssen

    2011-07-01

    Full Text Available The striatum is a key structure for movement control, but the mechanisms that dictate the output of distinct subpopulations of medium spiny projection neurons (MSNs, striatonigral projecting and dopamine D1 receptor- (D1+ or striatopallidal projecting and dopamine D2 receptor- (D2+ expressing neurons, remains poorly understood. GABA-mediated tonic inhibition largely controls neuronal excitability and action potential firing rates, and we previously suggested with pharmacological analysis that the GABAA receptor β3 subunit plays a large role in the basal tonic current seen in D2+ MSNs from young mice (Ade et al, 2008; Janssen et al, 2009. In this study, we demonstrated the essential role of the β3 GABAA receptor subunit in mediating MSN tonic currents using conditional β3 subunit knock-out (β3f/fDrd2 mice. Cre-lox genetics were used to generate conditional knock-out animals where Cre recombinase was expressed under the D2 receptor (Drd2 promoter. We show that while the wild-type MSN tonic current pattern demonstrates a high degree of variability, tonic current patterns from β3f/fDrd2 mice are narrow, suggesting that the β3 subunit is essential to striatal MSN GABA-mediated tonic current. Our data also suggest that a distinct population of synaptic receptors upregulate due to β3 subunit removal. Further, deletion of this subunit significantly decreases the D2+ MSN excitability. These results offer insight for target mechanisms in Parkinson’s disease, where symptoms arise due to the imbalance in striatal D1+ and D2+ MSN excitability and output.

  19. Involvement of basolateral amygdala GABAA receptors in the effect of dexamethasone on memory in rats

    Institute of Scientific and Technical Information of China (English)

    Lotfollah KHAJEHPOUR; Acieh ALIZADEH-MAKVANDI; Mahnaz KESMATI; Hooman ESHAGH-HAROONI

    2011-01-01

    In this study we investigated whether GABAA receptors of the basolateral amygdala (BLA) interact with the effect of dexamethasone on the retrieval stage of memory.Adult male Wistar rats were bilaterally cannulated in the BLA by stereotaxic surgery.The animals were trained in step-through apparatus by induction of electric shock (1.5 mA,3 s) and were tested for memory retrieval after 1 d.The time of latency for entering the dark compartment of the instrument and the time spent by rats in this chamber were recorded for evaluation of the animals' retrieval in passive avoidance memory.Administration of dexamethasone (0.3 and 0.9 mg/kg,subcutaneously (s.c.)),immediately after training,enhanced memory retrieval.This effect was reduced by intra-BLA microinjection of muscimol (0.125,0.250 and 0.500 μg/rat),when administered before 0.9 mg/kg of dexamethasone.Microinjection of bicuculline (0.75 μg/rat,intra-BLA) with an ineffective dose of dexamethasone (0.1 mg/kg,s.c.) increased memory retrieval.However,the same doses of muscimol and bicuculline without dexamethasone did not affect memory processes.Our data support reports that dexamethasone enhances memory retrieval.It seems that GABAA receptors of the BLA mediate the effect of dexamethasone on memory retrieval in rats.

  20. Local and global ligand-induced changes in the structure of the GABA(A) receptor.

    Science.gov (United States)

    Muroi, Yukiko; Czajkowski, Cynthia; Jackson, Meyer B

    2006-06-13

    Ligand-gated channels mediate synaptic transmission through conformational transitions triggered by the binding of neurotransmitters. These transitions are well-defined in terms of ion conductance, but their structural basis is poorly understood. To probe these changes in structure, GABA(A) receptors were expressed in Xenopus oocytes and labeled at selected sites with environment-sensitive fluorophores. With labels at two different residues in the alpha1 subunit in loop E of the GABA-binding pocket, GABA elicited fluorescence changes opposite in sign. This pattern of fluorescence changes is consistent with a closure of the GABA-binding cavity at the subunit interface. The competitive antagonist SR-95531 inverted this pattern of fluorescence change, but the noncompetitive antagonist picrotoxin failed to elicit optical signals. In response to GABA (but not SR-95531), labels at the homologous residues in the beta2 subunit showed the same pattern of fluorescence change as the alpha1-subunit labels, indicating a global transition with comparable movements in homologous regions of different subunits. Incorporation of the gamma2 subunit altered the fluorescence changes of alpha1-subunit labels and eliminated them in beta2-subunit labels. Thus, the ligand-induced structural changes in the GABA(A) receptor can extend over considerable distances or remain highly localized, depending upon subunit composition and ligand.

  1. Heterologous Expression of Rat Testis GABAA Receptor β3t Splicing Variant in CHO Cells

    Institute of Scientific and Technical Information of China (English)

    Shi-feng LI; Yu-guang CHEN; Yuan-chang YAN; Yi-ping LI

    2004-01-01

    Objective To characterize a possible retention function of unique sequence in the 5'end of rat testis GABAA receptor β3t splicing variantMethods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method. Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined.Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively. The chimera product failed to be translocated into the cell surface when expressed in CHO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane.Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.

  2. GABAA receptors containing ρ1 subunits contribute to in vivo effects of ethanol in mice.

    Directory of Open Access Journals (Sweden)

    Yuri A Blednov

    Full Text Available GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR, and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ1" antagonist, when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests, but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ2" antagonist did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.

  3. Regulation of cognition and symptoms of psychosis: focus on GABA(A) receptors and glycine transporter 1.

    Science.gov (United States)

    Möhler, Hanns; Rudolph, Uwe; Boison, Detlev; Singer, Philipp; Feldon, Joram; Yee, Benjamin K

    2008-07-01

    Adaptive purposeful behaviour depends on appropriate modifications of synaptic connectivity that incorporate an organism's past experience. At least some forms of such synaptic plasticity are believed to be mediated by NMDA receptors (NMDARs). Complementary interaction with inhibitory neurotransmission mediated by GABA(A) receptors, and upstream control of the excitability of NMDARs by glycine availability can greatly influence the efficacy of NMDAR mediated neuroplasticity, and thereby exert significant effects on cognition. Memory, selective attention or sensorimotor gating functions can be modified in mice with a reduction of alpha(5)GABA(A) receptors in the hippocampus or a selective deletion of glycine transporter 1 (GlyT1) in the forebrain. Both genetic manipulations altered the formation or persistence of associative links leading to distinct phenotypes on trace conditioning, extinction learning, latent inhibition, working memory, and object recognition. Behavioural assays of latent inhibition, prepulse inhibition, working memory, and sensitivity to psychostimulants in particular suggest that alpha(3) and alpha(5) subunit-containing GABA(A) receptors as well as GlyT1 are potential sites for ameliorating psychotic-like behaviour. Taken together, these results qualify distinct GABA-A receptor subtypes and GlyT1 as molecular targets for the development of a new pharmacology in the treatment of cognitive decline and psychotic symptoms.

  4. GABAA receptor subtypes in the mouse brain: Regional mapping and diazepam receptor occupancy by in vivo [(18)F]flumazenil PET.

    Science.gov (United States)

    Müller Herde, Adrienne; Benke, Dietmar; Ralvenius, William T; Mu, Linjing; Schibli, Roger; Zeilhofer, Hanns Ulrich; Krämer, Stefanie D

    2017-02-10

    Classical benzodiazepines, which are widely used as sedatives, anxiolytics and anticonvulsants, exert their therapeutic effects through interactions with heteropentameric GABAA receptors composed of two α, two β and one γ2 subunit. Their high affinity binding site is located at the interface between the γ2 and the adjacent α subunit. The α-subunit gene family consists of six members and receptors can be homomeric or mixed with respect to the α-subunits. Previous work has suggested that benzodiazepine binding site ligands with selectivity for individual GABAA receptor subtypes, as defined by the benzodiazepine-binding α subunit, may have fewer side effects and may even be effective in diseases, such as schizophrenia, autism or chronic pain, that do not respond well to classical benzodiazepines. The distributions of the individual α subunits across the CNS have been extensively characterized. However, as GABAA receptors may contain two different α subunits, the distribution of the subunits does not necessarily reflect the distribution of receptor subtypes with respect to benzodiazepine pharmacology. In the present study, we have used in vivo [(18)F]flumazenil PET and in vitro [(3)H]flumazenil autoradiography in combination with GABAA receptor point-mutated mice to characterize the distribution of the two most prevalent GABAA receptor subtypes (α1 and α2) throughout the mouse brain. The results were in agreement with published in vitro data. High levels of α2-containing receptors were found in brain regions of the neuronal network of anxiety. The α1/α2 subunit combinations were predictable from the individual subunit levels. In additional experiments, we explored in vivo [(18)F]flumazenil PET to determine the degree of receptor occupancy at GABAA receptor subtypes following oral administration of diazepam. The dose to occupy 50% of sensitive receptors, independent of the receptor subtype(s), was 1-2mg/kg, in agreement with published data from ex vivo

  5. The voltage dependence of GABAA receptor gating depends on extracellular pH.

    Science.gov (United States)

    Pytel, Maria; Mercik, Katarzyna; Mozrzymas, Jerzy W

    2005-11-28

    Recent studies have indicated that changes in extracellular pH and in membrane voltage affect the gamma-amino-n-butyric acid type A receptor gating mainly by altering desensitization and binding. To test whether the effects of membrane potential and pH are additive, their combined actions were investigated. By analyzing the current responses to rapid gamma-amino-n-butyric acid applications, we found that the current to voltage relationship was close to linear at acid pH but the increasing pH induced an inward rectification. Desensitization was enhanced at depolarizing potentials, but this strongly depended on pH, being weak at acidic and strong at basic pH values. A similar trend was observed for the onset rate of responses to saturating gamma-amino-n-butyric acid concentration. These data provide evidence that the voltage sensitivity of GABAA receptors depends on extracellular pH.

  6. The voltage dependence of GABAA receptor gating depends on extracellular pH

    Science.gov (United States)

    Pytel, Maria; Mercik, Katarzyna; Mozrzymas, Jerzy W.

    2007-01-01

    Recent studies have indicated that changes in extracellular pH and in membrane voltage affect the γ-amino-n-butyric acid type A receptor gating mainly by altering desensitization and binding. To test whether the effects of membrane potential and pH are additive, their combined actions were investigated. By analyzing the current responses to rapid γ-amino-n-butyric acid applications, we found that the current to voltage relationship was close to linear at acid pH but the increasing pH induced an inward rectification. Desensitization was enhanced at depolarizing potentials, but this strongly depended on pH, being weak at acidic and strong at basic pH values. A similar trend was observed for the onset rate of responses to saturating γ-amino-n-butyric acid concentration. These data provide evidence that the voltage sensitivity of GABAA receptors depends on extracellular pH. PMID:16272885

  7. The ataxia (axJ mutation causes abnormal GABAA receptor turnover in mice.

    Directory of Open Access Journals (Sweden)

    Corinna Lappe-Siefke

    2009-09-01

    Full Text Available Ataxia represents a pathological coordination failure that often involves functional disturbances in cerebellar circuits. Purkinje cells (PCs characterize the only output neurons of the cerebellar cortex and critically participate in regulating motor coordination. Although different genetic mutations are known that cause ataxia, little is known about the underlying cellular mechanisms. Here we show that a mutated ax(J gene locus, encoding the ubiquitin-specific protease 14 (Usp14, negatively influences synaptic receptor turnover. Ax(J mouse mutants, characterized by cerebellar ataxia, display both increased GABA(A receptor (GABA(AR levels at PC surface membranes accompanied by enlarged IPSCs. Accordingly, we identify physical interaction of Usp14 and the GABA(AR alpha1 subunit. Although other currently unknown changes might be involved, our data show that ubiquitin-dependent GABA(AR turnover at cerebellar synapses contributes to ax(J-mediated behavioural impairment.

  8. Sex-dependent anti-stress effect of an α5 subunit containing GABAA receptor positive allosteric modulator

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    Sean C. Piantadosi

    2016-11-01

    Full Text Available Rationale: Current first-line treatments for stress-related disorders such as Major Depressive Disorder (MDD act on monoaminergic systems and take weeks to achieve a therapeutic effect with poor response and low remission rates. Recent research has implicated the GABAergic system in the pathophysiology of depression, including deficits in interneurons targeting the dendritic compartment of cortical pyramidal cells. Objectives: The present study evaluates whether SH-053-2'F-R-CH3 (denoted α5-PAM, a positive allosteric modulator selective for α5-subunit containing GABAA receptors found predominantly on cortical pyramidal cell dendrites has anti-stress effects. Methods: Female and male C57BL6/J mice were exposed to unpredictable chronic mild stress (UCMS and treated with α5-PAM acutely (30 minutes prior to assessing behavior or chronically before being assessed behaviorally. Results: Acute and chronic α5-PAM treatments produce a pattern of decreased stress-induced behaviors (denoted as behavioral emotionality across various tests in female, but not in male mice. Behavioral Z-scores calculated across a panel of tests designed to best model the range and heterogeneity of human symptomatology confirmed that acute and chronic α5-PAM treatments consistently produce significant decreases in behavioral emotionality in several independent cohorts of females. The behavioral responses to α5-PAM could not be completely accounted for by differences in drug brain disposition between female and male mice. In mice exposed to UCMS, expression of the Gabra5 gene was increased in the frontal cortex after acute treatment and in hippocampus after chronic treatment with α5-PAM in females only, and these expression changes correlated with behavioral emotionality. Conclusions: We showed that acute and chronic positive modulation of α5 subunit-containing GABAA receptors elicit anti-stress effects in a sex-dependent manner, suggesting novel therapeutic modalities.

  9. The novel anxiolytic ELB139 displays selectivity to recombinant GABA(A) receptors different from diazepam.

    Science.gov (United States)

    Rabe, Holger; Kronbach, Christiane; Rundfeldt, Chris; Lüddens, Hartmut

    2007-03-01

    A chemically heterogeneous group of compounds acts at the benzodiazepine (BZ) recognition site of the diverse gamma-aminobutyric acid type A (GABA(A)) receptor complexes which can assemble from more than 16 known subunits. Most 1,4-BZs like diazepam recognize all GABA(A)/BZ receptors containing the alpha1-3 or alpha5 together with any beta and the gamma2 subunit. Other compounds differentiate less, e.g. Ro15-4513, that additionally recognizes alpha4- and a6-containing receptors, or differentiate more, e.g. zolpidem, that recognizes preferentially alpha1-containing receptors. Here we describe the functional properties of 1-(4-chloro-phenyl)-4-piperidin-1-yl-1,5-dihydro-imidazol-2-on (ELB139) in the presence and absence of the BZ receptor antagonist flumazenil (Ro15-1788) on recombinant alphaibeta2gamma2 (i=1-5) receptor subtypes expressed in HEK 293 cells. The properties were measured with the whole-cell variation of the patch-clamp technique and compared to those of diazepam. Like the latter, ELB139 did not potentiate GABA-induced currents in alpha4-containing receptors, but it displays functional subtype specificity between alpha1, alpha2, alpha3, and alpha5beta2gamma2 receptors with highest potency in alpha3-containing receptors but highest efficacy in alpha1- or alpha2-containing receptors, respectively. ELB139 acted as a partial agonist on these receptor subtypes reaching 40-50% of the efficacy of diazepam.

  10. The neurosteroid dehydroepiandrosterone (DHEA) and its metabolites alter 5-HT neuronal activity via modulation of GABAA receptors.

    Science.gov (United States)

    Gartside, S E; Griffith, N C; Kaura, V; Ingram, C D

    2010-11-01

    Dehydroepiandrosterone (DHEA) and its metabolites, DHEA-sulphate (DHEA-S) and androsterone, have neurosteroid activity. In this study, we examined whether DHEA, DHEA-S and androsterone, can influence serotonin (5-HT) neuronal firing activity via modulation of γ-aminobutryic acid (GABA(A)) receptors. The firing of presumed 5-HT neurones in a slice preparation containing rat dorsal raphe nucleus was inhibited by the GABA(A) receptor agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridinyl-3-ol (THIP) (25 μM) and GABA (100 μM). DHEA (100 and 300 μM) and DHEA-S (1, 10 and 100 μM) caused a rapid and reversible attenuation of the response to THIP. DHEA (100 μM) and DHEA-S (100 μM) also attenuated the effect of GABA. Androsterone (10 and 30 μM) markedly enhanced the inhibitory response to THIP (25 μM). The effect was apparent during androsterone administration but persisted and even increased in magnitude after drug wash-out. The data indicate that GABA(A) receptor-mediated regulation of 5-HT neuronal firing is sensitive to negative modulation by DHEA and its metabolite DHEA-S is sensitive to positive modulation by the metabolite androsterone. The effects of these neurosteroids on GABA(A) receptor-mediated regulation of 5-HT firing may underlie some of the reported behavioural and psychological effects of endogenous and exogenous DHEA.

  11. Flavonoid Myricetin Modulates GABAA Receptor Activity through Activation of Ca2+ Channels and CaMK-II Pathway

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    Xiao Hu Zhang

    2012-01-01

    Full Text Available The flavonoid myricetin is found in several sedative herbs, for example, the St. John's Wort, but its influence on sedation and its possible mechanism of action are unknown. Using patch-clamp technique on a brain slice preparation, the present study found that myricetin promoted GABAergic activity in the neurons of hypothalamic paraventricular nucleus (PVN by increasing the decay time and frequency of the inhibitory currents mediated by GABAA receptor. This effect of myricetin was not blocked by the GABAA receptor benzodiazepine- (BZ- binding site antagonist flumazenil, but by KN-62, a specific inhibitor of the Ca2+/calmodulin-stimulated protein kinase II (CaMK-II. Patch clamp and live Ca2+ imaging studies found that myricetin could increase Ca2+ current and intracellular Ca2+ concentration, respectively, via T- and L-type Ca2+ channels in rat PVN neurons and hypothalamic primary culture neurons. Immunofluorescence staining showed increased phosphorylation of CaMK-II after myricetin incubation in primary culture of rat hypothalamic neurons, and the myricetin-induced CaMK-II phosphorylation was further confirmed by Western blotting in PC-12 cells. The present results suggest that myricetin enhances GABAA receptor activity via calcium channel/CaMK-II dependent mechanism, which is distinctively different from that of most existing BZ-binding site agonists of GABAA receptor.

  12. Triton X-100 inhibits agonist-induced currents and suppresses benzodiazepine modulation of GABA(A) receptors in Xenopus oocytes

    DEFF Research Database (Denmark)

    Søgaard, Rikke; Ebert, Bjarke; Klaerke, Dan

    2009-01-01

    Changes in lipid bilayer elastic properties have been proposed to underlie the modulation of voltage-gated Na(+) and L-type Ca(2+) channels and GABA(A) receptors by amphiphiles. The amphiphile Triton X-100 increases the elasticity of lipid bilayers at micromolar concentrations, assessed from its ...

  13. GABA(A) receptors in visual and auditory cortex and neural activity changes during basic visual stimulation.

    Science.gov (United States)

    Qin, Pengmin; Duncan, Niall W; Wiebking, Christine; Gravel, Paul; Lyttelton, Oliver; Hayes, Dave J; Verhaeghe, Jeroen; Kostikov, Alexey; Schirrmacher, Ralf; Reader, Andrew J; Northoff, Georg

    2012-01-01

    Recent imaging studies have demonstrated that levels of resting γ-aminobutyric acid (GABA) in the visual cortex predict the degree of stimulus-induced activity in the same region. These studies have used the presentation of discrete visual stimulus; the change from closed eyes to open also represents a simple visual stimulus, however, and has been shown to induce changes in local brain activity and in functional connectivity between regions. We thus aimed to investigate the role of the GABA system, specifically GABA(A) receptors, in the changes in brain activity between the eyes closed (EC) and eyes open (EO) state in order to provide detail at the receptor level to complement previous studies of GABA concentrations. We conducted an fMRI study involving two different modes of the change from EC to EO: an EO and EC block design, allowing the modeling of the haemodynamic response, followed by longer periods of EC and EO to allow the measuring of functional connectivity. The same subjects also underwent [(18)F]Flumazenil PET to measure GABA(A) receptor binding potentials. It was demonstrated that the local-to-global ratio of GABA(A) receptor binding potential in the visual cortex predicted the degree of changes in neural activity from EC to EO. This same relationship was also shown in the auditory cortex. Furthermore, the local-to-global ratio of GABA(A) receptor binding potential in the visual cortex also predicted the change in functional connectivity between the visual and auditory cortex from EC to EO. These findings contribute to our understanding of the role of GABA(A) receptors in stimulus-induced neural activity in local regions and in inter-regional functional connectivity.

  14. Ethanol Regulation of Synaptic GABAA α4 Receptors Is Prevented by Protein Kinase A Activation.

    Science.gov (United States)

    Carlson, Stephen L; Bohnsack, John Peyton; Morrow, A Leslie

    2016-04-01

    Ethanol alters GABAA receptor trafficking and function through activation of protein kinases, and these changes may underlie ethanol dependence and withdrawal. In this study, we used subsynaptic fraction techniques and patch-clamp electrophysiology to investigate the biochemical and functional effects of protein kinase A (PKA) and protein kinase C (PKC) activation by ethanol on synaptic GABAA α4 receptors, a key target of ethanol-induced changes. Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or kinase modulators for 4 hours, a paradigm that recapitulates GABAergic changes found after chronic ethanol exposure in vivo. PKA activation by forskolin or rolipram during ethanol exposure prevented increases in P2 fraction α4 subunit abundance, whereas inhibiting PKA had no effect. Similarly, in the synaptic fraction, activation of PKA by rolipram in the presence of ethanol prevented the increase in synaptic α4 subunit abundance, whereas inhibiting PKA in the presence of ethanol was ineffective. Conversely, PKC inhibition in the presence of ethanol prevented the ethanol-induced increases in synaptic α4 subunit abundance. Finally, we found that either activating PKA or inhibiting PKC in the presence of ethanol prevented the ethanol-induced decrease in GABA miniature inhibitory postsynaptic current decay τ1, whereas inhibiting PKA had no effect. We conclude that PKA and PKC have opposing effects in the regulation of synaptic α4 receptors, with PKA activation negatively modulating, and PKC activation positively modulating, synaptic α4 subunit abundance and function. These results suggest potential targets for restoring normal GABAergic functioning in the treatment of alcohol use disorders.

  15. Modulation of the input-output function by GABAA receptor-mediated currents in rat oculomotor nucleus motoneurons.

    Science.gov (United States)

    Torres-Torrelo, Julio; Torres, Blas; Carrascal, Livia

    2014-11-15

    The neuronal input-output function depends on recruitment threshold and gain of the firing frequency-current (f-I) relationship. These two parameters are positively correlated in ocular motoneurons (MNs) recorded in alert preparation and inhibitory inputs could contribute to this correlation. Phasic inhibition mediated by γ-amino butyric acid (GABA) occurs when a high concentration of GABA at the synaptic cleft activates postsynaptic GABAA receptors, allowing neuronal information transfer. In some neuronal populations, low concentrations of GABA activate non-synaptic GABAA receptors and generate a tonic inhibition, which modulates cell excitability. This study determined how ambient GABA concentrations modulate the input-output relationship of rat oculomotor nucleus MNs. Superfusion of brain slices with GABA (100 μm) produced a GABAA receptor-mediated current that reduced the input resistance, increased the recruitment threshold and shifted the f-I relationship rightward without any change in gain. These modifications did not depend on MN size. In absence of exogenous GABA, gabazine (20 μm; antagonist of GABAA receptors) abolished spontaneous inhibitory postsynaptic currents and revealed a tonic current in MNs. Gabazine increased input resistance and decreased recruitment threshold mainly in larger MNs. The f-I relationship shifted to the left, without any change in gain. Gabazine effects were chiefly due to MN tonic inhibition because tonic current amplitude was five-fold greater than phasic. This study demonstrates a tonic inhibition in ocular MNs that modulates cell excitability depending on cell size. We suggest that GABAA tonic inhibition acting concurrently with glutamate receptors activation could reproduce the positive covariation between threshold and gain reported in alert preparation.

  16. Neonatal domoic acid increases receptor density of α2 adrenoceptors and GABAA α5 receptors in limbic brain regions of adult rats

    DEFF Research Database (Denmark)

    Thomsen, Majken; Lillethorup, Thea Pinholt; Wegener, Gregers

    autoradiography was performed using tracers of the α5 subtype of the GABAA receptor ([11C]Ro15-4513) and the α2 adrenoceptors ([3H]RX821002) to determine the total binding of these receptors in the hippocampus, amygdala and hypothalamus. High concentrations of unlabeled Ro15-4513 and phentolamine were used...

  17. Increased receptor density of α2 adrenoceptors and GABAA α5 receptors in limbic brain regions in the domoic acid rat model of epilepsy

    DEFF Research Database (Denmark)

    Thomsen, Majken; Lillethorup, Thea Pinholt; Wegener, Gregers

    , fresh frozen and cut into 20 µM thick slices. Autoradiography was performed using tracers of the α5 subtype of the GABAA receptor ([11C]Ro15-4513) and the α2 adrenoceptors ([3H]RX821002) to determine the binding in limbic brain regions. Results: The binding of postsynaptic GABA receptors...

  18. Activation of Glycine and Extrasynaptic GABAA Receptors by Taurine on the Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis

    Science.gov (United States)

    Bhattarai, Janardhan Prasad; Park, Soo Joung; Han, Seong Kyu

    2013-01-01

    The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been known for the processing and transmission of orofacial nociceptive information. Taurine, one of the most plentiful free amino-acids in humans, has proved to be involved in pain modulation. In this study, using whole-cell patch clamp technique, we investigated the direct membrane effects of taurine and the action mechanism behind taurine-mediated responses on the SG neurons of the Vc. Taurine showed non-desensitizing and repeatable membrane depolarizations and inward currents which remained in the presence of amino-acid receptors blocking cocktail (AARBC) with tetrodotoxin, indicating that taurine acts directly on the postsynaptic SG neurons. Further, application of taurine at different doses (10 μM to 3 mM) showed a concentration dependent depolarizations and inward currents with the EC50 of 84.3 μM and 723 μM, respectively. Taurine-mediated responses were partially blocked by picrotoxin (50 μM) and almost completely blocked by strychnine (2 μM), suggesting that taurine-mediated responses are via glycine receptor (GlyR) activation. In addition, taurine (1 mM) activated extrasynaptic GABAA receptor (GABAAR)-mediated currents. Taken together, our results indicate that taurine can be a target molecule for orofacial pain modulation through the activation of GlyRs and/or extrasynaptic GABAARs on the SG neurons. PMID:24379976

  19. Activation of Glycine and Extrasynaptic GABAA Receptors by Taurine on the Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis

    Directory of Open Access Journals (Sweden)

    Thi Thanh Hoang Nguyen

    2013-01-01

    Full Text Available The substantia gelatinosa (SG of the trigeminal subnucleus caudalis (Vc has been known for the processing and transmission of orofacial nociceptive information. Taurine, one of the most plentiful free amino-acids in humans, has proved to be involved in pain modulation. In this study, using whole-cell patch clamp technique, we investigated the direct membrane effects of taurine and the action mechanism behind taurine-mediated responses on the SG neurons of the Vc. Taurine showed non-desensitizing and repeatable membrane depolarizations and inward currents which remained in the presence of amino-acid receptors blocking cocktail (AARBC with tetrodotoxin, indicating that taurine acts directly on the postsynaptic SG neurons. Further, application of taurine at different doses (10 μM to 3 mM showed a concentration dependent depolarizations and inward currents with the EC50 of 84.3 μM and 723 μM, respectively. Taurine-mediated responses were partially blocked by picrotoxin (50 μM and almost completely blocked by strychnine (2 μM, suggesting that taurine-mediated responses are via glycine receptor (GlyR activation. In addition, taurine (1 mM activated extrasynaptic GABAA receptor (GABAAR-mediated currents. Taken together, our results indicate that taurine can be a target molecule for orofacial pain modulation through the activation of GlyRs and/or extrasynaptic GABAARs on the SG neurons.

  20. Receptor subtype-dependent positive and negative modulation of GABA(A) receptor function by niflumic acid, a nonsteroidal anti-inflammatory drug.

    Science.gov (United States)

    Sinkkonen, Saku T; Mansikkamäki, Salla; Möykkynen, Tommi; Lüddens, Hartmut; Uusi-Oukari, Mikko; Korpi, Esa R

    2003-09-01

    In addition to blocking cyclooxygenases, members of the fenamate group of nonsteroidal anti-inflammatory drugs have been proposed to affect brain GABAA receptors. Using quantitative autoradiography with GABAA receptor-associated ionophore ligand [35S]t-butylbicyclophosphorothionate (TBPS) on rat brain sections, one of the fenamates, niflumate, at micromolar concentration was found to potentiate GABA actions in most brain areas, whereas being in the cerebellar granule cell layer an efficient antagonist similar to furosemide. With recombinant GABAA receptors expressed in Xenopus laevis oocytes, we found that niflumate potentiated 3 microM GABA responses up to 160% and shifted the GABA concentration-response curve to the left in alpha1beta2gamma2 receptors, the predominant GABAA receptor subtype in the brain. This effect needed the gamma2 subunit, because on alpha1beta2 receptors, niflumate exhibited solely an antagonistic effect at high concentrations. The potentiation was not abolished by the specific benzodiazepine site antagonist flumazenil. Niflumate acted as a potent antagonist of alpha6beta2 receptors (with or without gamma2 subunit) and of alphaXbeta2gamma2 receptors containing a chimeric alpha1 to alpha6 subunit, which suggests that niflumate antagonism is dependent on the same transmembrane domain 1- and 2-including fragment of the alpha6 subunit as furosemide antagonism. This antagonism was noncompetitive because the maximal GABA response, but not the potency, was reduced by niflumate. These data show receptor subtype-dependent positive and negative modulatory actions of niflumate on GABAA receptors at clinically relevant concentrations, and they suggest the existence of a novel positive modulatory site on alpha1beta2gamma2 receptors that is dependent on the gamma2 subunit but not associated with the benzodiazepine binding site.

  1. Subregion-specific modulation of excitatory input and dopaminergic output in the striatum by tonically activated glycine and GABAA receptors

    Directory of Open Access Journals (Sweden)

    Louise eAdermark

    2011-10-01

    Full Text Available The flow of cortical information through the basal ganglia is a complex spatiotemporal pattern of increased and decreased firing. The striatum is the biggest input nucleus to the basal ganglia and the aim of this study was to assess the role of inhibitory GABAA and glycine receptors in regulating synaptic activity in the dorsolateral (DLS and ventral striatum (nucleus accumbens, nAc. Local field potential recordings from coronal brain slices of juvenile and adult Wistar rats showed that GABAA receptors and strychnine-sensitive glycine receptors are tonically activated and inhibit excitatory input to the DLS and to the nAc. Strychnine-induced disinhibition of glutamatergic transmission was insensitive to the muscarinic receptor inhibitor scopolamine (10 µM, inhibited by the nicotinic acetylcholine receptor antagonist mecamylamine (10 µM and blocked by GABAA receptor inhibitors, suggesting that tonically activated glycine receptors depress excitatory input to the striatum through modulation of cholinergic and GABAergic neurotransmission. As an end-product example of striatal GABAergic output in vivo we measured dopamine release in the DLS and nAc by microdialysis in the awake and freely moving rat. Reversed dialysis of bicuculline (50 μM in perfusate only increased extrasynaptic dopamine levels in the nAc, while strychnine administered locally (200 μM in perfusate decreased dopamine output by 60% in both the DLS and nAc. Our data suggest that GABAA and glycine receptors are tonically activated and modulate striatal transmission in a partially sub-region specific manner.

  2. Membrane voltage modulates the GABA(A) receptor gating in cultured rat hippocampal neurons.

    Science.gov (United States)

    Pytel, Maria; Mercik, Katarzyna; Mozrzymas, Jerzy W

    2006-02-01

    The kinetics of GABAergic currents in neurons is known to be modulated by the membrane voltage but the underlying mechanisms have not been fully explored. In particular, the impact of membrane potential on the GABA(A) receptor gating has not been elucidated. In the present study, the effect of membrane voltage on current responses elicited by ultrafast GABA applications was studied in cultured hippocampal neurons. The current to voltage relationship (I-V) for responses to saturating [GABA] (10 mM) showed an inward rectification (slope conductance at positive voltages was 0.62 +/- 0.05 of that at negative potentials). On the contrary, I-V for currents evoked by low [GABA] (1 microM) showed an outward rectification. The onset of currents elicited by saturating [GABA] was significantly accelerated at positive potentials. Analysis of currents evoked by prolonged applications of saturating [GABA] revealed that positive voltages significantly increased the rate and extent of desensitization. The onsets of current responses to non-saturating [GABA] were significantly accelerated at positive voltages indicating an enhancement of the binding rate. However, at low [GABA] at which the onset rate is expected to approach an asymptote set by opening/closing and unbinding rates, no significant modification of current onset by voltage was observed. Quantitative analysis based on model simulations indicated that the major effect of membrane depolarization was to increase the rates of binding, desensitization and of opening as well as to slightly reduce the rate of exit from desensitization. In conclusion, we provide evidence that membrane voltage affects the GABA(A) receptor microscopic gating.

  3. Effect of a GABA agonist on the expression and distribution of GABAA receptors in the plasma membrane of cultured cerebellar granule cells: an immunocytochemical study

    DEFF Research Database (Denmark)

    Hansen, G H; Belhage, B; Schousboe, A

    1991-01-01

    The effect of the gamma-aminobutyric acid (GABA) agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 microM) on the localization and density of GABAA receptors in the plasma membrane of rat cerebellar granule cells in primary cultures was studied at the electron microscope (EM) level...... by preembedding immunogold staining using the monoclonal antibody bd-17 directed against the beta-subunit of the GABAA receptor complex. In THIP-treated as well as untreated control cultures, GABAA receptors were found to be evenly distributed in the plasma membrane of cell bodies as well as processes. However...... at the EM level using the preembedding immunogold technique. It is likely that low-affinity GABAA receptors are preferentially located in the cell processes and to a considerable extent in the form of 'hot spots'. However, these 'hot spots' also contain high-affinity receptors....

  4. The role of NMDA and GABAA receptors in the inhibiting effect of 3 MPa nitrogen on striatal dopamine level.

    Science.gov (United States)

    Lavoute, Cécile; Weiss, Michel; Rostain, Jean-Claude

    2007-10-24

    Nitrogen pressure exposure, in rats, resulted in a decreased dopamine (DA) level by the striatal terminals of the substantia nigra pars compacta (SNc) dopaminergic neurons, due to the narcotic potency of nitrogen. In the SNc, the nigrostriatal pathway is under glutamatergic and GABAergic control mediated by ion-channel NMDA and GABA(A) receptors, main targets of volatile anesthetics. The aim of this study was to investigate the role of these receptors in the regulation of striatal dopamine level under nitrogen narcosis. Under general anesthesia, male Sprague-Dawley rats were bilaterally implanted in the striatum with dopamine-sensitive electrodes and, in the SNc, with guide cannulae for drug injections. After recovery from surgery, the striatal dopamine level was quantified using differential pulse voltammetric measurements in freely moving rats. Focal injections of agonists (NMDA/muscimol) and antagonists (AP7/gabazine) of NMDA/GABA(A) receptors were made within SNc. Both normobaric condition and 3 MPa nitrogen pressure were studied. Control experiments confirmed a direct glutamatergic control on the striatal DA level through NMDA receptors. Both direct and indirect GABAergic control through two different types of GABA(A) receptors located on GABAergic interneurons and on DA cells were indicated. Under nitrogen pressure, the decrease in dopamine level (20%) was suppressed by both NMDA and GABA(A) agonist infusion. There was an unexpected increasing DA level, induced by AP7 (about 10%) and gabazine (about 30%). These results indicate that NMDA receptors remain functional and suggest a decreased glutamate release. The findings also describe an increase of GABA(A) receptor-mediated inhibition on DA cells under nitrogen pressure exposure.

  5. Effects of central histamine receptors blockade on GABA(A) agonist-induced food intake in broiler cockerels.

    Science.gov (United States)

    Morteza, Zendehdel; Vahhab, Babapour; Hossein, Jonaidi

    2008-02-01

    In this study, the effect of intracerebroventricular (i.c.v) injection of H1, H2 and H3 antagonists on feed intake induced by GABA(A) agonist was evaluated. In Experiment 1, the animals received chloropheniramine, a H1 antagonist and then muscimol, a GABA(A) agonist. In Experiment 2, chickens received famotidine, a H2 receptor antagonist, prior to injection of muscimol. Finally in Experiment 3, the birds were injected with thioperamide, a H3 receptor antagonist and muscimol. Cumulative food intake was measured 15, 30, 45, 60, 90, 120, 150 and 180 min after injections. The results of this study indicated that effects of muscimol on food intake inhibited by pretreatment with chloropheneramine maleate (p histamine-GABA(A) receptor interaction on food intake in broiler cockerels.

  6. α4βδ GABA(A) receptors are high-affinity targets for γ-hydroxybutyric acid (GHB).

    Science.gov (United States)

    Absalom, Nathan; Eghorn, Laura F; Villumsen, Inge S; Karim, Nasiara; Bay, Tina; Olsen, Jesper V; Knudsen, Gitte M; Bräuner-Osborne, Hans; Frølund, Bente; Clausen, Rasmus P; Chebib, Mary; Wellendorph, Petrine

    2012-08-14

    γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA(A) receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA(A) receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC(50) = 140 nM) over α4β(2/3)δ (EC(50) = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [(3)H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA(A) receptors and postulate a role for extrasynaptic α4δ-containing GABA(A) receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.

  7. Generation of functional inhibitory synapses incorporating defined combinations of GABA(A or glycine receptor subunits

    Directory of Open Access Journals (Sweden)

    Christine Laura Dixon

    2015-12-01

    Full Text Available Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR and glycine receptor (GlyR isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of inhibitory postsynaptic currents mediated by individual isoforms in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2 – 5 weeks.

  8. Evaluation of native GABA(A) receptors containing an alpha 5 subunit.

    Science.gov (United States)

    Li, M; Szabo, A; Rosenberg, H C

    2001-02-09

    The type A receptor for gamma-aminobutyric acid (GABA), or GABA(A) receptor, is a pentamer of highly variable quaternary structure. It includes two alpha subunits, drawn from a pool of six genes, which largely determine benzodiazepine pharmacology of the receptor. In brain sections, both [(3)H]RY-80 (ethyl-8-acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylate) and [(3)H]L-655,708 (ethyl (S)-11,12,13,13a-tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate), which are selective for the benzodiazepine site of alpha 5 subunit-containing receptors, showed high-affinity, specific binding, but to fewer regions than did the nonselective benzodiazepine, [(3)H]flunitrazepam. The pattern mirrored alpha 5 mRNA distribution, and was similar to that previously reported for [(3)H]L-655,708 binding. Displacement of [(3)H]RY-80 bound to hippocampal homogenates, and of [(3)H]flunitrazepam bound to cerebellar and hippocampal homogenates showed comparable displacement by flumazenil (K(i)'s 5--7 nM). However, the K(i)'s for diazepam and for clobazam to displace [(3)H]RY-80 binding in hippocampus were about fourfold higher than for [(3)H]flunitrazepam, and the K(i) for clonazepam was sixfold larger, suggesting that these benzodiazepine receptor agonists bind with relatively lower affinity at hippocampal alpha 5-containing receptors.

  9. Association between Alcoholism and the Genetic Polymorphisms of the GABAA Receptor Genes on Chromosome 5q33-34 in Korean Population

    OpenAIRE

    Park, Chul-Soo; Park, So-Young; Lee, Chul-Soon; Sohn, Jin-Wook; Hahn, Gyu-Hee; Kim, Bong-Jo

    2006-01-01

    Family, twin, and adoption studies have demonstrated that genes play an important role in the development of alcoholism. We investigated the association between alcoholism and the genetic polymorphisms of the GABAA receptor genes on chromosome 5q33-34 in Korean population. The genotype of the GABAA receptor gene polymorphisms were determined by performing polymerase chain reaction genotyping for 172 normal controls and 162 male alcoholics who are hospitalized in alcoholism treatment institute...

  10. An altered GABA-A receptor function in spinocerebellar ataxia type 6 and familial hemiplegic migraine type 1 associated with the CACNA1A gene mutation

    Directory of Open Access Journals (Sweden)

    Satoshi Kono

    2014-12-01

    General significance: An altered GABA-A receptor function has previously been reported in models of inherited murine cerebellar ataxia caused by a mutation in the CACNA1A gene. This study showed novel clinical characteristics of alteration in the GABA-A receptor in vivo, which may provide clinical evidence indicating a pathological mechanism common to neurological disorders associated with CACNA1A gene mutation.

  11. Increased Motor-Impairing Effects of the Neuroactive Steroid Pregnanolone in Mice with Targeted Inactivation of the GABAA Receptor γ2 Subunit in the Cerebellum

    Science.gov (United States)

    Leppä, Elli; Linden, Anni-Maija; Aller, Maria I.; Wulff, Peer; Vekovischeva, Olga; Luscher, Bernhard; Lüddens, Hartmut; Wisden, William; Korpi, Esa R.

    2016-01-01

    Endogenous neurosteroids and neuroactive steroids have potent and widespread actions on the brain via inhibitory GABAA receptors. In recombinant receptors and genetic mouse models their actions depend on the α, β, and δ subunits of the receptor, especially on those that form extrasynaptic GABAA receptors responsible for non-synaptic (tonic) inhibition, but they also act on synaptically enriched γ2 subunit-containing receptors and even on αβ binary receptors. Here we tested whether behavioral sensitivity to the neuroactive steroid agonist 5β-pregnan-3α-ol-20-one is altered in genetically engineered mouse models that have deficient GABAA receptor-mediated synaptic inhibition in selected neuronal populations. Mouse lines with the GABAA receptor γ2 subunit gene selectively deleted either in parvalbumin-containing cells (including cerebellar Purkinje cells), cerebellar granule cells, or just in cerebellar Purkinje cells were trained on the accelerated rotating rod and then tested for motor impairment after cumulative intraperitoneal dosing of 5β-pregnan-3α-ol-20-one. Motor-impairing effects of 5β-pregnan-3α-ol-20-one were strongly increased in all three mouse models in which γ2 subunit-dependent synaptic GABAA responses in cerebellar neurons were genetically abolished. Furthermore, rescue of postsynaptic GABAA receptors in Purkinje cells normalized the effect of the steroid. Anxiolytic/explorative effects of the steroid in elevated plus maze and light:dark exploration tests in mice with Purkinje cell γ2 subunit inactivation were similar to those in control mice. The results suggest that, when the deletion of γ2 subunit has removed synaptic GABAA receptors from the specific cerebellar neuronal populations, the effects of neuroactive steroids solely on extrasynaptic αβ or αβδ receptors lead to enhanced changes in the cerebellum-generated behavior. PMID:27833556

  12. GABA(A) receptor physiology and its relationship to the mechanism of action of the 1,5-benzodiazepine clobazam.

    Science.gov (United States)

    Sankar, Raman

    2012-03-01

    Clobazam was initially developed in the early 1970s as a nonsedative anxiolytic agent, and is currently available as adjunctive therapy for epilepsy and anxiety disorders in more than 100 countries. In October 2011, clobazam (Onfi™; Lundbeck Inc., Deerfield, IL, USA) was approved by the US FDA for use as adjunctive therapy for the treatment of seizures associated with Lennox-Gastaut syndrome in patients aged 2 years and older. It is a long-acting 1,5-benzodiazepine whose structure distinguishes it from the classic 1,4-benzodiazepines, such as diazepam, lorazepam and clonazepam. Clobazam is well absorbed, with peak concentrations occurring linearly 1-4 hours after administration. Both clobazam and its active metabolite, N-desmethylclobazam, are metabolized in the liver via the cytochrome P450 pathway. The mean half-life of N-desmethylclobazam (67.5 hours) is nearly double the mean half-life of clobazam (37.5 hours). Clobazam was synthesized with the anticipation that its distinct chemical structure would provide greater efficacy with fewer benzodiazepine-associated adverse effects. Frequently reported adverse effects of clobazam therapy include dizziness, sedation, drowsiness and ataxia. Evidence gathered from approximately 50 epilepsy clinical trials in adults and children indicated that the sedative effects observed with clobazam treatment were less severe than those reported with 1,4-benzodiazepines. In several studies of healthy volunteers and patients with anxiety, clobazam appeared to enhance participants' performance in cognitive tests, further distinguishing it from the 1,4-benzodiazepines. The anxiolytic and anticonvulsant effects of clobazam are associated with allosteric activation of the ligand-gated GABA(A) receptor. GABA(A) receptors are found extensively throughout the CNS, occurring synaptically and extrasynaptically. GABA(A) receptors are composed of five protein subunits, two copies of a single type of α subunit, two copies of one type of

  13. Acute phenobarbital administration induces hyperalgesia: pharmacological evidence for the involvement of supraspinal GABA-A receptors

    Directory of Open Access Journals (Sweden)

    C.M. Yokoro

    2001-03-01

    Full Text Available The aim of the present study was to determine if phenobarbital affects the nociception threshold. Systemic (1-20 mg/kg phenobarbital administration dose dependently induced hyperalgesia in the tail-flick, hot-plate and formalin tests in rats and in the abdominal constriction test in mice. Formalin and abdominal constriction tests were the most sensitive procedures for the detection of hyperalgesia in response to phenobarbital compared with the tail-flick and hot-plate tests. The hyperalgesia induced by systemic phenobarbital was blocked by previous administration of 1 mg/kg ip picrotoxin or either 1-2 mg/kg sc or 10 ng icv bicuculline. Intracerebroventricular phenobarbital administration (5 µg induced hyperalgesia in the tail-flick test. In contrast, intrathecal phenobarbital administration (5 µg induced antinociception and blocked systemic-induced hyperalgesia in this test. We suggest that phenobarbital may mediate hyperalgesia through GABA-A receptors at supraspinal levels and antinociception through the same kind of receptors at spinal levels.

  14. Calcium-Modulating Cyclophilin Ligand Regulates Membrane Trafficking of Postsynaptic GABAA Receptors

    Science.gov (United States)

    Yuan, Xu; Yao, Jun; Norris, David; Tran, David D.; Bram, Richard J.; Chen, Gong; Luscher, Bernhard

    2008-01-01

    Accumulation of GABAA receptors (GABAARs) at GABAergic synapses requires the cytoplasmic loop region and C-terminal transmembrane domain of the receptor γ2 subunit. We here report a novel interaction of γ2 with Calcium-Modulating cyclophilin Ligand (CAML), an integral membrane protein that regulates this mechanism. Interaction of GABAARs with CAML depends on both the cytoplasmic region and fourth transmembrane domain of the γ2 subunit, CAML immunoprecipitates with GABAARs from transfected cells and brain lysates and colocalizes with γ2 in ER vesicles in soma and dendrites of neurons. CAML shRNA treatment results in reduced expression of postsynaptic GABAARs, along with significant reductions in GABA-evoked whole-cell currents and GABAergic synaptic function, while glutamatergic transmission is unaffected. Reduced surface expression of GABAARs in CAML mutant neurons is associated with selective deficits in recycling of endocytosed GABAARs to the cell surface. Our results indicate a specific role of CAML in functional expression and endocytic recycling of postsynaptic GABAARs. PMID:18424167

  15. Ethanol activation of protein kinase A regulates GABA-A receptor subunit expression in the cerebral cortex and contributes to ethanol-induced hypnosis

    Directory of Open Access Journals (Sweden)

    Sandeep eKumar

    2012-04-01

    Full Text Available Protein kinases are implicated in neuronal cell functions such as modulation of ion channel function, trafficking and synaptic excitability. Both protein kinase C (PKC and A (PKA are involved in regulation of γ-aminobutyric acid type A (GABA-A receptors through phosphorylation. However, the role of PKA in regulating GABA-A receptors following acute ethanol exposure is not known. The present study investigated the role of PKA in ethanol effects on GABA-A receptor α1 subunit expression in the P2 synaptosomal fraction of the rat cerebral cortex. Additionally, GABA-related behaviors were also examined. Rats were administered ethanol (2.0 – 3.5 g/kg or saline and PKC, PKA and GABA-A receptor α1 subunit levels were measured by Western blot analysis. Ethanol (3.5 g/kg transiently increased GABA-A receptor α1 subunit expression and PKA RIIβ subunit expression at similar time points whereas PKA RIIα was increased at later time points. In contrast, PKC isoform expression remained unchanged. Notably, the moderate ethanol dose (2.0g/kg had no effect on GABA-A α1 subunit levels although PKA RIIα and RIIβ were increased at 10 and 60 minutes, when PKC isozymes are also known to be elevated. To determine if PKA activation was responsible for the ethanol-induced elevation of GABA-A α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABA-A receptor α1 subunit expression. Moreover, increasing PKA activity intracerebroventricularly with Sp-cAMP prior to a hypnotic dose of ethanol increased ethanol-induced loss of righting reflex duration. This effect appears to be mediated in part by GABA-A receptors as increasing PKA activity also increased the duration of muscimol-induced loss of righting reflex. Overall these data suggest that PKA mediates ethanol-induced GABA-A receptor expression and contributes to ethanol behavioral effects involving GABA-A receptors.

  16. 5-HT1A receptor blockade reverses GABA(A) receptor alpha(3) subunit-mediated anxiolytic effects on stress-induced hyperthermia

    NARCIS (Netherlands)

    Vinkers, Christiaan H.; van Oorschot, Ruud; Korte, S. Mechiel; Olivier, Berend; Groenink, Lucianne

    2010-01-01

    Stress-related disorders are associated with dysfunction of both serotonergic and GABAergic pathways, and clinically effective anxiolytics act via both neurotransmitter systems. As there is evidence that the GABA(A) and the serotonin receptor system interact, a serotonergic component in the anxiolyt

  17. Exercise combined with low-level GABAA receptor inhibition up-regulates the expression of neurotrophins in the motor cortex.

