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Sample records for gaba uptake inhibitors

  1. Design, synthesis and SAR studies of GABA uptake inhibitors derived from 2-substituted pyrrolidine-2-yl-acetic acids.

    PubMed

    Steffan, Tobias; Renukappa-Gutke, Thejavathi; Höfner, Georg; Wanner, Klaus T

    2015-03-15

    In this paper, we disclose the design and synthesis of a series of 2-substituted pyrrolidine-2-yl-acetic acid as core structures and the N-arylalkyl derivatives thereof as potential GABA transport inhibitors. The 2-position in the side chain of pyrrolidine-2-yl-acetic acid derivatives was substituted with alkyl, hydroxy and amino groups to modulate the activity and selectivity to mGAT1 and mGAT4 proteins. SAR studies of the compounds performed for the four mouse GABA transporter proteins (mGAT1-mGAT4) implied significant potencies and subtype selectivities for 2-hydroxy-2-pyrrolidine-2-yl-acetic acid derivatives. The racemate rac-(u)-13c exhibited the highest potency (pIC50 5.67) at and selectivity for mGAT1 in GABA uptake assays. In fact, the potency of rac-(u)-13c at hGAT-1 (pIC50 6.14) was even higher than its potency at mGAT1. These uptake results for rac-(u)-13c are in line with the binding affinities to the aforesaid proteins mGAT1 (pKi 6.99) and hGAT-1 (pKi 7.18) determined by MS Binding Assay based on NO711 as marker quantified by LC-ESI-MS-MS analysis. Interestingly, the 2-hydroxy-2-pyrrolidine-2-yl-acetic acid rac-(u)-13d containing 2-{[tris(4-methoxyphenyl)]methoxy} ethyl group at the nitrogen atom of the pyrrolidine ring showed high potency at mGAT4 and a comparatively better selectivity for this protein (>15 against mGAT3) than the well known mGAT4 uptake inhibitor (S)-SNAP-5114.

  2. Parawixin2, a novel non-selective GABA uptake inhibitor from Parawixia bistriata spider venom, inhibits pentylenetetrazole-induced chemical kindling in rats.

    PubMed

    Gelfuso, Erica A; Liberato, José L; Cunha, Alexandra O S; Mortari, Márcia R; Beleboni, Renê O; Lopes, Norberto P; Dos Santos, Wagner F

    2013-05-24

    The aims of the present work were to investigate the effects of the repeated administration of Parawixin2 (2-amino-5-ureidopentanamide; formerly FrPbAII), a novel GABA and glycine uptake inhibitor, in rats submitted to PTZ-induced kindling. Wistar rats were randomly divided in groups (n=6-8) for different treatments. Systemic injections of PTZ were administered every 48 h in the dose of 33 mg/kg; i.p., that is sufficient to induce fully kindled seizures in saline i.c.v. treated rats in a short period of time (28 days). Treatments in two types of positive controls (diazepam - DZP and nipecotic acid - NA groups) consisted in daily systemic injections of DZP (2mg/kg; i.p.) or i.c.v. injections of NA (12 μg/μL), while in experimental groups in daily i.c.v. injections of different doses of Parawixin2 (0.15; 0.075; 0.015 μg/μL). Seizures were analyzed using the Lamberty & Klitgaard score and kindling was considered as established after at least three consecutive seizures of score 4 or 5. Cumulative seizure scores for each group were analyzed using repeated measures of ANOVA followed by Tukey test. PTZ induced 4 and 5-score seizures after 12 injections in saline treated rats, whereas daily injection of Parawixin2 inhibited the onset of seizures in a dose dependent manner. Also, the challenging administration of PTZ did not raise seizure score in animals treated with the highest dose of Parawixin2 or those treated with DZP or NA. These findings together with previous data from our laboratory show that Parawixin2 could be a useful probe to design new antiepileptic drugs.

  3. GAT-1 mediated GABA uptake in rat oligodendrocytes.

    PubMed

    Fattorini, Giorgia; Melone, Marcello; Sánchez-Gómez, María Victoria; Arellano, Rogelio O; Bassi, Silvia; Matute, Carlos; Conti, Fiorenzo

    2017-03-01

    Stimulated by the results of a recent paper on the effects of tiagabine, a selective inhibitor of the main GABA transporter GAT-1, on oligodendrogenesis, we verified the possibility that GAT-1 may be expressed in oligodendrocytes using immunocytochemical methods and functional assays. Light microscopic analysis of the subcortical white matter of all animals revealed the presence of numerous GAT-1+ cells of different size (from 3 to 29 µm) and morphology. An electron microscope analysis revealed that, besides fibrous astrocytes and interstitial neurons, GAT-1 immunoreactivity was present in immature and mature oligodendrocytes. Co-localization studies between GAT-1 and markers specific for oligodendrocytes (NG2 and RIP) showed that about 12% of GAT-1 positive cells in the white matter were immature oligodendrocytes, while about 15% were mature oligodendrocytes. In vitro functional assays showed that oligodendrocytes exhibit tiagabine-sensitive Na(+) -dependent GABA uptake. Although relationships between GABA and oligodendrocytes have been known for many years, this is the first demonstration that GAT-1 is expressed in oligodendrocytes. The present results on the one hand definitely closes the era of "neuronal" and "glial" GABA transporters, on the other they suggest that oligodendrocytes may contribute to pathophysiology of the several diseases in which GAT-1 have been implicated to date. GLIA 2017;65:514-522.

  4. Effect of centrally acting drugs on the uptake of γ-aminobutyric acid (GABA) by slices of rat cerebral cortex

    PubMed Central

    Harris, M.; Hopkin, Judy M.; Neal, M. J.

    1973-01-01

    1. The effects of centrally acting drugs on the uptake of 3H-γ-aminobutyric acid (GABA) by slices of rat cerebral cortex have been studied. 2. Many centrally acting drugs at concentrations of 0·1-1·0 mM significantly inhibited the uptake of 3H-GABA by cortical slices, but the only classes of drugs in which all members consistently produced inhibition of uptake were the phenothiazines, tricyclic antidepressants, and butyrophenones. 3. The receptor blocking drugs; phentolamine, propranolol, thymoxamine, mepyramine, and diphenhydramine at concentrations of 0·5-1 mM also significantly reduced the uptake of 3H-GABA. However, atropine, hexamethonium and (+)-tubocurarine had little effect on the uptake of 3H-GABA by cortical slices. 4. Centrally acting drugs, which did not significantly inhibit 3H-GABA uptake, included barbiturates, local anaesthetics, hallucinogens, monoamine oxidase inhibitors, anticonvulsants, and convulsants (except picrotoxin). 5. Chlorpromazine, prochlorperazine, L-2,4,diaminobutyric acid, desmethylimipramine, and iprindole inhibited the uptake of 3H-GABA by 50% (IC50) at concentrations of 30-100 μM. The most potent inhibitor of 3H-GABA uptake was p-chloromercuriphenylsulphonate (IC50 = 18 μM). 6. With the exception of L-2,4,diaminobutyric acid, an outstanding characteristic of these drugs was their complete lack of specificity. Thus at the IC50 for GABA, p-chloromercuriphenylsulphonate, chlorpromazine, prochlorperazine, iprindole, desmethylimipramine, apomorphine and diphenylhydramine also inhibited the uptake of radioactive glycine, alanine, noradrenaline, and 5-hydroxytryptamine. The uptake of the latter two compounds was often inhibited to a greater extent than GABA, glycine and alanine. 7. Kinetic analysis indicated that the inhibition of 3H-GABA by p-chloromercuriphenylsulphonate, chlorpromazine, and desmethylimipramine was noncompetitive. L-2,4,Diaminobutyric acid reduced the uptake of 3H-GABA by a `mixed' type of inhibition. 8. The

  5. Distribution of 3H-GABA uptake sites in the nematode Ascaris

    SciTech Connect

    Guastella, J.; Stretton, A.O. )

    1991-05-22

    The distribution of uptake sites for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the nematode Ascaris suum was examined by autoradiography of 3H-GABA uptake. Single neural processes in both the ventral and dorsal nerve cords were labeled with 3H-GABA. Serial section analysis identified the cells of origin of these processes as the RMEV-like and RMED-like neurons. These cells belong to a set of four neurons in the nerve ring, all of which are labeled by 3H-GABA. 3H-GABA labeling of at least two other sets of cephalic neurons was seen. One of these pairs consists of medium-sized lateral ganglia neurons, located at the level of the amphid commissure bundle. A second pair is located in the lateral ganglia at the level of the deirid commissure bundle. The position and size of these lateral ganglia cells suggest that they are the GABA-immunoreactive lateral ganglia cells frequently seen in whole-mount immunocytochemical preparations. Four neuronal cell bodies located in the retrovesicular ganglion were also labeled with 3H-GABA. These cells, which are probably cholinergic excitatory motor neurons, do not contain detectable GABA-like immunoreactivity. Heavy labeling of muscle cells was also observed. The ventral and dorsal nerve cord inhibitory motor neurons, which are known to contain GABA-like immunoreactivity, were not labeled above background with 3H-GABA. Together with the experiments reported previously, these results define three classes of GABA-associated neurons in Ascaris: (1) neurons that contain endogenous GABA and possess a GABA uptake system; (2) neurons that contain endogenous GABA, but that either lack a GABA uptake system or possess a GABA uptake system of low activity; (3) neurons that possess a GABA uptake system, but that lack endogenous GABA.

  6. Characterization of GABA/sub A/ receptor-mediated /sup 36/chloride uptake in rat brain synaptoneurosomes

    SciTech Connect

    Luu, M.D.; Morrow, A.L.; Paul, S.M.; Schwartz, R.D.

    1987-09-07

    ..gamma..-Aminobutyric acid (GABA) receptor-mediated /sup 36/chloride (/sup 36/Cl/sup -/) uptake was measured in synaptoneurosomes from rat brain. GABA and GABA agonists stimulated /sup 36/Cl/sup -/ uptake in a concentration-dependent manner with the following order of potency: Muscimol>GABA>piperidine-4-sulfonic acid (P4S)>4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP)=3-aminopropanesulfonic acid (3APS)>>taurine. Both P4S and 3APS behaved as partial agonists, while the GABA/sub B/ agonist, baclofen, was ineffective. The response to muscimol was inhibited by bicuculline and picrotoxin in a mixed competitive/non-competitive manner. Other inhibitors of GABA receptor-opened channels or non-neuronal anion channels such as penicillin, picrate, furosemide and disulfonic acid stilbenes also inhibited the response to muscimol. A regional variation in muscimol-stimulated /sup 36/Cl/sup -/ uptake was observed; the largest responses were observed in the cerebral cortex, cerebellum and hippocampus, moderate responses were obtained in the striatum and hypothalamus and the smallest response was observed in the pons-medulla. GABA receptor-mediated /sup 36/Cl/sup -/ uptake was also dependent on the anion present in the media. The muscinol response varied in media containing the following anions: Br/sup -/>Cl/sup -/greater than or equal toNO/sub 3//sup -/>I/sup -/greater than or equal toSCN/sup -/>>C/sub 3/H/sub 5/OO/sup -/greater than or equal toClO/sub 4//sup -/>F/sup -/, consistent with the relative anion permeability through GABA receptor-gated anion channels and the enhancement of convulsant binding to the GABA receptor-gated Cl/sup -/ channel. 43 references, 4 figures, 3 tables.

  7. [Pharmacological influences on the brain level and transport of GABA. II) Effect of various psychoactive drugs on brain level and uptake of GABA].

    PubMed

    Gabana, M A; Varotto, M; Saladini, M; Zanchin, G; Battistin, L

    1981-04-30

    The effects of some psychoactive drugs on the level and uptake of GABA in the mouse brain was studied using well standardized procedures, mainely the silica-gel cromatography for determining the GABA content and the brain slices for measuring GABA uptake. It was found that levomepromazine, sulpiride, haloperidol and amytryptiline were without effects on the cerebral level of GABA; it was also found that these drugs do not influence the rates of uptake of GABA by mouse brain slices. Such results do indicate that the psychoactive drugs studied are without effects on the level and uptake of GABA in the brain.

  8. 3H-GABA uptake selectively labels identifiable neurons in the leech central nervous system

    SciTech Connect

    Cline, H.T.

    1983-04-10

    Segmental ganglia of the leech ventral nerve cord synthesize the neurotransmitter gamma-aminobutyric acid (GABA) when incubated in the presence of the precursor glutamate, suggesting that there may be GABA-ergic neurons in the leech nerve cord. GABA-accumulating neurons of the two taxonomically distant leech species, Haementeria ghilianii and Hirudo medicinalis, have been labeled by taking advantage of their high-affinity uptake system for the neurotransmitter. Autoradiography of sectioned segmental ganglia previously exposed to 3H-GABA reveals a reproducible pattern of about thirty 3H-GABA-labeled neuronal cell bodies per ganglion. The majority of 3H-GABA-labeled neuronal cell bodies are bilaterally paired, although some apparently unpaired cell bodies also accumulate label. Neuronal processes were reproducibly labeled by GABA uptake and could be traced in the neuropil through commissures and fiber tracts into the segmental nerve roots and interganglionic connectives, respectively.

  9. Hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.

    PubMed

    Yahara, Tohru; Tachikawa, Masanori; Akanuma, Shin-ichi; Hosoya, Ken-ichi

    2010-01-01

    We have reported previously that taurine transporter (TauT) mediates γ-aminobutyric acid (GABA) as a substrate in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). This study investigates how TauT-mediated GABA transport is regulated in TR-iBRB2 cells under hypertonic conditions. [³H]GABA uptake by TR-iBRB2 cells exposed to 12 h- to 24 h-hypertonic culture medium was significantly greater than that of isotonic culture medium. [³H]GABA uptake by TR-iBRB2 cells was Na(+)-, Cl(-)-, and concentration-dependent with a Michaelis-Menten (K(m)) constant of 3.5 mM under isotonic conditions and K(m) of 0.324 and 5.48 mM under hypertonic conditions. Under hypertonic conditions, [³H]GABA uptake by TR-iBRB2 cells was more potently inhibited by substrates of TauT, such as taurine and β-alanine, than those of GABA transporters such as GABA, nipecotic acid, and betaine. These results suggest that an unknown high-affinity GABA transport process and TauT-mediated GABA transport are enhanced under hypertonic conditions. In conclusion, hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.

  10. The activation of cannabinoid receptors in striatonigral GABAergic neurons inhibited GABA uptake.

    PubMed

    Romero, J; de Miguel, R; Ramos, J A; Fernández-Ruiz, J J

    1998-01-01

    Cannabinoid receptors (CNRs) in basal ganglia are located on striatal efferent neurons which are gamma-aminobutiric acid (GABA)-containing neurons. Recently, we have demonstrated that CN-induced motor inhibition is reversed by GABA-B, but not GABA-A, receptor antagonists, presumably indicating that the activation of CNRs in striatal outflow nuclei, mainly in the substantia nigra, should be followed by an increase of GABA concentrations into the synaptic cleft of GABA-B receptor synapses. The present study was designed to examine whether this was originated by increasing GABA synthesis and/or release or by decreasing GABA uptake. We analyzed: (i) GABA synthesis, by measuring the activity of glutamic acid decarboxylase (GAD) and GABA contents in brain regions that contain striatonigral GABAergic neurons, after in vivo administration of CNs and/or the CNR antagonist SR141716; (ii) [3H]GABA release in vitro in the presence or the absence of a synthetic CN agonist, HU-210, by using perifusion of small fragments of substantia nigra; and (iii) [3H]GABA uptake in vitro in the presence or the absence of WIN-55,212-2, by using synaptosomes obtained from either globus pallidus or substantia nigra. Results were as follows. Delta9-tetrahydrocannabinol (delta9-THC) and HU-210, did not alter neither GAD activity nor GABA contents in both the striatum and the ventral midbrain at any of the two times tested, thus suggesting that CNs apparently failed to change GABA synthesis in striatonigral GABAergic neurons. A similar lack of effect of HU-210 on in vitro [3H]GABA release, both basal and K+-evoked, was seen when this CN was added to perifused substantia nigra fragments, also suggesting no changes at the level of GABA release. However, when synaptosome preparations obtained from the substantia nigra were incubated in the presence of WIN-55,212-2, a decrease in [3H]GABA uptake could be measured. This lowering effect was specific of striatonigral GABAergic neurons since it was not

  11. Treatment of Huntington disease with gamma-acetylenic GABA an irreversible inhibitor of GABA-transaminase: increased CSF GABA and homocarnosine without clinical amelioration.

    PubMed

    Tell, G; Böhlen, P; Schechter, P J; Koch-Weser, J; Agid, Y; Bonnet, A M; Coquillat, G; Chazot, G; Fischer, C

    1981-02-01

    gamma-Acetylenic GABA (GAG, RMI 71.645), a potent irreversible inhibitor of gamma-aminobutyric acid transaminase, was given orally in various dosage schedules to 14 patients with Huntington disease. The biochemical effects of the drug on cerebrospinal fluid (CSF) concentrations of gamma-aminobutyric acid (GABA) and the GABA-containing dipeptide, homocarnosine, were measured in 10 of 14 patients. Treatment with GAG increased CSF concentrations of GABA and homocarnosine as compared to pretreatment values, suggesting that the drug increased brain GABA concentration. Despite this neurochemical effect, the clinical state was not improved. Except for single seizure episodes in five patients, GAG therapy was well tolerated. These results do not exclude the possibility that agents that augment CNS GABAergic function may prove useful in therapy of Huntington disease.

  12. Uptake of taurine, GABA, 5-HT, and dopamine by blood platelets in progressive myoclonus epilepsy.

    PubMed

    Airaksinen, E M

    1979-10-01

    The uptakes of four neurotransmitters (taurine, GABA, 5-HT, and dopamine) by blood platelets from patients with degenerative-type progressive myoclonus epilepsy (PME) and from controls were studied using different incubation times and different concentrations. Only the uptakes of taurine differed significantly between patients and controls: patients' uptakes were 70% to 80% of control values at 10, 30, 60, and 120 min of incubation time. Km values were approximately the same, but Vmax values in PME patients were lower, showing quantitative but not qualitative differences in taurine uptake by platelets in PME. These results suggest that a defect or an inhibitory mechanism of some factor needed in the transport or binding of taurine (but not of GABA, 5-HT, and dopamine) is present in PME.

  13. Midbrain dopamine neurons sustain inhibitory transmission using plasma membrane uptake of GABA, not synthesis

    PubMed Central

    Tritsch, Nicolas X; Oh, Won-Jong; Gu, Chenghua; Sabatini, Bernardo L

    2014-01-01

    Synaptic transmission between midbrain dopamine neurons and target neurons in the striatum is essential for the selection and reinforcement of movements. Recent evidence indicates that nigrostriatal dopamine neurons inhibit striatal projection neurons by releasing a neurotransmitter that activates GABAA receptors. Here, we demonstrate that this phenomenon extends to mesolimbic afferents, and confirm that the released neurotransmitter is GABA. However, the GABA synthetic enzymes GAD65 and GAD67 are not detected in midbrain dopamine neurons. Instead, these cells express the membrane GABA transporters mGAT1 (Slc6a1) and mGAT4 (Slc6a11) and inhibition of these transporters prevents GABA co-release. These findings therefore indicate that GABA co-release is a general feature of midbrain dopaminergic neurons that relies on GABA uptake from the extracellular milieu as opposed to de novo synthesis. This atypical mechanism may confer dopaminergic neurons the flexibility to differentially control GABAergic transmission in a target-dependent manner across their extensive axonal arbors. DOI: http://dx.doi.org/10.7554/eLife.01936.001 PMID:24843012

  14. GABA uptake into astrocytes is not associated with significant metabolic cost: implications for brain imaging of inhibitory transmission.

    PubMed

    Chatton, Jean-Yves; Pellerin, Luc; Magistretti, Pierre J

    2003-10-14

    Synaptically released glutamate has been identified as a signal coupling excitatory neuronal activity to increased glucose utilization. The proposed mechanism of this coupling involves glutamate uptake into astrocytes resulting in increased intracellular Na+ (Nai+) and activation of the Na+/K+-ATPase. Increased metabolic demand linked to disruption of Nai+ homeostasis activates glucose uptake and glycolysis in astrocytes. Here, we have examined whether a similar neurometabolic coupling could operate for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), also taken up by Na+-dependent transporters into astrocytes. Thus, we have compared the Nai+ response to GABA and glutamate in mouse astrocytes by microspectrofluorimetry. The Nai+ response to GABA consisted of a rapid rise of 4-6 mM followed by a plateau that did not, however, significantly activate the pump. Indeed, the GABA transporter-evoked Na+ influxes are transient in nature, almost totally shutting off within approximately 30 sec of GABA application. The metabolic consequences of the GABA-induced Nai+ response were evaluated by monitoring cellular ATP changes indirectly in single cells and measuring 2-deoxyglucose uptake in astrocyte populations. Both approaches showed that, whereas glutamate induced a robust metabolic response in astrocytes (decreased ATP levels and glucose uptake stimulation), GABA did not cause any measurable metabolic response, consistent with the Nai+ measurements. Results indicate that GABA does not couple inhibitory neuronal activity with glucose utilization, as does glutamate for excitatory neurotransmission, and suggest that GABA-mediated synaptic transmission does not contribute directly to brain imaging signals based on deoxyglucose.

  15. Receptor regulation of the glutamate, GABA and taurine high-affinity uptake into astrocytes in primary culture.

    PubMed

    Hansson, E; Rönnbäck, L

    1991-05-10

    From experiments using dissociated primary astroglial cultures from newborn rat cerebral cortex, the stimulation of monoamine receptors (alpha, beta and 5HT) was shown to affect the high-affinity uptake kinetics of glutamate, GABA and taurine. In the presence of the alpha 1 agonist phenylephrine, there was an increased uptake (Vmax) of glutamate, while beta adrenoceptor activation slightly inhibited the glutamate uptake and stimulated the GABA and taurine uptakes. 5HT2 receptor stimulation caused a slight inhibition of the taurine uptake. The uptake rate of GABA was not affected by 5HT, alpha 1 or alpha 2 receptor agonists and the glutamate uptake was not affected by 5HT or alpha 2 receptor agonists. Nor was the taurine uptake affected by alpha 1 or alpha 2 receptor agonists. The active uptake of aspartate was unaffected by the presence of any of the monoamine receptor agonists used in this study. When the mechanisms behind these effects were studied, the GABA uptake seemed to be mediated via the G protein-adenylate cyclase complex in the receptor domain. Moreover, the K+ channels seemed to be involved. The taurine uptake, however, did not seem to be regulated by the same mechanism. It seems more probable that there is a direct interaction between the receptor and carrier of taurine at the membrane level. The mechanism underlying the receptor-regulated glutamate uptake is at present unclear, although it does not seem to involve protein kinase C.

  16. GABA (γ-Aminobutyric Acid) Uptake Via the GABA Permease GabP Represses Virulence Gene Expression in Pseudomonas syringae pv. tomato DC3000.

    PubMed

    McCraw, S L; Park, D H; Jones, R; Bentley, M A; Rico, A; Ratcliffe, R G; Kruger, N J; Collmer, A; Preston, G M

    2016-12-01

    The nonprotein amino acid γ-aminobutyric acid (GABA) is the most abundant amino acid in the tomato (Solanum lycopersicum) leaf apoplast and is synthesized by Arabidopsis thaliana in response to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (hereafter called DC3000). High levels of exogenous GABA have previously been shown to repress the expression of the type III secretion system (T3SS) in DC3000, resulting in reduced elicitation of the hypersensitive response (HR) in the nonhost plant tobacco (Nicotiana tabacum). This study demonstrates that the GABA permease GabP provides the primary mechanism for GABA uptake by DC3000 and that the gabP deletion mutant ΔgabP is insensitive to GABA-mediated repression of T3SS expression. ΔgabP displayed an enhanced ability to elicit the HR in young tobacco leaves and in tobacco plants engineered to produce increased levels of GABA, which supports the hypothesis that GABA uptake via GabP acts to regulate T3SS expression in planta. The observation that P. syringae can be rendered insensitive to GABA through loss of gabP but that gabP is retained by this bacterium suggests that GabP is important for DC3000 in a natural setting, either for nutrition or as a mechanism for regulating gene expression. [Formula: see text] Copyright © 2016 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

  17. Inhibition of GABA uptake potentiates the conductance increase produced by GABA-mimetic compounds on single neurones in isolated olfactory cortex slices of the guinea-pig.

    PubMed

    Brown, D A; Scholfield, C N

    1984-09-01

    Membrane potential and input conductance were recorded in single neurones in slices of guinea-pig olfactory cortex in vitro. gamma-Aminobutyric acid (GABA) and GABA-mimetic compounds were applied by bath-perfusion. Potency was measured as the concentration required to double the input conductance. The potency of GABA was increased (i.e. the equi-effective concentrations were reduced) by 15.5 +/- 2.3 times (mean +/- s.e. mean) on reducing external [Na+] from 144 to 20 mmol l-1, by replacement with Mg2+. Corresponding potency changes for other agonists were + 10.8 +/- 2.5 for 3-aminopropanesulphonic acid (3-APS); 3.25 +/- 1.06 for isoguvacine and 2.43 +/- 0.69 for muscimol. Nipecotic acid (0.5 mM) produced the following increases in potency: GABA 2.68 +/- 0.02; 3-aminopropanesulphonic acid, 3.11 +/- 0.07; isoguvacine, 1.92 +/- 0.34; muscimol, 2.24 +/- 0.17. The concentration of GABA in the bathing fluid necessary to double input conductance increased with increasing depth of the recording site from the cut surface. The apparent potency fell 10 times for each 60 micron depth increment up to 150 micron. The recording depth also affected the apparent potency of muscimol and 3-APS but to a lesser extent. Reduction of external [Na+] reduced the depth-dependence of both GABA and 3-APS potency. No clear change in the duration of the recurrent inhibitory postsynaptic conductance could be detected in the presence of 0.5 mmol l-1 nipecotic acid. 6 It is suggested that agonist uptake by a Na+-dependent, nipecotic acid-sensitive mechanism severely attenuates the responses of olfactory neurones to exogenous GABA and to its analogues 3-APS, muscimol and isoguvacine, but has little immediate influence on the duration of the GABA-mediated inhibitory postsynaptic conductance.

  18. (R)-roscovitine, a cyclin-dependent kinase inhibitor, enhances tonic GABA inhibition in rat hippocampus.

    PubMed

    Ivanov, A; Tyzio, R; Zilberter, Y; Ben-Ari, Yehezkel

    2008-10-02

    Pharmacological agents that mediate a persistent GABAergic conductance are of considerable interest for treatment of epilepsy. (R)-roscovitine is a membrane permeable cyclin-dependent kinase inhibitor, designed to block cell division. It is currently undergoing a phase II clinical trial as an anticancer drug. We show that (R)-roscovitine increases a tonic GABA-mediated current in rat hippocampal neurons. This enhanced tonic current appears independent of synaptic GABA release and requires functional transmembrane GABA transport. The effect of (R)-roscovitine is associated with neither modification of GABAA receptors nor protein kinase activity, but is associated with a significant increase in intracellular GABA concentration in hippocampal GABAergic neurons. (R)-roscovitine-induced tonic inhibition significantly suppresses spontaneous spiking activity of hippocampal pyramidal cells. Therefore, (R)-roscovitine is a potent modulator of neuronal activity in rat hippocampus and may provide a tool for preventing paroxysmal activity.

  19. Distribution and ultrastructure of neurons in opossum piriform cortex displaying immunoreactivity to GABA and GAD and high-affinity tritiated GABA uptake

    SciTech Connect

    Haberly, L.B.; Hansen, D.J.; Feig, S.L.; Presto, S.

    1987-12-08

    GABAergic neurons have been identified in the piriform cortex of the opossum at light and electron microscopic levels by immunocytochemical localization of GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase and by autoradiographic visualization of high-affinity /sup 3/H-GABA uptake. Four major neuron populations have been distinguished on the basis of soma size, shape, and segregation at specific depths and locations: large horizontal cells in layer Ia of the anterior piriform cortex, small globular cells with thin dendrites concentrated in layers Ib and II of the posterior piriform cortex, and multipolar and fusiform cells concentrated in the deep part of layer III in anterior and posterior parts of the piriform cortex and the subjacent endopiriform nucleus. All four populations were well visualized with both antisera, but the large layer Ia horizontal cells displayed only very light /sup 3/H-GABA uptake, thus suggesting a lack of local axon collaterals or lack of high-affinity GABA uptake sites. The large, ultrastructurally distinctive somata of layer Ia horizontal cells receive a very small number of symmetrical synapses; the thin, axonlike dendrites of small globular cells are exclusively postsynaptic and receive large numbers of both symmetrical and asymmetrical synapses, in contrast to somata which receive a small number of both types; and the deep multipolar and fusiform cells receive a highly variable number of symmetrical and asymmetrical synapses on somata and proximal dendrites. Labeled puncta of axon terminal dimensions were found in large numbers in the neuropil surrounding pyramidal cell somata in layer II and in the endopiriform nucleus. Moderately large numbers of labeled puncta were found in layer I at the depth of pyramidal cell apical dendrites with greater numbers in layer Ia at the depth of distal apical segments than in layer Ib.

  20. Enhanced astroglial GABA uptake attenuates tonic GABAA inhibition of the presympathetic hypothalamic paraventricular nucleus neurons in heart failure.

    PubMed

    Pandit, Sudip; Jo, Ji Yoon; Lee, Sang Ung; Lee, Young Jae; Lee, So Yeong; Ryu, Pan Dong; Lee, Jung Un; Kim, Hyun-Woo; Jeon, Byeong Hwa; Park, Jin Bong

    2015-08-01

    γ-Aminobutyric acid (GABA) generates persistent tonic inhibitory currents (Itonic) and conventional inhibitory postsynaptic currents in the hypothalamic paraventricular nucleus (PVN) via activation of GABAA receptors (GABAARs). We investigated the pathophysiological significance of astroglial GABA uptake in the regulation of Itonic in the PVN neurons projecting to the rostral ventrolateral medulla (PVN-RVLM). The Itonic of PVN-RVLM neurons were significantly reduced in heart failure (HF) compared with sham-operated (SHAM) rats. Reduced Itonic sensitivity to THIP argued for the decreased function of GABAAR δ subunits in HF, whereas similar Itonic sensitivity to benzodiazepines argued against the difference of γ2 subunit-containing GABAARs in SHAM and HF rats. HF Itonic attenuation was reversed by a nonselective GABA transporter (GAT) blocker (nipecotic acid, NPA) and a GAT-3 selective blocker, but not by a GAT-1 blocker, suggesting that astroglial GABA clearance increased in HF. Similar and minimal Itonic responses to bestrophin-1 blockade in SHAM and HF neurons further argued against a role for astroglial GABA release in HF Itonic attenuation. Finally, the NPA-induced inhibition of spontaneous firing was greater in HF than in SHAM PVN-RVLM neurons, whereas diazepam induced less inhibition of spontaneous firing in HF than in SHAM neurons. Overall, our results showed that combined with reduced GABAARs function, the enhanced astroglial GABA uptake-induced attenuation of Itonic in HF PVN-RVLM neurons explains the deficit in tonic GABAergic inhibition and increased sympathetic outflow from the PVN during heart failure.

  1. Impaired GABA synthesis, uptake and release are associated with depression-like behaviors induced by chronic mild stress

    PubMed Central

    Ma, K; Xu, A; Cui, S; Sun, M-R; Xue, Y-C; Wang, J-H

    2016-01-01

    Major depression is a prevalent emotion disorder. Chronic stressful life in genetically susceptible individuals is presumably a major etiology that leads to neuron and synapse atrophy in the limbic system. Molecular mechanisms underlying the pathological changes remain elusive. Mice were treated by chronic unpredictable mild stress (CUMS) until they demonstrated depression-like behavior. GABA release in the medial prefrontal cortex was evaluated by cell electrophysiology and imaging. Molecular profiles related to GABA synthesis and uptake were investigated by the high-throughput sequencings of microRNAs and mRNAs as well as western blot analysis in this cortical area. In CUMS-induced depression mice, there appear the decreases in the innervation and function of GABAergic axons and in the levels of mRNAs and proteins of glutamate decarboxylase-67, vesicular GABA transporter and GABA transporter-3. miRNA-15b-5p, miRNA-144-3p, miRNA-582-5p and miRNA-879-5p that directly downregulate such mRNAs increase in this cortex. Our results suggest that chronic mild stress impairs GABA release and uptake by upregulating miRNAs and downregulating mRNAs and proteins, which may constitute the subcellular and molecular mechanisms for the lowered GABA tone in major depression. PMID:27701406

  2. GABA-ergic neurons in the leach central nervous system

    SciTech Connect

    Cline, H.T.

    1985-01-01

    GABA is a candidate for an inhibitory neurotransmitter in the leech central nervous system because of the well-documented inhibitory action of GABA in other invertebrates. To demonstrate that GABA meets the criteria used to identify a substance as a neurotransmitter, the author examined GABA metabolism and synaptic interactions of inhibitory motor neurons in two leech species, Hirudo medicinalis and Haementeria ghilianii. Segmental ganglia of the leech ventral nerve cord and identified inhibitors have the capacity to synthesize GABA when incubated in the presence of the precursor glutamate. Application of GABA to cell bodies of excitatory motor neurons or muscle fibers innervated by the inhibitors hyperpolarizes the membrane potential of the target cell and activates a chloride ion conductance channel, similar to the inhibitory membrane response following intracellular stimulation of the inhibitor. Bicuculline methiodide (5 x 10/sup -5/M), GABA receptor antagonist, blocks reversibly the response to applied GABA and the inhibitory synaptic inputs onto the postsynaptic neurons or muscle fibers without interfering with their excitatory inputs. Furthermore, the inhibitors are included among approximately 25 neurons per segmental ganglion that take up GABA by a high affinity uptake system, as revealed by /sup 3/H-GABA-autoradiography. The development of the capacities to synthesize and to take up GABA were examined in leech embryos. The embryos are able to synthesize GABA at early stages of the development of the nervous system, before any neurons have extended neutrites.

  3. Manganese accumulation in membrane fractions of primary astrocytes is associated with decreased γ-aminobutyric acid (GABA) uptake, and is exacerbated by oleic acid and palmitate.

    PubMed

    Fordahl, Steve C; Erikson, Keith M

    2014-05-01

    Manganese (Mn) exposure interferes with GABA uptake; however, the effects of Mn on GABA transport proteins (GATs) have not been identified. We sought to characterize how Mn impairs GAT function in primary rat astrocytes. Astrocytes exposed to Mn (500 μM) had significantly reduced (3)H-GABA uptake despite no change in membrane or cytosolic GAT3 protein levels. Co-treatment with 100 μM oleic or palmitic acids (both known to be elevated in Mn neurotoxicity), exacerbated the Mn-induced decline in (3)H-GABA uptake. Mn accumulation in the membrane fraction of astrocytes was enhanced with fatty acid administration, and was negatively correlated with (3)H-GABA uptake. Furthermore, control cells exposed to Mn only during the experimental uptake had significantly reduced (3)H-GABA uptake, and the addition of GABA (50 μM) blunted cytosolic Mn accumulation. These data indicate that reduced GAT function in astrocytes is influenced by Mn and fatty acids accumulating at or interacting with the plasma membrane.

  4. The taurine uptake inhibitor guanidinoethyl sulphonate is an agonist at gamma-aminobutyric acid(A) receptors in cultured murine cerebellar granule cells.

    PubMed

    Mellor, J R; Gunthorpe, M J; Randall, A D

    2000-05-26

    In patch clamp experiments the beta-amino acid uptake inhibitor guanidinoethyl sulphonate (GES) activated currents in intact cultured murine cerebellar granule neurones. These responses could be attenuated by the gamma-aminobutyric acid(A) (GABA(A)) receptor antagonists bicuculline and picrotoxin. With intracellular chloride concentrations of either 20 or 130 mM, GES-induced current responses reversed polarity near the chloride equilibrium potential. When fast applications of agonist were made to excised granule cell macropatches GES responses were dose-dependent and exhibited significant outward rectification. Like taurine (but unlike GABA and beta-alanine) responses, macroscopic desensitisation of GES-induced currents was slow. Our data indicate that care should be exercised when using GES as a taurine uptake inhibitor in systems that also contain GABA(A) receptors.

  5. A novel GABA(A) alpha 5 receptor inhibitor with therapeutic potential.

    PubMed

    Ling, István; Mihalik, Balázs; Etherington, Lori-An; Kapus, Gábor; Pálvölgyi, Adrienn; Gigler, Gábor; Kertész, Szabolcs; Gaál, Attila; Pallagi, Katalin; Kiricsi, Péter; Szabó, Éva; Szénási, Gábor; Papp, Lilla; Hársing, László G; Lévay, György; Spedding, Michael; Lambert, Jeremy J; Belelli, Delia; Barkóczy, József; Volk, Balázs; Simig, Gyula; Gacsályi, István; Antoni, Ferenc A

    2015-10-05

    Novel 2,3-benzodiazepine and related isoquinoline derivatives, substituted at position 1 with a 2-benzothiophenyl moiety, were synthesized to produce compounds that potently inhibited the action of GABA on heterologously expressed GABAA receptors containing the alpha 5 subunit (GABAA α5), with no apparent affinity for the benzodiazepine site. Substitutions of the benzothiophene moiety at position 4 led to compounds with drug-like properties that were putative inhibitors of extra-synaptic GABAA α5 receptors and had substantial blood-brain barrier permeability. Initial characterization in vivo showed that 8-methyl-5-[4-(trifluoromethyl)-1-benzothiophen-2-yl]-1,9-dihydro-2H-[1,3]oxazolo[4,5-h][2,3]benzodiazepin-2-one was devoid of sedative, pro-convulsive or motor side-effects, and enhanced the performance of rats in the object recognition test. In summary, we have discovered a first-in-class GABA-site inhibitor of extra-synaptic GABAA α5 receptors that has promising drug-like properties and warrants further development.

  6. Fluvoxamine, a specific 5-hydroxytryptamine uptake inhibitor.

    PubMed

    Claassen, V; Davies, J E; Hertting, G; Placheta, P

    1977-08-01

    1. On the basis of both in vitro and in vivo experiments fluvoxamine has been characterized as a potential anti-depressant drug with almost exclusively 5-hydroxytryptamine (5-HT) uptake inhibiting properties. 2. Fluvoxamine is effective in inhibiting 5-ht uptake by blood platelets and brain synaptosomes. Due to inhibition of the membrane pump the compound prevents 5-HT depletion by the tyramine-derivatives H 75/12 and H 77/77. As a result of the interference with the neuronal re-uptake mechanism for 5-HT, fluvoxamine produces a decreased 5-HT turnover in the brain. Effects of 5-hydroxytryptophan (5-HTP) are potentiated in mice and in combination with pargyline, fluvoxamine induces 5-HT-like behavioural effects. 3. In contrast to tricyclic antidepressants, noradrenaline uptake processes are either unaffected or only slightly inhibited by fluvoxamine. The noradrenaline depleting effects of tyramine derivates are not influenced by fluvoxamine. Reserpine effects, such as ptosis are affected only at very high doses of the test compound. The antagonism by fluvoxamine of the reserpine-induced lowering of the pentamethylenetetrazole convulsive threshold can be regarded as due to an effect upon 5-HT uptake. In contrast to the effects of desmethylimipramine and imipramine, no stimulatory effects are found in rats when rapidly acting reserpine-like compounds are given following a dose of fluvoxamine.

  7. Fluvoxamine, a specific 5-hydroxytryptamine uptake inhibitor.

    PubMed Central

    Claassen, V; Davies, J E; Hertting, G; Placheta, P

    1977-01-01

    1. On the basis of both in vitro and in vivo experiments fluvoxamine has been characterized as a potential anti-depressant drug with almost exclusively 5-hydroxytryptamine (5-HT) uptake inhibiting properties. 2. Fluvoxamine is effective in inhibiting 5-ht uptake by blood platelets and brain synaptosomes. Due to inhibition of the membrane pump the compound prevents 5-HT depletion by the tyramine-derivatives H 75/12 and H 77/77. As a result of the interference with the neuronal re-uptake mechanism for 5-HT, fluvoxamine produces a decreased 5-HT turnover in the brain. Effects of 5-hydroxytryptophan (5-HTP) are potentiated in mice and in combination with pargyline, fluvoxamine induces 5-HT-like behavioural effects. 3. In contrast to tricyclic antidepressants, noradrenaline uptake processes are either unaffected or only slightly inhibited by fluvoxamine. The noradrenaline depleting effects of tyramine derivates are not influenced by fluvoxamine. Reserpine effects, such as ptosis are affected only at very high doses of the test compound. The antagonism by fluvoxamine of the reserpine-induced lowering of the pentamethylenetetrazole convulsive threshold can be regarded as due to an effect upon 5-HT uptake. In contrast to the effects of desmethylimipramine and imipramine, no stimulatory effects are found in rats when rapidly acting reserpine-like compounds are given following a dose of fluvoxamine. PMID:302726

  8. Effect of Uptake-one inhibitors on the uptake of norepinephrine and metaiodobenzylguanidine

    SciTech Connect

    Tobes, M.C.; Jaques, S. Jr.; Wieland, D.M.; Sisson, J.C.

    1985-08-01

    The mechanisms underlying the uptake of the radiopharmaceutical metaiodobenzylguanidine (MIBG) and the catecholamine norepinephrine (NE) were studied using cultured bovine adrenomedullary cells as an in vitro model system. Sodium-dependent and sodium-independent uptake systems have been identified and characterized for both MIBG and NE. The sodium-dependent uptake of Ne and MIBG was inhibited by the selective Uptake-one inhibitors, desmethylimipramine (DMI) and cocaine, whereas the sodium-independent uptake for NE and MIBG was much less sensitive to inhibition by these agents. The sodium-dependent uptake system fulfills the criteria for the neuronal Uptake-one system, and the sodium-independent uptake system fulfills the criteria for a passive diffusion mechanism. Arterial concentrations proximal to the dog adrenal were very small suggesting that the sodium-dependent (Uptake-one) system is predominant in vivo. Consistent with the in vitro observations, the in vivo uptake of MIBG and NE into dog adrenal medullae was effectively blocked by pretreatment with DMI or cocaine. Therefore, iodine-131 MIBG scintigraphy of the adrenal appears to reflect uptake by way of the Uptake-one system.

  9. Brofaromine: a monoamine oxidase-A and serotonin uptake inhibitor.

    PubMed

    Waldmeier, P C; Glatt, A; Jaekel, J; Bittiger, H

    1993-01-01

    Brofaromine is a tight-binding, reversible inhibitor of monoamine oxidase-A (MAO-A), with concomitant serotonin (5-HT) uptake-inhibiting properties. In psychopharmacologic investigations, the compound shows the properties expected of an MAO inhibitor, antagonizing the effects of reserpine, tetrabenazine, and 5-hydroxytryptophan in rats and mice, and suppressing rapid eye movement sleep in cats. Brofaromine showed antidepressant-like activity in a rat social conflict test. In radioligand binding assays, brofaromine exhibited weak or no interaction with alpha 1- and alpha 2-noradrenergic, 5-HT1, 5-HT2, 5-HT3, cholinergic, histamine H1 and H2, mu-opiate, GABAA, benzodiazepine, adenosine, neurotensin, and substance P receptors. Comparison of in vitro and in vivo potencies to inhibit 5-HT uptake with those of reference drugs, and direct evidence in patients and volunteers suggest that 5-HT uptake inhibition plays a role in the clinical profile of brofaromine.

  10. Cl− uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1

    PubMed Central

    Yamada, Junko; Okabe, Akihito; Toyoda, Hiroki; Kilb, Werner; Luhmann, Heiko J; Fukuda, Atsuo

    2004-01-01

    GABA is the principal inhibitory neurotransmitter in the mature brain, but during early postnatal development the elevated [Cl−]i in immature neocortical neurones causes GABAA receptor activation to be depolarizing. The molecular mechanisms underlying this intracellular Cl− accumulation remain controversial. Therefore, the GABA reversal potential (EGABA) or [Cl−]i in early postnatal rat neocortical neurones was measured by the gramicidin-perforated patch-clamp method, and the relative expression levels of the cation−Cl− cotransporter mRNAs (in the same cells) were examined by semiquantitative single-cell multiplex RT-PCR to look for statistical correlations with [Cl−]i. The mRNA expression levels were positively (the Cl− accumulating Na+,K+−2Cl− cotransporter NKCC1) or negatively (the Cl− extruding K+−Cl− cotransporter KCC2) correlated with [Cl−]i. NKCC1 mRNA expression was high in early postnatal days, but decreased during postnatal development, whereas KCC2 mRNA expression displayed the opposite pattern. [Cl−]i and NKCC1 mRNA expression were each higher in cortical plate (CP) neurones than in the presumably older layer V/VI pyramidal neurones in a given slice. The pharmacological effects of bumetanide on EGABA were consistent with the different expression levels of NKCC1 mRNA. These data suggest that NKCC1 may play a pivotal role in the generation of GABA-mediated depolarization in immature CP cells, while KCC2 promotes the later maturation of GABAergic inhibition in the rat neocortex. PMID:15090604

  11. Attenuation of malonate toxicity in primary mesencephalic cultures using the GABA transport blocker, NO-711.

    PubMed

    Stokes, A H; Bernard, L P; Nicklas, W J; Zeevalk, G D

    2001-04-01

    Cultured rat mesencephalic neurons were used to assess the effects of gamma-aminobutyric acid (GABA) transport blockers on toxicity caused by malonate, a reversible, competitive inhibitor of succinate dehydrogenase. Previous studies utilizing an ex vivo chick retinal preparation have shown that GABA release and cell swelling are early consequences of acute energy impairment and that GABA transport blockers attenuate this toxicity. The present results demonstrate that the nonsubstrate GABA transport blocker, NO-711 (1 nM-1 microM), dose-dependently protected cultured mesencephalic dopamine (DA) and GABA neurons from malonate-induced toxicity. Similar protection was demonstrated with nipecotic acid (1 mM) and SKF89976A (100 nM), substrate and nonsubstrate GABA transport blockers, respectively. These compounds by themselves produced no signs of toxicity, although nipecotic acid caused a long-term decrease in GABA uptake not associated with toxicity. Compounds which decrease intracellular reactive oxygen species (ROS) are protective in this model, but NO-711 did not prevent the rise in intracellular ROS induced by malonate, indicating its protective effects were downstream of ROS production. Supplementation of malonate treated cultures with the GABA(A) agonist, muscimol (10 microM), increased the toxicity toward the DA and GABA neuron populations. Antagonists at the GABA(A) and glycine receptors provided partial protection to both the GABA and DA neurons. These findings suggest that the GABA transporter, GABA(A), and/or glycine channels contribute to cell damage associated with energy impairment in this model.

  12. A functional role for both γ-aminobutyric acid (GABA) transporter-1 and GABA transporter-3 in the modulation of extracellular GABA and GABAergic tonic conductances in the rat hippocampus

    PubMed Central

    Kersanté, Flavie; Rowley, Samuel C S; Pavlov, Ivan; Gutièrrez-Mecinas, María; Semyanov, Alexey; Reul, Johannes M H M; Walker, Matthew C; Linthorst, Astrid C E

    2013-01-01

    Tonic γ-aminobutyric acid (GABA)A receptor-mediated signalling controls neuronal network excitability in the hippocampus. Although the extracellular concentration of GABA (e[GABA]) is critical in determining tonic conductances, knowledge on how e[GABA] is regulated by different GABA transporters (GATs) in vivo is limited. Therefore, we studied the role of GATs in the regulation of hippocampal e[GABA] using in vivo microdialysis in freely moving rats. Here we show that GAT-1, which is predominantly presynaptically located, is the major GABA transporter under baseline, quiescent conditions. Furthermore, a significant contribution of GAT-3 in regulating e[GABA] was revealed by administration of the GAT-3 inhibitor SNAP-5114 during simultaneous blockade of GAT-1 by NNC-711. Thus, the GABA transporting activity of GAT-3 (the expression of which is confined to astrocytes) is apparent under conditions in which GAT-1 is blocked. However, sustained neuronal activation by K+-induced depolarization caused a profound spillover of GABA into the extrasynaptic space and this increase in e[GABA] was significantly potentiated by sole blockade of GAT-3 (i.e. even when uptake of GAT-1 is intact). Furthermore, experiments using tetrodotoxin to block action potentials revealed that GAT-3 regulates extrasynaptic GABA levels from action potential-independent sources when GAT-1 is blocked. Importantly, changes in e[GABA] resulting from both GAT-1 and GAT-3 inhibition directly precipitate changes in tonic conductances in dentate granule cells as measured by whole-cell patch-clamp recording. Thus, astrocytic GAT-3 contributes to the regulation of e[GABA] in the hippocampus in vivo and may play an important role in controlling the excitability of hippocampal cells when network activity is increased. PMID:23381899

  13. Activity-dependent transport of GABA analogues into specific cell types demonstrated at high resolution using a novel immunocytochemical strategy.

    PubMed

    Pow, D V; Baldridge, W; Crook, D K

    1996-08-01

    We have raised antisera against the GABA analogues gamma-vinyl GABA, diaminobutyric acid and gabaculine. These analogues are thought to be substrates for high-affinity GABA transporters. Retinae were exposed to micromolar concentrations of these analogues in the presence or absence of uptake inhibitors and then fixed and processed for immunocytochemistry at the light and electron microscopic levels. Immunolabelling for gamma-vinyl GABA revealed specific labelling of GABAergic amacrine cells and displaced amacrine cells in retinae of rabbits, cats, chickens, fish and a monkey. GABA-containing horizontal cells of cat and monkey retinae failed to exhibit labelling for gamma-vinyl GABA, suggesting that they lacked an uptake system for this molecule. In light-adapted fish, gamma-vinyl GABA was readily detected in H1 horizontal cells; similar labelling was also observed in light-adapted chicken retinae. The pattern of labelling in the fish and chicken retinae was modified by dark adaptation, when labelling was greatly reduced in the horizontal cells, indicating the activity dependence of GABA (analogue) transport. Intraperitoneal injection of gamma-vinyl GABA into rats resulted in its transport across the blood-brain barrier and subsequent uptake into populations of GABAergic neurons. The other analogues investigated in this study exhibited different patterns of transport; gabaculine was taken up into glial cells, whilst diaminobutyric acid was taken up into neurons, glial cells and retinal pigment epithelia. Thus, these analogues are probably substrates for different GABA transporters. We conclude that immunocytochemical detection of the high-affinity uptake of gamma-vinyl GABA permits the identification of GABAergic neurons which are actively transporting GABA, and suggest that this novel methodology will be a useful tool in rapidly assessing the recent activity of GABAergic neurons at the cellular level.