    Science.gov (United States)

    Takahashi, Kazuma; Maejima, Hiroshi; Ikuta, Gaku; Mani, Hiroki; Asaka, Tadayoshi

    2017-01-01

    Neurotrophins play a crucial role in neuroplasticity, neurogenesis, and neuroprotection in the central nervous system. Aerobic exercise is known to increase the expression of BDNF in the cerebral cortex. Several animal studies have evaluated the tonic inhibition of GABAergic synapses to enhance hippocampal plasticity as well as learning and memory, whereas the effects of GABAergic inhibition on plasticity in the cerebral cortex related to motor learning are not well characterized. The objective of the present study was to examine the interactive effect of low-level GABAA receptor inhibition and exercise on the expression of neurotrophins including BDNF in the murine motor cortex. ICR mice were randomly distributed among 4 groups based on two factors of GABAA receptor inhibition and exercise, i.e. control group, an exercise group, a bicuculline group, and an exercise plus bicuculline group. We administered GABAA receptor antagonist, bicuculline intraperitoneally to the mice (bicuculline and exercise plus bicuculline group) at a non-epileptic dose of 0.25mg/kg, whereas the mice (exercise and exercise plus bicuculline group) were exercised on a treadmill for 1h every day. After two week intervention, the expression of mRNA and protein abundance of neurotrophins in the motor cortex was assayed using Real time PCR and ELISA. BDNF gene expression was significantly increased by approximately 3-fold in the bicuculline group relative to the control, exercise, and bicuculline plus exercise groups. Protein abundance of BDNF expression was significantly increased by approximately 3-fold in the bicuculline plus exercise group relative to other groups. Therefore, the present study revealed that combined GABAA receptor inhibition and moderate aerobic exercise up-regulated BDNF protein expression in the motor cortex without producing side effects on motor or cognitive functions. Alterations in BDNF expression could positively contribute to plasticity by regulating the balance

  18. The effects of GABAA and NMDA receptors in the shell-accumbens on spatial memory of METH-treated rats.

    Science.gov (United States)

    Heysieattalab, Soomaayeh; Naghdi, Nasser; Zarrindast, Mohammad-Reza; Haghparast, Abbas; Mehr, Shahram Ejtemaei; Khoshbouei, Habibeh

    2016-03-01

    Methamphetamine (METH) is a highly addictive and neurotoxic psychostimulant. Its use in humans is often associated with neurocognitive impairment and deficits in hippocampal plasticity. Striatal dopamine system is one of the main targets of METH. The dopamine neurons in the striatum directly or indirectly regulate the GABA and glutamatergic signaling in this region and thus their outputs. This is consistent with previous reports showing modification of neuronal activity in the striatum modulates the expression of hippocampal LTP and hippocampal-dependent memory tasks such as Morris water maze (MWM). Therefore, reversing or preventing METH-induced synaptic modifications via pharmacological manipulations of the shell-nucleus accumbens (shell-NAc) may introduce a viable therapeutic target to attenuate the METH-induced memory deficits. This study is designed to investigate the role of intra-shell NAc manipulation of GABAA and NMDA receptors and their interaction with METH on memory performance in MWM task. Pharmacological manipulations were performed in rats received METH or saline. We found systemic saline plus intra-shell NAc infusions of muscimol dose-dependently impaired performance, while bicuculline had no effect. Surprisingly, the intra-NAc infusions of 0.005μg/rat muscimol that has no effect on memory performance (ineffective dose) prevented METH-induced memory impairment. In the contrary, the intra-NAc infusions of bicuculline (0.2μg/rat) increased METH-induced memory impairment. However, pre-training intra-NAc infusions of D-AP5 dose-dependently impaired performance, while NMDA had no effect in rats received systemic saline (control group). The intra-NAc infusions with an ineffective dose of NMDA (0.1μg/rat) increased METH-induced memory impairment. Furthermore, intra-NAc infusions of D-AP5 with an ineffective dose (0.1μg/rat) prevented METH-induced memory impairment. Our result is consistent with the interpretation that METH-mediated learning deficit

  19. Mechanism of the cardiovascular effects of the GABAA receptors of the ventral tegmental area of the rat brain.

    Science.gov (United States)

    Yeganeh, Fahimeh; Ranjbar, Afsaneh; Hatam, Masoumeh; Nasimi, Ali

    2015-07-23

    The ventral tegmental area (VTA) contains GABA terminals involved in the regulation of the cardiovascular system. Previously, we demonstrated that blocking GABAA but not GABAB receptors produced a pressor response accompanied by marked bradycardia. This study was performed to find the possible mechanisms involved in these responses by blocking ganglionic nicotinic receptors, peripheral muscarinic receptors or peripheral V1 vasopressin receptors. Experiments were performed on urethane anesthetized male Wistar rats. Drugs were microinjected unilaterally into the VTA (100 nl). The average changes in mean arterial pressure (MAP) and heart rate (HR) were compared between pre- and post-treatment using paired t-test. Injection of bicuculline methiodide (BMI), a GABAA antagonist, into the VTA caused a significant increase in MAP and a decrease in HR. Administration (i.v.) of the nicotinic receptor blocker, hexamethonium, enhanced the pressor response but abolished the bradycardic response to BMI, which ruled out involvement of the sympathetic nervous system. Blockade of the peripheral muscarinic receptors by homatropine (i.v.) abolished the bradycardic effect of BMI, but had no effect on the pressor response, indicating that bradycardia was produced by the parasympathetic outflow to the heart. Both the pressor and bradycardic responses to BMI were blocked by V1 receptor antagonist (i.v.), indicating that administration of BMI in the VTA disinhibited the release of vasopressin into the circulation. In conclusion, we demonstrated that GABAergic mechanism of the VTA exerts a tonic inhibition on vasopressin release through activation of GABAA receptors. The sympathetic system is not involved in the decrease of blood pressure by GABA of the VTA. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  20. The effects of serotonin1A receptor on female mice body weight and food intake are associated with the differential expression of hypothalamic neuropeptides and the GABAA receptor.

    Science.gov (United States)

    Butt, Isma; Hong, Andrew; Di, Jing; Aracena, Sonia; Banerjee, Probal; Shen, Chang-Hui

    2014-10-01

    Both common eating disorders anorexia nervosa and bulimia nervosa are characteristically diseases of women. To characterize the role of the 5-HT1A receptor (5-HT1A-R) in these eating disorders in females, we investigated the effect of saline or 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) treatment on feeding behavior and body weight in adult WT female mice and in adult 5-HT1A-R knockout (KO) female mice. Our results showed that KO female mice have lower food intake and body weight than WT female mice. Administration of 8-OH-DPAT decreased food intake but not body weight in WT female mice. Furthermore, qRT-PCR was employed to analyze the expression levels of neuropeptides, γ-aminobutyric acid A receptor subunit β (GABAA β subunits) and glutamic acid decarboxylase in the hypothalamic area. The results showed the difference in food intake between WT and KO mice was accompanied by differential expression of POMC, CART and GABAA β2, and the difference in body weight between WT and KO mice was associated with significantly different expression levels of CART and GABAA β2. As such, our data provide new insight into the role of 5-HT1A-R in both feeding behavior and the associated expression of neuropeptides and the GABAA receptor.

  1. SB-205384 Is a Positive Allosteric Modulator of Recombinant GABAA Receptors Containing Rat α3, α5, or α6 Subunit Subtypes Coexpressed with β3 and γ2 Subunits

    OpenAIRE

    Heidelberg, Laura S.; Warren, James W.; Fisher, Janet L.

    2013-01-01

    Many drugs used to treat anxiety are positive modulators of GABAA receptors, which mediate fast inhibitory neurotransmission. The GABAA receptors can be assembled from a combination of at least 16 different subunits. The receptor’s subunit composition determines its pharmacologic and functional properties, and subunit expression varies throughout the brain. A primary goal for new treatments targeting GABAA receptors is the production of subunit-selective modulators acting upon a discrete popu...

  2. γ-glutamyl transpeptidase 1 specifically suppresses green-light avoidance via GABAA receptors in Drosophila.

    Science.gov (United States)

    Liu, Jiangqu; Gong, Zhefeng; Liu, Li

    2014-08-01

    Drosophila larvae innately show light avoidance behavior. Compared with robust blue-light avoidance, larvae exhibit relatively weaker green-light responses. In our previous screening for genes involved in larval light avoidance, compared with control w(1118) larvae, larvae with γ-glutamyl transpeptidase 1 (Ggt-1) knockdown or Ggt-1 mutation were found to exhibit higher percentage of green-light avoidance which was mediated by Rhodopsin6 (Rh6) photoreceptors. However, their responses to blue light did not change significantly. By adjusting the expression level of Ggt-1 in different tissues, we found that Ggt-1 in malpighian tubules was both necessary and sufficient for green-light avoidance. Our results showed that glutamate levels were lower in Ggt-1 null mutants compared with controls. Feeding Ggt-1 null mutants glutamate can normalize green-light avoidance, indicating that high glutamate concentrations suppressed larval green-light avoidance. However, rather than directly, glutamate affected green-light avoidance indirectly through GABA, the level of which was also lower in Ggt-1 mutants compared with controls. Mutants in glutamate decarboxylase 1, which encodes GABA synthase, and knockdown lines of the GABAA receptor, both exhibit elevated levels of green-light avoidance. Thus, our results elucidate the neurobiological mechanisms mediating green-light avoidance, which was inhibited in wild-type larvae. © 2014 International Society for Neurochemistry.

  3. The GABA(A) receptor RDL acts in peptidergic PDF neurons to promote sleep in Drosophila.

    Science.gov (United States)

    Chung, Brian Y; Kilman, Valerie L; Keath, J Russel; Pitman, Jena L; Allada, Ravi

    2009-03-10

    Sleep is regulated by a circadian clock that times sleep and wake to specific times of day and a homeostat that drives sleep as a function of prior wakefulness. To analyze the role of the circadian clock, we have used the fruit fly Drosophila. Flies display the core behavioral features of sleep, including relative immobility, elevated arousal thresholds, and homeostatic regulation. We assessed sleep-wake modulation by a core set of circadian pacemaker neurons that express the neuropeptide PDF. We find that disruption of PDF function increases sleep during the late night in light:dark and the first subjective day of constant darkness. Flies deploy genetic and neurotransmitter pathways to regulate sleep that are similar to those of their mammalian counterparts, including GABA. We find that RNA interference-mediated knockdown of the GABA(A) receptor gene, Resistant to dieldrin (Rdl), in PDF neurons reduces sleep, consistent with a role for GABA in inhibiting PDF neuron function. Patch-clamp electrophysiology reveals GABA-activated picrotoxin-sensitive chloride currents on PDF+ neurons. In addition, RDL is detectable most strongly on the large subset of PDF+ pacemaker neurons. These results suggest that GABAergic inhibition of arousal-promoting PDF neurons is an important mode of sleep-wake regulation in vivo.

  4. Spatial and Temporal Dynamics in the Ionic Driving Force for GABAA Receptors

    Directory of Open Access Journals (Sweden)

    R. Wright

    2011-01-01

    Full Text Available It is becoming increasingly apparent that the strength of GABAergic synaptic transmission is dynamic. One parameter that can establish differences in the actions of GABAergic synapses is the ionic driving force for the chloride-permeable GABAA receptor (GABAAR. Here we review some of the sophisticated ways in which this ionic driving force can vary within neuronal circuits. This driving force for GABAARs is subject to tight spatial control, with the distribution of Cl− transporter proteins and channels generating regional variation in the strength of GABAAR signalling across a single neuron. GABAAR dynamics can result from short-term changes in their driving force, which involve the temporary accumulation or depletion of intracellular Cl−. In addition, activity-dependent changes in the expression and function of Cl− regulating proteins can result in long-term shifts in the driving force for GABAARs. The multifaceted regulation of the ionic driving force for GABAARs has wide ranging implications for mature brain function, neural circuit development, and disease.

  5. Adenosine A1 Receptor Suppresses Tonic GABAA Receptor Currents in Hippocampal Pyramidal Cells and in a Defined Subpopulation of Interneurons.

    Science.gov (United States)

    Rombo, Diogo M; Dias, Raquel B; Duarte, Sofia T; Ribeiro, Joaquim A; Lamsa, Karri P; Sebastião, Ana M

    2016-03-01

    Adenosine is an endogenous neuromodulator that decreases excitability of hippocampal circuits activating membrane-bound metabotropic A1 receptor (A1R). The presynaptic inhibitory action of adenosine A1R in glutamatergic synapses is well documented, but its influence on inhibitory GABAergic transmission is poorly known. We report that GABAA receptor (GABAAR)-mediated tonic, but not phasic, transmission is suppressed by A1R in hippocampal neurons. Adenosine A1R activation strongly inhibits GABAAR agonist (muscimol)-evoked currents in Cornu Ammonis 1 (CA1) pyramidal neurons and in a specific subpopulation of interneurons expressing axonal cannabinoid receptor type 1. In addition, A1R suppresses tonic GABAAR currents measured in the presence of elevated ambient GABA as well as in naïve slices. The inhibition of GABAergic currents involves both protein kinase A (PKA) and protein kinase C (PKC) signaling pathways and decreases GABAAR δ-subunit expression. On the contrary, no A1R-mediated modulation was detected in phasic inhibitory postsynaptic currents evoked either by afferent electrical stimulation or by spontaneous quantal release. The results show that A1R modulates extrasynaptic rather than synaptic GABAAR-mediated signaling, and that this modulation selectively occurs in hippocampal pyramidal neurons and in a specific subpopulation of inhibitory interneurons. We conclude that modulation of tonic GABAAR signaling by adenosine A1R in specific neuron types may regulate neuronal gain and excitability in the hippocampus.

  6. Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors

    Science.gov (United States)

    Hoffmann, Katrin M.; Herbrechter, Robin; Ziemba, Paul M.; Lepke, Peter; Beltrán, Leopoldo; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2016-01-01

    Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications. PMID:27524967

  7. Gamma-aminobutyric acid and GABA_A receptors are involved in directional selectivity of pretectal neurons in pigeons

    Institute of Scientific and Technical Information of China (English)

    肖泉; 付煜西; 胡婧; 高洪峰; 王书荣

    2000-01-01

    The present study describes the effects of gamma-aminobutyric acid (GABA) and its antagonists, bicuculline and 2-hydroxysaclofen, on visual responses of neurons in the pigeon nucleus lentiformis mesencephali (nLM). The results indicate that GABA significantly reduces both spontaneous activity and visual responsiveness, and GABAA antagonist bicuculline but not GABAB antagonist 2-hydroxysaclofen enhances visual responses of nLM cells examined. Furthermore, inhibition produced by motion in the null-direction of pretectal neurons is diminished by bicuculline but not by 2-hydroxysaclofen. It is therefore concluded that the null-direction inhibition of directional cells in the pigeon nLM is predominantly mediated by GABA and GABAA receptors. This inhibition may at least in part underlie directional asymmetry of optokinetic responses.

  8. Gamma-aminobutyric acid and GABAA receptors are involved in directional selectivity of pretectal neurons in pigeons

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The present study describes the effects of gamma-aminobutyric acid (GABA) and itsantagonists, bicuculline and 2-hydroxysaclofen, on visual responses of neurons in the pigeon nucleuslentiformis mesencephali (nLM). The results indicate that GABA significantly reduces bothspontaneous activity and visual responsiveness, and GABAA antagonist bicuculline but not GABABantagonist 2-hydroxysaclofen enhances visual responses of nLM cells examined. Furthermore,inhibition produced by motion in the null-direction of pretectal neurons is diminished by bicucullinebut not by 2-hydroxysaclofen. It is therefore concluded that the null-direction inhibition of directionalcells in the pigeon nLM is predominantly mediated by GABA and GABAA receptors. This inhibitionmay at least in part underlie directional asymmetry of optokinetic responses.

  9. Traumatic brain injury and the effects of diazepam, diltiazem, and MK-801 on GABA-A receptor subunit expression in rat hippocampus

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    Meyer Rebecca C

    2010-05-01

    Full Text Available Abstract Background Excitatory amino acid release and subsequent biochemical cascades following traumatic brain injury (TBI have been well documented, especially glutamate-related excitotoxicity. The effects of TBI on the essential functions of inhibitory GABA-A receptors, however, are poorly understood. Methods We used Western blot procedures to test whether in vivo TBI in rat altered the protein expression of hippocampal GABA-A receptor subunits α1, α2, α3, α5, β3, and γ2 at 3 h, 6 h, 24 h, and 7 days post-injuy. We then used pre-injury injections of MK-801 to block calcium influx through the NMDA receptor, diltiazem to block L-type voltage-gated calcium influx, or diazepam to enhance chloride conductance, and re-examined the protein expressions of α1, α2, α3, and γ2, all of which were altered by TBI in the first study and all of which are important constituents in benzodiazepine-sensitive GABA-A receptors. Results Western blot analysis revealed no injury-induced alterations in protein expression for GABA-A receptor α2 or α5 subunits at any time point post-injury. Significant time-dependent changes in α1, α3, β3, and γ2 protein expression. The pattern of alterations to GABA-A subunits was nearly identical after diltiazem and diazepam treatment, and MK-801 normalized expression of all subunits 24 hours post-TBI. Conclusions These studies are the first to demonstrate that GABA-A receptor subunit expression is altered by TBI in vivo, and these alterations may be driven by calcium-mediated cascades in hippocampal neurons. Changes in GABA-A receptors in the hippocampus after TBI may have far-reaching consequences considering their essential importance in maintaining inhibitory balance and their extensive impact on neuronal function.

  10. Functional and molecular plasticity of gamma and alpha-1 GABAA receptor subunits in the dorsal motor nucleus of the vagus after experimentally-induced diabetes.

    Science.gov (United States)

    Boychuk, Carie R; Smith, Katalin Cs; Smith, Bret N

    2017-08-23

    Chronic experimentally-induced hyperglycemia augments subunit specific gamma-aminobutyric acid A (GABAA) receptor-mediated inhibition of parasympathetic preganglionic motor neurons in the dorsal motor nucleus of the vagus (DMV). However, the contribution of α1 or γ GABAA receptor subunits, which are ubiquitously expressed on central nervous system neurons, to this elevation in inhibitory tone have not been determined. This study investigated the effect of chronic hyperglycemia/hypoinsulinemia on α1- and γ-subunit specific GABAA receptor-mediated inhibition using electrophysiological recordings in vitro and quantitative (q)RT-PCR. DMV neurons from streptozotocin-treated mice demonstrated enhancement of both phasic and tonic inhibitory currents in response to application of the α1-subunit selective GABAA receptor positive allosteric modulator, zolpidem. Responses to low concentrations of the GABAA receptor antagonist, gabazine suggested an additional increased contribution of γ-subunit-containing receptors to tonic currents in DMV neurons. Consistent with the functional elevation in α1- and γ-subunit-dependent activity, transcription of both the α1- and γ2-subunits was increased in the dorsal vagal complex of streptozotocin-treated mice. Overall these findings suggest an increased sensitivity to both zolpidem and gabazine after several days of hyperglycemia/hypoinsulinemia, which could contribute to altered parasympathetic output from DMV neurons in diabetes. Copyright © 2017, Journal of Neurophysiology.

  11. GABAA receptors: post-synaptic co-localization and cross-talk with other receptors

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    Amulya Nidhi Shrivastava

    2011-06-01

    Full Text Available γ-aminobutyric acid type A receptors (GABAARs are the major inhibitory neurotransmitter receptors in the central nervous system (CNS, and importantly contribute to the functional regulation of the nervous system. Several studies in the last few decades have convincingly shown that GABA can be co-localized with other neurotransmitters in the same synapse, and can be co-released with these neurotransmitters either from the same vesicles or from different vesicle pools. The co-released transmitters may act on post-synaptically co-localized receptors resulting in a simultaneous activation of both receptors. Most of the studies investigating such co-activation observed a reduced efficacy of GABA for activating GABAARs and thus, a reduced inhibition of the postsynaptic neuron. Similarly, in several cases activation of GABAARs has been reported to suppress the response of the associated receptors. Such a receptor cross-talk is either mediated via a direct coupling between the two receptors or via the activation of intracellular signaling pathways and is used for fine tuning of inhibition in the nervous system. Recently, it was demonstrated that a direct interaction of different receptors might already occur in intracellular compartments and might also be used to specifically target the receptors to the cell membrane. In this article, we provide an overview on such cross-talks between GABAARs and several other neurotransmitter receptors and briefly discuss their possible physiological and clinical importance.

  12. Dopamine D3 receptor-dependent changes in alpha6 GABAA subunit expression in striatum modulate anxiety-like behaviour: Responsiveness and tolerance to diazepam.

    Science.gov (United States)

    Leggio, Gian Marco; Torrisi, Sebastiano Alfio; Castorina, Alessandro; Platania, Chiara Bianca Maria; Impellizzeri, Agata Antonia Rita; Fidilio, Annamaria; Caraci, Filippo; Bucolo, Claudio; Drago, Filippo; Salomone, Salvatore

    2015-09-01

    Increasing evidence indicates that central dopamine (DA) neurotransmission is involved in pathophysiology of anxiety, in particular the DA receptor subtype 3 (D3R). We previously reported that D3R null mice (D3R(-/-)) exhibit low baseline anxiety levels and that acutely administrated diazepam is more effective in D3R(-/-) than in wild type (WT) when tested in the elevated plus maze test (EPM). Here we tested the hypothesis that genetic deletion or pharmacological blockade of D3R affect GABAA subunit expression, which in turn modulates anxiety-like behaviour as well as responsiveness and tolerance to diazepam. D3R(-/-) mice exhibited tolerance to diazepam (0.5mg/kg, i.p.), assessed by EPM, as fast as after 3 day-treatment, performing similarly to untreated D3R(-/-) mice; conversely, WT exhibited tolerance to diazepam after a 14-21 day-treatment. Analysis of GABAA α6 subunit mRNA expression by qPCR in striatum showed that it was about 15-fold higher in D3R(-/-) than in WT. Diazepam treatment did not modify α6 expression in D3R(-/-), but progressively increased α6 expression in WT, to the level of untreated D3R(-/-) after 14-21 day-treatment. BDNF mRNA expression in striatum was remarkably (>10-fold) increased after 3 days of diazepam-treatment in both WT and D3R(-/-); such expression level, however, slowly declined below control levels, by 14-21 days. Following a 7 day-treatment with the selective D3R antagonist SB277011A, WT exhibited a fast tolerance to diazepam accompanied by a robust increase in α6 subunit expression. In conclusion, genetic deletion or pharmacological blockade of D3R accelerate the development of tolerance to repeated administrations of diazepam and increase α6 subunit expression, a GABAA subunit that has been linked to diazepam insensitivity. Modulation of GABAA receptor by DA transmission may be involved in the mechanisms of anxiety and, if occurring in humans, may have therapeutic relevance following repeated use of drugs targeting D3R.

  13. Amyloid β-protein differentially affects NMDA receptor- and GABAA receptor-mediated currents in rat hippocampal CA1 neurons

    Institute of Scientific and Technical Information of China (English)

    Junfang Zhang; Lei Hou; Xiuping Gao; Fen Guo; Wei Jing; Jinshun Qi; Jiantian Qiao

    2009-01-01

    Although the aggregated amyloid β-protein (Aβ) in senile plaques is one of the key neuropathological features of Alzheimer's disease (AD), soluble forms of Aβ also interfere with synaptic plasticity at the early stage of AD. The suppressive action of acute application of Aβ on hippocampal long-term potentiation (LTP) has been reported widely, whereas the mechanism underlying the effects of Aβ is still mostly unknown. The present study, using the whole-cell patch clamp technique, investigated the effects of Aβ fragments (Aβ25-35 and Aβ31-35) on the LTP induction-related postsynaptic ligand-gated channel currents in isolated hippocampal CA1 neurons. The results showed a rapid but opposite action of both peptides on excitatory and inhibitory receptor currents. Glutamate application-induced currents were suppressed by A β25-35 in a dose-dependent manner, and further N-methyl-I>aspartate (NMDA) receptor-mediated currents were selec-tively inhibited. In contrast, pretreatment with Aβ fragments potentiated γ-aminobutyric acid (GABA)-induced whole-cell currents. As a control, Aβ35-31 the reversed sequence of Aβ35-31 showed no effect on the currents induced by glutamate, NMDA or GABA. These results may partly explain the impaired effects of Aβ on hippocampal LTP, and suggest that the functional down-regulation of N M DA receptors and up-regulation of GABAA receptors may play an important role in remodeling the hippocampal synaptic plasticity in early AD.

  14. RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A) receptor-mediated currents in the amygdala: a mechanism for RDX-induced seizures.

    Science.gov (United States)

    Williams, Larry R; Aroniadou-Anderjaska, Vassiliki; Qashu, Felicia; Finne, Huckelberry; Pidoplichko, Volodymyr; Bannon, Desmond I; Braga, Maria F M

    2011-03-01

    Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high-energy, trinitrated cyclic compound that has been used worldwide since World War II as an explosive in both military and civilian applications. RDX can be released in the environment by way of waste streams generated during the manufacture, use, and disposal of RDX-containing munitions and can leach into groundwater from unexploded munitions found on training ranges. For > 60 years, it has been known that exposure to high doses of RDX causes generalized seizures, but the mechanism has remained unknown. We investigated the mechanism by which RDX induces seizures. By screening the affinity of RDX for a number of neurotransmitter receptors, we found that RDX binds exclusively to the picrotoxin convulsant site of the γ-aminobutyric acid type A (GABA(A)) ionophore. Whole-cell in vitro recordings in the rat basolateral amygdala (BLA) showed that RDX reduces the frequency and amplitude of spontaneous GABA(A) receptor-mediated inhibitory postsynaptic currents and the amplitude of GABA-evoked postsynaptic currents. In extracellular field recordings from the BLA, RDX induced prolonged, seizure-like neuronal discharges. These results suggest that binding to the GABA(A) receptor convulsant site is the primary mechanism of seizure induction by RDX and that reduction of GABAergic inhibitory transmission in the amygdala is involved in the generation of RDX-induced seizures. Knowledge of the molecular site and the mechanism of RDX action with respect to seizure induction can guide therapeutic strategies, allow more accurate development of safe thresholds for exposures, and help prevent the development of new explosives or other munitions that could pose similar health risks.

  15. No association of the GABAA receptor genes on chromosome 5 with alcoholism in the collaborative study on the genetics of alcoholism sample.

    Science.gov (United States)

    Dick, Danielle M; Edenberg, Howard J; Xuei, Xiaoling; Goate, Alison; Hesselbrock, Victor; Schuckit, Marc; Crowe, Raymond; Foroud, Tatiana

    2005-01-05

    A substantial body of literature suggests that gamma-aminobutyric acid (GABA) may be involved in the neurochemical pathways contributing to alcohol use and related disorders. Chromosome 5 contains a cluster of GABA(A) receptor genes, GABRA1, GABRA6, GABRB2, and GABRG2, which have been among the most extensively studied in relation to alcohol use. These studies have yielded mixed results. Using data from large, multiplex alcoholic families collected as part of the Collaborative Study on the Genetics of Alcoholism (COGA), we sought to provide more conclusive evidence regarding the role of the GABA(A) receptor genes on chromosome 5. Multiple single nucleotide polymorphisms (SNPs) were tested in each of the four chromosome 5q GABA(A) receptor genes, and we conducted both classic trio-based association analyzes and extended pedigree analyzes. We found no consistent evidence of association with alcohol dependence or alcohol dependence comorbid with antisocial personality disorder (ASPD) for any of the regions tested in the chromosome 5 GABA(A) receptor genes. These analyses suggest that the GABA(A) receptor genes on chromosome 5 do not play a strong role in alcohol dependence. Future studies are planned to test whether these genes are more important in influencing behavioral endophenotypes related to the risk of alcohol dependence.

  16. Inactivation of GABAA receptor is related to heat shock stress response in organism model Caenorhabditis elegans.

    Science.gov (United States)

    Camargo, Gabriela; Elizalde, Alejandro; Trujillo, Xochitl; Montoya-Pérez, Rocío; Mendoza-Magaña, María Luisa; Hernandez-Chavez, Abel; Hernandez, Leonardo

    2016-09-01

    The mechanisms underlying oxidative stress (OS) resistance are not completely clear. Caenorhabditis elegans (C. elegans) is a good organism model to study OS because it displays stress responses similar to those in mammals. Among these mechanisms, the insulin/IGF-1 signaling (IIS) pathway is thought to affect GABAergic neurotransmission. The aim of this study was to determine the influence of heat shock stress (HS) on GABAergic activity in C. elegans. For this purpose, we tested the effect of exposure to picrotoxin (PTX), gamma-aminobutyric acid (GABA), hydrogen peroxide, and HS on the occurrence of a shrinking response (SR) after nose touch stimulus in N2 (WT) worms. Moreover, the effect of HS on the expression of UNC-49 (GABAA receptor ortholog) in the EG1653 strain and the effect of GABA and PTX exposure on HSP-16.2 expression in the TJ375 strain were analyzed. PTX 1 mM- or H2O2 0.7 mM-exposed worms displayed a SR in about 80 % of trials. GABA exposure did not cause a SR. HS prompted the occurrence of a SR as did PTX 1 mM or H2O2 0.7 mM exposure. In addition, HS increased UNC-49 expression, and PTX augmented HSP-16.2 expression. Thus, the results of the present study suggest that oxidative stress, through either H2O2 exposure or application of heat shock, inactivates the GABAergic system, which subsequently would affect the oxidative stress response, perhaps by enhancing the activity of transcription factors DAF-16 and HSF-1, both regulated by the IIS pathway and related to hsp-16.2 expression.

  17. Modification of Male Courtship Motivation by Olfactory Habituation via the GABAA Receptor in Drosophila melanogaster.

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    Shin-Ichiro Tachibana

    Full Text Available A male-specific component, 11-cis-vaccenyl acetate (cVA works as an anti-aphrodisiac pheromone in Drosophila melanogaster. The presence of cVA on a male suppresses the courtship motivation of other males and contributes to suppression of male-male homosexual courtship, while the absence of cVA on a female stimulates the sexual motivation of nearby males and enhances the male-female interaction. However, little is known how a male distinguishes the presence or absence of cVA on a target fly from either self-produced cVA or secondhand cVA from other males in the vicinity. In this study, we demonstrate that male flies have keen sensitivity to cVA; therefore, the presence of another male in the area reduces courtship toward a female. This reduced level of sexual motivation, however, could be overcome by pretest odor exposure via olfactory habituation to cVA. Real-time imaging of cVA-responsive sensory neurons using the neural activity sensor revealed that prolonged exposure to cVA decreased the levels of cVA responses in the primary olfactory center. Pharmacological and genetic screening revealed that signal transduction via GABAA receptors contributed to this olfactory habituation. We also found that the habituation experience increased the copulation success of wild-type males in a group. In contrast, transgenic males, in which GABA input in a small subset of local neurons was blocked by RNAi, failed to acquire the sexual advantage conferred by habituation. Thus, we illustrate a novel phenomenon in which olfactory habituation positively affects sexual capability in a competitive environment.

  18. Interaction between cyclodextrin and neuronal membrane results in modulation of GABA(A) receptor conformational transitions.

    Science.gov (United States)

    Pytel, Maria; Mercik, Katarzyna; Mozrzymas, Jerzy W

    2006-06-01

    Cyclodextrins (CDs) are nanostructures widely applied in biotechnology and chemistry. Owing to partially hydrophobic character, CDs interact with biological membranes. While the mechanisms of CDs interactions with lipids were widely studied, their effects on proteins are less understood. In the present study we investigated the effects of beta cyclodextrin (betaCD) on GABA(A) receptor (GABA(A)R) gating. To reliably resolve the kinetics of conformational transitions, currents were elicited by ultrafast gamma-aminobutyric acid (GABA) applications to outside-out patches from rat cultured hippocampal neurons. betaCD increased the amplitude of responses to saturating GABA concentration ([GABA]) in a dose-dependent manner and this effect was accompanied by profound alterations in the current kinetics. Current deactivation was slowed down by betaCD but this effect was biphasic with a maximum at around 0.5 mM betaCD. While the fast deactivation time constant was monotonically slowed down within considered betaCD concentration range, the slow component first increased and then, at millimolar betaCD concentration, decreased. The rate and extent of desensitization was decreased by betaCD in a dose-dependent manner. The analysis of current responses to nonsaturating [GABA] indicated that betaCD affected the GABA(A)R agonist binding site by slowing down the unbinding rate. Modulation of GABA(A)R desensitization and binding showed different concentration-dependence suggesting different modualtory sites with higher affinity of the latter one. All the betaCD effects were fully reversible indicating that cholesterol uptake into betaCD was not the primary mechanism. We conclude that betaCD is a strong modulator of GABA(A)R conformational transitions.

  19. Interaction between cyclodextrin and neuronal membrane results in modulation of GABAA receptor conformational transitions

    Science.gov (United States)

    Pytel, Maria; Mercik, Katarzyna; Mozrzymas, Jerzy W

    2006-01-01

    Cyclodextrins (CDs) are nanostructures widely applied in biotechnology and chemistry. Owing to partially hydrophobic character, CDs interact with biological membranes. While the mechanisms of CDs interactions with lipids were widely studied, their effects on proteins are less understood. In the present study we investigated the effects of beta cyclodextrin (βCD) on GABAA receptor (GABAAR) gating. To reliably resolve the kinetics of conformational transitions, currents were elicited by ultrafast gamma-aminobutyric acid (GABA) applications to outside-out patches from rat cultured hippocampal neurons. βCD increased the amplitude of responses to saturating GABA concentration ([GABA]) in a dose-dependent manner and this effect was accompanied by profound alterations in the current kinetics. Current deactivation was slowed down by βCD but this effect was biphasic with a maximum at around 0.5 mM βCD. While the fast deactivation time constant was monotonically slowed down within considered βCD concentration range, the slow component first increased and then, at millimolar βCD concentration, decreased. The rate and extent of desensitization was decreased by βCD in a dose-dependent manner. The analysis of current responses to nonsaturating [GABA] indicated that βCD affected the GABAAR agonist binding site by slowing down the unbinding rate. Modulation of GABAAR desensitization and binding showed different concentration-dependence suggesting different modualtory sites with higher affinity of the latter one. All the βCD effects were fully reversible indicating that cholesterol uptake into βCD was not the primary mechanism. We conclude that βCD is a strong modulator of GABAAR conformational transitions. PMID:16702996

  20. Modification of Male Courtship Motivation by Olfactory Habituation via the GABAA Receptor in Drosophila melanogaster

    Science.gov (United States)

    Tachibana, Shin-Ichiro; Touhara, Kazushige; Ejima, Aki

    2015-01-01

    A male-specific component, 11-cis-vaccenyl acetate (cVA) works as an anti-aphrodisiac pheromone in Drosophila melanogaster. The presence of cVA on a male suppresses the courtship motivation of other males and contributes to suppression of male-male homosexual courtship, while the absence of cVA on a female stimulates the sexual motivation of nearby males and enhances the male-female interaction. However, little is known how a male distinguishes the presence or absence of cVA on a target fly from either self-produced cVA or secondhand cVA from other males in the vicinity. In this study, we demonstrate that male flies have keen sensitivity to cVA; therefore, the presence of another male in the area reduces courtship toward a female. This reduced level of sexual motivation, however, could be overcome by pretest odor exposure via olfactory habituation to cVA. Real-time imaging of cVA-responsive sensory neurons using the neural activity sensor revealed that prolonged exposure to cVA decreased the levels of cVA responses in the primary olfactory center. Pharmacological and genetic screening revealed that signal transduction via GABAA receptors contributed to this olfactory habituation. We also found that the habituation experience increased the copulation success of wild-type males in a group. In contrast, transgenic males, in which GABA input in a small subset of local neurons was blocked by RNAi, failed to acquire the sexual advantage conferred by habituation. Thus, we illustrate a novel phenomenon in which olfactory habituation positively affects sexual capability in a competitive environment. PMID:26252206

  1. Spontaneous thermal motion of the GABA(A) receptor M2 channel-lining segments.

    Science.gov (United States)

    Bera, Amal K; Akabas, Myles H

    2005-10-21

    The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.

  2. The blockade of GABAA receptors attenuates the inhibitory effect of orexin type 1 receptors antagonist on morphine withdrawal syndrome in rats.

    Science.gov (United States)

    Davoudi, Mahnaz; Azizi, Hossein; Mirnajafi-Zadeh, Javad; Semnanian, Saeed

    2016-03-23

    The aim of present study was to investigate the involvement of orexin-A neuropeptide in naloxone-induced morphine withdrawal syndrome via modulating neurons bearing GABAA receptors. The locus coeruleus (LC) is a sensitive site for expression of the somatic aspects of morphine withdrawal. Intra-LC microinjection of GABAA receptor agonist attenuates morphine withdrawal signs in rats. Here we studied the influence of LC orexin type 1 receptors blockade by SB-334867 in presence of bicuculline, a GABAA receptor antagonist, on naloxone-induced morphine withdrawal syndrome. Adult male Wistar rats, weighing 250-300 g, were rendered dependent on morphine by subcutaneous (s.c.) injection of increasing morphine doses (6, 16, 26, 36, 46, 56 and 66 mg/kg, 2 ml/kg) at set intervals of 24 h for 7 days. On 8th day, naloxone (3 mg/kg, s.c.) was injected and the somatic signs of morphine withdrawal were evaluated. Intra-LC microinjections (0.2 μl) of either bicuculline (15 μM) or SB-334867 (3 mM) or a combination of both chemicals were done immediately before naloxone injection. Intra-LC microinjection of bicuculline (15 μM) had no significant effect on morphine withdrawal signs, whereas intra-LC microinjection of SB-334867 considerably attenuated morphine withdrawal signs. However, the effect of SB-334867 in attenuating naloxone-induced morphine withdrawal signs was blocked in presence of bicuculline. This finding, for the first time, indicated that orexin-A may participate in expression of naloxone-induced morphine withdrawal syndrome partly through decreasing the activity of neurons bearing GABAA receptors.

  3. Alterations in nigral NMDA and GABAA receptor control of the striatal dopamine level after repetitive exposures to nitrogen narcosis.

    Science.gov (United States)

    Lavoute, Cécile; Weiss, Michel; Rostain, Jean-Claude

    2008-07-01

    Nitrogen pressure exposure in rats results in decreased dopamine (DA) release at the striatal terminals of the substantia nigra pars compacta (SNc) dopaminergic neurons, demonstrating the narcotic potency of nitrogen. This effect is attributed to decreased excitatory and increased inhibitory inputs to dopaminergic neurons, involving a change in NMDA and GABA(A) receptor function. We investigated whether repetitive exposures to nitrogen modify the excitatory and inhibitory control of the dopaminergic nigro-striatal pathway. We used voltammetry to measure dopamine levels in freely-moving rats, implanted with dopamine-sensitive electrodes in the striatum. NMDA/GABA(A) receptor agonists (NMDA/muscimol) and antagonists (AP7/gabazine) were administered through a guide-cannula into the SNc, and their effects on striatal dopamine levels were measured under normobaric conditions, before and after five repetitive exposures to 1 MPa nitrogen. NMDA-mediated dopamine release was greater following repetitive exposures, AP7-mediated inhibition of glutamatergic input was blocked, suggesting that NMDA receptor sensitivity was increased and glutamate release reduced. Muscimol did not modify dopamine levels following repetitive exposures, whereas the effect of gabazine was greater after exposures than before. This suggested that interneuronal GABA(A) receptors were desensitized, leading to an increased GABAergic input at dopaminergic cells. Thus, repetitive nitrogen exposure induced persistent changes in glutamatergic and GABAergic control of dopaminergic neurons, resulting in decreased activity of the nigrostriatal pathway.

  4. The GLP-1 Receptor Agonist Exendin-4 and Diazepam Differentially Regulate GABAA Receptor-Mediated Tonic Currents in Rat Hippocampal CA3 Pyramidal Neurons.

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    Sergiy V Korol

    Full Text Available Glucagon-like peptide-1 (GLP-1 is a metabolic hormone that is secreted in a glucose-dependent manner and enhances insulin secretion. GLP-1 receptors are also found in the brain where their signalling affects neuronal activity. We have previously shown that the GLP-1 receptor agonists, GLP-1 and exendin-4 enhanced GABA-activated synaptic and tonic currents in rat hippocampal CA3 pyramidal neurons. The hippocampus is the centre for memory and learning and is important for cognition. Here we examined if exendin-4 similarly enhanced the GABA-activated currents in the presence of the benzodiazepine diazepam. In whole-cell recordings in rat brain slices, diazepam (1 μM, an allosteric positive modulator of GABAA receptors, alone enhanced the spontaneous inhibitory postsynaptic current (sIPSC amplitude and frequency by a factor of 1.3 and 1.6, respectively, and doubled the tonic GABAA current normally recorded in the CA3 pyramidal cells. Importantly, in the presence of exendin-4 (10 nM plus diazepam (1 μM, only the tonic but not the sIPSC currents transiently increased as compared to currents recorded in the presence of diazepam alone. The results suggest that exendin-4 potentiates a subpopulation of extrasynaptic GABAA receptors in the CA3 pyramidal neurons.

  5. Chronic flumazenil alters GABA(A) receptor subunit mRNA expression, translation product assembly and channel function in neuronal cultures.

    Science.gov (United States)

    Zheng, T M; Caruncho, H J; Zhu, W J; Vicini, S; Ikonomovic, S; Grayson, D R; Costa, E

    1996-04-01

    Flumazenil competitively blocks the pharmacological effects of both positive and negative allosteric modulators acting at the benzodiazepine binding sites of gamma-aminobutyric acid (GABA(A)) receptors. Using quantitative reverse transcription polymerase chain reaction, label-fracture immunocytochemistry and whole-cell patch-clamp recordings, we determined changes in the contents of selected GABA(A) receptor subunit mRNA(s), in their translation products and in the electrophysiological characteristics of the receptor channels in cultured cerebellar granule cells treated daily with flumazenil (10 microM) for 4 days in vitro. The contents of the alpha1 and alpha6 receptor subunit mRNAs were significantly increased in the flumazenil-treated group as compared with the dimethyl sulfoxide vehicle-treated control group, whereas there were no significant differences in the absolute amounts of the beta2, beta3, gamma2S, gamma2L++ + and delta receptor subunit mRNAs. The gold immunolabeling densities of the alpha1 and delta receptor subunits were significantly increased, whereas those of the alpha6, beta2/beta3 and gamma2 receptor subunits were decreased. Double-immunolabeling experiments using 5- and 10-nm gold particles suggest that after chronic flumazenil treatment, receptor subunit assemblies containing the alpha1/gamma2 and alpha6/delta subunits may be replaced by a receptor assembly containing the alpha1/delta subunits. The GABA potency in eliciting Cl- channel activity decreased significantly, as indicated by the elevated EC50 values, and the positive modulation of GABA action by diazepam also decreased. These results suggest that flumazenil, perhaps by blocking the action of endogenous allosteric modulators of GABA(A) receptors, may trigger a change in the expression and assembly of the subunits of the GABA(A) receptor. This implies that there might be a dynamic state in the regulation of GABA(A) receptor structure.

  6. Removal of GABA(A receptor γ2 subunits from parvalbumin neurons causes wide-ranging behavioral alterations.

    Directory of Open Access Journals (Sweden)

    Elli Leppä

    Full Text Available We investigated the behavioral significance of fast synaptic inhibition by αβγ2-type GABA(A receptors on parvalbumin (Pv cells. The GABA(A receptor γ2 subunit gene was selectively inactivated in Pv-positive neurons by Cre/loxP recombination. The resulting Pv-Δγ2 mice were relatively healthy in the first postnatal weeks; but then as Cre started to be expressed, the mice progressively developed wide-ranging phenotypic alterations including low body weight, motor deficits and tremor, decreased anxiety levels, decreased pain sensitivity and deficient prepulse inhibition of the acoustic startle reflex and impaired spatial learning. Nevertheless, the deletion was not lethal, and mice did not show increased mortality even after one year. Autoradiography with t-butylbicyclophosphoro[(35S]thionate suggested an increased amount of GABA(A receptors with only α and β subunits in central nervous system regions that contained high levels of parvalbumin neurons. Using BAC-transgenesis, we reduced some of the Pv-Δγ2 phenotype by selectively re-expressing the wild-type γ2 subunit back into some Pv cells (reticular thalamic neurons and cerebellar Pv-positive neurons. This produced less severe impairments of motor skills and spatial learning compared with Pv-Δγ2 mice, but all other deficits remained. Our results reveal the widespread significance of fast GABAergic inhibition onto Pv-positive neurons for diverse behavioral modalities, such as motor coordination, sensorimotor integration, emotional behavior and nociception.

  7. Reinforcing Effects Of Compounds Lacking Intrinsic Efficacy At α1 Subunit-Containing GABAA Receptor Subtypes in Midazolam- But Not Cocaine-Experienced Rhesus Monkeys

    Science.gov (United States)

    Shinday, Nina M; Sawyer, Eileen K; Fischer, Bradford D; Platt, Donna M; Licata, Stephanie C; Atack, John R; Dawson, Gerard R; Reynolds, David S; Rowlett, James K

    2013-01-01

    Benzodiazepines are prescribed widely but their utility is limited by unwanted side effects, including abuse potential. The mechanisms underlying the abuse-related effects of benzodiazepines are not well understood, although α1 subunit-containing GABAA receptors have been proposed to have a critical role. Here, we examine the reinforcing effects of several compounds that vary with respect to intrinsic efficacy at α2, α3, and α5 subunit-containing GABAA receptors but lack efficacy at α1 subunit-containing GABAA receptors (‘α1-sparing compounds'): MRK-623 (functional selectivity for α2/α3 subunit-containing receptors), TPA023B (functional selectivity for α2/α3/α5 subunit-containing receptors), and TP003 (functional selectivity for α3 subunit-containing receptors). The reinforcing effects of the α1-sparing compounds were compared with those of the non-selective benzodiazepine receptor partial agonist MRK-696, and non-selective benzodiazepine receptor full agonists, midazolam and lorazepam, in rhesus monkeys trained to self-administer midazolam or cocaine, under a progressive-ratio schedule of intravenous (i.v.) drug injection. The α1-sparing compounds were self-administered significantly above vehicle levels in monkeys maintained under a midazolam baseline, but not under a cocaine baseline over the dose ranges tested. Importantly, TP003 had significant reinforcing effects, albeit at lower levels of self-administration than non-selective benzodiazepine receptor agonists. Together, these results suggest that α1 subunit-containing GABAA receptors may have a role in the reinforcing effects of benzodiazepine-type compounds in monkeys with a history of stimulant self-administration, whereas α3 subunit-containing GABAA receptors may be important mediators of the reinforcing effects of benzodiazepine-type compounds in animals with a history of sedative-anxiolytic/benzodiazepine self-administration. PMID:23303046

  8. Successful combination immunotherapy of anti-gamma aminobutyric acid (GABA)A receptor antibody-positive encephalitis with extensive multifocal brain lesions.

    Science.gov (United States)

    Fukami, Yuki; Okada, Hiroaki; Yoshida, Mari; Yamaguchi, Keiji

    2017-08-31

    A 78-year old woman who presented with akinetic mutism was admitted to our hospital. Brain MRI showed multifocal increased T2/FLAIR signal with extensive cortical-subcortical involvement. We suspected autoimmune encephalitis and the patient received methylprednisolone pulse. Her conscious level gradually recovered, but later relapsed again and presented with refractory status epilepticus. We treated her with intravenous immunoglobulin, plasma exchange and pulsed cyclophosphamide, with satisfactory response. A brain biopsy showed perivascular lymphocytic infiltrates and reactive gliosis. Anti-gamma aminobutyric acid (GABA)A receptor antibodies test came back to be positive after her recovery, and the diagnosis of anti-GABAA receptor antibody-positive encephalitis was made. This is a very rare case where brain biopsies were performed in a patient with anti-GABAA receptor antibody-positive encephalitis.

  9. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin

    DEFF Research Database (Denmark)

    Eghorn, Laura Friis; Høstgaard-Jensen, Kirsten; Kongstad, Kenneth Thermann

    2014-01-01

    conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed......γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate...... whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive...

  10. Ovarian cycle-linked plasticity of δ-GABAA receptor subunits in hippocampal interneurons affects γ oscillations in vivo

    Directory of Open Access Journals (Sweden)

    Albert Miklos Barth

    2014-08-01

    Full Text Available GABAA receptors containing δ subunits (δ-GABAARs are GABA-gated ion channels with extra- and perisynaptic localization, strong sensitivity to neurosteroids (NS, and a high degree of plasticity. In selective brain regions they are expressed on specific principal cells and interneurons (INs, and generate a tonic conductance that controls neuronal excitability and oscillations. Plasticity of δ-GABAARs in principal cells has been described during states of altered NS synthesis including acute stress, puberty, ovarian cycle, pregnancy and the postpartum period, with direct consequences on neuronal excitability and network dynamics. The defining network events implicated in cognitive function, memory formation and encoding are γ oscillations (30-120 Hz, a well-timed loop of excitation and inhibition between principal cells and PV-expressing INs (PV+INs. The δ-GABAARs of INs can modify γ oscillations, and a lower expression of δ-GABAARs on INs during pregnancy alters γ frequency recorded in vitro. The ovarian cycle is another physiological event with large fluctuations in NS levels and δ-GABAARs. Stages of the cycle are paralleled by swings in memory performance, cognitive function, and mood in both humans and rodents. Here we show δ-GABAARs changes during the mouse ovarian cycle in hippocampal cell types, with enhanced expression during diestrus in principal cells and specific INs. The plasticity of δ-GABAARs on PV-INs decreases the magnitude of γ oscillations continuously recorded in area CA1 throughout several days in vivo during diestrus and increases it during estrus. Such recurring changes in γ magnitude were not observed in non-cycling wild-type (WT females, cycling females lacking δ-GABAARs only on PV-INs (PV-Gabrd-/-, and in male mice during a time course equivalent to the ovarian cycle. Our findings may explain the impaired memory and cognitive performance experienced by women with premenstrual syndrome (PMS or premenstrual

  11. NPY Y1 receptors differentially modulate GABAA and NMDA receptors via divergent signal-transduction pathways to reduce excitability of amygdala neurons.

    Science.gov (United States)

    Molosh, Andrei I; Sajdyk, Tammy J; Truitt, William A; Zhu, Weiguo; Oxford, Gerry S; Shekhar, Anantha

    2013-06-01

    Neuropeptide Y (NPY) administration into the basolateral amygdala (BLA) decreases anxiety-like behavior, mediated in part through the Y1 receptor (Y1R) isoform. Activation of Y1Rs results in G-protein-mediated reduction of cAMP levels, which results in reduced excitability of amygdala projection neurons. Understanding the mechanisms linking decreased cAMP levels to reduced excitability in amygdala neurons is important for identifying novel anxiolytic targets. We studied the intracellular mechanisms of activation of Y1Rs on synaptic transmission in the BLA. Activating Y1Rs by [Leu(31),Pro(34)]-NPY (L-P NPY) reduced the amplitude of evoked NMDA-mediated excitatory postsynaptic currents (eEPSCs), without affecting AMPA-mediated eEPSCs, but conversely increased the amplitude of GABAA-mediated evoked inhibitory postsynaptic currents (eIPSCs). Both effects were abolished by the Y1R antagonist, PD160170. Intracellular GDP-β-S, or pre-treatment with either forskolin or 8Br-cAMP, eliminated the effects of L-P NPY on both NMDA- and GABAA-mediated currents. Thus, both the NMDA and GABAA effects of Y1R activation in the BLA are G-protein-mediated and cAMP-dependent. Pipette inclusion of protein kinase A (PKA) catalytic subunit blocked the effect of L-P NPY on GABAA-mediated eIPSCs, but not on NMDA-mediated eEPSCs. Conversely, activating the exchange protein activated by cAMP (Epac) with 8CPT-2Me-cAMP blocked the effect of L-P NPY on NMDA-mediated eEPSCs, but not on GABAA-mediated eIPSCs. Thus, NPY regulates amygdala excitability via two signal-transduction events, with reduced PKA activity enhancing GABAA-mediated eIPSCs and Epac deactivation reducing NMDA-mediated eEPSCs. This multipathway regulation of NMDA- and GABAA-mediated currents may be important for NPY plasticity and stress resilience in the amygdala.

  12. Molecular basis of the alternative recruitment of GABA(A) versus glycine receptors through gephyrin

    DEFF Research Database (Denmark)

    Maric, Hans-Michael; Kasaragod, Vikram Babu; Hausrat, Torben Johann;

    2014-01-01

    γ-Aminobutyric acid type A and glycine receptors (GABA(A)Rs, GlyRs) are the major inhibitory neurotransmitter receptors and contribute to many synaptic functions, dysfunctions and human diseases. GABA(A)Rs are important drug targets regulated by direct interactions with the scaffolding protein ge...... of GABA(A)Rs offers a framework for future investigations into the regulation of inhibitory synaptic strength and for the development of mechanistically and therapeutically relevant compounds targeting the gephyrin-GABA(A)R interaction.......γ-Aminobutyric acid type A and glycine receptors (GABA(A)Rs, GlyRs) are the major inhibitory neurotransmitter receptors and contribute to many synaptic functions, dysfunctions and human diseases. GABA(A)Rs are important drug targets regulated by direct interactions with the scaffolding protein...

  13. Demonstration of the dynamic mass redistribution label-free technology as a useful cell-based pharmacological assay for endogenously expressed GABAA receptors

    DEFF Research Database (Denmark)

    Klein, Anders B.; Nittegaard-Nielsen, Mia; Christensen, Julie T.

    2016-01-01

    Within the continuous quest for the discovery of pharmacol. interesting compds., the development of new and superior drug screening assays is desired. In recent years, the use of label-free techniques has paved the way for an alternative high-throughput screening method. An example is the Epic...... IMR-32 neuroblastoma cell line, which expresses relatively high levels of several endogenous GABAA receptor subunits, we show that GABA produces concn.-dependent cellular responses that can be measured and quantified in real-time. With the aid of the GABAA receptor-specific agonist muscimol...

  14. Phytochemical profiling of Curcuma kwangsiensis rhizome extract, and identification of labdane diterpenoids as positive GABAA receptor modulators.

    Science.gov (United States)

    Schramm, Anja; Ebrahimi, Samad Nejad; Raith, Melanie; Zaugg, Janine; Rueda, Diana C; Hering, Steffen; Hamburger, Matthias

    2013-12-01

    An ethyl acetate extract of Curcuma kwangsiensis S.G. Lee & C.F. Liang (Zingiberaceae) rhizomes (100 μg/ml) enhanced the GABA-induced chloride current (IGABA) through GABAA receptors of the α1β2γ2S subtype by 79.0±7.0%. Potentiation of IGABA was measured using the two-microelectrode voltage-clamp technique and Xenopus laevis oocytes. HPLC-based activity profiling of the crude extract led to the identification of 11 structurally related labdane diterpenoids, including four new compounds. Structure elucidation was achieved by comprehensive analysis of on-line (LC-PDA-ESI-TOF-MS) and off-line (microprobe 1D and 2D NMR) spectroscopic data. The absolute configuration of the compounds was established by comparison of experimental and calculated ECD spectra. Labdane diterpenes represent a new class of plant secondary metabolites eliciting positive GABAA receptor modulation. The highest efficiency was observed for zerumin A (maximum potentiation of IGABA by 309.4±35.6%, and EC50 of 24.9±8.8 μM).

  15. Effects of Chronic Ethanol Consumption on Rat GABAA and Strychnine-sensitive Glycine Receptors Expressed by Lateral/Basolateral Amygdala Neurons

    Science.gov (United States)

    McCool, Brian A.; Frye, Gerald D.; Pulido, Marisa D.; Botting, Shaleen K.

    2010-01-01

    It is well known that the anxiolytic potential of ethanol is maintained during chronic exposure. We have confirmed this using a light-dark box paradigm following chronic ethanol ingestion via a liquid diet. However, cessation from chronic ethanol exposure is known to cause severe withdrawal anxiety. These opposing effects on anxiety likely result from neuro-adaptations of neurotransmitter systems within the brain regions regulating anxiety. Recent work highlights the importance of amygdala ligand-gated chloride channels in the expression of anxiety. We have therefore examined the effects of chronic ethanol exposure on GABAA and strychnine-sensitive glycine receptors expressed by acutely isolated adult rat lateral/basolateral amygdala neurons. Chronic ethanol exposure increased the functional expression of GABAA receptors in acutely isolated basolateral amygdala neurons without altering strychnine-sensitive glycine receptors. Neither the acute ethanol nor benzodiazepine sensitivity of either receptor system was affected. We explored the likelihood that subunit composition might influence each receptor’s response to chronic ethanol. Importantly, when expressed in a mammalian heterologous system, GABAA receptors composed of unique α subunits were differentially sensitive to acute ethanol. Likewise, the presence of the β subunit appeared to influence the acute ethanol sensitivity of glycine receptors containing the α2 subunit. Our results suggest that the facilitation of GABAA receptors during chronic ethanol exposure may help explain the maintenance of ethanol’s anti-anxiety effects during chronic ethanol exposure. Furthermore, the subunit composition of GABAA and strychnine-sensitive glycine receptors may ultimately influence the response of each system to chronic ethanol exposure. PMID:12560122

  16. Differential Modulation of GABAA Receptors Underlies Postsynaptic Depolarization- and Purinoceptor-Mediated Enhancement of Cerebellar Inhibitory Transmission: A Non-Stationary Fluctuation Analysis Study

    Science.gov (United States)

    Ono, Yumie; Saitow, Fumihito; Konishi, Shiro

    2016-01-01

    Cerebellar GABAergic inhibitory transmission between interneurons and Purkinje cells (PCs) undergoes a long-lasting enhancement following different stimulations, such as brief depolarization or activation of purinergic receptors of postsynaptic PCs. The underlying mechanisms, however, are not completely understood. Using a peak-scaled non-stationary fluctuation analysis, we therefore aimed at characterizing changes in the electrophysiological properties of GABAA receptors in PCs of rat cerebellar cortex during depolarization-induced “rebound potentiation (RP)” and purinoceptor-mediated long-term potentiation (PM-LTP), because both RP and PM-LTP likely depend on postsynaptic mechanisms. Stimulation-evoked inhibitory postsynaptic currents (eIPSCs) were recorded from PCs in neonatal rat cerebellar slices. Our analysis showed that postsynaptic membrane depolarization induced RP of eIPSCs in association with significant increase in the number of synaptic GABAA receptors without changing the channel conductance. By contrast, bath application of ATP induced PM-LTP of eIPSCs with a significant increase of the channel conductance of GABAA receptors without affecting the receptor number. Pretreatment with protein kinase A (PKA) inhibitors, H-89 and cAMPS-Rp, completely abolished the PM-LTP. The CaMKII inhibitor KN-62 reported to abolish RP did not alter PM-LTP. These results suggest that the signaling mechanism underlying PM-LTP could involve ATP-induced phosphorylation of synaptic GABAA receptors, thereby resulting in upregulation of the channel conductance by stimulating adenylyl cyclase-PKA signaling cascade, possibly via activation of P2Y11 purinoceptor. Thus, our findings reveal that postsynaptic GABAA receptors at the interneuron-PC inhibitory synapses are under the control of two distinct forms of long-term potentiation linked with different second messenger cascades. PMID:26930485

  17. The General Anesthetic Propofol Excites Nociceptors by Activating TRPV1 and TRPA1 Rather than GABAA Receptors*

    Science.gov (United States)

    Fischer, Michael J. M.; Leffler, Andreas; Niedermirtl, Florian; Kistner, Katrin; Eberhardt, Mirjam; Reeh, Peter W.; Nau, Carla

    2010-01-01

    Anesthetic agents can induce a paradox activation and sensitization of nociceptive sensory neurons and, thus, potentially facilitate pain processing. Here we identify distinct molecular mechanisms that mediate an activation of sensory neurons by 2,6-diisopropylphenol (propofol), a commonly used intravenous anesthetic known to elicit intense pain upon injection. Clinically relevant concentrations of propofol activated the recombinant transient receptor potential (TRP) receptors TRPA1 and TRPV1 heterologously expressed in HEK293t cells. In dorsal root ganglion (DRG) neurons, propofol-induced activation correlated better to expression of TRPA1 than of TRPV1. However, pretreatment with the protein kinase C activator 4β-phorbol 12-myristate 13-acetate (PMA) resulted in a significantly sensitized propofol-induced activation of TRPV1 in DRG neurons as well as in HEK293t cells. Pharmacological and genetic silencing of both TRPA1 and TRPV1 only partially abrogated propofol-induced responses in DRG neurons. The remaining propofol-induced activation was abolished by the selective γ-aminobutyric acid, type A (GABAA) receptor antagonist picrotoxin. Propofol but not GABA evokes a release of calcitonin gene-related peptide, a key component of neurogenic inflammation, from isolated peripheral nerves of wild-type but not TRPV1 and TRPA1-deficient mice. Moreover, propofol but not GABA induced an intense pain upon intracutaneous injection. As both the release of calcitonin gene-related peptide and injection pain by propofol seem to be independent of GABAA receptors, our data identify TRPV1 and TRPA1 as key molecules for propofol-induced excitation of sensory neurons. This study warrants further investigations into the role of anesthetics to induce nociceptor sensitization and to foster postoperative pain. PMID:20826794

  18. Driving Human Motor Cortical Oscillations Leads to Behaviorally Relevant Changes in Local GABAA Inhibition: A tACS-TMS Study.

    Science.gov (United States)

    Nowak, Magdalena; Hinson, Emily; van Ede, Freek; Pogosyan, Alek; Guerra, Andrea; Quinn, Andrew; Brown, Peter; Stagg, Charlotte J

    2017-04-26

    Beta and gamma oscillations are the dominant oscillatory activity in the human motor cortex (M1). However, their physiological basis and precise functional significance remain poorly understood. Here, we used transcranial magnetic stimulation (TMS) to examine the physiological basis and behavioral relevance of driving beta and gamma oscillatory activity in the human M1 using transcranial alternating current stimulation (tACS). tACS was applied using a sham-controlled crossover design at individualized intensity for 20 min and TMS was performed at rest (before, during, and after tACS) and during movement preparation (before and after tACS). We demonstrated that driving gamma frequency oscillations using tACS led to a significant, duration-dependent decrease in local resting-state GABAA inhibition, as quantified by short interval intracortical inhibition. The magnitude of this effect was positively correlated with the magnitude of GABAA decrease during movement preparation, when gamma activity in motor circuitry is known to increase. In addition, gamma tACS-induced change in GABAA inhibition was closely related to performance in a motor learning task such that subjects who demonstrated a greater increase in GABAA inhibition also showed faster short-term learning. The findings presented here contribute to our understanding of the neurophysiological basis of motor rhythms and suggest that tACS may have similar physiological effects to endogenously driven local oscillatory activity. Moreover, the ability to modulate local interneuronal circuits by tACS in a behaviorally relevant manner provides a basis for tACS as a putative therapeutic intervention.SIGNIFICANCE STATEMENT Gamma oscillations have a vital role in motor control. Using a combined tACS-TMS approach, we demonstrate that driving gamma frequency oscillations modulates GABAA inhibition in the human motor cortex. Moreover, there is a clear relationship between the change in magnitude of GABAA inhibition induced

  19. Gender and age differences in expression of GABAA receptor subunits in rat somatosensory thalamus and cortex in an absence epilepsy model.

    Science.gov (United States)

    Li, Huifang; Huguenard, John R; Fisher, Robert S

    2007-03-01

    Absence epilepsy is more prevalent in females, but reasons for this gender asymmetry are unknown. We reported previously that perinatal treatment of Long-Evans Hooded rats with the cholesterol synthesis inhibitor (CSI) AY9944 causes a life-long increase in EEG spike-wave discharges (SWDs), correlated with decreased expression of GABA(A) receptor subunit gamma2 protein levels in thalamic reticular and ventrobasal nuclei (SS thalamus) [Li, H., Kraus, A., Wu, J., Huguenard, J.R., Fisher, R.S., 2006. Selective changes in thalamic and cortical GABA(A) receptor subunits in a model of acquired absence epilepsy in the rat. Neuropharmacology 51, 121-128]. In this study, we explored time course and gender different effects of perinatal AY9944 treatment on expression of GABA(A) receptor alpha1 and gamma2 subunits in SS thalamus and SS cortex. Perinatal AY9944 treatment-induced decreases in GABA(A) gamma2 receptor subunits in rat SS thalamus and increases in SS cortex are gender and age specific. The findings suggest a mechanism for the higher prevalence of absence epilepsy in female patients.

  20. Positive allosteric modulation of the GHB high-affinity binding site by the GABAA receptor modulator monastrol and the flavonoid catechin.

    Science.gov (United States)

    Eghorn, Laura F; Hoestgaard-Jensen, Kirsten; Kongstad, Kenneth T; Bay, Tina; Higgins, David; Frølund, Bente; Wellendorph, Petrine

    2014-10-05

    γ-Hydroxybutyric acid (GHB) is a metabolite of γ-aminobutyric acid (GABA) and a proposed neurotransmitter in the mammalian brain. We recently identified α4βδ GABAA receptors as possible high-affinity GHB targets. GABAA receptors are highly sensitive to allosteric modulation. Thus to investigate whether GHB high-affinity binding sites are also sensitive to allosteric modulation, we screened both known GABAA receptor ligands and a library of natural compounds in the rat cortical membrane GHB specific high-affinity [3H]NCS-382 binding assay. Two hits were identified: Monastrol, a positive allosteric modulator of GABA function at δ-containing GABAA receptors, and the naturally occurring flavonoid catechin. These compounds increased [3H]NCS-382 binding to 185-272% in high micromolar concentrations. Monastrol and (+)-catechin significantly reduced [3H]NCS-382 dissociation rates and induced conformational changes in the binding site, demonstrating a positive allosteric modulation of radioligand binding. Surprisingly, binding of [3H]GHB and the GHB high-affinity site-specific radioligands [125I]BnOPh-GHB and [3H]HOCPCA was either decreased or only weakly increased, indicating that the observed modulation was critically probe-dependent. Both monastrol and (+)-catechin were agonists at recombinant α4β3δ receptors expressed in Xenopus laevis oocytes. When monastrol and GHB were co-applied no changes were seen compared to the individual responses. In summary, we have identified the compounds monastrol and catechin as the first allosteric modulators of GHB high-affinity binding sites. Despite their relatively weak affinity, these compounds may aid in further characterization of the GHB high-affinity sites that are likely to represent certain GABAA receptors.

  1. Regulation of Extrasynaptic GABAA α4 Receptors by Ethanol-Induced Protein Kinase A, but Not Protein Kinase C Activation in Cultured Rat Cerebral Cortical Neurons.

    Science.gov (United States)

    Carlson, Stephen L; Bohnsack, J Peyton; Patel, Vraj; Morrow, A Leslie

    2016-01-01

    Ethanol produces changes in GABAA receptor trafficking and function that contribute to ethanol dependence symptomatology. Extrasynaptic γ-aminobutyric acid A receptors (GABAA-R) mediate inhibitory tonic current and are of particular interest because they are potentiated by physiologically relevant doses of ethanol. Here, we isolate GABAA α4δ receptors by western blotting in subsynaptic fractions to investigate protein kinase A (PKA) and protein kinase C (PKC) modulation of ethanol-induced receptor trafficking, while extrasynaptic receptor function is determined by measurement of tonic inhibition and responses evoked by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or PKA/PKC modulators. Ethanol exposure (1 hour) did not alter GABAA α4 receptor abundance, but it increased tonic current amplitude, an effect that was prevented by inhibiting PKA, but not PKC. Direct activation of PKA, but not PKC, increased the abundance and tonic current of extrasynaptic α4δ receptors. In contrast, prolonged ethanol exposure (4 hours) reduced α4δ receptor abundance as well as tonic current, and this effect was also PKA dependent. Finally, PKC activation by ethanol or phorbol-12,13-dibutyrate (PdBu) had no effect on extrasynaptic α4δ subunit abundance or activity. We conclude that ethanol alters extrasynaptic α4δ receptor function and expression in cortical neurons in a PKA-dependent manner, but ethanol activation of PKC does not influence these receptors. These results could have clinical relevance for therapeutic strategies to restore normal GABAergic functioning for the treatment of alcohol use disorders.

  2. Quantitative Electroencephalography Within Sleep/Wake States Differentiates GABAA Modulators Eszopiclone and Zolpidem From Dual Orexin Receptor Antagonists in Rats

    Science.gov (United States)

    Fox, Steven V; Gotter, Anthony L; Tye, Spencer J; Garson, Susan L; Savitz, Alan T; Uslaner, Jason M; Brunner, Joseph I; Tannenbaum, Pamela L; McDonald, Terrence P; Hodgson, Robert; Yao, Lihang; Bowlby, Mark R; Kuduk, Scott D; Coleman, Paul J; Hargreaves, Richard; Winrow, Christopher J; Renger, John J

    2013-01-01

    Dual orexin receptor antagonists (DORAs) induce sleep by blocking orexin 1 and orexin 2 receptor-mediated activities responsible for regulating wakefulness. DORAs represent a potential alternative mechanism to the current standard of care that includes the γ-aminobutyric acid (GABA)A receptor-positive allosteric modulators, eszopiclone and zolpidem. This work uses an innovative method to analyze electroencephalogram (EEG) spectral frequencies within sleep/wake states to differentiate the effects of GABAA modulators from DORA-22, an analog of the DORA MK-6096, in Sprague–Dawley rats. The effects of low, intermediate, and high doses of eszopiclone, zolpidem, and DORA-22 were examined after first defining each compound's ability to promote sleep during active-phase dosing. The EEG spectral frequency power within specific sleep stages was calculated in 1-Hz intervals from 1 to 100 Hz within each sleep/wake state for the first 4 h after the dose. Eszopiclone and zolpidem produced marked, dose-responsive disruptions in sleep stage-specific EEG spectral profiles compared with vehicle treatment. In marked contrast, DORA-22 exhibited marginal changes in the spectral profile, observed only during rapid eye movement sleep, and only at the highest dose tested. Moreover, while eszopiclone- and zolpidem-induced changes were evident in the inactive period, the EEG spectral responses to DORA-22 were absent during this phase. These results suggest that DORA-22 differs from eszopiclone and zolpidem whereby DORA-22 promotes somnolence without altering the neuronal network EEG activity observed during normal sleep. PMID:23722242

  3. Quantitative electroencephalography within sleep/wake states differentiates GABAA modulators eszopiclone and zolpidem from dual orexin receptor antagonists in rats.

    Science.gov (United States)

    Fox, Steven V; Gotter, Anthony L; Tye, Spencer J; Garson, Susan L; Savitz, Alan T; Uslaner, Jason M; Brunner, Joseph I; Tannenbaum, Pamela L; McDonald, Terrence P; Hodgson, Robert; Yao, Lihang; Bowlby, Mark R; Kuduk, Scott D; Coleman, Paul J; Hargreaves, Richard; Winrow, Christopher J; Renger, John J

    2013-11-01

    Dual orexin receptor antagonists (DORAs) induce sleep by blocking orexin 1 and orexin 2 receptor-mediated activities responsible for regulating wakefulness. DORAs represent a potential alternative mechanism to the current standard of care that includes the γ-aminobutyric acid (GABA)A receptor-positive allosteric modulators, eszopiclone and zolpidem. This work uses an innovative method to analyze electroencephalogram (EEG) spectral frequencies within sleep/wake states to differentiate the effects of GABAA modulators from DORA-22, an analog of the DORA MK-6096, in Sprague-Dawley rats. The effects of low, intermediate, and high doses of eszopiclone, zolpidem, and DORA-22 were examined after first defining each compound's ability to promote sleep during active-phase dosing. The EEG spectral frequency power within specific sleep stages was calculated in 1-Hz intervals from 1 to 100 Hz within each sleep/wake state for the first 4 h after the dose. Eszopiclone and zolpidem produced marked, dose-responsive disruptions in sleep stage-specific EEG spectral profiles compared with vehicle treatment. In marked contrast, DORA-22 exhibited marginal changes in the spectral profile, observed only during rapid eye movement sleep, and only at the highest dose tested. Moreover, while eszopiclone- and zolpidem-induced changes were evident in the inactive period, the EEG spectral responses to DORA-22 were absent during this phase. These results suggest that DORA-22 differs from eszopiclone and zolpidem whereby DORA-22 promotes somnolence without altering the neuronal network EEG activity observed during normal sleep.

  4. How microelectrode array-based chick forebrain neuron biosensors respond to glutamate NMDA receptor antagonist AP5 and GABAA receptor antagonist musimol

    Directory of Open Access Journals (Sweden)

    Serena Y. Kuang

    2016-09-01

    Full Text Available We have established a long-term, stable primary chick forebrain neuron (FBN culture on a microelectrode array platform as a biosensor system for neurotoxicant screening and for neuroelectrophysiological studies for multiple purposes. This paper reports some of our results, which characterize the biosensor pharmacologically. Dose-response experiments were conducted using NMDA receptor antagonist AP5 and GABAA receptor agonist musimol (MUS. The chick FBN biosensor (C-FBN-biosensor responds to the two agents in a pattern similar to that of rodent counterparts; the estimated EC50s (the effective concentration that causes 50% inhibition of the maximal effect are 2.3 μM and 0.25 μM, respectively. Intercultural and intracultural reproducibility and long-term reusability of the C-FBN-biosensor are addressed and discussed. A phenomenon of sensitization of the biosensor that accompanies intracultural reproducibility in paired dose-response experiments for the same agent (AP5 or MUS is reported. The potential application of the C-FBN-biosensor as an alternative to rodent biosensors in shared sensing domains (NMDA receptor and GABAA receptor is suggested.

  5. Negative modulation of GABAA α5 receptors by RO4938581 attenuates discrete sub-chronic and early postnatal phencyclidine (PCP)-induced cognitive deficits in rats.

    Science.gov (United States)

    Redrobe, John P; Elster, Lisbeth; Frederiksen, Kristen; Bundgaard, Christoffer; de Jong, Inge E M; Smith, Garrick P; Bruun, Anne Techau; Larsen, Peter H; Didriksen, Michael

    2012-06-01

    A growing body of evidence suggests that negative modulation of γ-aminobutyric acid (GABA) GABA(A) α5 receptors may be a promising strategy for the treatment of certain facets of cognitive impairment; however, selective modulators of GABA(A) α5 receptors have not yet been tested in "schizophrenia-relevant" cognitive assay/model systems in animals. The objectives of this study were to investigate the potential of RO4938581, a negative modulator of GABA(A) α5 receptors, and to attenuate cognitive impairments induced following sub-chronic (sub-PCP) and early postnatal PCP (neo-PCP) administration in the novel object recognition (NOR) and intra-extradimensional shift (ID/ED) paradigms in rats. Complementary in vitro, ex vivo and in vivo studies were performed to confirm negative modulatory activity of RO4938581 and to investigate animal model validity, concept validity and potential side effect issues, respectively. In vitro studies confirmed the reported negative modulatory activity of RO4938581, whilst immunohistochemical analyses revealed significantly reduced parvalbumin-positive cells in the prefrontal cortex of sub-PCP- and neo-PCP-treated rats. RO4938581 (1 mg/kg) ameliorated both sub-PCP- and neo-PCP-induced cognitive deficits in NOR and ID/ED performance, respectively. In contrast, QH-II-066 (1 and 3 mg/kg), a GABA(A) α5 receptor positive modulator, impaired cognitive performance in the NOR task when administered to vehicle-treated animals. Additional studies revealed that both RO4938581 (1 mg/kg) and QH-II-066 (1 and 3 mg/kg) attenuated amphetamine-induced hyperactivity in rats. Taken together, these novel findings suggest that negative modulation of GABA(A) α5 receptors may represent an attractive treatment option for the cognitive impairments, and potentially positive symptoms, associated with schizophrenia.

  6. Functional expression of the GABAA receptor alpha2 and alpha3 subunits at synapses between intercalated medial paracapsular neurons of mouse amygdala

    Directory of Open Access Journals (Sweden)

    Raffaella eGeracitano

    2012-05-01

    Full Text Available In the amygdala, GABAergic neurons in the intercalated medial paracapsular cluster (Imp have been suggested to play a key role in fear learning and extinction. These neurons project to the central amygdaloid nucleus and to other areas within and outside the amygdala. In addition, they give rise to local collaterals that innervate other neurons in the Imp. Several drugs, including benzodiazepines, are allosteric modulators of GABA-A receptors. Benzodiazepines have both anxiolytic and sedative actions, which are mediated through GABA-A receptors containing alpha2/3 and alpha1 subunits, respectively. To establish whether alpha1 or alpha2/3 subunits are expressed at Imp cell synapses, we used paired recordings of anatomically-identified Imp neurons and high resolution immunocytochemistry in the mouse. We observed that a selective alpha3 subunit agonist, TP003 (100 nM, significantly increased the decay time constant of the unitary IPSCs. A similar effect was also induced by zolpidem (10 microM or by diazepam (1 microM. In contrast, lower doses of zolpidem (0.1-1 microM did not significantly alter the kinetics of the unitary IPSCs. Accordingly, immunocytochemical experiments established that the alpha2 and alpha3, but not the alpha1 subunits of the GABA-A receptors, were present at Imp cell synapses of the mouse amygdala. These results define, for the first time, some of the functional GABA-A receptor subunits expressed at synapses of Imp cells. The data also provide an additional rationale to prompt the search of GABA-A receptor alpha3 selective ligands as improved anxiolytic drugs.

  7. GABA(A) receptors in the pontine reticular formation of C57BL/6J mouse modulate neurochemical, electrographic, and behavioral phenotypes of wakefulness.

    Science.gov (United States)

    Flint, RaShonda R; Chang, Theresa; Lydic, Ralph; Baghdoyan, Helen A

    2010-09-15

    Drugs that potentiate transmission at GABA(A) receptors are widely used to enhance sleep and to cause general anesthesia. The mechanisms underlying these effects are unknown. This study tested the hypothesis that GABA(A) receptors in the pontine reticular nucleus, oral part (PnO) of mouse modulate five phenotypes of arousal: sleep and wakefulness, cortical electroencephalogram (EEG) activity, acetylcholine (ACh) release in the PnO, breathing, and recovery time from general anesthesia. Microinjections into the PnO of saline (vehicle control), the GABA(A) receptor agonist muscimol, muscimol with the GABA(A) receptor antagonist bicuculline, and bicuculline alone were performed in male C57BL/6J mice (n = 33) implanted with EEG recording electrodes. Muscimol caused a significant increase in wakefulness and decrease in rapid eye movement (REM) and non-REM (NREM) sleep. These effects were reversed by coadministration of bicuculline. Bicuculline administered alone caused a significant decrease in wakefulness and increase in NREM sleep and REM sleep. Muscimol significantly increased EEG power in the delta range (0.5-4 Hz) during wakefulness and in the theta range (4-9 Hz) during REM sleep. Dialysis delivery of bicuculline to the PnO of male mice (n = 18) anesthetized with isoflurane significantly increased ACh release in the PnO, decreased breathing rate, and increased anesthesia recovery time. All drug effects were concentration dependent. The effects on phenotypes of arousal support the conclusion that GABA(A) receptors in the PnO promote wakefulness and suggest that increasing GABAergic transmission in the PnO may be one mechanism underlying the phenomenon of paradoxical behavioral activation by some benzodiazepines.

  8. Disruption of brain MEK-ERK sequential phosphorylation and activation during midazolam-induced hypnosis in mice: Roles of GABAA receptor, MEK1 inactivation, and phosphatase MKP-3.

    Science.gov (United States)

    Álvaro-Bartolomé, María; Salort, Glòria; García-Sevilla, Jesús A

    2017-04-03

    Midazolam is a positive allosteric modulator at GABAA receptor that induces a short hypnosis and neuroplasticity, in which the sequential phosphorylation of MEK1/2 and ERK1/2 was shown to play a role. This study investigated the parallel activation of p-MEK and p-ERK and regulatory mechanisms induced by midazolam through the stimulation of GABAA receptors in the mouse brain. During the time course of midazolam (60mg/kg)-induced sleep in mice (lasting for about 2h) p-Ser217/221 MEK1/2 was increased (+146% to +258%) whereas, unexpectedly, p-Tyr204/Thr202 ERK1/2 was found decreased (-16% to -38%), revealing uncoupling of MEK to ERK signals in various brain regions. Midazolam-induced p-MEK1/2 upregulation was prevented by pretreatment (30min) with flumazenil (10mg/kg), indicating the involvement of GABAA receptors. Also unexpectedly, midazolam-induced p-ERK1/2 downregulation was not prevented by flumazenil (10 or 30mg/kg). Notably, during midazolam-induced sleep the content of inactivated p-Thr286 MEK1, which can dampen ERK1/2 activation, was increased (+33% to +149%) through a mechanism sensitive to flumazenil (10mg/kg). Midazolam also increased MKP-3 (+13% to +73%) content and this upregulation was prevented by flumazenil (10mg/kg); an effect suggesting ERK inactivation because MKP-3 is the phosphatase selective for ERK1/2 dephosphorylation. The results indicate that during midazolam-induced sleep in mice there is an uncoupling of p-MEK (increased) to p-ERK (decreased) signals. p-ERK1/2 downregulation (not involving GABAA receptors) is the result of increased inactivated MEK1 and phosphatase MKP-3 (both effects involving GABAA receptors). These findings are relevant for the neurobiology and clinical use of benzodiazepines. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. GABAA receptor γ2 subunit knockdown mice have enhanced anxiety-like behavior but unaltered hypnotic response to benzodiazepines

    Directory of Open Access Journals (Sweden)

    De Blas Angel L

    2005-04-01

    Full Text Available Abstract Background Gamma-aminobutyric acid type A receptors (GABAA-Rs are the major inhibitory receptors in the mammalian brain and are modulated by a number of sedative/hypnotic drugs including benzodiazepines and anesthetics. The significance of specific GABAA-Rs subunits with respect to behavior and in vivo drug responses is incompletely understood. The γ2 subunit is highly expressed throughout the brain. Global γ2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous γ2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the γ2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated γ2 expression, i.e., γ2 knockdown mice. Results Knockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the γ2 gene. Knockdown mice, on average, showed a 65% reduction of γ2 subunit mRNA compared to controls; however γ2 gene expression was highly variable in these mice, ranging from 10–95% of normal. Immunohistochemical studies demonstrated that γ2 protein levels were also variably reduced. Pharmacological studies using autoradiography on frozen brain sections demonstrated that binding of the benzodiazepine site ligand Ro15-4513 was decreased in mutant mice compared to controls. Behaviorally, knockdown mice displayed enhanced anxiety-like behaviors on the elevated plus maze and forced novelty exploration tests. Surprisingly, mutant mice had an unaltered response to hypnotic doses of the benzodiazepine site ligands diazepam, midazolam and zolpidem as well as ethanol and pentobarbital. Lastly, we demonstrated that the γ2 knockdown mouse line can be used to create γ2 global knockout mice by crossing to a general deleter cre-expressing mouse line. Conclusion We conclude that: 1 insertion of a neomycin resistance gene into intron 8 of the γ2 gene variably

  10. Antinociceptive effects of fisetin against diabetic neuropathic pain in mice: Engagement of antioxidant mechanisms and spinal GABAA receptors.

    Science.gov (United States)

    Zhao, Xin; Li, Xin-Lin; Liu, Xin; Wang, Chuang; Zhou, Dong-Sheng; Ma, Qing; Zhou, Wen-Hua; Hu, Zhen-Yu

    2015-12-01

    Peripheral painful neuropathy is one of the most common complications in diabetes and necessitates improved treatment. Fisetin, a naturally occurring flavonoid, has been reported to exert antidepressant-like effect in previous studies. As antidepressant drugs are employed clinically to treat neuropathic pain, this work aimed to investigate whether fisetin possess beneficial effect on diabetic neuropathic pain and explore the mechanism(s). We subjected mice to diabetes by a single intraperitoneal (i.p.) injection of streptozotocin (200mg/kg), and von Frey test or Hargreaves test was used to assess mechanical allodynia or thermal hyperalgesia, respectively. Chronic treatment of diabetic mice with fisetin not only ameliorated the established symptoms of thermal hyperalgesia and mechanical allodynia, but also arrested the development of neuropathic pain when given at low doses. Although chronic fisetin administration did not impact on the symptom of hyperglycemia in diabetic mice, it reduced exacerbated oxidative stress in tissues of spinal cord, dorsal root ganglion (DRG) and sciatic verve. Furthermore, the analgesic actions of fisetin were abolished by repetitive co-treatment with the reactive oxygen species (ROS) donor tert-butyl hydroperoxide (t-BOOH), but potentiated by the ROS scavenger phenyl-N-tert-butylnitrone (PBN). Finally, acute blockade of spinal GABAA receptors by bicuculline totally counteracted such fisetin analgesia. These findings indicate that chronic fisetin treatment can delay or correct neuropathic hyperalgesia and allodynia in mice with type 1 diabetes. Mechanistically, the present fisetin analgesia may be associated with its antioxidant activity, and spinal GABAA receptors are likely rendered as downstream targets. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Quantification of GABAA receptors in the rat brain with [(123)I]Iomazenil SPECT from factor analysis-denoised images.

    Science.gov (United States)

    Tsartsalis, Stergios; Moulin-Sallanon, Marcelle; Dumas, Noé; Tournier, Benjamin B; Ghezzi, Catherine; Charnay, Yves; Ginovart, Nathalie; Millet, Philippe

    2014-02-01

    In vivo imaging of GABAA receptors is essential for the comprehension of psychiatric disorders in which the GABAergic system is implicated. Small animal SPECT provides a modality for in vivo imaging of the GABAergic system in rodents using [(123)I]Iomazenil, an antagonist of the GABAA receptor. The goal of this work is to describe and evaluate different quantitative reference tissue methods that enable reliable binding potential (BP) estimations in the rat brain to be obtained. Five male Sprague-Dawley rats were used for [(123)I]Iomazenil brain SPECT scans. Binding parameters were obtained with a one-tissue compartment model (1TC), a constrained two-tissue compartment model (2TCc), the two-step Simplified Reference Tissue Model (SRTM2), Logan graphical analysis and analysis of delayed-activity images. In addition, we employed factor analysis (FA) to deal with noise in data. BPND obtained with SRTM2, Logan graphical analysis and delayed-activity analysis was highly correlated with BPF values obtained with 2TCc (r=0.954 and 0.945 respectively, p<0.0001). Equally significant correlations were found between values obtained with 2TCc and SRTM2 in raw and FA-denoised images (r=0.961 and 0.909 respectively, p<0.0001). Scans of at least 100min are required to obtain stable BPND values from raw images while scans of only 70min are sufficient from FA-denoised images. These images are also associated with significantly lower standard errors of 2TCc and SRTM2 BP values. Reference tissue methods such as SRTM2 and Logan graphical analysis can provide equally reliable BPND values from rat brain [(123)I]Iomazenil SPECT. Acquisitions, however, can be much less time-consuming either with analysis of delayed activity obtained from a 20-minute scan 50min after tracer injection or with FA-denoising of images. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. CB1 cannabinoid receptor-mediated anandamide signalling reduces the defensive behaviour evoked through GABAA receptor blockade in the dorsomedial division of the ventromedial hypothalamus.

    Science.gov (United States)

    Dos Anjos-Garcia, Tayllon; Ullah, Farhad; Falconi-Sobrinho, Luiz Luciano; Coimbra, Norberto Cysne

    2017-02-01

    The effects of cannabinoids in brain areas expressing cannabinoid receptors, such as hypothalamic nuclei, are not yet well known. Several studies have demonstrated the role of hypothalamic nuclei in the organisation of behavioural responses induced through innate fear and panic attacks. Panic-prone states are experimentally induced in laboratory animals through a reduction in the GABAergic activity. The aim of the present study was to examine panic-like elaborated defensive behaviour evoked by GABAA receptor blockade with bicuculline (BIC) in the dorsomedial division of the ventromedial hypothalamus (VMHdm). We also aimed to characterise the involvement of endocannabinoids and the CB1 cannabinoid receptor in the modulation of elaborated defence behavioural responses organised with the VMHdm. The guide-cannula was stereotaxicaly implanted in VMHdm and the animals were treated with anandamide (AEA) at different doses, and the effective dose was used after the pre-treatment with the CB1 receptor antagonist AM251, followed by GABAA receptor blockade in VMHdm. The results showed that the intra-hypothalamic administration of AEA at an intermediate dose (5 pmol) attenuated defence responses induced through the intra-VMHdm microinjection of bicuculline (40 ng). This effect, however, was inhibited when applied central microinjection of the CB1 receptor antagonist AM251 in the VMHdm. Moreover, AM251 potentiates de non-oriented escape induced by bicuculline, effect blocked by pre-treatment with the TRPV1 channel antagonist 6-I-CPS. These results indicate that AEA modulates the pro-aversive effects of intra-VMHdm-bicuculline treatment, recruiting CB1 cannabinoid receptors and the TRPV1 channel is involved in the AM251-related potentiation of bicuculline effects on non-oriented escape behaviour.

  13. Allopregnanolone microinjected into the lateral septum or dorsal hippocampus reduces immobility in the forced swim test: participation of the GABAA receptor.

    Science.gov (United States)

    Rodríguez-Landa, Juan Francisco; Contreras, Carlos M; García-Ríos, Rosa Isela

    2009-10-01

    Allopregnanolone is a 5α-reduced metabolite of progesterone with actions on γ-aminobutyric acid-A (GABAA) receptors that produce antidepressant-like effects. However, little is known about the target brain regions that mediate its antidepressant-like effects. In this study, allopregnanolone (2.0 μg/0.3 μl/rat) or its vehicle (35% cyclodextrin solution) were microinjected into the lateral septum, septofimbrial, or dorsal hippocampus of male Wistar rats that had previously received intraperitoneal injections of either saline or the GABAA antagonist bicuculline (1.0 mg/kg), and its effects were evaluated in the open field and forced swim tests. Allopregnanolone microinjected into the lateral septum or dorsal hippocampus, but not septofimbrial nucleus, induced a longer latency to the first immobility and a shorter total immobility time in the forced swim test compared with vehicle. Bicuculline pretreatment reversed the effect of allopregnanolone. None of the treatments produced significant changes in crossings in the open field test. In conclusion, allopregnanolone produces an antidepressant-like effect in rats submitted to the forced swim test through actions on GABAA receptors located in the lateral septum and dorsal hippocampus, which is consistent with the antistress effect of GABAA agonists in these particular brain structures.

  14. Expression of functional receptors by the human γ-aminobutyric acid A γ2 subunit

    Science.gov (United States)

    Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2004-01-01

    γ-Aminobutyric acid A (GABAA) receptors are heteromeric membrane proteins formed mainly by various combinations of α, β, and γ subunits; and it is commonly thought that the γ2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the γ2L subunit of the human GABAA receptor, injected alone into Xenopus oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique. GABA elicited desensitizing membrane currents that recovered after a few minutes' wash. Repetitive applications of GABA induced a “run-up” of GABA currents that nearly doubled the amplitude of the first response. The GABA currents inverted direction at about -30 mV, indicating that they are carried mainly by Cl- ions. The homomeric γ2L receptors were also activated by β-alanine > taurine > glycine, and, like some types of heteromeric GABAA receptors, the γ2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human γ2L subunit is capable of forming fully functional GABA receptors by itself in Xenopus oocytes and suggest that the roles proposed for the various subunits that make up the heteromeric GABAA receptors in situ require further clarification. PMID:14981251

  15. The muscle relaxant thiocolchicoside is an antagonist of GABAA receptor function in the central nervous system.

    Science.gov (United States)

    Carta, Mario; Murru, Luca; Botta, Paolo; Talani, Giuseppe; Sechi, GianPietro; De Riu, PierLuigi; Sanna, Enrico; Biggio, Giovanni

    2006-09-01

    Thiocolchicoside (TCC) is used clinically for its muscle relaxant, anti-inflammatory, and analgesic properties, and it has been shown to interact with gamma-aminobutyric acid (GABA) type A receptors (GABAARs) and strychnine-sensitive glycine receptors in the rat central nervous system. In contrast to a proposed agonistic action at these two types of inhibitory receptors, pharmacological evidence has shown that, under certain conditions, TCC manifests convulsant activity in animals and humans. We now show that the phasic and tonic GABAAR-mediated currents recorded from Purkinje cells and granule neurons, respectively, in parasagittal cerebellar slices from adult male rats were inhibited by TCC in a concentration-dependent manner. The median inhibitory concentrations of TCC for these effects were approximately 0.15 and approximately 0.9 microM, respectively. TCC did not potentiate GABABR-mediated currents in hippocampal slices, suggesting that its muscle relaxant action is not mediated by GABABRs. Intraperitoneal injection of TCC in rats either alone or in combination with negative modulators of GABAergic transmission revealed convulsant and proconvulsant actions of this drug. Our data, consistent with clinical observations of the epileptogenic effect of this compound, suggest that TCC is a potent competitive antagonist of GABAAR function.

  16. The parvalbumin-positive interneurons in the mouse dentate gyrus express GABAA receptor subunits α1, β2, and δ along their extrasynaptic cell membrane.

    Science.gov (United States)

    Milenkovic, I; Vasiljevic, M; Maurer, D; Höger, H; Klausberger, T; Sieghart, W

    2013-12-19

    Neuronal circuitries in the hippocampus are involved in navigation and memory and are controlled by major networks of GABAergic interneurons. Parvalbumin (PV)-expressing interneurons in the dentate gyrus (DG) are identified as fast-spiking cells, playing a crucial role in network oscillation and synchrony. The inhibitory modulation of these interneurons is thought to be mediated mainly through GABAA receptors, the major inhibitory neurotransmitter receptors in the brain. Here we show that all PV-positive interneurons in the granular/subgranular layer (GL/SGL) of the mouse DG express high levels of the GABAA receptor δ subunit. PV-containing interneurons in the hilus and the molecular layer, however, express the δ subunit to a lower extent. Only 8% of the somatostatin-containing interneurons express the δ subunit, whereas calbindin- or calretinin-containing interneurons in the DG seem not to express the GABAA receptor δ subunit at all. Hence, these cells receive a GABAergic control different from that of PV-containing interneurons in the GL/SGL. Experiments investigating a possible co-expression of GABAA receptor α1, α2, α3, α4, α5, β1, β2, β3, or γ2 subunits with PV and δ subunits indicated that α1 and β2 subunits are co-expressed with δ subunits along the extrasynaptic membranes of PV-interneurons. These results suggest a robust tonic GABAergic control of PV-containing interneurons in the GL/SGL of the DG via δ subunit-containing receptors. Our data are important for better understanding of the neuronal circuitries in the DG and the role of specific cell types under pathological conditions.

  17. The anti-convulsant stiripentol acts directly on the GABA(A) receptor as a positive allosteric modulator.

    Science.gov (United States)

    Fisher, Janet L

    2009-01-01

    Stiripentol (STP) has been used as co-therapy for treatment of epilepsy for many years. Its mechanism of action has long been considered to be indirect, as it inhibits the enzymes responsible for metabolism of other anti-convulsant agents. However, a recent report suggested that STP might also act at the neuronal level, increasing inhibitory GABAergic neurotransmission. We examined the effect of STP on the functional properties of recombinant GABA(A) receptors (GABARs) and found that it was a positive allosteric modulator of these ion channels. Its activity showed some dependence on subunit composition, with greater potentiation of alpha3-containing receptors and reduced potentiation when the beta1 or epsilon subunits were present. STP caused a leftward shift in the GABA concentration-response relationship, but did not increase the peak response of the receptors to a maximal GABA concentration. Although STP shares some functional characteristics with the neurosteroids, its activity was not inhibited by a neurosteroid site antagonist and was unaffected by a mutation in the alpha3 subunit that reduced positive modulation by neurosteroids. The differential effect of STP on beta1- and beta2/beta3-containing receptors was not altered by mutations within the second transmembrane domain that affect modulation by loreclezole. These findings suggest that STP acts as a direct allosteric modulator of the GABAR at a site distinct from many commonly used anti-convulsant, sedative and anxiolytic drugs. Its higher activity at alpha3-containing receptors as well as its activity at delta-containing receptors may provide a unique opportunity to target selected populations of GABARs.

  18. Effect of extracellular pH on recombinant alpha1beta2gamma2 and alpha1beta2 GABAA receptors.

    Science.gov (United States)

    Mercik, Katarzyna; Pytel, Maria; Cherubini, Enrico; Mozrzymas, Jerzy W

    2006-08-01

    Recently, we have reported that extracellular protons allosterically modulated neuronal GABA(A) receptors [Mozrzymas, J.W., Zarnowska, E.D., Pytel, M., Mercik, K., 2003a. Modulation of GABA(A) receptors by hydrogen ions reveals synaptic GABA transient and a crucial role of desensitiztion process. Journal of Neuroscience 23, 7981-7992]. However, GABAARs in neurons are heterogeneous and the effect of hydrogen ions depends on the receptor subtype. In particular, gamma2 subunit sets the receptor sensibility to several modulators including protons. However, the mechanisms whereby protons modulate gamma2-containing and gamma2-free GABAARs have not been fully elucidated. To this end, current responses to ultrafast GABA applications were recorded for alpha1beta2gamma2 and alpha1beta2 receptors at different pH values. For both receptor types, increase in pH induced a decrease in amplitudes of currents elicited by saturating [GABA] but this effect was stronger for alpha1beta2 receptors. In the case of alpha1beta2gamma2 receptors, protons strongly affected the current time course due to a down regulation of binding and desensitization rates. This effect was qualitatively similar to that described in neurons. Protons strongly influenced the amplitude of alpha1beta2 receptor-mediated currents but the effect on their kinetics was weak suggesting a predominant direct non-competitive inhibition with a minor allosteric modulation. In conclusion, we provide evidence that extracellular protons strongly affect GABAA receptors and that, depending on the presence of the gamma2 subunit, the modulatory mechanisms show profound quantitative and qualitative differences.

  19. Liver biomarker and in vitro assessment confirm the hepatic origin of aminotransferase elevations lacking histopathological correlate in beagle dogs treated with GABAA receptor antagonist NP260.

    Science.gov (United States)

    Harrill, Alison H; Eaddy, John S; Rose, Kelly; Cullen, John M; Ramanathan, Lakshmi; Wanaski, Stephen; Collins, Stephen; Ho, Yu; Watkins, Paul B; Lecluyse, Edward L

    2014-06-01

    NP260 was designed as a first-in-class selective antagonist of α4-subtype GABAA receptors that had promising efficacy in animal models of pain, epilepsy, psychosis, and anxiety. However, development of NP260 was complicated following a 28-day safety study in dogs in which pronounced elevations of serum aminotransferase levels were observed, although there was no accompanying histopathological indication of hepatocellular injury. To further investigate the liver effects of NP260, we assayed stored serum samples from the 28-day dog study for liver specific miRNA (miR-122) as well as enzymatic biomarkers glutamate dehydrogenase and sorbitol dehydrogenase, which indicate liver necrosis. Cytotoxicity assessments were conducted in hepatocytes derived from dog, rat, and human liver samples to address the species specificity of the liver response to NP260. All biomarkers, except ALT, returned toward baseline by Day 29 despite continued drug treatment, suggesting adaptation to the initial injury. In vitro analysis of the toxicity potential of NP260 to primary hepatocytes indicated a relative sensitivity of dog>human>rat, which may explain, in part, why the liver effects were not evident in the rodent safety studies. Taken together, the data indicate that a diagnostic biomarker approach, coupled with sensitive in vitro screening strategies, may facilitate interpretation of toxicity potential when an adaptive event masks the underlying toxicity.

  20. Effects of essential amino acid deficiency: down-regulation of KCC2 and the GABAA receptor; disinhibition in the anterior piriform cortex.

    Science.gov (United States)

    Sharp, James W; Ross-Inta, Catherine M; Baccelli, Irène; Payne, John A; Rudell, John B; Gietzen, Dorothy W

    2013-11-01

    The anterior piriform cortex (APC) is activated by, and is the brain area most sensitive to, essential (indispensable) amino acid (IAA) deficiency. The APC is required for the rapid (20 min) behavioral rejection of IAA deficient diets and increased foraging, both crucial adaptive functions supporting IAA homeostasis in omnivores. The biochemical mechanisms signaling IAA deficiency in the APC block initiation of translation in protein synthesis via uncharged tRNA and the general amino acid control kinase, general control nonderepressing kinase 2. Yet, how inhibition of protein synthesis activates the APC is unknown. The neuronal K(+) Cl(-) cotransporter, neural potassium chloride co-transporter (KCC2), and GABAA receptors are essential inhibitory elements in the APC with short plasmalemmal half-lives that maintain control in this highly excitable circuitry. After a single IAA deficient meal both proteins were reduced (vs. basal diet controls) in western blots of APC (but not neocortex or cerebellum) and in immunohistochemistry of APC. Furthermore, electrophysiological analyses support loss of inhibitory elements such as the GABAA receptor in this model. As the crucial inhibitory function of the GABAA receptor depends on KCC2 and the Cl(-) transmembrane gradient it establishes, these results suggest that loss of such inhibitory elements contributes to disinhibition of the APC in IAA deficiency. The circuitry of the anterior piriform cortex (APC) is finely balanced between excitatory (glutamate, +) and inhibitory (GABA, -) transmission. GABAA receptors use Cl(-), requiring the neural potassium chloride co-transporter (KCC2). Both are rapidly turning-over proteins, dependent on protein synthesis for repletion. In IAA (indispensable amino acid) deficiency, within 20 min, blockade of protein synthesis prevents restoration of these inhibitors; they are diminished; disinhibition ensues. GCN2 = general control non-derepressing kinase 2, eIF2α = α-subunit of the eukaryotic

  1. Duration of treatment and activation of α1-containing GABAA receptors variably affect the level of anxiety and seizure susceptibility after diazepam withdrawal in rats.