  14. Significant reductions in human visual gamma frequency by the gaba reuptake inhibitor tiagabine revealed by robust peak frequency estimation

    PubMed Central

    Muthukumaraswamy, Suresh D.; Campbell, Anne E.; Hamandi, Khalid; Lingford‐Hughes, Anne; Myers, Jim F. M.; Nutt, David J.; Sumner, Petroc; Wilson, Sue J.; Singh, Krish D.

    2016-01-01

    Abstract The frequency of visual gamma oscillations is determined by both the neuronal excitation–inhibition balance and the time constants of GABAergic processes. The gamma peak frequency has been linked to sensory processing, cognitive function, cortical structure, and may have a genetic contribution. To disentangle the intricate relationship among these factors, accurate and reliable estimates of peak frequency are required. Here, a bootstrapping approach that provides estimates of peak frequency reliability, thereby increasing the robustness of the inferences made on this parameter was developed. The method using both simulated data and real data from two previous pharmacological MEG studies of visual gamma with alcohol and tiagabine was validated. In particular, the study by Muthukumaraswamy et al. [2013a] (Neuropsychopharmacology 38(6):1105–1112), in which GABAergic enhancement by tiagabine had previously demonstrated a null effect on visual gamma oscillations, contrasting with strong evidence from both animal models and very recent human studies was re‐evaluated. After improved peak frequency estimation and additional exclusion of unreliably measured data, it was found that the GABA reuptake inhibitor tiagabine did produce, as predicted, a marked decrease in visual gamma oscillation frequency. This result demonstrates the potential impact of objective approaches to data quality control, and provides additional translational evidence for the mechanisms of GABAergic transmission generating gamma oscillations in humans. Hum Brain Mapp 37:3882–3896, 2016. © 2016 Wiley Periodicals, Inc. PMID:27273695

  15. Significant reductions in human visual gamma frequency by the gaba reuptake inhibitor tiagabine revealed by robust peak frequency estimation.

    PubMed

    Magazzini, Lorenzo; Muthukumaraswamy, Suresh D; Campbell, Anne E; Hamandi, Khalid; Lingford-Hughes, Anne; Myers, Jim F M; Nutt, David J; Sumner, Petroc; Wilson, Sue J; Singh, Krish D

    2016-11-01

    The frequency of visual gamma oscillations is determined by both the neuronal excitation-inhibition balance and the time constants of GABAergic processes. The gamma peak frequency has been linked to sensory processing, cognitive function, cortical structure, and may have a genetic contribution. To disentangle the intricate relationship among these factors, accurate and reliable estimates of peak frequency are required. Here, a bootstrapping approach that provides estimates of peak frequency reliability, thereby increasing the robustness of the inferences made on this parameter was developed. The method using both simulated data and real data from two previous pharmacological MEG studies of visual gamma with alcohol and tiagabine was validated. In particular, the study by Muthukumaraswamy et al. [] (Neuropsychopharmacology 38(6):1105-1112), in which GABAergic enhancement by tiagabine had previously demonstrated a null effect on visual gamma oscillations, contrasting with strong evidence from both animal models and very recent human studies was re-evaluated. After improved peak frequency estimation and additional exclusion of unreliably measured data, it was found that the GABA reuptake inhibitor tiagabine did produce, as predicted, a marked decrease in visual gamma oscillation frequency. This result demonstrates the potential impact of objective approaches to data quality control, and provides additional translational evidence for the mechanisms of GABAergic transmission generating gamma oscillations in humans. Hum Brain Mapp 37:3882-3896, 2016. © 2016 Wiley Periodicals, Inc.

  16. Measuring the serotonin uptake site using (/sup 3/H)paroxetine--a new serotonin uptake inhibitor

    SciTech Connect

    Gleiter, C.H.; Nutt, D.J.

    1988-01-01

    Serotonin is an important neurotransmitter that may be involved in ethanol preference and dependence. It is possible to label the serotonin uptake site in brain using the tricyclic antidepressant imipramine, but this also binds to other sites. We have used the new high-affinity uptake blocker paroxetine to define binding to this site and report it to have advantages over imipramine as a ligand.

  17. Taurine-like GABA aminotransferase inhibitors prevent rabbit brain slices against oxygen-glucose deprivation-induced damage.

    PubMed

    Ricci, Lorenzo; Valoti, Massimo; Sgaragli, Giampietro; Frosini, Maria

    2012-06-01

    The activation of the GABAergic system has been shown to protect brain tissues against the damage that occurs after cerebral ischaemia. On the other hand, the taurine analogues (±)Piperidine-3-sulphonic- (PSA), 2-aminoethane phosphonic- (AEP), 2-(N-acetylamino) cyclohexane sulfonic-acids (ATAHS) and 2-aminobenzene sulfonate-acids (ANSA) have been reported to block GABA metabolism by inhibiting rabbit brain GABA aminotransferase and to increase GABA content in rabbit brain slices. The present investigation explored the neuroprotection provided by GABA, Vigabatrin (VIGA) and taurine analogues in the course of oxygen-glucose deprivation and reperfusion induced damage of rabbit brain slices. Tissue damage was assessed by measuring the release of glutamate and lactate dehydrogenase (LDH) during reperfusion and by determining final tissue water gain, measured as the index of cell swelling. GABA (30-300 μM) and VIGA (30-300 μM) significantly antagonised LDH and glutamate release, as well as tissue water gain caused by oxygen-glucose deprivation and reperfusion. Lower (1-10 μM) or higher concentrations (up to 3,000 μM) were ineffective. ANSA, PSA and ATAHS significantly reduced glutamate and LDH release and tissue water gain in a range of concentrations between 30 and 300 μM. Lower (0-10 μM) or higher (up to 3,000 μM) concentrations were ineffective. Both mechanisms suggest hormetic ("U-shaped") effects. These results indicate that the GABAergic system activation performed directly by GABA or indirectly through GABA aminotransferase inhibition is a promising approach for protecting the brain against ischemia and reperfusion-induced damage.

  18. Endogenous dopamine increases extracellular concentrations of glutamate and GABA in striatum of the freely moving rat: involvement of D1 and D2 dopamine receptors.

    PubMed

    Expósito, I; Del Arco, A; Segovia, G; Mora, F

    1999-07-01

    Interactions between endogenous dopamine, glutamate, GABA, and taurine were investigated in striatum of the freely moving rat by using microdialysis. Intrastriatal infusions of the selective dopamine uptake inhibitor nomifensine (NMF) were used to increase the endogenous extracellular dopamine. NMF produced a dose-related increase in extracellular dopamine and also increased extracellular concentrations of glutamate, GABA, and taurine. Extracellular increases of dopamine were significantly correlated with extracellular increases of glutamate and GABA, but not taurine. To investigate whether the increased extracellular dopamine produced by NMF was responsible for the concomitant increase of glutamate and GABA, D1, and D2 receptor antagonists were used. Dopamine receptor antagonists D1 (SCH23390) and D2 (sulpiride) significantly attenuated the increases of glutamate and GABA produced by NMF. These data suggest that endogenous dopamine, through both D1 and D2 dopamine receptors, plays a role in releasing glutamate and GABA in striatum of the freely moving rat.

  19. Effects of polyamine inhibitors on zinc uptake by COMMA-1D mammary epithelial cells

    SciTech Connect

    Allen, J.C.; Haedrich, L.H. )

    1991-03-15

    Zn uptake or transport is stimulated by glucocorticoids in many types of epithelial cells, including the COMMA-1D mouse mammary cell line. The current objective was to determine whether polyamines also mediate glucocorticoid stimulation of Zn-uptake. Initially, cells grown in lactogenic hormone supplemented-media had approximately 65% greater {sup 65}Zn-uptake over 24 h than cells in nonsupplemented growth media (GM). {sup 65}Zn-uptake from HM with 10{sup {minus}5}M methylglyoxal-bis(guanylhydrazone) (MGBG) (s-adenosyl-methionine decarboxylase inhibitor to block polyamine synthesis) added was less than from GM. Exogenous spermidine added to the MGBG-HM media increased {sup 65}Zn-uptake. However, up to 10mM difluoromethylornithine (DFMO), a more specific inhibitor of sperimidine synthesis, had no significant effect on 24-h {sup 65}Zn-uptake by cells in HM. In GM, DFMO caused a slight dose-dependent decrease in {sup 65}Zn-uptake over the range 10{sup {minus}6} to 5 {times} 10{sup 3}M. Also, with 8 h of incubation, DFMO tended to decrease {sup 65}Zn-uptake in HM-stimulated cells. These data cannot yet distinguish between the possibilities that DFMO is inactivated during the 24-h incubation or that the dramatic effects of MGBG on {sup 65}Zn-uptake in these mammary-derived cells is not related to its inhibition of polyamine synthesis. Because COMMA-1D cells alter Zn uptake in response to lactogenic hormones and MGBG, the model system is suitable for further studies of the mechanisms of zinc transport in epithelia.

  20. On the origin of extracellular GABA collected by brain microdialysis and assayed by a simplified on-line method.

    PubMed

    Westerink, B H; de Vries, J B

    1989-06-01

    The present study describes a simplified on-line system for determination of GABA in brain dialysates. GABA was determined with an isocratic HPLC method after derivatization with o-phthaldialdehyde. One peristaltic pump was sufficient to transport both the perfusion fluid and the derivatizing reagent. The basal release of GABA was stimulated by infusion with either elevated K+ or the GABA uptake inhibitor (-)-nipecotic acid. Basal as well as stimulated GABA release were investigated for possible calcium-dependency by infusing submmolar amounts of the potent calcium antagonist cadmium. Infusion of cadmium did not modify the dialysate concentrations of GABA. In addition basal as well as nipecotic acid enhanced release of GABA dialysate concentrations were investigated for nerve-impulse dependency by infusing mumolar amounts of tetrodotoxin. No change in the GABA output was observed during infusion of TTX. From these results it is concluded that the basal as well as the nipecotic acid induced release of GABA did not fulfill the criteria for classic exocytotic release. Possible explanations for these unexpected findings are discussed.

  1. Iodine status in Turkish populations and exposure to iodide uptake inhibitors.

    PubMed

    Ozpinar, Aysel; Kelestimur, Fahrettin; Songur, Yildiran; Can, Ozge; Valentin, Liza; Caldwell, Kathleen; Arikan, Ender; Unsal, Ibrahim; Serteser, Mustafa; Inal, Tamer; Erdemgil, Yigit; Coskun, Abdurrahman; Bakirci, Nadi; Sezgin, Ozlem; Blount, Ben

    2014-01-01

    Perchlorate, nitrate, and thiocyanate are competitive inhibitors of the sodium iodide symporter of the thyroid membrane. These inhibitors can decrease iodine uptake by the symporter into the thyroid gland and may disrupt thyroid function. This study assesses iodine status and exposure to iodide uptake inhibitors of non-pregnant and non-lactating adult women living in three different cities in Turkey (Istanbul, Isparta and Kayseri). We measured iodine and iodide uptake inhibitors in 24-hr urines collected from study participants (N = 255). All three study populations were mildly iodine deficient, with median urinary iodine (UI) levels of 77.5 µg/L in Istanbul, 58.8 µg/L in Isparta, and 69.8 µg/L in Kayseri. Perchlorate doses were higher in the study population (median 0.13 µg/kg/day), compared with a reference population (median 0.059 µg/kg/day), but lower than the U.S. EPA reference dose (0.7 µg/kg/day). Urinary thiocyanate levels increased with increasing exposure to tobacco smoke, with non-smokers (268 µg/L) significantly lower than light smokers (1110 µg/L), who were significantly lower than heavy smokers (2410 µg/L). This pilot study provides novel data indicating that study participants were moderately iodine deficient and had higher intakes of the iodide uptake inhibitor perchlorate compared with a reference population. Further investigation is needed to characterize the thyroid impact resulting from iodine deficiency coupled with exposure to iodide uptake inhibitors such as perchlorate, thiocyanate and nitrate.

  2. Iodine Status in Turkish Populations and Exposure to Iodide Uptake Inhibitors

    PubMed Central

    Ozpinar, Aysel; Kelestimur, Fahrettin; Songur, Yildiran; Can, Ozge; Valentin, Liza; Caldwell, Kathleen; Arikan, Ender; Unsal, Ibrahim; Serteser, Mustafa; Inal, Tamer; Erdemgil, Yigit; Coskun, Abdurrahman; Bakirci, Nadi; Sezgin, Ozlem; Blount, Ben

    2014-01-01

    Perchlorate, nitrate, and thiocyanate are competitive inhibitors of the sodium iodide symporter of the thyroid membrane. These inhibitors can decrease iodine uptake by the symporter into the thyroid gland and may disrupt thyroid function. This study assesses iodine status and exposure to iodide uptake inhibitors of non-pregnant and non-lactating adult women living in three different cities in Turkey (Istanbul, Isparta and Kayseri). We measured iodine and iodide uptake inhibitors in 24-hr urines collected from study participants (N = 255). All three study populations were mildly iodine deficient, with median urinary iodine (UI) levels of 77.5 µg/L in Istanbul, 58.8 µg/L in Isparta, and 69.8 µg/L in Kayseri. Perchlorate doses were higher in the study population (median 0.13 µg/kg/day), compared with a reference population (median 0.059 µg/kg/day), but lower than the U.S. EPA reference dose (0.7 µg/kg/day). Urinary thiocyanate levels increased with increasing exposure to tobacco smoke, with non-smokers (268 µg/L) significantly lower than light smokers (1110 µg/L), who were significantly lower than heavy smokers (2410 µg/L). This pilot study provides novel data indicating that study participants were moderately iodine deficient and had higher intakes of the iodide uptake inhibitor perchlorate compared with a reference population. Further investigation is needed to characterize the thyroid impact resulting from iodine deficiency coupled with exposure to iodide uptake inhibitors such as perchlorate, thiocyanate and nitrate. PMID:24505430

  3. 2,5-Disubstituted tetrahydrofurans as selective serotonin re-uptake inhibitors.

    PubMed

    Voelker, Troy; Xia, Haiji; Fandrick, Keith; Johnson, Robert; Janowsky, Aaron; Cashman, John R

    2009-03-01

    Enhancement of 5-hydroxytryptamine (5-HT, serotonin) neurotransmission is a viable means of treating depression. On the basis of this observation, agents that inhibit re-uptake of 5-HT were prepared based on (-)-cocaine and aryltropanes as lead compounds because they are reasonably potent 5-HT re-uptake inhibitors. Molecular dissection of an aryltropane provided a series of 5- and 6-membered ring compounds. From among this library of compounds a series of disubstituted tetrahydrofurans bearing 2-alkyl aryl and 5-alkyl amino groups were identified as having highly potent and selective 5-HT re-uptake inhibition. The compounds were evaluated for their ability to compete with radiolabeled RTI-55 binding and to inhibit re-uptake of neurotransmitters at the human dopamine, serotonin and norepinephrine transporters. Based on potency (e.g., K(i)=800 pM) and significant functional selectivity (e.g., IC(50) ratios for human dopamine:serotonin or norepinephrine:serotonin, >or=1397) highly potent and selective serotonin re-uptake inhibitors were identified. Optimal features playing a dominant role in binding affinity and re-uptake inhibition included lipophilic substitution on the aromatic moiety, trans relative stereochemistry of the 2,5-disubstituted tetrahydrofuran ring, and a total of four or five methylene groups between the alkyl amine and the alkyl aryl moiety and the tetrahydrofuran group. A number of the most potent serotonin re-uptake inhibitors were tested in Balb/c mice in the forced-swim test (FST), a behavioral test used to measure the effects of antidepressant agents. Acute administration of 32c (10mg/kg), or 32d (10mg/kg) ip tended to decrease the duration of mouse immobility in the FST although the effect was not statistically significant.

  4. Correlating FAAH and anandamide cellular uptake inhibition using N-alkylcarbamate inhibitors: from ultrapotent to hyperpotent.

    PubMed

    Nicolussi, Simon; Chicca, Andrea; Rau, Mark; Rihs, Sabine; Soeberdt, Michael; Abels, Christoph; Gertsch, Jürg

    2014-12-15

    Besides the suggested role of a putative endocannabinoid membrane transporter mediating the cellular uptake of the endocannabinoid anandamide (AEA), this process is intrinsically coupled to AEA degradation by the fatty acid amide hydrolase (FAAH). Differential blockage of each mechanism is possible using specific small-molecule inhibitors. Starting from the natural product-derived 2E,4E-dodecadiene scaffold previously shown to interact with the endocannabinoid system (ECS), a series of diverse N-alkylcarbamates were prepared with the aim of generating novel ECS modulators. While being inactive at cannabinoid receptors and monoacylglycerol lipase, these N-alkylcarbamates showed potent to ultrapotent picomolar FAAH inhibition in U937 cells. Overall, a highly significant correlation (Spearman's rho=0.91) was found between the inhibition of FAAH and AEA cellular uptake among 54 compounds. Accordingly, in HMC-1 cells lacking FAAH expression the effect on AEA cellular uptake was dramatically reduced. Unexpectedly, 3-(4,5-dihydrothiazol-2-yl)phenyl carbamates and the 3-(1,2,3-thiadiazol-4-yl)phenyl carbamates WOBE490, WOBE491 and WOBE492 showed a potentiation of cellular AEA uptake inhibition in U937 cells, resulting in unprecedented femtomolar (hyperpotent) IC50 values. Potential methodological issues and the role of cellular accumulation of selected probes were investigated. It is shown that albumin impacts the potency of specific N-alkylcarbamates and, more importantly, that accumulation of FAAH inhibitors can significantly increase their effect on cellular AEA uptake. Taken together, this series of N-alkylcarbamates shows a FAAH-dependent inhibition of cellular AEA uptake, which can be strongly potentiated using specific head group modifications. These findings provide a rational basis for the development of hyperpotent AEA uptake inhibitors mediated by ultrapotent FAAH inhibition.

  5. Enhancement of GABA release through endogenous activation of axonal GABA(A) receptors in juvenile cerebellum.

    PubMed

    Trigo, Federico F; Chat, Mireille; Marty, Alain

    2007-11-14

    Recent evidence indicates the presence of presynaptic GABA(A) receptors (GABA(A)Rs) in the axon domain of several classes of central neurons, including cerebellar basket and stellate cells. Here, we investigate the possibility that these receptors could be activated in the absence of electrical or chemical stimulation. We find that low concentrations of GABA increase the frequency of miniature GABAergic synaptic currents. Submaximal concentrations of a GABA(A)R blocker, gabazine, decrease both the miniature current frequency and the probability of evoked GABA release. Zolpidem, an agonist of the benzodiazepine binding site, and NO-711 (1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride), a blocker of GABA uptake, both increase the frequency of miniature currents. These effects occur up to postnatal day 14, but not later. Immunohistochemistry indicates the presence of alpha1-containing GABA(A)Rs in interneuron presynaptic terminals with a similar age dependence. We conclude that, under resting conditions, axonal GABA(A)Rs are significantly activated, that this activation results in enhanced GABA release, and that it can be augmented by increasing the affinity of GABA(A)Rs or reducing GABA uptake. Our findings suggest the existence of a positive-feedback mechanism involving presynaptic GABA(A)Rs that maintains a high release rate and a high local GABA concentration in the immature cerebellar network.

  6. The Design, Synthesis and Structure-Activity Relationship of Mixed Serotonin, Norepinephrine and Dopamine Uptake Inhibitors

    NASA Astrophysics Data System (ADS)

    Chen, Zhengming; Yang, Ji; Skolnick, Phil

    The evolution of antidepressants over the past four decades has involved the replacement of drugs with a multiplicity of effects (e.g., TCAs) by those with selective actions (i.e., SSRIs). This strategy was employed to reduce the adverse effects of TCAs, largely by eliminating interactions with certain neurotransmitters or receptors. Although these more selective compounds may be better tolerated by patients, selective drugs, specifically SSRIs, are not superior to older drugs in treating depressed patients as measured by response and remission rates. It may be an advantage to increase synaptic levels of both serotonin and norepinephrine, as in the case of dual uptake inhibitors like duloxetine and venlafaxine. An important recent development has been the emergence of the triple-uptake inhibitors (TUIs/SNDRIs), which inhibit the uptake of the three neurotransmitters most closely linked to depression: serotonin, norepinephrine, and dopamine. Preclinical studies and clinical trials indicate that a drug inhibiting the reuptake of all three of these neurotransmitters could produce more rapid onset of action and greater efficacy than traditional antidepressants. This review will detail the medicinal chemistry involved in the design, synthesis and discovery of mixed serotonin, norepinephrine and dopamine transporter uptake inhibitors.

  7. Fluoxetine at anorectic doses does not have properties of a dopamine uptake inhibitor.

    PubMed

    Fuller, R W; Hemrick-Luecke, S K; Snoddy, H D

    1994-01-01

    Although fluoxetine is a highly selective inhibitor of serotonin uptake in vitro and in vivo, some investigators have suggested that dopamine uptake inhibition may contribute to anorectic actions of fluoxetine. The present experiments were done to determine fluoxetine's effects in some animal protocols in which dopamine uptake inhibitors have characteristic actions. Mazindol prevented the depletion of striatal dopamine and its metabolites by amphetamine in iprindole-pretreated rats, but fluoxetine had no effect. Mazindol prevented the depletion of striatal dopamine and its metabolites by 6-hydroxydopamine injected intracerebroventricularly into rats, but fluoxetine had no effect. Mazindol enhanced the elevation of 3,4-dihydroxyphenylacetic acid concentration in rat brain after spiperone injection, but fluoxetine did not cause that effect. Fluoxetine did not mimic amfonelic acid in antagonizing the retention of alpha-methyl-m-tyramine invant striatum after the injection of alpha-methyl-m-tyrosine. These results show that fluoxetine, at doses that are effective in blocking the serotonin uptake carrier and causing anorexia, does not block the dopamine uptake carrier.

  8. Extracellular GABA in the ventrolateral thalamus of rats exhibiting spontaneous absence epilepsy: a microdialysis study.

    PubMed

    Richards, D A; Lemos, T; Whitton, P S; Bowery, N G

    1995-10-01

    There is compelling evidence that excessive GABA-mediated inhibition may underlie the abnormal electrical activity, initiated in the thalamus, associated with epileptic absence seizures. In particular, the GABAB receptor subtype seems to play a critical role, because its antagonists are potent inhibitors of absence seizures, whereas its agonists exacerbate seizure activity. Using a validated rat model of absence epilepsy, we have previously found no evidence of abnormal GABAB receptor density or affinity in thalamic tissue. In the present study, we have used in vivo microdialysis to monitor changes in levels of extracellular GABA and other amino acids in this brain region. We have shown that basal extracellular levels of GABA and, to a lesser extent, taurine are increased when compared with values in nonepileptic controls. However, modifying GABAergic transmission with the GABAB agonist (-)-baclofen (2 mg/kg i.p.), the GABAB antagonist CGP-35348 (200 mg/kg i.p.), or the GABA uptake inhibitor tiagabine (100 microM) did not produce any further alteration in extracellular GABA levels, despite the ability of these compounds to increase (baclofen and tiagabine) or decrease (CGP-35348) seizure activity. These findings suggest that the increased basal GABA levels observed in this animal model are not simply a consequence of seizure activity but may contribute to the initiation of absence seizures.

  9. An investigation of the origin of extracellular GABA in rat nucleus accumbens measured in vivo by microdialysis.

    PubMed

    Smith, S E; Sharp, T

    1994-01-01

    GABA transmission in the nucleus accumbens is believed to play a central role in motivational processes and the expression of psychostimulant drug action. Here we report measurements of extracellular GABA in nucleus accumbens of the rat and investigate its origin. Extracellular GABA was detected using microdialysis in combination with a novel HPLC-based assay. In the awake rat, GABA in the microdialysates (1) increased 10-fold following perfusion with 0.5 mM nipecotic acid, a GABA releasing agent and uptake blocker, (2) increased 7-fold following local perfusion with 50 mM KCl, (3) decreased 50% following perfusion with tetrodotoxin, (4) decreased 50% following perfusion with a Ca(2+(-free medium and (5) decreased 40% following perfusion with high (12.5 mM) MgCl. Finally, in the anaesthetized rat, GABA in the microdialysates decreased 50% following i.p. injection of 100 mg/kg 3-mercaptoproprionic acid, a GABA synthesis inhibitor. We conclude that GABA in microdialysates from nucleus accumbens of the rat (awake) responds appropriately to selected pharmacological agents and derives at least in part (50%) from neurones.

  10. The presence of a serotonin uptake inhibitor alters pharmacological manipulations of serotonin release.

    PubMed

    Kreiss, D S; Wieland, S; Lucki, I

    1993-01-01

    The present study investigated the effects of the presence of the serotonin uptake inhibitor citalopram in the perfusion medium on pharmacological manipulations which increased and decreased striatal serotonin release using in vivo microdialysis. A high performance liquid chromatography detection system equipped with a microbore column was used which reduced the detection limit to 0.5 fmol serotonin/5 microliters sample and enabled basal striatal serotonin release to be measured without the addition of a serotonin uptake inhibitor to the perfusion medium. Although serotonin uptake inhibitors have frequently been used to enhance the serotonin content of dialysate samples, the effects of the presence of serotonin uptake inhibitors on pharmacological manipulations which increased and decreased the release of serotonin have not yet been characterized. Serotonin release was reduced by the systemic administration of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). Although 5-HT release was reduced by 8-OH-DPAT after the addition of citalopram, the 5-HT1A receptor agonist did not reduce absolute levels of extracellular serotonin below basal values of serotonin measured in the absence of citalopram. In addition, citalopram dramatically prevented the four-fold increase in the release of serotonin produced by the systemic administration of the serotonin-releasing agent fenfluramine. The blockade of fenfluramine's effects by citalopram supports the hypothesis that transport of fenfluramine into serotonergic neurons is necessary to increase serotonin release. This study demonstrates that the use of an HPLC detection system equipped with a microbore column can reliably measure basal serotonin release using in vivo microdialysis.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Revisiting serotonin reuptake inhibitors and the therapeutic potential of "uptake-2" in psychiatric disorders.

    PubMed

    Daws, Lynette C; Koek, Wouter; Mitchell, Nathan C

    2013-01-16

    Depression is among the most common psychiatric disorders, and in many patients a disorder for which available medications provide suboptimal or no symptom relief. The most commonly prescribed class of antidepressants, the selective serotonin reuptake inhibitors (SSRIs), are thought to act by increasing extracellular serotonin in brain by blocking its uptake via the high-affinity serotonin transporter (SERT). However, the relative lack of therapeutic efficacy of SSRIs has brought into question the utility of increasing extracellular serotonin for the treatment of depression. In this Viewpoint, we discuss why increasing extracellular serotonin should not be written off as a therapeutic strategy. We describe how "uptake-2" transporters may explain the relative lack of therapeutic efficacy of SSRIs, as well as why "uptake-2" transporters might be useful therapeutic targets.

  12. Uptake inhibitors but not substrates induce protease resistance in extracellular loop two of the dopamine transporter.

    PubMed

    Gaffaney, Jon D; Vaughan, Roxanne A

    2004-03-01

    Changes in protease sensitivity of extracellular loop two (EL2) of the dopamine transporter (DAT) during inhibitor and substrate binding were examined using trypsin proteolysis and epitope-specific immunoblotting. In control rat striatal membranes, proteolysis of DAT in a restricted region of EL2 was produced by 0.001 to 10 microg/ml trypsin. However, in the presence of the dopamine uptake blockers [2-(diphenylmethoxyl) ethyl]-4-(3phenylpropyl) piperazine (GBR 12909), mazindol, 2beta-carbomethoxy-3beta-(4-flourophenyl)tropane (beta-CFT), nomifensine, benztropine, or (-)-cocaine, 100- to 1000-fold higher concentrations of trypsin were required to produce comparable levels of proteolysis. Protease resistance induced by ligands was correlated with their affinity for DAT binding, was not observed with Zn2+, (+)-cocaine, or inhibitors of norepinephrine or serotonin transporters, and was not caused by altered catalytic activity of trypsin. Together, these results support the hypothesis that the interaction of uptake inhibitors with DAT induces a protease-resistant conformation in EL2. In contrast, binding of substrates did not induce protease resistance in EL2, suggesting that substrates and inhibitors interact with DAT differently during binding. To assess the effects of EL2 proteolysis on DAT function, the binding and transport properties of trypsin-digested DAT were assayed with [3H]CFT and [3H]dopamine. Digestion decreased the Bmax for binding and the Vmax for uptake in amounts that were proportional to the extent of proteolysis, indicating that the structural integrity of EL2 is required for maintenance of both DAT binding and transport functions. Together this data provides novel information about inhibitor and substrate interactions at EL2, possibly relating the protease resistant DAT conformation to a mechanism of transport inhibition.

  13. Modulation of thyroidal radioiodide uptake by oncological pipeline inhibitors and Apigenin.

    PubMed

    Lakshmanan, Aparna; Scarberry, Daniel; Green, Jill A; Zhang, Xiaoli; Selmi-Ruby, Samia; Jhiang, Sissy M

    2015-10-13

    Targeted radioiodine therapy for thyroid cancer is based on selective stimulation of Na+/I- Symporter (NIS)-mediated radioactive iodide uptake (RAIU) in thyroid cells by thyrotropin. Patients with advanced thyroid cancer do not benefit from radioiodine therapy due to reduced or absent NIS expression. To identify inhibitors that can be readily translated into clinical care, we examined oncological pipeline inhibitors targeting Akt, MEK, PI3K, Hsp90 or BRAF in their ability to increase RAIU in thyroid cells expressing BRAFV600E or RET/PTC3 oncogene. Our data showed that (1) PI3K inhibitor GDC-0941 outperformed other inhibitors in RAIU increase mainly by decreasing iodide efflux rate to a great extent; (2) RAIU increase by all inhibitors was extensively reduced by TGF-β, a cytokine secreted in the invasive fronts of thyroid cancers; (3) RAIU reduction by TGF-β was mainly mediated by NIS reduction and could be reversed by Apigenin, a plant-derived flavonoid; and (4) In the presence of TGF-β, GDC-0941 with Apigenin co-treatment had the highest RAIU level in both BRAFV600E expressing cells and RET/PTC3 expressing cells. Taken together, Apigenin may serve as a dietary supplement along with small molecule inhibitors to improve radioiodine therapeutic efficacy on invasive tumor margins thereby minimizing future metastatic events.

  14. Voltammetric characterization of the effect of monoamine uptake inhibitors and releasers on dopamine and serotonin uptake in mouse caudate-putamen and substantia nigra slices

    PubMed Central

    John, Carrie E.; Jones, Sara R.

    2007-01-01

    Summary Fast scan cyclic voltammetry is an electrochemical technique used to measure dynamics of transporter-mediated monoamine uptake in real time and provides a tool to evaluate the detailed effects of monoamine uptake inhibitors and releasers on dopamine and serotonin transporter function. We measured the effects of cocaine, methylphenidate, 2β-propanoyl–3β-(4tolyl) tropane (PTT), fluoxetine, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), phentermine and fenfluramine on dopamine and serotonin uptake following electrically stimulated release in mouse caudate-putamen and substantia nigra pars reticulata slices. We determined rank orders of uptake inhibition effects based on two variables; increases in apparent Km for dopamine and serotonin uptake and inhibition constant (Ki) values. For example, the rank order of uptake inhibition based on apparent Km values at the dopamine transporter was amphetamine ≥ PTT ≥ methylphenidate ≫ methamphetamine = phentermine = MDMA > cocaine ≫ fluoxetine = fenfluramine, and at the serotonin transporter was fluoxetine = methamphetamine = fenfluramine = MDMA > amphetamine = cocaine = PTT ≥ methylphenidate > phentermine. Additionally, changes in electrically stimulated release were documented. This is the first study using voltammetry to measure the effects of a wide range of monoamine uptake inhibitors and releasers on dopamine and serotonin uptake in mouse brain slices. These studies also highlight methodological considerations for comparison of effects between heterogeneous brain regions. PMID:17459426

  15. Conjugation of spermine enhances cellular uptake of the stapled peptide-based inhibitors of p53-Mdm2 interaction.

    PubMed

    Muppidi, Avinash; Li, Xiaolong; Chen, Jiandong; Lin, Qing

    2011-12-15

    We report the first synthesis of the C-terminally spermine-conjugated stapled peptide-based inhibitors of the p53-Mdm2 interaction. Subsequent biological, biophysical and cellular uptake assays with the spermine-conjugated stapled peptides revealed that spermine conjugation minimally affects biological activity while significantly increases peptide helicity and cellular uptake without apparent cytotoxicity.

  16. Use of inhibitors for coastal bacteria and phytoplankton: Application to nitrogen uptake measurement

    NASA Astrophysics Data System (ADS)

    Trottet, Aurore; Fouilland, Eric; Leboulanger, Christophe; Lanouguère, Elodie; Bouvy, Marc

    2011-06-01

    For several decades, prokaryotic and eukaryotic inhibitors have been used to exclude bacteria from microalgal cultures and for investigating prey-predator relationships. Recently there has been considerable interest in using specific inhibitors for studying the interactions between bacteria and phytoplankton, by selective repression of either organism's activity. The effectiveness of chemical inhibitors must be tested before applying them to natural communities to partition metabolic activities between functional groups. Six different antibiotics selected from the most commonly reported in the literature were tested, at concentrations varying from 12.5 to 100 mg L -1, for their effect on bacterial growth and functional diversity of natural communities from Mediterranean coastal waters. Penicillin and streptomycin each at a final concentration of 100 mg L -1 significantly reduced bacterial growth within 2 h. There was a greater impact on bacterial functional diversity when both antibiotics were mixed together. This mixture did not have any significant effect on the growth of selected cultured phytoplankton strains, whereas the eukaryote inhibitor cycloheximide at 100 mg L -1 reduced growth within 2 h of incubation. The penicillin-streptomycin mixture and cycloheximide alone successfully partitioned NH 4+ and NO 3- uptake between bacteria and phytoplankton bi-weekly sampled in a coastal lagoon in Autumn, where bacterial contribution to total NH 4+ and NO 3- uptake averaged 46 and 41%, respectively. The use of specific inhibitors may be a valuable method for studying interactions, such as competition and mutualism, or lack of interaction between the different components of microbial communities and could be used to study their relative importance in biogeochemical fluxes.

  17. Design, characterization and cellular uptake studies of fluorescence-labeled prototypic cathepsin inhibitors.

    PubMed

    Kohl, Franziska; Schmitz, Janina; Furtmann, Norbert; Schulz-Fincke, Anna-Christina; Mertens, Matthias D; Küppers, Jim; Benkhoff, Marcel; Tobiasch, Edda; Bartz, Ulrike; Bajorath, Jürgen; Stirnberg, Marit; Gütschow, Michael

    2015-11-07

    Besides their extracellular activity crucial for several pathophysiological conditions, human cysteine cathepsins, in particular cathepsins K and S, represent important intracellular targets for drug development. In the present study, a prototypic dipeptide nitrile inhibitor structure was equipped with a coumarin moiety to function as a fluorescent reporter group. In a second inhibitor, a PEG linker was introduced between the dipeptide nitrile and the fluorophore. These tool compounds 6 and 7 were characterized by kinetic investigations as covalent reversible inhibitors of human cathepsins L, S, K and B. Probe 6 showed a pronounced inhibitory activity against cathepsins K and S, which was corroborated by modeling of inhibition modes. Probe 7 was highly potent (Ki = 93 nM) and selective for cathepsin S. To examine the ability of both probes to enter living cells, human embryonic kidney 293 cells were targeted. At a concentration of 10 μM, cellular uptake of probe 6 was demonstrated by fluorescence measurement after an incubation time of 30 min and 3 h, respectively. The probe's concentration in cell lysates was ascertained on the basis of the emission at 492 nm upon excitation at 450 nm, and the results were expressed as concentrations of probe 6 relative to the protein concentration originating from the lysate. After incubation of 10 μM of probe 6 for 3 h, the cellular uptake was confirmed by fluorescence microscopy. HPLC was used to assess the probes’ lipophilicity, and the obtained

  18. Reviewing the serotonin reuptake inhibitors (SSRIs) footprint in the aquatic biota: uptake, bioaccumulation and ecotoxicology.

    PubMed

    Silva, Liliana J G; Pereira, André M P T; Meisel, Leonor M; Lino, Celeste M; Pena, Angelina

    2015-02-01

    Selective serotonin re-uptake inhibitors (SSRIs) antidepressants are amongst the most prescribed pharmaceutical active substances throughout the world. Their presence, already described in different environmental compartments such as wastewaters, surface, ground and drinking waters, and sediments, and their remarkable effects on non-target organisms justify the growing concern about these emerging environmental pollutants. A comprehensive review of the literature data with focus on their footprint in the aquatic biota, namely their uptake, bioaccumulation and both acute and chronic ecotoxicology is presented. Long-term multigenerational exposure studies, at environmental relevant concentrations and in mixtures of related compounds, such as oestrogenic endocrine disruptors, continue to be sparse and are imperative to better know their environmental impact.

  19. GABA localization in the nematode Ascaris

    SciTech Connect

    Guastella, J.

    1988-01-01

    A histochemical approach was used to examine the distribution of GABA-associated neurons in the nematode Ascaris, an organism whose small number of morphologically simple neurons make it an excellent preparation for analyzing neuronal phenotypes. Two GABAergic markers were examined: GABA-like immunoreactivity (GLIR), a marker for endogenous stores of GABA; and ({sup 3}H)-GABA uptake, a marker for GABA uptake sites. Strong GLIR was present in the cell bodies, neurites and commissures of dorsal and ventral inhibitory motorneurons present in this region. Strong GLIR was also present in the cell bodies and processes of the four RME neurons in the nerve ring and in several other ganglionic neurons. Staining was absent in excitatory motorneurons, in ventral cord interneurons and in muscle cells and hypodermis. GABA uptake sites were found in single neural processes in both the ventral and dorsal nerve cords. ({sup 3}H)-GABA labeling was also observed in the other two RME cells and several other cephalic neurons. Four putative cholinergic excitatory motorneurons in the retrovesicular ganglion (RVG) were heavily labeled. Ventral and dorsal nerve cord inhibitory motorneurons did not take up ({sup 3}H)-GABA. Labeling of the ventral cord excitatory motorneuron somata and cell bodies was at or slightly above background. Heavy labeling of muscle cells was also observed.

  20. Modulation of extracellular neurotransmitter levels in the nucleus accumbens by a taurine uptake inhibitor.

    PubMed

    Olive, M F; Mehmert, K K; Hodge, C W

    2000-12-15

    Using in vivo microdialysis, we examined the effect of local perfusion of the taurine uptake inhibitor guanidinoethyl sulfonate on extracellular levels of various neurotransmitters in the rat nucleus accumbens. Guanidinoethyl sulfonate (500 microM-50 mM) produced a concentration-dependent increase in extracellular taurine levels. While 500 microM and 5 mM concentrations of guanidinoethyl sulfonate were largely without effect, 50 mM guanidinoethyl sulfonate produced a significant decrease in extracellular levels of aspartate, glutamate and glycine, with no effect on extracellular dopamine levels. These results indicate that guanidinoethyl sulfonate can modulate extracellular amino acid levels in the nucleus accumbens.

  1. Selective serotonin re-uptake inhibitors (SSRIs) in the aquatic environment: an ecopharmacovigilance approach.

    PubMed

    Silva, Liliana J G; Lino, Celeste M; Meisel, Leonor M; Pena, Angelina

    2012-10-15

    Selective serotonin re-uptake inhibitors (SSRIs) antidepressants are among the most prescribed pharmaceutical active substances throughout the world. The occurrence of these widely used compounds in different environmental compartments (wastewaters, surface, ground and drinking waters, and sediments), justify the growing concern about these emerging environmental pollutants. Viewing an ecopharmacovigilance approach, a comprehensive discussion of the state of the art regarding different contamination sources, fate, degradation and occurrence is presented. Information on the current distribution levels and fate in different environmental matrices continues to be sparse and measures are imperative to improve awareness and encourage precautionary actions to minimize SSRIs' environmental impact.

  2. Basic aspects of GABA-transmission in alcoholism, with particular reference to GABA-transaminase.

    PubMed

    Sherif, F M; Tawati, A M; Ahmed, S S; Sharif, S I

    1997-02-01

    Neuronal dysfunction is the neurobiological basis for alcoholic behaviour, and ethanol craving seems related to hypofunction of the GABA-ergic activity. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). In several studies, GABA has been shown to be an important target of ethanol in the CNS, partly, as a consequence of damage to membrane-bound enzymes and receptors. GABA is involved in mediating pre- and post-synaptic inhibition of neuronal activity. It is speculated that the initial excitatory effects of ethanol may be due to inhibition of GABA-ergic activity whereas the sedative effects of the higher doses may be mediated by the activation of this inhibitory system. In the CNS, GABA is synthesised from glutamic acid by the enzyme glutamate decarboxylase (GAD) and catabolized into succinic semialdehyde by the enzyme GABA-transaminase (GABA-T), which are pyridoxal phosphate (PLP) dependent enzymes. Platelet GABA-T was characterized as being similar to central GABA-T. Inhibition of GABA-T with certain potent and selective compounds markedly increases the levels of brain GABA. Experimentally, acute ethanol treatment does not alter GABA-T activity whereas chronic treatment produces an increase in the activity, though, with some reservations since a bimodal effect has been found in chronically ethanol-treated rats. Thus, as it will be discussed below, it may be suggested that GABA-T inhibitors (e.g. vigabatrin) could have a potential role in the treatment of alcoholism and in some of the problems of ethanol withdrawal and of other drugs of abuse. Related studies on metabolism and concentrations of GABA are also promising and show a greater increase in our understanding of the aetiology and treatment of ethanol dependence and withdrawal. In general, this article also reviews both the animal and clinical observations in the field of alcoholism with regard to the GABA system.

  3. High-affinity uptake of gamma-aminobutyric acid in cultured glial and neuronal cells.

    PubMed

    Balcar, V J; Mark, J; Borg, J; Mandel, P

    1979-06-01

    Both glial and neuronal cells maintained in primary culture were found to accumulate [3H]GABA by an efficient "high-affinity" uptake system (apparent Km = 9 muM, Vmax = 0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1mM) also strongly inhibited uptake of [3H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, beta-alanine, and 2,4-diaminobutyrate) inhibited uptake of [3H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine, L-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (Km = 92 muM, Vmax = 0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [3H]GABA against a concentration gradient. Apparent Km of this uptake was relatively high (819 muM), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, non of the nontransformed continuous cell lines of either tumoral (glioma, C6; neuroblastoma, M1; M1NN) or normal (NN;I6) origin actively accumulated [3H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.

  4. Kinetic studies on the inhibition of GABA-T by gamma-vinyl GABA and taurine.

    PubMed

    Sulaiman, Saba A J; Suliman, Fakhr Eldin O; Barghouthi, Samira

    2003-08-01

    Gamma-aminobutyric acid transaminase (GABA-T, EC 2.6.1.19) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the degradation of gamma-aminobutyric acid. The kinetics of this reaction are studied in vitro, both in the absence, and in the presence of two inhibitors: gamma-vinyl GABA (4-aminohex-5-enoic acid), and a natural product, taurine (ethylamine-2-sulfonic acid). A kinetic model that describes the transamination process is proposed. GABA-T from Pseudomonas fluorescens is inhibited by gamma-vinyl GABA and taurine at concentrations of 51.0 and 78.5 mM. Both inhibitors show competitive inhibition behavior when GABA is the substrate and the inhibition constant (Ki) values for gamma-vinyl GABA and taurine were found to be 26 +/- 3 mM and 68 +/- 7 mM respectively. The transamination process of alpha-ketoglutarate was not affected by the presence of gamma-vinyl GABA, whereas, taurine was a noncompetitive inhibitor of GABA-T when alpha-ketoglutarate was the substrate. The inhibition dissociation constant (Kii) for this system was found to be 96 +/- 10 mM. The Michaelis-Menten constant (Km) in the absence of inhibition, was found to be 0.79 +/- 0.11 mM, and 0.47 +/- 0.10 mM for GABA and alpha-ketoglutarate respectively.

  5. Interaction between tricyclic and nontricyclic 5-hydroxytryptamine uptake inhibitors and the presynaptic 5-hydroxytryptamine inhibitory autoreceptors in the rat hypothalamus.

    PubMed

    Galzin, A M; Moret, C; Verzier, B; Langer, S Z

    1985-10-01

    In slices of the rat hypothalamus prelabeled with [3H]-5-hydroxytryptamine [( 3H]-5-HT), exposure to lysergic acid diethylamide or 5-methoxytryptamine decreased, in a concentration-dependent manner, the release of 3H-transmitter elicited by electrical stimulation. These inhibitory effects were antagonized by the 5-HT receptor antagonist methiothepin (1 microM). Exposure to methiothepin on its own increased in a concentration-dependent manner the electrically evoked overflow of [3H]-5-HT. Exposure to tricyclic antidepressants, like imipramine and amitriptyline, and to nontricyclic 5-HT uptake inhibitors, like paroxetine and citalopram, did not modify by themselves the electrically evoked overflow of [3H]-5-HT. Yet, the four inhibitors of neuronal uptake of 5-HT, antagonized the inhibition by lysergic acid diethylamide or 5-methoxytryptamine of the electrically induced release of [3H]-5-HT. After depletion of endogenous stores of 5-HT by pretreatment with para-chlorophenylalanine (300 mg/kg i.p.), the inhibitors of 5-HT uptake increased the electrically evoked release of [3H]-5-HT in a concentration-dependent manner. Their order of potency to enhance 5-HT overflow after pretreatment with parachlorophenylalanine paralleled their potency at inhibiting neuronal uptake of 5-HT (paroxetine = citalopram greater than imipramine greater than amitriptyline). In para-chlorophenylalanine-treated rat hypothalamic slices, these inhibitors of 5-HT uptake antagonized the inhibition by 5-HT autoreceptor agonists of the electrically evoked release of [3H]-5-HT to a similar extent than was observed in control rats. It is concluded that inhibition of 5-HT uptake reduces the effectiveness of 5-HT autoreceptor agonists to inhibit the electrically evoked release of [3H]-5-HT, irrespective of the chemical structure of the uptake inhibitor or of the levels of endogenous 5-HT achieved in the synaptic gap.

  6. Evidence for GABA-Induced Systemic GABA Accumulation in Arabidopsis upon Wounding

    PubMed Central

    Scholz, Sandra S.; Malabarba, Jaiana; Reichelt, Michael; Heyer, Monika; Ludewig, Frank; Mithöfer, Axel

    2017-01-01

    The non-proteinogenic amino acid γ-aminobutyric acid (GABA) is present in all plant species analyzed so far. Its synthesis is stimulated by either acidic conditions occurring after tissue disruption or higher cytosolic calcium level. In mammals, GABA acts as inhibitory neurotransmitter but its function in plants is still not well understood. Besides its involvement in abiotic stress resistance, GABA has a role in the jasmonate-independent defense against invertebrate pests. While the biochemical basis for GABA accumulation in wounded leaves is obvious, the underlying mechanisms for wounding-induced GABA accumulation in systemic leaves remained unclear. Here, the Arabidopsis thaliana knock-out mutant lines pop2-5, unable to degrade GABA, and tpc1-2, lacking a wounding-induced systemic cytosolic calcium elevation, were employed for a comprehensive investigation of systemic GABA accumulation. A wounding-induced systemic GABA accumulation was detected in tpc1-2 plants demonstrating that an increased calcium level was not involved. Similarly, after both mechanical wounding and Spodoptera littoralis feeding, GABA accumulation in pop2-5 plants was significantly higher in local and systemic leaves, compared to wild-type plants. Consequently, larvae feeding on these GABA-enriched mutant plants grew significantly less. Upon exogenous application of a D2-labeled GABA to wounded leaves of pop2-5 plants, its uptake but no translocation to unwounded leaves was detected. In contrast, an accumulation of endogenous GABA was observed in vascular connected systemic leaves. These results suggest that the systemic accumulation of GABA upon wounding does not depend on the translocation of GABA or on an increase in cytosolic calcium. PMID:28382046

  7. Effects of phenylethanolamine N-methyltransferase inhibitors on uptake and release of norepinephrine and dopamine from rat brain

    SciTech Connect

    Liang, N.Y.; Hower, J.A.; Borchardt, R.T.

    1985-09-01

    Inhibitors of phenylethanolamine N-methyltransferase (PNMT) and amphetamine were evaluated for their effects on the uptake of (TH)-norepinephrine (TH-NE) and the release of endogenous NE and dopamine (DA) from chopped rat brain tissues. Unlike amphetamine, all of PNMT inhibitors tested produced only slight inhibition of (TH)-NE uptake into chopped cerebral cortex. 2,3-Dichloro-alpha-methylbenzylamine (DCMB) and 7,8-dichloro-1,2,3,4-tetrahydroisoquinoline (SKF64139), but not 2-cyclooctyl-2-hydroxyethylamine (CONH) and 1-aminomethylcycloundecanol (CUNH) produced slight release of endogenous NE and DA from chopped hypothalami, but their effects were less pronounced than those produced by amphetamine.