    Science.gov (United States)

    Kovačević, Jovana; Timić, Tamara; Tiruveedhula, Veera V; Batinić, Bojan; Namjoshi, Ojas A; Milić, Marija; Joksimović, Srđan; Cook, James M; Savić, Miroslav M

    2014-05-01

    Long-term use of benzodiazepine-type drugs may lead to physical dependence, manifested by withdrawal syndrome after abrupt cessation of treatment. The aim of the present study was to investigate the influence of duration of treatment, as well as the role of α1-containing GABAA receptors, in development of physical dependence to diazepam, assessed through the level of anxiety and susceptibility to pentylenetetrazole (PTZ)-induced seizures, 24h after withdrawal from protracted treatment in rats. Withdrawal of 2mg/kg diazepam after 28, but not after 14 or 21 days of administration led to an anxiety-like behavior in the elevated plus maze. Antagonism of the diazepam effects at α1-containing GABAA receptors, achieved by daily administration of the neutral modulator βCCt (5mg/kg), did not affect the anxiety level during withdrawal. An increased susceptibility to PTZ-induced seizures was observed during diazepam withdrawal after 21 and 28 days of treatment. Daily co-administration of βCCt further decreased the PTZ-seizure threshold after 21 days of treatment, whilst it prevented the diazepam withdrawal-elicited decrease of the PTZ threshold after 28 days of treatment. In conclusion, the current study suggests that the role of α1-containing GABAA receptors in mediating the development of physical dependence may vary based on the effect being studied and duration of protracted treatment. Moreover, the present data supports previous findings that the lack of activity at α1-containing GABAA receptors is not sufficient to eliminate physical dependence liability of ligands of the benzodiazepine type.

  2. Differential distribution of GABAA receptor subunits in soma and processes of cerebellar granule cells: effects of maturation and a GABA agonist

    DEFF Research Database (Denmark)

    Elster, L; Hansen, Gert Helge; Belhage, B;

    1995-01-01

    or absence of the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridin-3-ol (THIP). THIP (150 microM) induced a 2-fold increase in the number of alpha 1 and beta 2/3 subunits in both cell bodies and processes in 4-day-old cultures. Extending the culture period to 8 days led to a polarization...... composition. Interestingly, receptor subunit clusters, consisting of alpha 1 alone, were more frequently observed than composite (alpha 1; beta 2/3) clusters. This substantiates the view that receptors not having alpha 1 and beta 2/3 subunits in the same complex may exist....

  3. Glycine and GABAA receptors mediate tonic and phasic inhibitory processes that contribute to prepulse inhibition in the goldfish startle network

    Directory of Open Access Journals (Sweden)

    Paul C.P. Curtin

    2015-03-01

    Full Text Available Prepulse inhibition (PPI is understood as an inhibitory process that attenuates sensory flow during early stages (20-1000ms of information processing. Here, we applied in vivo electrophysiology and pharmacology to determine if prepulse inhibition (PPI is mediated by glycine receptors (GlyRs and/or GABAA receptors (GABAARs in the goldfish auditory startle circuit. Specifically, we used selective antagonists to dissect the contributions of target receptors on sound-evoked postsynaptic potentials (PSPs recorded in the neurons that initiate startle, the Mauthner-cells (M-cell. We found that strychnine, a GlyR antagonist, disrupted a fast-activated (5 ms and rapidly (< 50ms decaying (feed-forward inhibitory process that disrupts PPI at 20 ms prepulse/pulse inter-stimulus intervals (ISI. Additionally we observed increases of the evoked postsynaptic potential (PSP peak amplitude (+87.43 ± 21.53%; N=9 and duration (+204 ± 48.91%, N=9. In contrast, treatment with bicuculline, a GABAAR antagonist, caused a general reduction in PPI across all tested ISIs (20-500 ms, essentially eliminating PPI at ISIs from 20-100 ms. Bicuculline also increased PSP peak amplitude (+133.8 ± 10.3%, N=5 and PSP duration (+284.95 ± 65.64%, N=5. Treatment with either antagonist also tonically increased post-synaptic excitability in the M-cells, reflected by an increase in the magnitude of antidromically-evoked action potentials (APs by 15.07 ± 3.21%, N=7 and 16.23 ± 7.08%, N=5 for strychnine and bicuculline, respectively. These results suggest that GABAARs and GlyRs are functionally segregated to short- and longer-lasting sound-evoked (phasic inhibitory processes that contribute to PPI, with the mediation of tonic inhibition by both receptor systems being critical for gain control within the M-cell startle circuit.

  4. GABA(A receptors containing the α2 subunit are critical for direction-selective inhibition in the retina.

    Directory of Open Access Journals (Sweden)

    Olivia Nicola Auferkorte

    Full Text Available Far from being a simple sensor, the retina actively participates in processing visual signals. One of the best understood aspects of this processing is the detection of motion direction. Direction-selective (DS retinal circuits include several subtypes of ganglion cells (GCs and inhibitory interneurons, such as starburst amacrine cells (SACs. Recent studies demonstrated a surprising complexity in the arrangement of synapses in the DS circuit, i.e. between SACs and DS ganglion cells. Thus, to fully understand retinal DS mechanisms, detailed knowledge of all synaptic elements involved, particularly the nature and localization of neurotransmitter receptors, is needed. Since inhibition from SACs onto DSGCs is crucial for generating retinal direction selectivity, we investigate here the nature of the GABA receptors mediating this interaction. We found that in the inner plexiform layer (IPL of mouse and rabbit retina, GABA(A receptor subunit α2 (GABA(AR α2 aggregated in synaptic clusters along two bands overlapping the dendritic plexuses of both ON and OFF SACs. On distal dendrites of individually labeled SACs in rabbit, GABA(AR α2 was aligned with the majority of varicosities, the cell's output structures, and found postsynaptically on DSGC dendrites, both in the ON and OFF portion of the IPL. In GABA(AR α2 knock-out (KO mice, light responses of retinal GCs recorded with two-photon calcium imaging revealed a significant impairment of DS responses compared to their wild-type littermates. We observed a dramatic drop in the proportion of cells exhibiting DS phenotype in both the ON and ON-OFF populations, which strongly supports our anatomical findings that α2-containing GABA(ARs are critical for mediating retinal DS inhibition. Our study reveals for the first time, to the best of our knowledge, the precise functional localization of a specific receptor subunit in the retinal DS circuit.

  5. Die physiologische Funktion der GABAA Autorezeptoren im Kleinhirn

    OpenAIRE

    Chavas, Joël

    1999-01-01

    Diplomarbeit from Joel Chavas at the university of Goettingen. Supervisor: Dr Alain Marty; The physiological effect of GABAA autoreceptors in the cerebellum. In this Diplomarbeit, I measured the reversal potential of GABAA receptors (-56 mV) in the interneurons of the rat cerebellum, using gramicidin perforated patch, . I then determined that the GABAA autoreceptors can be excitatory. Last, I proved that a tonic activation of the GABAA receptors increases the intracellular calcium concentrati...

  6. Allopregnanolone suppresses diabetes-induced neuropathic pain and motor deficit through inhibition of GABAA receptor down-regulation in the spinal cord of diabetic rats

    Directory of Open Access Journals (Sweden)

    Samira Afrazi

    2014-05-01

    Full Text Available Objective(s:Painful diabetic neuropathy is associated with hyperexcitability and hyperactivity of spinal cord neurons. However, its underlying pathophysiological mechanisms have not been fully clarified. Induction of excitatory/inhibitory neurotransmission imbalance at the spinal cord seems to account for the abnormal neuronal activity in diabetes. Protective properties of neurosteroids have been demonstrated in numerous cellular and animal models of neurodegeneration. Materials and Methods: Here, the protective effects of allopregnanolone, a neurosteroid were investigated in an in vivo model of diabetic neuropathy. The tail-flick test was used to assess the nociceptive threshold. Diabetes was induced by injection of 50 mg/kg (IP streptozotocin. Seven weeks after the induction of diabetes, the dorsal half of the lumbar spinal cord was assayed for the expression of γ2 subunit of GABAA receptor using semiquantitative RT-PCR. Results: The data shows that allopregnanolone (5 and 20 mg/kg markedly ameliorated diabetes-induced thermal hyperalgesia and motor deficit. The weights of diabetic rats that received 5 and 20 mg/kg allopregnanolone did not significantly reduce during the time course of study. Furthermore, this neurosteroid could inhibit GABAA receptor down-regulation induced by diabetes in the rat spinal cord. Conclusion: The data revealed that allopregnanolone has preventive effects against hyperglycemic-induced neuropathic pain and motor deficit which are related to the inhibition of GABAA receptor down-regulation.

  7. The influence of stress at puberty on mood and learning: role of the α4βδ GABAA receptor.

    Science.gov (United States)

    Smith, S S

    2013-09-26

    It is well-known that the onset of puberty is associated with changes in mood as well as cognition. Stress can have an impact on these outcomes, which in many cases, can be more influential in females, suggesting that gender differences exist. The adolescent period is a vulnerable time for the onset of certain psychopathologies, including anxiety disorders, depression and eating disorders, which are also more prevalent in females. One factor which may contribute to stress-triggered anxiety at puberty is the GABAA receptor (GABAR), which is known to play a pivotal role in anxiety. Expression of α4βδ GABARs increases on the dendrites of CA1 pyramidal cells at the onset of puberty in the hippocampus, part of the limbic circuitry which governs emotion. This receptor is a sensitive target for the stress steroid 3α-OH-5[α]β-pregnan-20-one or [allo]pregnanolone, which paradoxically reduces inhibition and increases anxiety during the pubertal period (post-natal day ∼35-44) of female mice in contrast to its usual effect to enhance inhibition and reduce anxiety. Spatial learning and synaptic plasticity are also adversely impacted at puberty, likely a result of increased expression of α4βδ GABARs on the dendritic spines of CA1 hippocampal pyramidal cells, which are essential for consolidation of memory. This review will focus on the role of these receptors in mediating behavioral changes at puberty. Stress-mediated changes in mood and cognition in early adolescence may have relevance for the expression of psychopathologies in adulthood.

  8. Anticonvulsive Activity in Audiogenic DBA/2 Mice of 1,4-Benzodiazepines and 1,5-Benzodiazepines with Different Activities at Cerebellar Granule Cell GABAA Receptors.

    Science.gov (United States)

    Gatta, Elena; Cupello, Aroldo; Di Braccio, Mario; Grossi, Giancarlo; Robello, Mauro; Scicchitano, Francesca; Russo, Emilio; De Sarro, Giovambattista

    2016-12-01

    Herein, we tested in a model of generalized reflex epilepsy in mice different 1,4-benzodiazepines and 1,5-benzodiazepines with agonistic activity at the GABAA receptor population contributing to the peak component of the chloride current elicited by GABA in cerebellar granule cells (CGCs) in culture. The substances have all higher lipophilia than clobazam, an antiepileptic drug well known and used in human therapy. This ensures that they all can pass relatively easily the blood-brain barrier (BBB). The benzodiazepines were administered intraperitoneally (i.p.) and tested for their activity against sound-induced tonic and clonic seizures in a genetic model of experimental epilepsy, the DBA/2 mouse. Our data demonstrates an interesting inverse correlation between the ED50s and the efficacy (E %) of the drugs in increasing the peak chloride current elicited by GABA in cerebellar granule cells in culture. There is indication of the existence of a threshold of E % above which the increase of ED50 with increasing E % becomes linear. This is statistically significant for the clonic phase, whereas it is at the limit of significance for the tonic one. A possible interpretation of these results is that in this epilepsy model, projections from the cerebellum exert a convulsion prevention activity.

  9. Molecular cloning and expression analysis of GABA(A) receptor-associated protein (GABARAP) from small abalone, Haliotis diversicolor.

    Science.gov (United States)

    Bai, Rongyao; You, Weiwei; Chen, Jun; Huang, Heqing; Ke, Caihuan

    2012-10-01

    GABA(A) receptor-associated protein (GABARAP), a multifunctional protein participating in autophagy process, is evolutionarily conserved and involves in innate immunity in eukaryotic cells, but currently there is no research on the relationship between GABARAP and innate immunity in mollusc. In the present study, the GABARAP full-length cDNA and its genomic DNA were firstly cloned from small abalone (Haliotis diversicolor), which was named as saGABARAP. Its full-length cDNA is 963 bp with a 354 bp open reading frame encoding a protein of 117 aa, a 276 bp 5'-UTR, and a 333 bp 3'-UTR including a poly(A) tail, two typical polyadenylation signals (AATAA) and two RNA instability motifs (ATTTA). The deduced protein has an estimated molecular weight of 13.9 kDa and a predicted PI of 8.73. Its genomic DNA comprises 4352 bp, containing three exons and two introns. Quantitative real-time PCR analysis revealed that saGABARAP was constitutively expressed in all examined tissues, with the highest expression level in hepatopancreas, and was upregulated in hepatopancreas and hemocytes after bacterial challenge. In addition, saGABARAP was ubiquitously expressed at all examined embryonic and larval development stages. These results suggested that saGABARAP could respond to bacteria challenge and may play a vital role in the adult innate immune system against pathogens and the development process of abalone embryo and larvae.

  10. Activity blockade and GABAA receptor blockade produce synaptic scaling through chloride accumulation in embryonic spinal motoneurons and interneurons.

    Directory of Open Access Journals (Sweden)

    Casie Lindsly

    Full Text Available Synaptic scaling represents a process whereby the distribution of a cell's synaptic strengths are altered by a multiplicative scaling factor. Scaling is thought to be a compensatory response that homeostatically controls spiking activity levels in the cell or network. Previously, we observed GABAergic synaptic scaling in embryonic spinal motoneurons following in vivo blockade of either spiking activity or GABAA receptors (GABAARs. We had determined that activity blockade triggered upward GABAergic scaling through chloride accumulation, thus increasing the driving force for these currents. To determine whether chloride accumulation also underlies GABAergic scaling following GABAAR blockade we have developed a new technique. We expressed a genetically encoded chloride-indicator, Clomeleon, in the embryonic chick spinal cord, which provides a non-invasive fast measure of intracellular chloride. Using this technique we now show that chloride accumulation underlies GABAergic scaling following blockade of either spiking activity or the GABAAR. The finding that GABAAR blockade and activity blockade trigger scaling via a common mechanism supports our hypothesis that activity blockade reduces GABAAR activation, which triggers synaptic scaling. In addition, Clomeleon imaging demonstrated the time course and widespread nature of GABAergic scaling through chloride accumulation, as it was also observed in spinal interneurons. This suggests that homeostatic scaling via chloride accumulation is a common feature in many neuronal classes within the embryonic spinal cord and opens the possibility that this process may occur throughout the nervous system at early stages of development.

  11. The contribution of delta subunit-containing GABAA receptors to phasic and tonic conductance changes in cerebellum, thalamus and neocortex.

    Directory of Open Access Journals (Sweden)

    Stephen G Brickley

    2013-12-01

    Full Text Available We have made use of the delta subunit-selective allosteric modulator DS2 (4-chloro-N-[2-(2-thienylimidazo[1,2-a]pyridine-3-yl benzamide to assay the contribution of delta-GABAARs to tonic and phasic conductance changes in the cerebellum, thalamus and neocortex. In cerebellar granule cells, an enhancement of the tonic conductance was observed for DS2 and the orthosteric agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. As expected, DS2 did not alter the properties of GABAA receptor-mediated inhibitory postsynaptic synaptic currents (IPSCs supporting a purely extrasynaptic role for delta-GABAARs in cerebellar granule cells. DS2 also enhanced the tonic conductance recorded from thalamic relay neurons of the visual thalamus with no alteration in IPSC properties. However, in addition to enhancing the tonic conductance DS2 also slowed the decay of IPSCs recorded from layer II/III neocortical neurons. A slowing of the IPSC decay also occurred in the presence of the voltage-gated sodium channel blocker TTX. Moreover, under conditions of reduced GABA release the ability of DS2 to enhance the tonic conductance was attenuated. These results indicate that delta-GABAARs can be activated following vesicular GABA release onto neocortical neurons and that the actions of DS2 on the tonic conductance may be influenced by the ambient GABA levels present in particular brain regions.

  12. GABAA Receptor Binding Assays of Standardized Leonurus cardiaca and Leonurus japonicus Extracts as Well as Their Isolated Constituents.

    Science.gov (United States)

    Rauwald, Hans Wilhelm; Savtschenko, Alex; Merten, Alexander; Rusch, Christian; Appel, Kurt; Kuchta, Kenny

    2015-08-01

    A main traditional use of European Leonurus cardiaca and East Asian Leonurus japonicus is in the treatment of neurological disorders such as anxiety, depression, nervousness, and as a sedative for insomnia. However, their mechanism of action is still under discussion. As anxiety and depressive disorders are increasingly being recognized as connected to dysfunctions of the gamma-aminobutyric acid system, the in vitro effects of standardized L. cardiaca and L japonicus extracts as well as five of their isolated constituents, namely, the labdane-type isoleosibirin, the novel iridoid 7R-chloro-6-desoxy-harpagide, the phenylethanoid lavandulifolioside, and the N-containing compounds stachydrine and leonurine, on this type of neuronal receptor were investigated for the first time. Extracts of L. cardiaca and L. japonicus, characterized by reversed-phase high-performance liquid chromatography determination, as well as their above named isolated, possible active constituents of different chemical nature were tested in several receptor binding assays at rat GABAA receptors using [(3)H]-SR95 531 and [(3)H]-Ro-15-1788 (flumazenil)/diazepam control. The L. cardiaca and L. japonicus extracts as well as leonurine inhibited the concentration-dependent binding of [(3)H]-SR95 531 to the gamma-aminobutyric acid site of the gamma-aminobutyric acid type A receptor with a high binding affinity: IC50s 21 µg/ml, 46 µg/ml, and 15 µg/ml, respectively. In contrast, binding to the benzodiazepine site of the rat gamma-aminobutyric acid type A receptor had a 15 to 30 times lower binding affinity than to the gamma-aminobutyric acid site. The presented experiments provide hints that the neurological mechanism of action of L. cardiaca and L. japonicus may essentially be based on their interaction to the gamma-aminobutyric acid site of the gamma-aminobutyric acid type A receptor, while the benzodiazepine site most probably does not contribute to this effect. In the case of L

  13. A Unified Model of the GABA(A) Receptor Comprising Agonist and Benzodiazepine Binding Sites

    DEFF Research Database (Denmark)

    Kongsbak, Kristine Grønning; Bergmann, Rikke; Sørensen, Pernille Louise

    2013-01-01

    We present a full-length a1b2c2 GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate...

  14. Simultaneous optical recording in multiple cells by digital holographic microscopy of chloride current associated to activation of the ligand-gated chloride channel GABA(A) receptor.

    Science.gov (United States)

    Jourdain, Pascal; Boss, Daniel; Rappaz, Benjamin; Moratal, Corinne; Hernandez, Maria-Clemencia; Depeursinge, Christian; Magistretti, Pierre Julius; Marquet, Pierre

    2012-01-01

    Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABA(A) mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABA(A) receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABA(A) receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM.

  15. Simultaneous optical recording in multiple cells by digital holographic microscopy of chloride current associated to activation of the ligand-gated chloride channel GABA(A receptor.

    Directory of Open Access Journals (Sweden)

    Pascal Jourdain

    Full Text Available Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM, allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABA(A mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABA(A receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABA(A receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM.

  16. Kampo medicine: Evaluation of the pharmacological activity of 121 herbal drugs on GABA(A and 5 HT3A receptors

    Directory of Open Access Journals (Sweden)

    Katrin M Hoffmann

    2016-07-01

    Full Text Available Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM. During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and its constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, we performed a broad screening of Kampo remedies to look for pharmacologically relevant 5 HT3A and GABA(A receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness or insomnia. Therefore, we analyzed the pharmacological effects of 121 herbal drugs from Kampo medicine as ethanol tinctures on heterologously expressed 5 HT3A and GABA(A receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix and Leonurus japonicus (herba were the most effective inhibitory compounds on the 5 HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5 HT3A receptor antagonist. We also identified several potentiating herbs (e.g., Magnolia officinalis (cortex, Syzygium aromaticum (flos and Panax ginseng (radix for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects, for instance Salvia miltiorrhiza (radix were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing a better understanding of their physiological effects and clinical applications.

  17. PWZ-029, an inverse agonist selective for α₅ GABAA receptors, improves object recognition, but not water-maze memory in normal and scopolamine-treated rats.

    Science.gov (United States)

    Milić, Marija; Timić, Tamara; Joksimović, Srđan; Biawat, Poonam; Rallapalli, Sundari; Divljaković, Jovana; Radulović, Tamara; Cook, James M; Savić, Miroslav M

    2013-03-15

    Inverse agonism at the benzodiazepine site of α(5) subunit-containing GABA(A) receptors is an attractive approach for the development of putative cognition-enhancing compounds, which are still far from clinical application. Several ligands with binding and/or functional selectivity for α(5) GABA(A) receptors have been synthesized and tested in a few animal models. PWZ-029 is an α(5) GABA(A) selective inverse agonist whose memory enhancing effects were demonstrated in the passive avoidance task in rats and in Pavlovian fear conditioning in mice. In the present study we investigated the effects of PWZ-029 administration in novel object recognition test and Morris water maze, in normal and scopolamine-treated rats. All the three doses of PWZ-029 (2, 5 and 10 mg/kg) improved object recognition after the 24-h delay period, as shown by significant differences between the exploration times of the novel and old object, and the respective discrimination indices. PWZ-029 (2 mg/kg) also successfully reversed the 0.3 mg/kg scopolamine-induced deficit in recognition memory after the 1-h delay. In the Morris water maze test, PWZ-029 (5, 10 and 15 mg/kg) did not significantly influence swim patterns, either during five acquisition days or during the treatment-free probe trial. PWZ-029 (2, 5 and 10 mg/kg) also proved to be ineffective in the reversal of the 1mg/kg scopolamine-induced memory impairment in the water maze. The present mixed results encourage use of a variety of tests and experimental conditions in order to increase the predictability of preclinical testing of selective α(5) GABA(A) inverse agonists.

  18. Control of Spike Transfer at Hippocampal Mossy Fiber Synapses In Vivo by GABAA and GABAB Receptor-Mediated Inhibition.

    Science.gov (United States)

    Zucca, Stefano; Griguoli, Marilena; Malézieux, Meryl; Grosjean, Noëlle; Carta, Mario; Mulle, Christophe

    2017-01-18

    Despite extensive studies in hippocampal slices and incentive from computational theories, the synaptic mechanisms underlying information transfer at mossy fiber (mf) connections between the dentate gyrus (DG) and CA3 neurons in vivo are still elusive. Here we used an optogenetic approach in mice to selectively target and control the activity of DG granule cells (GCs) while performing whole-cell and juxtacellular recordings of CA3 neurons in vivo In CA3 pyramidal cells (PCs), mf-CA3 synaptic responses consisted predominantly of an IPSP at low stimulation frequency (0.05 Hz). Upon increasing the frequency of stimulation, a biphasic response was observed consisting of a brief mf EPSP followed by an inhibitory response lasting on the order of 100 ms. Spike transfer at DG-CA3 interneurons recorded in the juxtacellular mode was efficient at low presynaptic stimulation frequency and appeared insensitive to an increased frequency of GC activity. Overall, this resulted in a robust and slow feedforward inhibition of spike transfer at mf-CA3 pyramidal cell synapses. Short-term plasticity of EPSPs with increasing frequency of presynaptic activity allowed inhibition to be overcome to reach spike discharge in CA3 PCs. Whereas the activation of GABAA receptors was responsible for the direct inhibition of light-evoked spike responses, the slow inhibition of spiking activity required the activation of GABAB receptors in CA3 PCs. The slow inhibitory response defined an optimum frequency of presynaptic activity for spike transfer at ∼10 Hz. Altogether these properties define the temporal rules for efficient information transfer at DG-CA3 synaptic connections in the intact circuit.

  19. Block of GABA(A) receptor ion channel by penicillin: electrophysiological and modeling insights toward the mechanism.

    Science.gov (United States)

    Rossokhin, Alexey V; Sharonova, Irina N; Bukanova, Julia V; Kolbaev, Sergey N; Skrebitsky, Vladimir G

    2014-11-01

    GABA(A) receptors (GABA(A)R) mainly mediate fast inhibitory neurotransmission in the central nervous system. Different classes of modulators target GABA(A)R properties. Penicillin G (PNG) belongs to the class of noncompetitive antagonists blocking the open GABA(A)R and is a prototype of β-lactam antibiotics. In this study, we combined electrophysiological and modeling approaches to investigate the peculiarities of PNG blockade of GABA-activated currents recorded from isolated rat Purkinje cells and to predict the PNG binding site. Whole-cell patch-сlamp recording and fast application system was used in the electrophysiological experiments. PNG block developed after channel activation and increased with membrane depolarization suggesting that the ligand binds within the open channel pore. PNG blocked stationary component of GABA-activated currents in a concentration-dependent manner with IC50 value of 1.12mM at -70mV. The termination of GABA and PNG co-application was followed by a transient tail current. Protection of the tail current from bicuculline block and dependence of its kinetic parameters on agonist affinity suggest that PNG acts as a sequential open channel blocker that prevents agonist dissociation while the channel remains blocked. We built the GABA(A)R models based on nAChR and GLIC structures and performed an unbiased systematic search of the PNG binding site. Monte-Carlo energy minimization was used to find the lowest energy binding modes. We have shown that PNG binds close to the intracellular vestibule. In both models the maximum contribution to the energy of ligand-receptor interactions revealed residues located on the level of 2', 6' and 9' rings formed by a bundle of M2 transmembrane segments, indicating that these residues most likely participate in PNG binding. The predicted structural models support the described mechanism of PNG block.

  20. The neuroplastic index p-FADD/FADD and phosphoprotein PEA-15, interacting at GABAA receptor, are upregulated in brain cortex during midazolam-induced hypnosis in mice.

    Science.gov (United States)

    Álvaro-Bartolomé, María; García-Sevilla, Jesús A

    2015-11-01

    Fas-associated death domain (FADD) adaptor is involved in the signaling of metabotropic G protein-coupled receptors, whose agonists stimulate its phosphoryaltion (p) increasing p-FADD/FADD ratio in brain. Whether FADD might also participate in the activation of dissimilar receptors such as the ligand-gated ion channels is not known. This study investigated the role of FADD and phosphoprotein-enriched in astrocytes of 15 kDa (PEA-15, a FADD partner) in the activation of γ-aminobutyric acid-A (GABAA) receptor, which mediates the hypnotic effect of midazolam. The main findings revealed that during the time course of midazolam (60 mg/kg)-induced hypnosis in mice (about 2 h) p-FADD (and p-FADD/FADD ratio) as well as p-PEA (and its phosphorylating Akt1 kinase) were markedly increased (36-80%) in brain cortex, and these effects were partially (only p-FADD) or fully prevented by flumazenil (a neutral allosteric ligand) and FG 7142 (a partial negative allosteric ligand) acting at GABAA receptors. The upregulation of cortical p-FADD/FADD was exclusively observed in the nucleus (up to 2.8-fold), where the transciption factor NF-κB was also increased (up to 46%), and that of p-PEA/p-Akt1 only in the cytosol (up to 53%), suggesting that p-FADD/p-PEA/p-Akt1 are involved in sleep-induced neuroplasticity. Repeated treatment with midazolam (60 mg/kg, 4 days) induced behavioral (prolonged sleep latency and reduced sleeping time) and neurochemical (reduced p-FADD/p-PEA contents) tolerance. These findings indicated that p-FADD/p-PEA are novel molecules in GABAA receptor signaling and that cortical p-PEA and p-FADD, working in tandem, are involved in the complex molecular processes leading to the hypnotic effect of midazolam in mice.

  1. GABAA receptor associated protein (GABARAP) modulates TRPV1 expression and channel function and desensitization.

    NARCIS (Netherlands)

    Lainez, S.; Valente, P.; Ontoria-Oviedo, I.; Estevez-Herrera, J.; Camprubi-Robles, M.; Ferrer-Montiel, A.; Planells-Cases, R.

    2010-01-01

    Transient receptor potential vanilloid (TRPV1) transduces noxious chemical and physical stimuli in high-threshold nociceptors. The pivotal role of TRPV1 in the physiopathology of pain transduction has thrust the identification and characterization of interacting partners that modulate its cellular f

  2. GABAA受体转运及其在神经性疾病中的作用%Transportation and role of GABAA receptor in neurological diseases

    Institute of Scientific and Technical Information of China (English)

    李翠限; 王莹; 周春; 沈晓燕

    2011-01-01

    GABA (γ-aminobutyric acid ) type A receptors ( GABAA Rs ) are part of a ligand-gated ion-channel superfamily.They mediate most fast synaptic inhibition in the mammalian brain. In this paper, we discuss recent progress in our understanding of the proteins facilitating GABAAR transportation, endocytosis and post-endocytic GABAA R sorting, and a number of neurological disorders, including epilepsy and schizophrenia, in which alterations in GABAA R transportation occur.%γ-氨基丁酸A型受体(GABAARs)是配体门控离子通道超家族的成员之一,介导哺乳动物大脑中大多数的快速型突触抑制.该文对促使GABAAR运转的相关蛋白,内吞和内吞后的分拣过程,以及癫痫和精神分裂症等神经系统疾病中发生的GABAAR的转运改变进行讨论.

  3. The essential role of hippocampal alpha6 subunit-containing GABAA receptors in maternal separation stress-induced adolescent depressive behaviors.

    Science.gov (United States)

    Yang, Linjie; Xu, Ting; Zhang, Ke; Wei, Zhisheng; Li, Xuran; Huang, Mingfa; Rose, Gregory M; Cai, Xiang

    2016-10-15

    Exposure to early stressful adverse life events such as maternal separation severely impacts the development of the nervous system. Using immunohistochemistry, quantitative PCR and Western blot approaches, we found that alpha6 subunit-containing GABAA receptors (Gabra6-containing GABAA Rs) were expressed on hippocampal interneurons of adolescent rats. Maternal separation stress (MS) from postnatal day 2 to15 significantly reduced Gabra6 expression and provoked depressive behaviors such as anhedonia. Furosemide, the selective antagonist of Gabra6-containing GABAARs, strongly increased peak amplitude of evoked IPSCs at CA3-CA1 synapses and the frequency of miniature IPSPs recorded from CA1 pyramidal cells in naive control animals, and this effect was occluded in MS animals. Knockdown of Gabra6 expression in hippocampus mimicked furosemide's effect and was sufficient to produce similar depressive symptoms that were observed in MS animals. These results indicate that the Gabra6-containing GABAA R is a key modulator of hippocampal synaptic transmission and likely plays a crucial role in the pathophysiology of maternal separation-induced depression.

  4. The aqueous crude extract of Montanoa frutescens produces anxiolytic-like effects similarly to diazepam in Wistar rats: involvement of GABAA receptor.

    Science.gov (United States)

    Carro-Juárez, Miguel; Rodríguez-Landa, Juan Francisco; Rodríguez-Peña, María de Lourdes; Rovirosa-Hernández, María de Jesús; García-Orduña, Francisco

    2012-09-28

    Cihuapatli is the Nahuatl name assigned to some medicinal plants grouped in the genus Montanoa, where Montanoa frutescens (Family: Asteraceae, Tribe: Heliantheae) is included. The crude extract from these plants has been used for centuries in the Mexican traditional medicine as a remedy for reproductive impairments and mood disorders. Experimental studies have systematically corroborated the traditional use of cihuapatli on reproductive impairments and sexual motivation, however, the effect on mood and "nervous" disorders, remains to be explored. The anxiolytic-like effect of aqueous crude extract of M. frutescens (25, 50 and 75 mg/kg) was investigated in male Wistar rats evaluated in the elevated plus-maze and compared with several doses of diazepam (1, 2 and 4 mg/kg) as a reference anxiolytic drug. Picrotoxin (1 mg/kg), a noncompetitive antagonist of the GABA(A) receptor, was used in experimental procedures to evaluate if this receptor could be involved in the anxiolytic-like effects produced by M. frutescens. To discard hypoactivity, hyperactivity, or no changes associated with treatments, which could interfere with the behavioral activity in the elevated plus-maze, rats were subjected to the open field test. M. frutescens at 50 mg/kg showed anxiolytic-like activity similarly to 2 mg/kg of diazepam, without disrupts in general motor activity. The anxiolytic-like effect of M. frutescens detected in the elevated plus-maze was blocked by picrotoxin, indicating that GABA(A) receptors are involved in the modulation of this effect. The results corroborate the use of M. frutescens in folk Mexican ethnomedicine as a potential anxiolytic agent and suggest that this effect is mediated by the GABA(A) receptors. Additionally, some sedative effects with high doses of M. frutescens were detected in the present study. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  5. Identification of an Inhibitory Alcohol Binding Site in GABAA ρ1 Receptors.

    Science.gov (United States)

    Borghese, Cecilia M; Ruiz, Carlos I; Lee, Ui S; Cullins, Madeline A; Bertaccini, Edward J; Trudell, James R; Harris, R Adron

    2016-01-20

    Alcohols inhibit γ-aminobutyric acid type A ρ1 receptor function. After introducing mutations in several positions of the second transmembrane helix in ρ1, we studied the effects of ethanol and hexanol on GABA responses using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. The 6' mutations produced the following effects on ethanol and hexanol responses: small increase or no change (T6'M), increased inhibition (T6'V), and small potentiation (T6'Y and T6'F). The 5' mutations produced mainly increases in hexanol inhibition. Other mutations produced small (3' and 9') or no changes (2' and L277 in the first transmembrane domain) in alcohol effects. These results suggest an inhibitory alcohol binding site near the 6' position. Homology models of ρ1 receptors based on the X-ray structure of GluCl showed that the 2', 5', 6', and 9' residues were easily accessible from the ion pore, with 5' and 6' residues from neighboring subunits facing each other; L3' and L277 also faced the neighboring subunit. We tested ethanol through octanol on single and double mutated ρ1 receptors [ρ1(I15'S), ρ1(T6'Y), and ρ1(T6'Y,I15'S)] to further characterize the inhibitory alcohol pocket in the wild-type ρ1 receptor. The pocket can only bind relatively short-chain alcohols and is eliminated by introducing Y in the 6' position. Replacing the bulky 15' residue with a smaller side chain introduced a potentiating binding site, more sensitive to long-chain than to short-chain alcohols. In conclusion, the net alcohol effect on the ρ1 receptor is determined by the sum of its actions on inhibitory and potentiating sites.

  6. GABAA increases calcium in subventricular zone astrocyte-like cells through L- and T-type voltage-gated calcium channels

    Directory of Open Access Journals (Sweden)

    Stephanie Z Young

    2010-04-01

    Full Text Available In the adult neurogenic subventricular zone (SVZ, the behavior of astrocyte-like cells and some of their functions depend on changes in intracellular Ca2+ levels and tonic GABAA receptor activation. However, it is unknown whether, and if so how, GABAA receptor activity regulates intracellular Ca2+ dynamics in SVZ astrocytes. To monitor Ca2+ activity selectively in astrocyte-like cells, we used two lines of transgenic mice expressing either GFP fused to a Gq-coupled receptor or DsRed under the human glial fibrillary acidic protein (hGFAP promoter. GABAA receptor activation induced Ca2+ increases in 40-50% of SVZ astrocytes. GABAA-induced Ca2+ increases were prevented with nifedipine and mibefradil, blockers of L- and T-type voltage-gated calcium channels (VGCC. The L-type Ca2+ channel activator BayK 8644 increased the percentage of GABAA-responding astrocyte-like cells to 75%, suggesting that the majority of SVZ astrocytes express functional VGCCs. SVZ astrocytes also displayed spontaneous Ca2+ activity, the frequency of which was regulated by tonic GABAA receptor activation. These data support a role for ambient GABA in tonically regulating intracellular Ca2+ dynamics through GABAA receptors and VGCC in a subpopulation of astrocyte-like cells in the postnatal SVZ.

  7. Long-term inhibition of Rho-kinase restores the LTP impaired in chronic forebrain ischemia rats by regulating GABAA and GABAB receptors.

    Science.gov (United States)

    Huang, L; Zhao, L B; Yu, Z Y; He, X J; Ma, L P; Li, N; Guo, L J; Feng, W Y

    2014-09-26

    We previously demonstrated that inactivation of Rho-kinase by hydroxyfasudil could impact N-methyl-d-aspartate (NMDA) excitatory interneurons in the hippocampus and attenuate the spatial learning and memory dysfunction of rats caused by chronic forebrain hypoperfusion ischemia. Complementary interactions between the excitatory neurotransmitter glutamate and the inhibitory neurotransmitter GABA form the molecular basis of synaptic plasticity and cognitive performance. However, whether the GABAergic inhibitory interneurons are involved in the mechanisms underlying these processes remains unclear. Here, we further examined the role of GABAergic interneurons in the neuroprotective effect of the Rho-kinase inhibitor. Chronic forebrain ischemia was induced in Wistar rats by bilateral common carotid artery occlusion (BCAO). The general synaptic transmission and long-term potentiation (LTP) of hippocampal CA3 neurons were evaluated at 30 days after sham surgery or BCAO. Real-time PCR and Western blot analyses were conducted to determine the effect of the Rho-kinase inhibitor hydroxyfasudil on GABAergic inhibitory interneuron expression and function after ischemia. Hydroxyfasudil showed no significant effect on general synaptic transmission, but it could abolish the inhibition of LTP induced by chronic forebrain ischemia. Moreover, the mRNA and protein levels of GABAA and GABAB in three brain regions after ischemia were markedly decreased, and hydroxyfasudil could up-regulate all mRNA and protein expression levels in these areas except for GABAA mRNA in the cerebral cortex and striatum. Using phosphorylation antibodies against specific sites on the GABAA and GABAB receptors, we further demonstrated that hydroxyfasudil could inhibit GABAergic interneuron phosphorylation triggered by the theta burst stimulation. In summary, our results indicated that the inactivation of Rho-kinase could enhance GABAA and GABAB expressions by different mechanisms to guarantee the induction of

  8. Distinct roles of the hippocampus and perirhinal cortex in GABAA receptor blockade-induced enhancement of object recognition memory.

    Science.gov (United States)

    Kim, Jong Min; Kim, Dong Hyun; Lee, Younghwan; Park, Se Jin; Ryu, Jong Hoon

    2014-03-13

    It is well known that the hippocampus plays a role in spatial and contextual memory, and that spatial information is tightly regulated by the hippocampus. However, it is still highly controversial whether the hippocampus plays a role in object recognition memory. In a pilot study, the administration of bicuculline, a GABAA receptor antagonist, enhanced memory in the passive avoidance task, but not in the novel object recognition task. In the present study, we hypothesized that these different results are related to the characteristics of each task and the different roles of hippocampus and perirhinal cortex. A region-specific drug-treatment model was employed to clarify the role of the hippocampus and perirhinal cortex in object recognition memory. After a single habituation in the novel object recognition task, intra-perirhinal cortical injection of bicuculline increased and intra-hippocampal injection decreased the exploration time ratio to novel object. In addition, when animals were repeatedly habituated to the context, intra-perirhinal cortical administration of bicuculline still increased exploration time ratio to novel object, but the effect of intra-hippocampal administration disappeared. Concurrent increases of c-Fos expression and ERK phosphorylation were observed in the perirhinal cortex of the object with context-exposed group either after single or repeated habituation to the context, but no changes were noted in the hippocampus. Altogether, these results suggest that object recognition memory formation requires the perirhinal cortex but not the hippocampus, and that hippocampal activation interferes with object recognition memory by the information encoding of unfamiliar environment. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. In vitro gamma oscillations following partial and complete ablation of δ subunit-containing GABAA receptors from parvalbumin interneurons.

    Science.gov (United States)

    Ferando, Isabella; Mody, Istvan

    2015-01-01

    Perisynaptic and extrasynaptic δ subunit-containing GABAA receptors (δ-GABAARs) mediate tonic conductances in many neurons. On principal cells of the neocortex and hippocampus they comprise α4 subunits, whereas they usually contain α1 on various interneurons. Specific characteristics of δ-GABAARs are their pharmacology and high plasticity. In particular δ-GABAARs are sensitive to low concentrations of neurosteroids (NS) and during times of altered NS production (stress, puberty, ovarian cycle and pregnancy) δ-GABAARs expression varies in many neurons regardless of the α subunits they contain, with direct consequences for neuronal excitability and network synchrony. For example δ-GABAARs plasticity on INs underlies modifications in hippocampal γ oscillations during pregnancy or over the ovarian cycle. Most δ-GABAAR-expressing INs in CA3 stratum pyramidale (SP) are parvalbumin (PV) + INs, whose fundamental role in γ oscillations generation and control has been extensively investigated. In this study we reduced or deleted δ-subunits in PV + INs, with the use of a PV/Cre-Gabrd/floxed genetic system. We find that in vitro CA3 γ oscillations of both PV-Gabrd(+/-)and PV-Gabrd(-/-) mice are characterized by higher frequencies than WT controls. The increased frequencies could be lowered to control levels in PV-Gabrd(+/-) by the NS allopregnanolone (3α,5α-tetrahydroprogesterone, 100 nM) but not the synthetic δ-GABAAR positive allosteric modulator 4-Chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl] benzamide (DS-2, 10 μM). This is consistent with the idea that DS-2, in contrast to ALLO, selectively targets α4/δ-GABAARs but not the α1/δ-GABAARs found on INs. Therefore, development of drugs selective for IN-specific α1/δ-GABAARs may be useful in neurological and psychiatric conditions correlated with altered PV + IN function and aberrant γ oscillations.

  10. Delta-subunit containing GABAA-receptors mediate tonic inhibition in paracapsular cells of the mouse amygdala

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    Anne eMarowsky

    2014-03-01

    Full Text Available The intercalated paracapsular cells (pcs are small GABAergic interneurons that form densely populated clusters surrounding the basolateral (BLA complex of the amygdala. Their main task in the amygdala circuitry appears to be the control of information flow, as they act as an inhibitory interface between input and output nuclei. Modulation of their activity is thus thought to affect amygdala output and the generation of fear and anxiety. Recent evidence indicates that pcs express benzodiazepine (BZ-sensitive GABAA receptor (GABAAR variants containing the α2- and α3-subunit for transmission of postsynaptic currents, yet little is known about the expression of extrasynaptic GABAARs, mediating tonic inhibition and regulating neuronal excitability. Here, we show that pcs from the lateral and medial intercalated cell cluster (l- and mITC, respectively express a tonic GABAergic conductance that could be significantly increased in a concentration-dependent manner by the δ-preferring GABAAR agonist THIP (0.5-10 µM, but not by the BZ diazepam (1 µM. The neurosteroid THDOC (300 nM also increased tonic currents in pcs significantly, but only in the presence of additional GABA (5 µM. Immunohistochemical stainings revealed that both the δ-GABAAR and the α4-GABAAR subunit are expressed throughout all ITCs, while no staining for the α5-GABAAR subunit could be detected. Moreover, 1 µM THIP dampened excitability in pcs most likely by increasing shunting inhibition. In line with this, THIP significantly decreased lITC-generated inhibition in target cells residing in the BLA nucleus by 30%. Taken together these results demonstrate for the first time that pcs express a tonic inhibitory conductance mediated most likely by α4/δ-containing GABAARs. This data also suggest that δ-GABAAR targeting compounds might possibly interfere with pcs-related neuronal processes such as fear extinction.

  11. Involvement of AMPA/kainate and GABAA receptors in topiramate neuroprotective effects against methylphenidate abuse sequels involving oxidative stress and inflammation in rat isolated hippocampus.

    Science.gov (United States)

    Motaghinejad, Majid; Motevalian, Manijeh

    2016-08-01

    Abuses of methylphenidate (MPH) as psychostimulant cause neural damage of brain cells. Neuroprotective properties of topiramate (TPM) have been indicated in several studies but its exact mechanism of action remains unclear. The current study evaluates protective role of various doses of TPM and its mechanism of action in MPH induced oxidative stress and inflammation. The neuroprotective effects of various doses of TPM against MPH induced oxidative stress and inflammation were evaluated and then the action of TPM was studied in presence of domoic acid (DOM), as AMPA/kainate receptor agonist and bicuculline (BIC) as GABAA receptor antagonist, in isolated rat hippocampus. Open Field Test (OFT) was used to investigate motor activity changes. Oxidative, antioxidant and inflammatory factors were measured in isolated hippocampus. TPM (70 and 100mg/kg) decreased MPH induced motor activity disturbances and inhibit MPH induced oxidative stress and inflammation. On the other hand pretreatment of animals with DOM or BIC, inhibit this effect of TPM and potentiate MPH induced motor activity disturbances and increased lipid peroxidation, mitochondrial oxidized form of glutathione (GSSG) level, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in isolated hippocampal cells and decreased reduced form of glutathione (GSH) level, superoxide dismutase, glutathione peroxidase and glutathione reductase activity. It seems that TPM can protect cells of hippocampus from oxidative stress and neuroinflammation and it could be partly by activation of GABAA receptor and inhibition of AMPA/kainite receptor.

  12. Expression of 10 GABA(A) receptor subunit messenger RNAs in the motor-related thalamic nuclei and basal ganglia of Macaca mulatta studied with in situ hybridization histochemistry.

    Science.gov (United States)

    Kultas-Ilinsky, K; Leontiev, V; Whiting, P J

    1998-07-01

    In situ hybridization histochemistry technique with [35S]UTP-labelled riboprobes was used to study the expression pattern of 10 GABA(A) receptor subunit messenger RNAs in the basal ganglia and motor thalamic nuclei of rhesus monkey. Human transcripts were used for the synthesis of alpha2, alpha4, beta2, beta3, gamma1 and delta subunit messenger RNA probes. Rat complementary DNAs were used for generating alpha1, alpha3, beta1 and gamma2 subunit messenger RNA probes. Nigral, pallidal and cerebellar afferent territories in the ventral tier thalamic nuclei all expressed alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3, delta and gamma2 subunit messenger RNAs but at different levels. Each intralaminar nucleus displayed its own unique expression pattern. In the thalamus, gamma1 subunit messenger RNA was detected only in the parafascicular nucleus. Comparison of the expression patterns with the known organization of GABA(A) connections in thalamic nuclei suggests that (i) the composition of the receptor associated with reticulothalamic synapses, except for those in the intralaminar nuclei, may be alpha1alpha4beta2delta, (ii) receptors of various other subunit compositions may operate in the local GABAergic circuits, and (iii) the composition of receptors at nigro- and pallidothalamic synapses may differ, with those at nigrothalamic probably containing beta1 and gamma2 subunits. In the medial and lateral parts of the globus pallidus, the subthalamic nucleus and the substantia nigra pars reticularis, the alpha1, beta2 and gamma2 messenger RNAs were co-expressed at a high level suggesting that this subunit composition was associated with all GABAergic synapses in the direct and indirect striatal output pathways. Various other subunit messenger RNAs were also expressed but at a lower level. In the substantia nigra pars compacta the most highly expressed messenger RNAs were alpha3, alpha4 and beta3; all other subunit messenger RNAs studied, except for gamma1, alpha1 and

  13. Anticonvulsive evaluation of THIP in the murine pentylenetetrazole kindling model: lack of anticonvulsive effect of THIP despite functional δ-subunit-containing GABAA receptors in dentate gyrus granule cells.

    Science.gov (United States)

    Simonsen, Charlotte; Boddum, Kim; von Schoubye, Nadia L; Kloppenburg, Alissa; Sønderskov, Kasper; Hansen, Suzanne L; Kristiansen, Uffe

    2017-08-01

    THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a GABAA receptor agonist with varying potencies and efficacies at γ-subunit-containing receptors. More importantly, THIP acts as a selective superagonist at δ-subunit-containing receptors (δ-GABAA Rs) at clinically relevant concentrations. Evaluation of THIP as a potential anticonvulsant has given contradictory results in different animal models and for this reason, we reevaluated the anticonvulsive properties of THIP in the murine pentylenetetrazole (PTZ) kindling model. As loss of δ-GABAA R in the dentate gyrus has been associated with several animal models of epilepsy, we first investigated the presence of functional δ-GABAA receptors. Both immunohistochemistry and Western blot data demonstrated that δ-GABAA R expression is not only present in the dentate gyrus, but also the expression level was enhanced in the early phase after PTZ kindling. Whole-cell patch-clamp studies in acute hippocampal brain slices revealed that THIP was indeed able to induce a tonic inhibition in dentate gyrus granule cells. However, THIP induced a tonic current of similar magnitude in the PTZ-kindled mice compared to saline-treated animals despite the observed upregulation of δ-GABAA Rs. Even in the demonstrated presence of functional δ-GABAA Rs, THIP (0.5-4 mg/kg) showed no anticonvulsive effect in the PTZ kindling model using a comprehensive in vivo evaluation of the anticonvulsive properties. © 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

  14. Paired burst stimulation causes GABAA receptor-dependent spike firing facilitation in CA1 of rat hippocampal slices

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    Takashi eTominaga

    2016-01-01

    Full Text Available The theta oscillation (4–8 Hz is a pivotal form of oscillatory activity in the hippocampus that is intermittently concurrent with gamma (25–100 Hz burst events. In in vitro preparation, a stimulation protocol that mimics the theta oscillation, theta burst stimulation (TBS, is used to induce long-term potentiation. Thus, TBS is thought to have a distinct role in the neural network of the hippocampal slice preparation. However, the mechanisms that make TBS uniquely induce such neural circuit modifications are still unknown. Using electrophysiology and voltage-sensitive dye imaging (VSDI, we have found that TBS induces augmentation of spike firing. The augmentation was apparent in the first couple of brief burst stimulation (100Hz four pulses on a TBS-train in a presence of NMDA receptor blocker (APV 50 µM. In this study, we focused on the characterises of the NMDA independent augmentation caused by a pair of the brief burst stimulation (the first pair of the TBS; PBS. We found that PBS enhanced membrane potential responses on VSDI signal and intracellular recordings while it was absent in the current recording under whole-cell clamp condition. The enhancement of the response accompanied the augmentation of excitatory postsynaptic potential (EPSP to spike firing (E-S coupling. The paired burst facilitation (PBF reached a plateau when the number of the first burst stimulation (priming burst exceeds three. The interval between the bursts of 150 ms resulted in the maximum PBF. Gabazine (a GABAA receptor antagonist abolished PBF. The threshold for spike generation of the postsynaptic cells measured with a current injection to cells was not lowered by the priming burst of PBS. These results indicate that PBS activates the GABAergic system to cause short-term E-S augmentation without raising postsynaptic excitability. We propose that a GABAergic system of area CA1 of the hippocampus produce the short-term E-S plasticity that could cause exaggerated

  15. Probing the orthosteric binding site of GABAA receptors with heterocyclic GABA carboxylic acid bioisosteres

    DEFF Research Database (Denmark)

    Petersen, Jette G; Bergmann, Rikke; Krogsgaard-Larsen, Povl;

    2013-01-01

    selective and potent GABAAR agonists. This review investigates the use of heterocyclic carboxylic acid bioisosteres within the GABAAR area. Several heterocycles including 3-hydroxyisoxazole, 3-hydroxyisoxazoline, 3-hydroxyisothiazole, and the 1- and 3-hydroxypyrazole rings have been employed in order to map...... the orthosteric binding site. The physicochemical properties of the heterocyclic moieties making them suitable for bioisosteric replacement of the carboxylic acid in the molecule of GABA are discussed. A variety of synthetic strategies for synthesis of the heterocyclic scaffolds are available. Likewise, methods...... for introduction of substituents into specific positions of the heterocyclic scaffolds facilitate the investigation of different regions in the orthosteric binding pocket in close vicinity of the core scaffolds of muscimol/GABA. The development of structural models, from pharmacophore models to receptor homology...

  16. Prodynorphin gene deletion increased anxiety-like behaviours, impaired the anxiolytic effect of bromazepam and altered GABAA receptor subunits gene expression in the amygdala.

    Science.gov (United States)

    Femenía, Teresa; Pérez-Rial, Sandra; Urigüen, Leyre; Manzanares, Jorge

    2011-01-01

    This study evaluated the role of prodynorphin gene in the regulation of anxiety and associated molecular mechanisms. Emotional responses were assessed using the light-dark test, elevated plus maze and social interaction tests in prodynorphin knockout and wild-type mice. Corticotrophin releasing factor and proopiomelanocortin gene expressions in the hypothalamus were evaluated after restraint stress using in situ hybridization. The anxiolytic efficacy of bromazepam and GABA(A) receptor subunits gene expression in the amygdala were also assessed in both genotypes. The deletion of prodynorphin increased anxiety-like behaviours and proopiomelanocortin gene expression in the arcuate nucleus (two-fold). Moreover, the anxiolytic action of bromazepam was significantly attenuated in the mutant mice. Decreased GABA(A)γ(2) and increased GABA(A)β(2) gene expression receptor subunits were found in the amygdala of prodynorphin knockout mice. These results indicate that deletion of prodynorphin gene is associated with increased anxiety-like behaviours, enhanced sensibility response to stress stimuli, reduced anxiolytic efficacy of bromazepam and altered expression of the GABA(A) receptor subunits.

  17. Alternative-splicing in the exon-10 region of GABA(A receptor beta(2 subunit gene: relationships between novel isoforms and psychotic disorders.

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    Cunyou Zhao

    Full Text Available BACKGROUND: Non-coding single nucleotide polymorphisms (SNPs in GABRB2, the gene for beta(2-subunit of gamma-aminobutyric acid type A (GABA(A receptor, have been associated with schizophrenia (SCZ and quantitatively correlated to mRNA expression and alternative splicing. METHODS AND FINDINGS: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1 and beta(2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1 expression and decreased beta(2S2 expression in both SCZ and bipolar disorder (BPD compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1 and beta(2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2 expression. Moreover, site-directed mutagenesis indicated that Thr(365, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. CONCLUSION: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2-subunit splicing diversity and the etiologies of SCZ and BPD.

  18. alpha2-gamma-Aminobutyric acid (GABA)A receptors are the molecular substrates mediating precipitation of narcosis but not of sedation by the combined use of diazepam and alcohol in vivo.

    Science.gov (United States)

    Täuber, Marcus; Calame-Droz, Elisabeth; Prut, Laetitia; Rudolph, Uwe; Crestani, Florence

    2003-11-01

    Classical benzodiazepines such as diazepam are widely used tranquillisers and hypnotics in various neuropsychiatric diseases including alcohol-related disorders. One of the major drawbacks of benzodiazepine therapy, however, is an exacerbation of the sedative and hypnotic effects associated with alcohol intake, even at low doses. Even though the gamma-aminobutyric acid (GABA)A receptor complex is a common target for the actions of both classes of drugs, the molecular mechanisms underlying the enhanced pharmacological properties of the combined use of benzodiazepines and alcohol remain to be identified. The present experiments aimed at clarifying which of the GABAA receptor subtypes mediate the augmented hypnotic-like and sedative effects of combined diazepam and alcohol using the righting reflex and motor activity assays, respectively, in histidine-to-arginine point mutated mice that possess diazepam-insensitive alpha1-, alpha2-, alpha3- or alpha5-GABAA receptors. The combination of diazepam (2 or 3 mg/kg) and ethanol (3 g/kg) induced loss of righting reflex with a significantly dose-dependent increase of the latency to its full recovery in wild-type, alpha1(H101R), alpha3(H126R) and alpha5(H105R) but not in alpha2(H101R) mice. A combined treatment with diazepam (1 mg/kg) and ethanol (2.5 g/kg) precipitated motor inhibition similarly in wild-type and alpha2(H101R) mice. Responsiveness of the alpha2(H101R) mice to ethanol alone was similar to that of wild-type mice. These results demonstrate that induction of loss of righting reflex by combined diazepam and alcohol is closely dependent on the activation of the alpha2-GABAA receptors by the benzodiazepine whereas precipitation of sedation involves GABAA receptors other than the alpha2-GABAA receptors.

  19. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A receptor-mediated signaling.

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    Anna Kakehashi

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

  20. GABAA-benzodiazepine receptor availability in smokers and nonsmokers: relationship to subsyndromal anxiety and depression.

    Science.gov (United States)

    Esterlis, Irina; Cosgrove, Kelly P; Batis, Jeffery C; Bois, Frederic; Kloczynski, Tracy A; Stiklus, Stephanie M; Perry, Edward; Tamagnan, Gilles D; Seibyl, John P; Makuch, Robert; Krishnan-Sarin, Suchitra; O'Malley, Stephanie; Staley, Julie K

    2009-12-01

    Many smokers experience subsyndromal anxiety symptoms while smoking and during acute abstinence, which may contribute to relapse. We hypothesized that cortical gamma aminobutyric acid(A)-benzodiazepine receptor (GABA(A)-BZR) availability in smokers and nonsmokers might be related to the expression of subsyndromal anxiety, depressive, and pain symptoms. Cortical GABA(A)-BZRs were imaged in 15 smokers (8 men and 7 women), and 15 healthy age and sex-matched nonsmokers, and 4 abstinent tobacco smokers (3 men; 1 woman) using [(123)I]iomazenil and single photon emission computed tomography (SPECT). Anxiety and depressive symptoms were measured using the Spielberger's State-Trait Anxiety Index (STAI) and the Center for Epidemiology Scale for Depressive Symptoms (CES-D). The cold pressor task was administered to assess pain tolerance and sensitivity. The relationship between cortical GABA(A)-BZR availability, smoking status, and subsyndromal depression and anxiety symptoms, as well as pain tolerance and sensitivity, were evaluated. Surprisingly, there were no statistically significant differences in overall GABA(A)-BZR availability between smokers and nonsmokers or between active and abstinent smokers; however, cortical GABA(A)-BZR availability negatively correlated with subsyndromal state anxiety symptoms in nonsmokers but not in smokers. In nonsmokers, the correlation was seen across many brain areas with state anxiety [parietal (r = -0.47, P = 0.03), frontal (r = -0.46, P = 0.03), anterior cingulate (r = -0.47, P = 0.04), temporal (r = -0.47, P = 0.03), occipital (r = -0.43, P = 0.05) cortices, and cerebellum (r = -0.46, P = 0.04)], trait anxiety [parietal (r = -0.72, P = 0.02), frontal (r = -0.72, P = 0.02), and occipital (r = -0.65, P = 0.04) cortices] and depressive symptoms [parietal (r = -0.68; P = 0.02), frontal (r = -0.65; P = 0.03), anterior cingulate (r = -0.61; P = 0.04), and temporal (r = -0.66; P = 0.02) cortices]. The finding that a similar relationship

  1. Inhibitory nature of tiagabine-augmented GABAA receptor-mediated depolarizing responses in hippocampal pyramidal cells.

    Science.gov (United States)

    Jackson, M F; Esplin, B; Capek, R

    1999-03-01

    . Independently of the stimulus intensity with which they were evoked, the charge transferred to the soma by excitatory postsynaptic currents (EPSCs), elicited in the presence of tiagabine (20 microM) during the large (1,428 +/- 331 pA) inward currents that underlie the DRs, was decreased on the average by 90.8 +/- 1.7%. Such inhibition occurred despite the presence of the GABAB receptor antagonist, CGP 52 432 (10 microM), indicating that GABAB heteroreceptors, located on glutamatergic terminals, do not mediate the observed reduction in the amplitude of excitatory postsynaptic responses. The present results suggest that despite facilitating the induction of GABA-mediated depolarizations, tiagabine application may nevertheless increase the effectiveness of synaptic inhibition during the synchronous high-frequency activation of inhibitory interneurons by enhanced shunting.

  2. Spontaneous mobility of GABAA receptor M2 extracellular half relative to noncompetitive antagonist action.

    Science.gov (United States)

    Chen, Ligong; Durkin, Kathleen A; Casida, John E

    2006-12-15

    The gamma-aminobutyric acid type A receptor beta(3) homopentamer is spontaneously open and highly sensitive to many noncompetitive antagonists(NCAs) and Zn(2+). Our earlier study of the M2 cytoplasmic half (-1' to 10') established a model in which NCAs bind at pore-lining residues Ala(2)', Thr(6)', and Leu(9)'. To further define transmembrane 2 (M2) structure relative to NCA action, we extended the Cys scanning to the extra cellular half of the beta(3) homopentamer (11' to 20'). Spontaneous disulfides formed with T13'C, L18'C, and E20'C from M2/M2 cross-linking and with I14'C (weak), H17'C, and R19'Con bridging M2/M3 intersubunits, based on single (M2 Cys only) and dual (M2 Cys plus M3 C289S) mutations. Induced disulfides also formed with T16'C, but there were few or none with M11'C, T12'C, and N15'C. These findings show conformational flexibility/mobility in the M2 extracellular half 17' to 20' region interpreted as a deformed beta-like conformation in the open channel. The NCA radioligands used were [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane ([(3)H]EBOB) and [(3)H]3,3-bis-trifluoromethylbicyclo[2.2.1]heptane-2,2-dicarbonitrile with essentially the same results. NCA binding was disrupted by individual Cys substitutions at 13',14',16',17', and 19'. The inactivity of T13'C/T13'S may have been due to disturbance of the channel gate; I14'S and T16'S showed much better binding activity than their Cys counterparts, and the low activities of H17'C and R19'C were reversed by dithiothreitol. Zn(2+) potency for inhibition of [(3)H]EBOB binding was lowered 346-fold by the mutation H17'A. We propose that NCAs enter their binding site both directly, through the channel pore, and indirectly, through the water cavity of adjacent subunits.

  3. Tonic Inhibitory Control of Dentate Gyrus Granule Cells by α5-Containing GABAA Receptors Reduces Memory Interference.

    Science.gov (United States)

    Engin, Elif; Zarnowska, Ewa D; Benke, Dietmar; Tsvetkov, Evgeny; Sigal, Maksim; Keist, Ruth; Bolshakov, Vadim Y; Pearce, Robert A; Rudolph, Uwe

    2015-10-07

    Interference between similar or overlapping memories formed at different times poses an important challenge on the hippocampal declarative memory system. Difficulties in managing interference are at the core of disabling cognitive deficits in neuropsychiatric disorders. Computational models have suggested that, in the normal brain, the sparse activation of the dentate gyrus granule cells maintained by tonic inhibitory control enables pattern separation, an orthogonalization process that allows distinct representations of memories despite interference. To test this mechanistic hypothesis, we generated mice with significantly reduced expression of the α5-containing GABAA (α5-GABAARs) receptors selectively in the granule cells of the dentate gyrus (α5DGKO mice). α5DGKO mice had reduced tonic inhibition of the granule cells without any change in fast phasic inhibition and showed increased activation in the dentate gyrus when presented with novel stimuli. α5DGKO mice showed impairments in cognitive tasks characterized by high interference, without any deficiencies in low-interference tasks, suggesting specific impairment of pattern separation. Reduction of fast phasic inhibition in the dentate gyrus through granule cell-selective knock-out of α2-GABAARs or the knock-out of the α5-GABAARs in the downstream CA3 area did not detract from pattern separation abilities, which confirms the anatomical and molecular specificity of the findings. In addition to lending empirical support to computational hypotheses, our findings have implications for the treatment of interference-related cognitive symptoms in neuropsychiatric disorders, particularly considering the availability of pharmacological agents selectively targeting α5-GABAARs. Interference between similar memories poses a significant limitation on the hippocampal declarative memory system, and impaired interference management is a cognitive symptom in many disorders. Thus, understanding mechanisms of successful

  4. GABAA receptor partially mediated propofol-induced hyperalgesia at superspinal level and analgesia at spinal cord level in rats

    Institute of Scientific and Technical Information of China (English)

    Qin-yun WANG; Jun-li CAO; Yin-ming ZENG; Ti-jun DAI

    2004-01-01

    AIM: To observe effects of propofol on nociceptive response at superspinal and spinal level in rats. METHODS:Two hundreds and fifty-eight Sprague-Dawley male rats were randomized into thirty-two groups. Propofol and bicuculline were microinjected into lateral ventricle (icv), ventrolateral periaqueductal gray (vlPAG), intrathecal (ith), and intraperitoneal (ip). The noxious responses were evaluated by hot plate and formalin test. RESULTS: In hot-plate test, systemic and superspinal administration of propofol (40 mg·kg-1 ip, 100μg in 10μL, icy, and 4μg in 0.4μL vlPAG microinjection) produced hyperalgesia (P<0.01). Hyperalgesia induced by vlPAG microinjection of propofol was significantly antagonized by 69.8%, 71.2%, 98.8% at 10, 20, and 30 min by microinjection of bicuculline (10 ng in 0.4μL, vlPAG) (P<0.01). Analgesia induced by ith propofol (100μg·10μL-1) was antagonized about 81.3%, 54.8%, 80.8%, and 97.4% at 10, 20, 30 and 40 min by ith bicuculline (P<0.05). In formalin test,systemic and superspinal administration of propofol (40 mg·kg-1 ip, 4μg in 0.4μL, vlPAG) also produced hyperalgesia (P<0.01). The increased formalin pain scores were antagonized about 57.1% by bicuculline (10ng, vlPAG)(P<0.05) at 60 min after formalin injection. The decreased formalin pain scores induced by ith propofol (100μg in 10μL) were antagonized about 66.7% at 30 min by ith bicuculline (P<0.05) after formalin injection. Hyperalgesia produced by ip propofol in both hot plate and formalin test could not be antagonized by vlPAG administration of bicuculline. CONCLUSION: GABAA receptor partly mediated propofol-induced hyperalgesia at superspinal and analgesia at spinal cord in rats.

  5. GABA(A) receptor M2-M3 loop secondary structure and changes in accessibility during channel gating.

    Science.gov (United States)

    Bera, Amal K; Chatav, Maya; Akabas, Myles H

    2002-11-08

    The gamma-aminobutyric acid type A (GABA(A)) receptor M2-M3 loop structure and its role in gating were investigated using the substituted cysteine accessibility method. Residues from alpha(1)Arg-273 to alpha(1)Ile-289 were mutated to cysteine, one at a time. MTSET(+) or MTSES(-) reacted with all mutants from alpha(1)R273C to alpha(1)Y281C, except alpha(1)P277C, in the absence and presence of GABA. The MTSET(+) closed-state reaction rate was >1000 liters/mol-s at alpha(1)N274C, alpha(1)S275C, alpha(1)K278C, and alpha(1)Y281C and was <300 liters/mol-s at alpha(1)R273C, alpha(1)L276C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C. These two groups of residues lie on opposite sides of an alpha-helix. The fast reacting group lies on a continuation of the M2 segment channel-lining helix face. This suggests that the M2 segment alpha-helix extends about two helical turns beyond alpha(1)N274 (20'), aligned with the extracellular ring of charge. At alpha(1)S275C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C the reaction rate was faster in the presence of GABA. The reagents had no functional effect on the mutants from alpha(1)A282C to alpha(1)I289C, except alpha(1)A284C. Access may be sterically hindered possibly by close interaction with the extracellular domain. We suggest that the M2 segment alpha-helix extends beyond the predicted extracellular end of the M2 segment and that gating induces a conformational change in and/or around the N-terminal half of the M2-M3 loop. Implications for coupling ligand-evoked conformational changes in the extracellular domain to channel gating in the membrane-spanning domain are discussed.

  6. GR3027 antagonizes GABAA receptor-potentiating neurosteroids and restores spatial learning and motor coordination in rats with chronic hyperammonemia and hepatic encephalopathy.

    Science.gov (United States)

    Johansson, Maja; Agusti, Ana; Llansola, Marta; Montoliu, Carmina; Strömberg, Jessica; Malinina, Evgenya; Ragagnin, Gianna; Doverskog, Magnus; Bäckström, Torbjörn; Felipo, Vicente

    2015-09-01

    Hepatic encephalopathy (HE) is one of the primary complications of liver cirrhosis. Current treatments for HE, mainly directed to reduction of ammonia levels, are not effective enough because they cannot completely eliminate hyperammonemia and inflammation, which induce the neurological alterations. Studies in animal models show that overactivation of GABAA receptors is involved in cognitive and motor impairment in HE and that reducing this activation restores these functions. We have developed a new compound, GR3027, that selectively antagonizes the enhanced activation of GABAA receptors by neurosteroids such as allopregnanolone and 3α,21-dihydroxy-5α-pregnan-20-one (THDOC). This work aimed to assess whether GR3027 improves motor incoordination, spatial learning, and circadian rhythms of activity in rats with HE. GR3027 was administered subcutaneously to two main models of HE: rats with chronic hyperammonemia due to ammonia feeding and rats with portacaval shunts (PCS). Motor coordination was assessed in beam walking and spatial learning and memory in the Morris water maze and the radial maze. Circadian rhythms of ambulatory and vertical activity were also assessed. In both hyperammonemic and PCS rats, GR3027 restores motor coordination, spatial memory in the Morris water maze, and spatial learning in the radial maze. GR3027 also partially restores circadian rhythms of ambulatory and vertical activity in PCS rats. GR3027 is a novel approach to treatment of HE that would normalize neurological functions altered because of enhanced GABAergic tone, affording more complete normalization of cognitive and motor function than current treatments for HE.

  7. Synthesis, Modelling, and Anticonvulsant Studies of New Quinazolines Showing Three Highly Active Compounds with Low Toxicity and High Affinity to the GABA-A Receptor.

    Science.gov (United States)

    Zayed, Mohamed F; Ihmaid, Saleh K; Ahmed, Hany E A; El-Adl, Khaled; Asiri, Ahmed M; Omar, Abdelsattar M

    2017-01-24

    Some novel fluorinated quinazolines (5a-j) were designed and synthesized to be evaluated for their anticonvulsant activity and their neurotoxicity. Structures of all newly synthesized compounds were confirmed by their infrared (IR), mass spectrometry (MS) spectra, ¹H nuclear magnetic resonance (NMR), (13)C-NMR, and elemental analysis (CHN). The anticonvulsant activity was evaluated by a subcutaneous pentylenetetrazole (scPTZ) test and maximal electroshock (MES)-induced seizure test, while neurotoxicity was evaluated by a rotorod test. The molecular docking was performed for all newly-synthesized compounds to assess their binding affinities to the GABA-A receptor in order to rationalize their anticonvulsant activities in a qualitative way. The data obtained from the molecular modeling was correlated with that obtained from the biological screening. These data showed considerable anticonvulsant activity for all newly-synthesized compounds. Compounds 5b, 5c, and 5d showed the highest binding affinities toward the GABA-A receptor, along with the highest anticonvulsant activities in experimental mice. These compounds also showed low neurotoxicity and low toxicity in the median lethal dose test compared to the reference drugs. A GABA enzymatic assay was performed for these highly active compounds to confirm the obtained results and explain the possible mechanism for anticonvulsant action. The most active compounds might be used as leads for future modification and optimization.

  8. Memory impairment due to fipronil pesticide exposure occurs at the GABAA receptor level, in rats.

    Science.gov (United States)

    Godinho, Antonio Francisco; de Oliveira Souza, Ana Carolina; Carvalho, Caio Cristóvão; Horta, Daniel França; De Fraia, Daniel; Anselmo, Fabio; Chaguri, João Leandro; Faria, Caique Aparecido

    2016-10-15

    Fipronil (F) a pesticide considered of second generation cause various toxic effects in target and non-target organisms including humans in which provoke neurotoxicity, having the antagonism of gamma-amino butyric acid (GABA) as their main mechanism for toxic action. GABAergic system has been involved in processes related to the memory formation and consolidation. The present work studied the importance of GABA to the mechanisms involved in the very early development of fipronil-induced memory impairment in rats. Memory behavior was assessed using new object recognition task (ORT) and eight radial arm maze task (8-RAM) to study effects on cognitive and spatial memory. Locomotor behavior was assessed using open field task (OF). The dose of fipronil utilized was studied through a pilot experiment. The GABA antagonist picrotoxin (P) was used to enhance fipronil effects on GABAergic system. Fipronil or picrotoxin decrease memory studied in ORT and 8-RAM tasks. Additionally, F and P co-exposure enhanced effects on memory compared to controls, F, and P, suggesting strongly a GABAergic effect. Weight gain modulation and fipronil in blood were utilized as animal's intoxication indicators. In conclusion, here we report that second-generation pesticides, such as fipronil, can have toxic interactions with the CNS of mammals and lead to memory impairment by modulating the GABAergic system.

  9. Evidence for Tonic Control by the GABAA Receptor of Extracellular D-Serine Concentrations in the Medial Prefrontal Cortex of Rodents

    Directory of Open Access Journals (Sweden)

    Asami Umino

    2017-08-01

    Full Text Available Endogenous D-serine is a putative dominant co-agonist for the N-methyl-D-aspartate glutamate receptor (NMDAR in the mammalian forebrain. Although the NMDAR regulates the higher order brain functions by interacting with various neurotransmitter systems, the possible interactions between D-serine and an extra-glutamatergic system largely remain elusive. For the first time, we show in the rat and mouse using an in vivo microdialysis technique that the extracellular D-serine concentrations are under tonic increasing control by a major inhibitory transmitter, GABA, via the GABAA (GABAAR in the medial prefrontal cortex (mPFC. Thus, an intra-mPFC infusion of a selective GABAAR antagonist, bicuculline (BIC, caused a concentration-dependent and reversible decrease in the extracellular levels of D-serine in the rat mPFC without affecting those of another intrinsic NMDAR coagonist, glycine and an NMDAR agonist, L-glutamate. The decreasing effects of BIC were eliminated by co-infusion of a selective GABAA agonist, muscimol (MUS and were mimicked by a GABAA antagonist, gabazine (GBZ. In contrast, selective blockade of the GABAB or homomeric ρGABAA (formerly GABAC receptor by saclofen or (1,2,5,6-tetrahydropyridin-4-yl-methylphosphinic acid (TPMPA, respectively, failed to downregulate the prefrontal extracellular D-serine levels. Moreover, the local BIC application attenuated the ability of NMDA given to the mPFC to increase the cortical extracellular concentrations of taurine, indicating the hypofunction of the NMDAR. Finally, in the mouse mPFC, the reduction of the extracellular D-serine levels by a local injection of BIC into the prefrontal portion was replicated, and was precluded by inhibition of the neuronal or glial activity by co-local injection with tetrodotoxin (TTX or fluorocitrate (Fluo, respectively. These findings suggest that the GABAAR-mediated regulation of the D-serine signaling may exert fine-tuning of the NMDAR function and require both

  10. Low nanomolar GABA effects at extrasynaptic a4ß1/ß3delta GABAA receptor subtypes indicate a different binding mode for GABA at these receptors

    DEFF Research Database (Denmark)

    Karim, Nasiara; Wellendorph, Petrine; Absalom, Nathan;

    2012-01-01

    Ionotropic GABA(A) receptors are a highly heterogenous population of receptors assembled from a combination of multiple subunits. The aims of this study were to characterize the potency of GABA at human recombinant d-containing extrasynaptic GABA(A) receptors expressed in Xenopus oocytes using th...

  11. Allopregnanolone prevents dieldrin-induced NMDA receptor internalization and neurotoxicity by preserving GABA(A) receptor function.

    Science.gov (United States)

    Briz, Víctor; Parkash, Jyoti; Sánchez-Redondo, Sara; Prevot, Vincent; Suñol, Cristina

    2012-02-01

    Dieldrin is an endocrine disruptor that accumulates in mammalian adipose tissue and brain. It induces convulsions due to its antagonism of the γ-aminobutyric acid A receptor (GABA(A)R). We have previously reported that long-term exposure to dieldrin causes the internalization of the N-methyl-D-aspartate receptor (NMDAR) as a result of persistent GABA(A)R inhibition. Because the neurosteroids 17β-estradiol (E2) and allopregnanolone are known to modulate the function and trafficking of GABA(A)R and NMDAR, we examined the effects of E2 and allopregnanolone on dieldrin-induced GABA(A)R inhibition, NMDAR internalization, and neuronal death in cortical neurons. We found that 1 nM E2 increased the membrane expression of NR1/NR2B receptors and postsynaptic density 95 but did not induce their physical association. In contrast, 10 nM E2 had no effect on these proteins but reduced NR2A membrane expression. We also found that exposure to 60 nM dieldrin for 6 d in vitro caused the internalization of NR1 and NR2B but not NR2A. Treatment with either 1 nM E2 or 10 μM allopregnanolone prevented the dieldrin-induced reduction in membrane levels of the NR1/NR2B receptors. Furthermore, prolonged exposure to 200 nM dieldrin down-regulated the expression of NR2A; this was inhibited only by allopregnanolone. Although both hormones restored NMDAR function, as measured by the NMDA-induced rise in intracellular calcium, allopregnanolone (but not E2) reversed the inhibition of GABA(A)R and neuronal death caused by prolonged exposure to dieldrin. Our results indicate that allopregnanolone protects cortical neurons against the neurotoxicity caused by long-term exposure to dieldrin by maintaining GABA(A)R and NMDAR functionality.

  12. Different forms of glycine- and GABAA-receptor mediated inhibitory synaptic transmission in mouse superficial and deep dorsal horn neurons

    Directory of Open Access Journals (Sweden)

    Brichta Alan M

    2009-11-01

    Full Text Available Abstract Background Neurons in superficial (SDH and deep (DDH laminae of the spinal cord dorsal horn receive sensory information from skin, muscle, joints and viscera. In both regions, glycine- (GlyR and GABAA-receptors (GABAARs contribute to fast synaptic inhibition. For rat, several types of GABAAR coexist in the two regions and each receptor type provides different contributions to inhibitory tone. Recent work in mouse has discovered an additional type of GlyR, (containing alpha 3 subunits in the SDH. The contribution of differing forms of the GlyR to sensory processing in SDH and DDH is not understood. Methods and Results Here we compare fast inhibitory synaptic transmission in mouse (P17-37 SDH and DDH using patch-clamp electrophysiology in transverse spinal cord slices (L3-L5 segments, 23°C. GlyR-mediated mIPSCs were detected in 74% (25/34 and 94% (25/27 of SDH and DDH neurons, respectively. In contrast, GABAAR-mediated mIPSCs were detected in virtually all neurons in both regions (93%, 14/15 and 100%, 18/18. Several Gly- and GABAAR properties also differed in SDH vs. DDH. GlyR-mediated mIPSC amplitude was smaller (37.1 ± 3.9 vs. 64.7 ± 5.0 pA; n = 25 each, decay time was slower (8.5 ± 0.8 vs. 5.5 ± 0.3 ms, and frequency was lower (0.15 ± 0.03 vs. 0.72 ± 0.13 Hz in SDH vs. DDH neurons. In contrast, GABAAR-mediated mIPSCs had similar amplitudes (25.6 ± 2.4, n = 14 vs. 25. ± 2.0 pA, n = 18 and frequencies (0.21 ± 0.08 vs. 0.18 ± 0.04 Hz in both regions; however, decay times were slower (23.0 ± 3.2 vs. 18.9 ± 1.8 ms in SDH neurons. Mean single channel conductance underlying mIPSCs was identical for GlyRs (54.3 ± 1.6 pS, n = 11 vs. 55.7 ± 1.8, n = 8 and GABAARs (22.7 ± 1.7 pS, n = 10 vs. 22.4 ± 2.0 pS, n = 11 in both regions. We also tested whether the synthetic endocanabinoid, methandamide (methAEA, had direct effects on Gly- and GABAARs in each spinal cord region. MethAEA (5 μM reduced GlyR-mediated mIPSC frequency in SDH

  13. Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors

    OpenAIRE

    Katrin M Hoffmann; Herbrechter, Robin; Ziemba, Paul M.; Lepke, Peter; Beltrán, Leopoldo; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2016-01-01

    Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA r...

  14. Excitotoxic death induced by released glutamate in depolarized primary cultures of mouse cerebellar granule cells is dependent on GABAA receptors and niflumic acid-sensitive chloride channels.

    Science.gov (United States)

    Babot, Zoila; Cristòfol, Rosa; Suñol, Cristina

    2005-01-01

    Excitotoxic neuronal death has been linked to neurological and neurodegenerative diseases. Several studies have sought to clarify the involvement of Cl(-) channels in neuronal excitotoxicity using either N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainic acid agonists. In this work we induced excitotoxic death in primary cultures of cerebellar granule cells by means of endogenously released glutamate. Excitotoxicity was provoked by exposure to high extracellular K(+) concentrations ([K(+)](o)) for 5 min. Under these conditions, a Ca(2+)-dependent release of glutamate was evoked. When extracellular glutamate concentration rose to between 2 and 4 microM, cell viability was significantly reduced by 30-40%. The NMDA receptor antagonists (MK-801 and D-2-amino-5-phosphonopentanoic acid) prevented cell death. Exposure to high [K(+)](o) produced a (36)Cl(-) influx which was significantly reduced by picrotoxinin. In addition, the GABA(A) receptor antagonists (bicuculline, picrotoxinin and SR 95531) protected cells from high [K(+)](o)-triggered excitotoxicity and reduced extracellular glutamate concentration. The Cl(-) channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid also exerted a neuroprotective effect and reduced extracellular glutamate concentration, even though they did not reduce high [K(+)](o)-induced (36)Cl(-) influx. Primary cultures of cerebellar granule cells also contain a population of GABAergic neurons that released GABA in response to high [K(+)](o). Chronic treatment of primary cultures with kainic acid abolished GABA release and rendered granule cells insensitive to high [K(+)](o) exposure, even though NMDA receptors were functional. Altogether, these results demonstrate that, under conditions of membrane depolarization, low micromolar concentrations of extracellular glutamate might induce an excitotoxic process through both NMDA and GABA(A) receptors and niflumic acid-sensitive Cl

  15. The differential role of alpha1- and alpha5-containing GABA(A) receptors in mediating diazepam effects on spontaneous locomotor activity and water-maze learning and memory in rats.

    Science.gov (United States)

    Savić, Miroslav M; Milinković, Marija M; Rallapalli, Sundari; Clayton, Terry; Joksimović, Sroan; Van Linn, Michael; Cook, James M

    2009-10-01

    The clinical use of benzodiazepines (BZs) is hampered by sedation and cognitive deterioration. Although genetic and pharmacological studies suggest that alpha1- and alpha5-containing GABA(A) receptors mediate and/or modulate these effects, their molecular substrate is not fully elucidated. By the use of two selective ligands: the alpha1-subunit affinity-selective antagonist beta-CCt, and the alpha5-subunit affinity- and efficacy-selective antagonist XLi093, we examined the mechanisms of behavioural effects of diazepam in the tests of spontaneous locomotor activity and water-maze acquisition and recall, the two paradigms indicative of sedative- and cognition-impairing effects of BZs, respectively. The locomotor-activity decreasing propensity of diazepam (significant at 1.5 and 5 mg/kg) was antagonized by beta-CCt (5 and 15 mg/kg), while it tended to be potentiated by XLi093 in doses of 10 mg/kg, and especially 20 mg/kg. Diazepam decreased acquisition and recall in the water maze, with a minimum effective dose of 1.5 mg/kg. Both antagonists reversed the thigmotaxis induced by 2 mg/kg diazepam throughout the test, suggesting that both GABA(A) receptor subtypes participate in BZ effects on the procedural component of the task. Diazepam-induced impairment in the declarative component of the task, as assessed by path efficiency, the latency and distance before finding the platform across acquisition trials, and also by the spatial parameters in the probe trial, was partially prevented by both, 15 mg/kg beta-CCt and 10 mg/kg XLi093. Combining a BZ with beta-CCt results in the near to control level of performance of a cognitive task, without sedation, and may be worth testing on human subjects.

  16. Eugenol inhibits the GABAA current in trigeminal ganglion neurons.

    Science.gov (United States)

    Lee, Sang Hoon; Moon, Jee Youn; Jung, Sung Jun; Kang, Jin Gu; Choi, Seung Pyo; Jang, Jun Ho

    2015-01-01

    Eugenol has sedative, antioxidant, anti-inflammatory, and analgesic effects, but also serves as an irritant through the regulation of a different set of ion channels. Activation of gamma aminobutyric acid (GABA) receptors on sensory neurons leads to the stabilization of neuronal excitability but contributes to formalin-induced inflammatory pain. In this study, we examined the effect of eugenol on the GABA-induced current in rat trigeminal ganglia (TG) neurons and in human embryonic kidney (HEK) 293 cells expressing the GABAA receptor α1β2γ2 subtype using the whole-cell patch clamp technique. RT-PCR and Western blot analysis were used to confirm the expression of GABAA receptor γ2 subunit mRNA and protein in the TG and hippocampus. Eugenol decreased the amplitude ratio of the GABA-induced current to 27.5 ± 3.2% (p eugenol inhibited GABA-induced currents in a dose-dependent manner. Application of eugenol also decreased the GABA response in the presence of a G-protein blocker. Eugenol pretreatment with different concentrations of GABA resulted in similar inhibition of the GABA-induced current in a non-competitive manner. In conclusion, eugenol inhibits the GABA-induced current in TG neurons and HEK 293 cells expressing the GABAA receptor in a reversible, dose-dependent, and non-competitive manner, but not via the G-protein pathway. We suggest that the GABAA receptor could be a molecular target for eugenol in the modulation of nociceptive information.

  17. Zolpidem, a selective GABA(A) receptor alpha1 subunit agonist, induces comparable Fos expression in oxytocinergic neurons of the hypothalamic paraventricular and accessory but not supraoptic nuclei in the rat

    DEFF Research Database (Denmark)

    Kiss, Alexander; Søderman, Andreas; Bundzikova, Jana;

    2006-01-01

    Functional activation of oxytocinergic (OXY) cells in the hypothalamic paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei was investigated in response to acute treatment with Zolpidem (a GABA(A) receptor agonist with selectivity for alpha(1) subunits) utilizing dual Fos/OXY immun...

  18. In vivo neurochemical evidence that newly synthesised GABA activates GABA(B), but not GABA(A), receptors on dopaminergic nerve endings in the nucleus accumbens of freely moving rats

    NARCIS (Netherlands)

    Saigusa, T.; Aono, Y.; Sekino, R.; Uchida, T.; Takada, K.; Oi, Y.; Koshikawa, N.; Cools, A.R.

    2012-01-01

    GABA released from accumbal GABAergic interneurons plays an inhibitory role in the regulation of dopamine efflux through GABA(B) and GABA(A) receptors located on accumbal dopaminergic nerve endings. The cytosolic newly synthesised GABA alters vesicular GABA levels and, accordingly, the amount of GAB

  19. Zolpidem, a selective GABA(A) receptor alpha1 subunit agonist, induces comparable Fos expression in oxytocinergic neurons of the hypothalamic paraventricular and accessory but not supraoptic nuclei in the rat

    DEFF Research Database (Denmark)

    Kiss, Alexander; Søderman, Andreas; Bundzikova, Jana

    2006-01-01

    Functional activation of oxytocinergic (OXY) cells in the hypothalamic paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei was investigated in response to acute treatment with Zolpidem (a GABA(A) receptor agonist with selectivity for alpha(1) subunits) utilizing dual Fos/OXY immun...

  20. Central PGE2 exhibits anxiolytic-like activity via EP1 and EP4 receptors in a manner dependent on serotonin 5-HT1A, dopamine D1 and GABAA receptors.

    Science.gov (United States)

    Suzuki, Chihiro; Miyamoto, Chihiro; Furuyashiki, Tomoyuki; Narumiya, Shuh; Ohinata, Kousaku

    2011-07-21

    We found that centrally administered prostaglandin (PG) E(2) exhibited anxiolytic-like activity in the elevated plus-maze and open field test in mice. Agonists selective for EP(1) and EP(4) receptors, among four receptor subtypes for PGE(2), mimicked the anxiolytic-like activity of PGE(2). The anxiolytic-like activity of PGE(2) was blocked by an EP(1) or EP(4) antagonist, as well as in EP(4) but not EP(1) knockout mice. Central activation of either EP(1) or EP(4) receptors resulted in anxiolytic-like activity. The PGE(2)-induced anxiolytic-like activity was inhibited by antagonists for serotonin 5-HT(1A), dopamine D(1) and GABA(A) receptors. Taken together, PGE(2) exhibits anxiolytic-like activity via EP(1) and EP(4) receptors, with downstream involvement of 5-HT(1A), D(1) and GABA(A) receptor systems. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  1. Study of the Interaction of 1,4- and 1,5-Benzodiazepines with GABAA Receptors of Rat Cerebellum Granule Cells in Culture.

    Science.gov (United States)

    Nikas, Periklis; Gatta, Elena; Cupello, Aroldo; Di Braccio, Mario; Grossi, Giancarlo; Pellistri, Francesca; Robello, Mauro

    2015-08-01

    The effects of a classical 1,4-benzodiazepine agonist, such as diazepam, its catabolite N-desmethyl-diazepam (nordiazepam), and 1,5-benzodiazepines such as clobazam and RL 214 (a triazolobenzodiazepine previously synthesized in our labs) were evaluated on native GABAA receptors of cerebellar granule cells in culture. The parameter studied was the increase of GABA-activated chloride currents caused by these substances. The contributions of α6 β2/3 γ2 and α1 α6 β2/3 γ2 receptor subtypes to the increase of GABA-activated chloride current were investigated by comparing the effects of such substances in the presence vs. the absence of furosemide. Furosemide is in fact able to block such receptors. It was found that the percent enhancement of peak GABA-activated current doubled for diazepam, clobazam, and RL 214. However, it did not change for N-desmethyl-diazepam. These results indicate that diazepam, clobazam, and RL 214 interact exclusively with α1 β2/3 γ2 receptors, while N-desmethyl-diazepam seems to interact with not only α1- but also α6-containing receptors.

  2. GABAA receptor-mediated feedforward and feedback inhibition differentially modulate the gain and the neural code transformation in hippocampal CA1 pyramidal cells.

    Science.gov (United States)

    Jang, Hyun Jae; Park, Kyerl; Lee, Jaedong; Kim, Hyuncheol; Han, Kyu Hun; Kwag, Jeehyun

    2015-12-01

    Diverse variety of hippocampal interneurons exists in the CA1 area, which provides either feedforward (FF) or feedback (FB) inhibition to CA1 pyramidal cell (PC). However, how the two different inhibitory network architectures modulate the computational mode of CA1 PC is unknown. By investigating the CA3 PC rate-driven input-output function of CA1 PC using in vitro electrophysiology, in vitro-simulation of inhibitory network, and in silico computational modeling, we demonstrated for the first time that GABAA receptor-mediated FF and FB inhibition differentially modulate the gain, the spike precision, the neural code transformation and the information capacity of CA1 PC. Recruitment of FF inhibition buffered the CA1 PC spikes to theta-frequency regardless of the input frequency, abolishing the gain and making CA1 PC insensitive to its inputs. Instead, temporal variability of the CA1 PC spikes was increased, promoting the rate-to-temporal code transformation to enhance the information capacity of CA1 PC. In contrast, the recruitment of FB inhibition sub-linearly transformed the input rate to spike output rate with high gain and low spike temporal variability, promoting the rate-to-rate code transformation. These results suggest that GABAA receptor-mediated FF and FB inhibitory circuits could serve as network mechanisms for differentially modulating the gain of CA1 PC, allowing CA1 PC to switch between different computational modes using rate and temporal codes ad hoc. Such switch will allow CA1 PC to efficiently respond to spatio-temporally dynamic inputs and expand its computational capacity during different behavioral and neuromodulatory states in vivo.

  3. GABA(A) receptor activation in the CA1 area of the dorsal hippocampus impairs consolidation of conditioned contextual fear in C57BL/6J mice.

    Science.gov (United States)

    Misane, Ilga; Kruis, Ayla; Pieneman, Anton W; Ögren, Sven Ove; Stiedl, Oliver

    2013-02-01

    Local infusion of the GABA(A) receptor agonist muscimol is used for reversible inactivation of septohippocampal brain structures associated with cognitive functions. However, information on the effective duration, affected processes and site(s) of action of muscimol in the hippocampus is lacking. Therefore, the dose- and time-dependent effects of bilateral dorsohippocampal infusion of muscimol (0.01-2.0 μg/mouse) below the CA1 area were examined on processing of fear memory in male C57BL/6J mice. Infusion of muscimol 15 min-6 h but not 9 h or 24 h before training impaired conditioned context-dependent fear tested 24 h or 48 h after training. Post-training infusion of muscimol also impaired context-dependent fear when applied either 4 h or 6 h after training, although with lower efficacy. Muscimol was ineffective when administered immediately, 1 h or 24 h after training. Infusion of muscimol 15 min before training impaired context-dependent fear 4-6 h after training indicating preserved short-term but impaired long-term memory. Regardless of infusion time and dose, muscimol had no effect on tone-dependent (cued) fear memory. The impairment by the fluorescently-labeled muscimol-bodipy (5.3 μg/mouse) were similar to those of an equimolar dose of muscimol (1 μg/mouse). The distribution profile after local infusion indicated that muscimol-bodipy (5.3 μg/mouse) was confined to the CA1 area of the dorsal hippocampus. These results demonstrated that GABA(A) receptor activation in the CA1 area of the dorsal hippocampus causes a long-term memory impairment of conditioned context-dependent fear mediated by a long-lasting (≥6 h) muscimol action most likely affecting consolidation processes.

  4. Unique insecticide specificity of human homomeric rho 1 GABA(C) receptor.

    Science.gov (United States)

    Ratra, Gurpreet S; Erkkila, Brian E; Weiss, David S; Casida, John E

    2002-03-24

    Several convulsants and major insecticides block the gamma-aminobutyric acid (GABA)-gated chloride channel in brain on binding to the GABA(A) receptor. The GABA(C) receptor, important in retina and present in brain, is also coupled to a chloride channel and is therefore a potential target for toxicant action examined here in radioligand binding and electrophysiological experiments. Human homomeric rho 1 GABA(C) receptor expressed in human embryonic kidney cells (HEK293) undergoes specific and saturable high-affinity binding of 4-n-[3H]propyl-4' -ethynylbicycloorthobenzoate ([3H]EBOB) using a cyano analog (CNBOB) to determine non-specific binding. This GABA(C) rho 1 receptor is very sensitive to CNBOB and lindane relative to alpha-endosulfan, tert-butylbicyclophosphorothionate, picrotoxinin and fipronil (IC(50) values of 23, 91, 800, 1080, 4000 and >10000 nM, respectively, in displacing [3H]EBOB). A similar potency sequence (except for picrotoxinin) is observed for inhibition of GABA-induced currents of rho 1 receptor expressed in Xenopus oocytes. The present study does not consider rho 2 homomeric and rho 1 rho 2 heteromeric GABA(C) receptors which are known to be more sensitive than rho 1 to picrotoxinin. The inhibitor sensitivity and specificity of this rho 1 GABA(C) receptor differ greatly from those of human homomeric beta 3 and native GABA(A) receptors.

  5. Complex control of GABA(A receptor subunit mRNA expression: variation, covariation, and genetic regulation.

    Directory of Open Access Journals (Sweden)

    Megan K Mulligan

    Full Text Available GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly variable and heritable among the large cohort of BXD strains derived from crosses of fully sequenced parents--C57BL/6J and DBA/2J. Genetic control of these subunits is complex and highly dependent on tissue and mRNA region. Remarkably, this high variation is generally not linked to phenotypic differences. The single exception is Gabrb3, a locus that is linked to anxiety. We identified upstream genetic loci that influence subunit expression, including three unlinked regions of chromosome 5 that modulate the expression of nine subunits in hippocampus, and that are also associated with multiple phenotypes. Candidate genes within these loci include, Naaa, Nos1, and Zkscan1. We confirmed a high level of coexpression for subunits comprising the major channel--Gabra1, Gabrb2, and Gabrg2--and identified conserved members of this expression network in mice and humans. Gucy1a3, Gucy1b3, and Lis1 are novel and conserved associates of multiple subunits that are involved in inhibitory signaling. Finally, proximal and distal regions of the 3' UTRs of single subunits have remarkably independent expression patterns in both species. However, corresponding regions of different subunits often show congruent genetic control and coexpression (proximal-to-proximal or distal-to-distal, even in the absence of sequence homology. Our findings identify novel sources of variation that modulate subunit expression and highlight the extraordinary capacity of biological networks to buffer

  6. Human presynaptic receptors.

    Science.gov (United States)

    Schlicker, Eberhard; Feuerstein, Thomas

    2017-04-01

    Presynaptic receptors are sites at which transmitters, locally formed mediators or hormones inhibit or facilitate the release of a given transmitter from its axon terminals. The interest in the identification of presynaptic receptors has faded in recent years and it may therefore be justified to give an overview of their occurrence in the autonomic and central nervous system; this review will focus on presynaptic receptors in human tissues. Autoreceptors are presynaptic receptors at which a given transmitter restrains its further release, though in some instances may also increase its release. Inhibitory autoreceptors represent a typical example of a negative feedback; they are tonically activated by the respective endogenous transmitter and/or are constitutively active. Autoreceptors also play a role under pathophysiological conditions, e.g. by limiting the massive noradrenaline release occurring during congestive heart failure. They can be used for therapeutic purposes; e.g., the α2-adrenoceptor antagonist mirtazapine is used as an antidepressant and the inverse histamine H3 receptor agonist pitolisant has been marketed as a new drug for the treatment of narcolepsy in 2016. Heteroreceptors are presynaptic receptors at which transmitters from adjacent neurons, locally formed mediators (e.g. endocannabinoids) or hormones (e.g. adrenaline) can inhibit or facilitate transmitter release; they may be subject to an endogenous tone. The constipating effect of the sympathetic nervous system or of the antihypertensive drug clonidine is related to the activation of inhibitory α2-adrenoceptors on postganglionic parasympathetic neurons. Part of the stimulating effect of adrenaline on the sympathetic nervous system during stress is related to its facilitatory effect on noradrenaline release via β2-adrenoceptors.

  7. Participation of GABAA Chloride Channels in the Anxiolytic-Like Effects of a Fatty Acid Mixture

    Directory of Open Access Journals (Sweden)

    Juan Francisco Rodríguez-Landa

    2013-01-01

    Full Text Available Human amniotic fluid and a mixture of eight fatty acids (FAT-M identified in this maternal fluid (C12:0, lauric acid, 0.9 μg%; C14:0, myristic acid, 6.9 μg%; C16:0, palmitic acid, 35.3 μg%; C16:1, palmitoleic acid, 16.4 μg%; C18:0, stearic acid, 8.5 μg%; C18:1cis, oleic acid, 18.4 μg%; C18:1trans, elaidic acid, 3.5 μg%; C18:2, linoleic acid, 10.1 μg% produce anxiolytic-like effects that are comparable to diazepam in Wistar rats, suggesting the involvement of γ-aminobutyric acid-A (GABAA receptors, a possibility not yet explored. Wistar rats were subjected to the defensive burying test, elevated plus maze, and open field test. In different groups, three GABAA receptor antagonists were administered 30 min before FAT-M administration, including the competitive GABA binding antagonist bicuculline (1 mg/kg, GABAA benzodiazepine antagonist flumazenil (5 mg/kg, and noncompetitive GABAA chloride channel antagonist picrotoxin (1 mg/kg. The FAT-M exerted anxiolytic-like effects in the defensive burying test and elevated plus maze, without affecting locomotor activity in the open field test. The GABAA antagonists alone did not produce significant changes in the behavioral tests. Picrotoxin but not bicuculline or flumazenil blocked the anxiolytic-like effect of the FAT-M. Based on the specific blocking action of picrotoxin on the effects of the FAT-M, we conclude that the FAT-M exerted its anxiolytic-like effects through GABAA receptor chloride channels.

  8. Neuroprotection of GluK1 kainate receptor agonist ATPA against ischemic neuronal injury through inhibiting GluK2 kainate receptor-JNK3 pathway via GABA(A) receptors.

    Science.gov (United States)

    Lv, Qian; Liu, Yong; Han, Dong; Xu, Jing; Zong, Yan-Yan; Wang, Yao; Zhang, Guang-Yi

    2012-05-25

    It is well known that GluK2-containing kainate receptors play essential roles in seizure and cerebral ischemia-induced neuronal death, while GluK1-containing kainate receptors could increase tonic inhibition of post-synaptic pyramidal neurons. This research investigated whether GluK1 could inhibit activation of c-Jun N-terminal kinase 3 (JNK3) signaling pathway mediated by the GluK2 in cerebral ischemia-reperfusion. The results show that GluK1 activation by (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA) at 1nmol per rat could inhibit the assembly of GluK2·Postsynaptic density 95·mixed lineage kinase 3 signaling module, activation of JNK3 and its downstream signal molecules. However, the inhibition of ATPA could be prevented by GluK1 antagonist NS3763, GluK1 antisense, and GABA(A) receptor antagonist bicuculline. In addition, ATPA played a neuroprotective role against cerebral ischemia. In sum, the findings indicate that activation of GluK1 by ATPA at specific dosages may promote GABA release, which then suppresses post-synaptic GluK2-JNK3 signaling-mediated cerebral ischemic injury via GABA(A)R.