  8. Uptake of L-tri-iodothyronine by isolated rat liver cells. A process partially inhibited by metabolic inhibitors; attempts to distinguish between uptake and binding to intracellular proteins.

    PubMed Central

    Eckel, J; Rao, G S; Rao, M L; Breuer, H

    1979-01-01

    1. Rat liver cells obtained by dispersion with collagenase were used to investigate the mode of entry of L-tri-iodothyronine into the cell. 2. The hormone was taken up very rapidly at 23 degrees C; the linear phase of uptake lasted for up to approx. 20 s. 3. A plot of the initial rates of uptake against different concentrations of L-tri-iodothyronine yielded a sigmoidal curve. The Eadie--Hofstee plot (v/[S]2 versus v) yielded two straight lines. The uptake component with an apparent Kt value of 86 +/- 15 pM was designated as system I, and the second uptake component with an apparent Kt of 726 +/- 11 pM as system II. The Hill plot for system I was not linear; the apparent Hill coefficient for system II was calculated to be 2.1.4. Uptake of L-tri-iodothyronine by system I was higher at pH 6.4 than at pH 7.4; system II was relatively insensitive to changes in the pH of the external medium. 5. Both systems exhibited a transition temperature at about 16 degrees C in the Arrhenius plot. The activation energies of the two systems below and above 16 degrees C were 72.8 and 47.7 and 54.4 and 33.1 J/mol respectively. 6. Inhibitors of cellular energy reduced the uptake by system I to a larger extent than that by system II. 7. Replacement of Na+ in the external medium by either K+ or choline led to uptake that followed normal Michaelis--Menten kinetics. 8. Thiol-group-blocking agents reduced the uptake of the hormone by both systems. 9. Treatment of liver cells with beta-glucosidase, Pronase and neuraminidase led to a decrease in the uptake of L-tri-iodothyronine by system I, whereas uptake by system II was decreased after treatment with phospholipase A2, beta-galactosidase. Pronase and neuraminidase. 10. The stereoisomer D-tri-iodothyronine (100--3000 pM) did not affect system I, but uptake by system II decreased with increasing concentration of D-tri-iodothyronine. Reverse L-tri-iodothyronine (2--100 pM) and L-thyroxine (100--3000 pM) did not influence uptake by either

  9. Manganese exposure inhibits the clearance of extracellular GABA and influences taurine homeostasis in the striatum of developing rats.

    PubMed

    Fordahl, Steve C; Anderson, Joel G; Cooney, Paula T; Weaver, Tara L; Colyer, Christa L; Erikson, Keith M

    2010-12-01

    Manganese (Mn) accumulation in the brain has been shown to alter the neurochemistry of the basal ganglia. Mn-induced alterations in dopamine biology are fairly well understood, but recently more evidence has emerged characterizing the role of γ-aminobutyric acid (GABA) in this dysfunction. The purpose of this study was to determine if the previously observed Mn-induced increase in extracellular GABA (GABA(EC)) was due to altered GABA transporter (GAT) function, and whether Mn perturbs other amino acid neurotransmitters, namely taurine and glycine (known modulators of GABA). Extracellular GABA, taurine, and glycine concentrations were collected from the striatum of control (CN) or Mn-exposed Sprague-Dawley rats using in vivo microdialysis, and the GAT inhibitor nipecotic acid (NA) was used to probe GAT function. Tissue and extracellular Mn levels were significantly increased, and the Fe:Mn ratio was decreased 36-fold in the extracellular space due to Mn-exposure. NA led to a 2-fold increase in GABA(EC) of CNs, a response that was attenuated by Mn. Taurine responded inversely to GABA, and a novel 10-fold increase in taurine was observed after the removal of NA in CNs. Mn blunted this response and nearly abolished extracellular taurine throughout collection. Striatal taurine transporter (Slc6a6) mRNA levels were significantly increased with Mn-exposure, and Mn significantly increased (3)H-Taurine uptake after 3-min exposure in primary rat astrocytes. These data suggest that Mn increases GABA(EC) by inhibiting the function of GAT, and that perturbed taurine homeostasis potentially impacts neural function by jeopardizing the osmoregulatory and neuromodulatory functions of taurine in the brain.

  10. Atypical Dopamine Uptake Inhibitors that Provide Clues About Cocaine's Mechanism at the Dopamine Transporter

    NASA Astrophysics Data System (ADS)

    Hauck Newman, Amy; Katz, Jonathan L.

    The dopamine transporter (DAT) has been a primary target for cocaine abuse/addiction medication discovery. However predicted addiction liability and limited clinical evaluation has provided a formidable challenge for development of these agents for human use. The unique and atypical pharmacological profile of the benztropine (BZT) class of dopamine uptake inhibitors, in preclinical models of cocaine effects and abuse, has encouraged further development of these agents. Moreover, in vivo studies have challenged the original DAT hypothesis and demonstrated that DAT occupancy and subsequent increases in dopamine produced by BZT analogues are significantly delayed and long lasting, as compared to cocaine. These important and distinctive elements are critical to the lack of abuse liability among BZT analogues, and improve their potential for development as treatments for cocaine abuse and possibly other neuropsychiatric disorders.

  11. [Effects of urease/nitrification inhibitors on soil available N and microbial biomass N and on N uptake of wheat].

    PubMed

    Jiao, Xiaoguang; Liang, Wenju; Chen, Lijun; Jiang, Yong; Wen, Dazhong

    2004-10-01

    With an aquic brown earth as test soil, this paper studied the effects of urease inhibitor (NBPT), nitrification inhibitor (DCD) and their combinations on the dynamics of soil available N and microbial biomass N. The results showed that the treatments of inhibitors, especially the combined application of NBPT and DCD, could increase soil NH4+-N by 2%-53%, inhibit NH4+ oxidation, decrease soil NO3(-)-N concentration, increase soil total available N by 34%-44%, and increase wheat N uptake by 0.26%-6.79%. The best treatment was urease inhibitor combined with nitrification inhibitor. The application of inhibitors increased soil microbial biomass N immobilization at the early growth stage of wheat, and promoted soil N mineralization at filling stage.

  12. Invariance of the density of dopamine uptake sites and dopamine metabolism in the rat brain after a chronic treatment with the dopamine uptake inhibitor GBR 12783.

    PubMed

    Boulay, D; Leroux-Nicollet, I; Duterte-Boucher, D; Naudon, L; Costentin, J

    1994-01-01

    A chronic treatment (10 mg/kg, twice daily during 9 days) with the dopamine uptake inhibitor GBR 12783 was performed in rats at a dose increasing their locomotor activity. Forty-eight hours after the last administration, animals were sacrificed and 3H mazindol binding was performed on brain slices. Autoradiographic analysis revealed no change in this binding relatively to control animals in regions with high dopamine contents: striatum, nucleus accumbens, olfactory tubercle, substantia nigra and ventral tegmentum area. The treatment did not either modify the levels of dopamine (DA) and metabolites (HVA, DOPAC) both in the striatum and the nucleus accumbens. Thus, early after the end of the treatment, the chronic blockade of the dopamine uptake complex regulates neither the dopamine uptake complex nor the dopamine metabolism.

  13. Blocking the GABA transporter GAT-1 ameliorates spinal GABAergic disinhibition and neuropathic pain induced by paclitaxel

    PubMed Central

    Yadav, Ruchi; Yan, Xisheng; Maixner, Dylan W.; Gao, Mei; Weng, Han-Rong

    2015-01-01

    Paclitaxel is a chemotherapeutic agent widely used for treating carcinomas. Patients receiving paclitaxel often develop neuropathic pain and have a reduced quality of life which hinders the use of this life-saving drug. In this study, we determined the role of GABA transporters in the genesis of paclitaxel-induced neuropathic pain using behavioral tests, electrophysiology, and biochemical techniques. We found that tonic GABA receptor activities in the spinal dorsal horn were reduced in rats with neuropathic pain induced by paclitaxel. In normal controls, tonic GABA receptor activities were mainly controlled by the GABA transporter GAT-1 but not GAT-3. In the spinal dorsal horn, GAT-1 was expressed at presynaptic terminals and astrocytes while GAT-3 was only expressed in astrocytes. In rats with paclitaxel-induced neuropathic pain, the protein expression of GAT-1 was increased while GAT-3 was decreased. This was concurrently associated with an increase of global GABA uptake. The paclitaxel-induced attenuation of GABAergic tonic inhibition was ameliorated by blocking GAT-1 but not GAT-3 transporters. Paclitaxel-induced neuropathic pain was significantly attenuated by the intrathecal injection of a GAT-1 inhibitor. These findings suggest that targeting GAT-1 transporters for reversing disinhibition in the spinal dorsal horn may be a useful approach for treating paclitaxel-induced neuropathic pain. PMID:25827582

  14. GABA and glycine actions on spinal motoneurons.

    PubMed

    Krnjević, K; Puil, E; Werman, R

    1977-06-01

    Applied microiontophoretically in the spinal cord of cats, glycine is consistently more powerful than gamma-aminobutyric acid (GABA) in raising the membrane conductance of lumbosacral motoneurons (mean ratio of equipotent iontophoretic currents tested on same cells is 5.6:1). This is the reverse of the situation in cerebral cortex. The effect of glycine is well maintained during applications lasting about 1 min, but that of GABA, after an early peak, drops to a much lower plateau (mean plateau-over-peak ratio is 0.23). The reversal potentials for the action of GABA and glycine are initially similar but they behave differently during a prolonged application; that for glycine usually remains constant or becomes more negative whereas that for GABA tends to shift in the positive direction. Various explanations of these phenomena are considered. It is suggested that a single process, electrogenic uptake of GABA, may account for both desensitization (by removing GABA from its site of action) and the positive shift in GABA reversal potential (became uptake is probably associated with an influx of Na+).

  15. Not All Antidepressants Are Created Equal: Differential Effects of Monoamine Uptake Inhibitors on Effort-Related Choice Behavior

    PubMed Central

    Yohn, Samantha E; Collins, Samantha L; Contreras-Mora, Hector M; Errante, Emily L; Rowland, Margaret A; Correa, Merce; Salamone, John D

    2016-01-01

    Motivated behavior can be characterized by behavioral activation and high work output. Moreover, people with depression and other disorders show effort-related motivational symptoms, such as anergia, psychomotor retardation, and fatigue. Effort-based decision making is studied using tasks offering choices between high effort options leading to highly valued reinforcers vs low effort/low reward options, and such tasks could be useful as animal models of motivational symptoms. In the present studies the effort-related effects of the vesicular monoamine transport (VMAT-2) inhibitor tetrabenazine (TBZ) were investigated. TBZ blocks vesicular storage and also produces depressive symptoms in humans. Moreover, TBZ alters effort-based choice in rats, biasing animals toward low effort alternatives. The present studies investigated the ability of acute administration of various monoamine uptake inhibitors to reverse the effects of TBZ. Effort-related effects of TBZ were attenuated by the catecholamine uptake inhibitor and antidepressant bupropion, and this effect of bupropion was reversed by either D1 or D2 family antagonism. The effort-related effects of TBZ were also attenuated by the selective dopamine uptake blocker GBR12909. The 5-HT uptake inhibitor fluoxetine and the norepinephrine uptake inhibitor desipramine failed to reverse the effects of TBZ, and higher doses of these drugs, given alone or in combination with TBZ, led to further behavioral impairments. These results indicate that drugs acting on dopamine transmission are relatively effective at reversing the effort-related effects of TBZ, and are consistent with the hypothesis that drugs that enhance dopamine transmission may be effective at treating effort-related psychiatric symptoms in humans. PMID:26105139

  16. Not All Antidepressants Are Created Equal: Differential Effects of Monoamine Uptake Inhibitors on Effort-Related Choice Behavior.

    PubMed

    Yohn, Samantha E; Collins, Samantha L; Contreras-Mora, Hector M; Errante, Emily L; Rowland, Margaret A; Correa, Merce; Salamone, John D

    2016-02-01

    Motivated behavior can be characterized by behavioral activation and high work output. Moreover, people with depression and other disorders show effort-related motivational symptoms, such as anergia, psychomotor retardation, and fatigue. Effort-based decision making is studied using tasks offering choices between high effort options leading to highly valued reinforcers vs low effort/low reward options, and such tasks could be useful as animal models of motivational symptoms. In the present studies the effort-related effects of the vesicular monoamine transport (VMAT-2) inhibitor tetrabenazine (TBZ) were investigated. TBZ blocks vesicular storage and also produces depressive symptoms in humans. Moreover, TBZ alters effort-based choice in rats, biasing animals toward low effort alternatives. The present studies investigated the ability of acute administration of various monoamine uptake inhibitors to reverse the effects of TBZ. Effort-related effects of TBZ were attenuated by the catecholamine uptake inhibitor and antidepressant bupropion, and this effect of bupropion was reversed by either D1 or D2 family antagonism. The effort-related effects of TBZ were also attenuated by the selective dopamine uptake blocker GBR12909. The 5-HT uptake inhibitor fluoxetine and the norepinephrine uptake inhibitor desipramine failed to reverse the effects of TBZ, and higher doses of these drugs, given alone or in combination with TBZ, led to further behavioral impairments. These results indicate that drugs acting on dopamine transmission are relatively effective at reversing the effort-related effects of TBZ, and are consistent with the hypothesis that drugs that enhance dopamine transmission may be effective at treating effort-related psychiatric symptoms in humans.

  17. Protection of malonate-induced GABA but not dopamine loss by GABA transporter blockade in rat striatum.

    PubMed

    Zeevalk, Gail D; Manzino, Lawrence; Sonsalla, Patricia K

    2002-07-01

    Previous work has shown that overstimulation of GABA(A) receptors can potentiate neuronal cell damage during excitotoxic or metabolic stress in vitro and that GABA(A) antagonists or GABA transport blockers are neuroprotective under these situations. Malonate, a reversible succinate dehydrogenase/mitochondrial complex II inhibitor, is frequently used in animals to model cell loss in neurodegenerative diseases such as Parkinson's and Huntington's diseases. To determine if GABA transporter blockade during mitochondrial impairment can protect neurons in vivo as compared with in vitro studies, rats received a stereotaxic infusion of malonate (2 micromol) into the left striatum to induce a metabolic stress. The nonsubstrate GABA transport blocker, NO711 (20 nmol) was infused in some rats 30 min before and 3 h following malonate infusion. After 1 week, dopamine and GABA levels in the striata were measured. Malonate caused a significant loss of striatal dopamine and GABA. Blockade of the GABA transporter significantly attenuated GABA, but not dopamine loss. In contrast with several in vitro reports, GABA(A) receptors were not a downstream mediator of protection by NO711. Intrastriatal infusion of malonate (2 micromol) plus or minus the GABA(A) receptor agonist muscimol (1 micromol), the GABA(A) Cl- binding site antagonist picrotoxin (50 nmol) or the GABA(B) receptor antagonist saclofen (33 nmol) did not modify loss of striatal dopamine or GABA when examined 1 week following infusion. These data show that GABA transporter blockade during mitochondrial impairment in the striatum provides protection to GABAergic neurons. GABA transporter blockade, which is currently a pharmacological strategy for the treatment of epilepsy, may thus also be beneficial in the treatment of acute and chronic conditions involving energy inhibition such as stroke/ischemia or Huntington's disease. These findings also point to fundamental differences between immature and adult neurons in the

  18. A possible role of the non-GAT1 GABA transporters in transfer of GABA from GABAergic to glutamatergic neurons in mouse cerebellar neuronal cultures.

    PubMed

    Suñol, C; Babot, Z; Cristòfol, R; Sonnewald, U; Waagepetersen, H S; Schousboe, A

    2010-09-01

    Cultures of dissociated cerebellum from 7-day-old mice were used to investigate the mechanism involved in synthesis and cellular redistribution of GABA in these cultures consisting primarily of glutamatergic granule neurons and a smaller population of GABAergic Golgi and stellate neurons. The distribution of GAD, GABA and the vesicular glutamate transporter VGlut-1 was assessed using specific antibodies combined with immunofluorescence microscopy. Additionally, tiagabine, SKF 89976-A, betaine, beta-alanine, nipecotic acid and guvacine were used to inhibit the GAT1, betaine/GABA (BGT1), GAT2 and GAT3 transporters. Only a small population of cells were immuno-stained for GAD while many cells exhibited VGlut-1 like immuno-reactivity which, however, never co-localized with GAD positive neurons. This likely reflects the small number of GABAergic neurons compared to the glutamatergic granule neurons constituting the majority of the cells. GABA uptake exhibited the kinetics of high affinity transport and could be partly (20%) inhibited by betaine (IC(50) 142 microM), beta-alanine (30%) and almost fully (90%) inhibited by SKF 89976-A (IC(50) 0.8 microM) or nipecotic acid and guvacine at 1 mM concentrations (95%). Essentially all neurons showed GABA like immunostaining albeit with differences in intensity. The results indicate that GABA which is synthesized in a small population of GAD-positive neurons is redistributed to essentially all neurons including the glutamatergic granule cells. GAT1 is not likely involved in this redistribution since addition of 15 microM tiagabine (GAT1 inhibitor) to the culture medium had no effect on the overall GABA content of the cells. Likewise the BGT1 transporter cannot alone account for the redistribution since inclusion of 3 mM betaine in the culture medium had no effect on the overall GABA content. The inhibitory action of beta-alanine and high concentrations of nipecotic acid and guvacine on GABA transport strongly suggests that also

  19. Generation of a 3D model for human GABA transporter hGAT-1 using molecular modeling and investigation of the binding of GABA.

    PubMed

    Wein, Thomas; Wanner, Klaus T

    2010-01-01

    A three-dimensional model of the human Na(+)/Cl(-)-dependent gamma-aminobutyric acid (GABA) transporter hGAT-1 was developed by homology modeling and refined by subsequent molecular modeling using the crystal structure of a bacterial homologue leucine transporter from Aquifex aeolicus (LeuT(Aa)) as the template. Protein structure quality checks show that the resulting structure is particularly suited for the analysis of the substrate binding pocket and virtual screening experiments. Interactions of GABA and the substrate binding pocket were investigated using docking studies. The difference of 6 out of 13 substrate interacting side chains between hGAT-1 and LeuT(Aa) lead to the different substrate preference which can be explained using our three-dimensional model of hGAT-1. In particular the replacement of serine 256 and isoleucine 359 in LeuT(Aa) with glycine and threonine in hGAT-1 seems to facilitate the selection of GABA as the main substrate by changing the hydrogen bonding pattern in the active site to the amino group of the substrate. For a set of 12 compounds flexible docking experiments were performed using LigandFit in combination with the Jain scoring function. With few exceptions the obtained rank order of potency was in line with experimental data. Thus, the method can be assumed to give at least a rough estimate of the potency of the potential of GABA uptake inhibitors.

  20. A multivalent inhibitor of the DC-SIGN dependent uptake of HIV-1 and Dengue virus.

    PubMed

    Varga, Norbert; Sutkeviciute, Ieva; Ribeiro-Viana, Renato; Berzi, Angela; Ramdasi, Rasika; Daghetti, Anna; Vettoretti, Gerolamo; Amara, Ali; Clerici, Mario; Rojo, Javier; Fieschi, Franck; Bernardi, Anna

    2014-04-01

    DC-SIGN is a C-type lectin receptor on antigen presenting cells (dendritic cells) which has an important role in some viral infection, notably by HIV and Dengue virus (DV). Multivalent presentation of carbohydrates on dendrimeric scaffolds has been shown to inhibit DC-SIGN binding to HIV envelope glycoprotein gp120, thus blocking viral entry. This approach has interesting potential applications for infection prophylaxis. In an effort to develop high affinity inhibitors of DC-SIGN mediated viral entry, we have synthesized a group of glycodendrimers of different valency that bear different carbohydrates or glycomimetic DC-SIGN ligands and have studied their DC-SIGN binding activity and antiviral properties both in an HIV and a Dengue infection model. Surface Plasmon Resonance (SPR) competition studies have demonstrated that the materials obtained bind efficiently to DC-SIGN with IC50s in the μm range, which depend on the nature of the ligand and on the valency of the scaffold. In particular, a hexavalent presentation of the DC-SIGN selective antagonist 4 displayed high potency, as well as improved accessibility and chemical stability relative to previously reported dendrimers. At low μm concentration the material was shown to block both DC-SIGN mediated uptake of DV by Raji cells and HIV trans-infection of T cells.

  1. Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells

    SciTech Connect

    Saperstein, R.; Vicario, P.P.; Strout, H.V.; Brady, E.; Slater, E.E.; Greenlee, W.J.; Onedyka, D.L.; Patchett, A.A.; Hangauer, D.G. )

    1989-06-27

    An inhibitor of the insulin receptor tyrosine kinase (IRTK), (hydroxy-2-napthalenylmethyl)phosphonic acid, was designed and synthesized and was shown to be an inhibitor of the biological effects of insulin in vitro. With a wheat germ purified human placental insulin receptor preparation, this compound inhibited the insulin-stimulated autophosphorylation of the 95-kDa {beta}-subunit of the insulin receptor. The ability of the kinase to phosphorylate an exogenous peptide substrate, angiotensin II, was also inhibited. Half-maximal inhibition of basal and insulin-stimulated human placental IRTK activity was found at concentrations of 150 and 100 {mu}M, respectively, with 2 mM angiotensin II as the peptide substrate. The inhibitor was found to be specific for tyrosine kinases over serine kinases and noncompetitive with ATP. The inhibitor was converted into various (acyloxy)methyl prodrugs in order to achieve permeability through cell membranes. These prodrugs inhibited insulin-stimulated autophosphorylation of the insulin receptor 95-kDa {beta}-subunit in intact CHO cells transfected with human insulin receptor. Inhibition of insulin-stimulated glucose oxidation in isolated rat adipocytes and 2-deoxyglucose uptake into CHO cells was observed with these prodrugs. The data provide additional evidence for the involvement of the insulin receptor tyrosine kinase in the regulation of glucose uptake and metabolism. These results and additional data reported herein suggest that this class of prodrugs and inhibitors will be useful for modulating the activity of a variety of tyrosine kinases.

  2. Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells.

    PubMed

    Tomi, Masatoshi; Tajima, Ayumi; Tachikawa, Masanori; Hosoya, Ken-ichi

    2008-10-01

    The purpose of this study was to identify the uptake mechanism of gamma-aminobutyric acid (GABA) via taurine transporter (Slc6a6/TauT) and its relationship with GABA transport at the inner BRB. Rat Slc6a6/TauT-transfected HeLa cells exhibited Na(+)-, Cl(-)-, and concentration-dependent [3H]GABA uptake with a Km of 1.5 mM. Taurine, beta-alanine, and GABA markedly inhibited Slc6a6/TauT-mediated uptake of [3H]GABA. The uptake of [3H]GABA by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) was Na(+)-, Cl(-)-, and concentration-dependent with a Km of 2.0 mM. This process was more potently inhibited by substrates of Slc6a6/TauT, taurine and beta-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a Ki of 74 microM. [3H]Taurine also exhibited competitive inhibition with a Ki of 1.8 mM in the presence of GABA. In conclusion, rat Slc6a6/TauT has the ability to use GABA as a substrate and Slc6a6/TauT-mediated GABA transport appears to be present at the inner BRB.

  3. Revisiting Serotonin Reuptake Inhibitors and the Therapeutic Potential of “Uptake-2” in Psychiatric Disorders

    PubMed Central

    2013-01-01

    Depression is among the most common psychiatric disorders, and in many patients a disorder for which available medications provide suboptimal or no symptom relief. The most commonly prescribed class of antidepressants, the selective serotonin reuptake inhibitors (SSRIs), are thought to act by increasing extracellular serotonin in brain by blocking its uptake via the high-affinity serotonin transporter (SERT). However, the relative lack of therapeutic efficacy of SSRIs has brought into question the utility of increasing extracellular serotonin for the treatment of depression. In this Viewpoint, we discuss why increasing extracellular serotonin should not be written off as a therapeutic strategy. We describe how “uptake-2” transporters may explain the relative lack of therapeutic efficacy of SSRIs, as well as why “uptake-2” transporters might be useful therapeutic targets. PMID:23336039

  4. Synaptic GABA release prevents GABA transporter type-1 reversal during excessive network activity

    PubMed Central

    Savtchenko, Leonid; Megalogeni, Maria; Rusakov, Dmitri A.; Walker, Matthew C.; Pavlov, Ivan

    2015-01-01

    GABA transporters control extracellular GABA, which regulates the key aspects of neuronal and network behaviour. A prevailing view is that modest neuronal depolarization results in GABA transporter type-1 (GAT-1) reversal causing non-vesicular GABA release into the extracellular space during intense network activity. This has important implications for GABA uptake-targeting therapies. Here we combined a realistic kinetic model of GAT-1 with experimental measurements of tonic GABAA receptor currents in ex vivo hippocampal slices to examine GAT-1 operation under varying network conditions. Our simulations predict that synaptic GABA release during network activity robustly prevents GAT-1 reversal. We test this in the 0 Mg2+ model of epileptiform discharges using slices from healthy and chronically epileptic rats and find that epileptiform activity is associated with increased synaptic GABA release and is not accompanied by GAT-1 reversal. We conclude that sustained efflux of GABA through GAT-1 is unlikely to occur during physiological or pathological network activity. PMID:25798861

  5. Hazards and uptake of chitin synthesis inhibitors in bumblebees Bombus terrestris.

    PubMed

    Mommaerts, Veerle; Sterk, Guido; Smagghe, Guy

    2006-08-01

    This research project examined the potential hazards of a major class of insect growth regulators (IGRs) to survival, reproduction and larval growth in bumblebees Bombus terrestris L. Eight chitin synthesis inhibitors (CSIs) were tested: buprofezin, cyromazine, diflubenzuron, flucycloxuron, flufenoxuron, lufenuron, novaluron and teflubenzuron. These different IGRs, which are important in the control of pest insects in greenhouses, were applied via three different routes of exposure under laboratory conditions: dermal contact, and orally via the drinking of sugar/water and via pollen. The compounds were tested at their respective maximum field recommended concentrations (MFRC) and also in dose-response assays to calculate LC(50) values. In general, none of the CSIs showed acute worker toxicity. However, there was a dramatic reduction in brood production, especially after oral treatment with pollen and sugar/water. Conspicuously, egg fertility was reduced in all treatments with diflubenzuron and teflubenzuron. In addition to egg mortality, the worker bumblebees removed larvae from the treated nest, and in most cases these individuals were dead first-second instars. Under a binocular microscope, such larvae showed an abnormally formed cuticle leading to mechanical weakness and death. In another series of experiments using (14)C-diflubenzuron and (14)C-flufenoxuron, cuticular penetration in workers was studied for a better understanding of the differences in toxicity. With (14)C-diflubenzuron, transovarial transport and accumulation in the deposited eggs supported the strong reproductive effects. Overall, the present results suggest that CSIs should be applied with caution in combination with bumblebees. The compatibility of each compound to be used in combination with B. terrestris is discussed in relation to calculated LC(50) values, routes of uptake and effects.

  6. Neuronal gamma-aminobutyric acid (GABA) type A receptors undergo cognate ligand chaperoning in the endoplasmic reticulum by endogenous GABA

    PubMed Central

    Wang, Ping; Eshaq, Randa S.; Meshul, Charles K.; Moore, Cynthia; Hood, Rebecca L.; Leidenheimer, Nancy J.

    2015-01-01

    GABAA receptors mediate fast inhibitory neurotransmission in the brain. Dysfunction of these receptors is associated with various psychiatric/neurological disorders and drugs targeting this receptor are widely used therapeutic agents. Both the efficacy and plasticity of GABAA receptor-mediated neurotransmission depends on the number of surface GABAA receptors. An understudied aspect of receptor cell surface expression is the post-translational regulation of receptor biogenesis within the endoplasmic reticulum (ER). We have previously shown that exogenous GABA can act as a ligand chaperone of recombinant GABAA receptors in the early secretory pathway leading us to now investigate whether endogenous GABA facilitates the biogenesis of GABAA receptors in primary cerebral cortical cultures. In immunofluorescence labeling experiments, we have determined that neurons expressing surface GABAA receptors contain both GABA and its degradative enzyme GABA transaminase (GABA-T). Treatment of neurons with GABA-T inhibitors, a treatment known to increase intracellular GABA levels, decreases the interaction of the receptor with the ER quality control protein calnexin, concomittantly increasing receptor forward-trafficking and plasma membrane insertion. The effect of GABA-T inhibition on the receptor/calnexin interaction is not due to the activation of surface GABAA or GABAB receptors. Consistent with our hypothesis that GABA acts as a cognate ligand chaperone in the ER, immunogold-labeling of rodent brain slices reveals the presence of GABA within the rough ER. The density of this labeling is similar to that present in mitochondria, the organelle in which GABA is degraded. Lastly, the effect of GABA-T inhibition on the receptor/calnexin interaction was prevented by pretreatment with a GABA transporter inhibitor. Together, these data indicate that endogenous GABA acts in the rough ER as a cognate ligand chaperone to facilitate the biogenesis of neuronal GABAA receptors. PMID

  7. Neuronal release and successful astrocyte uptake of aminoacidergic neurotransmitters after spinal cord injury in lampreys.

    PubMed

    Fernández-López, Blanca; Valle-Maroto, Silvia María; Barreiro-Iglesias, Antón; Rodicio, María Celina

    2014-08-01

    In contrast to mammals, the spinal cord of lampreys spontaneously recovers from a complete spinal cord injury (SCI). Understanding the differences between lampreys and mammals in their response to SCI could provide valuable information to propose new therapies. Unique properties of the astrocytes of lampreys probably contribute to the success of spinal cord regeneration. The main aim of our study was to investigate, in the sea lamprey, the release of aminoacidergic neurotransmitters and the subsequent astrocyte uptake of these neurotransmitters during the first week following a complete SCI by detecting glutamate, GABA, glycine, Hu and cytokeratin immunoreactivities. This is the first time that aminoacidergic neurotransmitter release from neurons and the subsequent astrocytic response after SCI are analysed by immunocytochemistry in any vertebrate. Spinal injury caused the immediate loss of glutamate, GABA and glycine immunoreactivities in neurons close to the lesion site (except for the cerebrospinal fluid-contacting GABA cells). Only after SCI, astrocytes showed glutamate, GABA and glycine immunoreactivity. Treatment with an inhibitor of glutamate transporters (DL-TBOA) showed that neuronal glutamate was actively transported into astrocytes after SCI. Moreover, after SCI, a massive accumulation of inhibitory neurotransmitters around some reticulospinal axons was observed. Presence of GABA accumulation significantly correlated with a higher survival ability of these neurons. Our data show that, in contrast to mammals, astrocytes of lampreys have a high capacity to actively uptake glutamate after SCI. GABA may play a protective role that could explain the higher regenerative and survival ability of specific descending neurons of lampreys.

  8. Differing effects of transport inhibitor on glutamate uptake by nerve terminals before and after exposure of rats to artificial gravity.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Krisanova, N.; Himmelreich, N.

    Glutamate is the major excitatory neurotransmitter in the brain. Subsequent to its release from glutamatergic neurons and activation of receptors, it is removed from extracellular space by high affinity Na^+-dependent glutamate transporters, which utilize the Na^+/K^+ electrochemical gradient as a driving force and located in nerve terminals and astrocytes. The glutamate transporters may modify the time course of synaptic events. Like glutamate itself, glutamate transporters are somehow involved in almost all aspects of normal and abnormal brain activity (e.g. cerebral ischemia, amyotrophic lateral sclerosis, Alzheimer's disease, traumatic brain injury, epilepsy and schizophrenia). The present study assessed transporter inhibitor for the ability to inhibit glutamate uptake by synaptosomes at the normal and hypergravity conditions (rats were rotated in a long-arm centrifuge at ten-G during one-hour period). DL-threo-beta-benzyloxyaspartate (DL-TBOA) is a newly developed competitive inhibitor of the high-affinity, Na^+-dependent glutamate transporters. As a potent, non- transported inhibitor of glutamate transporters, DL-TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms. We demonstrated that DL-TBOA inhibited glutamate uptake ( 100 μM glutamate, 30 sec incubation period) in dose-dependent manner as in control as in hypergravity. The effect of this transport inhibitor on glutamate uptake by control synaptosomes and synaptosomes prepared of animals exposed to hypergravity was different. IC50 values calculated on the basis of curves of non-linear regression kinetic analysis was 18±2 μM and 11±2 μM ((P≤0,05) before and after exposure to artificial gravity, respectively. Inhibition caused by 10 μM DL-TBOA was significantly increased from 38,0±3,8 % in control group to 51,0±4,1 % in animals, exposed to hypergravity (P≤0,05). Thus, DL-TBOA had complex effect on glutamate uptake process and perhaps, became more potent under

  9. Localization of glycine, GABA and neuropeptide containing neurons in tiger salamander retina

    SciTech Connect

    Yang, C.Y.

    1988-01-01

    Putative glycinergic and GABAergic neurons in the salamander retina were localized by a parallel analysis of high affinity {sup 3}H-glycine uptake and glycine-like immunoreactivity (Gly-IR) and a comparative analysis of high affinity {sup 3}H-GABA uptake, GABA, like immunoreactivity (GABA-IR), and glutamate decarboxylase immunoreactivity (GAD-IR) at the light microscopic level. Good correspondence of labeling of {sup 3}H-glycine uptake and Gly-IR as well as that of {sup 3}H-GABA uptake and GABA-IR were observed. In addition, GAD immunoreactive neurons contained GABA-IR as well. Extensive colocalization of {sup 3}H-glycine uptake and Gly-IR and that of {sup 3}H-GABA uptake, GABA-IR and perhaps GAD-IR were indicated by the similarities in the distribution, morphology and labeling frequency of neurons and lamination in the inner plexiform layer (IPL). However, the Gly-IR and the GABA-IR probes appeared to be more sensitive and can thus be a reliable marker for glycine and GABA containing neurons respectively.

  10. Endogenous glutamate increases extracellular concentrations of dopamine, GABA, and taurine through NMDA and AMPA/kainate receptors in striatum of the freely moving rat: a microdialysis study.

    PubMed

    Segovia, G; Del Arco, A; Mora, F

    1997-10-01

    Interactions between glutamate (Glu), dopamine (DA), GABA, and taurine (Tau) were investigated in striatum of the freely moving rat by using microdialysis. Intrastriatal infusions of the selective Glu uptake inhibitor L-trans-pyrrolidine-3,4-dicarboxylic acid (PDC) were used to increase the endogenous extracellular [Glu]. Correlations between extracellular [Glu] and extracellular [DA], [GABA], and [Tau], and the effects of a selective blockade of ionotropic Glu receptors, were studied. PDC (1, 2, and 4 mM) produced a dose-related increase in extracellular [Glu]. At the highest dose of PDC, [Glu] increased from 1.55 +/- 0.35 to 6.11 +/- 0.88 microM. PDC also increased extracellular [DA], [GABA], and [Tau]. The increasing [Glu] was correlated significantly with increasing [DA], [GABA], and [Tau]. PDC also decreased extracellular concentrations of DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (HVA). Perfusion with the NMDA-receptor antagonist 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (1 mM) or the AMPA/kainate-receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) (1 mM) attenuated the increases produced by PDC (4 mM) on [DA], [GABA], and [Tau], and decreases in [DOPAC] and [HVA]. DNQX also attenuated the increases in [Glu] induced by PDC. These data show that endogenous Glu plays a role in modulating the extracellular concentrations of DA, GABA, and Tau in striatum of the freely moving rat.

  11. Synthetic Methods for the Preparation of a Functional Analogue of Ru360, a Potent Inhibitor of Mitochondrial Calcium Uptake.

    PubMed

    Nathan, Sarah R; Pino, Nicholas W; Arduino, Daniela M; Perocchi, Fabiana; MacMillan, Samantha N; Wilson, Justin J

    2017-03-20

    The mixed-valent oxo-bridged ruthenium complex [(HCO2)(NH3)4Ru(μ-O)Ru(NH3)4(O2CH)](3+), known as Ru360, is a selective inhibitor of mitochondrial calcium uptake. Although this compound is useful for studying the role of mitochondrial calcium in biological processes, its widespread availability is limited because of challenges in purification and characterization. Here, we describe our investigations of three different synthetic methods for the preparation of a functional analogue of this valuable compound. We demonstrate that this analogue, isolated from our procedures, exhibits potent mitochondrial calcium uptake inhibitory properties in permeabilized HeLa cells and in isolated mitochondria.

  12. Immunocytochemical localization of the GABA transporter in rat brain.

    PubMed

    Radian, R; Ottersen, O P; Storm-Mathisen, J; Castel, M; Kanner, B I

    1990-04-01

    Polyclonal antibodies were raised against the GABA transporter (GABA-Tp) purified from rat brain tissue (Radian et al., 1986) and used for immunocytochemical localization of the antigen in several rat brain areas, including the cerebellum, hippocampus, substantia nigra, and cerebral cortex. Light microscopic studies with the peroxidase-antiperoxidase and biotin-avidin-peroxidase techniques suggested that GABA-Tp is localized in the same types of axons and terminals that contain endogenous GABA, as judged by comparison with parallel sections incubated with antibodies against glutaraldehyde-conjugated GABA. However, as expected from biochemical results, different neurons differed in their relative contents of GABA-Tp and GABA; thus, GABA-Tp was relatively low in striatonigral and Purkinje axon terminals and relatively high in nerve plexus around the bases of cerebellar Purkinje cells and hippocampal pyramidal and granule cells. The GABA-Tp antiserum did not produce detectable labeling of nerve cell bodies. Electron microscopic studies supported the light microscopic observations and provided direct evidence of cellular co-localization of GABA-Tp and GABA (as visualized by the peroxidase-antiperoxidase technique and postembedding immunogold labeling, respectively). The ultrastructural studies indicated the presence of GABA-Tp also in glial processes but not in glial cell bodies. The relative intensity of the neuronal and glial staining varied among regions: glial staining predominated over neuronal staining in the substantia nigra, whereas the converse was true in the cerebellum and hippocampus. The present immunocytochemical data demonstrate directly what has previously been inferred from biochemical and autoradiographic evidence: that the mechanisms for high-affinity GABA uptake is selectively and differentially localized in GABAergic neurons and in glial cells.

  13. Relative vulnerability of dopamine and GABA neurons in mesencephalic culture to inhibition of succinate dehydrogenase by malonate and 3-nitropropionic acid and protection by NMDA receptor blockade.

    PubMed

    Zeevalk, G D; Derr-Yellin, E; Nicklas, W J

    1995-12-01

    The effects of different severities of metabolic stress on dopamine (DA) and gamma-aminobutyric acid (GABA) cell loss were examined in rat mesencephalic culture. Partial metabolic inhibition was induced in 12-day-old cultures by a 24-hr treatment with various concentrations of 3-nitropropionic acid(3-NPA, 0.1-0.5 mM) or malonate (10-50 mM), irreversible and reversible inhibitors of the Krebs cycle enzyme, succinate dehydrogenase. Cell damage to the DA and GABA populations was assessed after a 48-hr recovery period by simultaneous measurement of high affinity uptake for 3H-DA and 14C-GABA. 3-NPA or malonate caused a dose-dependent loss of DA uptake (EC50 0.21 or 42 mM, respectively). 3-NPA treatment was equally detrimental to the GABA population, whereas malonate exposure did not cause any significant loss of GABA uptake. The presence of the NMDA antagonist, MK-801 (1 microM), during 24 hr of 3-NPA or malonate treatment fully protected against DA and GABA loss with 50 mM malonate or 0.25 mM 3-NPA and partially protected versus 0.5 mM 3-NPA. To determine the degree of metabolic stress imposed by 3-NPA and malonate, 12-day-old cultures were treated with 0.5 mM 3-NPA or 50 mM malonate for 3 hr and the rate of lactate formation was measured. lactate was increased nearly 2-fold at 3 hr of treatment with 3-NPA, but was not significantly elevated above basal with malonate treatment. SDH activity was decreased by 48 or 58% after 3 hr of treatment with 0.25 and 0.5 mM 3-NPA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Effects of the monoamine uptake inhibitors RTI-112 and RTI-113 on cocaine- and food-maintained responding in rhesus monkeys.

    PubMed

    Negus, S S; Mello, N K; Kimmel, H L; Howell, L L; Carroll, F I

    2009-01-01

    Cocaine blocks uptake of the monoamines dopamine, serotonin and norepinephrine, and monoamine uptake inhibitors constitute one class of drugs under consideration as candidate "agonist" medications for the treatment of cocaine abuse and dependence. The pharmacological selectivity of monoamine uptake inhibitors to block uptake of dopamine, serotonin and norepinephrine is one factor that may influence the efficacy and/or safety of these compounds as drug abuse treatment medications. To address this issue, the present study compared the effects of 7-day treatment with a non-selective monoamine uptake inhibitor (RTI-112) and a dopamine-selective uptake inhibitor (RTI-113) on cocaine- and food-maintained responding in rhesus monkeys. Monkeys (N=3) were trained to respond for cocaine injections (0.01 mg/kg/inj) and food pellets under a second-order schedule [FR2(VR16:S)] during alternating daily components of cocaine and food availability. Both RTI-112 (0.0032-0.01 mg/kg/hr) and RTI-113 (0.01-0.056 mg/kg/h) produced dose-dependent, sustained and nearly complete elimination of cocaine self-administration. However, for both drugs, the potency to reduce cocaine self-administration was similar to the potency to reduce food-maintained responding. These findings do not support the hypothesis that pharmacological selectivity to block dopamine uptake is associated with behavioral selectivity to decrease cocaine- vs. food-maintained responding in rhesus monkeys.

  15. A mitochondrial GABA permease connects the GABA shunt and the TCA cycle, and is essential for normal carbon metabolism.

    PubMed

    Michaeli, Simon; Fait, Aaron; Lagor, Kelly; Nunes-Nesi, Adriano; Grillich, Nicole; Yellin, Ayelet; Bar, Dana; Khan, Munziba; Fernie, Alisdair R; Turano, Frank J; Fromm, Hillel

    2011-08-01

    In plants, γ-aminobutyric acid (GABA) accumulates in the cytosol in response to a variety of stresses. GABA is transported into mitochondria, where it is catabolized into TCA cycle or other intermediates. Although there is circumstantial evidence for mitochondrial GABA transporters in eukaryotes, none have yet been identified. Described here is an Arabidopsis protein similar in sequence and topology to unicellular GABA transporters. The expression of this protein complements a GABA-transport-deficient yeast mutant. Thus the protein was termed AtGABP to indicate GABA-permease activity. In vivo localization of GABP fused to GFP and immunobloting of subcellular fractions demonstrate its mitochondrial localization. Direct [(3) H]GABA uptake measurements into isolated mitochondria revealed impaired uptake into mitochondria of a gabp mutant compared with wild-type (WT) mitochondria, implicating AtGABP as a major mitochondrial GABA carrier. Measurements of CO(2) release, derived from radiolabeled substrates in whole seedlings and in isolated mitochondria, demonstrate impaired GABA-derived input into the TCA cycle, and a compensatory increase in TCA cycle activity in gabp mutants. Finally, growth abnormalities of gabp mutants under limited carbon availability on artificial media, and in soil under low light intensity, combined with their metabolite profiles, suggest an important role for AtGABP in primary carbon metabolism and plant growth. Thus, AtGABP-mediated transport of GABA from the cytosol into mitochondria is important to ensure proper GABA-mediated respiration and carbon metabolism. This function is particularly essential for plant growth under conditions of limited carbon.

  16. Selective serotonin re-uptake inhibitors potentiate gene blunting induced by repeated methylphenidate treatment: Zif268 versus Homer1a.

    PubMed

    Van Waes, Vincent; Vandrevala, Malcolm; Beverley, Joel; Steiner, Heinz

    2014-11-01

    There is a growing use of psychostimulants, such as methylphenidate (Ritalin; dopamine re-uptake inhibitor), for medical treatments and as cognitive enhancers in the healthy. Methylphenidate is known to produce some addiction-related gene regulation. Recent findings in animal models show that selective serotonin re-uptake inhibitors (SSRIs), including fluoxetine, can potentiate acute induction of gene expression by methylphenidate, thus indicating an acute facilitatory role for serotonin in dopamine-induced gene regulation. We investigated whether repeated exposure to fluoxetine, in conjunction with methylphenidate, in adolescent rats facilitated a gene regulation effect well established for repeated exposure to illicit psychostimulants such as cocaine-blunting (repression) of gene inducibility. We measured, by in situ hybridization histochemistry, the effects of a 5-day repeated treatment with methylphenidate (5 mg/kg), fluoxetine (5 mg/kg) or a combination on the inducibility (by cocaine) of neuroplasticity-related genes (Zif268, Homer1a) in the striatum. Repeated methylphenidate treatment alone produced minimal gene blunting, while fluoxetine alone had no effect. In contrast, fluoxetine added to methylphenidate robustly potentiated methylphenidate-induced blunting for both genes. This potentiation was widespread throughout the striatum, but was most robust in the lateral, sensorimotor striatum, thus mimicking cocaine effects. For illicit psychostimulants, blunting of gene expression is considered part of the molecular basis of addiction. Our results thus suggest that SSRIs, such as fluoxetine, may increase the addiction liability of methylphenidate.

  17. The Janus kinase 2 inhibitor fedratinib inhibits thiamine uptake: a putative mechanism for the onset of Wernicke's encephalopathy.

    PubMed

    Zhang, Qiang; Zhang, Yan; Diamond, Sharon; Boer, Jason; Harris, Jennifer J; Li, Yu; Rupar, Mark; Behshad, Elham; Gardiner, Christine; Collier, Paul; Liu, Phillip; Burn, Timothy; Wynn, Richard; Hollis, Gregory; Yeleswaram, Swamy

    2014-10-01

    The clinical development of fedratinib, a Janus kinase (JAK2) inhibitor, was terminated after reports of Wernicke's encephalopathy in myelofibrosis patients. Since Wernicke's encephalopathy is induced by thiamine deficiency, investigations were conducted to probe possible mechanisms through which fedratinib may lead to a thiamine-deficient state. In vitro studies indicate that fedratinib potently inhibits the carrier-mediated uptake and transcellular flux of thiamine in Caco-2 cells, suggesting that oral absorption of dietary thiamine is significantly compromised by fedratinib dosing. Transport studies with recombinant human thiamine transporters identified the individual human thiamine transporter (hTHTR2) that is inhibited by fedratinib. Inhibition of thiamine uptake appears unique to fedratinib and is not shared by marketed JAK inhibitors, and this observation is consistent with the known structure-activity relationship for the binding of thiamine to its transporters. The results from these studies provide a molecular basis for the development of Wernicke's encephalopathy upon fedratinib treatment and highlight the need to evaluate interactions of investigational drugs with nutrient transporters in addition to classic xenobiotic transporters.

  18. Acute psychological effects of 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") are attenuated by the serotonin uptake inhibitor citalopram.

    PubMed

    Liechti, M E; Baumann, C; Gamma, A; Vollenweider, F X

    2000-05-01

    3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") is a recreational drug that has been shown to release serotonin (5-HT) and dopamine (DA) in animals. The effect of MDMA on 5-HT release can be blocked by 5-HT uptake inhibitors such as citalopram, suggesting that MDMA interacts with the 5-HT uptake site. It is unknown whether this mechanism is also responsible for the psychological effects of MDMA in humans. We investigated the effect of citalopram pretreatment (40 mg iv) on the psychological effects of MDMA (1.5 mg/kg po) in a double-blind placebo-controlled psychometric study in 16 healthy human volunteers. MDMA produced an emotional state with heightened mood, increased self-confidence and extroversion, moderate derealization, and an intensification of sensory perception. Most of these effects were markedly reduced by citalopram. This finding suggests that the psychological effects of MDMA are mediated via action at the 5-HT uptake site to increase 5-HT release through the carrier, as expected from animal studies.

  19. Non-carrier-mediated uptake of the mannosidase I inhibitor 1-deoxymannojirimycin by K562 erythroleukemic cells

    SciTech Connect

    Neefjes, J.J.; Lindhout, J.; Broxterman, H.J.; van der Marel, G.A.; van Boom, J.H.; Ploegh, H.L.

    1989-06-15

    A /sup 3/H label was introduced at the C-1 position of the mannosidase I inhibitor 1-deoxymannojirimycin (dMM) by catalytic hydrogenolysis of benzyl-2,3-O-isopropylidene-5-N-benzyl-6-O-benzyl-alpha-D-mannofurano side with /sup 3/H/sub 2/. 1-(/sup 3/H)dMM as well as its precursor 1-(/sup 3/H)2,3-O-isopropylidene-dMM had identical Rf as the nonradioactive compounds on TLC. Furthermore, alpha 1-antitrypsin secreted by HepG2 cells was modified indistinguishably by treatment of the cells with dMM and 1-(/sup 3/H)dMM. Thus, 1-(/sup 3/H)dMM had chemical and biological properties identical with authentic dMM. Uptake of (/sup 14/C)mannose by K562 cells could be inhibited by glucose but not by the mannose analogue dMM. Thus, dMM does not enter the cell through hexose transporter(s). Uptake of 1-(/sup 3/H)dMM by K562 cells could not be inhibited by increasing concentrations of nonradioactive dMM (from 1-32,000 microM), showing transport of dMM into cells through nonfacilitated diffusion. Furthermore, uptake of 1-(/sup 3/H)dMM by K562 cells was observed at 0/degree/C.

  20. Interaction between neuronal uptake inhibitors and presynaptic serotonin autoreceptors in rat hypothalamic slices: comparison of K+ and electrical depolarization.