  9. Changes in mACh, NMDA and GABA(A) receptor binding after lateral fluid-percussion injury: in vitro autoradiography of rat brain frozen sections.

    Science.gov (United States)

    Sihver, S; Marklund, N; Hillered, L; Långström, B; Watanabe, Y; Bergström, M

    2001-08-01

    Adult rats were subjected to a moderate lateral fluid percussion injury (FPI), followed by survival periods of 2 and 12 h. Regional NMDA subtype glutamate, muscarinic acetylcholine and GABA(A) receptor binding in various brain regions was analysed by quantitative in vitro autoradiography and short-lived positron emission tomography tracers [11C]cyano-dizocilpine, 4-N-[11C]methylpiperidylbenzilate (4-N-[11C]MPB), and [11C]flumazenil, respectively. The binding potential (BP, Bmax/KD) was calculated. The data with [11C]cyano-dizocilpine showed a significant decrease in BP bilaterally for the frontoparietal cortex and hippocampus at both time points, in comparison with that of the sham-operated controls. At 12 h the decrease was significantly more prominent for the ipsilateral cortex and hippocampus than for the contralateral side. The BP of 4-N-[11C]MPB was significantly decreased after 2 h for the trauma-side hippocampus, and after 12 h it had decreased for the trauma-site cortex and the bilateral hippocampus. The [11C]flumazenil exhibited a significant decrease in BP for the trauma-site cortex and the underlying hippocampus by 2 h after the traumatic brain injury. After 12 h a significantly decreased BP was observed only for the trauma-site cortex. The finding of a decreased BP demonstrates the involvement of these receptor systems in the development of cellular dysfunction, which is widespread and not limited to the site of lateral FPI.

  10. GABA(A) Increases Calcium in Subventricular Zone Astrocyte-Like Cells Through L- and T-Type Voltage-Gated Calcium Channels

    DEFF Research Database (Denmark)

    Young, Stephanie Z; Platel, Jean-Claude; Nielsen, Jakob V;

    2010-01-01

    intracellular Ca(2+) dynamics in SVZ astrocytes. To monitor Ca(2+) activity selectively in astrocyte-like cells, we used two lines of transgenic mice expressing either GFP fused to a Gq-coupled receptor or DsRed under the human glial fibrillary acidic protein (hGFAP) promoter. GABA(A) receptor activation......-like cells to 75%, suggesting that the majority of SVZ astrocytes express functional VGCCs. SVZ astrocytes also displayed spontaneous Ca(2+) activity, the frequency of which was regulated by tonic GABA(A) receptor activation. These data support a role for ambient GABA in tonically regulating intracellular Ca...

  11. Receptores GABAA (ácido γ-aminobutírico y su relación con la dependencia al alcohol Receptores GABAA (ácido γ-aminobutírico y su relación con la dependencia al alcohol

    Directory of Open Access Journals (Sweden)

    Aura Rengifo

    2005-04-01

    Full Text Available El síndrome de dependencia al alcohol es una enfermedad multifactorial, ambiental, cultural, social y genética. Son innumerables las investigaciones que han intentado elu- cidar los posibles mecanismos neurobiológicos de este síndrome para entender los cambios fisiológicos y comportamentales del individuo, de tal manera que se puedan brindar alter- nativas farmacológicas que ayuden a controlar este tipo dependencia.Esta revisión describe el papel de la neurotransmisión involucrada en el síndrome de de- pendencia al alcohol, principalmente la relacionada con el glutamato, la dopamina, los opioides y en especial el ácido γ-aminobutírico (GABA, porque la activación del receptor GABAA (receptor canal de cloro desencadena un efecto inhibitorio a nivel neuronal, y en individuos alcohólicos presenta hipofunción, la cual no le permite modular la recurrente excitación neuronal obteniendo como resultado un estado de desinhibición/hiperexcitación típico de alcohólicos. Por otra parte, los genes que codifican para las subunidades de los receptores GABAA ubicados en los cromosomas 4 y 15 han sido asociados con alcoholismo, al presentarse fuertes relaciones entre el desequilibrio de unión entre estos genes y el fenotipo de las ondas β en los electroencefalogramas de los individuos con el síndrome de dependencia al alcohol y sus descendientes.De acuerdo a las investigaciones en el tema, finalmente proponemos las posibles vías y sus mecanismos de control implicados en el síndrome de dependencia al alcohol.The alcohol dependence syndrome is a multifactorial, enviromental, cultural, social, and genetic dissease. The investigations are countless that have tried to elucidate the possible neurobiologic mechanisms of this syndrome to understand the individual’s physiologic and behavioral changes, in such way that it can be offered pharmacological alternatives that help to control this type of dependence. This revision describes the

  12. Do N-arachidonyl-glycine (NA-glycine and 2-arachidonoyl glycerol (2-AG share mode of action and the binding site on the β2 subunit of GABAA receptors?

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    Roland Baur

    2013-09-01

    Full Text Available NA-glycine is an endogenous lipid molecule with analgesic properties, which is structurally similar to the endocannabinoids 2-AG and anandamide but does not interact with cannabinoid receptors. NA-glycine has been suggested to act at the G-protein coupled receptors GPR18 and GPR92. Recently, we have described that NA-glycine can also modulate recombinant α1β2γ2 GABAA receptors. Here we characterize in more detail this modulation and investigate the relationship of its binding site with that of the endocannabinoid 2-AG.

  13. Anxiolytic effects of the GABA(A) receptor partial agonist, L-838,417: impact of age, test context familiarity, and stress.

    Science.gov (United States)

    Morales, Melissa; Varlinskaya, Elena I; Spear, Linda P

    2013-08-01

    The partial α2,3,5 GABA(A) receptor agonist, L-838,417 has been reported to have anxiolytic effects in adult rodents. Although maturational differences exist for the GABA(A) receptor subunits, the anxiolytic effects of L-838,417 have not been tested in younger animals. The goal of the present experiments was to determine whether L-838,417 reverses anxiety-like behavior induced by either an unfamiliar environment (Experiment 1) or repeated restraint stress (Experiment 2) differentially in adolescent and adult, male and female Sprague-Dawley rats using a modified social interaction test. In Experiment 1, rats were injected with 0, 0.5, 1.0, 2.0, or 4.0 mg/kg L-838,417, i.p. and tested 30 min later in an unfamiliar test context for 10 min. In Experiment 2, rats were exposed to restraint stress (90 min daily for 5 days). Immediately after the last restraint session, animals were injected with L-838,417 and placed alone for 30 min in the test apparatus to familiarize them to this context prior to the 10 min social interaction test. In Experiment 1, L-838,417 produced anxiolytic effects in adults at 1.0 mg/kg, as indexed by a transformation of social avoidance into preference and an increase in social investigation. In adolescents, a dose of 2.0 mg/kg eliminated social avoidance, but had no anxiolytic effects on social investigation. Testing under familiar circumstances (Experiment 2) after repeated restraint stress eliminated age differences in sensitivity to L-838,417, with 0.5 mg/kg reversing the anxiogenic effects of prior stress regardless of age, but with doses ≥ 1 mg/kg decreasing social investigation, an effect possibly due in part to locomotor-impairing effects of this compound. Although locomotor activity was suppressed in both experiments, higher doses of L-838,417 were necessary to suppress locomotor activity in Experiment 1. Thus, anxiolytic effects of L-838,417 were found to be context-, age-, and stress-dependent.

  14. Memantine treatment reduces the expression of the K(+)/Cl(-) cotransporter KCC2 in the hippocampus and cerebral cortex, and attenuates behavioural responses mediated by GABA(A) receptor activation in mice.

    Science.gov (United States)

    Molinaro, Gemma; Battaglia, Giuseppe; Riozzi, Barbara; Di Menna, Luisa; Rampello, Liborio; Bruno, Valeria; Nicoletti, Ferdinando

    2009-04-10

    A 7-day treatment with memantine (25 mg/kg, i.p.), a drug that is currently prescribed for the treatment of Alzheimer's disease, increased the levels of brain-derived neurotrophic factor (BDNF) and reduced the expression of the neuron-specific K(+)/Cl(-) co-transporter, KCC2, in the hippocampus and cerebral cortex of mice. Knowing that KCC2 maintains low intracellular Cl(-) concentrations, which drive Cl(-) influx in response to GABA(A) receptor activation, we monitored the behavioural response to the GABA(A) receptor enhancer, diazepam, in mice pre-treated for 7 days with saline or 25 mg/kg of memantine. Memantine treatment substantially attenuated motor impairment induced by an acute challenge with diazepam (6 mg/kg, i.p.), as assessed by the rotarod test and the horizontal wire test. We suggest that a prolonged treatment with memantine induces changes in the activity of GABA(A) receptors that might contribute to the therapeutic and/or toxic effects of the drug.

  15. The anxioselective agent 7-(2-chloropyridin-4-yl)pyrazolo-[1,5-a]-pyrimidin-3-yl](pyridin-2-yl)methanone (DOV 51892) is more efficacious than diazepam at enhancing GABA-gated currents at alpha1 subunit-containing GABAA receptors.

    Science.gov (United States)

    Popik, Piotr; Kostakis, Emmanuel; Krawczyk, Martyna; Nowak, Gabriel; Szewczyk, Bernadeta; Krieter, Philip; Chen, Zhengming; Russek, Shelley J; Gibbs, Terrell T; Farb, David H; Skolnick, Phil; Lippa, Arnold S; Basile, Anthony S

    2006-12-01

    Studies using mice with point mutations of GABA(A) receptor alpha subunits suggest that the sedative and anxiolytic properties of 1,4-benzodiazepines are mediated, respectively, by GABA(A) receptors bearing the alpha(1) and alpha(2) subunits. This hypothesis predicts that a compound with high efficacy at GABA(A) receptors containing the alpha(1) subunit would produce sedation, whereas an agonist acting at alpha(2) subunit-containing receptors (with low or null efficacy at alpha(1)-containing receptors) would be anxioselective. Electrophysiological studies using recombinant GABA(A) receptors expressed in Xenopus oocytes indicate that maximal potentiation of GABA-stimulated currents by the pyrazolo-[1,5-a]-pyrimidine, DOV 51892, at alpha(1)beta(2)gamma(2S) constructs of the GABA(A) receptor was significantly higher (148%) than diazepam. In contrast, DOV 51892 was considerably less efficacious and/or potent than diazepam in enhancing GABA-stimulated currents mediated by constructs containing alpha(2), alpha(3), or alpha(5) subunits. In vivo, DOV 51892 increased punished responding in the Vogel conflict test, an effect blocked by flumazenil, and increased the percentage of time spent in the open arms of the elevated plus-maze. However, DOV 51892 had no consistent effects on motor function or muscle relaxation at doses more than 1 order of magnitude greater than the minimal effective anxiolytic dose. Although the mutant mouse data predict that the high-efficacy potentiation of GABA(A1a) receptor-mediated currents by DOV 51892 would be sedating, behavioral studies demonstrate that DOV 51892 is anxioselective, indicating that GABA potentiation mediated by alpha(1) subunit-containing GABA(A) receptors may be neither the sole mechanism nor highly predictive of the sedative properties of benzodiazepine recognition site modulators.

  16. Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

    Science.gov (United States)

    Vela, J; Vitorica, J; Ruano, D

    2001-12-01

    We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.

  17. Allopregnanolone reduces immobility in the forced swimming test and increases the firing rate of lateral septal neurons through actions on the GABAA receptor in the rat.

    Science.gov (United States)

    Rodrìguez-Landa, Juan Francisco; Contreras, Carlos M; Bernal-Morales, Blandina; Gutièrrez-Garcìa, Ana G; Saavedra, Margarita

    2007-01-01

    Since allopregnanolone reduces the total time of immobility in rats submitted to the forced swimming test, we decided to explore whether this neuroactive steroid shares other antidepressant-like actions, such as increasing the neuronal firing rate in the lateral septal nucleus (LSN). In order to discard the influence of the oestrous cycle on immobility and on the firing rate of LSN neurons, all Wistar rats used in the study underwent ovariectomy before treatments. A group of rats received different doses of allopregnanolone (0.5, 1.0, 2.0 and 3.0 mg/kg, i.p.) 1 hour before being forced to swim in order to identify the minimum effective dose diminishing immobility. None of the tested doses of allopregnanolone produced significant changes in motor activity in the open-field test. The minimum dose of allopregnanolone producing a significant reduction in the total time of immobility (pimmobility (pimmobility in the forced swimming test (1.0 mg/kg) significantly (p immobility and LSN firing rate. In conclusion, allopregnanolone produces an antidepressant-like effect in the forced swimming test, associated with an increase in the LSN neuronal firing rate, seemingly mediated by the GABAA receptor.

  18. Regulation of GABA(A and glutamate receptor expression, synaptic facilitation and long-term potentiation in the hippocampus of prion mutant mice.

    Directory of Open Access Journals (Sweden)

    Alejandra Rangel

    Full Text Available BACKGROUND: Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrP(sc of the natural cellular prion protein (PrP(c encoded by the Prnp gene. Although several roles have been attributed to PrP(c, its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrP(c studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation. METHODOLOGY/PRINCIPAL FINDINGS: Here we explore the role of PrP(c expression in neurotransmission and neural excitability using wild-type, Prnp -/- and PrP(c-overexpressing mice (Tg20 strain. By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp -/- mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp -/- and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABA(A and AMPA-kainate receptors are co-regulated in both Prnp -/- and Tg20 mice. CONCLUSIONS/SIGNIFICANCE: Present results demonstrate that PrP(c is necessary for the

  19. α4-containing GABAA receptors are required for antagonism of ethanol-induced motor incoordination and hypnosis by the imidazobenzodiazepine Ro15-4513

    Directory of Open Access Journals (Sweden)

    Sangeetha V. Iyer

    2011-04-01

    Full Text Available Alcohol (ethanol is widely consumed for its desirable effects but unfortunately has strong addiction potential. Some imidazobenzodiazepines such as Ro15-4513 are able to antagonize many ethanol-induced behaviors. Controversial biochemical and pharmacological evidence suggest that the effects of these ethanol antagonists and ethanol are mediated specifically via overlapping binding sites on α4/δ-containing GABAA-Rs. To investigate the requirement of α4-containing GABAA-Rs in the mechanism of action of Ro15-4513 on behavior, wildtype (WT and α4 knockout (KO mice were compared for antagonism of ethanol-induced motor incoordination and hypnosis. Motor effects of ethanol were tested in two different fixed speed rotarod assays. In the first experiment, mice were injected with 2.0 g/kg ethanol followed 5 min later by 10 mg/kg Ro15-4513 (or vehicle and tested on a rotarod at 8 rpm. In the second experiment, mice received a single injection of 1.5 g/kg ethanol +/- 3 mg/kg Ro15-4513 and were tested on a rotarod at 12 rpm. In both experiments, the robust Ro15-4513 antagonism of ethanol-induced motor ataxia that was observed in WT mice was absent in KO mice. A loss of righting reflex (LORR assay was used to test Ro15-4513 (20 mg/kg antagonism of ethanol (3.5 g/kg-induced hypnosis. An effect of sex was observed on the LORR assay, so males and females were analyzed separately. In male mice, Ro15-4513 markedly reduced ethanol-induced LORR in WT controls, but α4 KO mice were insensitive to this effect of Ro15-4513. In contrast, female KO mice did not differ from WT controls in the antagonistic effects of Ro15-4513 on ethanol-induced LORR. We conclude that Ro15-4513 requires α4-containing receptors for antagonism of ethanol-induced LORR (in males and motor ataxia.

  20. Topiramate reduces excitability in the basolateral amygdala by selectively inhibiting GluK1 (GluR5) kainate receptors on interneurons and positively modulating GABAA receptors on principal neurons.

    Science.gov (United States)

    Braga, Maria F M; Aroniadou-Anderjaska, Vassiliki; Li, He; Rogawski, Michael A

    2009-08-01

    Topiramate [2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate] is a structurally novel antiepileptic drug that has broad efficacy in epilepsy, but the mechanisms underlying its therapeutic activity are not fully understood. We have found that topiramate selectively inhibits GluK1 (GluR5) kainate receptor-mediated excitatory postsynaptic responses in rat basolateral amygdala (BLA) principal neurons and protects against seizures induced by the GluK1 kainate receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid (ATPA). Here, we demonstrate that topiramate also modulates inhibitory function in the BLA. Using whole-cell recordings in rat amygdala slices, we found that 0.3 to 10 microM topiramate 1) inhibited ATPA-evoked postsynaptic currents recorded from BLA interneurons; 2) suppressed ATPA-induced enhancement of spontaneous inhibitory postsynaptic currents (IPSCs) recorded from BLA pyramidal cells; and 3) blocked ATPA-induced suppression of evoked IPSCs, which is mediated by presynaptic GluK1 kainate receptors present on BLA interneurons. Topiramate (10 microM) had no effect on the AMPA [(R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid]-induced enhancement of spontaneous activity of BLA neurons. Thus, although topiramate inhibits GluK1 kainate receptor-mediated enhancement of interneuron firing, it promotes evoked GABA release, leading to a net inhibition of circuit excitability. In addition, we found that topiramate (0.3-10 microM) increased the amplitude of evoked, spontaneous, and miniature IPSCs in BLA pyramidal neurons, indicating an enhancement of postsynaptic GABA(A) receptor responses. Taken together with our previous findings, we conclude that topiramate protects against hyperexcitability in the BLA by suppressing the GluK1 kainate receptor-mediated excitation of principal neurons by glutamatergic afferents, blocking the suppression of GABA release from interneurons mediated by presynaptic GluK1

  1. Ectopic Expression of α6 and δ GABAA Receptor Subunits in Hilar Somatostatin Neurons Increases Tonic Inhibition and Alters Network Activity in the Dentate Gyrus.

    Science.gov (United States)

    Tong, Xiaoping; Peng, Zechun; Zhang, Nianhui; Cetina, Yliana; Huang, Christine S; Wallner, Martin; Otis, Thomas S; Houser, Carolyn R

    2015-12-09

    The role of GABAA receptor (GABAAR)-mediated tonic inhibition in interneurons remains unclear and may vary among subgroups. Somatostatin (SOM) interneurons in the hilus of the dentate gyrus show negligible expression of nonsynaptic GABAAR subunits and very low tonic inhibition. To determine the effects of ectopic expression of tonic GABAAR subtypes in these neurons, Cre-dependent viral vectors were used to express GFP-tagged GABAAR subunits (α6 and δ) selectively in hilar SOM neurons in SOM-Cre mice. In single-transfected animals, immunohistochemistry demonstrated strong expression of either the α6 or δ subunit; in cotransfected animals, both subunits were consistently expressed in the same neurons. Electrophysiology revealed a robust increase of tonic current, with progressively larger increases following transfection of δ, α6, and α6/δ subunits, respectively, indicating formation of functional receptors in all conditions and likely coassembly of the subunits in the same receptor following cotransfection. An in vitro model of repetitive bursting was used to determine the effects of increased tonic inhibition in hilar SOM interneurons on circuit activity in the dentate gyrus. Upon cotransfection, the frequency of GABAAR-mediated bursting in granule cells was reduced, consistent with a reduction in synchronous firing among hilar SOM interneurons. Moreover, in vivo studies of Fos expression demonstrated reduced activation of α6/δ-cotransfected neurons following acute seizure induction by pentylenetetrazole. The findings demonstrate that increasing tonic inhibition in hilar SOM interneurons can alter dentate gyrus circuit activity during strong stimulation and suggest that tonic inhibition of interneurons could play a role in regulating excessive synchrony within the network. In contrast to many hippocampal interneurons, somatostatin (SOM) neurons in the hilus of the dentate gyrus have very low levels of nonsynaptic GABAARs and exhibit very little tonic

  2. Pro-brain-derived neurotrophic factor inhibits GABAergic neurotransmission by activating endocytosis and repression of GABAA receptors.

    Science.gov (United States)

    Riffault, Baptiste; Medina, Igor; Dumon, Camille; Thalman, Carine; Ferrand, Nadine; Friedel, Perrine; Gaiarsa, Jean-Luc; Porcher, Christophe

    2014-10-01

    GABA is the canonical inhibitory neurotransmitter in the CNS. This inhibitory action is largely mediated by GABA type A receptors (GABAARs). Among the many factors controlling GABAergic transmission, brain-derived neurotrophic factor (BDNF) appears to play a major role in regulating synaptic inhibition. Recent findings have demonstrated that BDNF can be released as a precursor (proBDNF). Although the role of mature BDNF on GABAergic synaptogenesis and maintenance has been well studied, an important question still unanswered is whether secreted proBDNF might affect GABAergic neurotransmission. Here, we have used 14 d in vitro primary culture of hippocampal neurons and ex vivo preparations from rats to study the function of proBDNF in regulation of GABAAR trafficking and activity. We demonstrate that proBDNF impairs GABAergic transmission by the activation of two distinct pathways: (1) a RhoA-Rock-PTEN pathway that decreases the phosphorylation levels of GABAAR, thus affecting receptor function and triggering endocytosis and degradation of internalized receptors, and (2) a JAK-STAT-ICER pathway leading to the repression of GABAARs synthesis. These effects lead to the diminution of GABAergic synapses and are correlated with a decrease in GABAergic synaptic currents. These results revealed new functions for proBDNF-p75 neurotrophin receptor signaling pathway in the control of the efficacy of GABAergic synaptic activity by regulating the trafficking and synthesis of GABAARs at inhibitory synapses.

  3. Roles of taurine-mediated tonic GABAA receptor activation in the radial migration of neurons in the fetal mouse cerebral cortex

    Directory of Open Access Journals (Sweden)

    Tomonori eFurukawa

    2014-03-01

    Full Text Available γ-Aminobutyric acid (GABA depolarizes embryonic cerebrocortical neurons and continuous activation of the GABAA receptor (GABAAR contributes to their tonic depolarization. Although multiple reports have demonstrated a role of GABAAR activation in neocortical development, including in migration, most of these studies have used pharmacological blockers. Herein, we performed in utero electroporation in GABA synthesis-lacking homozygous GAD67-GFP knock-in mice (GAD67GFP/GFP to label neurons born in the ventricular zone. Three days after electroporation, there were no differences in the distribution of labeled cells between the genotypes. The dose-response properties of cells labeled to detect GABA were equivalent among genotypes. However, continuous blockade of GABAAR with the GABAAR antagonist SR95531 accelerated radial migration. This effect of GABAAR blockade in GAD67GFP/GFP mice suggested a role for alternative endogenous GABAAR agonists. Thus, we tested the role of taurine, which is derived from maternal blood but is abundant in the fetal brain. The taurine-evoked currents in labeled cells were mediated by GABAAR. Taurine uptake was blocked by a taurine transporter inhibitor, 2-(guanidinoethanesulfonic acid (GES, and taurine release was blocked by a volume-sensitive anion channel blocker, 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl oxobutyric acid (DCPIB, as examined through high-performance liquid chromatography (HPLC. GES increased the extracellular taurine concentration and induced an inward shift of the holding current, which was reversed by SR95531. In a taurine-deficient mouse model, the GABAAR-mediated tonic currents were greatly reduced, and radial migration was accelerated. As the tonic currents were equivalent among the genotypes of GAD67-GFP knock-in mice, taurine, rather than GABA, might play a major role as an endogenous agonist of embryonic tonic GABAAR conductance, regulating the radial migration of neurons in the

  4. Inhibitory synapse formation in a co-culture model incorporating GABAergic medium spiny neurons and HEK293 cells stably expressing GABAA receptors.

    Science.gov (United States)

    Brown, Laura E; Fuchs, Celine; Nicholson, Martin W; Stephenson, F Anne; Thomson, Alex M; Jovanovic, Jasmina N

    2014-11-14

    Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed

  5. Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABA(A) receptor δ subunit in cerebellar granule neurons and delays motor development in rats.

    Science.gov (United States)

    Diaz, Marvin R; Vollmer, Cyndel C; Zamudio-Bulcock, Paula A; Vollmer, William; Blomquist, Samantha L; Morton, Russell A; Everett, Julie C; Zurek, Agnieszka A; Yu, Jieying; Orser, Beverley A; Valenzuela, C Fernando

    2014-04-01

    Exposure to ethanol (EtOH) during fetal development can lead to long-lasting alterations, including deficits in fine motor skills and motor learning. Studies suggest that these are, in part, a consequence of cerebellar damage. Cerebellar granule neurons (CGNs) are the gateway of information into the cerebellar cortex. Functionally, CGNs are heavily regulated by phasic and tonic GABAergic inhibition from Golgi cell interneurons; however, the effect of EtOH exposure on the development of GABAergic transmission in immature CGNs has not been investigated. To model EtOH exposure during the 3rd trimester-equivalent of human pregnancy, neonatal pups were exposed intermittently to high levels of vaporized EtOH from postnatal day (P) 2 to P12. This exposure gradually increased pup serum EtOH concentrations (SECs) to ∼60 mM (∼0.28 g/dl) during the 4 h of exposure. EtOH levels gradually decreased to baseline 8 h after the end of exposure. Surprisingly, basal tonic and phasic GABAergic currents in CGNs were not significantly affected by postnatal alcohol exposure (PAE). However, PAE increased δ subunit expression at P28 as detected by immunohistochemical and western blot analyses. Also, electrophysiological studies with an agonist that is highly selective for δ-containing GABA(A) receptors, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THIP), showed an increase in THIP-induced tonic current. Behavioral studies of PAE rats did not reveal any deficits in motor coordination, except for a delay in the acquisition of the mid-air righting reflex that was apparent at P15 to P18. These findings demonstrate that repeated intermittent exposure to high levels of EtOH during the equivalent of the last trimester of human pregnancy has significant but relatively subtle effects on motor coordination and GABAergic transmission in CGNs in rats.

  6. CoMFA analysis on a set of new noncompetitive GABAA receptor antagonists: fipronil and related analogs%两类促肾上腺皮质释放因子(CRF)抑制剂的CoMFA研究

    Institute of Scientific and Technical Information of China (English)

    郝艳丽; 巨修练; 陈向阳; 柯霞芳

    2005-01-01

    Fipronil and related analogs, a set of new noncompetitive GABAA receptor antagonists, were investigated using comparative molecular field analysis (CoMFA) to explore their three-dimensional quantitative structure-activity relationships (3D-QSAR).Considering the structural complexity of molecules of fipronil and related analogs, three different alignments were performed in this paper. CoMFA model for housefly receptor yield the leave-one-out and cross-validated correlation coefficient q2 value of 0.511 and the conventional correlation coefficient r2 value of 0.997. The new compounds with higher activity would be designed from this model.CoMFA model for rat receptor was not successful using all these three alignments, the reason of which maybe that some molecules adopt different conformations for rat receptor.

  7. Ligand- and subunit-specific conformational changes in the ligand-binding domain and the TM2-TM3 linker of {alpha}1 {beta}2 {gamma}2 GABAA receptors

    DEFF Research Database (Denmark)

    Wang, Qian; Pless, Stephan Alexander; Lynch, Joseph W

    2010-01-01

    changes are essential for gating. Here we used voltage clamp fluorometry to investigate the roles of loops C and F in gating the α1 β2 γ2 GABA(A) receptor. Voltage clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements......Cys-loop receptor ligand binding sites are located at subunit interfaces where they are lined by loops A-C from one subunit and loops D-F from the adjacent subunit. Agonist binding induces large conformational changes in loops C and F. However, it is controversial as to whether these conformational...... from ligand-induced fluorescence changes. Previous attempts to define the roles of loops C and F using this technique have focused on homomeric Cys-loop receptors. However, the problem with studying homomeric receptors is that it is difficult to eliminate the possibility of bound ligands interacting...

  8. The Neuropeptide Orexin-A Inhibits the GABAA Receptor by PKC and Ca(2+)/CaMKII-Dependent Phosphorylation of Its β1 Subunit.

    Science.gov (United States)

    Sachidanandan, Divya; Reddy, Haritha P; Mani, Anitha; Hyde, Geoffrey J; Bera, Amal Kanti

    2017-04-01

    Orexin-A and orexin-B (Ox-A, Ox-B) are neuropeptides produced by a small number of neurons that originate in the hypothalamus and project widely in the brain. Only discovered in 1998, the orexins are already known to regulate several behaviours. Most prominently, they help to stabilise the waking state, a role with demonstrated significance in the clinical management of narcolepsy and insomnia. Orexins bind to G-protein-coupled receptors (predominantly postsynaptic) of two subtypes, OX1R and OX2R. The primary effect of Ox-OXR binding is a direct depolarising influence mediated by cell membrane cation channels, but a wide variety of secondary effects, both pre- and postsynaptic, are also emerging. Given that inhibitory GABAergic neurons also influence orexin-regulated behaviours, crosstalk between the two systems is expected, but at the cellular level, little is known and possible mechanisms remain unidentified. Here, we have used an expression system approach to examine the feasibility, and nature, of possible postsynaptic crosstalk between Ox-A and the GABAA receptor (GABAAR), the brain's main inhibitory neuroreceptor. When HEK293 cells transfected with OX1R and the α1, β1, and γ2S subunits of GABAAR were exposed to Ox-A, GABA-induced currents were inhibited, in a calcium-dependent manner. This inhibition was associated with increased phosphorylation of the β1 subunit of GABAAR, and the inhibition could itself be attenuated by (1) kinase inhibitors (of protein kinase C and CaM kinase II) and (2) the mutation, to alanine, of serine 409 of the β1 subunit, a site previously identified in phosphorylation-dependent regulation in other pathways. These results are the first to directly support the feasibility of postsynaptic crosstalk between Ox-A and GABAAR, indicating a process in which Ox-A could promote phosphorylation of the β1 subunit, reducing the GABA-induced, hyperpolarising current. In this model, Ox-A/GABAAR crosstalk would cause the depolarising

  9. RDX binds to the convulsant site of the GABAA receptor and increases spontaneous firing rates of cortical neurons in vitro

    Science.gov (United States)

    RDX (hexahydro-1 ,3,5-trinitro-1 ,3,5-triazine, hexogen, Royal Demolition eXplosive) is an explosive widely used by the military and has been found in soil and ground water in and surrounding training ranges, creating potential hazards to the environment and human health. Oral RD...

  10. Laminar distribution of GABAA- and glycine-receptor mediated tonic inhibition in the dorsal horn of the rat lumbar spinal cord: effects of picrotoxin and strychnine on expression of Fos-like immunoreactivity.

    Science.gov (United States)

    Cronin, John N; Bradbury, Elizabeth J; Lidierth, Malcolm

    2004-11-01

    Inhibitory mechanisms are essential in suppressing the development of allodynia and hyperalgesia in the normal animal and there is evidence that loss of inhibition can lead to the development of neuropathic pain. We used Fos expression to map the distribution of tonically inhibited cells in the healthy rat lumbar spinal cord. In a control group, Fos-like immunoreactive (Fos-LI) cells were rare, averaging 7.5+/-2.2 cells (mean+/-SEM; N=13 sections) per 20 microm thick section of dorsal horn. This rose to 103+/-11 (mean+/-SEM; N=20) in picrotoxin-treated rats and to 88+/-11 (mean+/-SEM; N=18) in strychnine-treated rats. These changes were significant (ANOVA; Pstrychnine-treated animals. Picrotoxin induced a significant increase in the number of Fos-LI cells throughout the dorsal horn (lamina I-VI) while strychnine significantly elevated Fos-like immunoreactivity only in deep laminae (III-VI). For both picrotoxin and strychnine, the increase in Fos-like immunoreactivity peaked in lamina V (at 3579+/-319 and 3649+/-375% of control, respectively; mean+/-SEM) but for picrotoxin an additional peak was observed in the outer part of lamina II (1959+/-196%). Intrathecal administration of both GABAA and glycine receptor antagonists has been shown elsewhere to induce tactile allodynia. The present data suggest that this allodynia could arise due to blockade of tonic GABAA and glycine-receptor mediated inhibition in the deep dorsal horn. GABAA antagonists also induce hypersensitivity to noxious inputs. The blockade of tonic inhibition in the superficial dorsal horn shown here may underlie this hyperalgesia.

  11. Altered gamma oscillations during pregnancy through loss of δ subunit-containing GABA(A) receptors on parvalbumin interneurons.

    Science.gov (United States)

    Ferando, Isabella; Mody, Istvan

    2013-01-01

    Gamma (γ) oscillations (30-120 Hz), an emergent property of neuronal networks, correlate with memory, cognition and encoding. In the hippocampal CA3 region, locally generated γ oscillations emerge through feedback between inhibitory parvalbumin-positive basket cells (PV+BCs) and the principal (pyramidal) cells. PV+BCs express δ-subunit-containing GABA(A)Rs (δ-GABA(A)Rs) and NMDA receptors (NMDA-Rs) that balance the frequency of γ oscillations. Neuroactive steroids (NS), such as the progesterone-derived (3α,5α)-3-hydroxy-pregnan-20-one (allopregnanolone; ALLO), modulate the expression of δ-GABA(A)Rs and the tonic conductance they mediate. Pregnancy produces large increases in ALLO and brain-region-specific homeostatic changes in δ-GABA(A)Rs expression. Here we show that in CA3, where most PV+ interneurons (INs) express δ-GABA(A)Rs, expression of δ-GABA(A)Rs on INs diminishes during pregnancy, but reverts to control levels within 48 h postpartum. These anatomical findings were corroborated by a pregnancy-related increase in the frequency of kainate-induced CA3 γ oscillations in vitro that could be countered by the NMDA-R antagonists D-AP5 and PPDA. Mimicking the typical hormonal conditions during pregnancy by supplementing 100 nM ALLO lowered the γ frequencies to levels found in virgin or postpartum mice. Our findings show that states of altered NS levels (e.g., pregnancy) may provoke perturbations in γ oscillatory activity through direct effects on the GABAergic system, and underscore the importance of δ-GABA(A)Rs homeostatic plasticity in maintaining constant network output despite large hormonal changes. Inaccurate coupling of NS levels to δ-GABA(A)R expression may facilitate abnormal neurological and psychiatric conditions such as epilepsy, post-partum depression, and post-partum psychosis, thus providing insights into potential new treatments.

  12. Increased GABA(A receptor ε-subunit expression on ventral respiratory column neurons protects breathing during pregnancy.

    Directory of Open Access Journals (Sweden)

    Keith B Hengen

    Full Text Available GABAergic signaling is essential for proper respiratory function. Potentiation of this signaling with allosteric modulators such as anesthetics, barbiturates, and neurosteroids can lead to respiratory arrest. Paradoxically, pregnant animals continue to breathe normally despite nearly 100-fold increases in circulating neurosteroids. ε subunit-containing GABA(ARs are insensitive to positive allosteric modulation, thus we hypothesized that pregnant rats increase ε subunit-containing GABA(AR expression on brainstem neurons of the ventral respiratory column (VRC. In vivo, pregnancy rendered respiratory motor output insensitive to otherwise lethal doses of pentobarbital, a barbiturate previously used to categorize the ε subunit. Using electrode array recordings in vitro, we demonstrated that putative respiratory neurons of the preBötzinger Complex (preBötC were also rendered insensitive to the effects of pentobarbital during pregnancy, but unit activity in the VRC was rapidly inhibited by the GABA(AR agonist, muscimol. VRC unit activity from virgin and post-partum females was potently inhibited by both pentobarbital and muscimol. Brainstem ε subunit mRNA and protein levels were increased in pregnant rats, and GABA(AR ε subunit expression co-localized with a marker of rhythm generating neurons (neurokinin 1 receptors in the preBötC. These data support the hypothesis that pregnancy renders respiratory motor output and respiratory neuron activity insensitive to barbiturates, most likely via increased ε subunit-containing GABA(AR expression on respiratory rhythm-generating neurons. Increased ε subunit expression may be critical to preserve respiratory function (and life despite increased neurosteroid levels during pregnancy.

  13. Altered gamma oscillations during pregnancy through loss of δ subunit-containing GABAA receptors on parvalbumin interneurons

    Directory of Open Access Journals (Sweden)

    Isabella eFerando

    2013-09-01

    Full Text Available Gamma (γ oscillations (30-120 Hz, an emergent property of neuronal networks, correlate with memory, cognition and encoding. In the hippocampal CA3 region, locally generated γ oscillations emerge through feedback between inhibitory parvalbumin-positive basket cells (PV+BCs and the principal (pyramidal cells. PV+BCs express δ-subunit-containing GABAARs (-GABAARs and NMDA receptors (NMDA-Rs that balance the frequency of γ oscillations. Neuroactive steroids (NS, such as the progesterone-derived (3α,5α-3-hydroxy-pregnan-20-one (allopregnanolone; ALLO, modulate the expression of δ-GABAARs and the tonic conductance they mediate. Pregnancy produces large increases in ALLO and brain-region-specific homeostatic changes in δ-GABAARs expression. Here we show that in CA3, where most PV+ interneurons (INs express δ-GABAARs, expression of δ-GABAARs on INs diminishes during pregnancy, but reverts to control levels within 48 hours postpartum. These anatomical findings were corroborated by a pregnancy-related increase in the frequency of kainate-induced CA3 γ oscillations in vitro that could be countered by the NMDA-R antagonists D-AP5 and PPDA. Mimicking the typical hormonal conditions during pregnancy by supplementing 100 nM ALLO lowered the γ frequencies to levels found in virgin or postpartum mice. Our findings show that states of altered NS levels (e.g., pregnancy may provoke perturbations in γ oscillatory activity through direct effects on the GABAergic system, and underscore the importance of δ-GABAARs homeostatic plasticity in maintaining constant network output despite large hormonal changes. Inaccurate coupling of NS levels to δ-GABAAR expression may facilitate abnormal neurological and psychiatric conditions such as epilepsy, post-partum depression, and post-partum psychosis, thus providing insights into potential new treatments.

  14. GABA-A receptor modulators alter emotionality and hippocampal theta rhythm in an animal model of long-lasting anxiety.

    Science.gov (United States)

    Hoeller, Alexandre Ademar; Duzzioni, Marcelo; Duarte, Filipe Silveira; Leme, Leandro Rinaldi; Costa, Ana Paula Ramos; Santos, Evelyn Cristina da Silva; de Pieri, Claudini Honório; dos Santos, Alessandra Antunes; Naime, Aline Aita; Farina, Marcelo; de Lima, Thereza Christina Monteiro

    2013-09-26

    The cholinergic system is implicated in emotional regulation. The injection of non-convulsant doses of the muscarinic receptor agonist pilocarpine (PILO) induces long-lasting anxiogenic responses in rats evaluated at different time-points (24h to 3 months). To investigate the underlying mechanisms, rats treated with PILO (150mg/kg) were injected 24h or 1 month later with an anxiolytic (diazepam, 1mg/kg, DZP) or anxiogenic (pentylenetetrazole, 15mg/kg, PTZ) drug and evaluated in the elevated plus-maze (EPM). Prefrontal cortex (PFC) and hippocampal (HIP) electroencephalographic recordings and acetylcolinesterase (AChE) activity were also analyzed after PILO treatment. Anxiogenic responses observed in the EPM 24h or 1 month after PILO treatment (e.g., decreased time spent and number of entries into the open arms of the maze) were blocked by DZP but not affected by PTZ. No epileptiform events were registered in the HIP or PFC at 24h or 1 month after PILO injection, but enhanced theta activity was observed in the HIP. DZP decreased hippocampal theta of PILO-treated rats in contrast with PTZ, which increased this parameter in saline- and PILO-treated rats. The HIP and PFC AChE activity did not change after PILO treatment. Our findings demonstrate that the long-term effects on the emotionality of rats induced by PILO are associated with electrophysiological changes in the HIP and sensitive to pharmacological manipulation of the GABAergic system. The present work may support this new research model of long-lasting anxiety, while also highlighting the muscarinic system as a potential target involved in anxiety disorders.

  15. A linkage study between the GABAA beta2 and GABAA gamma2 subunit genes and major psychoses.

    Science.gov (United States)

    Ambrósio, Alda M; Kennedy, James L; Macciardi, Fabio; King, Nicole; Azevedo, Maria H; Oliveira, Catarina R; Pato, Carlos N

    2005-01-01

    Alterations of the gamma-aminobutyric acid (GABA) system have been implicated in the pathophysiology of major psychoses. Restriction fragment length polymorphisms associated with the human gamma-aminobutyric acid type A (GABAA) beta2 and GABAA gamma2 subunit genes on chromosome 5q32-q35 were tested to determine whether they confer susceptibility to major psychoses. Thirty-two schizophrenic families and 25 bipolar families were tested for linkage. Nonparametric linkage (NPL) analysis performed by GENEHUNTER showed no significant NPL scores for both genes in schizophrenia (GABAA beta2: NPL narrow= -0.450; NPL broad= -0.808; GABAA gamma2: NPL narrow=0.177; NPL broad= -0.051) or bipolar disorder (GABAA beta2: NPL narrow=0.834; NPL broad=0.783; GABAA gamma2: NPL narrow= -0.159; NPL broad=0.070). Linkage analysis does not support the hypothesis that variants within the GABAA beta2 and GABAA gamma2 genes are significantly linked to major psychoses in a Portuguese population.

  16. The function study of entorhinal cortex neuron GABAA receptor in an epilepsia rat model%癫痫大鼠内嗅皮层神经元GABAA受体功能的研究

    Institute of Scientific and Technical Information of China (English)

    李桀; 孙杨; 刘备; 王超; 井晓荣; 梁秦川; 李焕发; 张华; 高国栋

    2011-01-01

    Objective Established the lithium chloride-pilocarpine inducing epilepsia rat model, and initially study about the entorhinal cortex neuron GABAA receptor by patch clamp of whole-cell mode. Methods The SD rats were divided into control group and experiment group randomly. Intraperitoneal injection of lithium chloride-pilocarpine have been used in experiment group, and physiologic saline in control group. Observed the ethology characteristic and recorded the attenuation tendency of GABAa receptor electric current in whole cell mode. Results The e-lectric current attenuation tendency of GABAA Receptor of Epilepsia Rat greatly intensify in the experiment group. The group diveded have significant difference by variance test and interclass analyse (P<0. 05), and the datas of every point of time in experiment group and control group also have significant difference by t-text analyse(P<0. 05). Conclusion The electric current attenuation tendency of Entorhinal Cortex Neuron GABAA Receptor of lithium chloride-pilocarpine inducing epilepsia rat have intensified. This phenomenon could be the possible mechanism of lithium chloride-pilocarpine inducing epilepsia rat and hint that GABAA receptor may contribute to the epileptic attack and brain injured after epileptic attack.%目的 介绍建立氯化锂-匹鲁卡品致痫大鼠模型的方法,并且通过全细胞膜片钳记录,初步研究其内嗅皮层神经元GABAA受体功能.方法 将所有SD大鼠随机分为对照组和实验组.实验组大鼠腹腔注射氯化锂以及匹鲁卡品,对照组注射生理盐水,观察其行为学特征,并用全细胞膜片钳记录GABAA受体电流的衰减趋势.结果 与对照组相比,实验组癫痫大鼠内嗅皮层神经元的GABAA受体电流的衰减加剧.方差检验进行组间分析,分组的作用是有差异的(P<0.001),;固定时间,对每个时间点上的处理组和对照组进行t检验,分组都有统计学意义(P<0.001).结论 锂-匹罗卡品致病大鼠

  17. Kampo medicine: Evaluation of the pharmacological activity of 121 herbal drugs on GABA(A) and 5 HT3A receptors

    OpenAIRE

    Katrin M Hoffmann; Robin Herbrechter; Ziemba, Paul M.; Peter Lepke; Leopoldo Raul Beltran; Hanns Hatt; Markus Werner; Guenter Gisselmann

    2016-01-01

    Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and its constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, we performed a broad screening of Kampo remedies to look for pharmacologically relevant 5 HT3A and GABA(A) re...

  18. Progesterone Exerts a Neuromodulatory Effect on Turning Behavior of Hemiparkinsonian Male Rats: Expression of 3α-Hydroxysteroid Oxidoreductase and Allopregnanolone as Suggestive of GABAA Receptors Involvement

    Directory of Open Access Journals (Sweden)

    Roberto Yunes

    2015-01-01

    Full Text Available There is a growing amount of evidence for a neuroprotective role of progesterone and its neuroactive metabolite, allopregnanolone, in animal models of neurodegenerative diseases. By using a model of hemiparkinsonism in male rats, injection of the neurotoxic 6-OHDA in left striatum, we studied progesterone’s effects on rotational behavior induced by amphetamine or apomorphine. Also, in order to find potential explanatory mechanisms, we studied expression and activity of nigrostriatal 3α-hydroxysteroid oxidoreductase, the enzyme that catalyzes progesterone to its active metabolite allopregnanolone. Coherently, we tested allopregnanolone for a possible neuromodulatory effect on rotational behavior. Also, since allopregnanolone is known as a GABAA modulator, we finally examined the action of GABAA antagonist bicuculline. We found that progesterone, in addition to an apparent neuroprotective effect, also increased ipsilateral expression and activity of 3α-hydroxysteroid oxidoreductase. It was interesting to note that ipsilateral administration of allopregnanolone reversed a clear sign of motor neurodegeneration, that is, contralateral rotational behavior. A possible GABAA involvement modulated by allopregnanolone was shown by the blocking effect of bicuculline. Our results suggest that early administration of progesterone possibly activates genomic mechanisms that promote neuroprotection subchronically. This, in turn, could be partially mediated by fast, nongenomic, actions of allopregnanolone acting as an acute modulator of GABAergic transmission.

  19. GABAρ1/GABAAα1 receptor chimeras to study receptor desensitization

    Science.gov (United States)

    Martínez-Torres, Ataúlfo; Demuro, Angelo; Miledi, Ricardo

    2000-01-01

    γ-Aminobutyrate type C (GABAC) receptors are ligand-gated ion channels that are expressed preponderantly in the vertebrate retina and are characterized, among other things, by a very low rate of desensitization and resistance to the specific GABAA antagonist bicuculline. To examine which structural elements determine the nondesensitizing character of the human homomeric ρ1 receptor, we used a combination of gene chimeras and electrophysiology of receptors expressed in Xenopus oocytes. Two chimeric genes were constructed, made up of portions of the ρ1-subunit and of the α1-subunit of the GABAA receptor. When expressed in Xenopus oocytes, one chimeric gene (ρ1/α1) formed functional homooligomeric receptors that were fully resistant to bicuculline and were blocked by the specific GABAC antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid and by zinc. Moreover, these chimeric receptors had a fast-desensitizing component, even faster than that of heterooligomeric GABAA receptors, in striking contrast to the almost nil desensitization of wild-type ρ1 (wt ρ1) receptors. To see whether the fast-desensitizing characteristic of the chimera was determined by the amino acids forming the ion channels, we replaced the second transmembrane segment (TM2) of ρ1 by that of the α1-subunit of GABAA. Although the α1-subunit forms fast-desensitizing receptors when coexpressed with other GABAA subunits, the sole transfer of the α1TM2 segment to ρ1 was not sufficient to form desensitizing receptors. All this suggests that the slow-desensitizing trait of ρ1 receptors is determined by a combination of several interacting domains along the molecule. PMID:10725369

  20. Muscarinic Long-Term Enhancement of Tonic and Phasic GABAA Inhibition in Rat CA1 Pyramidal Neurons

    Science.gov (United States)

    Domínguez, Soledad; Fernández de Sevilla, David; Buño, Washington

    2016-01-01

    Acetylcholine (ACh) regulates network operation in the hippocampus by controlling excitation and inhibition in rat CA1 pyramidal neurons (PCs), the latter through gamma-aminobutyric acid type-A receptors (GABAARs). Although, the enhancing effects of ACh on GABAARs have been reported (Dominguez et al., 2014, 2015), its role in regulating tonic GABAA inhibition has not been explored in depth. Therefore, we aimed at determining the effects of the activation of ACh receptors on responses mediated by synaptic and extrasynaptic GABAARs. Here, we show that under blockade of ionotropic glutamate receptors ACh, acting through muscarinic type 1 receptors, paired with post-synaptic depolarization induced a long-term enhancement of tonic GABAA currents (tGABAA) and puff-evoked GABAA currents (pGABAA). ACh combined with depolarization also potentiated IPSCs (i.e., phasic inhibition) in the same PCs, without signs of interactions of synaptic responses with pGABAA and tGABAA, suggesting the contribution of two different GABAA receptor pools. The long-term enhancement of GABAA currents and IPSCs reduced the excitability of PCs, possibly regulating plasticity and learning in behaving animals. PMID:27833531

  1. Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

    Directory of Open Access Journals (Sweden)

    Florian Wegner

    Full Text Available BACKGROUND: Human fetal midbrain-derived neural progenitor cells (NPCs may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+-K(+-Cl(- co-transporter 1 (NKCC1-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. CONCLUSIONS/SIGNIFICANCE: These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

  2. Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

    Science.gov (United States)

    Wegner, Florian; Kraft, Robert; Busse, Kathy; Härtig, Wolfgang; Ahrens, Jörg; Leffler, Andreas; Dengler, Reinhard; Schwarz, Johannes

    2012-01-01

    Human fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A) receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+)-K(+)-Cl(-) co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

  3. Topiramate via NMDA, AMPA/kainate, GABAA and Alpha2 receptors and by modulation of CREB/BDNF and Akt/GSK3 signaling pathway exerts neuroprotective effects against methylphenidate-induced neurotoxicity in rats.

    Science.gov (United States)

    Motaghinejad, Majid; Motevalian, Manijeh; Fatima, Sulail; Beiranvand, Tabassom; Mozaffari, Shiva

    2017-08-09

    Chronic abuse of methylphenidate (MPH) often causes neuronal cell death. Topiramate (TPM) carries neuroprotective effects, but its exact mechanism of action remains unclear. In the present study, the role of various doses of TPM and its possible mechanisms, receptors and signaling pathways involved against MPH-induced hippocampal neurodegeneration were evaluated in vivo. Thus, domoic acid (DOM) was used as AMPA/kainate receptor agonist, bicuculline (BIC) as GABAA receptor antagonist, ketamine (KET) as NMDA receptor antagonist, yohimbine (YOH) as α2 adrenergic receptor antagonist and haloperidol (HAL) was used as dopamine D2 receptor antagonist. Open field test (OFT) was used to investigate the disturbances in motor activity. Hippocampal neurodegenerative parameters were evaluated. Protein expressions of CREB/BDNF and Akt/GSK3 signaling pathways were also evaluated. Cresyl violet staining was performed to show and confirm the changes in the shape of the cells. TPM (70 and 100 mg/kg) reduced MPH-induced rise in lipid peroxidation, oxidized form of glutathione (GSSG), IL-1β and TNF-α levels, Bax expression and motor activity disturbances. In addition, TPM treatment increased Bcl-2 expression, the level of reduced form of glutathione (GSH) and the levels and activities of superoxide dismutase, glutathione peroxidase and glutathione reductase enzymes. TPM also inhibited MPH-induced hippocampal degeneration. Pretreatment of animals with DOM, BIC, KET and YOH inhibited TPM-induced neuroprotection and increased oxidative stress, neuroinflammation, neuroapoptosis and neurodegeneration while reducing CREB, BDNF and Akt protein expressions. Also pretreatment with DOM, BIC, KET and YOH inhibited TPM-induced decreases in GSK3. It can be concluded that the mentioned receptors by modulation of CREB/BDNF and Akt/GSK3 pathways, are involved in neuroprotection of TPM against MPH-induced neurodegeneration.

  4. Agonist-dependent endocytosis of γ-aminobutyric acid type A (GABAA) receptors revealed by a γ2(R43Q) epilepsy mutation.

    Science.gov (United States)

    Chaumont, Severine; André, Caroline; Perrais, David; Boué-Grabot, Eric; Taly, Antoine; Garret, Maurice

    2013-09-27

    GABA-gated chloride channels (GABAARs) trafficking is involved in the regulation of fast inhibitory transmission. Here, we took advantage of a γ2(R43Q) subunit mutation linked to epilepsy in humans that considerably reduces the number of GABAARs on the cell surface to better understand the trafficking of GABAARs. Using recombinant expression in cultured rat hippocampal neurons and COS-7 cells, we showed that receptors containing γ2(R43Q) were addressed to the cell membrane but underwent clathrin-mediated dynamin-dependent endocytosis. The γ2(R43Q)-dependent endocytosis was reduced by GABAAR antagonists. These data, in addition to a new homology model, suggested that a conformational change in the extracellular domain of γ2(R43Q)-containing GABAARs increased their internalization. This led us to show that endogenous and recombinant wild-type GABAAR endocytosis in both cultured neurons and COS-7 cells can be amplified by their agonists. These findings revealed not only a direct relationship between endocytosis of GABAARs and a genetic neurological disorder but also that trafficking of these receptors can be modulated by their agonist.

  5. Similarity of Bovine Rotavirus Receptor and Human Rotavirus Receptor

    Institute of Scientific and Technical Information of China (English)

    苏琦华; 訾自强; 潘菊芬; 徐燕

    2004-01-01

    The monoclonal antibody against bovine rotavirus (BRV) receptor (BRV-R-mAb) was used to explore the similarity between the receptors of BRV and human rotavirus (HRV). ELISA, dot immunobinding assay, cell protection assay, solid-phase assay and immunohistochemistry method were applied. BRV-R-mAb bound both anti-BRV IgG and anti-HRV IgG respectively and could protect MA 104 cells against BRV and HRV challenges. Immunohistochemistry test showed that there were rotavirus receptors on the surfaces of foetal intestinal, tracheal mucosa and MA 104 cells membrane. We purified the rotavirus receptors on MA 104 ceils, which could bind both BRV and HRV in vitro. It is concluded that BRV receptor and HRV receptor are homogenous proteins and can be recognized by both BRV and HRV.

  6. Role of spinal GABAA receptor in antinociceptive effect of alphadolone in rats%大鼠脊髓GABAA受体在乙酯羟孕双酮抗伤害性效应中的作用

    Institute of Scientific and Technical Information of China (English)

    严春燕; 张小梅; 徐建国

    2008-01-01

    目的 探讨脊髓γ氨基丁酸A(GABAA)受体在乙酯羟孕双酮抗伤害性效应中的作用.方法 雄性Wistar大鼠,体重176~181 g,经L2,3行蛛网膜下腔置管.取置管成功的大鼠27只,随机分为4组:荷包牡丹碱0.01 pmol组(Ⅰ组,n=9)、0.1 pmol组(Ⅱ组,n=7)、1 pmol组(Ⅲ组,n=5)和10pmol组(Ⅳ组,n=6).采用电刺激法测定大鼠颈部和尾部痛阈,记录诱发尾部和颈部伤害性反应的最小电流,测定3次,间隔5 min,取其平均值作为基础痛阈(TH1).腹腔注射乙酯羟孕双酮1 mg/kg,5min后测定痛阈(TH2).经蛛网膜下腔导管注入重比重荷包牡丹碱溶液(溶于5 μl 6%葡萄糖溶液),5min后测定痛阈(TH3).结果 与TH1比较,各组颈部TH2和TH3、尾部TH2和Ⅰ组尾部TH3升高(P<0.05);与TH2比较,Ⅱ组~Ⅳ组尾部TH3降低(P<0.05),各组颈部TH3,差异无统计学意义(P0.05).结论 乙酯羟孕双酮可通过激活脊髓GABAA受体产生抗伤害性效应.%Objective To investigate the role of spinal GABAA receptor in the antinociceptive effect of. alphadolone in rats. Methods Male Wistsr rats weighing 176-181 g were studied. A catheter was inserted into suharachnoid space via L2,3. Twenty-seven rats successfully catheterized were randomly divided into 4 groups: bicuculline 0.01 pmol group (Ⅰ , n = 9), bicuculline 0.1 pmol group (Ⅱ, n = 7), bicuculline 1 pmol group (Ⅲ, n = 5) and bicuculline 10 pmol group (Ⅳ, n = 6) .The pain threshold in the neck and tail was measured using electrical stimulation. The minimum current of antinociceptive effects caused by stimulating the tail and neck were recorded (3 times every 5 min ) and the mean value was calculated and defined as the baseline value (TH1 ). Alphadolone 1.0mg/kg was then injected intraperitoneally and 5 min later the pain threshold (TH2) was measured. Hyperbaric bicuculline (in 6% glucose 5 μl) was then injected through the catheter into the]umber region of the subarachnoid space and 5 min later the pain threshold (TH

  7. Molecular pharmacology of human NMDA receptors

    DEFF Research Database (Denmark)

    Hedegaard, Maiken; Hansen, Kasper Bø; Andersen, Karen Toftegaard

    2012-01-01

    current knowledge of the relationship between NMDA receptor structure and function. We summarize studies on the biophysical properties of human NMDA receptors and compare these properties to those of rat orthologs. Finally, we provide a comprehensive pharmacological characterization that allows side......-by-side comparison of agonists, un-competitive antagonists, GluN2B-selective non-competitive antagonists, and GluN2C/D-selective modulators at recombinant human and rat NMDA receptors. The evaluation of biophysical properties and pharmacological probes acting at different sites on the receptor suggest...... that the binding sites and conformational changes leading to channel gating in response to agonist binding are highly conserved between human and rat NMDA receptors. In summary, the results of this study suggest that no major detectable differences exist in the pharmacological and functional properties of human...

  8. Human Neuroepithelial Cells Express NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Cappell B

    2003-11-01

    Full Text Available Abstract L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1 cerebral endothelial barrier and 2 cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR expression via immunohistochemistry and murine neuroepithelial cell line (V1 were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.

  9. GABA type a receptor trafficking and the architecture of synaptic inhibition.

    Science.gov (United States)

    Lorenz-Guertin, Joshua M; Jacob, Tija C

    2017-09-13

    Ubiquitous expression of GABA type A receptors (GABAA R) in the central nervous system establishes their central role in coordinating most aspects of neural function and development. Dysregulation of GABAergic neurotransmission manifests in a number of human health disorders and conditions that in certain cases can be alleviated by drugs targeting these receptors. Precise changes in the quantity or activity of GABAA Rs localized at the cell surface and at GABAergic postsynaptic sites directly impact the strength of inhibition. The molecular mechanisms constituting receptor trafficking to and from these compartments therefore dictate the efficacy of GABAA R function. Here we review the current understanding of how GABAA Rs traffic through biogenesis, plasma membrane transport, and degradation. Emphasis is placed on discussing novel GABAergic synaptic proteins, receptor and scaffolding post-translational modifications, activity-dependent changes in GABAA R confinement, and neuropeptide and neurosteroid mediated changes. We further highlight modern techniques currently advancing the knowledge of GABAA R trafficking and clinically relevant neurodevelopmental diseases connected to GABAergic dysfunction. © 2017 Wiley Periodicals, Inc. Develop Neurobiol, 2017. © 2017 Wiley Periodicals, Inc.

  10. Use-dependent shift from inhibitory to excitatory GABAA receptor action in SP-O interneurons in the rat hippocampal CA3 area.

    Science.gov (United States)

    Lamsa, Karri; Taira, Tomi

    2003-09-01

    Cortical inhibitory interneurons set the pace of synchronous neuronal oscillations implicated in synaptic plasticity and various cognitive functions. The hyperpolarizing nature of inhibitory postsynaptic potentials (IPSPs) in interneurons has been considered crucial for the generation of oscillations at beta (15-30 Hz) and gamma (30-100 Hz) frequency. Hippocampal basket cells and axo-axonic cells in stratum pyramidale-oriens (S-PO) play a central role in the synchronization of the local interneuronal network as well as in pacing of glutamatergic principal cell firing. A lack of conventional forms of plasticity in excitatory synapses onto interneurons facilitates their function as stable neuronal oscillators. We have used gramicidin-perforated and whole cell clamp recordings to study properties of GABAAR-mediated transmission in CA3 SP-O interneurons and in CA3 pyramidal cells in rat hippocampal slices during electrical 5- to 100-Hz stimulation and during spontaneous activity. We show that GABAergic synapses onto SP-O interneurons can easily switch their mode from inhibitory to excitatory during heightened activity. This is based on a depolarizing shift in the GABAA reversal potential (EGABA-A), which is much faster and more pronounced in interneurons than in pyramidal cells. We also found that the shift in interneuronal function was frequency dependent, being most prominent at 20- to 40-Hz activation of the GABAergic synapses. After 40-Hz tetanic stimulation (100 pulses), GABAA responses remained depolarizing for approximately 45 s in the interneurons, promoting bursting in the GABAergic network. Hyperpolarizing EGABA-A was restored >60 s after the stimulus train. Similar but spontaneous GABAergic bursting was induced by application of 4-aminopyridine (100 microM) to slices. A shift to depolarizing IPSPs by the GABAAR permeant weak acid anion formate provoked interneuronal population bursting, supporting the role of GABAergic excitation in burst generation

  11. A comparison of the effects of a subtype selective and non-selective benzodiazepine receptor agonist in two CO(2) models of experimental human anxiety.

    Science.gov (United States)

    Bailey, J E; Papadopoulos, A; Seddon, K; Nutt, D J

    2009-03-01

    Studies in human volunteers that can demonstrate proof of concept are attractive in that possible mechanisms and potential new drug treatments can be examined. We have been developing models of anxiety disorders using the inhalation of 7.5% CO(2) for 20 min to model generalised anxiety disorder, as well as using the previously reported 35% CO(2) as a model for panic anxiety. In a double-blind, placebo-controlled, three-way crossover study in 12 healthy volunteer subjects, we compared a full agonist at the benzodiazepine receptor that binds to four alpha-subtypes of the receptor (alpha-1,-2,-3,-5) (alprazolam 1 mg), with zolpidem (5 mg), an agonist selective for the alpha-1 subtype of the gamma amino butyric acid-receptor subtype A (GABA-A) receptor, which is a widely used hypnotic drug. Compared with placebo, both drugs significantly attenuated peak CO(2)-induced changes in subjective feelings after the inhalation of 7.5% CO(2) for 20 min. However, there were fewer significant differences after a single vital capacity inhalation of 35% CO(2), where zolpidem was less efficacious than alprazolam at reducing CO(2)-induced symptoms. In conclusion, our results show that zolpidem shows some anxiolytic efficacy in the 7.5% CO(2) model, similar to alprazolam, and this is the first report of such an effect of zolpidem in a model of anxiety. These and other studies of benzodiazepines in clinical and volunteer studies suggest a definite role of the GABA-A receptor in CO(2)-induced anxiety, and it would be of interest to examine other GABA-A receptor subtype selective drugs, which are now in early phase clinical studies and are showing selective efficacy in pharmacodynamic studies.

  12. Gene expression changes in serotonin, GABA-A receptors, neuropeptides and ion channels in the dorsal raphe nucleus of adolescent alcohol-preferring (P) rats following binge-like alcohol drinking.

    Science.gov (United States)

    McClintick, Jeanette N; McBride, William J; Bell, Richard L; Ding, Zheng-Ming; Liu, Yunlong; Xuei, Xiaoling; Edenberg, Howard J

    2015-02-01

    Alcohol binge-drinking during adolescence is a serious public health concern with long-term consequences. We used RNA sequencing to assess the effects of excessive adolescent ethanol binge-drinking on gene expression in the dorsal raphe nucleus (DRN) of alcohol preferring (P) rats. Repeated binges across adolescence (three 1h sessions across the dark-cycle per day, 5 days per week for 3 weeks starting at 28 days of age; ethanol intakes of 2.5-3 g/kg/session) significantly altered the expression of approximately one-third of the detected genes. Multiple neurotransmitter systems were altered, with the largest changes in the serotonin system (21 of 23 serotonin-related genes showed decreased expression) and GABA-A receptors (8 decreased and 2 increased). Multiple neuropeptide systems were also altered, with changes in the neuropeptide Y and corticotropin-releasing hormone systems similar to those associated with increased drinking and decreased resistance to stress. There was increased expression of 21 of 32 genes for potassium channels. Expression of downstream targets of CREB signaling was increased. There were also changes in expression of genes involved in inflammatory processes, axonal guidance, growth factors, transcription factors, and several intracellular signaling pathways. These widespread changes indicate that excessive binge drinking during adolescence alters the functioning of the DRN and likely its modulation of many regions of the central nervous system, including the mesocorticolimbic system.

  13. A multilevel prediction of physiological response to challenge: Interactions among child maltreatment, neighborhood crime, endothelial nitric oxide synthase gene (eNOS), and GABA(A) receptor subunit alpha-6 gene (GABRA6).

    Science.gov (United States)

    Lynch, Michael; Manly, Jody Todd; Cicchetti, Dante

    2015-11-01

    Physiological response to stress has been linked to a variety of healthy and pathological conditions. The current study conducted a multilevel examination of interactions among environmental toxins (i.e., neighborhood crime and child maltreatment) and specific genetic polymorphisms of the endothelial nitric oxide synthase gene (eNOS) and GABA(A) receptor subunit alpha-6 gene (GABRA6). One hundred eighty-six children were recruited at age 4. The presence or absence of child maltreatment as well as the amount of crime that occurred in their neighborhood during the previous year were determined at that time. At age 9, the children were brought to the lab, where their physiological response to a cognitive challenge (i.e., change in the amplitude of the respiratory sinus arrhythmia) was assessed and DNA samples were collected for subsequent genotyping. The results confirmed that complex Gene × Gene, Environment × Environment, and Gene × Environment interactions were associated with different patterns of respiratory sinus arrhythmia reactivity. The implications for future research and evidence-based intervention are discussed.

  14. α2 Subunit-Containing GABAA Receptor Subtypes Are Upregulated and Contribute to Alcohol-Induced Functional Plasticity in the Rat Hippocampus.

    Science.gov (United States)

    Lindemeyer, A Kerstin; Shen, Yi; Yazdani, Ferin; Shao, Xuesi M; Spigelman, Igor; Davies, Daryl L; Olsen, Richard W; Liang, Jing

    2017-08-01

    Alcohol (EtOH) intoxication causes changes in the rodent brain γ-aminobutyric acid receptor (GABAAR) subunit composition and function, playing a crucial role in EtOH withdrawal symptoms and dependence. Building evidence indicates that withdrawal from acute EtOH and chronic intermittent EtOH (CIE) results in decreased EtOH-enhanced GABAAR δ subunit-containing extrasynaptic and EtOH-insensitive α1βγ2 subtype synaptic GABAARs but increased synaptic α4βγ2 subtype, and increased EtOH sensitivity of GABAAR miniature postsynaptic currents (mIPSCs) correlated with EtOH dependence. Here we demonstrate that after acute EtOH intoxication and CIE, upregulation of hippocampal α4βγ2 subtypes, as well as increased cell-surface levels of GABAAR α2 and γ1 subunits, along with increased α2β1γ1 GABAAR pentamers in hippocampal slices using cell-surface cross-linking, followed by Western blot and coimmunoprecipitation. One-dose and two-dose acute EtOH treatments produced temporal plastic changes in EtOH-induced anxiolysis or withdrawal anxiety, and the presence or absence of EtOH-sensitive synaptic currents correlated with cell surface peptide levels of both α4 and γ1(new α2) subunits. CIE increased the abundance of novel mIPSC patterns differing in activation/deactivation kinetics, charge transfer, and sensitivity to EtOH. The different mIPSC patterns in CIE could be correlated with upregulated highly EtOH-sensitive α2βγ subtypes and EtOH-sensitive α4βγ2 subtypes. Naïve α4 subunit knockout mice express EtOH-sensitive mIPSCs in hippocampal slices, correlating with upregulated GABAAR α2 (and not α4) subunits. Consistent with α2, β1, and γ1 subunits genetically linked to alcoholism in humans, our findings indicate that these new α2-containing synaptic GABAARs could mediate the maintained anxiolytic response to EtOH in dependent individuals, rat or human, contributing to elevated EtOH consumption. Copyright © 2017 by The American Society for Pharmacology

  15. Prostanoid Receptors in the Human Vascular Wall

    Directory of Open Access Journals (Sweden)

    Xavier Norel

    2007-01-01

    Full Text Available The mechanisms involved in vascular homeostasis and disease are mostly dependent on the interactions between blood, vascular smooth muscle, and endothelial cells. There is an accumulation of evidence for the involvement of prostanoids, the arachidonic acid metabolites derived from the cyclooxygenase enzymatic pathway, in physiological and/or pathophysiological conditions. In humans, the prostanoids activate different receptors. The classical prostanoid receptors (DP, EP1–4, FP, IP, and TP are localized at the cell plasma or nuclear membrane. In addition, CRTH2 and the nuclear PPAR receptors are two other targets for prostanoids, namely, prostacyclin (PGI2 or the natural derivatives of prostaglandin D2. While there is little information on the role of CRTH2, there are many reports on PPAR activation and the consecutive expression of genes involved in the human vascular system. The role of the classical prostanoid receptors stimulated by PGI2 and thromboxane in the control of the vascular tone has been largely documented, whereas the other receptor subtypes have been overlooked. There is now increasing evidence that suggests a role of PGE2 and the EP receptor subtypes in the control of the human vascular tone and remodeling of the vascular wall. These receptors are also present on leukocytes and platelets, and they are implicated in most of the inflammatory processes within the vascular wall. Consequently, the EP receptor subtypes or isoforms would provide a novel and specific cardiovascular therapeutic approach in the near future.

  16. Blocking GABA(A) inhibition reveals AMPA- and NMDA-receptor-mediated polysynaptic responses in the CA1 region of the rat hippocampus.

    Science.gov (United States)

    Crépel, V; Khazipov, R; Ben-Ari, Y

    1997-04-01

    We have investigated the conditions required to evoke polysynaptic responses in the isolated CA1 region of hippocampal slices from Wistar adult rats. Experiments were performed with extracellular and whole cell recording techniques. In the presence of bicuculline (10 microM), 6-cyano-7-nitroquinoxaline-2-3-dione (10 microM), glycine (10 microM), and a low external concentration of Mg2+ (0.3 mM), electrical stimulation of the Schaffer collaterals/commissural pathway evoked graded N-methyl-D-aspartate (NMDA)-receptor-mediated late field potentials in the stratum radiatum of the CA1 region. These responses were generated via polysynaptic connections because their latency varied strongly and inversely with the stimulation intensity and they were abolished by a high concentration of divalent cations (7 mM Ca2+). These responses likely were driven by local collateral branches of CA1 pyramidal cell axons because focal application of tetrodotoxin (30 microM) in the stratum oriens strongly reduced the late synaptic component and antidromic stimulation of CA1 pyramidal cells could evoke the polysynaptic response. Current-source density analysis suggested that the polysynaptic response was generated along the proximal part of the apical dendrites of CA1 pyramidal cells (50-150 microm below the pyramidal cell layer in the stratum radiatum). In physiological concentration of Mg2+ (1.3 mM), the pharmacologically isolated NMDA-receptor-mediated polysynaptic response was abolished. In control artificial cerebrospinal fluid (with physiological concentration of Mg2+), bicuculline ( 10 microM) generated a graded polysynaptic response. Under these conditions, this response was mediated both by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/NMDA receptors. In the presence of D-2-amino-5-phosphonovalerate (50 microM), the polysynaptic response could be mediated by AMPA receptors, although less efficiently. In conclusion, suppression of gamma-aminobutyric acid

  17. Enhanced human receptor binding by H5 haemagglutinins

    OpenAIRE

    Xiong, Xiaoli; Xiao, Haixia; Martin, Stephen R.; Coombs, Peter J.; Liu, Junfeng; Collins, Patrick J.; Vachieri, Sebastien G.; Walker, Philip A.; Lin, Yi Pu; McCauley, John W.; Gamblin, Steven J.; John J Skehel

    2014-01-01

    Mutant H5N1 influenza viruses have been isolated from humans that have increased human receptor avidity. We have compared the receptor binding properties of these mutants with those of wild-type viruses, and determined the structures of their haemagglutinins in complex with receptor analogues. Mutants from Vietnam bind tighter to human receptor by acquiring basic residues near the receptor binding site. They bind more weakly to avian receptor because they lack specific interactions between As...

  18. Elevated potassium elicits recurrent surges of large GABAA-receptor-mediated post-synaptic currents in hippocampal CA3 pyramidal neurons.

    Science.gov (United States)

    Shin, Damian Seung-Ho; Yu, Wilson; Sutton, Alex; Calos, Megan; Carlen, Peter Louis

    2011-03-01

    Previously, we found that rat hippocampal CA3 interneurons become hyperactive with increasing concentrations of extracellular K(+) up to 10 mM. However, it is unclear how this enhanced interneuronal activity affects pyramidal neurons. Here we voltage-clamped rat hippocampal CA3 pyramidal neurons in vitro at 0 mV to isolate γ-aminobutyric acid (GABA)-activated inhibitory post-synaptic currents (IPSCs) and measured these in artificial cerebrospinal fluid (aCSF) and with 10 mM K(+) bath perfusion. In aCSF, small IPSCs were present with amplitudes of 0.053 ± 0.007 nA and a frequency of 0.27 ± 0.14 Hz. With 10 mM K(+) perfusion, IPSCs increased greatly in frequency and amplitude, culminating in surge events with peak amplitudes of 0.56 ± 0.08 nA, that appeared and disappeared cyclically with durations lasting 2.02 ± 0.37 min repeatedly, up to 10 times over a 30-min bath perfusion of elevated K(+). These large IPSCs were GABA(A)-receptor mediated and did not involve significant desensitization of this receptor. Perfusion of a GABA transporter inhibitor (NO-711), glutamate receptor inhibitors CNQX and APV, or a gap junctional blocker (carbenoxolone) prevented the resurgence of large IPSCs. Pressure ejected sucrose resulted in the abolishment of subsequent surges. No elevated K(+)-mediated surges were observed in CA3 interneurons from the stratum oriens layer. In conclusion, these cyclic large IPSC events observable in CA3 pyramidal neurons in 10 mM KCl may be due to transient GABA depletion from continuously active interneuronal afferents.

  19. Changes in the sensitivity of GABAA current rundown to drug treatments in a model of temporal lobe epilepsy

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    Pierangelo eCifelli

    2013-07-01

    Full Text Available The pharmacological treatment of mesial temporal lobe epilepsy (mTLE, the most common epileptic syndrome in adults, is still unsatisfactory, as one third of the patients are or become refractory to antiepileptic agents. Refractoriness may depend upon drug-induced alterations, but the disease per se may also undergo a progressive evolution that affects the sensitivity to drugs. mTLE has been shown to be associated with a dysfunction of the inhibitory signaling mediated by GABAA receptors. In particular, the repetitive activation of GABAA receptors produces a use-dependent decrease (rundown of the evoked currents (IGABA, which is markedly enhanced in the hippocampus and cortex of drug-resistant mTLE patients. This phenomenon has been also observed in the pilocarpine model, where the increased IGABA rundown is observed in the hippocampus at the time of the first spontaneous seizure, then extends to the cortex and remains constant in the chronic phase of the disease. Here, we examined the sensitivity of IGABA to pharmacological modulation. We focused on the antiepileptic agent levetiracetam and on the neurotrophin BDNF, which were previously reported to attenuate mTLE-induced increased rundown in the chronic human tissue. In the pilocarpine model, BDNF displayed a paramount effect, decreasing rundown in the hippocampus at the time of the first seizure, as well as in the hippocampus and cortex in the chronic period. In contrast, levetiracetam did not affect rundown in the hippocampus, but attenuated it in the cortex. Interestingly, this effect of levetiracetam was also observed on the still unaltered rundown observed in the cortex at the time of the first spontaneous seizure. These data suggest that the sensitivity of GABAA receptors to pharmacological interventions undergoes changes during the natural history of mTLE, implicating that the site of seizure initiation and the timing of treatment may highly affect the therapeutic outcome.

  20. Chloride Accumulators NKCC1 and AE2 in Mouse GnRH Neurons: Implications for GABAA Mediated Excitation.

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    Carol Taylor-Burds

    Full Text Available A developmental "switch" in chloride transporters occurs in most neurons resulting in GABAA mediated hyperpolarization in the adult. However, several neuronal cell subtypes maintain primarily depolarizing responses to GABAA receptor activation. Among this group are gonadotropin-releasing hormone-1 (GnRH neurons, which control puberty and reproduction. NKCC1 is the primary chloride accumulator in neurons, expressed at high levels early in development and contributes to depolarization after GABAA receptor activation. In contrast, KCC2 is the primary chloride extruder in neurons, expressed at high levels in the adult and contributes to hyperpolarization after GABAA receptor activation. Anion exchangers (AEs are also potential modulators of responses to GABAA activation since they accumulate chloride and extrude bicarbonate. To evaluate the mechanism(s underlying GABAA mediated depolarization, GnRH neurons were analyzed for 1 expression of chloride transporters and AEs in embryonic, pre-pubertal, and adult mice 2 responses to GABAA receptor activation in NKCC1-/- mice and 3 function of AEs in these responses. At all ages, GnRH neurons were immunopositive for NKCC1 and AE2 but not KCC2 or AE3. Using explants, calcium imaging and gramicidin perforated patch clamp techniques we found that GnRH neurons from NKCC1-/- mice retained relatively normal responses to the GABAA agonist muscimol. However, acute pharmacological inhibition of NKCC1 with bumetanide eliminated the depolarization/calcium response to muscimol in 40% of GnRH neurons from WT mice. In the remaining GnRH neurons, HCO3- mediated mechanisms accounted for the remaining calcium responses to muscimol. Collectively these data reveal mechanisms responsible for maintaining depolarizing GABAA mediated transmission in GnRH neurons.

  1. Expression of GABAA Receptor γ2 Subunit in Hippocampus of Rats with KA-induced Temporal Lobe Epilepsy%GABAA 受体γ2亚单位在海人酸颞叶癫痫大鼠海马内的表达

    Institute of Scientific and Technical Information of China (English)

    戚月凤; 汤继宏; 李岩; 顾琴

    2015-01-01

    目的:探讨癫痫发作后 GABAA 受体γ2亚单位在海马各区的动态表达以及氯硝西泮干预对其表达的影响。方法:健康成年雄性 SD 大鼠40只,随机分为对照组5只,致痫组15只,干预组15只,干预对照组5只。大鼠海马 CA3区注射海人酸建立颞叶癫痫模型,干预组大鼠在致痫前予以氯硝西泮灌胃。于致痫后6 h、12 h和1 d 采用免疫组化法检测各组大鼠海马 CA1及 CA3区 γ-氨基丁酸 A 受体γ2亚单位(GABAARγ2)的动态表达水平。结果:致痫组在海人酸给药后6 h、12 h 及1 d,海马 CA3区 GABAARγ2蛋白表达均显著低于对照组(P<0.01);CA1区 GABAARγ2蛋白表达也下降,注射后1 d 显著低于对照组(P<0.01)。干预组在海人酸注射后1 d CA1和 CA3区 GABAARγ2蛋白表达低于对照组(P<0.05);海人酸注射后6 h、12 h 及1 d,CA3区 GABAARγ2蛋白表达均高于同时间点致痫组(P<0.05),CA1区于海人酸注射后1 d ,GABAARγ2蛋白表达显著高于同时间点致痫组(P<0.01)。结论:海人酸诱导的颞叶癫痫模型中,海马 GABAARγ2蛋白表达减少,氯硝西泮可缓解颞叶癫痫导致的 GABAARγ2蛋白表达减少。%Objective: To study the expression of GABAA receptor γ2 subunit (GABAARγ2) protein in hippocam-pus of rats with KA-induced temporal lobe epilepsy. To investigate the effect of clonazepam (CZP) administration on expression of GABAARγ2. Methods: Forty male SD rats were randomly divided into control group (n=5), epilepsy group (n=15), intervention group (n=15), and intervention control group (n=5). Temporal lobe epilepsy model was established by injecting kainic acid into CA3 region of the hippocampus in rats in both the epilepsy and intervention groups. Rats in the intervention group were treated with CZP. The expression of GABAARγ2 protein was investigated by immunohistochemistry at 6 h, 12 h and 1 d after operation respectively. Results: The

  2. Impaired surface αβγ GABA(A) receptor expression in familial epilepsy due to a GABRG2 frameshift mutation.

    Science.gov (United States)

    Tian, Mengnan; Mei, Davide; Freri, Elena; Hernandez, Ciria C; Granata, Tiziana; Shen, Wangzhen; Macdonald, Robert L; Guerrini, Renzo

    2013-02-01

    The purpose of the study was to explore the pathogenic mechanisms underlying generalized epilepsy and febrile seizures plus (GEFS+) in a family with a novel γ2 subunit gene (GABRG2) frameshift mutation. Four affected and one unaffected individuals carried a c.1329delC GABRG2 mutation resulting in a subunit [γ2S(S443delC)] with a modified and elongated carboxy-terminus that is different from that of the wildtype γ2S subunit. We expressed the wildtype γ2S subunit and the predicted mutant γ2S(S443delC) subunit cDNAs in HEK293T cells and performed immunoblotting, flow cytometry and electrophysiology studies. The mutant subunit was translated as a stable protein that was larger than the wildtype γ2S subunit and was retained in the ER and not expressed on the cell surface membrane, suggesting GABRG2 haploinsufficiency. Peak GABA-evoked currents recorded from cells cotransfected with wildtype α1 and β2 subunits and mutant γ2S subunits were significantly decreased and were comparable to α1β2 receptor currents. S443delC is the first GABR epilepsy mutation predicted to abolish the natural stop codon and produce a stop codon in the 3' UTR that leads to translation of an extended peptide. The GEFS+ phenotype observed in this family is likely caused by γ2S subunit loss-of-function and possibly to dominant-negative suppression of α1β2γ2 receptors. Many GABRG2 truncation mutations result in GEFS+, but the spectrum of phenotypic severity is wider, ranging from asymptomatic individuals to the Dravet syndrome. Mechanisms influencing the severity of the phenotype are therefore complex and difficult to correlate with its demonstrable functional effects.

  3. 咪达唑仑对大鼠背根神经节神经元GABAA受体激活电流的影响%Effects of midazolam on GABAA receptor-activated currents in isolated dorsal root ganglion neurons in rats

    Institute of Scientific and Technical Information of China (English)

    樊超; 马克涛; 杨越; 成洪聚; 王洋; 李丽; 司军强

    2012-01-01

    目的 咪达唑仑对大鼠背根神经节(DRG)神经元GABAA受体激活电流的影响.方法 健康SD大鼠,体重200 ~ 250 g,4周龄,雌雄不拘,分离DRG神经元,采用全细胞膜片钳技术记录GABAA受体激活电流.采用无糖细胞外液进行药物配制.记录咪达唑仑(终浓度3.00 μmol/L,)与不同浓度(终浓度0.03、0.10、1.00、10.00、100.00、1000.00 μmol/L) GABA混合液作用下的GABAA受体激活电流、不同浓度(终浓度0.03、0.10、1.00、3.00、10.00、100.00 μmol/L)咪达唑仑作用下的GABAA受体激活电流、不同浓度(终浓度0.03、0.10、1.00、3.00、10.00、100.00 μmol/L)咪达唑仑与GABA(终浓度100.00 μmol/L)混合液作用下的GABAA受体激活电流和不同咪达唑仑预灌注时间(灌注即刻、20、40、60、120 s)时咪达唑仑(终浓度1.00 μmol/L)与GABA(终浓度100.00 μmol/L)混合液作用下的GABAA受体激活电流.计算咪达唑仑·给药前后电流的增强率.结果 对GABA敏感的神经元灌注咪达唑仑均未记录到可检测的膜电流变化;各浓度GABA下,咪达唑仑给药后GABAA受体激活电流均较给药前增强(P<0.01);各浓度咪达唑仑给药后DRG神经元GABAA受体激活电流均较给药前增强,且随咪达唑仑浓度升高,对DRG神经元GABAA受体激活电流的增强率逐渐升高,咪达唑仑3.00 μmol/L时达峰值(P <0.05或0.01);随咪达唑仑预灌流时间延长,DRG神经元GABAA受体激活电流增强率逐渐升高,预灌流40 s时达峰值(β<0.05或0.01).结论 咪达唑仑可增强DRG神经元GABAA受体激活电流,提示咪达唑仑可通过增强GABAA受体活性,从而增强GABA的作用,在脊髓水平产生镇痛作用.%Objective To investigate the effects of midazolam on GABAA receptor-activated currents in isolated dorsal root ganglion (DRG) neurons in rats.Methods Sprague-Dawley rats of both sexes,weighing 200-250 g,aged 4 weeks,were used in the study.The DRG neurons were isolated and GABAA receptor

  4. Pattern-recognition receptors in human eosinophils.

    Science.gov (United States)

    Kvarnhammar, Anne Månsson; Cardell, Lars Olaf

    2012-05-01

    The pattern-recognition receptor (PRR) family includes Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD) -like receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) and the receptor for advanced glycation end products (RAGE). They recognize various microbial signatures or host-derived danger signals and trigger an immune response. Eosinophils are multifunctional leucocytes involved in the pathogenesis of several inflammatory processes, including parasitic helminth infection, allergic diseases, tissue injury and tumour immunity. Human eosinophils express several PRRs, including TLR1-5, TLR7, TLR9, NOD1, NOD2, Dectin-1 and RAGE. Receptor stimulation induces survival, oxidative burst, activation of the adhesion system and release of cytokines (interleukin-1β, interleukin-6, tumour necrosis factor-α and granulocyte-macrophage colony-stimulating factor), chemokines (interleukin-8 and growth-related oncogene-α) and cytotoxic granule proteins (eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase and major basic protein). It is also evident that eosinophils play an immunomodulatory role by interacting with surrounding cells. The presence of a broad range of PRRs in eosinophils indicates that they are not only involved in defence against parasitic helminths, but also against bacteria, viruses and fungi. From a clinical perspective, eosinophilic PRRs seem to be involved in both allergic and malignant diseases by causing exacerbations and affecting tumour growth, respectively.

  5. Distinct α subunit variations of the hypothalamic GABAA receptor triplets (αβγ are linked to hibernating state in hamsters

    Directory of Open Access Journals (Sweden)

    Alò Raffaella

    2010-09-01

    Full Text Available Abstract Background The structural arrangement of the γ-aminobutyric acid type A receptor (GABAAR is known to be crucial for the maintenance of cerebral-dependent homeostatic mechanisms during the promotion of highly adaptive neurophysiological events of the permissive hibernating rodent, i.e the Syrian golden hamster. In this study, in vitro quantitative autoradiography and in situ hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR tests were performed for specific GABAAR receptor subunit gene primers synthases of non-hibernating (NHIB and hibernating (HIB hamsters. Attempts were made to identify the type of αβγ subunit combinations operating during the switching ON/OFF of neuronal activities in some hypothalamic nuclei of hibernators. Results Both autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p 1 ratio (over total α subunits considered in the present study in the medial preoptic area (MPOA and arcuate nucleus (Arc of NHIBs with respect to HIBs. At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%. Regarding the other two subunits (β and γ, elevated β3 and γ3 mRNAs levels mostly characterized MPOA of HIBs, while prevalently elevated expression concentrations of the same subunits were also typical of Sch, even though this time during the awakening state. In the case of Arc, notably elevated levels were obtained for β3 and γ2 during hibernating conditions. Conclusion We conclude that different αβγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster. The identification of a brain regional

  6. Ciprofloxacin and sparfloxacin penetration into human brain tissue and their activity as antagonists of GABAA receptor of rat vagus nerve.

    OpenAIRE

    Davey, P G; Charter, M; Kelly, S; Varma, T R; Jacobson, I.; A. Freeman; Precious, E; Lambert, J

    1994-01-01

    Patients undergoing elective surgery for removal of brain tumors, aneurysms, or other vascular malformations were administered a single oral dose of sparfloxacin (400 mg; 16 patients) or ciprofloxacin (750 mg; 5 patients) either 3 to 5 h or 22 to 26 h before surgery. Serum samples were taken from all patients at 0, 1, 3 to 5, 7 to 9, and 22 to 26 h after dosing; an additional serum sample was obtained at 48 h from patients who received sparfloxacin. A single sample of brain tissue was taken f...

  7. Optogenetic Evocation of Field Inhibitory Postsynaptic Potentials in Hippocampal Slices: A Simple and Reliable Approach for Studying Pharmacological Effects on GABAA and GABAB Receptor-Mediated Neurotransmission

    Directory of Open Access Journals (Sweden)

    Julien eDine

    2014-01-01

    Full Text Available The GABAergic system is the main source of inhibition in the mammalian brain. Consequently, much effort is still made to develop new modulators of GABAergic synaptic transmission. In contrast to glutamatergic postsynaptic potentials (PSPs, accurate monitoring of GABA receptor-mediated PSPs (GABAR-PSPs and their pharmacological modulation in brain tissue invariably requires the use of intracellular recording techniques. However, these techniques are expensive, time- and labor-consuming, and, in case of the frequently employed whole-cell patch-clamp configuration, impact on intracellular ion concentrations, signaling cascades, and pH buffering systems. Here, we describe a novel approach to circumvent these drawbacks. In particular, we demonstrate in mouse hippocampal slices that selective optogenetic activation of interneurons leads to prominent field inhibitory GABAAR- and GABABR-PSPs in area CA1 which are easily and reliably detectable by a single extracellular recording electrode. The field PSPs exhibit typical temporal and pharmacological characteristics, display pronounced paired-pulse depression, and remain stable over many consecutive evocations. Additionally validating the methodological value of this approach, we further show that the neuroactive steroid 5-THDOC (5 µM shifts the inhibitory GABAAR-PSPs towards excitatory ones.

  8. Association of SNPs and haplotypes in GABA(A) receptor beta(2) gene with schizophrenia

    Institute of Scientific and Technical Information of China (English)

    WSLau; CFXuan; ZChan; CFFeng; GYHe; LCao; ZCLiu; HLuan; QMXue

    2005-01-01

    Disturbances in GABAergic system have been observed in schizophrenics.(1-3) In the present study, population association analysis was performed on 19 SNPs in the alpha(l), beta(2), gamma(2), epsilon and pi subunit genes of GABA(A) receptor. Five SNPs in GABRB2, namely B217G1584T, rs1816071, rs194072, rs252944 and rs187269,were found to be significantly associated, and their haplotypes in linkage disequilibrium, with schizophrenia. This represents the first report on any disease association of SNPs in the human GABA(A) receptor genes, and focuses attention on the GABAergic hypothesis of schizophrenia etiology.(3,4)

  9. Excitatory synapses on dendritic shafts of the caudal basal amygdala exhibit elevated levels of GABAA receptor α4 subunits following the induction of activity-based anorexia.

    Science.gov (United States)

    Wable, Gauri S; Barbarich-Marsteller, Nicole C; Chowdhury, Tara G; Sabaliauskas, Nicole A; Farb, Claudia R; Aoki, Chiye

    2014-01-01

    Anorexia nervosa (AN) is an eating disorder characterized by self-imposed severe starvation, excessive exercise, and anxiety. The onset of AN is most often at puberty, suggesting that gonadal hormonal fluctuations may contribute to AN vulnerability. Activity-based anorexia (ABA) is an animal model that reproduces some of the behavioral phenotypes of AN, including the paradoxical increase in voluntary exercise following food restriction. The basal amygdala as well as the GABAergic system regulate trait anxiety. We therefore examined the subcellular distribution of GABA receptors (GABARs) in the basal amygdala of female pubertal rats and specifically of their α4 subunits, because expression of α4-containing GABARs is regulated by gonadal hormone fluctuations. Moreover, because these GABARs reduce neuronal excitability through shunting of EPSPs, we quantified the frequency of occurrence of these GABARs adjacent to excitatory synapses. Electron microscopic immunoctychemistry revealed no change in the frequency of association of α4 subunits with excitatory synapses on dendritic spines, whether in the anterior (Bregma -2.8 mm) or caudal (Bregma -3.8 mm) portion of the basal amygdala. Sholl analysis of golgi-stained neurons also revealed no change in the extent of dendritic branching by these densely spiny, pyramidal-like neurons. However, there was an increase of membranous α4 subunits near excitatory synapses on dendritic shafts, specifically in the caudal basal amygdala, and this was accompanied by a rise of α4 subunits intracellularly. Because most dendritic shafts exhibiting excitatory synapses are GABAergic interneurons, the results predict disinhibition, which would increase excitability of the amygdaloid network, in turn augmenting ABA animals' anxiety.

  10. Ethanol exposure during the third trimester equivalent does not affect GABAA or AMPA receptor-mediated spontaneous synaptic transmission in rat CA3 pyramidal neurons

    OpenAIRE

    Baculis, Brian Charles; Valenzuela, Carlos Fernando

    2015-01-01

    Background Ethanol exposure during the rodent equivalent to the 3rd trimester of human pregnancy (i.e., first 1–2 weeks of neonatal life) has been shown to produce structural and functional alterations in the CA3 hippocampal sub-region, which is involved in associative memory. Synaptic plasticity mechanisms dependent on retrograde release of brain-derived neurotrophic factor (BDNF) driven by activation of L-type voltage-gated Ca2+ channels (L-VGCCs) are thought to play a role in stabilization...

  11. Cellular receptors for human enterovirus species A

    Directory of Open Access Journals (Sweden)

    Yorihiro eNishimura

    2012-03-01

    Full Text Available Human enterovirus species A (HEV-A is one of the four species of HEV in the genus Enterovirus in the family Picornaviridae. Among HEV-A, coxsackievirus A16 (CVA16 and enterovirus 71 (EV71 are the major causative agents of hand, foot, and mouth disease (HFMD. Some other types of HEV-A are commonly associated with herpangina. Although HFMD and herpangina due to HEV-A are common febrile diseases among infants and children, EV71 can cause various neurological diseases, such as aseptic meningitis and fatal encephalitis.Recently, two human transmembrane proteins, P-selectin glycoprotein ligand-1 (PSGL-1 and scavenger receptor class B, member 2 (SCARB2, were identified as functional receptors for EV71 and CVA16. In in vitro infection experiments using the prototype HEV-A strains, PSGL-1 and SCARB2 could be responsible for the specific receptors for EV71 and CVA16. However, the involvement of both receptors in the in vitro and in vivo infections of clinical isolates of HEV-A has not been clarified yet. To elucidate a diverse array of the clinical outcome of HEV-A-associated diseases, the identification and characterization of HEV-A receptors may provide useful information in understanding the HEV-A pathogenesis at a molecular level.

  12. Beta adrenergic receptors in human cavernous tissue

    Energy Technology Data Exchange (ETDEWEB)

    Dhabuwala, C.B.; Ramakrishna, C.V.; Anderson, G.F.

    1985-04-01

    Beta adrenergic receptor binding was performed with /sup 125/I iodocyanopindolol on human cavernous tissue membrane fractions from normal tissue and transsexual procedures obtained postoperatively, as well as from postmortem sources. Isotherm binding studies on normal fresh tissues indicated that the receptor density was 9.1 fmoles/mg. with a KD of 23 pM. Tissue stored at room temperature for 4 to 6 hours, then at 4C in saline solution for 19 to 20 hours before freezing showed no significant changes in receptor density or affinity, and provided evidence for the stability of postmortem tissue obtained within the same time period. Beta receptor density of 2 cavernous preparations from transsexual procedures was not significantly different from normal control tissues, and showed that high concentrations of estrogen received by these patients had no effect on beta adrenergic receptor density. Displacement of /sup 125/iodocyanopindolol by 5 beta adrenergic agents demonstrated that 1-propranolol had the greatest affinity followed by ICI 118,551, zinterol, metoprolol and practolol. When the results of these displacement studies were subjected to Scatfit, non- linear regression line analysis, a single binding site was described. Based on the relative potency of the selective beta adrenergic agents it appears that these receptors were of the beta 2 subtype.

  13. Synthesis and biological evaluation of 4-(aminomethyl)-1-hydroxypyrazole analogues of muscimol as γ-aminobutyric acid(a) receptor agonists.

    Science.gov (United States)

    Petersen, Jette G; Bergmann, Rikke; Møller, Henriette A; Jørgensen, Charlotte G; Nielsen, Birgitte; Kehler, Jan; Frydenvang, Karla; Kristensen, Jesper; Balle, Thomas; Jensen, Anders A; Kristiansen, Uffe; Frølund, Bente

    2013-02-14

    A series of bioisosteric 4-(aminomethyl)-1-hydroxypyrazole (4-AHP) analogues of muscimol, a GABA(A) receptor agonist, has been synthesized and pharmacologically characterized at native and selected recombinant GABA(A) receptors. The unsubstituted 4-AHP analogue (2a) (EC(50) 19 μM, R(max) 69%) was a moderately potent agonist at human α(1)β(2)γ(2) GABA(A) receptors, and in SAR studies substitutions in the 3- and/or 5-position were found to be detrimental to binding affinities. Ligand-receptor docking in an α(1)β(2)γ(2) GABA(A) receptor homology model along with the obtained SAR indicate that 2a and muscimol share a common binding mode, which deviates from the binding mode of the structurally related antagonist series based on 4-(piperidin-4-yl)-1-hydroxypyrazole (4-PHP, 1). Selectivity for α(1)β(2)γ(2) over ρ(1) GABA(A) receptors was observed for the 5-chloro, 5-bromo, and 5-methyl substituted analogues of 2a illustrating that even small differences in structure can give rise to subtype selectivity.

  14. Carbamate Insecticides Target Human Melatonin Receptors.

    Science.gov (United States)

    Popovska-Gorevski, Marina; Dubocovich, Margarita L; Rajnarayanan, Rajendram V

    2017-02-20

    Carbaryl (1-naphthyl methylcarbamate) and carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) are among the most toxic insecticides, implicated in a variety of diseases including diabetes and cancer among others. Using an integrated pharmacoinformatics based screening approach, we have identified these insecticides to be structural mimics of the neurohormone melatonin and were able to bind to the putative melatonin binding sites in MT1 and MT2 melatonin receptors in silico. Carbaryl and carbofuran then were tested for competition with 2-[(125)I]-iodomelatonin (300 pM) binding to hMT1 or hMT2 receptors stably expressed in CHO cells. Carbaryl and carbofuran showed higher affinity for competition with 2-[(125)I]-iodomelatonin binding to the hMT2 compared to the hMT1 melatonin receptor (33 and 35-fold difference, respectively) as predicted by the molecular modeling. In the presence of GTP (100 μM), which decouples the G-protein linked receptors to modulate signaling, the apparent efficacy of carbaryl and carbofuran for 2-[(125)I]-iodomelatonin binding for the hMT1 melatonin receptor was not affected but significantly decreased for the hMT2 melatonin receptor compatible with receptor antagonist/inverse agonist and agonist efficacy, respectively. Altogether, our data points to a potentially new mechanism through which carbamate insecticides carbaryl and carbofuran could impact human health by altering the homeostatic balance of key regulatory processes by directly binding to melatonin receptors.