    PubMed

    Passarelli, F; Galzin, A M; Langer, S Z

    1987-09-01

    In rat hypothalamic slices prelabeled with [3H]-5-hydroxytryptamine ([3H]-5-HT), exposure to the 5-HT receptor agonist lysergic acid diethylamide (0.1-1 microM) or 5-methoxytryptamine (0.1-10 microM) decreased in a concentration-dependent manner the release of 3H-transmitter elicited by high K+ or electrical stimulation. Exposure to the 5-HT autoreceptor antagonist methiothepin (0.1-1 microM) increased in a concentration-dependent manner the K+ stimulation-evoked overflow of [3H]-5-HT and a similar increase was observed under conditions of electrical stimulation. In contrast, exposure to the nontricyclic 5-HT uptake inhibitor citalopram (0.1-1 microM) did not modify by itself the electrically evoked overflow of [3H]-5-HT, but increased in a concentration-dependent manner the release of 3H-transmitter elicited by K+ stimulation. This effect of citalopram on transmitter release was potentiated when the endogenous stores of 5-HT were depleted by pretreatment with para-chlorophenylalanine methyl ester (300 mg/kg i.p.). Citalopram was shown previously to antagonize the inhibition by lysergic acid diethylamide of the electrically evoked release of [3H]-5-HT in rat hypothalamic slices. Yet, this inhibitor of neuronal uptake of 5-HT did not antagonize the effects of lysergic acid diethylamide when the release of [3H]-5-HT was evoked by K+ depolarization. Electrical stimulation represents a more physiological experimental model for transmitter release than exposure to high K+, and therefore the interaction between 5-HT uptake blockade and presynaptic inhibitory 5-HT autoreceptors, observed in the hypothalamus with electrical stimulation but not with K+ depolarization, remains of biological relevance.

  1. The Urease Inhibitor NBPT Negatively Affects DUR3-mediated Uptake and Assimilation of Urea in Maize Roots.

    PubMed

    Zanin, Laura; Tomasi, Nicola; Zamboni, Anita; Varanini, Zeno; Pinton, Roberto

    2015-01-01

    Despite the widespread use of urease inhibitors in agriculture, little information is available on their effect on nitrogen (N) uptake and assimilation. Aim of this work was to study, at physiological and transcriptional level, the effects of N-(n-butyl) thiophosphoric triamide (NBPT) on urea nutrition in hydroponically grown maize plants. Presence of NBPT in the nutrient solution limited the capacity of plants to utilize urea as a N-source; this was shown by a decrease in urea uptake rate and (15)N accumulation. Noteworthy, these negative effects were evident only when plants were fed with urea, as NBPT did not alter (15)N accumulation in nitrate-fed plants. NBPT also impaired the growth of Arabidopsis plants when urea was used as N-source, while having no effect on plants grown with nitrate or ammonium. This response was related, at least in part, to a direct effect of NBPT on the high affinity urea transport system. Impact of NBPT on urea uptake was further evaluated using lines of Arabidopsis overexpressing ZmDUR3 and dur3-knockout; results suggest that not only transport but also urea assimilation could be compromised by the inhibitor. This hypothesis was reinforced by an over-accumulation of urea and a decrease in ammonium concentration in NBPT-treated plants. Furthermore, transcriptional analyses showed that in maize roots NBPT treatment severely impaired the expression of genes involved in the cytosolic pathway of ureic-N assimilation and ammonium transport. NBPT also limited the expression of a gene coding for a transcription factor highly induced by urea and possibly playing a crucial role in the regulation of its acquisition. This work provides evidence that NBPT can heavily interfere with urea nutrition in maize plants, limiting influx as well as the following assimilation pathway.

  2. The Urease Inhibitor NBPT Negatively Affects DUR3-mediated Uptake and Assimilation of Urea in Maize Roots

    PubMed Central

    Zanin, Laura; Tomasi, Nicola; Zamboni, Anita; Varanini, Zeno; Pinton, Roberto

    2015-01-01

    Despite the widespread use of urease inhibitors in agriculture, little information is available on their effect on nitrogen (N) uptake and assimilation. Aim of this work was to study, at physiological and transcriptional level, the effects of N-(n-butyl) thiophosphoric triamide (NBPT) on urea nutrition in hydroponically grown maize plants. Presence of NBPT in the nutrient solution limited the capacity of plants to utilize urea as a N-source; this was shown by a decrease in urea uptake rate and 15N accumulation. Noteworthy, these negative effects were evident only when plants were fed with urea, as NBPT did not alter 15N accumulation in nitrate-fed plants. NBPT also impaired the growth of Arabidopsis plants when urea was used as N-source, while having no effect on plants grown with nitrate or ammonium. This response was related, at least in part, to a direct effect of NBPT on the high affinity urea transport system. Impact of NBPT on urea uptake was further evaluated using lines of Arabidopsis overexpressing ZmDUR3 and dur3-knockout; results suggest that not only transport but also urea assimilation could be compromised by the inhibitor. This hypothesis was reinforced by an over-accumulation of urea and a decrease in ammonium concentration in NBPT-treated plants. Furthermore, transcriptional analyses showed that in maize roots NBPT treatment severely impaired the expression of genes involved in the cytosolic pathway of ureic-N assimilation and ammonium transport. NBPT also limited the expression of a gene coding for a transcription factor highly induced by urea and possibly playing a crucial role in the regulation of its acquisition. This work provides evidence that NBPT can heavily interfere with urea nutrition in maize plants, limiting influx as well as the following assimilation pathway. PMID:26635834

  3. Loss of cortical GABA terminals in Unverricht-Lundborg disease.

    PubMed

    Buzzi, Andrea; Chikhladze, Maia; Falcicchia, Chiara; Paradiso, Beatrice; Lanza, Giovanni; Soukupova, Marie; Marti, Matteo; Morari, Michele; Franceschetti, Silvana; Simonato, Michele

    2012-08-01

    Unverricht-Lundborg disease (ULD) is the most common progressive myoclonic epilepsy. Its etiology has been identified in a defect of a protease inhibitor, cystatin B (CSTB), but the mechanism(s) by which this defect translates in the clinical manifestations of the disease are still obscure. We tested the hypothesis that ULD is accompanied by a loss of cortical GABA inhibition in a murine model (the CSTB knockout mouse) and in a human case. Cortical GABA signaling has been investigated measuring VGAT immunohistochemistry (a histological marker of the density of GABA terminals), GABA release from synaptosomes and paired-pulse stimulation. In CSTB knockout mice, a progressive decrease in neocortex thickness was found, associated with a prevalent loss of GABA interneurons. A marked reduction in VGAT labeling was found in the cortex of both CSTB knockout mice and an ULD patient. This implicates a reduction in GABA synaptic transmission, which was confirmed in the mouse model as reduction in GABA release from isolated nerve terminals and as loss of electrophysiologically measured GABA inhibition. The alterations in VGAT immunolabeling progressed in time, paralleling the worsening of myoclonus. These results provide direct evidence that loss of cortical GABA input occurs in a relevant animal model and in a case of human ULD, leading to a condition of latent hyperexcitability that favors myoclonus and seizures. These findings contribute to the understanding of the pathogenic mechanism of ULD and of the neurobiological basis of the effect of currently employed drugs.

  4. Manganese exposure inhibits the clearance of extracellular GABA and influences taurine homeostasis in the striatum of developing rats

    PubMed Central

    Fordahl, Steve C.; Anderson, Joel G.; Cooney, Paula T.; Weaver, Tara L.; Colyer, Christa L.; Erikson, Keith M.

    2010-01-01

    Manganese (Mn) accumulation in the brain has been shown to alter the neurochemistry of the basal ganglia. Mn-induced alterations in dopamine biology are fairly well understood, but recently more evidence has emerged characterizing the role of γ-aminobutyric acid (GABA) in this dysfunction. The purpose of this study was to determine if the previously observed Mn-induced increase in extracellular GABA (GABAEC) was due to altered GABA transporter (GAT) function, and whether Mn perturbs other amino acid neurotransmitters, namely taurine and glycine (known modulators of GABA). Extracellular GABA, taurine, and glycine concentrations were collected from the striatum of control (CN) or Mn-exposed Sprague-Dawley rats using in vivo microdialysis, and the GAT inhibitor nipecotic acid (NA) was used to probe GAT function. Tissue and extracellular Mn levels were significantly increased, and the Fe:Mn ratio was decreased 36-fold in the extracellular space due to Mn exposure. NA led to a 2-fold increase in GABAEC of CNs, a response that was attenuated by Mn. Taurine responded inversely to GABA, and a novel 10-fold increase in taurine was observed after the removal of NA in CNs. Mn blunted this response and nearly abolished extracellular taurine throughout collection. Striatal taurine transporter (Slc6a6) mRNA levels were significantly increased with Mn exposure, and Mn significantly increased 3H-Taurine uptake after 3-minute exposure in primary rat astrocytes. These data suggest that Mn increases GABAEC by inhibiting the function of GAT, and that perturbed taurine homeostasis potentially impacts neural function by jeopardizing the osmoregulatory and neuromodulatory functions of taurine in the brain. PMID:20832424

  5. Enhancement of muscle glucose uptake by the vasopeptidase inhibitor, omapatrilat, is independent of insulin signaling and the AMP kinase pathway.

    PubMed

    Wong, Victor; Szeto, Linda; Uffelman, Kristine; Fantus, I George; Lewis, Gary F

    2006-08-01

    Omapatrilat (OMA), a vasopeptidase inhibitor (VPI), presently being tested in clinical trials for its antihypertensive properties, inhibits both angiotensin-converting enzyme and neutral endopeptidase, and raises tissue bradykinin levels. Recent studies from our laboratory and those of others have demonstrated that VPIs enhance muscle glucose uptake in animal models, and this effect is mediated by the bradykinin-nitric oxide pathway. The mechanism of the effect of OMA on muscle glucose uptake, however, is presently unknown. To investigate the effect of OMA on insulin signaling, soleus muscle was isolated 2 or 5 min after an i.v. bolus of insulin or saline from male Zucker fatty rats (8-10 weeks of age), following a 5-day treatment period of oral OMA (15 mg/kg per day) or drug vehicle (placebo). OMA resulted in significantly lower systolic blood pressure compared with the placebo-treated group (84.4+/- 7.52 mmHg in OMA vs 112+/-2.18 mmHg in controls, P<0.01). Immunoprecipitation and Western blot analysis of insulin receptor substrate 1 (IRS-1) revealed no changes in protein mass with OMA treatment. OMA did not enhance basal or insulin-stimulated IRS-1 tyrosine phosphorylation or its subsequent association with the p85 regulatory subunit of phosphatidylinositol 3-kinase. Under basal and insulin-stimulated conditions, OMA treatment did not alter the protein mass or the phosphorylation of Akt/protein kinase B, p42/44 extracellular signal-regulated kinase or adenosine monophosphate-activated protein kinase, or GLUT4 protein expression. We conclude that the ability of OMA to enhance whole body and specifically muscle glucose uptake in Zucker fatty rats is not mediated by enhancing insulin or AMPK signaling. Future studies should examine whether hemodynamic effects of the drug, independent of insulin signaling, enhance glucose uptake in insulin-resistant skeletal muscle.

  6. Extracellular conversion of guanine-based purines to guanosine specifically enhances astrocyte glutamate uptake.

    PubMed

    Frizzo, Marcos Emílio dos Santos; Antunes Soares, Félix Alexandre; Dall'Onder, Leonara Patrícia; Lara, Diogo Rizzato; Swanson, Raymond A; Souza, Diogo Onofre

    2003-05-16

    Guanosine (GUO) has been shown to stimulate glutamate uptake in primary astrocyte cultures. The purpose of this study was to determine the effect and specificity of guanine- or adenine-based purines on glutamate and GABA uptake in cultured astrocytes. Stimulatory effect on glutamate uptake was observed with GUO, GMP or GTP. Simultaneous exposure with these guanine-based purines did not show an additive effect. We also investigated a possible interconversion of guanine-based purines during incubation time. Action by GTP was excluded since the hydrolysis resistant GTP analog, GMP-PNP did not stimulate glutamate uptake. Addition of an ecto-5'-nucleotidase inhibitor abolished GMP-stimulatory effect on glutamate uptake, without affecting GUO action. Taken together, these results suggest that GUO is the guanine-based purines responsible for glutamate uptake activation. In addition, the stimulatory effect on glutamate uptake was not observed with adenine-based purines. Moreover, GABA uptake was not activated by GUO. These results point to specificity in the interaction between GUO and the astrocyte glutamate uptake system.

  7. Localization and expression of GABA transporters in the suprachiasmatic nucleus

    PubMed Central

    Moldavan, Michael; Cravetchi, Olga; Williams, Melissa; Irwin, Robert P.; Aicher, Sue A.; Allen, Charles N.

    2015-01-01

    GABA is a principal neurotransmitter in the suprachiasmatic hypothalamic nucleus (SCN), the master circadian clock. Despite the importance of GABA and GABA uptake for functioning of the circadian pacemaker, the localization and expression of GABA transporters (GATs) in the SCN has not been investigated. The present studies used Western blot analysis, immunohistochemistry, and electron microscopy to demonstrate the presence of GABA transporter 1 (GAT1) and GABA transporter 3 (GAT3) in the SCN. By light microscopy, GAT1 and GAT3 were co-localized throughout the SCN, but were not expressed in the perikarya of arginine vasopressin- or vasoactive intestinal peptide-immunoreactive (−ir) neurons of adult rats, nor in the neuronal processes labeled with the Neurofilament Heavy Chain. By electron microscopy, GAT1- and GAT3-ir was found in glial processes surrounding unlabeled neuronal perikarya, axons, dendrites, and enveloped symmetric and asymmetric axo-dendritic synapses. Glial Fibrillary Acidic Protein-ir astrocytes grown in cell culture were immunopositive for GAT1 and GAT3 – and both GATs could be observed in the same glial cell. These data demonstrate that synapses in the SCN function as “tripartite” synapses consisting of presynaptic axon terminals, postsynaptic membranes, and astrocytes that contain GABA transporters. This model suggests that astrocytes expressing both GATs may regulate the extracellular GABA, and thereby modulate the activity of neuronal networks in the SCN. PMID:26390912

  8. Treating depression in coronary artery disease and chronic heart failure: what's new in using selective serotonin re-uptake inhibitors?

    PubMed

    Paraskevaidis, Ioannis; Palios, John; Parissis, John; Filippatos, Gerasimos; Anastasiou-Nana, Maria

    2012-06-01

    Depression is a common co-morbidity in patients with cardiovascular diseases such as chronic coronary artery disease, acute coronary syndromes, post by-pass surgery and chronic heart failure. The presence of depression is independently associated with a decline in health status and an increase in the risk of hospitalization and death for patients with coronary artery disease or congestive heart failure. Novel treatment modalities such as selective serotonin re-uptake inhibitors (SSRIs) may improve depressive symptoms and prognosis of post-myocardial infarction and heart failure patients interacting with the common pathophysiological mechanisms of depression and cardiovascular disease. This review summarizes current experimental and clinical evidence regarding the effects of SSRIs on platelet functions, immune and neurohormonal activation, and cardiac rhythm disturbances in patients with cardiovascular disease.

  9. A defect in sodium-dependent amino acid uptake in diabetic rabbit peripheral nerve. Correction by an aldose reductase inhibitor or myo-inositol administration.

    PubMed Central

    Greene, D A; Lattimer, S A; Carroll, P B; Fernstrom, J D; Finegold, D N

    1990-01-01

    A myo-inositol-related defect in nerve sodium-potassium ATPase activity in experimental diabetes has been suggested as a possible pathogenetic factor in diabetic neuropathy. Because the sodium-potassium ATPase is essential for other sodium-cotransport systems, and because myo-inositol-derived phosphoinositide metabolites regulate multiple membrane transport processes, sodium gradient-dependent amino acid uptake was examined in vitro in endoneurial preparations derived from nondiabetic and 14-d alloxan diabetic rabbits. Untreated alloxan diabetes reduced endoneurial sodium-gradient dependent uptake of the nonmetabolized amino acid 2-aminoisobutyric acid by greater than 50%. Administration of an aldose reductase inhibitor prevented reductions in both nerve myo-inositol content and endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Myo-inositol supplementation that produced a transient pharmacological elevation in plasma myo-inositol concentration, but did not raise nerve myo-inositol content, reproduced the effect of the aldose reductase inhibitor on endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Phorbol myristate acetate, which acutely normalizes sodium-potassium ATPase activity in diabetic nerve, did not acutely correct 2-aminoisobutyric uptake when added in vitro. These data suggest that depletion of a small myo-inositol pool may be implicated in the pathogenesis of defects in amino acid uptake in diabetic nerve and that rapid correction of sodium-potassium ATPase activity with protein kinase C agonists in vitro does not acutely normalize sodium-dependent 2-aminoisobutyric acid uptake. PMID:2185278

  10. Cerebral uptake of mefloquine enantiomers with and without the P-gp inhibitor elacridar (GF1210918) in mice

    PubMed Central

    de Lagerie, Sylvie Barraud; Comets, Emmanuelle; Gautrand, Céline; Fernandez, Christine; Auchere, Daniel; Singlas, Eric; Mentre, France; Gimenez, François

    2004-01-01

    Mefloquine is a chiral neurotoxic antimalarial agent showing stereoselective brain uptake in humans and rats. It is a substrate and an inhibitor of the efflux protein P-glycoprotein. We investigated the stereoselective uptake and efflux of mefloquine in mice, and the consequences of the combination with an efflux protein inhibitor, elacridar (GF120918) on its brain transport. Racemic mefloquine (25 mg kg−1) was administered intraperitoneally with or without elacridar (10 mg kg−1). Six to seven mice were killed at each of 11 time-points between 30 min and 168 h after administration. Blood and brain concentrations of mefloquine enantiomers were determined using liquid chromatography. A three-compartment model with zero-order absorption from the injection site was found to best represent the pharmacokinetics of both enantiomers in blood and brain. (−)Mefloquine had a lower blood and brain apparent volume of distribution and a lower efflux clearance from the brain, resulting in a larger brain/blood ratio compared to (+)mefloquine. Elacridar did not modify blood concentrations or the elimination rate from blood for either enantiomers. However, cerebral AUCinf of both enantiomers were increased, with a stronger effect on (+)mefloquine. The efflux clearance from the brain decreased for both enantiomers, with a larger decrease for (+)mefloquine. After administration of racemic mefloquine in mice, blood and brain pharmacokinetics are stereoselective, (+)mefloquine being excreted from brain more rapidly than its antipode, showing that mefloquine is a substrate of efflux proteins and that mefloquine enantiomers undergo efflux in a stereoselective manner. Moreover, pretreatment with elacridar reduced the brain efflux clearances with a more pronounced effect on (+)mefloquine. PMID:15023856

  11. Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.

    PubMed

    Poutiainen, Pekka K; Rönkkö, Teemu; Hinkkanen, Ari E; Palvimo, Jorma J; Närvänen, Ale; Turhanen, Petri; Laatikainen, Reino; Weisell, Janne; Pulkkinen, Juha T

    2014-01-15

    In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 μM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.

  12. Multi-kinase inhibitors induce cutaneous toxicity through OAT6-mediated uptake and MAP3K7-driven cell death

    PubMed Central

    Zimmerman, Eric I.; Gibson, Alice A.; Hu, Shuiying; Vasilyeva, Aksana; Orwick, Shelley J.; Du, Guoqing; Mascara, Gerard P.; Ong, Su Sien; Chen, Taosheng; Vogel, Peter; Inaba, Hiroto; Maitland, Michael L.; Sparreboom, Alex; Baker, Sharyn D.

    2015-01-01

    The use of multi-kinase inhibitors (MKI) in oncology, such as sorafenib, is associated with a cutaneous adverse event called hand-foot skin reaction (HFSR) in which sites of pressure or friction become inflamed and painful, thus significantly impacting quality of life. The pathogenesis of MKI-induced HFSR is unknown, and the only available treatment options involve dose reduction or discontinuation of therapy, which have negative effects on primary disease management. To investigate the underlying mechanisms by which sorafenib promotes keratinocyte cytotoxicity and subsequent HFSR induction, we performed a transporter-directed RNAi screen in human epidermal keratinocytes and identified SLC22A20 (OAT6) as an uptake carrier of sorafenib. Further investigations into the intracellular mechanism of sorafenib activity through in situ kinome profiling identified the mitogen-activated protein kinase MAP3K7 (TAK1) as a target of sorafenib that induces cell death. Finally, we demonstrate that sorafenib induced keratinocyte injury in vivo, and that this effect could be reversed by co-treatment with the OAT6 inhibitor probenecid. Collectively, our findings reveal a novel pathway that regulates the entry of some MKIs into keratinocytes and explains the basis underlying sorafenib-induced skin toxicity, with important implications for the therapeutic management of HFSR. PMID:26677977

  13. Multikinase Inhibitors Induce Cutaneous Toxicity through OAT6-Mediated Uptake and MAP3K7-Driven Cell Death.

    PubMed

    Zimmerman, Eric I; Gibson, Alice A; Hu, Shuiying; Vasilyeva, Aksana; Orwick, Shelley J; Du, Guoqing; Mascara, Gerard P; Ong, Su Sien; Chen, Taosheng; Vogel, Peter; Inaba, Hiroto; Maitland, Michael L; Sparreboom, Alex; Baker, Sharyn D

    2016-01-01

    The use of multikinase inhibitors (MKI) in oncology, such as sorafenib, is associated with a cutaneous adverse event called hand-foot skin reaction (HFSR), in which sites of pressure or friction become inflamed and painful, thus significantly impacting quality of life. The pathogenesis of MKI-induced HFSR is unknown, and the only available treatment options involve dose reduction or discontinuation of therapy, which have negative effects on primary disease management. To investigate the underlying mechanisms by which sorafenib promotes keratinocyte cytotoxicity and subsequent HFSR induction, we performed a transporter-directed RNAi screen in human epidermal keratinocytes and identified SLC22A20 (OAT6) as an uptake carrier of sorafenib. Further investigations into the intracellular mechanism of sorafenib activity through in situ kinome profiling identified the mitogen-activated protein kinase MAP3K7 (TAK1) as a target of sorafenib that induces cell death. Finally, we demonstrate that sorafenib induced keratinocyte injury in vivo and that this effect could be reversed by cotreatment with the OAT6 inhibitor probenecid. Collectively, our findings reveal a novel pathway that regulates the entry of some MKIs into keratinocytes and explains the basis underlying sorafenib-induced skin toxicity, with important implications for the therapeutic management of HFSR.

  14. Selective CNS Uptake of the GCP-II Inhibitor 2-PMPA following Intranasal Administration

    PubMed Central

    Rais, Rana; Wozniak, Krystyna; Wu, Ying; Niwa, Minae; Stathis, Marigo; Alt, Jesse; Giroux, Marc; Sawa, Akira; Rojas, Camilo; Slusher, Barbara S.

    2015-01-01

    Glutamate carboxypeptidase II (GCP-II) is a brain metallopeptidase that hydrolyzes the abundant neuropeptide N-acetyl-aspartyl-glutamate (NAAG) to NAA and glutamate. Small molecule GCP-II inhibitors increase brain NAAG, which activates mGluR3, decreases glutamate, and provide therapeutic utility in a variety of preclinical models of neurodegenerative diseases wherein excess glutamate is presumed pathogenic. Unfortunately no GCP-II inhibitor has advanced clinically, largely due to their highly polar nature resulting in insufficient oral bioavailability and limited brain penetration. Herein we report a non-invasive route for delivery of GCP-II inhibitors to the brain via intranasal (i.n.) administration. Three structurally distinct classes of GCP-II inhibitors were evaluated including DCMC (urea-based), 2-MPPA (thiol-based) and 2-PMPA (phosphonate-based). While all showed some brain penetration following i.n. administration, 2-PMPA exhibited the highest levels and was chosen for further evaluation. Compared to intraperitoneal (i.p.) administration, equivalent doses of i.n. administered 2-PMPA resulted in similar plasma exposures (AUC0-t, i.n./AUC0-t, i.p. = 1.0) but dramatically enhanced brain exposures in the olfactory bulb (AUC0-t, i.n./AUC0-t, i.p. = 67), cortex (AUC0-t, i.n./AUC0-t, i.p. = 46) and cerebellum (AUC0-t, i.n./AUC0-t, i.p. = 6.3). Following i.n. administration, the brain tissue to plasma ratio based on AUC0-t in the olfactory bulb, cortex, and cerebellum were 1.49, 0.71 and 0.10, respectively, compared to an i.p. brain tissue to plasma ratio of less than 0.02 in all areas. Furthermore, i.n. administration of 2-PMPA resulted in complete inhibition of brain GCP-II enzymatic activity ex-vivo confirming target engagement. Lastly, because the rodent nasal system is not similar to humans, we evaluated i.n. 2-PMPA also in a non-human primate. We report that i.n. 2-PMPA provides selective brain delivery with micromolar concentrations. These studies support

  15. Different transporter systems regulate extracellular GABA from vesicular and non-vesicular sources

    PubMed Central

    Song, Inseon; Volynski, Kirill; Brenner, Tanja; Ushkaryov, Yuri; Walker, Matthew; Semyanov, Alexey

    2013-01-01

    Tonic GABA type A (GABAA) conductance is a key factor regulating neuronal excitability and computation in neuronal networks. The magnitude of the tonic GABAA conductance depends on the concentration of ambient GABA originating from vesicular and non-vesicular sources and is tightly regulated by GABA uptake. Here we show that the transport system regulating ambient GABA responsible for tonic GABAA conductances in hippocampal CA1 interneurons depends on its source. In mice, GABA from vesicular sources is regulated by mouse GABA transporter 1 (mGAT1), while that from non-vesicular sources by mouse GABA transporters 3/4 (mGAT3/4). This finding suggests that the two transporter systems do not just provide backup for each other, but regulate distinct signaling pathways. This allows individual tuning of the two signaling systems and indicates that drugs designed to act at specific transporters will have distinct therapeutic actions. PMID:23494150

  16. Modulation of GABA-stimulated chloride influx into membrane vesicles from rat cerebral cortex by triazolobenzodiazepines

    SciTech Connect

    Obata, T.; Yamamura, H.I.

    1988-01-01

    The effects of triazolobenzodiazepines of GABA-stimulated /sup 36/Cl/sup -/ uptake by membrane vesicles from rat cerebral cortex were examined. Triazolam and alprazolam showed a significant enhancement of GABA-stimulated /sup 36/Cl/sup -/ uptake at 0.01-10 uM. On the other hand, adinazolam showed a small enhancement at 0.1-1 uM followed by a significant inhibition of GABA-stimulated /sup 36/Cl/sup -/ uptake at 100 uM. The enhancement of GABA-stimulated /sup 36/Cl/sup -/ uptake by 1 uM alprazolam was antagonized by Ro15-1788, a benzodiazepine antagonist, but the inhibition of this response by 30 uM adinazolam was not antagonized by Ro15-1788. These results indicate that triazolobenzodiazepines enhanced GABA-stimulated /sup 36/Cl/sup -/ uptake through benzodiazepine receptors. High concentrations of adinazolam inhibit GABA-stimulated /sup 36/Cl/sup -/ uptake which may be due to the direct blockade of GABA-gated chloride channel. 23 references, 4 figures.

  17. Involvement of γ-aminobutyric acid transporter 2 in the hepatic uptake of taurine in rats.

    PubMed

    Ikeda, Saori; Tachikawa, Masanori; Akanuma, Shin-ichi; Fujinawa, Jun; Hosoya, Ken-ichi

    2012-08-01

    Taurine is essential for the hepatic synthesis of bile salts and, although taurine is synthesized mainly in pericentral hepatocytes, taurine and taurine-conjugated bile acids are abundant in periportal hepatocytes. One possible explanation for this discrepancy is that the active supply of taurine to hepatocytes from the blood stream is a key regulatory factor. The purpose of the present study is to investigate and identify the transporter responsible for taurine uptake by periportal hepatocytes. An in vivo bolus injection of [(3)H]taurine into the rat portal vein demonstrated that 25% of the injected [(3)H]taurine was taken up by the liver on a single pass. The in vivo uptake was significantly inhibited by GABA, taurine, β-alanine, and nipecotic acid, a GABA transporter (GAT) inhibitor, each at a concentration of 10 mM. The characteristics of Na(+)- and Cl(-)-dependent [(3)H]taurine uptake by freshly isolated rat hepatocytes were consistent with those of GAT2 (solute carrier SLC6A13). Indeed, the K(m) value of the saturable uptake (594 μM) was close to that of mouse SLC6A13-mediated taurine transport. Although GABA, taurine, and β-alanine inhibited the [(3)H]taurine uptake by > 50%, each at a concentration of 10 mM, GABA caused a marked inhibition with an IC(50) value of 95 μM. The [(3)H]taurine uptake exhibited a significant reduction when the GAT2 gene was silenced. Immunohistochemical analysis showed that GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. These results suggest that GAT2 is responsible for taurine transport from the circulating blood to hepatocytes predominantly in the periportal region.

  18. Amitifadine, a triple monoamine re-uptake inhibitor, reduces nicotine self-administration in female rats

    PubMed Central

    Levin, Edward D; Wells, Corinne; Johnson, Joshua E; Rezvani, Amir H.; Bymaster, Frank P.; Rose, Jed E.

    2016-01-01

    A wider diversity of drug treatments to aid smoking cessation is needed to help tailor the most efficacious treatment for different types of smokers. This study was conducted to determine whether amitifadine, which inhibits re-uptake of dopamine, norepinephrine and serotonin, would decrease nicotine self-administration at doses that do not cause adverse side effects. Adult female Sprague-Dawley rats were trained to self-administer nicotine intravenous (IV) and were given acute doses of amitifadine in a repeated measures counterbalanced design. Effects of amitifadine on locomotor activity and food motivated responding were also evaluated. Chronic amitifadine effects were also examined. The 30 mg/kg amitifadine dose significantly reduced nicotine self-administration. The 5 and 10 mg/kg doses reduced nicotine self-administration during the first 15 min. of the session when the greatest amount of nicotine was self-administered. The 30 mg/kg amitifadine dose, but not the lower doses caused a significant reduction in locomotor activity averaged over the 1-hour session and reduced food motivated responding. The 10 mg/kg dose caused hypoactivity at the beginning of the session, but 5 mg/kg did not cause any hypoactivity. The effects of chronic amitifadine treatment (10 mg/kg) over the course of 15 sessions was also determined. Amitifadine caused a significant reduction in nicotine self-administration, which was not seen to diminish over two consecutive weeks of treatment and a week after enforced abstinence. Amitifadine significantly reduced nicotine self-administration. This prompts further research to determine if amitifadine might be an effective treatment for smoking cessation. PMID:26101069

  19. Amitifadine, a triple monoamine re-uptake inhibitor, reduces nicotine self-administration in female rats.

    PubMed

    Levin, Edward D; Wells, Corinne; Johnson, Joshua E; Rezvani, Amir H; Bymaster, Frank P; Rose, Jed E

    2015-10-05

    A wider diversity of drug treatments to aid smoking cessation is needed to help tailor the most efficacious treatment for different types of smokers. This study was conducted to determine whether amitifadine, which inhibits re-uptake of dopamine, norepinephrine and serotonin, would decrease nicotine self-administration at doses that do not cause adverse side effects. Adult female Sprague-Dawley rats were trained to self-administer nicotine intravenous (IV) and were given acute doses of amitifadine in a repeated measures counterbalanced design. Effects of amitifadine on locomotor activity and food motivated responding were also evaluated. Chronic amitifadine effects were also examined. The 30 mg/kg amitifadine dose significantly reduced nicotine self-administration. The 5 and 10 mg/kg doses reduced nicotine self-administration during the first 15 min of the session when the greatest amount of nicotine was self-administered. The 30 mg/kg amitifadine dose, but not the lower doses caused a significant reduction in locomotor activity averaged over the one-hour session and reduced food motivated responding. The 10 mg/kg dose caused hypoactivity at the beginning of the session, but 5 mg/kg did not cause any hypoactivity. The effects of chronic amitifadine treatment (10 mg/kg) over the course of 15 sessions was also determined. Amitifadine caused a significant reduction in nicotine self-administration, which was not seen to diminish over two consecutive weeks of treatment and a week after enforced abstinence. Amitifadine significantly reduced nicotine self-administration. This prompts further research to determine if amitifadine might be an effective treatment for smoking cessation.

  20. Crucial interactions between selective serotonin uptake inhibitors and sigma-1 receptor in heart failure.

    PubMed

    Bhuiyan, Md Shenuarin; Tagashira, Hideaki; Fukunaga, Kohji

    2013-01-01

    Depression is associated with a substantial increase in the risk of developing heart failure and is independently associated with increased cardiovascular morbidity and mortality. Inversely, cardiovascular disease can lead to severe depression. Thus, therapy with selective serotonin reuptake inhibitors (SSRIs) is strongly recommended to reduce cardiovascular disease-induced morbidity and mortality. However, molecular mechanisms to support evidence-based SSRI treatment of cardiovascular disease have not been elucidated. We recently found very high expression of the sigma-1 receptor, an orphan receptor, in rat heart tissue and defined the cardiac sigma-1 receptor as a direct SSRI target in eliciting cardioprotection in both pressure overload (PO)induced and transverse aortic constriction (TAC)-induced myocardial hypertrophy models in rodents. Our findings suggest that SSRIs such as fluvoxamine protect against PO- and TAC-induced cardiac dysfunction by upregulating sigma-1 receptor expression and stimulating sigma-1 receptor-mediated Akt-eNOS signaling. Here, we discuss the association of depression and cardiovascular diseases, the protective mechanism of SSRIs in heart failure patients, and the pathophysiological relevance of sigma-1 receptors to progression of heart failure. These findings should promote development of clinical therapeutics targeting the sigma-1 receptor in cardiovascular diseases.

  1. Effects of various serotonin agonists, antagonists, and uptake inhibitors on the discriminative stimulus effects of methamphetamine in rats.

    PubMed

    Munzar, P; Laufert, M D; Kutkat, S W; Nováková, J; Goldberg, S R

    1999-10-01

    Neurochemical studies indicate that methamphetamine increases central serotonin (5-HT) levels more markedly than other psychomotor stimulants such as amphetamine or cocaine. In the present study, we investigated 5-HT involvement in the discriminative stimulus effects of methamphetamine. In Sprague-Dawley rats trained to discriminate 1.0 mg/kg methamphetamine i.p. from saline under a fixed-ratio schedule of food presentation, the effects of selected 5-HT agonists, antagonists, and uptake inhibitors were tested. Fluoxetine (1.8-18.0 mg/kg) and clomipramine (3.0-18.0 mg/kg), selective serotonin uptake inhibitors, did not produce any methamphetamine-like discriminative stimulus effects when administered alone, but fluoxetine (5.6 mg/kg), unlike clomipramine (5.6 mg/kg), significantly shifted the methamphetamine dose-response curve to the left. Both 8-hydroxy-2-dipropylaminotetralin (0.03-0.56 mg/kg), a full agonist, and buspirone (1.0-10.0 mg/kg), a partial agonist at 5-HT(1A) receptors, partially generalized to the training dose of methamphetamine but only at high doses that decreased response rate. This generalization was antagonized by the coadministration of the 5-HT(1A) antagonist WAY-100635 (1.0 mg/kg). WAY-100635 (1.0 mg/kg) also partially reversed the leftward shift of the methamphetamine dose-response curve produced by fluoxetine. (+/-)-1-(2, 5-Dimethoxy-4-iodophenyl)-2-aminopropane (0.3 mg/kg), a 5-HT(2A/2C) agonist, shifted the methamphetamine dose-response curve to the left, and this leftward shift was antagonized by the coadministration of ketanserin (3.0 mg/kg), a 5-HT(2A/2C) antagonist. Ketanserin (3.0 mg/kg) also produced a shift to the right in the methamphetamine dose-response curve and completely reversed the leftward shift in the methamphetamine dose-response curve produced by fluoxetine. In contrast, tropisetron (1.0 mg/kg), a 5-HT(3) antagonist, produced a shift to the left of the methamphetamine dose-response curve, and this effect of tropisetron

  2. Antagonism of fenfluramine-induced hyperthermia in rats by some, but not all, selective inhibitors of 5-hydroxytryptamine uptake.

    PubMed Central

    Sugrue, M. F.

    1984-01-01

    The injection of fenfluramine (7.5 mg kg-1,i.p.) to rats housed at 27-28 degrees C was associated with an elevation of core body temperature which peaked at approximately 1 h post-injection. One h pretreatment with citalopram (20 mg kg-1, i.p.), chlorimipramine (10 mg kg-1, i.p.), femoxetine (10 mg kg-1, i.p.) and fluoxetine (20 mg kg-1, i.p.) resulted in an attenuated response to fenfluramine. In contrast, Org 6582 (20 mg kg-1) and zimelidine (20 mg kg-1) were devoid of an effect on fenfluramine-induced hyperthermia. The response to fenfluramine was was also blocked by i.p. injections of metergoline (0.2 mg kg-1), methysergide (5 mg kg-1) and mianserin (0.5 mg kg-1). Rectal temperature was unaltered by both the 5-hydroxytryptamine (5-HT) uptake inhibitors and the 5-HT receptor antagonists. The IC50 values (nM) for in vitro inhibition of [3H]-5-HT uptake into rat hypothalamic synaptosomes were for citalopram 2.4, chlorimipramine 8.8, femoxetine 14, fluoxetine 16, Org 6582 75 and zimelidine 250. The injection of all six compounds (20 mg kg-1, i.p.) 1 h before death was associated with an inhibition of [3H]-5-HT uptake into rat hypothalamic synaptosomes which ranged from 47.2% for chlorimipramine to 83.3% for citalopram. Rat hypothalamic 5-HT levels were decreased by approximately 50% 3 h after the injection of fenfluramine (15 mg kg-1, i.p.). This effect was blocked by a 1 h pretreatment with fluoxetine, Org 6582 and zimelidine (all 20 mg kg-1, i.p.). Ki values for displacement of specifically bound [3H]-5-HT (1 nM) to rat hypothalamic membranes were for metergoline 26 nM, methysergide 1.1 microM, mianserin 3.6 microM, chlorimipramine 9.2 microM and fluoxetine 32.7 microM. Values for citalopram, femoxetine, Org 6582 and zimelidine were in excess of 65.4 microM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6722394

  3. Dopamine-dependent hyperactivity in the rat following manipulation of GABA mechanisms in the region of the nucleus accumbens.

    PubMed

    Pycock, C J; Horton, R W

    1979-01-01

    The effect of manipulation of GABA mechanisms in the region of the nucleus accumbens on dopamine-dependent locomotor hyperactivity in the rat has been studied. Two models of hyperactivity were used: (1) the injection of dopamine into the region of the nucleus accumbens in nialamide-pretreated animals and (2) the systemic administration of d-amphetamine. Both GABA and the GABA agonist 3-aminopropane sulphonic acid (3-APS) depressed hyperactivity in a dose-related manner. High concentrations of GABA (greater than 100 micrograms) were required to produce a significant effect and the response was short-lived possibly reflecting the efficient GABA inactivating mechanisms. 3-APS proved to be approximately 10 times more potent as compared to GABA in the dopamine-accumbens hyperactivity model. Conversely GABA receptor antagonism with low doses of either picrotoxin or bicuculline enhanced the mild locomotor response induced by a low dose of dopamine injected into the nucleus accumbens. However such results were difficult to evaluate fairly as higher doses of the GABA antagonists resulted in varying degrees of generalized seizures. Blockade of GABA uptake systems with cis-1, 3-aminocyclohexane carboxylic acid (ACHC), nipecotic acid or beta-alanine within the region of the nucleus accumbens produced dose-related depression of dopamine-dependent hyperactivity in both models. GABA uptake blockade (nipecotic acid) significantly enhanced the GABA-mediated depression of hyperactivity induced by bilateral injection of dopamine into the nucleus accumbens. The results demonstrate an inhibitory action of GABA and drugs facilitating GABA-ergic transmission on dopamine-dependent hyperactivity in the rat. Although open to criticisms of not being able to distinguish between true GABA effects and the results of non-specific neuronal depression the hyperactivity model underlines the potency of the GABA uptake blocking compounds and their possible potential for future clinical use.

  4. Preventative and therapeutic effects of a GABA transporter 1 inhibitor administered systemically in a mouse model of paclitaxel-induced neuropathic pain

    PubMed Central

    Parvathy, Subramanian S.

    2016-01-01

    Background There is a dearth of drugs to manage a dose-limiting painful peripheral neuropathy induced by paclitaxel in some patients during the treatment of cancer. Gamma-aminobutyric acid transporter-1 (GAT-1) whose expression is increased in the brain and spinal cord during paclitaxel-induced neuropathic pain (PINP) might be a potential therapeutic target for managing PINP. Thus, our aim was to evaluate if systemic administration of a GAT-1 inhibitor ameliorates PINP. Methods The reaction latency to thermal stimuli (hot plate test; at 55 °C) and cold stimuli (cold plate test; at 4 °C) of female BALB/c mice was recorded before and after intraperitoneal treatment with paclitaxel, its vehicle, and/or a selective GAT-1 inhibitor NO-711. The effects of NO-711 on motor coordination were evaluated using the rotarod test at a constant speed of 4 rpm or accelerating mode from 4 rpm to 40 rpm over 5 min. Results The coadministration of paclitaxel with NO-711 3 mg/kg prevented the development of paclitaxel-induced thermal hyperalgesia and cold allodynia at day 7 after drug treatment. NO-711 at 3 mg/kg produced antihyperalgesic activity up to 1 h and antiallodynic activity up to 2 h in mice with established paclitaxel-induced thermal hyperalgesia and cold allodynia. No motor deficits were observed with NO-711 at a dose of 3 mg/kg, whereas a higher dose 5 mg/kg caused motor impairment and reduced mean time spent on the rotarod at a constant speed of 4 rpm. However, at a rotarod accelerating mode from 4 rpm to 40 rpm over 5 min, NO-711 3 mg/kg caused motor impairment up to 1 h, but had recovered by 2 h. Conclusions These results show that systemic administration of the GAT-1 inhibitor NO-711 has preventative and therapeutic activity against paclitaxel-induced thermal hyperalgesia and cold allodynia. NO-711’s antiallodynic effects, but not antihyperalgesic effects, were independent of its motor impairment/sedation properties. Thus, low doses of GAT-1 inhibitors

  5. Catabolism of GABA, succinic semialdehyde or gamma-hydroxybutyrate through the GABA shunt impair mitochondrial substrate-level phosphorylation.

    PubMed

    Ravasz, Dora; Kacso, Gergely; Fodor, Viktoria; Horvath, Kata; Adam-Vizi, Vera; Chinopoulos, Christos

    2017-03-11

    GABA is catabolized in the mitochondrial matrix through the GABA shunt, encompassing transamination to succinic semialdehyde followed by oxidation to succinate by the concerted actions of GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH), respectively. Gamma-hydroxybutyrate (GHB) is a neurotransmitter and a psychoactive drug that could enter the citric acid cycle through transhydrogenation with α-ketoglutarate to succinic semialdehyde and d-hydroxyglutarate, a reaction catalyzed by hydroxyacid-oxoacid transhydrogenase (HOT). Here, we tested the hypothesis that the elevation in matrix succinate concentration caused by exogenous addition of GABA, succinic semialdehyde or GHB shifts the equilibrium of the reversible reaction catalyzed by succinate-CoA ligase towards ATP (or GTP) hydrolysis, effectively negating substrate-level phosphorylation (SLP). Mitochondrial SLP was addressed by interrogating the directionality of the adenine nucleotide translocase during anoxia in isolated mouse brain and liver mitochondria. GABA eliminated SLP, and this was rescued by the GABA-T inhibitors vigabatrin and aminooxyacetic acid. Succinic semialdehyde was an extremely efficient substrate energizing mitochondria during normoxia but mimicked GABA in abolishing SLP in anoxia, in a manner refractory to vigabatrin and aminooxyacetic acid. GHB could moderately energize liver but not brain mitochondria consistent with the scarcity of HOT expression in the latter. In line with these results, GHB abolished SLP in liver but not brain mitochondria during anoxia and this was unaffected by either vigabatrin or aminooxyacetic acid. It is concluded that when mitochondria catabolize GABA or succinic semialdehyde or GHB through the GABA shunt, their ability to perform SLP is impaired.

  6. Transport and equilibrium uptake of a peptide inhibitor of PACE4 into articular cartilage is dominated by electrostatic interactions.

    PubMed

    Byun, Sangwon; Tortorella, Micky D; Malfait, Anne-Marie; Fok, Kam; Frank, Eliot H; Grodzinsky, Alan J

    2010-07-01

    The availability of therapeutic molecules to targets within cartilage depends on transport through the avascular matrix. We studied equilibrium partitioning and non-equilibrium transport into cartilage of Pf-pep, a 760 Da positively charged peptide inhibitor of the proprotein convertase PACE4. Competitive binding measurements revealed negligible binding of Pf-pep to sites within cartilage. Uptake of Pf-pep depended on glycosaminoglycan charge density, and was consistent with predictions of Donnan equilibrium given the known charge of Pf-pep. In separate transport experiments, the diffusivity of Pf-pep in cartilage was measured to be approximately 1 x 10(-6) cm(2)/s, close to other similarly-sized non-binding solutes. These results suggest that small positively charged therapeutics will have a higher concentration within cartilage than in the surrounding synovial fluid, a desired property for local delivery; however, such therapeutics may rapidly diffuse out of cartilage unless there is additional specific binding to intra-tissue substrates that can maintain enhanced intra-tissue concentration for local delivery.

  7. Characteristics of the ambulation-increasing effect of GBR-12909, a selective dopamine uptake inhibitor, in mice.

    PubMed

    Hirate, K; Kuribara, H

    1991-04-01

    Behavioral effects of a dopamine uptake inhibitor, GBR-12909 (GBR), were evaluated by ambulatory activity in mice. The single administration of over 10 mg/kg of GBR, i.p. and p.o., significantly increased the ambulatory activity. The repeated administration of GBR, at only 10 mg/kg, produced a reverse tolerance to its ambulation-increasing effect. However, a cross-reverse tolerance was induced between GBR (10 and 20 mg/kg) and methamphetamine (2 mg/kg) in both directions. Furthermore, 5 mg/kg of GBR significantly enhanced the effects of methamphetamine, cocaine, imipramine, morphine, scopolamine and caffeine. R-THBP, a coenzyme of tyrosine hydroxylase, also enhanced the effect of GBR. In contrast, the ambulation-increasing effect of 10 mg/kg of GBR was markedly reduced by haloperidol, chlorpromazine, tetrabenazine, oxypertine, reserpine and alpha-methyl-p-tyrosine. On the other hand, the effect of GBR was only slightly and/or scarcely modified by apomorphine, caerulein, physostigmine, pilocarpine, N6-(L-2-phenylisopropyl)-adenosine and naloxone. The neurochemical experiment in rats, not in mice, revealed that GBR possessed more dominant action on dopaminergic systems than noradrenergic or serotonergic systems. However, the behavioral characteristics of GBR are similar to those of methamphetamine and cocaine, which possess less selective action than GBR on dopaminergic and noradrenergic systems.

  8. The serotonin uptake inhibitor citalopram reduces acute cardiovascular and vegetative effects of 3,4-methylenedioxymethamphetamine ('Ecstasy') in healthy volunteers.

    PubMed

    Liechti, M E; Vollenweider, F X

    2000-01-01

    MDMA (3,4-methylenedioxymethamphetamine) or 'Ecstasy' is a widely used recreational drug that produces a state of heightened mood but also cardiovascular and vegetative side-effects. In animals, MDMA releases serotonin and, to a lesser extent, dopamine and norepinephrine. The release of serotonin can be blocked by serotonin uptake inhibitors such as citalopram. It is unknown to what extent this mechanism is also responsible for the physiological side-effects of MDMA seen in humans. We investigated the effect of citalopram pretreatment (40 mg i.v.) on vegetative and cardiovascular effects of MDMA (1.5 mg/kg p.o.) in a double-blind placebo-controlled study in 16 healthy volunteers. MDMA moderately increased blood pressure and heart rate, slightly elevated body temperature and produced a broad range of acute and short-term side-effects. Citalopram reduced all these MDMA-induced physiological changes except for body temperature. These findings suggest that physiological effects of MDMA in humans are partially due to an interaction of MDMA with the serotonin carrier and a subsequent release of serotonin.