  15. Relationship between gene polymorphism of GABAA receptors gene and childhood autism%γ-氨基丁酸A受体基因多态性与儿童孤独症的关系

    Institute of Scientific and Technical Information of China (English)

    卢国斌; 余秋娟; 王章琼; 林元; 欧萍; 徐两蒲; 黄海龙; 成玲; 杨式薇; 钱沁芳; 黄艳; 谢燕钦

    2012-01-01

    Objective To explore the relationship between gene polymorphism of GABAA receptors and childhood autism by detecting rs140682,rs2081648 and rs140679 site of single nucleotide polymorphism (SNP) in GABAA receptors gene.Methods A total of 94 children with autism and 124 normal children were enrolled in a hospital from November 2010 to May 2011.Childhood autism rating scale(CARS) and autism behavior checklist(ABC) were used to evaluate or investigate the case group.After collecting venous blood and extracting the genome DNA,the allele and genotype of SNP rs140682,rs2081648 and rs140679 site in GABAA receptors gene were detected by PCR-RFLP.The allele and genotype of case group and control group were analized by χ2 test,while the score of scales was analized by Spearman rank conrelation analysis.Results The age of the case group was 5.12 ±0.32,and it was 5.25 ± 0.27 in the control group ( P <0.05 ).In case group,the frequency of genotype CC,CT and TT of rs140682 site was 44,41 and 9,while it was 48,65,and 11 in control group(P >0.05 ),respectively.The frequency of genotype AA,AG and GG of rs2081648 site was 8,58 and 28 in case group,while it was 12,49 and 63 in control group( P < 0.05),respectively.In case group,the frequency of genotype CC,CT and TT of rs140679 site was 15,36 and 43,while it was 18,59 and 47 in control group( P >0.05 ),respectively.It was revealed by Spearmanrank correlation analysis that of rs2081648 site,there was a positive correlation between genotype AG andsensation factor( S ),so cial intercourse factor ( R ),and language factor ( L ) of autism behavior checklist (ABC) (r values were 0.149,0.165 and 0.155,all P values <0.05 ).A negative correlation between genotype GG and S,R,L and self-help factor(V) was proved(r values were -0.140,-0.173,-0.158 and -0.135,all P values <0.05 ).There was a positive correlation between allele A and R and L factors(r values were 0.153 and 0.137,all P values < 0.05 ),while a negative correlation

  16. Fyn Kinase regulates GluN2B subunit-dominant NMDA receptors in human induced pluripotent stem cell-derived neurons.

    Science.gov (United States)

    Zhang, Wen-Bo; Ross, P Joel; Tu, YuShan; Wang, Yongqian; Beggs, Simon; Sengar, Ameet S; Ellis, James; Salter, Michael W

    2016-04-04

    NMDA receptor (NMDAR)-mediated fast excitatory neurotransmission is implicated in a broad range of physiological and pathological processes in the mammalian central nervous system. The function and regulation of NMDARs have been extensively studied in neurons from rodents and other non-human species, and in recombinant expression systems. Here, we investigated human NMDARs in situ by using neurons produced by directed differentiation of human induced pluripotent stem cells (iPSCs). The resultant cells showed electrophysiological characteristics demonstrating that they are bona fide neurons. In particular, human iPSC-derived neurons expressed functional ligand-gated ion channels, including NMDARs, AMPA receptors, GABAA receptors, as well as glycine receptors. Pharmacological and electrophysiological properties of NMDAR-mediated currents indicated that these were dominated by receptors containing GluN2B subunits. The NMDAR currents were suppressed by genistein, a broad-spectrum tyrosine kinase inhibitor. The NMDAR currents were also inhibited by a Fyn-interfering peptide, Fyn(39-57), but not a Src-interfering peptide, Src(40-58). Together, these findings are the first evidence that tyrosine phosphorylation regulates the function of NMDARs in human iPSC-derived neurons. Our findings provide a basis for utilizing human iPSC-derived neurons in screening for drugs targeting NMDARs in neurological disorders.

  17. Dopamine receptor repertoire of human granulosa cells

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    Kunz Lars

    2007-10-01

    Full Text Available Abstract Background High levels of dopamine (DA were described in human ovary and recently evidence for DA receptors in granulosa and luteal cells has been provided, as well. However, neither the full repertoire of ovarian receptors for DA, nor their specific role, is established. Human granulosa cells (GCs derived from women undergoing in vitro fertilization (IVF are an adequate model for endocrine cells of the follicle and the corpus luteum and were therefore employed in an attempt to decipher their DA receptor repertoire and functionality. Methods Cells were obtained from patients undergoing IVF and examined using cDNA-array, RT-PCR, Western blotting and immunocytochemistry. In addition, calcium measurements (with FLUO-4 were employed. Expression of two DA receptors was also examined by in-situ hybridization in rat ovary. Effects of DA on cell viability and cell volume were studied by using an ATP assay and an electronic cell counter system. Results We found members of the two DA receptor families (D1- and D2 -like associated with different signaling pathways in human GCs, namely D1 (as expected and D5 (both are Gs coupled and linked to cAMP increase and D2, D4 (Gi/Gq coupled and linked to IP3/DAG. D3 was not found. The presence of the trophic hormone hCG (10 IU/ml in the culture medium for several days did not alter mRNA (semiquantitative RT-PCR or protein levels (immunocytochemistry/Western blotting of D1,2,4,5 DA receptors. Expression of prototype receptors for the two families, D1 and D2, was furthermore shown in rat granulosa and luteal cells by in situ hybridization. Among the DA receptors found in human GCs, D2 expression was marked both at mRNA and protein levels and it was therefore further studied. Results of additional RT-PCR and Western blots showed two splice variants (D2L, D2S. Irrespective of these variants, D2 proved to be functional, as DA raised intracellular calcium levels. This calcium mobilizing effect of DA was observed

  18. IL-21 Receptor Expression in Human Tendinopathy

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    Campbell, Abigail L.; Smith, Nicola C.; Reilly, James H.; Kerr, Shauna C.; Leach, William J.; Fazzi, Umberto G.; Rooney, Brian P.; Murrell, George A. C.; Millar, Neal L.

    2014-01-01

    The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1β) in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy. PMID:24757284

  19. IL-21 Receptor Expression in Human Tendinopathy

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    Abigail L. Campbell

    2014-01-01

    Full Text Available The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1β in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy.

  20. Computer Modeling of Human Delta Opioid Receptor

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    Tatyana Dzimbova

    2013-04-01

    Full Text Available The development of selective agonists of δ-opioid receptor as well as the model of interaction of ligands with this receptor is the subjects of increased interest. In the absence of crystal structures of opioid receptors, 3D homology models with different templates have been reported in the literature. The problem is that these models are not available for widespread use. The aims of our study are: (1 to choose within recently published crystallographic structures templates for homology modeling of the human δ-opioid receptor (DOR; (2 to evaluate the models with different computational tools; and (3 to precise the most reliable model basing on correlation between docking data and in vitro bioassay results. The enkephalin analogues, as ligands used in this study, were previously synthesized by our group and their biological activity was evaluated. Several models of DOR were generated using different templates. All these models were evaluated by PROCHECK and MolProbity and relationship between docking data and in vitro results was determined. The best correlations received for the tested models of DOR were found between efficacy (erel of the compounds, calculated from in vitro experiments and Fitness scoring function from docking studies. New model of DOR was generated and evaluated by different approaches. This model has good GA341 value (0.99 from MODELLER, good values from PROCHECK (92.6% of most favored regions and MolProbity (99.5% of favored regions. Scoring function correlates (Pearson r = -0.7368, p-value = 0.0097 with erel of a series of enkephalin analogues, calculated from in vitro experiments. So, this investigation allows suggesting a reliable model of DOR. Newly generated model of DOR receptor could be used further for in silico experiments and it will give possibility for faster and more correct design of selective and effective ligands for δ-opioid receptor.

  1. Enhancement of GABAA-current run-down in the hippocampus occurs at the first spontaneous seizure in a model of temporal lobe epilepsy

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    Mazzuferi, Manuela; Palma, Eleonora; Martinello, Katiuscia; Maiolino, Francesca; Roseti, Cristina; Fucile, Sergio; Fabene, Paolo F.; Schio, Federica; Pellitteri, Michele; Sperk, Guenther; Miledi, Ricardo; Eusebi, Fabrizio; Simonato, Michele

    2010-01-01

    Refractory temporal lobe epilepsy (TLE) is associated with a dysfunction of inhibitory signaling mediated by GABAA receptors. In particular, the use-dependent decrease (run-down) of the currents (IGABA) evoked by the repetitive activation of GABAA receptors is markedly enhanced in hippocampal and cortical neurons of TLE patients. Understanding the role of IGABA 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 IGABA 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 α4-, relative to α1-containing GABAA 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. PMID:20133704

  2. Antihyperalgesic effect of the GABA(A) ligand clobazam in a neuropathic pain model in mice: a pharmacokinetic-pharmacodynamic study.

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    Besson, Marie; Daali, Youssef; Di Lio, Alessandra; Dayer, Pierre; Zeilhofer, Hanns Ulrich; Desmeules, Jules

    2013-03-01

    Facilitation of spinal GABAergic inhibition with benzodiazepines (BZDs) reverses pain sensitization in animals; however, the use of BZDs in man is limited by their sedative effect. The antihyperalgesic effects of GABA(A) agonists are mediated by GABA(A) receptors containing α2 subunits, whereas sedation is linked to α1 subunit-containing receptors. α2 and α3 selective GABA(A) receptor modulators have been tested in animals but are not yet available for use in human beings. Clobazam is a 1,5-BZD, which exhibits less cognitive side effects than other benzodiazepines. Here, we studied its antihyperalgesic effects in a mouse model of neuropathic pain. Clobazam showed a dose-dependent antihyperalgesic effect in the chronic constriction injury (CCI) model of neuropathic pain, peaking at 1 hr after administration and lasting for 4 hr with no relevant sedation at a dose of 3 mg/kg. At higher doses, the antihyperalgesic effect was stronger, but sedation became significant. The blood and brain kinetics of clobazam were linear over the range of doses tested with a short half-life of the parent compound and a ready penetration of the blood-brain barrier. Clobazam blood concentrations decreased rapidly, falling below the limit of detection at 120 min. after drug application. Its main metabolite, N-desmethyl-clobazam, showed more delayed and prolonged pharmacokinetics, partly explaining why antihyperalgesia persisted when clobazam was no longer detectable in the blood. Considering its therapeutic margin and its pharmacokinetic properties, clobazam would be a valuable compound to assess the role of the GABAergic pathway in pain transmission in human beings.

  3. NMDA and AMPA/kainate glutamatergic receptors in the prelimbic medial prefrontal cortex modulate the elaborated defensive behavior and innate fear-induced antinociception elicited by GABAA receptor blockade in the medial hypothalamus.

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    de Freitas, Renato Leonardo; Salgado-Rohner, Carlos José; Biagioni, Audrey Francisco; Medeiros, Priscila; Hallak, Jaime Eduardo Cecílio; Crippa, José Alexandre S; Coimbra, Norberto Cysne

    2014-06-01

    The aim of the present study was to investigate the involvement of N-methyl-d-aspartate (NMDA) and amino-3-hydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainate receptors of the prelimbic (PL) division of the medial prefrontal cortex (MPFC) on the panic attack-like reactions evoked by γ-aminobutyric acid-A receptor blockade in the medial hypothalamus (MH). Rats were pretreated with NaCl 0.9%, LY235959 (NMDA receptor antagonist), and NBQX (AMPA/kainate receptor antagonist) in the PL at 3 different concentrations. Ten minutes later, the MH was treated with bicuculline, and the defensive responses were recorded for 10 min. The antagonism of NMDA receptors in the PL decreased the frequency and duration of all defensive behaviors evoked by the stimulation of the MH and reduced the innate fear-induced antinociception. However, the pretreatment of the PL cortex with NBQX was able to decrease only part of defensive responses and innate fear-induced antinociception. The present findings suggest that the NMDA-glutamatergic system of the PL is critically involved in panic-like responses and innate fear-induced antinociception and those AMPA/kainate receptors are also recruited during the elaboration of fear-induced antinociception and in panic attack-related response. The activation of the glutamatergic neurotransmission of PL division of the MPFC during the elaboration of oriented behavioral reactions elicited by the chemical stimulation of the MH recruits mainly NMDA receptors in comparison with AMPA/kainate receptors.

  4. Differential modulatory actions of GABAA agonists on susceptibility to GABAA antagonists-induced seizures in morphine dependent rats: possible mechanisms in seizure propensity.

    Science.gov (United States)

    Joukar, Siyavash; Atapour, Nafiseh; Kalantaripour, Tajpari; Bashiri, Hamideh; Shahidi, Alireza

    2011-07-01

    In order to clarify the mechanisms involved in the susceptibility to GABA(A) antagonists-induced seizures in morphine dependent rats, we investigated how GABA(A) agonists modulate this vulnerability. Seizures were induced to animals by infusion of GABA(A) antagonists: pentylenetetrazole (PTZ), picrotoxin (PIC) and bicuculline (BIC). GABA(A) agonists, muscimol (MUS) and 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP), were administered intravenous (i.v.) before antagonists. Morphine-dependence significantly decreased the PTZ threshold dose (19.16±1.89 versus 25.74±1.25mg/kg) while, it had no effect on PIC induced seizures. BIC doses for both threshold and tonic-clonic seizures induction were significantly lower in morphine dependent rats (0.10±0.01 and 0.12±0.02 versus 0.25±0.02 and 0.39±0.07mg/kg respectively). In morphine-dependence, although pre-treatment with MUS significantly increased the required dose of PTZ for seizures threshold, THIP significantly decreased the required dose of PTZ for tonic-clonic convulsion. Moreover, MUS pretreatment completely recovered the effect of morphine dependency on BIC seizure activity. The results suggest that the capability of GABA(A) agonists on modulation of propensity to seizures induced by different antagonists in morphine-dependence is dissimilar. Therefore, it seems that long-term morphine alters some properties of GABA system so that the responsive rate of GABA(A) receptors not only to its antagonists, but also to its agonists will change differently.

  5. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    Science.gov (United States)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  6. Distribution of melatonin receptor in human fetal brain

    Institute of Scientific and Technical Information of China (English)

    WANG Guo-quan; SHAO Fu-yuan; ZHAO Ying; LIU Zhi-min

    2001-01-01

    Objective: To study the distribution of 2 kinds of melatonin receptor subtypes (mtl and MT2) in human fetal brain. Methods: The fetal brain tissues were sliced and the distribution ofmelatonin receptors in human fetal brain were detected using immunohistochemistry and in situ hybridization. Results: Melatonin receptor mtl existed in the cerebellun and hypothalamus, melatonin receptor MT2 exists in hypothalamus, occipital and medulla. Conclusion: Two kinds of melatonin receptors, mtl and MT2 exist in the membrane and cytosol of brain cells, indicating that human fetal brain is a target organ of melatonin.

  7. Human basophils express interleukin-4 receptors

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    Valent, P.; Besemer, J.; Kishi, K.; Di Padova, F.; Geissler, K.; Lechner, K.; Bettelheim, P. (Univ. of Vienna (Austria))

    1990-11-01

    Interleukin-4 (IL-4), a multipotential lymphokine reputed to play an important role in the regulation of immune responses, interacts with a variety of hemopoietic target cells through specific cell surface membrane receptors. The present study was designed to investigate whether human basophils express IL-4 binding sites. For this purpose, basophils were enriched to homogeneity (93% and 98% purity, respectively) from the peripheral blood of two chronic granulocytic leukemia (CGL) donors using a cocktail of monoclonal antibodies (MoAbs) and complement. Purified basophils bound 125I-radiolabeled recombinant human (rh) IL-4 in a specific manner. Quantitative binding studies and Scatchard plot analysis revealed the presence of a single class of high affinity IL-4 binding sites (280 +/- 40 sites per cell in donor 1 and 640 +/- 45 sites per cell in donor 2) with an apparent dissociation constant, kd, of 7.12 x 10(-11) +/- 2.29 x 10(-11) and 9.55 +/- 3.5 x 10(-11) mol/L, respectively. KU812-F, a human basophil precursor cell line, was found to express a single class of 810 to 1,500 high affinity IL-4 binding sites with a kd of 2.63 to 5.54 x 10(-10) mol/L. No change in the numbers or binding constants of IL-4 receptors was found after exposure of KU812-F cells to rhIL-3 (a potent activator of basophils) for 60 minutes. No effect of rhIL-4 on 3H-thymidine uptake, release or synthesis of histamine, or expression of basophil differentiation antigens (Bsp-1, CD11b, CD25, CD40, CD54) on primary human CGL basophils or KU812-F cells was observed.

  8. Bitter taste receptor polymorphisms and human aging.

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    Daniele Campa

    Full Text Available Several studies have shown that genetic factors account for 25% of the variation in human life span. On the basis of published molecular, genetic and epidemiological data, we hypothesized that genetic polymorphisms of taste receptors, which modulate food preferences but are also expressed in a number of organs and regulate food absorption processing and metabolism, could modulate the aging process. Using a tagging approach, we investigated the possible associations between longevity and the common genetic variation at the three bitter taste receptor gene clusters on chromosomes 5, 7 and 12 in a population of 941 individuals ranging in age from 20 to 106 years from the South of Italy. We found that one polymorphism, rs978739, situated 212 bp upstream of the TAS2R16 gene, shows a statistically significant association (p = 0.001 with longevity. In particular, the frequency of A/A homozygotes increases gradually from 35% in subjects aged 20 to 70 up to 55% in centenarians. These data provide suggestive evidence on the possible correlation between human longevity and taste genetics.

  9. The Human Laminin Receptor is a Member of the Integrin Family of Cell Adhesion Receptors

    Science.gov (United States)

    Gehlsen, Kurt R.; Dillner, Lena; Engvall, Eva; Ruoslahti, Erkki

    1988-09-01

    A receptor for the adhesive basement membrane protein, laminin, was isolated from human glioblastoma cells by affinity chromatography on laminin. This receptor has a heterodimeric structure similar to that of receptors for other extracellular matrix proteins such as fibronectin and vitronectin. Incorporation of the laminin receptor into liposomal membranes makes it possible for liposomes to attach to surfaces coated with laminin. The receptor liposomes also attached to some extent to surfaces coated with fibronectin, but not with other matrix proteins. These properties identify the laminin receptor as a member of the integrin family of cell adhesion receptors.

  10. PET imaging of human cardiac opioid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Villemagne, Patricia S.R.; Dannals, Robert F. [Department of Radiology, The Johns Hopkins University School of Medicine, 605 N Caroline St., Baltimore, Maryland (United States); Department of Environmental Health Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Ravert, Hayden T. [Department of Radiology, The Johns Hopkins University School of Medicine, 605 N Caroline St., Baltimore, Maryland (United States); Frost, James J. [Department of Radiology, The Johns Hopkins University School of Medicine, 605 N Caroline St., Baltimore, Maryland (United States); Department of Environmental Health Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States)

    2002-10-01

    The presence of opioid peptides and receptors and their role in the regulation of cardiovascular function has been previously demonstrated in the mammalian heart. The aim of this study was to image {mu} and {delta} opioid receptors in the human heart using positron emission tomography (PET). Five subjects (three females, two males, 65{+-}8 years old) underwent PET scanning of the chest with [{sup 11}C]carfentanil ([{sup 11}C]CFN) and [{sup 11}C]-N-methyl-naltrindole ([{sup 11}C]MeNTI) and the images were analyzed for evidence of opioid receptor binding in the heart. Either [{sup 11}C]CFN or [{sup 11}C]MeNTI (20 mCi) was injected i.v. with subsequent dynamic acquisitions over 90 min. For the blocking studies, either 0.2 mg/kg or 1 mg/kg of naloxone was injected i.v. 5 min prior to the injection of [{sup 11}C]CFN and [{sup 11}C]MeNTI, respectively. Regions of interest were placed over the left ventricle, left ventricular chamber, lung and skeletal muscle. Graphical analysis demonstrated average baseline myocardial binding potentials (BP) of 4.37{+-}0.91 with [{sup 11}C]CFN and 3.86{+-}0.60 with [{sup 11}C]MeNTI. Administration of 0.2 mg/kg naloxone prior to [{sup 11}C]CFN produced a 25% reduction in BP in one subject in comparison with baseline values, and a 19% decrease in myocardial distribution volume (DV). Administration of 1 mg/kg of naloxone before [{sup 11}C]MeNTI in another subject produced a 14% decrease in BP and a 21% decrease in the myocardial DV. These results demonstrate the ability to image these receptors in vivo by PET. PET imaging of cardiac opioid receptors may help to better understand their role in cardiovascular pathophysiology and the effect of abuse of opioids and drugs on heart function. (orig.)

  11. Sevoflurane neurotoxicity in neonatal rats is related to an increase in the GABAA R α1/ GABAA R α2 ratio.

    Science.gov (United States)

    Xie, Si-Ning; Ye, Hong; Li, Jun-Fa; An, Li-Xin

    2017-08-26

    Exposure of neonatal rat to sevoflurane leads to neurodegeneration and deficits of spatial learning and memory in adulthood. However, the underlying mechanisms remain unclear. The type A γ-aminobutyric acid receptor (GABAA R) is a target receptor for sevoflurane. The present study intends to investigate the changes in GABAA R α1/α2 expression and its relationship with the neurotoxicity effect due to sevoflurane in neonatal rats. After a dose-response curve was constructed to determine minimum alveolar concentration (MAC) and safety was guaranteed in our 7-day-old neonatal rat pup mode, we conducted two studies among the following groups: (A) the control group; (B) the sham anesthesia group; and (C) the sevoflurane anesthesia group and all three groups were treated in the same way as the model. First, poly(ADP-ribose) polymerase-1 protein (PARP-1) expression was determined in the different brain areas at 6 hr after anesthesia. Second, the expression of PARP-1 and GABAA R α1/GABAA R α2 in the hippocampus area was tested by Western blotting at 6 hr, 24 hr, and 72 hr after anesthesia in all three groups. After 4 hr, with 0.8 MAC (2.1%) sevoflurane anesthesia, the PARP-1 expression was significantly higher in the hippocampus than the other brain areas (p < .05). Compared with Groups A and B, the expression of PARP-1 in the hippocampus of Group C significantly increased at 6 hr after sevoflurane exposure (216% ± 15%, p < .05), and the ratio of the α1/α2 subunit of GABAA R surged at 6 hr (126% ± 6%), 24 hr (127% ± 8%), and 72 hr (183% ± 22%) after sevoflurane exposure in the hippocampus (p < .05). Our study showed that sevoflurane exposure of 0.8 MAC (2.1%)/4 hr was a suitable model for 7-day-old rats. And the exposure to sevoflurane could induce the apoptosis of neurons in the early stage, which may be related to the transmission from GABAA R α2 to GABAA R α1. © 2017 Wiley Periodicals, Inc.

  12. Pathophysiological power of improper tonic GABAA conductances in mature and immature models

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    Kiyoshi eEgawa

    2013-10-01

    Full Text Available High-affinity extrasynaptic gamma-aminobutyric acid A (GABAA receptors are tonically activated by low and consistent levels of ambient GABA, mediating chronic inhibition against neuronal excitability (tonic inhibition and the modulation of neural development. Synaptic (phasic inhibition is spatially and temporally precise compared with tonic inhibition, which provides blunt yet strong integral inhibitory force by shunting electrical signaling. Although effects of acute modification of tonic inhibition are known, its pathophysiological significance remains unclear because homeostatic regulation of neuronal excitability can compensate for long-term deficit of extrasynaptic GABAA receptor activation. Nevertheless, tonic inhibition is of great interest for its pathophysiological involvement in central nervous system (CNS diseases and thus as a therapeutic target. Together with the development of experimental models for various pathological states, recent evidence demonstrates such pathological involvements of tonic inhibition in neuronal dysfunction. This review focuses on the recent progress of tonic activation of GABAA conductance on the development and pathology of the CNS. Findings indicate that neuronal function in various brain regions are exacerbated with a gain or loss of function of tonic inhibition by GABA spillover. Disturbance of tonic GABAA conductance mediated by non-synaptic ambient GABA may result in brain mal-development. Therefore, various pathological states (epilepsy, motor dysfunctions, psychiatric disorders, and neurodevelopmental disorders may be partly attributable to abnormal tonic GABAA conductances. Thus, the tone of tonic conductance and level of ambient GABA may be precisely tuned to maintain the regular function and development of the CNS. Therefore, receptor expression and factors for regulating the ambient GABA concentration are highlighted to gain a deeper understanding of pathology and therapeutic strategy for CNS

  13. The pharmacology of spontaneously open alpha 1 beta 3 epsilon GABA A receptor-ionophores.

    Science.gov (United States)

    Maksay, Gábor; Thompson, Sally A; Wafford, Keith A

    2003-06-01

    Human alpha(1)beta(3) epsilon GABA(A) receptors were expressed in Xenopus oocytes and examined using the conventional two-electrode voltage-clamp technique and compared to alpha(1)beta(3)gamma(2) receptors. The effects of several GABA(A) agonists were studied, and the allosteric modulation of the channel by a number of GABAergic modulators investigated. The presence of the epsilon subunit increased the potency and efficacy of direct activation by partial GABA(A) agonists (piperidine-4-sulphonic acid and thio-4-PIOL), pentobarbital and neuro-steroids. Direct activation by 3-hydroxylated neurosteroids was restricted to 3alpha epimers, while chirality at C5 was indifferent. The 3beta-sulfate esters of pregnenolone and dehydroepiandrosterone inhibited the spontaneous currents with efficacies higher, while bicuculline methiodide and SR 95531 did so lower than picrotoxin and TBPS. Furosemide, fipronil, triphenylcyanoborate and Zn(2+) blocked the spontaneous currents of alpha(1)beta(3) epsilon receptors with different efficacies. Flunitrazepam and 4'-chlorodiazepam inhibited the spontaneous currents with micromolar potencies. In conclusion, spontaneously active alpha(1)beta(3) epsilon GABA(A) receptors can be potentiated and blocked by GABAergic agents within a broad range of efficacy.

  14. Selective increases of AMPA, NMDA and kainate receptor subunit mRNAs in the hippocampus and orbitofrontal cortex but not in prefrontal cortex of human alcoholics

    Directory of Open Access Journals (Sweden)

    Zhe eJin

    2014-01-01

    Full Text Available Glutamate is the main excitatory transmitter in the human brain. Drugs that affect the glutamatergic signaling will alter neuronal excitability. Ethanol inhibits glutamate receptors. We examined the expression level of glutamate receptor subunit mRNAs in human post-mortem samples from alcoholics and compared the results to brain samples from control subjects. RNA from hippocampal dentate gyrus (HP-DG, orbitofrontal cortex (OFC, and dorso-lateral prefrontal cortex (DL-PFC samples from 21 controls and 19 individuals with chronic alcohol dependence were included in the study. Total RNA was assayed using quantitative RT-PCR. Out of the 16 glutamate receptor subunits, mRNAs encoding two AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-ylpropanoic acid receptor subunits GluA2 and GluA3; three kainate receptor subunits GluK2, GluK3 and GluK5 and five NMDA (N-methyl-D-aspartate receptor subunits GluN1, GluN2A, GluN2C, GluN2D and GluN3A were significantly increased in the HP-DG region in alcoholics. In the OFC, mRNA encoding the NMDA receptor subunit GluN3A was increased, whereas in the DL-PFC, no differences in mRNA levels were observed. Our laboratory has previously shown that the expression of genes encoding inhibitory GABA-A receptors is altered in the HP-DG and OFC of alcoholics (Jin et al., 2011. Whether the changes in one neurotransmitter system drives changes in the other or if they change independently is currently not known. The results demonstrate that excessive long-term alcohol consumption is associated with altered expression of genes encoding glutamate receptors in a brain region-specific manner. It is an intriguing possibility that genetic predisposition to alcoholism may contribute to these gene expression changes.

  15. Relative neurotoxicity of ivermectin and moxidectin in Mdr1ab (-/-) mice and effects on mammalian GABA(A) channel activity.

    Science.gov (United States)

    Ménez, Cécile; Sutra, Jean-François; Prichard, Roger; Lespine, Anne

    2012-01-01

    The anthelmintics ivermectin (IVM) and moxidectin (MOX) display differences in toxicity in several host species. Entrance into the brain is restricted by the P-glycoprotein (P-gp) efflux transporter, while toxicity is mediated through the brain GABA(A) receptors. This study compared the toxicity of IVM and MOX in vivo and their interaction with GABA(A) receptors in vitro. Drug toxicity was assessed in Mdr1ab(-/-) mice P-gp-deficient after subcutaneous administration of increasing doses (0.11-2.0 and 0.23-12.9 µmol/kg for IVM and MOX in P-gp-deficient mice and half lethal doses (LD(50)) in wild-type mice). Survival was evaluated over 14-days. In Mdr1ab(-/-) mice, LD(50) was 0.46 and 2.3 µmol/kg for IVM and MOX, respectively, demonstrating that MOX was less toxic than IVM. In P-gp-deficient mice, MOX had a lower brain-to-plasma concentration ratio and entered into the brain more slowly than IVM. The brain sublethal drug concentrations determined after administration of doses close to LD(50) were, in Mdr1ab(-/-) and wild-type mice, respectively, 270 and 210 pmol/g for IVM and 830 and 740-1380 pmol/g for MOX, indicating that higher brain concentrations are required for MOX toxicity than IVM. In rat α1β2γ2 GABA channels expressed in Xenopus oocytes, IVM and MOX were both allosteric activators of the GABA-induced response. The Hill coefficient was 1.52±0.45 for IVM and 0.34±0.56 for MOX (pMOX relative to GABA alone was 413.7±66.1 and 257.4±40.6%, respectively (pMOX in the brain and in the interaction of IVM and MOX with GABA(A) receptors account for differences in neurotoxicity seen in intact and Mdr1-deficient animals. These differences in neurotoxicity of IVM and MOX are important in considering their use in humans.

  16. Estrogen receptors in human vaginal tissue

    NARCIS (Netherlands)

    Wiegerinck, M.A.H.M.; Poortman, J.; Agema, A.R.; Thijssen, J.H.H.

    1980-01-01

    The presence of specific estrogen receptors could be demonstrated in vaginal tissue, obtained during operation from 38 women, age 27–75 yr. In 23 premenopausal women the receptor concentration in the vaginal tissue varied between 12 and 91 fmol/mg protein, no significant difference in the receptor

  17. Differences in the interaction of acetylcholine receptor antibodies with receptor from normal, denervated and myasthenic human muscle.

    OpenAIRE

    Lefvert, A. K.

    1982-01-01

    The interaction of acetylcholine receptor antibodies with different kinds of human skeletal muscle receptor was investigated. The reaction of most receptor antibodies was strongest with receptor from a patient with myasthenia gravis and with receptor from denervated muscle. Results obtained with these receptors were well correlated. The binding of most receptor antibodies to receptor from functionally normal muscle was much weaker and also qualitatively different. In a few patients with moder...

  18. Discoidin Domain Receptors Role in Human Diseases

    Directory of Open Access Journals (Sweden)

    Iker BADIOLA

    2011-11-01

    Full Text Available Discoidin Domain Receptor 1 and Discodin Domain Receptor 2 are the two only members of the DDR family. The DDR family is a Tyrosine Kinase Receptor (TKR family with some peculiarities compared with other Tyrosine Kinase Receptors such as their natural ligand; which in this case is the fibrillar collagen; or the slow phosphorylation pattern. These peculiarities confer a special role to the receptors present in many diseases development processes as cancer, cirrhosis or lung fibrosis. In this review it is described the overview of the DDRs structure and their role in the different disease development and the possibility to consider them as therapeutic targets.

  19. Notch receptors in human choroid plexus tumors.

    Science.gov (United States)

    Beschorner, R; Waidelich, J; Trautmann, K; Psaras, T; Schittenhelm, J

    2013-08-01

    Notch signaling plays a role in development and formation of the normal choroid plexus (nCP), and in formation of various tumors in humans. Activation of Notch3 has been reported to promote tumor growth in invasive gliomas and to initiate formation of choroid plexus tumors (CPT) in mice. We investigated the expression of all currently known Notch receptors (Notch 1-4) in 55 samples of nCP and 88 CPT, including 61 choroid plexus papillomas (CPP), 22 atypical CPP and 5 choroid plexus carcinomas by immunohistochemistry. Notch expression was semiquantitatively evaluated separately for membranous/cytoplasmic and for nuclear staining. In addition, we examined Her2 expression (EGFR2, Her2/neu, ErbB2, CD340) because of its functional link to Notch signaling. All samples were negative for Notch3. Membranous/cytoplasmic expression of Notch1 (pnCP compared to CPT. Nuclear expression of Notch1, -2 and -4 was significantly higher in CPT compared to nCP (pnCP to a predominant nuclear expression in CPT. Her2 was weakly expressed in 42/84 CPT but only in 2/53 nCP (p=0.0001) and positively correlated with nuclear expression of Notch1, -2 and 4 in CPT. In summary, a shift between membranous/cytoplasmic (non-canonical signaling pathway) and nuclear expression (canonical signaling pathway) of Notch1, -2 and -4 and upregulation of Her2 indicate neoplastic transformation in human CP and may reveal new therapeutic approaches.

  20. Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts

    OpenAIRE

    1984-01-01

    The metabolism of the receptor for epidermal growth factor (EGF) has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. In human fibroblasts the rate of EGF receptor degradation (t1/2 = 10.1 h) was faster than the rate of degradation of total cell protein. When EGF was added to th...

  1. Expression of histamine receptors in the human endolymphatic sac

    DEFF Research Database (Denmark)

    Møller, M Nue; Kirkeby, S; Vikeså, J.

    2016-01-01

    in 2012. This leaves betahistine (Betaserc) as the only drug for potential prevention of the incapacitating attacks of dizziness, tinnitus and hearing loss. However, the histamine receptors targeted by betahistine have never been demonstrated in the human ES. Accordingly, this study aims to investigate...... the expression of histamine receptors of the human ES epithelium and sub-epithelial stroma. Following sampling of human endolymphatic sac tissue during translabyrinthine surgery, the expression of histamine receptor genes was determined by cDNA microarray analysis. Results were subsequently verified by immuno......-histochemistry. The combined results of microarrays and immuno-histochemistry showed expression of the histamine receptor HRH1 in the epithelial lining of the ES, whereas HRH3 was expressed exclusively in the sub-epithelial capillary network. Receptors HRH2 and -4 were not expressed. The present data provide the first direct...

  2. Expression of haemopexin receptors by cultured human cytotrophoblast

    NARCIS (Netherlands)

    H.P. van Dijk (Hans); M.J. Kroos; J.S. Starreveld; H.G. van Eijk (Henk); S.P. Tang; D.X. Song

    1995-01-01

    textabstractThe expression of cell-surface haemopexin (Hx) receptors on human cytotrophoblasts was assessed by using four different Hx species purified from plasma: human Hx isolated by wheatgerm-affinity chromatography, human Hx isolated by haem-agarose-affinity

  3. The GABAA Antagonist DPP-4-PIOL Selectively Antagonises Tonic over Phasic GABAergic Currents in Dentate Gyrus Granule Cells

    DEFF Research Database (Denmark)

    Boddum, Kim; Frølund, Bente; Kristiansen, Uffe

    2014-01-01

    that phasic and tonic GABAA receptor currents can be selectively inhibited by the antagonists SR 95531 and the 4-PIOL derivative, 4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide (DPP-4-PIOL), respectively. In dentate gyrus granule cells, SR 95531 was found approximately 4 times as potent...

  4. PARADOXICAL EFFECTS OF GABA-A MODULATORS MAY EXPLAIN SEX STEROID INDUCED NEGATIVE MOOD SYMPTOMS IN SOME PERSONS

    NARCIS (Netherlands)

    T. Backstrom; D. Haage; M. Lofgren; I.M. Johansson; J. Stromberg; S. Nyberg; L. Andreen; L. Ossewaarde; G.A. van Wingen; S. Turkmen; S.K. Bengtsson

    2011-01-01

    Some women have negative mood symptoms, caused by progestagens in hormonal contraceptives or sequential hormone therapy or by progesterone in the luteal phase of the menstrual cycle, which may be attributed to metabolites acting on the GABA-A receptor. The GABA system is the major inhibitory system

  5. Pattern of hormone receptors and human epidermal growth factor ...

    African Journals Online (AJOL)

    2015-02-05

    Feb 5, 2015 ... Key words: Breast cancer, human epidermal growth factor receptor 2/neu, immunohistochemistry, ... therapy.[6‑8] Of all these prognostic and predictive factors, ... one of the biggest private medical laboratories in Nigeria.

  6. GABA increases electrical excitability in a subset of human unmyelinated peripheral axons.

    Directory of Open Access Journals (Sweden)

    Richard W Carr

    Full Text Available BACKGROUND: A proportion of small diameter primary sensory neurones innervating human skin are chemosensitive. They respond in a receptor dependent manner to chemical mediators of inflammation as well as naturally occurring algogens, thermogens and pruritogens. The neurotransmitter GABA is interesting in this respect because in animal models of neuropathic pain GABA pre-synaptically regulates nociceptive input to the spinal cord. However, the effect of GABA on human peripheral unmyelinated axons has not been established. METHODOLOGY/PRINCIPAL FINDINGS: Electrical stimulation was used to assess the effect of GABA on the electrical excitability of unmyelinated axons in isolated fascicles of human sural nerve. GABA (0.1-100 microM increased electrical excitability in a subset (ca. 40% of C-fibres in human sural nerve fascicles suggesting that axonal GABA sensitivity is selectively restricted to a sub-population of human unmyelinated axons. The effects of GABA were mediated by GABA(A receptors, being mimicked by bath application of the GABA(A agonist muscimol (0.1-30 microM while the GABA(B agonist baclofen (10-30 microM was without effect. Increases in excitability produced by GABA (10-30 microM were blocked by the GABA(A antagonists gabazine (10-20 microM, bicuculline (10-20 microM and picrotoxin (10-20 microM. CONCLUSIONS/SIGNIFICANCE: Functional GABA(A receptors are present on a subset of unmyelinated primary afferents in humans and their activation depolarizes these axons, an effect likely due to an elevated intra-axonal chloride concentration. GABA(A receptor modulation may therefore regulate segmental and peripheral components of nociception.

  7. Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues

    Energy Technology Data Exchange (ETDEWEB)

    Waser, Beatrice; Reubi, Jean Claude [University of Berne, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, PO Box 62, Berne (Switzerland)

    2014-06-15

    Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the {sup 125}iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer {sup 125}I-GLP-1(7-36)amide. Receptor autoradiography studies with {sup 125}I-GLP-1(7-36)amide agonist or {sup 125}I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. The antagonist {sup 125}I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer {sup 125}I-GLP-1(7-36)amide. For comparison, {sup 125}I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. The GLP-1 receptor antagonist exendin(9-39) labelled with {sup 125}I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients. (orig.)

  8. Expression of the endocannabinoid receptors in human fascial tissue

    Directory of Open Access Journals (Sweden)

    C. Fede

    2016-06-01

    Full Text Available Cannabinoid receptors have been localized in the central and peripheral nervous system as well as on cells of the immune system, but recent studies on animal tissue gave evidence for the presence of cannabinoid receptors in different types of tissues. Their presence was supposed also in myofascial tissue, suggesting that the endocannabinoid system may help resolve myofascial trigger points and relieve symptoms of fibromyalgia. However, until now the expression of CB1 (cannabinoid receptor 1 and CB2 (cannabinoid receptor 2 in fasciae has not yet been established. Small samples of fascia were collected from volunteers patients during orthopedic surgery. For each sample were done a cell isolation, immunohistochemical investigation (CB1 and CB2 antibodies and real time RT-PCR to detect the expression of CB1 and CB2. Both cannabinoid receptors are expressed in human fascia and in human fascial fibroblasts culture cells, although to a lesser extent than the control gene. We can assume that the expression of mRNA and protein of CB1 and CB2 receptors in fascial tissue are concentrated into the fibroblasts. This is the first demonstration that the fibroblasts of the muscular fasciae express CB1 and CB2. The presence of these receptors could help to provide a description of cannabinoid receptors distribution and to better explain the role of fasciae as pain generator and the efficacy of some fascial treatments. Indeed the endocannabinoid receptors of fascial fibroblasts can contribute to modulate the fascial fibrosis and inflammation.

  9. Characterization of muscarinic receptor subtypes in human tissues

    Energy Technology Data Exchange (ETDEWEB)

    Giraldo, E.; Martos, F.; Gomez, A.; Garcia, A.; Vigano, M.A.; Ladinsky, H.; Sanchez de La Cuesta, F.

    1988-01-01

    The affinities of selective, pirenzepine and AF-DX 116, and classical, N-methylscopolamine and atropine, muscarinic cholinergic receptor antagonists were investigated in displacement binding experiments with (/sup 3/H)Pirenzepine and (/sup 3/H)N-methylscopolamine in membranes from human autoptic tissues (forebrain, cerebellum, atria, ventricle and submaxillary salivary glands). Affinity estimates of N-methylscopolamine and atropine indicated a non-selective profile. Pirenzepine showed differentiation between the M/sub 1/ neuronal receptor of the forebrain and the receptors in other tissues while AF-DX 116 clearly discriminated between muscarinic receptors of heart and glands. The results in human tissues confirm the previously described selectivity profiles of pirenzepine and AF-DX 116 in rat tissues. These findings thus reveal the presence also in man of three distinct muscarinic receptor subtypes: the neuronal M/sub 1/, the cardiac M/sub 2/ and the glandular M/sub 3/.

  10. Pentobarbital enhances GABAergic neurotransmission to cardiac parasympathetic neurons, which is prevented by expression of GABA(A) epsilon subunit.

    Science.gov (United States)

    Irnaten, Mustapha; Walwyn, Wendy M; Wang, Jijiang; Venkatesan, Priya; Evans, Cory; Chang, Kyoung S K; Andresen, Michael C; Hales, Tim G; Mendelowitz, David

    2002-09-01

    Pentobarbital decreases the gain of the baroreceptor reflex on the order of 50%, and this blunting is caused nearly entirely by decreasing cardioinhibitory parasympathetic activity. The most likely site of action of pentobarbital is the gamma-aminobutyric acid type A (GABA(A)) receptor. The authors tested whether pentobarbital augments the inhibitory GABAergic neurotransmission to cardiac parasympathetic neurons, and whether expression of the GABA(A) epsilon subunit prevents this facilitation. The authors used a novel approach to study the effect of pentobarbital on identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with a fluorescent tracer and were visually identified for patch clamp recording. The effects of pentobarbital on spontaneous GABAergic synaptic events were tested. An adenovirus was used to express the epsilon subunit of the GABA(A) receptor in cardiac parasympathetic neurons to examine whether this transfection alters pentobarbital-mediated changes in GABAergic neurotransmission. Pentobarbital increased the duration but not the frequency or amplitude of spontaneous GABAergic currents in cardiac parasympathetic neurons. Transfection of cardiac parasympathetic neurons with the epsilon subunit of the GABA(A) receptor prevented the pentobarbital-evoked facilitation of GABAergic currents. Pentobarbital, at clinically relevant concentrations, prolongs the duration of spontaneous inhibitory postsynaptic currents that impinge on cardiac parasympathetic neurons. This action would augment the inhibition of cardiac parasympathetic neurons, reduce parasympathetic cardioinhibitory activity, and increase heart rate. Expression of the GABA(A) receptor epsilon subunit in cardiac parasympathetic neurons renders the GABA receptors insensitive to pentobarbital.

  11. Evidence for Alpha Receptors in the Human Ureter

    Science.gov (United States)

    Madeb, Ralph; Knopf, Joy; Golijanin, Dragan; Bourne, Patricia; Erturk, Erdal

    2007-04-01

    An immunohistochemical and western blot expression analysis of human ureters was performed in order to characterize the alpha-1-adrenergic receptor distribution along the length of the human ureteral wall. Mapping the distribution will assist in understanding the potential role alpha -1-adrenergic receptors and their subtype density might have in the pathophysiology of ureteral colic and stone passage. Patients diagnosed with renal cancer or bladder cancer undergoing nephrectomy, nephroureterectomy, or cystectomy had ureteral specimens taken from the proximal, mid, distal and tunneled ureter. Tissues were processed for fresh frozen examination and fixed in formalin. None of the ureteral specimens were involved with cancer. Serial histologic sections and immunohistochemical studies were performed using antibodies specific for alpha-1-adrenergic receptor subtypes (alpha 1a, alpha 1b, alpha 1d). The sections were examined under a light microscope and scored as positive or negative. In order to validate and quantify the alpha receptor subtypes along the human ureter. Western blotting techniques were applied. Human ureter stained positively for alpha -1-adrenergic receptors. Immunostaining appeared red, with intense reaction in the smooth muscle of the ureter and endothelium of the neighboring blood vessels. There was differential expression between all the receptors with the highest staining for alpha-1D subtype. The highest protein expression for all three subtypes was in the renal pelvis and decreased with advancement along the ureter to the distal ureter. At the distal ureter, there was marked increase in expression as one progressed towards the ureteral orifice. The same pattern of protein expression was exhibited for all three alpha -1-adrenergic receptor subtypes. We provide preliminary evidence for the ability to detect and quantify the alpha-1-receptor subtypes along the human ureter which to the best of our knowledge has never been done with

  12. G protein-coupled receptor mutations and human genetic disease.

    Science.gov (United States)

    Thompson, Miles D; Hendy, Geoffrey N; Percy, Maire E; Bichet, Daniel G; Cole, David E C

    2014-01-01

    Genetic variations in G protein-coupled receptor genes (GPCRs) disrupt GPCR function in a wide variety of human genetic diseases. In vitro strategies and animal models have been used to identify the molecular pathologies underlying naturally occurring GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified that result in pathology. These receptor variants may alter ligand binding, G protein coupling, receptor desensitization and receptor recycling. Receptor systems discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, gonadotropin-releasing hormone (GNRHR), adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma (GPRA or neuropeptide S receptor 1 (NPSR1)). The role of activating and inactivating calcium-sensing receptor (CaSR) mutations is discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalemia (ADH). The CASR mutations have been associated with epilepsy. Diseases caused by the genetic disruption of GPCR functions are discussed in the context of their potential to be selectively targeted by drugs that rescue altered receptors. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: calcimimetics and calcilytics, therapeutics targeting melanocortin receptors in obesity, interventions that alter GNRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor congenital bleeding phenotype. De-orphanization projects have identified novel disease-associated receptors, such as NPSR1 and GPR35. The identification of variants in these receptors provides genetic reagents useful in drug screens. Discussion of the variety of GPCRs that are disrupted in monogenic Mendelian disorders provides the basis for examining the significance of common

  13. High expression of NPY receptors in the human testis.

    Science.gov (United States)

    Körner, Meike; Waser, Beatriche; Thalmann, George N; Reubii, Jean Claude

    2011-04-30

    NPY receptors represent novel molecular therapeutic targets in cancer and obesity. However, the extent of NPY receptor expression in normal human tissues is poorly investigated. Based on the role of NPY in reproductive functions, the NPY receptor expression was studied in 25 normal human testes and, additionally, 24 testicular tumors using NPY receptor autoradiography. In the normal testis, Leydig cells strongly expressed NPY receptor subtype Y2, and small arterial blood vessels Y1. Y2 receptors were found to be functional with agonist-stimulated [(35)S]GTPγS binding autoradiography. Full functional integrity of the NPY system was further suggested by the immunohistochemical detection of NPY peptide in nerve fibers directly adjacent to Leydig cells and arteries. Germ cell tumors expressed Y1 and Y2 on tumor cells in 33% and Y1 on intratumoral blood vessels in 50%. Based on its strong NPY receptor expression in Leydig cells and blood vessels, the normal human testis represents a potentially important physiological and pharmalogical NPY target.

  14. Distribution of cellular HSV-1 receptor expression in human brain.

    Science.gov (United States)

    Lathe, Richard; Haas, Juergen G

    2016-12-15

    Herpes simplex virus type 1 (HSV-1) is a neurotropic virus linked to a range of acute and chronic neurological disorders affecting distinct regions of the brain. Unusually, HSV-1 entry into cells requires the interaction of viral proteins glycoprotein D (gD) and glycoprotein B (gB) with distinct cellular receptor proteins. Several different gD and gB receptors have been identified, including TNFRSF14/HVEM and PVRL1/nectin 1 as gD receptors and PILRA, MAG, and MYH9 as gB receptors. We investigated the expression of these receptor molecules in different areas of the adult and developing human brain using online transcriptome databases. Whereas all HSV-1 receptors showed distinct expression patterns in different brain areas, the Allan Brain Atlas (ABA) reported increased expression of both gD and gB receptors in the hippocampus. Specifically, for PVRL1, TNFRFS14, and MYH9, the differential z scores for hippocampal expression, a measure of relative levels of increased expression, rose to 2.9, 2.9, and 2.5, respectively, comparable to the z score for the archetypical hippocampus-enriched mineralocorticoid receptor (NR3C2, z = 3.1). These data were confirmed at the Human Brain Transcriptome (HBT) database, but HBT data indicate that MAG expression is also enriched in hippocampus. The HBT database allowed the developmental pattern of expression to be investigated; we report that all HSV1 receptors markedly increase in expression levels between gestation and the postnatal/adult periods. These results suggest that differential receptor expression levels of several HSV-1 gD and gB receptors in the adult hippocampus are likely to underlie the susceptibility of this brain region to HSV-1 infection.

  15. Identification of agonists for a group of human odorant receptors

    Directory of Open Access Journals (Sweden)

    Daniela eGonzalez-Kristeller

    2015-03-01

    Full Text Available Olfaction plays a critical role in several aspects of the human life. Odorants are detected by hundreds of odorant receptors (ORs which belong to the superfamily of G protein-coupled receptors. These receptors are expressed in the olfactory sensory neurons of the nose. The information provided by the activation of different combinations of ORs in the nose is transmitted to the brain, leading to odorant perception and emotional and behavioral responses. There are ~400 intact human ORs, and to date only a small percentage of these receptors (~10% have known agonists. The determination of the specificity of the human ORs will contribute to a better understanding of how odorants are discriminated by the olfactory system. In this work, we aimed to identify human specific ORs, that is, ORs that are present in humans but absent from other species, and their corresponding agonists. To do this, we first selected 22 OR gene sequences from the human genome with no counterparts in the mouse, rat or dog genomes. Then we used a heterologous expression system to screen a subset of these human ORs against a panel of odorants of biological relevance, including foodborne aroma volatiles. We found that different types of odorants are able to activate some of these previously uncharacterized human ORs.

  16. Fluorescent ligand for human progesterone receptor imaging in live cells.

    Science.gov (United States)

    Weinstain, Roy; Kanter, Joan; Friedman, Beth; Ellies, Lesley G; Baker, Michael E; Tsien, Roger Y

    2013-05-15

    We employed molecular modeling to design and then synthesize fluorescent ligands for the human progesterone receptor. Boron dipyrromethene (BODIPY) or tetramethylrhodamine were conjugated to the progesterone receptor antagonist RU486 (Mifepristone) through an extended hydrophilic linker. The fluorescent ligands demonstrated comparable bioactivity to the parent antagonist in live cells and triggered nuclear translocation of the receptor in a specific manner. The BODIPY labeled ligand was applied to investigate the dependency of progesterone receptor nuclear translocation on partner proteins and to show that functional heat shock protein 90 but not immunophilin FKBP52 activity is essential. A tissue distribution study indicated that the fluorescent ligand preferentially accumulates in tissues that express high levels of the receptor in vivo. The design and properties of the BODIPY-labeled RU486 make it a potential candidate for in vivo imaging of PR by positron emission tomography through incorporation of (18)F into the BODIPY core.

  17. Glycomimetic ligands for the human asialoglycoprotein receptor.

    Science.gov (United States)

    Mamidyala, Sreeman K; Dutta, Sanjay; Chrunyk, Boris A; Préville, Cathy; Wang, Hong; Withka, Jane M; McColl, Alexander; Subashi, Timothy A; Hawrylik, Steven J; Griffor, Matthew C; Kim, Sung; Pfefferkorn, Jeffrey A; Price, David A; Menhaji-Klotz, Elnaz; Mascitti, Vincent; Finn, M G

    2012-02-01

    The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.

  18. Structure of a human rhinovirus complexed with its