  9. GABA receptor agonists: pharmacological spectrum and therapeutic actions.

    PubMed

    Bartholini, G

    1985-01-01

    From the data discussed in this review it appears that GABA receptor agonists exhibit a variety of actions in the central nervous system, some of which are therapeutically useful (Table V). GABA receptor agonists, by changing the firing rate of the corresponding neurons accelerate noradrenaline turnover without changes in postsynaptic receptor density and diminish serotonin liberation with an up-regulation of 5HT2 receptors. These effects differ from those of tricyclic antidepressants which primarily block monoamine re-uptake and cause down-regulation of beta-adrenergic and 5HT2 receptors. The GABA receptor agonist progabide has been shown to exert an antidepressant action which is indistinguishable from that of imipramine in patients with major affective disorders. The fact that: (a) GABA receptor agonists and tricyclic antidepressants affect noradrenergic and serotonergic transmission differently; and (b) tricyclic antidepressants alter GABA-related parameters challenges the classical monoamine hypothesis of depression and suggests that GABA-mediated mechanisms play a role in mood disorders. Decreases in cellular excitability produced by GABAergic stimulation leads to control of seizures in practically all animal models of epilepsy. GABA receptor agonists have a wide spectrum as they antagonize not only seizures which are dependent on decreased GABA synaptic activity but also convulsant states which are apparently independent of alterations in GABA-mediated events. These results in animals are confirmed in a wide range of human epileptic syndromes. GABA receptor agonists decrease dopamine turnover in the basal ganglia and antagonize neuroleptic-induced increase in dopamine release. On repeated treatment, progabide prevents or reverses the neuroleptic-induced up-regulation of dopamine receptors in the rat striatum and antagonizes the concomitant supersensitivity to dopaminomimetics. Behaviorally, GABA receptor agonists diminish the stereotypies induced by

  10. Release and effect of gamma-aminobutyric acid (GABA) on rat pineal melatonin production in vitro.

    PubMed

    Rosenstein, R E; Chuluyan, H E; Pereyra, E N; Cardinali, D P

    1989-06-01

    1. 3H-gamma-Aminobutyric acid (GABA) release elicited by a depolarizing K+ stimulus or by noradrenergic transmitter was examined in rat pineals in vitro. 2. The release of 3H-GABA was detectable at a 20 mM K+ concentration in medium and increased steadily up to 80 mM K+. 3. In a Ca2+-free medium 3H-GABA release elicited by 30 mM K+, but not that elicited by 50 mM K+, became blunted. 4. Norepinephrine (NE; 10(-6)-10(-4) M) stimulated 3H-GABA release from rat pineal explants in a dose-dependent manner. 5. The activity of 10(-5) M NE on pineal GABA release was suppressed by equimolecular amounts of prazosin or phentolamine (alpha 1- and alpha 1/alpha 2-adrenoceptor blockers, respectively) and was unaffected by propranolol (beta-adrenoceptor blocker). 6. The alpha 1-adrenoceptor agonist phenylephrine (10(-7)-10(-5) M) and the beta-adrenoceptor agonist isoproterenol (10(-5) M) mimicked the GABA releasing activity of NE, while 10(-7) M isoproterenol failed to affect it; the alpha 2-adrenoceptor agonist clonidine (10(-7)-10(-5) M) did not modify 3H-GABA release. 7. The addition of 10(-4) M GABA or of the GABA transaminase inhibitor gamma-acetylenic GABA or aminooxyacetic acid inhibited the melatonin content and/or release to the medium in rat pineal organotypic cultures. 8. GABA at concentrations of 10(-5) M or greater partially inhibited the NE-induced increase in melatonin production by pineal explants. 9. The depressant effect of GABA on melatonin production was inhibited by the GABA type A receptor antagonist bicuculline; bicuculline alone increased the pineal melatonin content. Baclofen, a GABA type B receptor agonist, did not affect the pineal melatonin content or release. 10. The decrease in serotonin (5-HT) content of rat pineal explants brought about by NE was not modified by GABA; GABA by itself increased 5-HT levels. 11. These results indicate that (a) GABA is released from rat pineals by a depolarizing stimulus of K+ through a mechanism which is partially Ca2

  11. Evidence for a Revised Ion/Substrate Coupling Stoichiometry of GABA Transporters.

    PubMed

    Willford, Samantha L; Anderson, Cynthia M; Spencer, Shelly R; Eskandari, Sepehr

    2015-08-01

    Plasma membrane γ-aminobutyric acid (GABA) transporters (GATs) are electrogenic transport proteins that couple the cotranslocation of Na(+), Cl(-), and GABA across the plasma membrane of neurons and glia. A fundamental property of the transporter that determines its ability to concentrate GABA in cells and, hence, regulate synaptic and extra-synaptic GABA concentrations, is the ion/substrate coupling stoichiometry. Here, we scrutinized the currently accepted 2 Na(+):1 Cl(-):1 GABA stoichiometry because it is inconsistent with the measured net charge translocated per co-substrate (Na(+), Cl(-), and GABA). We expressed GAT1 and GAT3 in Xenopus laevis oocytes and utilized thermodynamic and uptake under voltage-clamp measurements to determine the stoichiometry of the GABA transporters. Voltage-clamped GAT1-expressing oocytes were internally loaded with GABA, and the reversal potential (V rev) of the transporter-mediated current was recorded at different external concentrations of Na(+), Cl(-), or GABA. The shifts in V rev for a tenfold change in the external Na(+), Cl(-), and GABA concentration were 84 ± 4, 30 ± 1, and 29 ± 1 mV, respectively. To determine the net charge translocated per Na(+), Cl(-), and GABA, we measured substrate fluxes under voltage clamp in cells expressing GAT1 or GAT3. Charge flux to substrate flux ratios were 0.7 ± 0.1 charge/Na(+), 2.0 ± 0.2 charges/Cl(-), and 2.1 ± 0.1 charges/GABA. Altogether, our results strongly suggest a 3 Na(+):1 Cl(-):1 GABA coupling stoichiometry for the GABA transporters. The revised stoichiometry has important implications for understanding the contribution of GATs to GABAergic signaling in health and disease.

  12. Antidepressants and seizure-interactions at the GABA-receptor chloride-ionophore complex

    SciTech Connect

    Malatynska, E.; Knapp, R.J.; Ikeda, M.; Yamamura, H.I.

    1988-01-01

    Convulsive seizures are a potential side effect of antidepressant drug treatment and can be produced by all classes of antidepressants. It is also know that some convulsant and anticonvulsant drug actions are mediated by the GABA-receptor chloride-ionophore complex. Drugs acting at this complex appear to induce convulsions by inhibiting chloride conductance through the associated chloride channel. Using the method of GABA-stimulated /sup 36/Cl-uptake by rat cerebral cortical vesicles, we show that some antidepressant drugs can inhibit the GABA-receptor chloride uptake, and that the degree of chloride channel inhibition by these drugs correlates with the frequency of convulsive seizures induced by them.

  13. Disorders of GABA metabolism: SSADH and GABA-transaminase deficiencies.

    PubMed

    Parviz, Mahsa; Vogel, Kara; Gibson, K Michael; Pearl, Phillip L

    2014-11-25

    Clinical disorders known to affect inherited gamma-amino butyric acid (GABA) metabolism are autosomal recessively inherited succinic semialdehyde dehydrogenase and GABA-transaminase deficiency. The clinical presentation of succinic semialdehyde dehydrogenase deficiency includes intellectual disability, ataxia, obsessive-compulsive disorder and epilepsy with a nonprogressive course in typical cases, although a progressive form in early childhood as well as deterioration in adulthood with worsening epilepsy are reported. GABA-transaminase deficiency is associated with a severe neonatal-infantile epileptic encephalopathy.

  14. Ethanol, not metabolized in brain, significantly reduces brain metabolism, probably via specific GABA(A) receptors

    PubMed Central

    Rae, Caroline D.; Davidson, Joanne E.; Maher, Anthony D.; Rowlands, Benjamin D.; Kashem, Mohammed A.; Nasrallah, Fatima A.; Rallapalli, Sundari K.; Cook, James M; Balcar, Vladimir J.

    2014-01-01

    Ethanol is a known neuromodulatory agent with reported actions at a range of neurotransmitter receptors. Here, we used an indirect approach, measuring the effect of alcohol on metabolism of [3-13C]pyruvate in the adult Guinea pig brain cortical tissue slice and comparing the outcomes to those from a library of ligands active in the GABAergic system as well as studying the metabolic fate of [1,2-13C]ethanol. Ethanol (10, 30 and 60 mM) significantly reduced metabolic flux into all measured isotopomers and reduced all metabolic pool sizes. The metabolic profiles of these three concentrations of ethanol were similar and clustered with that of the α4β3δ positive allosteric modulator DS2 (4-Chloro-N-[2-(2-thienyl)imidazo[1,2a]-pyridin-3-yl]benzamide). Ethanol at a very low concentration (0.1 mM) produced a metabolic profile which clustered with those from inhibitors of GABA uptake, and ligands showing affinity for α5, and to a lesser extent, α1-containing GABA(A)R. There was no measureable metabolism of [1,2-13C]ethanol with no significant incorporation of 13C from [1,2-13C]ethanol into any measured metabolite above natural abundance, although there were measurable effects on total metabolite sizes similar to those seen with unlabeled ethanol. The reduction in metabolism seen in the presence of ethanol is therefore likely to be due to its actions at neurotransmitter receptors, particularly α4β3δ receptors, and not because ethanol is substituting as a substrate or because of the effects of ethanol catabolites acetaldehyde or acetate. We suggest that the stimulatory effects of very low concentrations of ethanol are due to release of GABA via GAT1 and the subsequent interaction of this GABA with local α5-containing, and to a lesser extent, α1-containing GABA(A)R. PMID:24313287

  15. Inhibition of recombinant N-type and native high voltage-gated neuronal Ca{sup 2+} channels by AdGABA: Mechanism of action studies

    SciTech Connect

    Martinez-Hernandez, Elizabeth; Sandoval, Alejandro; Gonzalez-Ramirez, Ricardo; Zoidis, Grigoris; Felix, Ricardo

    2011-02-01

    High-voltage activated Ca{sup 2+} (Ca{sub V}) channels play a key role in the regulation of numerous physiological events by causing transient changes in the intracellular Ca{sup 2+} concentration. These channels consist of a pore-forming Ca{sub V}{alpha}{sub 1} protein and three auxiliary subunits (Ca{sub V}{beta}, Ca{sub V}{alpha}{sub 2}{delta} and Ca{sub V}{gamma}). Ca{sub V}{alpha}{sub 2}{delta} is an important component of Ca{sub V} channels in many tissues and of great interest as a drug target. It is well known that anticonvulsant agent gabapentin (GBP) binds to Ca{sub V}{alpha}{sub 2}{delta} and reduces Ca{sup 2+} currents by modulating the expression and/or function of the Ca{sub V}{alpha}{sub 1} subunit. Recently, we showed that an adamantane derivative of GABA, AdGABA, has also inhibitory effects on Ca{sub V} channels. However, the importance of the interaction of AdGABA with the Ca{sub V}{alpha}{sub 2}{delta} subunit has not been conclusively demonstrated and the mechanism of action of the drug has yet to be elucidated. Here, we describe studies on the mechanism of action of AdGABA. Using a combined approach of patch-clamp recordings and molecular biology we show that AdGABA inhibits Ca{sup 2+} currents acting on Ca{sub V}{alpha}{sub 2}{delta} only when applied chronically, both in a heterologous expression system and in dorsal root-ganglion neurons. AdGABA seems to require uptake and be acting intracellularly given that its effects are prevented by an inhibitor of the L-amino acid transport system. Interestingly, a mutation in the Ca{sub V}{alpha}{sub 2}{delta} that abolishes GBP binding did not affect AdGABA actions, revealing that its mechanism of action is similar but not identical to that of GBP. These results indicate that AdGABA is an important Ca{sub V}{alpha}{sub 2}{delta} ligand that regulates Ca{sub V} channels.

  16. T-1032, a cyclic GMP phosphodiesterase-5 inhibitor, acutely blocks physiologic insulin-mediated muscle haemodynamic effects and glucose uptake in vivo.

    PubMed

    Mahajan, Hema; Richards, Stephen M; Rattigan, Stephen; Clark, Michael G

    2003-12-01

    1. Cyclic GMP phosphodiesterase-5 inhibitors have been shown to alter blood flow in specific tissues by potentiating local NO-dependent vasodilatory mechanisms. Since the haemodynamic effects of physiologic insulin, particularly capillary recruitment, may be critical for muscle glucose uptake in vivo and are blocked by inhibitors of nitric oxide synthase, we have explored the acute effects of the specific cGMP phosphodiesterase-5 inhibitor T-1032 on physiologic insulin action in anaesthetized healthy rats in vivo. 2. Whole-body glucose infusion (GIR), femoral blood flow (FBF), hind leg vascular resistance (VR), hind leg glucose uptake (HGU), 2-deoxyglucose uptake into muscles of the lower leg (R'g), hind leg metabolism of infused 1-methylxanthine (1-MX), a measure of capillary recruitment, and muscle cGMP were determined. The experimental groups were T-1032 (10 microg min-1 kg-1) infused for 1 h before and during a euglycaemic insulin clamp (3 mU min-1 kg-1 x 2 h), T-1032 infused for 3 h with saline, T-1032 during a 2 h clamp, T-1032 with saline for 2 h, and a 2 h saline control. 3. Insulin increased GIR from zero to 13 mg min-1 kg-1, HGU from 0.1+/-0.01 to 0.43+/-0.05 micromol min-1, R'g and 1-MX, marginally increased FBF, and had no effect on blood pressure or heart rate. T-1032 alone had no effect on blood pressure, heart rate, FBF, VR, HGU, R'g or 1-MX, but increased muscle cGMP. T-1032 1 h before and during insulin completely blocked GIR (1 h), HGU (2 h), R'g (2 h), and 1-MX (2 h). T-1032 commenced with insulin had only partial blocking activity against insulin. 4. We conclude that T-1032 is a potent acutely acting inhibitor of the muscle effects of physiologic insulin on capillary recruitment and glucose uptake in vivo. These, together with inhibition of whole-body glucose infusion during insulin, may caution against the use of isoenzyme-5-specific cyclic GMP phosphodiesterase inhibitors as therapeutic agents.

  17. Overlap between the ligand recognition properties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsaicin-like activity.

    PubMed

    De Petrocellis, L; Bisogno, T; Davis, J B; Pertwee, R G; Di Marzo, V

    2000-10-13

    Some synthetic agonists of the VR1 vanilloid (capsaicin) receptor also inhibit the facilitated transport into cells of the endogenous cannabinoid anandamide (arachidonoylethanolamide, AEA). Here we tested several AEA derivatives containing various derivatized phenyl groups or different alkyl chains as either inhibitors of the AEA membrane transporter (AMT) in intact cells or functional agonists of the VR1 vanilloid receptor in HEK cells transfected with the human VR1. We found that four known AMT inhibitors, AM404, arvanil, olvanil and linvanil, activate VR1 receptors at concentrations 400-10000-fold lower than those necessary to inhibit the AMT. However, we also found three novel AEA derivatives, named VDM11, VDM12 and VDM13, which inhibit the AMT as potently as AM404 but exhibit little or no agonist activity at hVR1. These compounds are weak inhibitors of AEA enzymatic hydrolysis and poor CB(1)/CB(2) receptor ligands. We show for the first time that, despite the overlap between the chemical moieties of AMT inhibitors and VR1 agonists, selective inhibitors of AEA uptake that do not activate VR1 (e.g. VDM11) can be developed.

  18. R59949, a diacylglycerol kinase inhibitor, inhibits inducible nitric oxide production through decreasing transplasmalemmal L-arginine uptake in vascular smooth muscle cells.

    PubMed

    Shimomura, Tomoko; Nakano, Tomoyuki; Goto, Kaoru; Wakabayashi, Ichiro

    2017-02-01

    Although diacylglycerol kinase (DGK) is known to be expressed in vascular smooth muscle cell, its functional significance remains to be clarified. We hypothesized that DGK is involved in the pathway of cytokine-induced nitric oxide (NO) production in vascular smooth muscle cells. The purpose of this study was to investigate the effects of R59949, a diacylglycerol kinase inhibitor, on inducible nitric oxide production in vascular smooth muscle cell. Cultured rat aortic smooth muscle cells (RASMCs) were used to elucidate the effects of R59949 on basal and interleukin-1β (IL-1β)-induced NO production. The effects of R59949 on protein and mRNA expression of induced nitric oxide synthase (iNOS) and on transplasmalemmal L-arginine uptake were also evaluated using RASMCs. Treatment of RASMCs with R59949 (10 μM) inhibited IL-1β (10 ng/ml)-induced NO production but not basal NO production. Neither protein nor mRNA expression level of iNOS after stimulation with IL-1β was significantly affected by R59949. Estimated enzymatic activities of iNOS in RASMCs were comparable in the absence and presence of R59949. Stimulation of RASMCs with IL-1β caused a marked increase in transplasmalemmal L-arginine uptake into RASMCs. L-Arginine uptake in the presence of IL-1β was markedly inhibited by R59949, while basal L-arginine uptake was not significantly affected by R59949. Both IL-1β-induced NO production and L-arginine uptake were abolished in the presence of cycloheximide (1 μM). The results indicate that R59949 inhibits inducible NO production through decreasing transplasmalemmal L-arginine uptake. DGK is suggested to be involved in cytokine-stimulated L-arginine transport and regulate its intracellular concentration in vascular smooth muscle cell.

  19. Identification and functional characterization of a dual GABA/taurine transporter in the bullfrog retinal pigment epithelium

    PubMed Central

    1995-01-01

    Intracellular microelectrodes, fluorescence imaging, and radiotracer flux techniques were used to investigate the physiological response of the retinal pigment epithelium (RPE) to the major retinal inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). GABA is released tonically in the dark by amphibian horizontal cells, but is not taken up by the nearby Muller cells. Addition of GABA to the apical bath produced voltage responses in the bullfrog RPE that were not blocked nor mimicked by any of the major GABA-receptor antagonists or agonists. Nipecotic acid, a substrate for GABA transport, inhibited the voltage effects of GABA. GABA and nipecotic acid also inhibited the voltage effects of taurine, suggesting that the previously characterized beta- alanine sensitive taurine carrier also takes up GABA. The voltage responses of GABA, taurine, nipecotic acid, and beta-alanine all showed first-order saturable kinetics with the following Km's: GABA (Km = 160 microM), beta-alanine (Km = 250 microM), nipecotic acid (Km = 420 microM), and taurine (Km = 850 microM). This low affinity GABA transporter is dependent on external Na, partially dependent on external Cl, and is stimulated in low [K]o, which approximates subretinal space [K]o during light onset. Apical GABA also produced a significant conductance increase at the basolateral membrane. These GABA-induced conductance changes were blocked by basal Ba2+, suggesting that GABA decreased basolateral membrane K conductance. In addition, the apical membrane Na/K ATPase was stimulated in the presence of GABA. A model for the interaction between the GABA transporter, the Na/K ATPase, and the basolateral membrane K conductance accounts for the electrical effects of GABA. Net apical-to-basal flux of [3H]-GABA was also observed in radioactive flux experiments. The present study shows that a high capacity GABA uptake mechanism with unique pharmacological properties is located at the RPE apical membrane and could play an

  20. Manganese exposure alters extracellular GABA, GABA receptor and transporter protein and mRNA levels in the developing rat brain.

    PubMed

    Anderson, Joel G; Fordahl, Steve C; Cooney, Paula T; Weaver, Tara L; Colyer, Christa L; Erikson, Keith M

    2008-11-01

    Unlike other essential trace elements (e.g., zinc and iron) it is the toxicity of manganese (Mn) that is more common in human populations than its deficiency. Data suggest alterations in dopamine biology may drive the effects associated with Mn neurotoxicity, though recently gamma-aminobutyric acid (GABA) has been implicated. In addition, iron deficiency (ID), a common nutritional problem, may cause disturbances in neurochemistry by facilitating accumulation of Mn in the brain. Previous data from our lab have shown decreased brain tissue levels of GABA as well as decreased (3)H-GABA uptake in synaptosomes as a result of Mn exposure and ID. These results indicate a possible increase in the concentration of extracellular GABA due to alterations in expression of GABA transport and receptor proteins. In this study weanling-male Sprague-Dawley rats were randomly placed into one of four dietary treatment groups: control (CN; 35mg Fe/kg diet), iron-deficient (ID; 6mg Fe/kg diet), CN with Mn supplementation (via the drinking water; 1g Mn/l) (CNMn), and ID with Mn supplementation (IDMn). Using in vivo microdialysis, an increase in extracellular GABA concentrations in the striatum was observed in response to Mn exposure and ID although correlational analysis reveals that extracellular GABA is related more to extracellular iron levels and not Mn. A diverse effect of Mn exposure and ID was observed in the regions examined via Western blot and RT-PCR analysis, with effects on mRNA and protein expression of GAT-1, GABA(A), and GABA(B) differing between and within the regions examined. For example, Mn exposure reduced GAT-1 protein expression by approximately 50% in the substantia nigra, while increasing mRNA expression approximately four-fold, while in the caudate putamen mRNA expression was decreased with no effect on protein expression. These data suggest that Mn exposure results in an increase in extracellular GABA concentrations via altered expression of transport and

  1. Neuronal calcium channel inhibitors. Synthesis of omega-conotoxin GVIA and effects on 45Ca uptake by synaptosomes

    SciTech Connect

    Rivier, J.; Galyean, R.; Gray, W.R.; Azimi-Zonooz, A.; McIntosh, J.M.; Cruz, L.J.; Olivera, B.M.

    1987-01-25

    We previously described a 27-amino acid peptide neurotoxin from the venom of Conus geographus, omega-conotoxin GVIA, which inhibits neuronal voltage-activated calcium channels. In this paper we describe the total synthesis of omega-conotoxin GVIA and demonstrate that it efficiently blocks voltage-activated uptake of /sup 45/Ca by standard synaptosomal preparations from chick brain. Dihydropyridines do not block /sup 45/Ca uptake under these conditions. Thus, the omega-conotoxin-sensitive, but dihydropyridine-insensitive uptake of /sup 45/Ca/sup 2 +/ by chick brain synaptosomes serves as a functional assay for a Ca channel target of omega-conotoxin. The use of synthetic GVIA should rapidly accelerate our understanding of the molecular biology of Ca/sup 2 +/ channels and their role in neuronal function.

  2. GABA deficiency in NF1

    PubMed Central

    Patricio, Miguel; Bernardino, Inês; Rebola, José; Abrunhosa, Antero J.; Ferreira, Nuno; Castelo-Branco, Miguel

    2016-01-01

    Objective: To provide a comprehensive investigation of the γ-aminobutyric acid (GABA) system in patients with neurofibromatosis type 1 (NF1) that allows understanding the nature of the GABA imbalance in humans at pre- and postsynaptic levels. Methods: In this cross-sectional study, we employed multimodal imaging and spectroscopy measures to investigate GABA type A (GABAA) receptor binding, using [11C]-flumazenil PET, and GABA concentration, using magnetic resonance spectroscopy (MRS). Fourteen adult patients with NF1 and 13 matched controls were included in the study. MRS was performed in the occipital cortex and in a frontal region centered in the functionally localized frontal eye fields. PET and MRS acquisitions were performed in the same day. Results: Patients with NF1 have reduced concentration of GABA+ in the occipital cortex (p = 0.004) and frontal eye fields (p = 0.026). PET results showed decreased binding of GABAA receptors in patients in the parieto-occipital cortex, midbrain, and thalamus, which are not explained by decreased gray matter levels. Conclusions: Abnormalities in the GABA system in NF1 involve both GABA concentration and GABAA receptor density suggestive of neurodevelopmental synaptopathy with both pre- and postsynaptic involvement. PMID:27473134

  3. Frequency-dependent depression of inhibition in guinea-pig neocortex in vitro by GABAB receptor feed-back on GABA release.

    PubMed Central

    Deisz, R A; Prince, D A

    1989-01-01

    induced constant responses; at higher frequencies, the responses sometimes declined. The current-voltage relationships of the two GABA responses were similar to each other and to the early IPSP. An apparently fivefold higher conductance was estimated at lower Ems, suggesting that the GABA response had a voltage sensitivity. The slope conductance of IPSPs was decreased by up to 50% for tens of seconds after postsynaptically detectable effects of GABA had dissipated. 6. Application of the GABA uptake inhibitor nipecotic acid (50-500 microM) reduced the conductance of both components of orthodromically evoked inhibition and shortened the IPSP at low frequencies, but had no additional effects at higher stimulation rates.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2557431

  4. Tesofensine, a novel triple monoamine re-uptake inhibitor with anti-obesity effects: dopamine transporter occupancy as measured by PET.

    PubMed

    Appel, Lieuwe; Bergström, Mats; Buus Lassen, Jørgen; Långström, Bengt

    2014-02-01

    Tesofensine (TE) is a novel triple monoamine re-uptake inhibitor inducing a potent inhibition of the re-uptake process in the synaptic cleft of the neurotransmitters dopamine, norepinephrine, and serotonin. In recent preclinical and clinical evaluations TE showed a robust anti-obesity effect, but the specific mechanism of this triple monoamine re-uptake inhibitor still needs to be further elucidated. This positron emission tomography (PET) study, using [¹¹C]βCIT-FE, aimed to assess the degree of the dopamine transporter (DAT) occupancy, at constant TE plasma levels, following different oral, multiple doses of TE during totally 8-12 days. In addition, the relationships between DAT occupancy and TE plasma concentrations, or doses, were investigated to enable assessment of DAT occupancies in subsequent clinical trials. The results demonstrated that TE induced a dose-dependent blockade of DAT following multiple doses of 0.125-1 mg TE at anticipated steady-state conditions. The mean striatal DAT occupancy varied dose-dependently between 18% and 77%. A sigmoid E(max) model well described the relationship between striatal DAT occupancy and TE plasma concentrations or doses. It was estimated that the maximum achievable DAT occupancy was about 80% and that half of this effect was accomplished by approximately 0.25 mg TE and a plasma drug concentration of 4 ng/ml. The results indicated an important mechanism of action of TE on DAT. Further, these results suggest that the previously reported dose-dependent weight loss, in TE treated subjects, was in part mediated by an up-regulation of dopaminergic pathways due to enhanced amounts of synaptic dopamine after blockade of DAT.

  5. Actions of insecticides on the insect GABA receptor complex

    SciTech Connect

    Bermudez, I.; Hawkins, C.A.; Taylor, A.M.; Beadle, D.J. )

    1991-01-01

    The actions of insecticides on the insect gamma-aminobutyric acid (GABA) receptor were investigated using (35S)t-butylbicyclophosphorothionate (( 35S)TBPS) binding and voltage-clamp techniques. Specific binding of (35S)TBPS to a membrane homogenate derived from the brain of Locusta migratoria locusts is characterised by a Kd value of 79.3 {plus minus} 2.9 nM and a Bmax value of 1770 {plus minus} 40 fmol/mg protein. (35S)TBPS binding is inhibited by mM concentrations of barbiturates and benzodiazepines. In contrast dieldrin, ivermectin, lindane, picrotoxin and TBPS are inhibitors of (35S)TBPS binding at the nanomolar range. Bicuculline, baclofen and pyrethroid insecticides have no effect on (35S)TBPS binding. These results are similar to those obtained in electrophysiological studies of the current elicited by GABA in both Locusta and Periplaneta americana central neurones. Noise analysis of the effects of lindane, TBPS, dieldrin and picrotoxin on the cockroach GABA responses reveals that these compounds decrease the variance of the GABA-induced current but have no effect on its mean open time. All these compounds, with the exception of dieldrin, significantly decrease the conductance of GABA-evoked single current.

  6. Combinational Spinal GAD65 Gene Delivery and Systemic GABA-Mimetic Treatment for Modulation of Spasticity

    PubMed Central

    Kakinohana, Osamu; Hefferan, Michael P.; Miyanohara, Atsushi; Nejime, Tetsuya; Marsala, Silvia; Juhas, Stefan; Juhasova, Jana; Motlik, Jan; Kucharova, Karolina; Strnadel, Jan; Platoshyn, Oleksandr; Lazar, Peter; Galik, Jan; Vinay, Laurent; Marsala, Martin

    2012-01-01

    Background Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABAB receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments. Methods/Principal Findings Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2–3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene. Conclusions/Significance These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel

  7. Evidence That GABA Mediates Dopaminergic and Serotonergic Pathways Associated with Locomotor Activity in Juvenile Chinook Salmon (Oncorhynchus tshawytscha)

    USGS Publications Warehouse

    Clements, S.; Schreck, C.B.

    2004-01-01

    The authors examined the control of locomotor activity in juvenile salmon (Oncorhynchus tshawytscha) by manipulating 3 neurotransmitter systems-gamma-amino-n-butyric acid (GABA), dopamine, and serotonin-as well as the neuropeptide corticotropin releasing hormone (CRH). Intracerebroventricular (ICV) injections of CRH and the GABAAagonist muscimol stimulated locomotor activity. The effect of muscimol was attenuated by administration of a dopamine receptor antagonist, haloperidol. Conversely, the administration of a dopamine uptake inhibitor (4???,4??? -difluoro-3-alpha-[diphenylmethoxy] tropane hydrochloride [DUI]) potentiated the effect of muscimol. They found no evidence that CRH-induced hyperactivity is mediated by dopaminergic systems following concurrent injections of haloperidol or DUI with CRH. Administration of muscimol either had no effect or attenuated the locomotor response to concurrent injections of CRH and fluoxetine, whereas the GABAA antagonist bicuculline methiodide potentiated the effect of CRH and fluoxetine.

  8. Selaginellin and biflavonoids as protein tyrosine phosphatase 1B inhibitors from Selaginella tamariscina and their glucose uptake stimulatory effects.

    PubMed

    Nguyen, Phi-Hung; Ji, Da-Jung; Han, Yu-Ran; Choi, Jae-Sue; Rhyu, Dong-Young; Min, Byung-Sun; Woo, Mi-Hee

    2015-07-01

    As part of an ongoing search for new antidiabetic agents from medicinal plants, the methanol extract of the aerial parts of Selaginella tamariscina was found to possess stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells. Thus, bioassay-guided isolation of this active extract yielded two new compounds (1 and 2) along with five known biflavonoids (3-7). Their structures were elucidated by extensive analysis of spectroscopic and physicochemical data. The absolute configuration of compound 2 was determined by specific rotation and CD data analysis. All isolates exhibited potent inhibitory effects on PTP1B enzyme with IC50 values ranging from 4.5±0.1 to 13.2±0.8μM. Furthermore, the isolates (1-7) showed significant stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. Of these, compounds (1, 6, and 7) which exhibited mixed-competitive inhibition modes against PTP1B, showed potent stimulatory effects on 2-NBDG uptake. This result indicated the potential of these biflavonoids as lead molecules for development of antidiabetic agents and the beneficial use of S. tamariscina against hyperglycemia.

  9. Does extracellular calcium determine what pool of GABA is the target for alpha-latrotoxin?

    PubMed

    Storchak, L G; Linetska, M V; Himmelreich, N H

    2002-04-01

    Presynaptic neurotoxin alpha-latrotoxin, from the venom of Latrodectus mactans tredecimguttatus, causes massive [(3)H]GABA release from rat brain synaptosomes, irrespective of calcium presence in the extracellular medium. Whether the binding of alpha-latrotoxin to Ca(2+)-dependent (neurexin 1 alpha) or to Ca(2+)-independent (latrophilin) receptor triggers [(3)H]GABA release by the same mechanisms or different ones, inducing either exocytotic process or outflow by mobile membrane GABA transporter, is unknown. We examined alpha-latrotoxin-evoked [(3)H]GABA release from synaptosomes which cytosolic [(3)H]GABA pool was depleted either by applying competitive inhibitors of the GABA transporter, nipecotic acid and 2,4-diaminobutyric acid, or by permeation with digitonin. We also compared the effect of the GABA transporter inhibitors on depolarisation-evoked and alpha-latrotoxin-evoked [(3)H]GABA release using as depolarising agents 4-aminopyridine and high KCl in the Ca(2+)-containing and in Ca(2+)-free medium, respectively. Incubation of synaptosomes with nipecotic acid induced the essential acceleration of unstimulated [(3)H]GABA release and deep inhibition of high KCl-evoked Ca(2+)-independent [(3)H]GABA release. In contrast, at the similar conditions the effect of alpha-latrotoxin was greatly augmented with respect to the control response. Another way to assay what GABA pool was involved in alpha-latrotoxin-induced release lays in an analysis of the effects of depolarisation and alpha-latrotoxin in consecutive order. The preliminary 4-aminopyridine-stimulated [(3)H]GABA release attenuated the toxin effect. But when depolarisation occurred in Ca(2+)-free medium, no influence on alpha-latrotoxin effect was revealed. Employing digitonin-permeated synaptosomes, we have shown that alpha-latrotoxin could stimulate [3H]GABA release in the medium with 1mM EGTA, this effect of the toxin was blocked by concanavalin A and was ATP-dependent. The latter suggests that alpha

  10. Participation of a GABA-ergic system in the processes of neuroimmunomodulation.

    PubMed

    Devoino, L; Idova, G; Beletskaya, I

    1992-01-01

    Participation of a GABA-ergic system in neuroimmunomodulation was established through the use of a large number of chemical compounds which selectively modulate the activity of the GABA-BD-receptor-ionophore complex. Activation of the GABA-receptors with muscimol or activation of the BD-receptors with diazepam or tazepam had stimulatory effects upon immunogenesis. A decrease in the GABA-BD-receptor-ionophore complex activity led to a suppression of the immune response. The effect was achieved with: a blockade of the complex with bicuculline--a competitive inhibitor of the GABA-receptors: administration of a specific antagonist of the BD-receptors flumazenil or Ro 15-3505: or with blockade of chloride channels with picrotoxin. Activation of the GABA-ergic system causes an increase in bone marrow content of T-helper cells marked by L3T4. The immunomodulatory action of the GABA-ergic system is of central origin and can occur only when the hypothalamo-pituitary system is intact. Section of the pituitary stalk prevents accumulation of the T-helper cells in the bone marrow. The result show that the influence of GABA-ergic system on immunogenesis requires participation of both dopaminergic and serotoninergic systems.

  11. Effects of nitrification inhibitors (DCD and DMPP) on nitrous oxide emission, crop yield and nitrogen uptake in a wheat-maize cropping system

    NASA Astrophysics Data System (ADS)

    Liu, C.; Wang, K.; Zheng, X.

    2013-04-01

    The application of nitrification inhibitors together with ammonium-based fertilizers is proposed as a potent method to decrease nitrous oxide (N2O) emission while promoting crop yield and nitrogen use efficiency in fertilized agricultural fields. To evaluate the effects of nitrification inhibitors, we conducted year-round measurements of N2O fluxes, yield, aboveground biomass, plant carbon and nitrogen contents, soil inorganic nitrogen and dissolved organic carbon contents and the main environmental factors for urea (U), urea + dicyandiamide (DCD) and urea + 3,4-dimethylpyrazol phosphate (DMPP) treatments in a wheat-maize rotation field. The cumulative N2O emissions were calculated to be 4.49 ± 0.21, 2.93 ± 0.06 and 2.78 ± 0.16 kg N ha-1 yr-1 for the U, DCD and DMPP treatments, respectively. Therefore, the DCD and DMPP treatments significantly decreased the annual emissions by 35% and 38%, respectively (p < 0.01). The variations of soil temperature, moisture and inorganic nitrogen content regulated the seasonal fluctuation of N2O emissions. When the emissions presented clearly temporal variations, high-frequency measurements or optimized sampling schedule for intermittent measurements would likely provide more accurate estimations of annual cumulative emission and treatment effect. The application of nitrification inhibitors significantly increased the soil inorganic nitrogen content (p < 0.01); shifted the main soil inorganic nitrogen form from nitrate to ammonium; and tended to increase the dissolved organic carbon content, crop yield, aboveground biomass and nitrogen uptake by aboveground plant. The results demonstrate the roles the nitrification inhibitors play in enhancing yield and nitrogen use efficiency and reducing N2O emission from the wheat-maize cropping system.

  12. Effects of nitrification inhibitors (DCD and DMPP) on nitrous oxide emission, crop yield and nitrogen uptake in a wheat-maize cropping system

    NASA Astrophysics Data System (ADS)

    Liu, C.; Wang, K.; Zheng, X.

    2013-01-01

    The application of nitrification inhibitors together with ammonium-based fertilizers is proposed as a potent method to decrease nitrous oxide (N2O) emission while promoting yield and nitrogen use efficiency in fertilized agricultural fields. To evaluate the effects of nitrification inhibitors, we conducted year-round measurements of N2O fluxes, yield, aboveground biomass, plant carbon and nitrogen contents, soil inorganic nitrogen and dissolved organic carbon contents and the main environmental factors for urea (U), urea + dicyandiamide (DCD) and urea + 3,4-dimethylpyrazol-phosphate (DMPP) treatments in a wheat-maize rotation field. The cumulative N2O emissions were calculated to be 4.49 ± 0.21, 2.93 ± 0.06 and 2.78 ± 0.16 kg N ha-1 yr-1 for the U, DCD and DMPP treatments, respectively. Therefore, the DCD and DMPP treatments decreased the annual emissions by 35% and 38%, respectively. The variations of soil temperature, moisture and inorganic nitrogen content regulated the seasonal fluctuation of N2O emissions. When the emissions presented clearly temporal variations, year-round and high-frequency measurements should be adopted to estimate annual cumulative emissions and treatment effects. The application of nitrification inhibitors increased the soil inorganic nitrogen and dissolved organic carbon availability and shifted the main soil inorganic nitrogen form from nitrate to ammonium. The annual yield, aboveground biomass and nitrogen uptake by aboveground plants increased by 8.5-9.1%, 8.6-9.7% and 10.9-13.2%, respectively, for the DCD and DMPP treatments compared with the U treatment. The results demonstrate the roles the nitrification inhibitors play in enhancing yield and nitrogen use efficiency and reducing N2O emission from the wheat-maize cropping system.

  13. Selective serotonin re-uptake inhibitors for the treatment of depression in coronary artery disease and chronic heart failure: evidence for pleiotropic effects.

    PubMed

    Paraskevaidis, Ioannis; Parissis, John T; Fountoulaki, Katerina; Filippatos, Gerasimos; Kremastinos, Dimitrios

    2006-10-01

    Depression is a common co-morbidity in patients with cardiovascular diseases such as chronic coronary artery disease, acute coronary syndromes, post by-pass surgery and chronic heart failure. There is a significant body of evidence suggesting that the presence of depression is independently associated with a decline in health status and an increase in the risk of hospitalization and death for patients with coronary artery disease or congestive heart failure. Novel treatment modalities such as selective serotonin re-uptake inhibitors (SSRIs) may improve depressive symptoms and prognosis of post-myocardial infarction and heart failure patients interacting with the common pathophysiologic mechanisms of depression and cardiovascular disease. This review summarizes current experimental and clinical evidence regarding the pleiotropic effects of SSRIs on platelet functions, immune and neurohormonal activation, and cardiac rhythm disturbances in patients with cardiovascular disease. These bio-modulatory properties of SSRIs may be translated into improvement of patient clinical outcomes beyond their anti-depressant action.

  14. Differential effects of GABA in modulating nociceptive vs. non-nociceptive synapses.

    PubMed

    Wang, Y; Summers, T; Peterson, W; Miiller, E; Burrell, B D

    2015-07-09

    GABA (γ-amino-butyric acid) -mediated signaling is normally associated with synaptic inhibition due to ionotropic GABA receptors that gate an inward Cl(-) current, hyperpolarizing the membrane potential. However, there are also situations where ionotropic GABA receptors trigger a Cl(-) efflux that results in depolarization. The well-characterized central nervous system of the medicinal leech was used to study the functional significance of opposing effects of GABA at the synaptic circuit level. Specifically, we focused on synapses made by the nociceptive N cell and the non-nociceptive P (pressure) cell that converge onto a common postsynaptic target. It is already known that GABA hyperpolarizes the P cell, but depolarizes the N cell and that inhibition of ionotropic GABA receptors by bicuculline (BIC) has opposing effects on the synapses made by these two inputs; enhancing P cell synaptic transmission, but depressing N cell synapses. The goal of the present study was to determine whether the opposing effects of GABA were due to differences in Cl(-) homeostasis between the two presynaptic neurons. VU 0240551 (VU), an inhibitor of the Cl(-) exporter K-Cl co-transporter isoform 2 (KCC2), attenuated GABA-mediated hyperpolarization of the non-nociceptive afferent while bumetanide (BUM), an inhibitor of the Cl(-) importer Na-K-Cl co-transporter isoform 1 (NKCC1), reduced GABA-mediated depolarization of the nociceptive neuron. VU treatment also enhanced P cell synaptic signaling, similar to the previously observed effects of BIC and consistent with the idea that GABA inhibits synaptic signaling at the presynaptic level. BUM treatment depressed N cell synapses, again similar to what is observed following BIC treatment and suggests that GABA has an excitatory effect on these synapses. The opposing effects of GABA could also be observed at the behavioral level with BIC and VU increasing responsiveness to non-nociceptive stimulation while BIC and BUM decreased responsiveness

  15. GAD67-mediated GABA Synthesis and Signaling Regulate Inhibitory Synaptic Innervation in the Visual Cortex

    PubMed Central

    Chattopadhyaya, Bidisha; Di Cristo, Graziella; Wu, Cai Zhi; Knott, Graham; Kuhlman, Sandra; Fu, Yu; Palmiter, Richard D.; Huang, Z. Josh

    2007-01-01

    The development of GABAergic inhibitory circuits is shaped by neural activity, but the underlying mechanisms are unclear. we demonstrate a novel function of GABA in regulating GABAergic innervation in the adolescent brain, when GABA is mainly known as an inhibitory transmitter. Conditional knockdown of the rate-limiting synthetic enzyme GAD67 in basket interneurons in adolescent visual cortex resulted in cell autonomous deficits in axon branching, perisomatic synapse formation around pyramidal neurons, and complexity of the innervation fields; the same manipulation had little influence on the subsequent maintenance of perisomatic synapses. These effects of GABA deficiency were rescued by suppressing GABA re-uptake and by GABA receptor agonists. Germ-line knockdown of GAD67 but not GAD65 showed similar deficits, suggesting a specific role of GAD67 in the maturation of perisomatic innervation. Since intracellular GABA levels are modulated by neuronal activity, our results implicate GAD67-mediated GABA synthesis in activity-dependent regulation of inhibitory innervation patterns. PMID:17582330

  16. Regulation of GABA and benzodiazepine receptors following neurotoxin-induced striatal and medial forebrain bundle lesions

    SciTech Connect

    Pan, H.S.I.

    1985-01-01

    GABA, a major inhibitory transmitter, is used by many projection neurons of the striatum. To investigate the role of GABA in striatal function, the GABA receptor complex was studied after lesions of the striatum or the nigrostriatal neurons. Quantitative receptor autoradiography using thaw-mounted tissue slices was developed for the study of GABA and benzodiazepine (BDZ) receptors. With the technique established, binding to GABA and BDZ receptors after unilateral striatal kainate lesions was examined. Subsequently, changes in GABA and BDZ receptors were studied following the destruction of dopaminergic nigrostriatal cells by unilateral 6-hydroxydopamine lesion of the medial forebrain bundle. In summary, quantitative receptor autoradiography allowed the detection of GABA and BDZ receptor changes in multiple small areas in each lesioned brain. This technique made it feasible to carry out kinetic saturation, and competition studies using less than 1 mg of tissue. The data suggest that dopamine is functionally inhibitory on striatopallidal neurons but is functionally excitatory on striatoentopeduncular and striatonigral cells which in turn inhibit the thalamus. This quantitative autoradiographic technique can be generalized to study other transmitter receptors and can be combined with 2-deoxyglucose uptake studies.

  17. A study on the involvement of GABA-transaminase in MCT induced pulmonary hypertension.

    PubMed

    Lingeshwar, Poorella; Kaur, Gurpreet; Singh, Neetu; Singh, Seema; Mishra, Akanksha; Shukla, Shubha; Ramakrishna, Rachumallu; Laxman, Tulsankar Sachin; Bhatta, Rabi Sankar; Siddiqui, Hefazat H; Hanif, Kashif

    2016-02-01

    Increased sympathetic nervous system (SNS) activity is associated with cardiovascular diseases but its role has not been completely explored in pulmonary hypertension (PH). Increased SNS activity is distinguished by elevated level of norepinephrine (NE) and activity of γ-Amino butyric acid Transminase (GABA-T) which degrades GABA, an inhibitory neurotransmitter within the central and peripheral nervous system. Therefore, we hypothesized that GABA-T may contribute in pathophysiology of PH by modulating level of GABA and NE. The effect of daily oral administration of GABA-T inhibitor, Vigabatrin (GVG, 50 and 75 mg/kg/day, 35 days) was studied following a single subcutaneous administration of monocrotaline (MCT, 60 mg/kg) in male SD rats. The pressure and hypertrophy of right ventricle (RV), oxidative stress, inflammation, pulmonary vascular remodelling were assessed after 35 days in MCT treated rats. The expression of GABA-T and HIF-1α was studied in lung tissue. The levels of plasma NE (by High performance liquid chromatography coupled with electrochemical detector; HPLC-ECD) and lung GABA (by liquid chromatography-mass spectrometry) were also estimated. GVG at both doses significantly attenuated increased in pressure (35.82 ± 4.80 mm Hg, p < 0.001; 28.37 ± 3.32 mm Hg, p < 0.001 respectively) and hypertrophy of RV, pulmonary vascular remodelling, oxidative stress and inflammation in lungs of MCT exposed rats. GVG also reduced the expression of GABA-T and HIF-1α in MCT treated rats. Increased NE level and decreased GABA level was also reversed by GVG in MCT exposed rats. GABA-T plays an important role in PH by modulating SNS activity and may be considered as a therapeutic target in PH.

  18. Gestational changes of GABA levels and GABA binding in the human uterus

    SciTech Connect

    Erdoe, S.L.; Villanyi, P.; Laszlo, A.

    1989-01-01

    The concentrations of gamma-aminobutyric acid (GABA), the activities of L-glutamate decarboxylase and GABA-transaminase, and the nature of the sodium-independent binding of GABA were examined in uterine tissue pieces obtained surgically from pregnant and non-pregnant women. GABA concentrations were reduced, while the activity of GABA-transaminase and the specific binding of (/sup 3/H)GABA significantly increased in specimens from pregnant subjects. These findings suggest some gestation-related functional role for the GABA system in the human uterus.

  19. Inhibition of fatty acid amide hydrolase and monoacylglycerol lipase by the anandamide uptake inhibitor VDM11: evidence that VDM11 acts as an FAAH substrate.

    PubMed

    Vandevoorde, Séverine; Fowler, Christopher J

    2005-08-01

    There is some dispute concerning the extent to which the uptake inhibitor VDM11 (N-(4-hydroxy-2-methylphenyl) arachidonoyl amide) is capable of inhibiting the metabolism of the endocannabinoid anandamide (AEA) by fatty acid amide hydrolase (FAAH). In view of a recent study demonstrating that the closely related compound AM404 (N-(4-hydroxyphenyl)arachidonylamide) is a substrate for FAAH, we re-examined the interaction of VDM11 with FAAH. In the presence of fatty acid-free bovine serum albumin (BSA, 0.125% w v(-1)), both AM404 and VDM11 inhibited the metabolism of AEA by rat brain FAAH with similar potencies (IC(50) values of 2.1 and 2.6 microM, respectively). The compounds were about 10-fold less potent as inhibitors of the metabolism of 2-oleoylglycerol (2-OG) by cytosolic monoacylglycerol lipase (MAGL). The potency of VDM11 towards FAAH was dependent upon the assay concentration of fatty acid-free bovine serum albumin (BSA). Thus, in the absence of fatty acid-free BSA, the IC(50) value for inhibition of FAAH was reduced by a factor of about two (from 2.9 to 1.6 microM). A similar reduction in the IC(50) value for the inhibition of membrane bound MAGL by both this compound (from 14 to 6 microM) and by arachidonoyl serinol (from 24 to 13 microM) was seen. An HPLC assay was set up to measure 4-amino-m-cresol, the hypothesised product of FAAH-catalysed VDM11 hydrolysis. 4-Amino-m-cresol was eluted with a retention time of approximately 2.4 min, but showed a time-dependent degradation to compounds eluting at peaks of approximately 5.6 and approximately 8 min. Peaks with the same retention times were also found following incubation of the membranes with VDM11, but were not seen when the membranes were preincubated with the FAAH inhibitors URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) and CAY10401 (1-oxazolo[4,5-b]pyridin-2-yl-9-octadecyn-1-one) prior to addition of VDM11. The rate of metabolism of VDM11 was estimated to be roughly 15-20% of that for

  20. Mutations in the GABA Transporter SLC6A1 Cause Epilepsy with Myoclonic-Atonic Seizures.

    PubMed

    Carvill, Gemma L; McMahon, Jacinta M; Schneider, Amy; Zemel, Matthew; Myers, Candace T; Saykally, Julia; Nguyen, John; Robbiano, Angela; Zara, Federico; Specchio, Nicola; Mecarelli, Oriano; Smith, Robert L; Leventer, Richard J; Møller, Rikke S; Nikanorova, Marina; Dimova, Petia; Jordanova, Albena; Petrou, Steven; Helbig, Ingo; Striano, Pasquale; Weckhuysen, Sarah; Berkovic, Samuel F; Scheffer, Ingrid E; Mefford, Heather C

    2015-05-07

    GAT-1, encoded by SLC6A1, is one of the major gamma-aminobutyric acid (GABA) transporters in the brain and is responsible for re-uptake of GABA from the synapse. In this study, targeted resequencing of 644 individuals with epileptic encephalopathies led to the identification of six SLC6A1 mutations in seven individuals, all of whom have epilepsy with myoclonic-atonic seizures (MAE). We describe two truncations and four missense alterations, all of which most likely lead to loss of function of GAT-1 and thus reduced GABA re-uptake from the synapse. These individuals share many of the electrophysiological properties of Gat1-deficient mice, including spontaneous spike-wave discharges. Overall, pathogenic mutations occurred in 6/160 individuals with MAE, accounting for ~4% of unsolved MAE cases.

  1. Effect of GABA agonists and GABA-A receptor modulators on cocaine- and food-maintained responding and cocaine discrimination in rats.

    PubMed

    Barrett, Andrew C; Negus, S Stevens; Mello, Nancy K; Caine, S Barak

    2005-11-01

    Recent studies indicate that GABAergic ligands modulate abuse-related effects of cocaine. The goal of this study was to evaluate the effects of a mechanistically diverse group of GABAergic ligands on the discriminative stimulus and reinforcing effects of cocaine in rats. One group of rats was trained to discriminate 5.6 mg/kg cocaine from saline in a two-lever, food-reinforced, drug discrimination procedure. In two other groups, responding was maintained by cocaine (0-3.2 mg/kg/injection) or liquid food (0-100%) under a fixed ratio 5 schedule. Six GABA agonists were tested: the GABA-A receptor agonist muscimol, the GABA-B receptor agonist baclofen, the GABA transaminase inhibitor gamma-vinyl-GABA (GVG), and three GABA-A receptor modulators (the barbiturate pentobarbital, the high-efficacy benzodiazepine midazolam, and the low-efficacy benzodiazepine enazenil). When tested alone, none of the compounds substituted fully for the discriminative stimulus effects of cocaine. As acute pretreatments, select doses of midazolam and pentobarbital produced 2.2- to 3.6-fold rightward shifts in the cocaine dose-effect function. In contrast, muscimol, baclofen, GVG, and enazenil failed to alter the discriminative stimulus effects of cocaine. In assays of cocaine- and food-maintained responding, midazolam and pentobarbital decreased cocaine self-administration at doses 9.6- and 3.3-fold lower, respectively, than those that decreased food-maintained responding. In contrast, muscimol, baclofen, and GVG decreased cocaine self-administration at doses that also decreased food-maintained responding. Enazenil failed to alter cocaine self-administration. Together with previous studies, these data suggest that among mechanistically diverse GABA agonists, high-efficacy GABA-A modulators may be the most effective for modifying the abuse-related effects of cocaine.

  2. Fluoxetine, a selective inhibitor of serotonin uptake, potentiates morphine analgesia without altering its discriminative stimulus properties or affinity for opioid receptors

    SciTech Connect

    Hynes, M.D.; Lochner, M.A.; Bemis, K.G.; Hymson, D.L.

    1985-06-17

    The analgesic effect of morphine in the rat tail jerk assay was enhanced by the serotonin uptake inhibitor, fluoxetine. Tail jerk latency was not affected by fluoxetine alone. Morphine's affinity for opioid receptors labeled in vitro with /sup 3/H-naloxone or /sup 3/H-D-Ala/sup 2/-D-Leu/sup 5/-enkephalin was not altered by fluoxetine, which has no affinity for these sites at concentrations as high as 1000 nM. In rats trained to discriminate morphine from saline, fluoxetine at doses of 5 or 10 mg/kg were recognized as saline. Increasing the fluoxetine dose to 20 mg/kg did not result in generalization to either saline or morphine. The dose response curve for morphine generalization was not significantly altered by fluoxetine doses of 5 or 10 mg/kg. Those rats treated with the combination of morphine and 20 mg/kg of fluoxetine did not exhibit saline or morphine appropriate responding. Fluoxetine potentiates the analgesic properties of morphine without enhancing its affinity for opioid receptors or its discriminative stimulus properties. 30 references, 2 figures, 2 tables.

  3. Novel dose-dependent alterations in excitatory GABA during embryonic development associated with lead (Pb) neurotoxicity.

    PubMed

    Wirbisky, Sara E; Weber, Gregory J; Lee, Jang-Won; Cannon, Jason R; Freeman, Jennifer L

    2014-08-17

    Lead (Pb) is a heavy metal that is toxic to numerous physiological processes. Its use in industrial applications is widespread and results in an increased risk of human environmental exposure. The central nervous system (CNS) is most sensitive to Pb exposure during early development due to rapid cell proliferation and migration, axonal growth, and synaptogenesis. One of the key components of CNS development is the Gamma-aminobutyric acid (GABA)-ergic system. GABA is the primary inhibitory neurotransmitter in the adult brain. However, during development GABA acts as an excitatory neurotrophic factor which contributes to these cellular processes. Multiple studies report effects of Pb on GABA in the mature brain; however, little is known regarding the adverse effects of Pb exposure on the GABAergic system during embryonic development. To characterize the effects of Pb on the GABAergic system during development, zebrafish embryos were exposed to 10, 50, or 100 ppb Pb or a control treatment. Tissue up-take, gross morphological alterations, gene expression, and neurotransmitter levels were analyzed. Analysis revealed that alterations in gene expression throughout the GABAergic system and GABA levels were dose and developmental time point specific. These data provide a framework for further analysis of the effects of Pb on the GABAergic system during the excitatory phase and as GABA transitions to an inhibitory neurotransmitter during development.

  4. Methamphetamine-evoked depression of GABA(B) receptor signaling in GABA neurons of the VTA.

    PubMed

    Padgett, Claire L; Lalive, Arnaud L; Tan, Kelly R; Terunuma, Miho; Munoz, Michaelanne B; Pangalos, Menelas N; Martínez-Hernández, José; Watanabe, Masahiko; Moss, Stephen J; Luján, Rafael; Lüscher, Christian; Slesinger, Paul A

    2012-03-08

    Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.

  5. GABA and Central Neuropathic Pain following Spinal Cord Injury

    PubMed Central

    Gwak, Young S.; Hulsebosch, Claire E.

    2012-01-01

    Spinal cord injury induces maladaptive synaptic transmission in the somatosensory system that results in chronic central neuropathic pain. Recent literature suggests that glial-neuronal interactions are important modulators in synaptic transmission following spinal cord injury. Neuronal hyperexcitability is one of the predominant phenomenon caused by maladaptive synaptic transmission via altered glial-neuronal interactions after spinal cord injury. In the somatosensory system, spinal inhibitory neurons counter balance the enhanced synaptic transmission from peripheral input. For a decade, the literature suggests that hypofunction of GABAergic inhibitory tone is an important factor in the enhanced synaptic transmission that often results in neuronal hyperexcitability in dorsal horn neurons following spinal cord injury. Neurons and glial cells synergistically control intracellular chloride ion gradients via modulation of chloride transporters, extracellular glutamate and GABA concentrations via uptake mechanisms. Thus, the intracellular “GABA-glutamate-glutamine cycle” is maintained for normal physiological homeostasis. However, hyperexcitable neurons and glial activation after spinal cord injury disrupts the balance of chloride ions, glutamate and GABA distribution in the spinal dorsal horn and results in chronic neuropathic pain. In this review, we address spinal cord injury induced mechanisms in hypofunction of GABAergic tone that results in chronic central neuropathic pain. PMID:21216257

  6. Stimulation of [3H] GABA and beta-[3H] alanine release from rat brain slices by cis-4-aminocrotonic acid.

    PubMed

    Chebib, M; Johnston, G A

    1997-02-01

    cis-4-Aminocrotonic acid (CACA; 100 microM), an analogue of GABA in a folded conformation, stimulated the passive release of [3H] GABA from slices of rat cerebellum, cerebral cortex, retina, and spinal cord and of beta-[3H]alanine from slices of cerebellum and spinal cord without influencing potassium-evoked release. In contrast, CACA (100 microM) did not stimulate the passive release of [3H]taurine from slices of cerebellum and spinal cord or of D-[3H]aspartate from slices of cerebellum and did not influence potassium-evoked release of [3H]-taurine from the cerebellum and spinal cord and D-[3H]-aspartate from the cerebellum. These results suggest that the effects of CACA on GABA and beta-alanine release are due to CACA acting as a substrate for a beta-alanine-sensitive GABA transport system, consistent with CACA inhibiting the uptake of beta-[3H]alanine into slices of rat cerebellum and cerebral cortex. The observed Ki for CACA against beta-[3H]alanine uptake in the cerebellum was 750 +/- 60 microM. CACA appears to be 10-fold weaker as a substrate for the transporter system than as an agonist for the GABAc receptor. The effects of CACA on GABA and beta-alanine release provide indirect evidence for a GABA transporter in cerebellum, cerebral cortex, retina, and spinal cord that transports GABA, beta-alanine, CACA, and nipecotic acid that has a similar pharmacological profile to that of the GABA transporter, GAT-3, cloned from rat CNS. The structural similarities of GABA, beta-alanine, CACA, and nipecotic acid are demonstrated by computer-aided molecular modeling, providing information on the possible conformations of these substances being transported by a common carrier protein.

  7. Anion transport and GABA signaling

    PubMed Central

    Hübner, Christian A.; Holthoff, Knut

    2013-01-01

    Whereas activation of GABAA receptors by GABA usually results in a hyperpolarizing influx of chloride into the neuron, the reversed chloride driving force in the immature nervous system results in a depolarizing efflux of chloride. This GABAergic depolarization is deemed to be important for the maturation of the neuronal network. The concept of a developmental GABA switch has mainly been derived from in vitro experiments and reliable in vivo evidence is still missing. As GABAA receptors are permeable for both chloride and bicarbonate, the net effect of GABA also critically depends on the distribution of bicarbonate. Whereas chloride can either mediate depolarizing or hyperpolarizing currents, bicarbonate invariably mediates a depolarizing current under physiological conditions. Intracellular bicarbonate is quickly replenished by cytosolic carbonic anhydrases. Intracellular bicarbonate levels also depend on different bicarbonate transporters expressed by neurons. The expression of these proteins is not only developmentally regulated but also differs between cell types and even subcellular regions. In this review we will summarize current knowledge about the role of some of these transporters for brain development and brain function. PMID:24187533

  8. PKCβ Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux.

    PubMed

    Zestos, Alexander G; Mikelman, Sarah R; Kennedy, Robert T; Gnegy, Margaret E

    2016-06-15

    Amphetamine abuse afflicts over 13 million people, and there is currently no universally accepted treatment for amphetamine addiction. Amphetamine serves as a substrate for the dopamine transporter and reverses the transporter to cause an increase in extracellular dopamine. Activation of the beta subunit of protein kinase C (PKCβ) enhances extracellular dopamine in the presence of amphetamine by facilitating the reverse transport of dopamine and internalizing the D2 autoreceptor. We previously demonstrated that PKCβ inhibitors block amphetamine-stimulated dopamine efflux in synaptosomes from rat striatum in vitro. In this study, we utilized in vivo microdialysis in live, behaving rats to assess the effect of the PKCβ inhibitors, enzastaurin and ruboxistaurin, on amphetamine-stimulated locomotion and increases in monoamines and their metabolites. A 30 min perfusion of the nucleus accumbens core with 1 μM enzastaurin or 1 μM ruboxistaurin reduced efflux of dopamine and its metabolite 3-methoxytyramine induced by amphetamine by approximately 50%. The inhibitors also significantly reduced amphetamine-stimulated extracellular levels of norepinephrine. The stimulation of locomotor behavior by amphetamine, measured simultaneously with the analytes, was comparably reduced by the PKCβ inhibitors. Using a stable isotope label retrodialysis procedure, we determined that ruboxistaurin had no effect on basal levels of dopamine, norepinephrine, glutamate, or GABA. In addition, normal uptake function through the dopamine transporter was unaltered by the PKCβ inhibitors, as measured in rat synaptosomes. Our results support the utility of using PKCβ inhibitors to reduce the effects of amphetamine.

  9. Unique pH dynamics in GABAergic synaptic vesicles illuminates the mechanism and kinetics of GABA loading

    PubMed Central

    Egashira, Yoshihiro; Takase, Miki; Watanabe, Shoji; Ishida, Junji; Fukamizu, Akiyoshi; Kaneko, Ryosuke; Yanagawa, Yuchio; Takamori, Shigeo

    2016-01-01

    GABA acts as the major inhibitory neurotransmitter in the mammalian brain, shaping neuronal and circuit activity. For sustained synaptic transmission, synaptic vesicles (SVs) are required to be recycled and refilled with neurotransmitters using an H+ electrochemical gradient. However, neither the mechanism underlying vesicular GABA uptake nor the kinetics of GABA loading in living neurons have been fully elucidated. To characterize the process of GABA uptake into SVs in functional synapses, we monitored luminal pH of GABAergic SVs separately from that of excitatory glutamatergic SVs in cultured hippocampal neurons. By using a pH sensor optimal for the SV lumen, we found that GABAergic SVs exhibited an unexpectedly higher resting pH (∼6.4) than glutamatergic SVs (pH ∼5.8). Moreover, unlike glutamatergic SVs, GABAergic SVs displayed unique pH dynamics after endocytosis that involved initial overacidification and subsequent alkalization that restored their resting pH. GABAergic SVs that lacked the vesicular GABA transporter (VGAT) did not show the pH overshoot and acidified further to ∼6.0. Comparison of luminal pH dynamics in the presence or absence of VGAT showed that VGAT operates as a GABA/H+ exchanger, which is continuously required to offset GABA leakage. Furthermore, the kinetics of GABA transport was slower (τ > 20 s at physiological temperature) than that of glutamate uptake and may exceed the time required for reuse of exocytosed SVs, allowing reuse of incompletely filled vesicles in the presence of high demand for inhibitory transmission. PMID:27601664

  10. Correlation between the enhancement of flunitrazepam binding by GABA and seizure susceptibility in mice

    SciTech Connect

    Marley, R.J.; Wehner, J.M.

    1987-06-08

    Various populations of mice exhibit differential sensitivity to seizure-inducing agents. The relationship of seizure susceptibility to alterations in the GABA receptor complex was investigated in six different populations of mice consisting of four inbred strains (C57BL, DBA, C3H, and BALB) and two selected lines (long sleep and short sleep). Seizure activity was induced by intraperitoneal administration of the GAD inhibitor, 3-mercaptopropionic acid, and latencies to seizure onset and tonus were measured. In naive mice of the same populations, GABA enhancement of TH-flunitrazepam binding was measured in extensively washed whole brain membranes at several GABA concentrations. Both differential seizure sensitivity to 3-mercaptopropionic acid and differential enhancement of TH-flunitrazepam binding by GABA were observed in these six populations of mice. Correlational analyses indicated a positive correlation between the degree of GABA enhancement of TH-flunitrazepam binding and resistance to the seizure-inducing properties of 3-mercaptopropionic acid. These data suggest that genetic differences in sensitivity to seizure-inducing agents that disrupt the GABAergic system may be related to differences in coupling between the various receptors associated with the GABA receptor complex.

  11. The effect of anaesthetics on the uptake and release of gamma-aminobutyrate and D-aspartate in rat brain slices.

    PubMed Central

    Minchin, M. C.

    1981-01-01

    1 The effect of various concentrations of thiopentone, pentobarbitone, methohexitone, hydroxydione, alphaxalone/alphadolone, ketamine, alpha-chloralose, and urethane on the transport of radiolabelled gamma-aminobutyric acid (GABA) and D-aspartate was investigated. 2 Uptake of the amino acids was weakly inhibited, if at all, by the anaesthetics and it is unlikely that such effects contribute significantly to their physiological function. 3 The spontaneous efflux of GABA and D-aspartate was not detectably altered by any of the drugs tested. 4 Thiopentone, pentobarbitone, methohexitone and hydroxydione inhibited K+-stimulated GABA and D-aspartate release. The other anaesthetics had no effect on K+-stimulated amino acid release. 5 The rank order of potency of the inhibitors of K+-stimulated amino acid release did not correlate with their anaesthetic potency. Furthermore not all inhibitors appeared to be very effective at anaesthetic concentrations. 6 It is concluded that although it is possible that inhibition of excitatory transmitter release may be involved in the anaesthetic action of some anaesthetics, for many of the substances tested in this study such as mechanism does not appear to be implicated. PMID:6265017

  12. Effects of the novel endocannabinoid uptake inhibitor, LY2183240, on fear-potentiated startle and alcohol-seeking behaviors in mice selectively bred for high alcohol preference

    PubMed Central

    Powers, Matthew S.; Barrenha, Gustavo D.; Mlinac, Nate S.; Barker, Eric L.; Chester, Julia A.

    2010-01-01

    Rationale Alcohol-use disorders often occur together with anxiety disorders in humans which may be partly due to common inherited genetic factors. Evidence suggests that the endocannabinoid system (ECS) is a promising therapeutic target for the treatment of individuals with anxiety and/or alcohol-use disorders. Objectives The present study assessed the effects of a novel endocannabinoid uptake inhibitor, LY2183240, on anxiety- and alcohol-seeking behaviors in a unique animal model that may represent increased genetic risk to develop comorbid anxiety and alcohol-use disorders in humans. Mice selectively bred for high alcohol preference (HAP) show greater fear-potentiated startle (FPS) than mice selectively bred for low alcohol preference (LAP). We examined the effects of LY2183240 on the expression of FPS in HAP and LAP mice and on alcohol-induced conditioned place preference (CPP) and limited-access alcohol drinking behavior in HAP mice. Results Repeated administration of LY2183240 (30 mg/kg) reduced the expression of FPS in HAP but not LAP mice when given prior to a second FPS test 48 h after fear conditioning. Both the 10 and 30 mg/kg doses of LY2183240 enhanced the expression of alcohol-induced CPP and this effect persisted in the absence of the drug. LY2183240 did not alter limited-access alcohol drinking behavior, unconditioned startle responding, or locomotor activity. Conclusions These findings suggest that ECS modulation influences both conditioned fear and conditioned alcohol reward behavior. LY2183240 may be an effective pharmacotherapy for individuals with anxiety disorders, such as post-traumatic stress disorder, but may not be appropriate for individuals with co-morbid anxiety and alcohol-use disorders. PMID:20838777

  13. GABA interaction with lipids in organic medium

    SciTech Connect

    Beltramo, D.; Kivatinitz, S.; Lassaga, E.; Arce, A.

    1987-08-10

    The interaction of TH-GABA and UC-glutamate with lipids in an aqueous organic partition system was studied. With this partition system TH-GABA and UC-glutamate were able to interact with sphingomyelin, sulfatide, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and phosphatidic acid but not with cholesterol or ceramide. In an homogeneous aqueous medium the authors could not demonstrate any interaction between TH-GABA-lipids. The apparent dissociation constants (K/sub d/) for TH-GABA-lipids or UC-glutamate-lipids interactions inorganic medium were in the millimolar range and maximal charge between 3 and 7 moles of GABA or glutamate by mole of lipid. Amino acids such as glutamic acid, US -alanine and glycine displaced TH-GABA with the same potency as GABA itself; thus these results show that the interaction lacks pharmacological specificity. To detect this interaction lipid concentrations higher than 2 M were required and in the partition system TH-GABA and lipid phosphorus were both concentrated at the interface. Therefore, lipids tested with a biphasic partition system do not fulfill the classical criteria for a neurotransmitter receptor at least not for GABA and glutamate. 15 references, 1 figure, 3 tables.

  14. Taurine is a potent activator of extrasynaptic GABA(A) receptors in the thalamus.

    PubMed

    Jia, Fan; Yue, Minerva; Chandra, Dev; Keramidas, Angelo; Goldstein, Peter A; Homanics, Gregg E; Harrison, Neil L

    2008-01-02

    Taurine is one of the most abundant free amino acids in the brain. In a number of studies, taurine has been reported to activate glycine receptors (Gly-Rs) at moderate concentrations (> or = 100 microM), and to be a weak agonist at GABA(A) receptors (GABA(A)-Rs), which are usually activated at high concentrations (> or = 1 mM). In this study, we show that taurine reduced the excitability of thalamocortical relay neurons and activated both extrasynaptic GABA(A)-Rs and Gly-Rs in neurons in the mouse ventrobasal (VB) thalamus. Low concentrations of taurine (10-100 microM) decreased neuronal input resistance and firing frequency, and elicited a steady outward current under voltage clamp, but had no effects on fast inhibitory synaptic currents. Currents elicited by 50 microM taurine were abolished by gabazine, insensitive to midazolam, and partially blocked by 20 microM Zn2+, consistent with the pharmacological properties of extrasynaptic GABA(A)-Rs (alpha4beta2delta subtype) involved in tonic inhibition in the thalamus. Tonic inhibition was enhanced by an inhibitor of taurine transport, suggesting that taurine can act as an endogenous activator of these receptors. Taurine-evoked currents were absent in relay neurons from GABA(A)-R alpha4 subunit knock-out mice. The amplitude of the taurine current was larger in neurons from adult mice than juvenile mice. Taurine was a more potent agonist at recombinant alpha4beta2delta GABA(A)-Rs than at alpha1beta2gamma2 GABA(A)-Rs. We conclude that physiological concentrations of taurine can inhibit VB neurons via activation of extrasynaptic GABA(A)-Rs and that taurine may function as an endogenous regulator of excitability and network activity in the thalamus.

  15. The GABA transaminase, ABAT, is essential for mitochondrial nucleoside metabolism

    PubMed Central

    Besse, Arnaud; Wu, Ping; Bruni, Francesco; Donti, Taraka; Graham, Brett H.; Craigen, William J.; McFarland, Robert; Moretti, Paolo; Lalani, Seema; Scott, Kenneth L.; Taylor, Robert W.; Bonnen, Penelope E.

    2015-01-01

    Summary ABAT is a key enzyme responsible for catabolism of principal inhibitory neurotransmitter gamma-aminobutyric acid (GABA). We report an essential role for ABAT in a seemingly unrelated pathway, mitochondrial nucleoside salvage, and demonstrate that mutations in this enzyme cause an autosomal recessive neurometabolic disorder and mtDNA depletion syndrome (MDS). We describe a family with encephalomyopathic MDS caused by a homozygous missense mutation in ABAT that results in elevated GABA in subjects’ brains as well as decreased mtDNA levels in subjects’ fibroblasts. Nucleoside rescue and co-IP experiments pinpoint that ABAT functions in the mitochondrial nucleoside salvage pathway to facilitate conversion of dNDPs to dNTPs. Pharmacological inhibition of ABAT through the irreversible inhibitor Vigabatrin caused depletion of mtDNA in photoreceptor cells that was prevented through addition of dNTPs in cell culture media. This work reveals ABAT as a connection between GABA metabolism and nucleoside metabolism and defines a neurometabolic disorder that includes MDS. PMID:25738457

  16. The putative P-gp inhibitor telmisartan does not affect the transcellular permeability and cellular uptake of the calcium channel antagonist verapamil in the P-glycoprotein expressing cell line MDCK II MDR1

    PubMed Central

    Saaby, Lasse; Tfelt-Hansen, Peer; Brodin, Birger

    2015-01-01

    Verapamil is used in high doses for the treatment of cluster headache. Verapamil has been described as a P-glycoprotein (P-gp, ABCB1) substrate. We wished to evaluate in vitro whether co administration of a P-gp inhibitor with verapamil could be a feasible strategy for increasing CNS uptake of verapamil. Fluxes of radiolabelled verapamil across MDCK II MDR1 monolayers were measured in the absence and presence of the putative P-gp inhibitor telmisartan (a clinically approved drug compound). Verapamil displayed a vectorial basolateral-to-apical transepithelial efflux across the MDCK II MDR1 monolayers with a permeability of 5.7 × 10−5 cm sec−1 compared to an apical to basolateral permeability of 1.3 × 10−5 cm sec-1. The efflux could be inhibited with the P-gp inhibitor zosuquidar. Zosuquidar (0.4 μmol/L) reduced the efflux ratio (PB-A/PA-B) for verapamil 4.6–1.6. The presence of telmisartan, however, only caused a slight reduction in P-gp-mediated verapamil transport to an efflux ratio of 3.4. Overall, the results of the present in vitro approach indicate, that clinical use of telmisartan as a P-gp inhibitor may not be an effective strategy for increasing brain uptake of verapamil by co-administration with telmisartan. PMID:26171231

  17. Altered GABA Signaling in Early Life Epilepsies

    PubMed Central

    Briggs, Stephen W.; Galanopoulou, Aristea S.

    2011-01-01

    The incidence of seizures is particularly high in the early ages of life. The immaturity of inhibitory systems, such as GABA, during normal brain development and its further dysregulation under pathological conditions that predispose to seizures have been speculated to play a major role in facilitating seizures. Seizures can further impair or disrupt GABAA signaling by reshuffling the subunit composition of its receptors or causing aberrant reappearance of depolarizing or hyperpolarizing GABAA receptor currents. Such effects may not result in epileptogenesis as frequently as they do in adults. Given the central role of GABAA signaling in brain function and development, perturbation of its physiological role may interfere with neuronal morphology, differentiation, and connectivity, manifesting as cognitive or neurodevelopmental deficits. The current GABAergic antiepileptic drugs, while often effective for adults, are not always capable of stopping seizures and preventing their sequelae in neonates. Recent studies have explored the therapeutic potential of chloride cotransporter inhibitors, such as bumetanide, as adjunctive therapies of neonatal seizures. However, more needs to be known so as to develop therapies capable of stopping seizures while preserving the age- and sex-appropriate development of the brain. PMID:21826277

  18. GABA maintains the proliferation of progenitors in the developing chick ciliary marginal zone and non-pigmented ciliary epithelium.

    PubMed

    Ring, Henrik; Mendu, Suresh Kumar; Shirazi-Fard, Shahrzad; Birnir, Bryndis; Hallböök, Finn

    2012-01-01

    GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABA(A) receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABA(A) receptor system. To quantify the effects on proliferation by GABA(A) receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABA(A) receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABA(A) receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl-transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABA(A) receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABA(A) receptors. This supported the depolarising role for the GABA(A) receptors. Inhibition of L-type voltage-gated Ca(2+) channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABA(A) receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27(KIP1), along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27(KIP1) after inhibition of either the GABA(A) receptors or the L-type VGCCs suggests a link between the GABA(A) receptors, membrane potential, and intracellular Ca

  19. Closing the loop on the GABA shunt in plants: are GABA metabolism and signaling entwined?

    PubMed Central

    Michaeli, Simon; Fromm, Hillel

    2015-01-01

    γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that is found in uni- and multi-cellular organisms and is involved in many aspects of plant life cycle. GABA metabolism occurs by the action of evolutionary conserved enzymes that constitute the GABA shunt, bypassing two steps of the TCA cycle. The central position of GABA in the interface between plant carbon and nitrogen metabolism is well established. In parallel, there is evidence to support a role for GABA as a signaling molecule in plants. Here we cover some of the recent findings on GABA metabolism and signaling in plants and further suggest that the metabolic and signaling aspects of GABA may actually be inseparable. PMID:26106401

  20. Structure, function, and plasticity of GABA transporters

    PubMed Central

    Scimemi, Annalisa

    2014-01-01

    GABA transporters belong to a large family of neurotransmitter:sodium symporters. They are widely expressed throughout the brain, with different levels of expression in different brain regions. GABA transporters are present in neurons and in astrocytes and their activity is crucial to regulate the extracellular concentration of GABA under basal conditions and during ongoing synaptic events. Numerous efforts have been devoted to determine the structural and functional properties of GABA transporters. There is also evidence that the expression of GABA transporters on the cell membrane and their lateral mobility can be modulated by different intracellular signaling cascades. The strength of individual synaptic contacts and the activity of entire neuronal networks may be finely tuned by altering the density, distribution and diffusion rate of GABA transporters within the cell membrane. These findings are intriguing because they suggest the existence of complex regulatory systems that control the plasticity of GABAergic transmission in the brain. Here we review the current knowledge on the structural and functional properties of GABA transporters and highlight the molecular mechanisms that alter the expression and mobility of GABA transporters at central synapses. PMID:24987330

  1. Amoxapine inhibition of GABA-stimulated chloride conductance: Investigations of potential sites of activity

    SciTech Connect

    Ikeda, M.; Knapp, R.J.; Yamamura, H.I. ); Malatynska, E. )

    1989-01-01

    Amoxapine inhibits GABA-stimulated chloride conductance by acting on the GABA{sub A}-receptor chloride-ionophore complex which can be studied using membrane vesicles prepared from rat cerebral cortex. Amoxapine produces a right shift in the GABA concentration-response curve for the stimulation of {sup 36}Cl{sup {minus}} uptake into these vesicles with no apparent change in the maximum response. Schild analysis of these data gave a pA{sub 2} value of 5.52 with a slope of 0.79. Amoxapine inhibits the binding of the GABA{sub A} receptor selective antagonist ({sup 3}H)SR 95531 with an IC{sub 50} value of 3.45 {mu}M and a pseudo Hill coefficient of 0.83. In contrast, 10 {mu}M amoxapine inhibits ({sup 3}H) flunitrazepam binding by less than 25% while the benzodiazepine antagonist Ro 15-1788 reduces the amoxapine inhibition of GABA-stimulated chloride conductance only at high concentrations.

  2. Cortical GABA Levels in Primary Insomnia

    PubMed Central

    Morgan, Peter T.; Pace-Schott, Edward F.; Mason, Graeme F.; Forselius, Erica; Fasula, Madonna; Valentine, Gerald W.; Sanacora, Gerard

    2012-01-01

    Study Objectives: GABA is increasingly recognized as an important neurotransmitter for the initiation and maintenance of sleep. We sought to measure cortical GABA content through proton magnetic resonance spectroscopy (MRS) in persons with and without primary insomnia, and relate brain GABA levels to polysomnographic sleep measures. Design: Two-group comparison study. Setting: Outpatient study at a university research clinic. Participants: Non-medicated persons with primary insomnia (N = 16) and no sleep complaints (N = 17). Interventions: Participants kept sleep diaries and a regular time-in-bed schedule for 9 days, culminating in 2 consecutive nights of ambulatory polysomnography and a single proton MRS session. The main outcome measure was occipital GABA/creatine ratios; secondary measures included sleep measurements and relationship between polysomnographically measured time awake after sleep onset and occipital GABA content. Measurements and Results: The primary insomnia group was distinguished from persons with no sleep complaints on self-reported and polysomnographically measured sleep. The two groups did not differ in age, sex, body mass index, habitual bed- and wake-times, napping, use of caffeine, or use of cigarettes. Mean occipital GABA level was 12% higher in persons with insomnia than in persons without sleep complaints (P < 0.05). In both groups, GABA levels correlated negatively with polysomnographically measured time awake after sleep onset (P < 0.05). Conclusions: Increased GABA levels in persons with insomnia may reflect an allostatic response to chronic hyperarousal. The preserved, negative relationship between GABA and time awake after sleep onset supports this notion, indicating that the possible allostatic response is adaptive. Citation: Morgan PT; Pace-Schott EF; Mason GF; Forselius E; Fasula M; Valentine GW; Sanacora G. Cortical GABA levels in primary insomnia. SLEEP 2012;35(6):807-814. PMID:22654200

  3. Neuronal GABA release and GABA inhibition of ACh release in guinea pig urinary bladder.

    PubMed

    Kusunoki, M; Taniyama, K; Tanaka, C

    1984-04-01

    gamma-Aminobutyric acid (GABA) and glutamate decarboxylase (GAD) are present in the urinary bladder of guinea pigs, and the possible correlation in regional distribution between GABA, GAD, and the number of vesical ganglion cells was studied. Electrical stimulation of the bladder strips produced an increase in the calcium-dependent and tetrodotoxin-sensitive [3H]GABA release and contractions in the strips preloaded with [3H]GABA. Nicotine, acetylcholine chloride (ACh), and hexamethonium did not significantly alter the release of [3H]GABA. Bicuculline significantly enhanced [3H]ACh release and cholinergic components of contractions evoked by electrical stimulation of the bladder strips preloaded with [3H]choline, thereby suggesting that this compound antagonizes the effect of endogenous GABA released during stimulation. GABA and muscimol but not baclofen reduced both the [3H]ACh release and contractions evoked by nicotine. These effects of GABA were antagonized by bicuculline and furosemide but not by alpha- and beta-adrenergic blockers. These findings suggest that GABA may be a noncholinergic nonadrenergic inhibitory neurotransmitter in the urinary bladder. The motility of the urinary bladder is thus inhibited by reducing the release of ACh from the postganglionic cholinergic neurons through bicuculline-sensitive GABA receptors probably associated with the chloride ion channel.

  4. Low nanomolar GABA effects at extrasynaptic α4β1/β3δ GABA(A) receptor subtypes indicate a different binding mode for GABA at these receptors.

    PubMed

    Karim, Nasiara; Wellendorph, Petrine; Absalom, Nathan; Bang, Line Haunstrup; Jensen, Marianne Lerbech; Hansen, Maja Michelle; Lee, Ho Joon; Johnston, Graham A R; Hanrahan, Jane R; Chebib, Mary

    2012-08-15

    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 δ-containing extrasynaptic GABA(A) receptors expressed in Xenopus oocytes using the two-electrode voltage clamp technique, and to investigate, using site-directed mutagenesis, the molecular determinants for GABA potency at α4β3δ GABA(A) receptors. α4/δ-Containing GABA(A) receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating α4β1δ (EC₅₀=24 nM) and α4β3δ (EC₅₀=12 nM) receptors. In the majority of oocytes expressing α4β3δ subtypes, GABA produced a biphasic concentration-response curve, and activated the receptor with low and high concentrations (EC₅₀(1)=16 nM; EC₅₀(2)=1.2 μM). At α4β2δ, GABA had low micromolar activity (EC₅₀=1 μM). An analysis of 10 N-terminal singly mutated α4β3δ receptors shows that GABA interacts with amino acids different to those reported for α1β2γ2 GABA(A) receptors. Residues Y205 and R207 of the β3-subunit significantly affected GABA potency, while the residue F71 of the α4- and the residue Y97 of the β3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the δ-subunit, equivalent to the GABA binding residue R207 of the β2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the δ-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic δ-containing GABA(A) receptors compared to their synaptic counterparts.

  5. PR-independent neurosteroid regulation of α2-GABA-A receptors in the hippocampus subfields.

    PubMed

    Reddy, Doodipala Samba; Gangisetty, Omkaram; Wu, Xin

    2017-03-15

    Progesterone (P) binding to the intracellular progesterone receptors (PRs) plays a key role in epilepsy via modulation of GABA-A receptor plasticity in the brain. This is thought to occur via conversion of P to neurosteroids such as allopregnanolone, an allosteric modulator of GABA-A receptors. In the female brain, the composition of GABA-A receptors is not static and undergoes dynamic spatial changes in response to fluctuations in P and neurosteroid levels. Synaptic α2-containing GABA-A receptors contribute to phasic neuronal excitability and seizure susceptibility. However, the mechanisms underlying α2-subunit plasticity remain unclear. Here, we utilized the neurosteroid synthesis inhibitor finasteride and PR knockout mice to investigate the role of PRs in α2-subunit in the hippocampus. α2-Subunit expression was significantly upregulated during the high-P state of diestrous stage and with P treatment in wildtype and PR knockout mice. In contrast, there was no change in α2-subunit expression when metabolism of P into neurosteroids was blocked by finasteride in both genotypes. These findings suggest that ovarian cycle-related P and neurosteroids regulate α2-GABA-A receptor expression in the hippocampus via a non-PR pathway, which may be relevant to menstrual-cycle related brain conditions.

  6. Interactions between dopamine and GABA in the control of ambulatory activity.

    PubMed

    Agmo, A; Belzung, C; Giordano, M

    1996-01-01

    Ambulatory activity of male rats was quantified in an open field. The subjects were treated with DL-amphetamine and amfonelic acid alone or combined with the GABA transaminase inhibitors gamma-acetylen GABA (GAG) and sodium valproate as well as with the GABAA agonist THIP and the GABAB agonist baclofen. Subeffective doses of the GABAergic drugs did not modify the effects of moderate doses of the dopaminergic stimulants whereas effective doses continued to reduce ambulatory activity just as in the absence of dopaminergic activation. When DL-amphetamine or amfonelic acid were administered in doses that strongly enhanced ambulatory activity, doses of the GABAergic drugs that were inhibitory in the absence of dopaminergic stimulation were no longer effective. The mixed D1/D2 dopamine antagonist pimozide, the D1 antagonist SCH 23390 and the D2 antagonist sulpiride were then combined with subeffective doses of the GABA agonists. GAG, sodium valproate and baclofen were potentiated by pimozide and SCH 23390 but not by sulpiride. THIP was ineffective. These data show that GABAergic drugs had a reduced effect after stimulation of dopaminergic neurotransmission. On the other hand, when dopamine D1 receptors were blocked, nonselective GABA agonists and the GABAB agonist baclofen were potentiated. This was not the case for the GABAA agonist THIP, suggesting that the GABAA receptor is of slight importance for the interactions between GABA and dopamine in the control of ambulatory activity. No potentiation of GABAergic agonists was obtained after treatment with a dopamine D2 antagonist.

  7. Muscimol as an ionotropic GABA receptor agonist.

    PubMed

    Johnston, Graham A R

    2014-10-01

    Muscimol, a psychoactive isoxazole from Amanita muscaria and related mushrooms, has proved to be a remarkably selective agonist at ionotropic receptors for the inhibitory neurotransmitter GABA. This historic overview highlights the discovery and development of muscimol and related compounds as a GABA agonist by Danish and Australian neurochemists. Muscimol is widely used as a ligand to probe GABA receptors and was the lead compound in the development of a range of GABAergic agents including nipecotic acid, tiagabine, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, (Gaboxadol(®)) and 4-PIOL.

  8. Synthesis and evaluation of novel heteroaromatic substrates of GABA aminotransferase

    PubMed Central

    Hawker, Dustin D.; Silverman, Richard B.

    2012-01-01

    Two principal neurotransmitters are involved in the regulation of mammalian neuronal activity, namely, γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, and L-glutamic acid, an excitatory neurotransmitter. Low GABA levels in the brain have been implicated in epilepsy and several other neurological diseases. Because of GABA’s poor ability to cross the blood-brain barrier (BBB), a successful strategy to raise brain GABA concentrations is the use of a compound that does cross the BBB and inhibits or inactivates GABA aminotransferase (GABA-AT), the enzyme responsible for GABA catabolism. Vigabatrin, a mechanism-based inactivator of GABA-AT, is currently a successful therapeutic for epilepsy, but has harmful side effects, leaving a need for improved GABA-AT inactivators. Here, we report the synthesis and evaluation of a series of heteroaromatic GABA analogues as substrates of GABA-AT, which will be used as the basis for the design of novel enzyme inactivators. PMID:22944334

  9. Presynaptic Na+-dependent transport and exocytose of GABA and glutamate in brain in hypergravity.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Pozdnyakova, N.; Krisanova, N.; Himmelreich, N.

    γ-Aminobutyric acid (GABA) and L-glutamate are the most widespread neurotransmitter amino acids in the mammalian central nervous system. GABA is now widely recognized as the major inhibitory neurotransmitter. L-glutamate mediates the most of excitatory synaptic neurotransmission in the brain. They involved in the main aspects of normal brain function. The nerve terminals (synaptosomes) offer several advantages as a model system for the study of general mechanisms of neurosecretion. Our data allowed to conclude that exposure of animals to hypergravity (centrifugation of rats at 10G for 1 hour) had a profound effect on synaptic processes in brain. Comparative analysis of uptake and release of GABA and glutamate have demonstrated that hypergravity loading evokes oppositely directed alterations in inhibitory and excitatory signal transmission. We studied the maximal velocities of [^3H]GABA reuptake and revealed more than twofold enhancement of GABA transporter activity (Vmax rises from 1.4 |pm 0.3 nmol/min/mg of protein in the control group to 3.3 ± 0.59 nmol/min/mg of protein for animals exposed to hypergravity (P ≤ 0.05)). Recently we have also demonstrated the significant lowering of glutamate transporter activity (Vmax of glutamate reuptake decreased from 12.5 ± 3.2 nmol/min/mg of protein in the control group to 5.6 ± 0.9 nmol/min/mg of protein in the group of animals, exposed to the hypergravity stress (P ≤ 0.05)). Significant changes occurred in release of neurotransmitters induced by stimulating exocytosis with the agents, which depolarized nerve terminal plasma membrane. Depolarization-evoked Ca2+-stimulated release was more abundant for GABA (7.2 ± 0.54% and 11,74 ±1,2 % of total accumulated label for control and hypergravity, respectively (P≤0.05)) and was essentially less for glutamate (14.4 ± 0.7% and 6.2 ± 1.9%) after exposure of animals to centrifuge induced artificial gravity. Changes observed in depolarization-evoked exocytotic release

  10. Guinea Pig Horizontal Cells Express GABA, the GABA-Synthesizing Enzyme GAD65, and the GABA Vesicular Transporter

    PubMed Central

    Guo, Chenying; Hirano, Arlene A.; Stella, Salvatore L.; Bitzer, Michaela; Brecha, Nicholas C.

    2013-01-01

    γ-Aminobutyric acid (GABA) is likely expressed in horizontal cells of all species, although conflicting physiological findings have led to considerable controversy regarding its role as a transmitter in the outer retina. This study has evaluated key components of the GABA system in the outer retina of guinea pig, an emerging retinal model system. The presence of GABA, its rate-limiting synthetic enzyme glutamic acid decarboxylase (GAD65 and GAD67 isoforms), the plasma membrane GABA transporters (GAT-1 and GAT-3), and the vesicular GABA transporter (VGAT) was evaluated by using immunohistochemistry with well-characterized antibodies. The presence of GAD65 mRNA was also evaluated by using laser capture microdissection and reverse transcriptase-polymerase chain reaction. Specific GABA, GAD65, and VGAT immunostaining was localized to horizontal cell bodies, as well as to their processes and tips in the outer plexiform layer. Furthermore, immunostaining of retinal whole mounts and acutely dissociated retinas showed GAD65 and VGAT immunoreactivity in both A-type and B-type horizontal cells. However, these cells did not contain GAD67, GAT-1, or GAT-3 immunoreactivity. GAD65 mRNA was detected in horizontal cells, and sequencing of the amplified GAD65 fragment showed approximately 85% identity with other mammalian GAD65 mRNAs. These studies demonstrate the presence of GABA, GAD65, and VGAT in horizontal cells of the guinea pig retina, and support the idea that GABA is synthesized from GAD65, taken up into synaptic vesicles by VGAT, and likely released by a vesicular mechanism from horizontal cells. PMID:20235161

  11. GABA Australis, some reflections on the history of GABA receptor research in Australia.

    PubMed

    Johnston, Graham A R

    2017-02-01

    Research on GABA receptors has a long history in Australia dating from 1958 with David Curtis and his colleagues in Canberra. This review traces many of the advances made in Australia guided by highly cited publications and some obscure ones. It covers the discovery of key chemicals with which to investigate GABA receptor function including bicuculline, muscimol, phaclofen, THIP and (+)-CAMP. Also described are findings relevant to the involvement of mutant GABA receptors in inherited epilepsy. The modulation of GABA receptors by a bewildering range of chemicals, especially by flavonoids and terpenoids, is discussed.

  12. Inhibition of GABA release by presynaptic ionotropic GABA receptors in hippocampal CA3.

    PubMed

    Axmacher, Nikolai; Draguhn, Andreas

    2004-02-09

    Vesicular transmitter release can be regulated by transmitter-gated ion channels at presynaptic axon terminals. The central inhibitory transmitter GABA acts on such presynaptic ionotropic receptors in various cells, including inhibitory interneurons. Here we report that GABA-mediated postsynaptic inhibitory currents in CA3 pyramidal cells of rat hippocampal slices are suppressed by agonists of GABAA receptors. The effect is present for both stimulus-induced and miniature IPSCs, indicating a reduction in the probability of vesicular release by presynaptic, action-potential-independent mechanisms. We conclude that the release of GABA from hippocampal CA3 interneurons is regulated by a negative feedback via presynaptic ionotropic GABA autoreceptors.

  13. Treatment of chronic pruritus with the selective serotonin re-uptake inhibitors paroxetine and fluvoxamine: results of an open-labelled, two-arm proof-of-concept study.

    PubMed

    Ständer, Sonja; Böckenholt, Barbara; Schürmeyer-Horst, Funda; Weishaupt, Carsten; Heuft, Gereon; Luger, Thomas A; Schneider, Gudrun

    2009-01-01

    Chronic pruritus is difficult to treat and requires the evaluation of new therapeutic modalities. We initiated an open-labelled, two-arm prospective, proof-of-concept study applying two selective serotonin re-uptake inhibitors on a long-term basis. Paroxetine and fluvoxamine were tested in a total of 72 pruritic patients (27 men, 45 women, age range 28-88 years, mean age 59.2 years). The reduction in pruritus was evaluated by analysis of visual analogue scores and determination of the maximal antipruritic effect (maximal percentual reduction in pruritus). Forty-nine of 72 patients (68.0%) experienced a weak (n=9), good (n=16) or very good (n=24) antipruritic effect. Statistical analysis proved the efficacy of paroxetine and fluvoxamine with no significant difference. The best response was observed in patients with pruritus due to atopic dermatitis, systemic lymphoma and solid carcinoma. Chronic scratch lesions healed completely in 14/31 patients and partially in 17/31 patients. Adverse drug effects were observed in 70.8% of patients, resulting in discontinuation of treatment in 18 patients. These results support previous reports of high antipruritic potency of selective serotonin re-uptake inhibitors, which are a good alternative treatment modality in chronic pruritus. This should be confirmed in future double-blind studies.

  14. Inositol uptake in rat aorta

    SciTech Connect

    Rapoport, R.M.; Van Gorp, C.; Chang, Ki-Churl )

    1990-01-01

    {sup 3}H-inositol uptake into deendothelialized aorta was linear for at least 2 h and was composed of both a saturable, Na{sup +}-dependent, and a nonsaturable, Na{sup +}-independent component. The Na{sup +}-dependent component of inositol uptake had a K{sub m} of 50 {mu}M and a V{sub max} of 289 pmol/mg prot/h. Exposure to LiCl, ouabain, or Ca{sup 2+} - free Krebs-Ringer bicarbonate solution inhibited uptake. Metabolic poisoning with dinitrophenol, as well as incubation with phloretin, an inhibitor of carrier-mediated hexose transport, also inhibited uptake. Exposure to norepinephrine decreased inositol uptake, while phorbol myristate acetate was without effect. Isobutylmethylxanthine significantly increased inositol uptake, while the increased uptake due to dibutyryl cyclic AMP and forskolin were not statistically significant. Sodium nitroprusside, and activator of guanylate cyclase, and 8-bromo cyclic GMP, were without effect on uptake, as was methylene blue, an inhibitor of guanylate cyclase. Inositol uptake into the aorta was increased when the endothelium was allowed to remain intact, although this effect was likely due to uptake in both the endothelial and smooth muscle cells.

  15. Novel GABA receptor pesticide targets.

    PubMed

    Casida, John E; Durkin, Kathleen A

    2015-06-01

    The γ-aminobutyric acid (GABA) receptor has four distinct but overlapping and coupled targets of pesticide action importantly associated with little or no cross-resistance. The target sites are differentiated by binding assays with specific radioligands, resistant strains, site-directed mutagenesis and molecular modeling. Three of the targets are for non-competitive antagonists (NCAs) or channel blockers of widely varied chemotypes. The target of the first generation (20th century) NCAs differs between the larger or elongated compounds (NCA-IA) including many important insecticides of the past (cyclodienes and polychlorocycloalkanes) or present (fiproles) and the smaller or compact compounds (NCA-IB) highly toxic to mammals and known as cage convulsants, rodenticides or chemical threat agents. The target of greatest current interest is designated NCA-II for the second generation (21st century) of NCAs consisting for now of isoxazolines and meta-diamides. This new and uniquely different NCA-II site apparently differs enough between insects and mammals to confer selective toxicity. The fourth target is the avermectin site (AVE) for allosteric modulators of the chloride channel. NCA pesticides vary in molecular surface area and solvent accessible volume relative to avermectin with NCA-IBs at 20-22%, NCA-IAs at 40-45% and NCA-IIs at 57-60%. The same type of relationship relative to ligand-docked length is 27-43% for NCA-IBs, 63-71% for NCA-IAs and 85-105% for NCA-IIs. The four targets are compared by molecular modeling for the Drosophila melanogaster GABA-R. The principal sites of interaction are proposed to be: pore V1' and A2' for NCA-IB compounds; pore A2', L6' and T9' for NCA-IA compounds; pore T9' to S15' in proximity to M1/M3 subunit interface (or alternatively an interstitial site) for NCA-II compounds; and M1/M3, M2 interfaces for AVE. Understanding the relationships of these four binding sites is important in resistance management and in the discovery and use

  16. Basic mechanism leading to stimulation of glycogenolysis by isoproterenol, EGF, elevated extracellular K+ concentrations, or GABA.

    PubMed

    Xu, Junnan; Song, Dan; Bai, Qiufang; Cai, Liping; Hertz, Leif; Peng, Liang

    2014-04-01

    Glycogenolysis, in brain parenchyma an astrocyte-specific process, has changed from being envisaged as an emergency procedure to playing central roles during brain response to whisker stimulation, memory formation, astrocytic K(+) uptake and stimulated release of ATP. It is activated by several transmitters and by even very small increases in extracellular K(+) concentration, and to be critically dependent upon an increase in free cytosolic Ca(2+) concentration ([Ca(2+)]i), whereas cAMP plays only a facilitatory role together with increased [Ca(2+)]i. Detailed knowledge about the signaling pathways eliciting glycogenolysis is therefore of interest and was investigated in the present study in well differentiated cultures of mouse astrocytes. The β-adrenergic agonist isoproterenol stimulated glycogenolysis by a β1-adrenergic effect, which initiated a pathway in which cAMP/protein kinase A activated a Gi/Gs shift, leading to Ca(2+)-activated glycogenolysis. Inhibition of this pathway downstream of cAMP but upstream of the Gi/Gs shift abolished the glycogenolysis. However, inhibitors operating downstream of the Ca(2+)-sensitive step, but preventing transactivation-mediated epidermal growth factor (EGF) receptor stimulation, a later step in the activated pathway, also caused inhibition of glycogenolysis. For this reason the effect of EGF was investigated and it was found to be glycogenolytic. Large increases in extracellular K(+) activated glycogenolysis by a nifedipine-inhibited L-channel opening allowing influx of Ca(2+), known to be glycogenolysis-dependent. Small increases (addition of 5 mM KCl) caused a smaller effect by a similarly glycogenolysis-reliant opening of an IP3 receptor-dependent ouabain signaling pathway. The same pathway could be activated by GABA (also in brain slices) due to its depolarizing effect in astrocytes.

  17. Differential effects of phosphonic analogues of GABA on GABA(B) autoreceptors in rat neocortical slices.

    PubMed

    Ong, J; Marino, V; Parker, D A; Kerr, D I

    1998-04-01

    The effects of five phosphonic derivatives of GABA on the release of [3H]-GABA from rat neocortical slices, preloaded with [3H]-GABA, were investigated. Phaclofen and 4-aminobutylphosphonic acid (4-ABPA) increased the overflow of [3H] evoked by electrical stimulation (2 Hz) in a concentration-dependent manner, with similar potencies (phaclofen EC50=0.3 mmol/l, 4-ABPA EC50=0.4 mmol/l). At 3 mmol/l, phaclofen increased the release of [3H]-GABA by 82.6+/-8.6%, and 4-ABPA increased the release by 81.3+/-9.0%. 2-Amino-ethylphosphonic acid (2-AEPA) increased the overflow of [3H] by 46.8+/-10.9% at the highest concentration tested (3 mmol/l). In contrast, the lower phosphonic homologue 3-aminopropylphosphonic acid (3-APPA), and 2-amino-2-(p-chlorophenyl)-ethylphosphonic acid (2-CPEPA), a baclofen analogue, did not modify the stimulated overflow. These results suggest that phaclofen, 4-ABPA and 2-AEPA are antagonists at GABA(B) autoreceptors, the latter being the weakest antagonist, whilst neither 3-APPA nor 2-CPEPA are active at these receptors. Since phaclofen, 4-ABPA and 2-CPEPA are antagonists and 3-APPA a partial agonist/antagonist on GABA(B) heteroreceptors, the lack of effect of 3-APPA and 2-CPEPA on [3H]-GABA release in this study suggests that GABA(B) autoreceptors may be pharmacologically distinct from the heteroreceptors.

  18. GABA(B2) is essential for g-protein coupling of the GABA(B) receptor heterodimer.

    PubMed

    Robbins, M J; Calver, A R; Filippov, A K; Hirst, W D; Russell, R B; Wood, M D; Nasir, S; Couve, A; Brown, D A; Moss, S J; Pangalos, M N

    2001-10-15

    GABA(B) receptors are unique among G-protein-coupled receptors (GPCRs) in their requirement for heterodimerization between two homologous subunits, GABA(B1) and GABA(B2), for functional expression. Whereas GABA(B1) is capable of binding receptor agonists and antagonists, the role of each GABA(B) subunit in receptor signaling is unknown. Here we identified amino acid residues within the second intracellular domain of GABA(B2) that are critical for the coupling of GABA(B) receptor heterodimers to their downstream effector systems. Our results provide strong evidence for a functional role of the GABA(B2) subunit in G-protein coupling of the GABA(B) receptor heterodimer. In addition, they provide evidence for a novel "sequential" GPCR signaling mechanism in which ligand binding to one heterodimer subunit can induce signal transduction through the second partner of a heteromeric complex.

  19. GABA and Glutamate Pathways Are Spatially and Developmentally Affected in the Brain of Mecp2-Deficient Mice

    PubMed Central

    Matagne, Valérie; Ghata, Adeline; Villard, Laurent; Roux, Jean-Christophe

    2014-01-01

    Proper brain functioning requires a fine-tuning between excitatory and inhibitory neurotransmission, a balance maintained through the regulation and release of glutamate and GABA. Rett syndrome (RTT) is a rare genetic disorder caused by mutations in the methyl-CpG binding protein 2 (MECP2) gene affecting the postnatal brain development. Dysfunctions in the GABAergic and glutamatergic systems have been implicated in the neuropathology of RTT and a disruption of the balance between excitation and inhibition, together with a perturbation of the electrophysiological properties of GABA and glutamate neurons, were reported in the brain of the Mecp2-deficient mouse. However, to date, the extent and the nature of the GABA/glutamate deficit affecting the Mecp2-deficient mouse brain are unclear. In order to better characterize these deficits, we simultaneously analyzed the GABA and glutamate levels in Mecp2-deficient mice at 2 different ages (P35 and P55) and in several brain areas. We used a multilevel approach including the quantification of GABA and glutamate levels, as well as the quantification of the mRNA and protein expression levels of key genes involved in the GABAergic and glutamatergic pathways. Our results show that Mecp2-deficient mice displayed regional- and age-dependent variations in the GABA pathway and, to a lesser extent, in the glutamate pathway. The implication of the GABA pathway in the RTT neuropathology was further confirmed using an in vivo treatment with a GABA reuptake inhibitor that significantly improved the lifespan of Mecp2-deficient mice. Our results confirm that RTT mouse present a deficit in the GABAergic pathway and suggest that GABAergic modulators could be interesting therapeutic agents for this severe neurological disorder. PMID:24667344

  20. Chronic lamotrigine treatment increases rat hippocampal GABA shunt activity and elevates cerebral taurine levels.

    PubMed

    Hassel, B; Taubøll, E; Gjerstad, L

    2001-02-01

    The mechanism of action of the antiepileptic drug lamotrigine has previously been investigated only in acute experiments and is thought to involve inhibition of voltage-dependent sodium channels. However, lamotrigine is effective against more forms of epilepsies than other antiepileptic drugs that also inhibit sodium channels. We investigated whether chronic lamotrigine treatment may affect cerebral amino acid levels. Rats received lamotrigine, 10 mg/kg/day, for 90 days. The hippocampal level of GABA increased 25%, and the activities of glutamate decarboxylase and succinic semialdehyde/GABA transaminase increased 12 and 21% (p< 0.05), respectively, indicating increased GABA turnover. The uptake of GABA and glutamate into proteoliposomes remained unaltered. The level of taurine increased 27% in the hippocampus and 16% in the frontal and parietal cortices. The activities of hexokinase and alpha-ketoglutarate dehydrogenase, remained at control values. Serum lamotrigine was 41.7+/-1.5 microM (mean+/-S.E.M.), which is within the range seen in epileptic patients. Acute experiments with 5, 20 or 100 mg lamotrigine/kg, caused no changes in brain amino acid levels. The results suggest that chronic lamotrigine treatment increases GABAergic activity in the hippocampus. The cerebral increase in taurine, which has neuromodulatory properties, may contribute to the antiepileptic effect of lamotrigine.

  1. My close encounter with GABA(B) receptors.

    PubMed

    Nicoll, Roger A

    2004-10-15

    In this review, I summarize the sequence of events involved in characterizing the functional role of GABA(B) receptors in the CNS and their involvement in synaptic transmission. The story was launched with the realization that baclofen was a selective agonist of GABA(B) receptors. This lead to the discovery in the CNS that GABA(B) receptor activation could result in a presynaptic inhibition of transmitter release as well as a postsynaptic increase in potassium conductance. Based on this information, it was found that GABA also activated a potassium conductance. A role for GABA(B) receptors in synaptic transmission was suggested by the fact that activation of GABAergic interneurons could generate a slow IPSP mediated by an increase in potassium conductance. To link this slow IPSP to GABA(B) receptors required a selective GABA(B) antagonist. Phaclofen was the first antagonist developed and was found to antagonize the action of baclofen and the GABA(A) independent action of GABA. Most importantly, it blocked the slow IPSP. The properties of GABA(A) and GABA(B) IPSPs are remarkably different. GABA(A) IPSPs powerfully inhibit neurons and rapidly curtail excitatory inputs. This greatly enhances the precision of excitatory synaptic transmission. GABA(B) IPSPs are recruited with repetitive and synchronous activity and are postulated to modulate the rhythmic network activity of cortical tissue.

  2. Production of gaba (γ - Aminobutyric acid) by microorganisms: a review.

    PubMed

    Dhakal, Radhika; Bajpai, Vivek K; Baek, Kwang-Hyun

    2012-10-01

    GABA (γ-aminobutyric acid) is a four carbon non-protein amino acid that is widely distributed in plants, animals and microorganisms. As a metabolic product of plants and microorganisms produced by the decarboxylation of glutamic acid, GABA functions as an inhibitory neurotransmitter in the brain that directly affects the personality and the stress management. A wide range of traditional foods produced by microbial fermentation contain GABA, in which GABA is safe and eco-friendly, and also has the possibility of providing new health-benefited products enriched with GABA. Synthesis of GABA is catalyzed by glutamate decarboxylase, therefore, the optimal fermentation condition is mainly based on the biochemical properties of the enzyme. Major GABA producing microorganisms are lactic acid bacteria (LAB), which make food spoilage pathogens unable to grow and act as probiotics in the gastrointestinal tract. The major factors affecting the production of GABA by microbial fermentation are temperature, pH, fermentation time and different media additives, therefore, these factors are summarized to provide the most up-dated information for effective GABA synthesis. There has been a huge accumulation of knowledge on GABA application for human health accompanying with a demand on natural GABA supply. Only the GABA production by microorganisms can fulfill the demand with GABA-enriched health beneficial foods.

  3. GABA blocks pathological but not acute TRPV1 pain signals.

    PubMed

    Hanack, Christina; Moroni, Mirko; Lima, Wanessa C; Wende, Hagen; Kirchner, Marieluise; Adelfinger, Lisa; Schrenk-Siemens, Katrin; Tappe-Theodor, Anke; Wetzel, Christiane; Kuich, P Henning; Gassmann, Martin; Roggenkamp, Dennis; Bettler, Bernhard; Lewin, Gary R; Selbach, Matthias; Siemens, Jan

    2015-02-12

    Sensitization of the capsaicin receptor TRPV1 is central to the initiation of pathological forms of pain, and multiple signaling cascades are known to enhance TRPV1 activity under inflammatory conditions. How might detrimental escalation of TRPV1 activity be counteracted? Using a genetic-proteomic approach, we identify the GABAB1 receptor subunit as bona fide inhibitor of TRPV1 sensitization in the context of diverse inflammatory settings. We find that the endogenous GABAB agonist, GABA, is released from nociceptive nerve terminals, suggesting an autocrine feedback mechanism limiting TRPV1 sensitization. The effect of GABAB on TRPV1 is independent of canonical G protein signaling and rather relies on close juxtaposition of the GABAB1 receptor subunit and TRPV1. Activating the GABAB1 receptor subunit does not attenuate normal functioning of the capsaicin receptor but exclusively reverts its sensitized state. Thus, harnessing this mechanism for anti-pain therapy may prevent adverse effects associated with currently available TRPV1 blockers.

  4. Taurine activates GABA(A) but not GABA(B) receptors in rat hippocampal CA1 area.

    PubMed

    del Olmo, N; Bustamante, J; del Río, R M; Solís, J M

    2000-05-12

    We investigated if taurine, an endogenous GABA analog, could mimic both hyperpolarizing and depolarizing GABA(A)-mediated responses as well as pre- and postsynaptic GABA(B)-mediated actions in the CA1 region of rat hippocampal slices. Taurine (10 mM) perfusion induced changes in membrane potential and input resistance that are compatible with GABA(A) receptor activation. Local pressure application of taurine and GABA from a double barrel pipette positioned along the dendritic shaft of pyramidal cells revealed that taurine evoked a very small change of membrane potential and resistance compared with the large changes induced by GABA in these parameters. Moreover, in the presence of GABA(A) antagonists, local application of GABA on the dendrites evoked a GABA(B)-mediated hyperpolarization while taurine did not induce any change. Taurine neither mimicked baclofen inhibitory actions on presynaptic release of glutamate and GABA as judging by the lack of taurine effect on paired-pulse facilitation ratio and slow inhibitory postsynaptic potentials, respectively. These results show that taurine mainly activates GABA(A) receptors located on the cell body, indicating therefore that if taurine has any action on the dendrites it will not be mediated by either GABA(A) or GABA(B) receptors activation.

  5. Advantages of an antagonist: bicuculline and other GABA antagonists

    PubMed Central

    Johnston, Graham AR

    2013-01-01

    The convulsant alkaloid bicuculline continues to be investigated more than 40 years after the first publication of its action as an antagonist of receptors for the inhibitory neurotransmitter GABA. This historical perspective highlights key aspects of the discovery of bicuculline as a GABA antagonist and the sustained interest in this and other GABA antagonists. The exciting advances in the molecular biology, pharmacology and physiology of GABA receptors provide a continuing stimulus for the discovery of new antagonists with increasing selectivity for the myriad of GABA receptor subclasses. Interesting GABA antagonists not structurally related to bicuculline include gabazine, salicylidene salicylhydrazide, RU5135 and 4-(3-biphenyl-5-(4-piperidyl)-3-isoxazole. Bicuculline became the benchmark antagonist for what became known as GABAA receptors, but not all ionotropic GABA receptors are susceptible to bicuculline. In addition, not all GABAA receptor antagonists are convulsants. Thus there are still surprises in store as the study of GABA receptors evolves. PMID:23425285

  6. Trophic actions of GABA on neuronal development.

    PubMed

    Represa, Alfonso; Ben-Ari, Yehezkel

    2005-06-01

    During brain development, transmitter-gated receptors are operative before synapse formation, suggesting that their action is not restricted to synaptic transmission. GABA, which is the principal excitatory transmitter in the developing brain, acts as an epigenetic factor to control processes including cell proliferation, neuroblast migration and dendritic maturation. These effects appear to be mediated through a paracrine, diffuse, non-synaptic mode of action that precedes the more focused, rapid mode of operation characteristic of synaptic connections. This sequential operation implies that GABA is used as an informative agent but in a unique context at an early developmental stage. This sequence also implies that by altering these effects, drugs acting on the GABA system could be pathogenic during pregnancy.

  7. An excitatory GABA loop operating in vivo

    PubMed Central

    Astorga, Guadalupe; Bao, Jin; Marty, Alain; Augustine, George J.; Franconville, Romain; Jalil, Abdelali; Bradley, Jonathan; Llano, Isabel

    2015-01-01

    While it has been proposed that the conventional inhibitory neurotransmitter GABA can be excitatory in the mammalian brain, much remains to be learned concerning the circumstances and the cellular mechanisms governing potential excitatory GABA action. Using a combination of optogenetics and two-photon calcium imaging in vivo, we find that activation of chloride-permeable GABAA receptors in parallel fibers (PFs) of the cerebellar molecular layer of adult mice causes parallel fiber excitation. Stimulation of PFs at submaximal stimulus intensities leads to GABA release from molecular layer interneurons (MLIs), thus creating a positive feedback loop that enhances excitation near the center of an activated PF bundle. Our results imply that elevated chloride concentration can occur in specific intracellular compartments of mature mammalian neurons and suggest an excitatory role for GABAA receptors in the cerebellar cortex of adult mice. PMID:26236197

  8. Glutamate and GABA in Appetite Regulation

    PubMed Central

    Delgado, Teresa C.

    2013-01-01

    Appetite is regulated by a coordinated interplay between gut, adipose tissue, and brain. A primary site for the regulation of appetite is the hypothalamus where interaction between orexigenic neurons, expressing Neuropeptide Y/Agouti-related protein, and anorexigenic neurons, expressing Pro-opiomelanocortin cocaine/Amphetamine-related transcript, controls energy homeostasis. Within the hypothalamus, several peripheral signals have been shown to modulate the activity of these neurons, including the orexigenic peptide ghrelin and the anorexigenic hormones insulin and leptin. In addition to the accumulated knowledge on neuropeptide signaling, presence and function of amino acid neurotransmitters in key hypothalamic neurons brought a new light into appetite regulation. Therefore, the principal aim of this review will be to describe the current knowledge of the role of amino acid neurotransmitters in the mechanism of neuronal activation during appetite regulation and the associated neuronal-astrocytic metabolic coupling mechanisms. Glutamate and GABA dominate synaptic transmission in the hypothalamus and administration of their receptors agonists into hypothalamic nuclei stimulates feeding. By using 13C High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance spectroscopy based analysis, the Cerdán group has shown that increased neuronal firing in mice hypothalamus, as triggered by appetite during the feeding-fasting paradigm, may stimulate the use of lactate as neuronal fuel leading to increased astrocytic glucose consumption and glycolysis. Moreover, fasted mice showed increased hypothalamic [2-13C]GABA content, which may be explained by the existence of GABAergic neurons in key appetite regulation hypothalamic nuclei. Interestingly, increased [2-13C]GABA concentration in the hypothalamus of fasted animals appears to result mainly from reduction in GABA metabolizing pathways, rather than increased GABA synthesis by augmented activity of the glutamate-glutamine-GABA

  9. Glutamate and GABA in Appetite Regulation.

    PubMed

    Delgado, Teresa C

    2013-01-01

    Appetite is regulated by a coordinated interplay between gut, adipose tissue, and brain. A primary site for the regulation of appetite is the hypothalamus where interaction between orexigenic neurons, expressing Neuropeptide Y/Agouti-related protein, and anorexigenic neurons, expressing Pro-opiomelanocortin cocaine/Amphetamine-related transcript, controls energy homeostasis. Within the hypothalamus, several peripheral signals have been shown to modulate the activity of these neurons, including the orexigenic peptide ghrelin and the anorexigenic hormones insulin and leptin. In addition to the accumulated knowledge on neuropeptide signaling, presence and function of amino acid neurotransmitters in key hypothalamic neurons brought a new light into appetite regulation. Therefore, the principal aim of this review will be to describe the current knowledge of the role of amino acid neurotransmitters in the mechanism of neuronal activation during appetite regulation and the associated neuronal-astrocytic metabolic coupling mechanisms. Glutamate and GABA dominate synaptic transmission in the hypothalamus and administration of their receptors agonists into hypothalamic nuclei stimulates feeding. By using (13)C High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance spectroscopy based analysis, the Cerdán group has shown that increased neuronal firing in mice hypothalamus, as triggered by appetite during the feeding-fasting paradigm, may stimulate the use of lactate as neuronal fuel leading to increased astrocytic glucose consumption and glycolysis. Moreover, fasted mice showed increased hypothalamic [2-(13)C]GABA content, which may be explained by the existence of GABAergic neurons in key appetite regulation hypothalamic nuclei. Interestingly, increased [2-(13)C]GABA concentration in the hypothalamus of fasted animals appears to result mainly from reduction in GABA metabolizing pathways, rather than increased GABA synthesis by augmented activity of the glutamate-glutamine-GABA

  10. Oxytocin regulates changes of extracellular glutamate and GABA levels induced by methamphetamine in the mouse brain.

    PubMed

    Qi, Jia; Han, Wen-Yan; Yang, Jing-Yu; Wang, Li-Hui; Dong, Ying-Xu; Wang, Fang; Song, Ming; Wu, Chun-Fu

    2012-07-01

    Oxytocin (OT), a neurohypophyseal neuropeptide, affects adaptive processes of the central nervous system. In the present study, we investigated the effects of OT on extracellular levels of glutamate (Glu) and γ-aminobutyric acid (GABA) induced by methamphetamine (MAP) in the medial prefrontal cortex (mPFC) and dorsal hippocampus (DHC) of freely moving mice, using in vivo microdialysis coupled to high-performance liquid chromatography and fluorescence detection. The results showed that OT had no effect on basal Glu levels, but attenuated MAP-induced Glu increase in the mPFC and decrease in the DHC. OT increased the basal levels of extracellular GABA in mPFC and DHC of mice, and inhibited the MAP-induced GABA decrease in DHC. Western blot results indicated that OT significantly inhibited the increased glutamatergic receptor (NR1 subunit) levels in the PFC after acute MAP administration, whereas OT further enhanced the elevated levels of glutamatergic transporter (GLT1) induced by MAP in the hippocampus of mice. Atosiban, a selective inhibitor of OT receptor, antagonized the effects of OT. The results provided the first neurochemical evidence that OT, which exerted its action via its receptor, decreased Glu release induced by MAP, and attenuated the changes in glutamatergic neurotransmission partially via regulation of NR1 and GLT1 expression. OT-induced extracellular GABA increase also suggests that OT acts potentially as an inhibitory neuromodulator in mPFC and DHC of mice.

  11. [GABA-ergic system in defense against excitatory kynurenines].

    PubMed

    Lapin, I P

    1997-01-01

    Protection against the excitatory action of L-kynurenine and quinolinic acid in mice is related to the activation of GABA-B and dopamine receptors of the brain and to much lesser degree to the activation of GABA-A receptors. It is hardly believable that the anticonvulsant effect of phenibut (beta-phenyl-GABA), baclofen (CL-phenibut), sodium hydroxybutyrate and taurine against seizures induced by these two kynurenines is determined by alterations in metabolism of GABA.

  12. Medicinal and aromatic plant materials as nitrification inhibitors for augmenting yield and nitrogen uptake of Japanese mint (Mentha arvensis L. Var. Piperascens).

    PubMed

    Kiran, Usha; Patra, D D

    2003-02-01

    Pot experiments were conducted to evaluate the relative performance of medicinal and aromatic plant materials and dicyandiamide (DCD) as nitrification inhibitors to regulate transformation of N from urea. Their effect on the efficiencies of use of N by Japanese mint (Mentha arvensis cv. Hy 77) was tested. Urea was coated with these materials viz., Mentha spicata, Artemisia annua or DCD at the rate of 5% (w/w) of fertilizer urea using an appropriate coating technique. Nimin (tetranortriterpenoids, an ethanol extract of neem (Azadirachta indica Juss) coating was done at the rate of 1% w/w of urea. Fertilizer nitrogen was applied at 100 and 200 mg kg(-1) soil. These natural coating materials significantly increased the herb and essential oil yields of the crop at both rates of fertilizer nitrogen compared to urea alone and were found to be as effective as DCD in retarding NO3- formation in soil. Herb yield increased by 6-81% when compared to uncoated urea. The increase in essential oil yield ranged between 3% and 68% due to coating. The effectiveness of the nitrification-inhibitor--coated urea, however, varied with the soils used and the rate of fertilizer nitrogen applied. The results suggest that the natural products could be potential nitrification inhibitors for increasing fertilizer N use efficiency.

  13. Effects of inhibitors of key enzymes of sphingolipid metabolism on insulin-induced glucose uptake and glycogen synthesis in liver cells of old rats.

    PubMed

    Babenko, N A; Kharchenko, V S

    2015-01-01

    Sphingolipids play an important role in the development of insulin resistance. Ceramides are the most potent inhibitors of insulin signal transduction. Ceramides are generated in response to stress stimuli and in old age. In this work, we studied the possible contribution of different pathways of sphingolipid metabolism in age-dependent insulin resistance development in liver cells. Inhibition of key enzymes of sphingolipid synthesis (serine palmitoyl transferase, ceramide synthase) and degradation (neutral and acidic SMases) by means of specific inhibitors (myriocin, fumonisin B1, imipramine, and GW4869) was followed with the reduction of ceramide level and partly improved insulin regulation of glucose metabolism in "old" hepatocytes. Imipramine and GW4869 decreased significantly the acidic and neutral SMase activities, respectively. Treatment of "old" cells with myriocin or fumonisin B1 reduced the elevated in old age ceramide and SM synthesis. Ceramide and SM levels and glucose metabolism regulation by insulin could be improved with concerted action of all tested inhibitors of sphingolipid turnover on hepatocytes. The data demonstrate that not only newly synthesized ceramide and SM but also neutral and acidic SMase-dependent ceramide accumulation plays an important role in development of age-dependent insulin resistance.

  14. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and gamma-vinyl-gamma-aminobutyric acid (gamma-vinyl GABA) alter neurotransmitter concentrations in the nervous tissue of the goldfish (Carassius auratus) but not the cockroach (Periplaneta americana).

    PubMed

    Sloley, B D; McKenna, K F

    1993-02-01

    1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium iodide (MPP+) and gamma-vinyl-gamma-aminobutyric acid (gamma-vinyl GABA) are drugs demonstrated to alter catecholamine or gamma-aminobutyric acid (GABA) concentrations in vertebrate nervous tissue. MPTP and MPP+, which are potent and selective vertebrate neurotoxins, are effective in depleting noradrenaline and dopamine concentrations in goldfish. However, only MPP+ depletes dopamine in the central nervous tissues of the cockroach, and only when injected directly into the nervous tissue. Systemic injection of gamma-vinyl GABA, a selective GABA transaminase inhibitor in vertebrates, increases GABA concentrations in goldfish but not cockroach nervous tissue. Incubations of both goldfish hypothalamus and cockroach nervous tissue demonstrated the presence of GABA transaminase activity in vitro. However, the GABA transaminase activity obtained from goldfish tissues was much more sensitive to inhibition by gamma-vinyl GABA than that obtained from cockroach nervous tissue. These results demonstrate that MPTP, MPP+ and gamma-vinyl GABA are useful pharmacological tools which can alter neurotransmitter concentrations in a lower vertebrate. Unfortunately, they possess limited effectiveness in the cockroach.

  15. Assignment of the human GABA transporter gene (GABATHG) locus to chromosome 3p24-p25

    SciTech Connect

    Huang, Fang; Fei, Jian; Guo, Li-He

    1995-09-01

    An essential regulatory process of synaptic transmission is the inactivation of released neurotransmitters by the transmitter-specific uptake mechanism, {gamma}-Aminobutyric acid (GABA) is an inhibitory transmitter in the vertebrate central nervous system; its activity is terminated by a high-affinity Na{sup +} and Cl{sup -} -dependent specific GABA transporter (GAT), which carries the neurotransmitter to the presynaptic neuron and/or glial elements surrounding the synaptic cleft. Deficiency of the transporter may cause epilepsy and some other nervous diseases. The human GAT gene (GABATHG), approximately 25 kb in length, has been cloned and sequenced by our colleagues (7). Here the results of the in situ hybridization mapping with the gene are presented. 10 refs., 1 fig.

  16. Development of potent dopamine-norepinephrine uptake inhibitors (DNRIs) based on a (2S,4R,5R)-2-benzhydryl-5-((4-methoxybenzyl)amino)tetrahydro-2H-pyran-4-ol molecular template.

    PubMed

    Santra, Soumava; Sharma, Horrick; Vedachalam, Seenuvasan; Antonio, Tamara; Reith, Maarten; Dutta, Aloke

    2015-02-15

    Current therapy of depression is less than ideal with remission rates of only 25-35% and response rates of 45-60%. It has been hypothesized that a dysfunctional dopaminergic system in the mesocorticolimbic pathway in depressive disorder may lead to development of anhedonia associated with loss of pleasure and interest along with loss of motivation. The current antidepressants do not address dopamine dysfunction which might explain their low efficacy. In this report, we have described an SAR study on our pyran-based triple reuptake inhibitors (TRIs) which are being investigated as the next-generation antidepressants. In the present work we demonstrate that our lead TRIs can be modified with appropriate aromatic substitutions to display a highly potent SSRI profile for compounds 2a and 4a (Ki (SERT); 0.71 and 2.68nM, respectively) or a potent DNRI profile for compounds 6b and 6h (Ki (DAT/NET); 8.94/4.76 and 13/7.37nM, respectively). Compounds 4g-4i exhibited potencies at all three monoamine transporters. The results provide insights into the structural requirements for developing selective dual- and triple-uptake inhibitors from a unique pyran molecular template for an effective management of depression and related disorders.

  17. Frontal GABA levels change during working memory.

    PubMed

    Michels, Lars; Martin, Ernst; Klaver, Peter; Edden, Richard; Zelaya, Fernando; Lythgoe, David J; Lüchinger, Rafael; Brandeis, Daniel; O'Gorman, Ruth L

    2012-01-01

    Functional neuroimaging metrics are thought to reflect changes in neurotransmitter flux, but changes in neurotransmitter levels have not been demonstrated in humans during a cognitive task, and the relationship between neurotransmitter dynamics and hemodynamic activity during cognition has not yet been established. We evaluate the concentration of the major inhibitory (GABA) and excitatory (glutamate + glutamine: Glx) neurotransmitters and the cerebral perfusion at rest and during a prolonged delayed match-to-sample working memory task. Resting GABA levels in the dorsolateral prefrontal cortex correlated positively with the resting perfusion and inversely with the change in perfusion during the task. Further, only GABA increased significantly during the first working memory run and then decreased continuously across subsequent task runs. The decrease of GABA over time was paralleled by a trend towards decreased reaction times and higher task accuracy. These results demonstrate a link between neurotransmitter dynamics and hemodynamic activity during working memory, indicating that functional neuroimaging metrics depend on the balance of excitation and inhibition required for cognitive processing.

  18. Plant reproduction: GABA gradient, guidance and growth.

    PubMed

    Ma, Hong

    2003-10-28

    How a pollen tube manages to navigate through the female tissues during plant reproduction has been a mystery. A new analysis of an Arabidopsis mutant has provided the strongest evidence yet that a GABA gradient may be a critical signal for correct targeting of the pollen tube.

  19. Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs cycle in rat pancreatic islets.

    PubMed

    Hernández-Fisac, Inés; Fernández-Pascual, Sergio; Ortsäter, Henrik; Pizarro-Delgado, Javier; Martín del Río, Rafael; Bergsten, Peter; Tamarit-Rodriguez, Jorge

    2006-11-15

    OMP (oxo-4-methylpentanoic acid) stimulates by itself a biphasic secretion of insulin whereas L-leucine requires the presence of L-glutamine. L-Glutamine is predominantly converted into GABA (gamma-aminobutyric acid) in rat islets and L-leucine seems to promote its metabolism in the 'GABA shunt' [Fernández-Pascual, Mukala-Nsengu-Tshibangu, Martín del Río and Tamarit-Rodríguez (2004) Biochem. J. 379, 721-729]. In the present study, we have investigated how 10 mM OMP affects L-glutamine metabolism to uncover possible differences with L-leucine that might help to elucidate whether they share a common mechanism of stimulation of insulin secretion. In contrast with L-leucine, OMP alone stimulated a biphasic insulin secretion in rat perifused islets and decreased the islet content of GABA without modifying its extracellular release irrespective of the concentration of L-glutamine in the medium. GABA was transaminated to L-leucine whose intracellular concentration did not change because it was efficiently transported out of the islet cells. The L-[U-14C]-Glutamine (at 0.5 and 10.0 mM) conversion to 14CO2 was enhanced by 10 mM OMP within 30% and 70% respectively. Gabaculine (250 microM), a GABA transaminase inhibitor, suppressed OMP-induced oxygen consumption but not L-leucine- or glucose-stimulated respiration. It also suppressed the OMP-induced decrease in islet GABA content and the OMP-induced increase in insulin release. These results support the view that OMP promotes islet metabolism in the 'GABA shunt' generating 2-oxo-glutarate, in the branched-chain alpha-amino acid transaminase reaction, which would in turn trigger GABA deamination by GABA transaminase. OMP, but not L-leucine, suppressed islet semialdehyde succinic acid reductase activity and this might shift the metabolic flux of the 'GABA shunt' from gamma-hydroxybutyrate to succinic acid production.

  20. Changes in antiviral susceptibility to entry inhibitors and endocytic uptake of dengue-2 virus serially passaged in Vero or C6/36 cells.

    PubMed

    Acosta, Eliana G; Piccini, Luana E; Talarico, Laura B; Castilla, Viviana; Damonte, Elsa B

    2014-05-12

    The aim of the present study was to analyze the influence of virus origin, mammalian or mosquito cell-derived, on antiviral susceptibility of DENV-2 to entry inhibitors and the association of this effect with any alteration in the mode of entry into the cell. To this end, ten serial passages of DENV-2 were performed in mosquito C6/36 cells or monkey Vero cells and the antiviral susceptibility of each virus passage to sulfated polysaccharides (SPs), like heparin and carrageenans, was evaluated by a virus plaque reduction assay. After serial passaging in Vero cells, DENV-2 became increasingly resistant to SP inhibition whereas the antiviral susceptibility was not altered in virus propagated in C6/36 cells. The change in antiviral susceptibility was associated to a differential mode of entry into the host cell. The route of endocytic entry for productive Vero cell infection was altered from a non-classical clathrin independent pathway for C6/36-grown virus to a clathrin-mediated endocytosis when the virus was serially propagated in Vero cells. Our results show the impact of the cellular system used for successive propagation of DENV on the initial interaction between the host cell and the virion in the next round of infection and the relevant consequences it might have during the in vitro evaluation of entry inhibitors.

  1. Comparative immunohistochemical localisation of GABA(B1a), GABA(B1b) and GABA(B2) subunits in rat brain, spinal cord and dorsal root ganglion.

    PubMed

    Charles, K J; Evans, M L; Robbins, M J; Calver, A R; Leslie, R A; Pangalos, M N

    2001-01-01

    GABA(B) receptors are G-protein-coupled receptors mediating the slow onset and prolonged synaptic actions of GABA in the CNS. The recent cloning of two genes, GABA(B1) and GABA(B2), has revealed a novel requirement for GABA(B) receptor signalling. Studies have demonstrated that the two receptor subunits associate as a GABA(B1)/GABA(B2) heterodimer to form a functional GABA(B) receptor. In this study we have developed polyclonal antisera specific to two splice variants of the GABA(B1) subunit, GABA(B1a) and GABA(B1b), as well as an antiserum to the GABA(B2) subunit. Using affinity-purified antibodies derived from these antisera we have mapped out the distribution profile of each subunit in rat brain, spinal cord and dorsal root ganglion. In brain the highest areas of GABA(B1a), GABA(B1b) and GABA(B2) subunit expression were found in neocortex, hippocampus, thalamus, cerebellum and habenula. In spinal cord, GABA(B1) and GABA(B2) subunits were expressed in the superficial layers of the dorsal horn, as well as in motor neurones in the deeper layers of the ventral horn. GABA(B) receptor subunit immunoreactivity in dorsal root ganglion suggested that expression of GABA(B1b) was restricted to the large diameter neurones, in contrast to GABA(B1a) and GABA(B2) subunits which were expressed in both large and small diameter neurones. Although expression levels of GABA(B1) and GABA(B2) subunits varied we found no areas in which GABA(B1) was expressed in the absence of GABA(B2). This suggests that most, if not all, GABA(B1) immunoreactivity may represent functional GABA(B) receptors. Although our data are in general agreement with functional studies, some discrepancies in GABA(B1) subunit expression occurred with respect to other immunohistochemical studies. Overall our data suggest that GABA(B) receptors are widely expressed throughout the brain and spinal cord, and that GABA(B1a) and GABA(B1b) subunits can associate with GABA(B2) to form both pre- and post-synaptic receptors.

  2. Drug interactions at GABA(A) receptors.

    PubMed

    Korpi, Esa R; Gründer, Gerhard; Lüddens, Hartmut

    2002-06-01

    Neurotransmitter receptor systems have been the focus of intensive pharmacological research for more than 20 years for basic and applied scientific reasons, but only recently has there been a better understanding of their key features. One of these systems includes the type A receptor for the gamma-aminobutyric acid (GABA), which forms an integral anion channel from a pentameric subunit assembly and mediates most of the fast inhibitory neurotransmission in the adult vertebrate central nervous system. Up to now, depending on the definition, 16-19 mammalian subunits have been cloned and localized on different genes. Their assembly into proteins in a poorly defined stoichiometry forms the basis of functional and pharmacological GABA(A) receptor diversity, i.e. the receptor subtypes. The latter has been well documented in autoradiographic studies using ligands that label some of the receptors' various binding sites, corroborated by recombinant expression studies using the same tools. Significantly less heterogeneity has been found at the physiological level in native receptors, where the subunit combinations have been difficult to dissect. This review focuses on the characteristics, use and usefulness of various ligands and their binding sites to probe GABA(A) receptor properties and to gain insight into the biological function from fish to man and into evolutionary conserved GABA(A) receptor heterogeneity. We also summarize the properties of the novel mouse models created for the study of various brain functions and review the state-of-the-art imaging of brain GABA(A) receptors in various human neuropsychiatric conditions. The data indicate that the present ligands are only partly satisfactory tools and further ligands with subtype-selective properties are needed for imaging purposes and for confirming the behavioral and functional results of the studies presently carried out in gene-targeted mice with other species, including man.

  3. Activation of GABA-A Receptor Ameliorates Homocysteine-Induced MMP-9 Activation by ERK Pathway

    PubMed Central

    TYAGI, NEETU; GILLESPIE, WILLIAM; VACEK, JONATHAN C.; SEN, UTPAL; TYAGI, SURESH C.; LOMINADZE, DAVID

    2010-01-01

    Hyperhomocysteinemia (HHcy) is a risk factor for neuroinflammatory and neurodegenerative diseases. Homocysteine (Hcy) induces redox stress, in part, by activating matrix metalloproteinase-9 (MMP-9), which degrades the matrix and leads to blood–brain barrier dysfunction. Hcy competitively binds to γ-aminbutyric acid (GABA) receptors, which are excitatory neurotransmitter receptors. However, the role of GABA-A receptor in Hcy-induced cerebrovascular remodeling is not clear. We hypothesized that Hcy causes cerebrovascular remodeling by increasing redox stress and MMP-9 activity via the extracellular signal-regulated kinase (ERK) signaling pathway and by inhibition of GABA-A receptors, thus behaving as an inhibitory neurotransmitter. Hcy-induced reactive oxygen species production was detected using the fluorescent probe, 2′–7′-dichlorodihydrofluorescein diacetate. Hcy increased nicotinamide adenine dinucleotide phosphate-oxidase-4 concomitantly suppressing thioredoxin. Hcy caused activation of MMP-9, measured by gelatin zymography. The GABA-A receptor agonist, muscimol ameliorated the Hcy-mediated MMP-9 activation. In parallel, Hcy caused phosphorylation of ERK and selectively decreased levels of tissue inhibitors of metalloproteinase-4 (TIMP-4). Treatment of the endothelial cell with muscimol restored the levels of TIMP-4 to the levels in control group. Hcy induced expression of iNOS and decreased eNOS expression, which lead to a decreased NO bioavailability. Furthermore muscimol attenuated Hcy-induced MMP-9 via ERK signaling pathway. These results suggest that Hcy competes with GABA-A receptors, inducing the oxidative stress transduction pathway and leading to ERK activation. PMID:19308943

  4. [Effects of agonists and antagonists of benzodiazepine, GABA and NMDA receptors, on caffeine-induced seizures in mice].

    PubMed

    Inano, S

    1992-08-01

    In mice, tonic convulsive seizure induced by intravenous administration of caffeine (adenosine A1, A2 receptors antagonist) was significantly potentiated by any one of L-PIA (adenosine A1 receptor agonist), NECA (adenosine A2 receptor agonist) and 2-ClAd (adenosine A1, A2 receptors agonist). The caffeine-induced seizure was unaffected by diazepam (benzodiazepine receptor agonist), but was inhibited by Ro 15-1788 (antagonist or partial agonist). beta-DMCM (antagonist or inverse agonist) increased the seizure. Muscimol (GABA-a receptor agonist), baclofen (GABA-b receptor agonist) and AOAA (GABA transaminase inhibitor) did not show significant effect on caffeine-induced convulsion. Bicuculline (GABA-a receptor antagonist) and picrotoxin (chloride channel blocker) significantly potentiated the convulsion at the doses which did not induce it. Caffeine-induced convulsion was potentiated by NMDA with its non-convulsive dose. CPP (competitive NMDA receptor antagonist) and MK-801 (non-competitive NMDA receptor antagonist) significantly inhibited the seizures. These results suggest that caffeine-induced seizure is not caused by blockade of adenosine receptors. Caffeine may act to beta-carboline sensitive benzodiazepine receptor (Type 1) which has no linkage with GABA-a receptor. Furthermore, it is implied that caffeine plays some role at NMDA receptor calcium ion channel complex.

  5. Effects of ABA and CaCl₂ on GABA accumulation in fava bean germinating under hypoxia-NaCl stress.

    PubMed

    Yang, Runqiang; Hui, Qianru; Gu, Zhenxin

    2016-01-01

    Effects of exogenous abscisic acid (ABA) and CaCl2 on γ-aminobutyric acid (GABA) accumulation of germinated fava bean under hypoxia-NaCl stress were investigated. Exogenous ABA resulted in the enhancement of glutamate decarboxylase (GAD) and diamine oxidase (DAO) activity as well as GABA content in cotyledon and shoot. CaCl2 increased both enzyme activities in shoot and GABA content in cotyledon and shoot. ABA downregulated GAD expression in cotyledon and radicle, while upregulated that in shoot; it also upregulated DAO expression in each organ. CaCl2 upregulated GAD expression in cotyledon, while downregulated that in radicle. However, it upregulated DAO expression in shoot, downregulated that in radicle. ABA inhibitor fluridon and ethylenediaminetetraacetic acid inhibited GAD and DAO activities significantly so that inhibited GABA accumulation through reducing ABA biosynthesis and chelating Ca(2+), respectively. However, they upregulated GAD and DAO expression in varying degrees. These results indicate that ABA and Ca(2+) participate in GABA biosynthesis in fava bean during germination under hypoxia-NaCl stress.

  6. Comparative metabolic profiling revealed limitations in xylose-fermenting yeast during co-fermentation of glucose and xylose in the presence of inhibitors.

    PubMed

    Wang, Xin; Jin, Mingjie; Balan, Venkatesh; Jones, A Daniel; Li, Xia; Li, Bing-Zhi; Dale, Bruce E; Yuan, Ying-Jin

    2014-01-01

    During lignocellulosic ethanol fermentation, yeasts are exposed to various lignocellulose-derived inhibitors, which disrupt the efficiency of hexose and pentose co-fermentation. To understand the metabolic response of fermentation microbes to these inhibitors, a comparative metabolomic investigation was performed on a xylose-fermenting Saccharomyces cerevisiae 424A (LNH-ST) and its parental strain 4124 with and without three typical inhibitors (furfural, acetic acid, and phenol). Three traits were uncovered according to fermentation results. First, the growth of strain 424A (LNH-ST) was more sensitive to inhibitors than strain 4124. Through metabolomic analysis, the variance of trehalose, cadaverine, glutamate and g-aminobutyric acid (GABA) suggested that strain 424A (LNH-ST) had a lower capability to buffer redox changes caused by inhibitors. Second, lower ethanol yield in glucose and xylose co-fermentation than glucose fermentation was observed in strain 424A (LNH-ST), which was considered to be correlated with the generation of xylitol, as well as the reduced levels of lysine, glutamate, glycine and isoleucine in strain 424A (LNH-ST). Accumulation of glycerol, galactinol and mannitol was also observed in strain 424A (LNH-ST) during xylose fermentation. Third, xylose utilization of strain 424A (LNH-ST) was more significantly disturbed by inhibitors than glucose utilization. Through the analysis of fermentation and metabolomic results, it was suggested that xylose catabolism and energy supply, rather than xylose uptake, were the limiting steps in xylose utilization in the presence of inhibitors.

  7. Excitatory GABA in rodent developing neocortex in vitro.

    PubMed

    Rheims, Sylvain; Minlebaev, Marat; Ivanov, Anton; Represa, Alfonso; Khazipov, Rustem; Holmes, Gregory L; Ben-Ari, Yehezkel; Zilberter, Yuri

    2008-08-01

    GABA depolarizes immature cortical neurons. However, whether GABA excites immature neocortical neurons and drives network oscillations as in other brain structures remains controversial. Excitatory actions of GABA depend on three fundamental parameters: the resting membrane potential (Em), reversal potential of GABA (E(GABA)), and threshold of action potential generation (Vthr). We have shown recently that conventional invasive recording techniques provide an erroneous estimation of these parameters in immature neurons. In this study, we used noninvasive single N-methyl-d-aspartate and GABA channel recordings in rodent brain slices to measure both Em and E(GABA) in the same neuron. We show that GABA strongly depolarizes pyramidal neurons and interneurons in both deep and superficial layers of the immature neocortex (P2-P10). However, GABA generates action potentials in layer 5/6 (L5/6) but not L2/3 pyramidal cells, since L5/6 pyramidal cells have more depolarized resting potentials and more hyperpolarized Vthr. The excitatory GABA transiently drives oscillations generated by L5/6 pyramidal cells and interneurons during development (P5-P12). The NKCC1 co-transporter antagonist bumetanide strongly reduces [Cl(-)]i, GABA-induced depolarization, and network oscillations, confirming the importance of GABA signaling. Thus a strong GABA excitatory drive coupled with high intrinsic excitability of L5/6 pyramidal neurons and interneurons provide a powerful mechanism of synapse-driven oscillatory activity in the rodent neocortex in vitro. In the companion paper, we show that the excitatory GABA drives layer-specific seizures in the immature neocortex.

  8. Effects of RHC 80267, a diglyceride lipase inhibitor, on prolactin secretion and calcium uptake in GH/sub 3/ pituitary cells

    SciTech Connect

    Camoratto, A.M.; Grandison, L.

    1987-01-19

    The effect of the diglyceride lipase inhibitor RHC 80267 on the prolactin secretory process was examined in clonal anterior pituitary GH/sub 3/ cells. This compound reduced basal prolactin secretion as well as secretion induced by TRH and phospholipase C but not that induced by phorbol myristate acetate. Although exogenous phospholipase C increased diglyceride, no increase in the products of diglyceride lipase was detected. Moreover, low doses of RHC 80267 were observed to effectively block potassium-stimulated /sup 45/calcium influx. It is unlikely that RHC 80267 inhibits prolactin release solely by inhibiting diglyceride lipase. These data suggest blockage of plasma membrane calcium channels as an alternate mechanism for the inhibitory actions of RHC 80267 on intact GH/sub 3/ cells. These observations may have implications for RHC 80267 action in other cell types.

  9. WAY-318068: a novel, potent and selective noradrenaline re-uptake inhibitor with activity in rodent models of pain and depression

    PubMed Central

    Whiteside, GT; Dwyer, JM; Harrison, JE; Beyer, CE; Cummons, T; Manzino, L; Mark, L; Johnston, GH; Strassle, BW; Adedoyin, A; Lu, P; Piesla, MJ; Pulicicchio, CM; Erve, JCL; Platt, BJ; Hughes, ZA; Rogers, KE; Deecher, DC; Trybulski, EJ; Kennedy, JD; Zhang, P; Leventhal, L

    2010-01-01

    Background and purpose: Antidepressants, which raise the CNS concentrations of 5-HT and noradrenaline, are frequently used in the treatment of chronic pain; however, it is not known if increasing CNS noradrenaline levels alone is sufficient for efficacy, in part resulting from a lack of small molecules with sufficient selectivity. Experimental approach: In this report, we present the in vitro pharmacological and in vivo pharmacokinetic and pharmacological properties of the novel, orally available and CNS penetrant inhibitor of the noradrenaline transporter (NET), WAY-318068 (1-[(1S,2R)-1-(3,5-difluorophenyl)-2-hydroxy-3-(methylamino)propyl]-7-fluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-one). Key results: WAY-318068 is a potent and effective inhibitor of the NET with a Ki of 8.7 nM in a binding assay, and an IC50 of 6.8 nM in an assay of transporter function, without significant binding to the dopamine transporter. Furthermore, the compound has only weak activity at the 5-HT transporter, leading to a functional selectivity of greater than 2500-fold. It is orally bioavailable with substantial quantities of the compound found in the CNS after oral dosing. As measured by microdialysis in rats, the compound causes a robust and significant increase in cortical noradrenaline levels without affecting 5-HT. WAY-318068 was effective in models of acute, visceral, inflammatory, osteoarthritic, neuropathic, diabetic and bone cancer pain, as well as in traditional models of depression at doses that do not cause motor deficits. Conclusions and implications: Collectively, the present results support the conclusion that selectively increasing CNS levels of noradrenaline is sufficient for efficacy in models of depression and pain. PMID:20590604

  10. Effects of nitrogen application rate and a nitrification inhibitor dicyandiamide on methanotroph abundance and methane uptake in a grazed pasture soil.

    PubMed

    Dai, Yu; Di, Hong J; Cameron, Keith C; He, Ji-Zheng

    2013-12-01

    Methane-oxidizing bacteria (methanotrophs) in the soil are a unique group of methylotrophic bacteria that utilize methane (CH4) as their sole source of carbon and energy which limit the flux of methane to the atmosphere from soils and consume atmospheric methane. A field experiment was conducted to determine the effect of nitrogen application rates and the nitrification inhibitor dicyandiamide (DCD) on the abundance of methanotrophs and on methane flux in a grazed pasture soil. Nitrogen (N) was applied at four different rates, with urea applied at 50 and 100 kg N ha(-1) and animal urine at 300 and 600 kg N ha(-1). DCD was applied at 10 kg ha(-1). The results showed that both the DNA and selected mRNA copy numbers of the methanotroph pmoA gene were not affected by the application of urea, urine or DCD. The methanotroph DNA and mRNA pmoA gene copy numbers were low in this soil, below 7.13 × 10(3) g(-1) soil and 3.75 × 10(3) μg(-1) RNA, respectively. Daily CH4 flux varied slightly among different treatments during the experimental period, ranging from -12.89 g CH4 ha(-1) day(-1) to -0.83 g CH4 ha(-1) day(-1), but no significant treatment effect was found. This study suggests that the application of urea fertilizer, animal urine returns and the use of the nitrification inhibitor DCD do not significantly affect soil methanotroph abundance or daily CH4 fluxes in grazed grassland soils.

  11. Impact of exogenous GABA treatments on endogenous GABA metabolism in anthurium cut flowers in response to postharvest chilling temperature.

    PubMed

    Aghdam, Morteza Soleimani; Naderi, Roohangiz; Jannatizadeh, Abbasali; Babalar, Mesbah; Sarcheshmeh, Mohammad Ali Askari; Faradonbe, Mojtaba Zamani

    2016-09-01

    Anthurium flowers are susceptible to chilling injury, and the optimum storage temperature is 12.5-20 °C. The γ-aminobutyric acid (GABA) shunt pathway may alleviate chilling stress in horticultural commodities by providing energy (ATP), reducing molecules (NADH), and minimizing accumulation of reactive oxygen species (ROS). In this experiment, the impact of a preharvest spray treatment with 1 mM GABA and postharvest treatment of 5 mM GABA stem-end dipping on GABA shunt pathway activity of anthurium cut flowers (cv. Sirion) in response to cold storage (4 °C for 21 days) was investigated. GABA treatments resulted in lower glutamate decarboxylase (GAD) and higher GABA transaminase (GABA-T) activities in flowers during cold storage, which was associated with lower GABA content and coincided with higher ATP content. GABA treatments also enhanced accumulation of endogenous glycine betaine (GB) in flowers during cold storage, as well as higher spathe relative water content (RWC). These findings suggest that GABA treatments may alleviate chilling injury of anthurium cut flowers by enhancing GABA shunt pathway activity leading to provide sufficient ATP and promoting endogenous GB accumulation.

  12. Interaction of GABA-mimetics with the taurine transporter (TauT, Slc6a6) in hyperosmotic treated Caco-2, LLC-PK1 and rat renal SKPT cells.

    PubMed

    Rasmussen, Rune Nørgaard; Lagunas, Candela; Plum, Jakob; Holm, René; Nielsen, Carsten Uhd

    2016-01-20

    The aim of the present study was to investigate if basic GABA-mimetics interact with the taurine transporter (TauT, Slc6a6), and to find a suitable cell based model that is robust towards extracellular changes in osmolality during uptake studies. Taurine uptake was measured in human Caco-2 cells, porcine LLC-PK1 cells, and rat SKPT cells using radiolabelled taurine. Hyperosmotic conditions were obtained by incubation with raffinose (final osmolality of 500mOsm) for 24h prior to the uptake experiments. Expression of the taurine transporter, TauT, was investigated at the mRNA level by real-time PCR. Uptake of the GABA-mimetics gaboxadol and vigabatrin was investigated in SKPT cells, and quantified by liquid scintillation or HPLC-MS/MS analysis, respectively. The uptake rate of [(3)H]-taurine was Na(+) and Cl(-) and concentration dependent with taurine with an apparent Vmax of 6.3±1.6pmolcm(-2)min(-1) and a Km of 24.9±15.0μM. β-alanine, nipecotic acid, gaboxadol, GABA, vigabatrin, δ-ALA and guvacine inhibited the taurine uptake rate in a concentration dependent manner. The order of affinity for TauT was β-alanine>GABA>nipecotic acid>guvacine>δ-ALA>vigabatrin>gaboxadol with IC50-values of 0.04, 1.07, 2.02, 4.19, 4.94, 31.4 and 39.9mM, respectively. In conclusion, GABA mimetics inhibited taurine uptake in hyperosmotic rat renal SKPT cells. SKPT cells, which seem to be a useful model for investigating taurine transport in the short-term presence of high concentrations of osmolytes. Furthermore, analogues of β-alanine appear to have higher affinities for TauT than GABA-analogues.

  13. Excitatory actions of GABA in developing rat hypothalamic neurones.

    PubMed Central

    Chen, G; Trombley, P Q; van den Pol, A N

    1996-01-01

    1. Gramicidin-perforated patch clamp recording was employed to study GABA-mediated responses in rat hypothalamic neurones (n = 102) with an intracellular Cl- concentration unaltered by the pipette solution. 2. In young cultures after 1-7 days in vitro (DIV), GABA induced depolarizing membrane potentials (+16.5 +/- 1.3 mV) that often surpassed the threshold for the firing of action potentials (-42 +/- 1 mV) and resulted in an increase in neuronal activity. The depolarizing responses to GABA in young cultures were dose dependent. The concentration of GABA necessary to evoke the half-maximal depolarization (EC50) was 2.8 microM. In contrast, GABA induced hyperpolarizing membrane potentials (-12.0 +/- 1.4 mV) and a decrease in neuronal activity in older neurones (20-33 DIV). Both the depolarization and the hyperpolarization induced by GABA were blocked by bicuculline, indicating a mediation by GABAA receptors. 3. The reversal potentials of the GABA-evoked currents were between -40 to -50 mV during the first week of culture, and shifted to below -70 mV after 3 weeks of culture. In parallel, neurones that were dissociated from older animals (postnatal day 5) had a more negative reversal potential for the GABA-evoked currents than cells from younger animals (embryonic day 15), suggesting that the negative shift of the reversal potential occurs both in vitro and in vivo. Our data suggest that the mechanism for GABA-induced depolarization is the depolarized Cl- reversal potential found in young but not older neurones. 4. Consistent with the depolarizing response to exogenous application of GABA, some spontaneous depolarizing postsynaptic potentials in young cultures were insensitive to AP5-CNQX, but were eliminated by bicuculline, indicating that synaptically released GABA mediated excitatory synaptic transmission in early development. 5. By combining a rapid computer-controlled delivery of GABA with subthreshold positive current injections into recorded neurones, we found

  14. Pharmacologically novel GABA receptor in human dorsal root ganglion neurons.

    PubMed

    Valeyev, A Y; Hackman, J C; Wood, P M; Davidoff, R A

    1996-11-01

    1. Whole cell voltage-clamp studies of gamma-aminobutyric acid (GABA) receptors were performed on large (> 80 microns) cultured human dorsal root ganglion (DRG) neurons. 2. GABA and pentobarbital sodium when applied in micromolar concentrations evoked inward Cl- currents in DRG neurons voltage clamped at negative membrane potentials. 3. Diazepam (10 microM) and pentobarbital (10 microM) upmodulated the GABA current by approximately 149 and 168%, respectively. 4. The GABA currents in human DRG cells were unaffected by the classical GABA antagonists picrotoxin and bicuclline (100 microM). In contrast, the GABA responses evoked in adult rat DRG cells cultured in an identical manner were inhibited by both antagonists. The glycine receptor antagonist strychnine (100 microM) did not alter GABA currents in human DRG cells. 5. Human DRG cells did not respond to glycine (10-100 microM) or taurine (10-100 microM). The GABAB agonist baclofen had no effect on the holding current when patch pipettes were filled with 130 mM KCl. The GABAB antagonists saclofen applied either alone or with GABA was without effect. 6. The differences between the GABA receptors described here and GABA receptors in other species may reflect the presence of receptor subunits unique to human DRG cells.

  15. Design and Mechanism of Tetrahydrothiophene-based GABA Aminotransferase Inactivators

    PubMed Central

    Le, Hoang V.; Hawker, Dustin D.; Wu, Rui; Doud, Emma; Widom, Julia; Sanishvili, Ruslan; Liu, Dali; Kelleher, Neil L.; Silverman, Richard B.

    2015-01-01

    Low levels of γ-aminobutyric acid (GABA), one of two major neurotransmitters that regulate brain neuronal activity, are associated with many neurological disorders, such as epilepsy, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and cocaine addiction. One of the main methods to raise the GABA level in human brain is to use small molecules that cross the blood-brain barrier and inhibit the activity of γ-aminobutyric acid aminotransferase (GABA-AT), the enzyme that degrades GABA. We have designed a series of conformationally-restricted, tetrahydrothiophene-based GABA analogs with a properly-positioned leaving group that could facilitate a ring-opening mechanism, leading to inactivation of GABA-AT. One compound in the series is eight times more efficient an inactivator of GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. Our mechanistic studies show that the compound inactivates GABA-AT by a new mechanism. The metabolite resulting from inactivation does not covalently bind to amino acid residues of GABA-AT but stays in the active site via H-bond interactions with Arg-192, a π-π interaction with Phe-189, and a weak nonbonded S···O=C interaction with Glu-270, thereby inactivating the enzyme. PMID:25781189

  16. Studies, using in vivo microdialysis, on the effect of the dopamine uptake inhibitor GBR 12909 on 3,4-methylenedioxymethamphetamine ('ecstasy')-induced dopamine release and free radical formation in the mouse striatum.

    PubMed

    Camarero, Jorge; Sanchez, Veronica; O'Shea, Esther; Green, A Richard; Colado, M Isabel

    2002-06-01

    The present study examined the mechanisms by which 3,4-methylenedioxymethamphetamine (MDMA) produces long-term neurotoxicity of striatal dopamine neurones in mice and the protective action of the dopamine uptake inhibitor GBR 12909. MDMA (30 mg/kg, i.p.), given three times at 3-h intervals, produced a rapid increase in striatal dopamine release measured by in vivo microdialysis (maximum increase to 380 +/- 64% of baseline). This increase was enhanced to 576 +/- 109% of baseline by GBR 12909 (10 mg/kg, i.p.) administered 30 min before each dose of MDMA, supporting the contention that MDMA enters the terminal by diffusion and not via the dopamine uptake site. This, in addition to the fact that perfusion of the probe with a low Ca(2+) medium inhibited the MDMA-induced increase in extracellular dopamine, indicates that the neurotransmitter may be released by a Ca(2+) -dependent mechanism not related to the dopamine transporter. MDMA (30 mg/kg x 3) increased the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) from salicylic acid perfused through a probe implanted in the striatum, indicating that MDMA increased free radical formation. GBR 12909 pre-treatment attenuated the MDMA-induced increase in 2,3-DHBA formation by approximately 50%, but had no significant intrinsic radical trapping activity. MDMA administration increased lipid peroxidation in striatal synaptosomes, an effect reduced by approximately 60% by GBR 12909 pre-treatment. GBR 12909 did not modify the MDMA-induced changes in body temperature. These data suggest that MDMA-induced toxicity of dopamine neurones in mice results from free radical formation which in turn induces an oxidative stress process. The data also indicate that the free radical formation is probably not associated with the MDMA-induced dopamine release and that MDMA does not induce dopamine release via an action at the dopamine transporter.

  17. Synthesis, cytotoxicity, cellular uptake and influence on eicosanoid metabolism of cobalt-alkyne modified fructoses in comparison to auranofin and the cytotoxic COX inhibitor Co-ASS.

    PubMed

    Ott, Ingo; Koch, Thao; Shorafa, Hashem; Bai, Zhenlin; Poeckel, Daniel; Steinhilber, Dieter; Gust, Ronald

    2005-06-21

    Propargylhexacarbonyldicobalt complexes with fructopyranose ligands were prepared and investigated for cytotoxicity in the MCF-7 human breast cancer cell line. The antiproliferative effects depended on the presence of isopropylidene protecting groups in the carbohydrate ligand and correlated with the cellular concentration of the complexes. IC(50) values of > 20 microM demonstrated that the fructose derivatives were only moderately active compared to the references auranofin and the aspirin (ASS) derivative [2-acetoxy(2-propynyl)benzoate]hexacarbonyldicobalt (Co-ASS). In continuation of our studies on the mode of action of cobalt-alkyne complexes we studied the influence of the compounds on the formation of 12-HHT (COX-1 product) and 12-HETE (12-LOX product) by human platelets as an indication of the interference in the eicosanoid metabolism, which is discussed as a target system of cytostatics. Co-ASS was an efficient COX-1 inhibitor without LOX inhibitory activity and auranofin inhibited both COX-1 and 12-LOX eicosanoid production. The missing activity of the fructopyranose complexes at the 12-LOX and the only moderate effects at COX-1 indicate that COX/LOX inhibition may be in part responsible for the pharmacological effects of auranofin and Co-ASS but not for those of the fructopyranose complexes.

  18. Astrocytic GABA transporter activity modulates excitatory neurotransmission

    PubMed Central

    Boddum, Kim; Jensen, Thomas P.; Magloire, Vincent; Kristiansen, Uffe; Rusakov, Dmitri A.; Pavlov, Ivan; Walker, Matthew C.

    2016-01-01

    Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na+ concentrations and a consequent increase in astrocytic Ca2+ through Na+/Ca2+ exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus. PMID:27886179

  19. GABA and Neuroactive Steroid Interactions in Glia: New Roles for Old Players?

    PubMed Central

    Magnaghi, Valerio

    2007-01-01

    In recent years it has becoming clear that glial cells of the central and peripheral nervous system play a crucial role from the earliest stages of development throughout adult life. Glial cells are important for neuronal plasticity, axonal conduction and synaptic transmission. In this respect, glial cells are able to produce, uptake and metabolize many factors that are essential for neuronal physiology, including classic neurotransmitters and neuroactive steroids. In particular, neuroactive steroids, which are mainly synthesized by glial cells, are able to modulate some neurotransmitter receptors affecting both glia and neurons. Among the signaling systems that are specialized for neuron-glial communication, we can include neurotransmitter GABA. The main focus of this review is to illustrate the cross-talk between neurons and glial cells in terms of GABA neurotransmission and actions of neuroactive steroids. To this purpose, we will review the presence of the different GABA receptors in the glial cells of the central and peripheral nervous system. Then, we will discuss their modulation by some neuroactive steroids. PMID:18615153

  20. [Effect of synaptosomal cytosolic [3H]GABA pool depletion on secretory ability of alpha-latrotoxin].

    PubMed

    Linets'ka, M V; Storchak, L H; Himmelreĭch, N H

    2002-01-01

    alpha-Latrotoxin, a presynaptic neurotoxin from the venom of Latrodectus mactans tredecimguttatus, induces massive [3H]GABA release from rat brain synaptosomes as a result of interaction with either Ca(2+)-dependent (neurexin 1 alpha or Ca(2+)-independent (latrophilin) membrane receptor. The main aim of the study was to elucidate whether the binding of alpha-latrotoxin to different types of receptors led to [3H]GABA secretion from one pool or in each case the source of neurotransmitter differs: in the presence of Ca2+ exocytosis is induced, while in the absence of Ca(2+)--outflow by mobile membrane GABA transporter from cytoplasm. We examined the effect of the depletion of cytosolic [3H]GABA pool by competitive inhibitors of the GABA transporter (nipecotic acid and 2,4-diaminobutyric acid) on the alpha-latrotoxin-stimulated neurotransmitter release. We also compared the influence of these agents on neurosecretion, evoked by depolarization with that evoked by alpha-latrotoxin. Depolarization was stimulated by 4-aminopyridine in the Ca(2+)-containing saline and high KCl in Ca(2+)-free medium. In synaptosomes treated with nipecotic acid unstimulated [3H]GABA release was significantly augmented and high KCl-evoked Ca(2+)-independent [3H]GABA release was essentially inhibited. But under the same conditions neurosecretion stimulated by alpha-latrotoxin greatly raised with respect to the control response. The similar results were obtained with the synaptosomes treated with 2,4-diaminobutyric acid. Another way to determine which of GABA pool is the target of alpha-latrotoxin action lay in analysis of the toxin effects on the preliminary depolarized synaptosomes. alpha-Latrotoxin influence was diminished by the preceding depolarization by 4-aminopyridine in Ca2+ presence. But after the high KCl stimulation effect of alpha-latrotoxin didn't change. These data suggest that alpha-latrotoxin triggers neurotransmitter release from synaptic vesicles via exocytosis. We suppose

  1. GABA and GABA receptors alterations in the primary visual cortex of concave lens-induced myopic model.

    PubMed

    Zhao, Wen; Bi, Ai-Ling; Xu, Chao-Li; Ye, Xiang; Chen, Mei-Qing; Wang, Xin-Ting; Zhang, Xiao-Yan; Guo, Jun-Guo; Jiang, Wen-Jun; Zhang, Jin; Bi, Hong-Sheng

    2017-02-02

    Until recently most researches on myopia mechanisms have mainly been focused on the eye ball and few investigations were explored on the upper visual pathway, such as the visual cortex. The roles of gamma-aminobutyric acid (GABA) in the retinal and in the upper visual pathway are inter-correlated. As the retinal glutamate decarboxylase (GAD), GABA, and the mRNA levels of GABA receptors increased during the concave lens induced myopia formation, however, whether GABA alterations also occurred in the visual cortex during the concave lens induction is still unknown. In the present study, using HPLC, Enzyme-Linked Immunosorbent Assay (ELISA) and Real-Time Quantitative-PCR (RT-PCR) methods, we observed the changing trends of GABA, glutamate decarboxylase (GAD), and GABA receptors in the visual cortex of concave lens-induced myopic guinea pigs. Similar to the changing patterns of retinal GABA, the concentrations of GAD, GABA and the mRNA levels of GABA receptors in the visual cortex also increased. These results indicate that the exploration on myopia mechanisms should possibly be investigated on the whole visual pathway and the detailed significance of cortical GABA alterations needs further investigation.

  2. GABA-BZD Receptor Modulating Mechanism of Panax quinquefolius against 72-h Sleep Deprivation Induced Anxiety like Behavior: Possible Roles of Oxidative Stress, Mitochondrial Dysfunction and Neuroinflammation

    PubMed Central

    Chanana, Priyanka; Kumar, Anil

    2016-01-01

    Rationale: Panax quinquefolius (American Ginseng) is known for its therapeutic potential against various neurological disorders, but its plausible mechanism of action still remains undeciphered. GABA (Gamma Amino Butyric Acid) plays an important role in sleep wake cycle homeostasis. Thus, there exists rationale in exploring the GABA-ergic potential of Panax quinquefolius as neuroprotective strategy in sleep deprivation induced secondary neurological problems. Objective: The present study was designed to explore the possible GABA-ergic mechanism in the neuro-protective effect of Panax quinquefolius against 72-h sleep deprivation induced anxiety like behavior, oxidative stress, mitochondrial dysfunction, HPA-axis activation and neuroinflammation. Materials and Methods: Male laca mice were sleep deprived for 72-h by using Grid suspended over water method. Panax quinquefolius (American Ginseng 50, 100, and 200 mg/kg) was administered alone and in combination with GABA modulators (GABA Cl− channel inhibitor, GABA-benzodiazepine receptor inhibitor and GABAA agonist) for 8 days, starting 5 days prior to 72-h sleep deprivation period. Various behavioral (locomotor activity, mirror chamber test), biochemical (lipid peroxidation, reduced glutathione, catalase, nitrite levels), mitochondrial complexes, neuroinflammation marker (Tumor Necrosis Factor, TNF-alpha), serum corticosterone, and histopathological sections of brains were assessed. Results: Seventy two hours sleep deprivation significantly impaired locomotor activity, caused anxiety-like behavior, conditions of oxidative stress, alterations in mitochondrial enzyme complex activities, raised serum corticosterone levels, brain TNFα levels and led to neuroinflammation like signs in discrete brain areas as compared to naive group. Panax quinquefolius (100 and 200 mg/kg) treatment restored the behavioral, biochemical, mitochondrial, molecular and histopathological alterations. Pre-treatment of GABA Cl− channel

  3. Suppression of γ-aminobutyric acid (GABA) transaminases induces prominent GABA accumulation, dwarfism and infertility in the tomato (Solanum lycopersicum L.).

    PubMed

    Koike, Satoshi; Matsukura, Chiaki; Takayama, Mariko; Asamizu, Erika; Ezura, Hiroshi

    2013-05-01

    Tomatoes accumulate γ-aminobutyric acid (GABA) at high levels in the immature fruits. GABA is rapidly converted to succinate during fruit ripening through the activities of GABA transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH). Although three genes encoding GABA-T and both pyruvate- and α-ketoglutarate-dependent GABA-T activities have been detected in tomato fruits, the mechanism underlying the GABA-T-mediated conversion of GABA has not been fully understood. In this work, we conducted loss-of-function analyses utilizing RNA interference (RNAi) transgenic plants with suppressed pyruvate- and glyoxylate-dependent GABA-T gene expression to clarify which GABA-T isoforms are essential for its function. The RNAi plants with suppressed SlGABA-T gene expression, particularly SlGABA-T1, showed severe dwarfism and infertility. SlGABA-T1 expression was inversely associated with GABA levels in the fruit at the red ripe stage. The GABA contents in 35S::SlGABA-T1(RNAi) lines were 1.3-2.0 times and 6.8-9.2 times higher in mature green and red ripe fruits, respectively, than the contents in wild-type fruits. In addition, SlGABA-T1 expression was strongly suppressed in the GABA-accumulating lines. These results indicate that pyruvate- and glyoxylate-dependent GABA-T is the essential isoform for GABA metabolism in tomato plants and that GABA-T1 primarily contributes to GABA reduction in the ripening fruits.

  4. Neuroprotection by estradiol: a role of aromatase against spine synapse loss after blockade of GABA(A) receptors.

    PubMed

    Zhou, Lepu; Lehan, Nadine; Wehrenberg, Uwe; Disteldorf, Erik; von Lossow, Richard; Mares, Ute; Jarry, Hubertus; Rune, Gabriele M

    2007-01-01

    Estrogen has been suggested to be pro-epileptic by reducing GABA synthesis, resulting in increased spine density and a decreased threshold for seizures in the hippocampus, which, once they occur, are characterized by a dramatic spine loss in the affected brain areas. As considerable amounts of estradiol are synthesized in the hippocampus, in this study we focused on aromatase, the rate-limiting enzyme in estrogen synthesis in order to examine the role of locally synthesized estrogens in epilepsy. To this end, we first examined the effects of letrozole, a potent aromatase inhibitor, on GABA metabolism in single interneurons of hippocampal dispersion cultures. Letrozole downregulated estradiol release into the medium, as well as glutamate decarboxylase (GAD) expression and GABA synthesis, and decreased the number of GAD positive cells in the cultures. Next, we counted spine synapses and measured estradiol release of hippocampal slice cultures, in which GABA(A) receptors had been blocked by bicuculline, in order to mimic epileptic activity. Treatment of slice cultures with bicuculline resulted in a dramatic decrease in the number of spine synapses and in a significant suppression of estrogen synthesis. The decrease in synapse number in response to bicuculline was restored by combined application of estradiol and bicuculline. Surprisingly, estradiol alone had no effect on either spine synapse number or on GAD expression and GABA synthesis. "Rescue" of synapse number in "epileptic slices" by estradiol and maintenance of GABA metabolism by hippocampus-derived estradiol points to a neuroprotective role of aromatase in epilepsy. Re-filling of estradiol stores after their depletion due to overexcitation may therefore add to therapeutical strategies in epilepsy.

  5. Target-specific suppression of GABA release from parvalbumin interneurons in the basolateral amygdala by dopamine.

    PubMed

    Chu, Hong-Yuan; Ito, Wataru; Li, Jiayang; Morozov, Alexei

    2012-10-17

    Dopamine (DA) in the basolateral amygdala (BLA) promotes fear learning by disinhibiting principal neurons (PNs) and enabling synaptic plasticity in their sensory inputs. While BLA interneurons (INs) are heterogeneous, it is unclear which interneuron subtypes decrease GABAergic input to PNs in the presence of DA. Here, using cell type-selective photostimulation by channelrhodopsin 2 in BLA slices from mouse brain, we examined the role of parvalbumin-positive INs (PV-INs), the major interneuronal subpopulation in BLA, in the disinhibitory effect of DA. We found that DA selectively suppressed GABAergic transmission from PV-INs to PNs by acting on presynaptic D(2) receptors, and this effect was mimicked by Rp-cAMP, an inhibitor of cAMP-dependent signaling. In contrast, DA did not alter GABA release from PV-INs to INs. Furthermore, neither suppressing cAMP-dependent signaling by Rp-cAMP nor enhancing it by forskolin altered GABA release from PV-INs to BLA INs. Overall, DA disinhibits BLA, at least in part, by suppressing GABA release from PV-INs in the target cell-specific manner that results from differential control of this release by cAMP-dependent signaling.

  6. Long-Lasting Effects of GABA Infusion Into the Cerebral Cortex of the Rat

    PubMed Central

    Montiel, Teresa; Almeida, Daniel; Arango, Iván; Calixto, Eduardo; Casasola, César; Brailowsky, Simón

    2000-01-01

    In electrophysiological terms, experimental models of durable information storage in the brain include long-term potentiation (LTP), long-term depression, and kindling. Protein synthesis correlates with these enduring processes. We propose a fourth example of long-lasting information storage in the brain, which we call the GABA-withdrawal syndrome (GWS). In rats, withdrawal of a chronic intracortical infusion of GABA, a ubiquitous inhibitory neurotransmitter, induced epileptogenesis at the infusion site. This overt GWS lasted for days. Anisomycin, a protein synthesis inhibitor, prevented the appearance of GWS in vivo. Hippocampal and neocortical slices showed a similar post-GABA hyperexcitability in vitro and an enhanced susceptibility to LTP induction. One to four months after the epileptic behavior disappeared, systemic administration of a subconvulsant dose of pentylenetetrazol produced the reappearance of paroxysmal activity. The long-lasting effects of tonic GABAA receptor stimulation may be involved in long-term information storage processes at the cortical level, whereas the cessation of GABAA receptor stimulation may be involved in chronic pathological conditions, such as epilepsy. Furthermore, we propose that GWS may represent a common key factor in the addiction to GABAergic agents (for example, barbiturates, benzodiazepines, and ethanol). GWS represents a novel form of neurono-glial plasticity. The mechanisms of this phenomenon remain to be understood. PMID:10709209

  7. Expression of spinal cord GABA transporter 1 in morphine-tolerant male Wistar rats.

    PubMed

    Shokoofeh, Siroosi; Homa, Manaheji; Leila, Dargahi; Samira, Daniali

    2015-11-15

    Chronic morphine exposure produces morphine tolerance. One of the mechanisms of morphine tolerance involves γ-aminobutric acid (GABA), whose level is regulated by GABA transporter 1 (GAT-1). The aim of this study was to investigate the expression of GAT-1 in the spinal cord during morphine treatment. Morphine was administrated to rats via drinking water for 21 days. On day 21, a single dose of morphine (10mg/kg) was injected, followed by the administration of 5% formalin after 30 min. Expression of GAT-1 in the lumbar spinal cord during morphine treatment was analyzed by Western blotting and immunohistochemistry assay. In another set of experiments, a morphine-tolerant group was treated with a GAT-1 inhibitor, ethyl nipecotate (60 mg/kg), 5 min prior to the formalin test. To assess a possible analgesic effect of the GAT-1 inhibitor, a non-tolerant group was injected only with ethyl nipecotate 5 min prior to the formalin test. Our results indicated that a chronic consumption of morphine led to morphine tolerance. Morphine tolerance was also concomitant with GAT-1 up-regulation in the lumbar spinal cord. The GAT-1 inhibitor ethyl nipecotate improved the antinociceptive effect of morphine in the morphine-tolerant group. Ethyl nipecotate also had an antinociceptive effect on the non-tolerant group. Thus, our data suggest that GAT-1 overexpression in the spinal cord plays an important role in morphine tolerance.

  8. Effects of Tityus serrulatus scorpion venom and its toxin TsTX-V on neurotransmitter uptake in vitro

    SciTech Connect

    Cecchini, Alessandra L.; Vasconcelos, Flavio; Giglio, Jose Roberto; Arantes, Eliane Candiani . E-mail: ecabraga@fcfrp.usp.br

    2006-12-01

    Scorpion neurotoxins targeting the Na{sub v} channel can be classified into two classes: {alpha}- and {beta}-neurotoxins and are reported as highly active in mammalian brain. In this work, we evaluate the effects of Tityus serrulatus venom (Ts venom) and its {alpha}-neurotoxin TsTX-V on {gamma}-aminobutyric acid (GABA), dopamine (DA) and glutamate (Glu) uptake in isolated rat brain synaptosomes. TsTX-V was isolated from Ts venom by ion exchange chromatography followed by reverse-phase (C18) high-performance liquid chromatography. Neither Ts venom nor TsTX-V was able to affect {sup 3}H-Glu uptake. On the other hand, Ts venom (0.13 {mu}g/mg) significantly inhibited both {sup 3}H-GABA and {sup 3}H-DA uptake ({approx} 50%). TsTX-V showed IC{sub 5} values of 9.37 {mu}M and 22.2 {mu}M for the inhibition of {sup 3}H-GABA and {sup 3}H-DA uptake, respectively. These effects were abolished by pre-treatment with tetrodotoxin (TTX, 1 {mu}M), indicating the involvement of voltage-gated Na{sup +} channels in this process. In the absence of Ca{sup 2+}, and at low Ts venom concentrations, the reduction of {sup 3}H-GABA uptake was not as marked as in the presence of Ca{sup 2+}. TsTX-V did not reduce {sup 3}H-GABA uptake in COS-7 cells expressing the GABA transporters GAT-1 and GAT-3, suggesting that this toxin indirectly reduces the transport. The reduced {sup 3}H-GABA uptake by synaptosomes might be due to rapid cell depolarization as revealed by confocal microscopy of C6 glioma cells. Thus, TsTX-V causes a reduction of {sup 3}H-GABA and {sup 3}H-DA uptake in a Ca{sup 2+}-dependent manner, not directly affecting GABA transporters, but, in consequence of depolarization, involving voltage-gated Na{sup +} channels.

  9. Sleep and GABA levels in the oral part of rat pontine reticular formation are decreased by local and systemic administration of morphine.

    PubMed

    Watson, C J; Lydic, R; Baghdoyan, H A

    2007-01-05

    Morphine, a mu-opioid receptor agonist, is a commonly prescribed treatment for pain. Although highly efficacious, morphine has many unwanted side effects including disruption of sleep and obtundation of wakefulness. One mechanism by which morphine alters sleep and wakefulness may be by modulating GABAergic signaling in brain regions regulating arousal, including the pontine reticular nucleus, oral part (PnO). This study used in vivo microdialysis in unanesthetized Sprague-Dawley rat to test the hypothesis that mu-opioid receptors modulate PnO GABA levels. Validation of the high performance liquid chromatographic technique used to quantify GABA was obtained by dialyzing the PnO (n=4 rats) with the GABA reuptake inhibitor nipecotic acid (500 microM). Nipecotic acid caused a 185+/-20% increase in PnO GABA levels, confirming chromatographic detection of GABA and demonstrating the existence of functional GABA transporters in rat PnO. Morphine caused a concentration-dependent decrease in PnO GABA levels (n=25 rats). Coadministration of morphine (100 microM) with naloxone (1 microM), a mu-opioid receptor antagonist, blocked the morphine-induced decrease in PnO GABA levels (n=5 rats). These results show for the first time that mu-opioid receptors in rat PnO modulate GABA levels. A second group of rats (n=6) was used to test the hypothesis that systemically administered morphine also decreases PnO GABA levels. I.v. morphine caused a significant (P<0.05) decrease (19%) in PnO GABA levels relative to control i.v. infusions of saline. Finally, microinjections followed by 2 h recordings of electroencephalogram and electromyogram tested the hypothesis that PnO morphine administration disrupts sleep (n=8 rats). Morphine significantly (P<0.05) increased the percent of time spent in wakefulness (65%) and significantly (P<0.05) decreased the percent of rapid eye movement (REM) sleep (-53%) and non-REM sleep (-69%). The neurochemical and behavioral data suggest that morphine may

  10. Norepinephrine turnover in the goldfish brain is modulated by sex steroids and GABA.

    PubMed

    Trudeau, V L; Sloley, B D; Peter, R E

    1993-10-08

    It is known that norepinephrine (NE) is important in the neuroendocrine control of pituitary gonadotropin II (GTH-II) and growth hormone (GH) release but very little is known about the factors regulating NE neurons in the goldfish brain. Female gonad-intact goldfish were implanted intraperitoneally (100 micrograms/g) with testosterone (T) or estradiol (E2) to elevate serum steroid levels. High-performance liquid chromatography measurements showed that steroid implantation had no effect on NE content in the telencephalon, including preoptic area (TEL-POA), or the hypothalamus (HYP). The turnover rate of NE was estimated from the rate of depletion of NE content from tissues following inhibition of tyrosine hydroxylase by alpha-methyl-p-tyrosine (240 micrograms/g). The present study demonstrates that E2 can decrease NE turnover rates in TEL-POA and HYP of sexually regressed goldfish (August). The results in recrudescent fish (November), however, indicate a more complex interaction of E2 with NE neurons since E2 increased NE turnover in TEL-POA and HYP in these animals. Testosterone (T) has less prominent effects on NE turnover rates in TEL-POA and HYP; the only significant effect of T-implantation was a small reduction of NE turnover in the TEL-POA of sexually recrudescent fish. Elevation of endogenous brain GABA concentrations by injection of the GABA transaminase inhibitor, gamma-vinyl-GABA (300 micrograms/g), significantly reduced NE turnover in TEL-POA. These data demonstrate that goldfish NE neurons in the TEL-POA are sensitive to regulation by changes in circulating sex steroids and by increases in brain GABA.

  11. Bicyclic γ-amino acids as inhibitors of γ-aminobutyrate aminotransferase.

    PubMed

    Pinto, Andrea; Tamborini, Lucia; Pennacchietti, Eugenia; Coluccia, Antonio; Silvestri, Romano; Cullia, Gregorio; De Micheli, Carlo; Conti, Paola; De Biase, Daniela

    2016-01-01

    The γ-aminobutyrate (GABA)-degradative enzyme GABA aminotransferase (GABA-AT) is regarded as an attractive target to control GABA levels in the central nervous system: this has important implications in the treatment of several neurological disorders and drug dependencies. We have investigated the ability of newly synthesized compounds to act as GABA-AT inhibitors. These compounds have a unique bicyclic structure: the carbocyclic ring bears the GABA skeleton, while the fused 3-Br-isoxazoline ring contains an electrophilic warhead susceptible of nucleophilic attack by an active site residue of the target enzyme. Out of the four compounds tested, only the one named (+)-3 was found to significantly inhibit mammalian GABA-AT in vitro. Docking studies, performed on the available structures of GABA-AT, support the experimental findings: out of the four tested compounds, only (+)-3 suitably orients the electrophilic 3-Br-isoxazoline warhead towards the active site nucleophilic residue Lys329, thereby explaining the irreversible inhibition of GABA-AT observed experimentally.

  12. The expression of GABA(B1) and GABA(B2) receptor subunits in the cNS differs from that in peripheral tissues.

    PubMed

    Calver, A R; Medhurst, A D; Robbins, M J; Charles, K J; Evans, M L; Harrison, D C; Stammers, M; Hughes, S A; Hervieu, G; Couve, A; Moss, S J; Middlemiss, D N; Pangalos, M N

    2000-01-01

    GABA(B) receptors are G-protein-coupled receptors that mediate the slow and prolonged synaptic actions of GABA in the CNS via the modulation of ion channels. Unusually, GABA(B) receptors form functional heterodimers composed of GABA(B1) and GABA(B2) subunits. The GABA(B1) subunit is essential for ligand binding, whereas the GABA(B2) subunit is essential for functional expression of the receptor dimer at the cell surface. We have used real-time reverse transcriptase-polymerase chain reaction to analyse expression levels of these subunits, and their associated splice variants, in the CNS and peripheral tissues of human and rat. GABA(B1) subunit splice variants were expressed throughout the CNS and peripheral tissues, whereas surprisingly GABA(B2) subunit splice variants were neural specific. Using novel antisera specific to individual GABA(B) receptor subunits, we have confirmed these findings at the protein level. Analysis by immunoblotting demonstrated the presence of the GABA(B1) subunit, but not the GABA(B2) subunit, in uterus and spleen. Furthermore, we have shown the first immunocytochemical analysis of the GABA(B2) subunit in the brain and spinal cord using a GABA(B2)-specific antibody. We have, therefore, identified areas of non-overlap between GABA(B1) and GABA(B2) subunit expression in tissues known to contain functional GABA(B) receptors. Such areas are of interest as they may well contain novel GABA(B) receptor subunit isoforms, expression of which would enable the GABA(B1) subunit to reach the cell surface and form functional GABA(B) receptors.

  13. Cloning and expression of a rat brain GABA transporter

    SciTech Connect

    Guastella, J.; Czyzyk, L.; Davidson, N.; Lester, H.A. ); Nelson, N.; Nelson, H.; Miedel, M.C. ); Keynan, S.; Kanner, B.I. )

    1990-09-14

    A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter {gamma}-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated ({sup 3}H)GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium- and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.

  14. A noncanonical release of GABA and glutamate modulates neuronal migration.

    PubMed

    Manent, Jean-Bernard; Demarque, Michaël; Jorquera, Isabel; Pellegrino, Christophe; Ben-Ari, Yehezkel; Aniksztejn, Laurent; Represa, Alfonso

    2005-05-11

    Immature neurons express GABA and glutamate receptors before synapse formation, and both transmitters are released at an early developmental stage. We have now tested the hypothesis that the ongoing release of GABA and glutamate modulates neuronal migration. Using 5-bromo-2'-deoxyuridine labeling and cocultures of hippocampal slices obtained from naive and green fluorescent protein-transgenic mice, we report that migration is severely affected by GABA(A) or NMDA receptor antagonist treatments. These effects were also present in munc18-1 knock-out slices in which soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicular secretion of transmitters has been deleted. GABA(A) antagonists were more efficient than NMDA antagonists to reduce cell migration, in keeping with the earlier maturation of GABAergic mechanisms. We conclude that GABA and, to a lesser degree, glutamate released in a SNARE-independent mechanism exert a paracrine action on neuronal migration.

  15. Laser photolysis of DPNI-GABA, a tool for investigating the properties and distribution of GABA receptors and for silencing neurons in situ.

    PubMed

    Trigo, Federico F; Papageorgiou, George; Corrie, John E T; Ogden, David

    2009-07-30

    Laser photolysis to release GABA at precisely defined times and locations permits investigation of the distribution of functional GABA(A) receptors in neuronal compartments, the activation kinetics and pharmacology of GABA(A) receptors in situ, and the role of individual neurons in neural circuits by selective silencing with low GABA concentrations. We describe the experimental evaluation and applications of a new nitroindoline-caged GABA, DPNI-GABA, modified to minimize the pharmacological interference commonly found with caged GABA reagents, but retaining the advantages of nitroindoline cages. Unlike the 5-methoxycarbonylmethyl-7-nitroindolinyl-GABA tested previously, DPNI-GABA inhibited GABA(A) receptors with much lower affinity, reducing peak GABA-evoked responses with an IC(50) of approximately 0.5 mM. Most importantly, the kinetics of receptor activation, determined as 10-90% rise-times, were comparable to synaptic events and were little affected by DPNI-GABA present at 1mM concentration, permitting photolysis of DPNI-GABA to mimic synaptic activation of GABA(A) receptors. With a laser spot of 1 microm applied to cerebellar molecular layer interneurons, the spatial resolution of uncaging DPNI-GABA in dendrites was estimated as 2 microm laterally and 7.5 microm focally. Finally, at low DPNI-GABA concentration, photorelease restricted to the area of the soma suppressed spiking in single Purkinje neurons or molecular layer interneurons for periods controlled by the flash intensity and duration. DPNI-GABA has properties better adapted for fast kinetic studies with laser photolysis at GABA(A) receptors than previously reported caged GABA reagents, and can be used in experiments where spatial resolution is determined by the dimensions of the laser light spot.

  16. Neurosteroids and GABA-A Receptor Function

    PubMed Central

    Wang, Mingde

    2011-01-01

    Neurosteroids represent a class of endogenous steroids that are synthesized in the brain, the adrenals, and the gonads and have potent and selective effects on the GABAA-receptor. 3α-hydroxy A-ring reduced metabolites of progesterone, deoxycorticosterone, and testosterone are positive modulators of GABAA-receptor in a non-genomic manner. Allopregnanolone (3α-OH-5α-pregnan-20-one), 5α-androstane-3α, 17α-diol (Adiol), and 3α5α-tetrahydrodeoxycorticosterone (3α5α-THDOC) enhance the GABA-mediated Cl- currents acting on a site (or sites) distinct from the GABA, benzodiazepine, barbiturate, and picrotoxin binding sites. 3α5α-P and 3α5α-THDOC potentiate synaptic GABAA-receptor function and activate δ-subunit containing extrasynaptic receptors that mediate tonic currents. On the contrary, 3β-OH pregnane steroids and pregnenolone sulfate (PS) are GABAA-receptor antagonists and induce activation-dependent inhibition of the receptor. The activities of neurosteroid are dependent on brain regions and types of neurons. In addition to the slow genomic action of the parent steroids, the non-genomic, and rapid actions of neurosteroids play a significant role in the GABAA-receptor function and shift in mood and memory function. This review describes molecular mechanisms underlying neurosteroid action on the GABAA-receptor, mood changes, and cognitive functions. PMID:22654809

  17. Glia of the cholinergic electromotor nucleus of Torpedo are the source of the cDNA encoding a GAT-1-like GABA transporter.

    PubMed

    Swanson, G T; Umbach, J A; Gundersen, C B

    1994-07-01

    A PCR-based strategy was used to clone DNAs encoding Na(+)- and Cl(-)-dependent cotransport proteins using DNA from the cholinergic electromotor nucleus of Torpedo californica. This cloning strategy resulted in the isolation of a cDNA clone that shows strong nucleotide sequence homology to the GABA transporter-1 (GAT-1) types of rat and human brain. When expressed in frog oocytes, this transporter mediates the uptake of GABA. Moreover, physiologically and pharmacologically, the Torpedo protein behaves very similarly to the rat and human GAT-1 proteins. However, in contrast to the predominantly neuronal localization of the mammalian GAT-1 proteins, the mRNA for the fish protein is found almost exclusively in glial elements of the electromotor nucleus. This unexpected discovery of a GABA transporter cDNA in a nucleus that has no previously characterized GABAergic innervation raises questions about the role of GABA and this transporter in the electromotor system. Several speculative models for GABA function are proposed.

  18. Study of GABA in Healthy Volunteers: Pharmacokinetics and Pharmacodynamics

    PubMed Central

    Li, Junfeng; Zhang, Zhaoyun; Liu, Xiaoxia; Wang, Yi; Mao, Fei; Mao, Junjun; Lu, Xiaolan; Jiang, Dongdong; Wan, Yun; Lv, Jia-Ying; Cao, Guoying; Zhang, Jing; Zhao, Naiqing; Atkinson, Mark; Greiner, Dale L.; Prud'homme, Gerald J.; Jiao, Zheng; Li, Yiming; Wang, Qinghua

    2015-01-01

    Preclinical studies show that GABA exerts anti-diabetic effects in rodent models of type 1 diabetes. Because little is known about its absorption and effects in humans, we investigated the pharmacokinetics and pharmacodynamics of GABA in healthy volunteers. Twelve subjects were subjected to an open-labeled, three-period trial involving sequential oral administration of placebo, 2 g GABA once, and 2 g GABA three times/day for 7 days, with a 7-day washout between each period. GABA was rapidly absorbed (Tmax: 0.5 ~ 1 h) with the half-life (t1/2) of 5 h. No accumulation was observed after repeated oral GABA administration for 7 days. Remarkably, GABA significantly increased circulating insulin levels in the subjects under either fasting (1.6-fold, single dose; 2.0-fold, repeated dose; p < 0.01) or fed conditions (1.4-fold, single dose; 1.6-fold, repeated dose; p < 0.01). GABA also increased glucagon levels only under fasting conditions (1.3-fold, single dose, p < 0.05; 1.5-fold, repeated dose, p < 0.01). However, there were no significant differences in the insulin-to-glucagon ratio and no significant change in glucose levels in these healthy subjects during the study period. Importantly, GABA significantly decreased glycated albumin levels in the repeated dosing period. Subjects with repeated dosing showed an elevated incidence of minor adverse events in comparison to placebo or the single dosing period, most notably transient discomforts such as dizziness and sore throat. However, there were no serious adverse events observed throughout the study. Our data show that GABA is rapidly absorbed and tolerated in human beings; its endocrine effects, exemplified by increasing islet hormonal secretion, suggest potential therapeutic benefits for diabetes. PMID:26617516

  19. The effects of agonists of ionotropic GABA(A) and metabotropic GABA(B) receptors on learning.

    PubMed

    Zyablitseva, Evgeniya A; Kositsyn, Nikolay S; Shul'gina, Galina I

    2009-05-01

    The research described here investigates the role played by inhibitory processes in the discriminations made by the nervous system of humans and animals between familiar and unfamiliar and significant and nonsignificant events. This research compared the effects of two inhibitory mediators of gamma-aminobutyric acid (GABA): 1) phenibut, a nonselective agonist of ionotropic GABA(A) and metabotropic GABA(B) receptors and 2) gaboxadol a selective agonist of ionotropic GABA(A) receptors on the process of developing active defensive and inhibitory conditioned reflexes in alert non-immobilized rabbits. It was found that phenibut, but not gaboxadol, accelerates the development of defensive reflexes at an early stage of conditioning. Both phenibut and gaboxadol facilitate the development of conditioned inhibition, but the effect of gaboxadol occurs at later stages of conditioning and is less stable than that of phenibut. The earlier and more stable effects of phenibut, as compared to gaboxadol, on storage in memory of the inhibitory significance of a stimulus may occur because GABA(B) receptors play the dominant role in the development of internal inhibition during an early stage of conditioning. On the other hand this may occur because the participation of both GABA(A) and GABA(B) receptors are essential to the process. We discuss the polyfunctionality of GABA receptors as a function of their structure and the positions of the relevant neurons in the brain as this factor can affect regulation of various types of psychological processes.

  20. GABA-A and GABA-B receptors in the cuneate nucleus of the rat in vivo.

    PubMed

    Orviz, P; Cecchini, B G; Andrés-Trelles, F

    1986-09-01

    Electric stimulation of the rat forepaw evokes a negative potential (N-wave) at the ipsilateral cuneate nucleus. The responses of the N-wave to microiontophoretically applied GABA agonists and antagonists have been studied. Applications of GABA-A agonists (3-amino-propanesulfonic acid and muscimol) reduce the amplitude of the N-wave. This effect decreases during prolonged application, suggesting a desensitization of GABA-A receptors. In addition the effect of muscimol is reduced by (-)-bicuculline methiodide. Baclofen (a GABA-B agonist) also depresses the N-wave but its action lasts longer, is less reversible, shows no desensitization and is not blocked by (-)-bicuculline methiodide. The different responses of the N-wave to GABA-A and GABA-B agonists are compatible with the existence of different types of functional receptors for them in the cuneate nucleus of the rat. The receptors activated by muscimol (GABA-A) are clearly not the same as the ones activated by baclofen (conceivably GABA-B).

  1. Interaction of pyracetam with specific /sup 3/H-imipramine binding sites and GABA-benzodiazepine receptor complex of brain membranes

    SciTech Connect

    Rozhanets, V.V.; Chakhbra, K.K.; Danchev, N.D.; Malin, K.M.; Rusakov, D.Yu.; Val'dman, A.V.

    1986-06-01

    This paper studies the effect of pyracetam on parameters of specific binding of tritium-imipramine and GABA-activated binding of tritium-flunitrazepam with rat brain membranes. The experimental method is described and it is shown that pyracetam and mebicar in experiments in vivo on normal animals can exert their anxiolytic action without the participation of bensodiazepine receptors. Either the interaction of pyracetam and mebicar with benzodiazeprine receptors has a different interpretation than competition of these compounds with specific binding sites of tritium-flunitrazepam, or in experiments on normal animals in vivo GABA-benzodiazepine receptor complex does not accept pyracetam and mebicar, for it contains endogenous inhibitors of GABA-modulating action.

  2. gamma-Hydroxybutyrate (GHB) induces GABA(B) receptor independent intracellular Ca2+ transients in astrocytes, but has no effect on GHB or GABA(B) receptors of medium spiny neurons in the nucleus accumbens.

    PubMed

    Molnár, T; Antal, K; Nyitrai, G; Emri, Z

    2009-08-18

    We report on cellular actions of the illicit recreational drug gamma-hydroxybutyrate (GHB) in the brain reward area nucleus accumbens. First, we compared the effects of GHB and the GABA(B) receptor agonist baclofen. Neither of them affected the membrane currents of medium spiny neurons in rat nucleus accumbens slices. GABAergic and glutamatergic synaptic potentials of medium spiny neurons, however, were reduced by baclofen but not GHB. These results indicate the lack of GHB as well as postsynaptic GABA(B) receptors, and the presence of GHB insensitive presynaptic GABA(B) receptors in medium spiny neurons. In astrocytes GHB induced intracellular Ca(2+) transients, preserved in slices from GABA(B) receptor type 1 subunit knockout mice. The effects of tetrodotoxin, zero added Ca(2+) with/without intracellular Ca(2+) store depletor cyclopiazonic acid or vacuolar H-ATPase inhibitor bafilomycin A1 indicate that GHB-evoked Ca(2+) transients depend on external Ca(2+) and intracellular Ca(2+) stores, but not on vesicular transmitter release. GHB-induced astrocytic Ca(2+) transients were not affected by the GHB receptor-specific antagonist NCS-382, suggesting the presence of a novel NCS-382-insensitive target for GHB in astrocytes. The activation of astrocytes by GHB implies their involvement in physiological actions of GHB. Our findings disclose a novel profile of GHB action in the nucleus accumbens. Here, unlike in other brain areas, GHB does not act on GABA(B) receptors, but activates an NCS-382 insensitive GHB-specific target in a subpopulation of astrocytes. The lack of either post- or presynaptic effects on medium spiny neurons in the nucleus accumbens distinguishes GHB from many drugs and natural rewards with addictive properties and might explain why GHB has only a weak reinforcing capacity.

  3. Presynaptic modulation by L-glutamate and GABA of sympathetic co-transmission in rat isolated vas deferens.

    PubMed

    Kwan, Y W; Ngan, M P; Tsang, K Y; Lee, H M; Chu, L A

    1996-06-01

    1. The modulatory effects of L-glutamate and its structural analogues, and of gamma-aminobutyric acid (GABA), on sympathetic co-transmission were studied in the rat isolated vas deferens exposed to electrical field stimulation (EFS). 2. Application of exogenous L-glutamate caused a concentration-dependent (1 microM-3 mM) inhibition of the rapid twitch component of the biphasic EFS contraction. However, L-glutamate (1 microM-3 mM) had a minimal effect on the phasic contraction induced by exogenous adenosine 5'-triphosphate (ATP, 150 microM) and noradrenaline (50 microM). Unlike L-glutamate, D-glutamate had no effect on the EFS contraction. 3. The L-glutamate-induced inhibition of the EFS contractions was significantly attenuated by the glutamate decarboxylase (GAD) inhibitor 3-mercapto-propionic acid (150 microM) and was abolished in the presence of the GABA transaminase (GABA-T) inhibitor, 2-aminoethyl hydrogen sulphate (500 microM). 4. The L-glutamate-induced inhibition of the electrically evoked contraction was not affected by the adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)(30 nM), reactive blue 2 (30 microM) or the GABAA receptor antagonist bicuculline (50 microM). However, the GABAB receptor antagonist 2-hydroxysaclofen (50 microM) significantly inhibited the L-glutamate effect. 5. Similar to L-glutamate, GABA also caused a concentration-dependent (0.1-100 microM) inhibition of the EFS contractions. This GABA-induced inhibition was not affected by either the GABAA receptor antagonist bicuculline (50 microM) or reactive blue 2 (30 microM). However, a significant attenuation of the GABA-mediated effect was recorded with the GABAB receptor antagonist 2-hydroxysaclofen (50 microM). Contractions of the vas deferens induced by exogenous ATP and noradrenaline were not affected by GABA (0.1-100 microM). 6. The L-glutamate analogues, N-methyl-D-aspartate (NMDA) (1 microM-1 mM) and quisqualate (Quis 0.1 microM-0.3 mM) had no effect

  4. Mechanisms of carbacholine and GABA action on resting membrane potential and Na+/K+-ATPase of Lumbricus terrestris body wall muscles.

    PubMed

    Volkov, Eugeny M; Nurullin, Leniz F; Volkov, Michael E; Nikolsky, Eugeny E; Vyskočil, Frantisek

    2011-04-01

    This work was aimed to identify the action of several ion channel and pump inhibitors as well as nicotinic, GABAergic, purinergic and serotoninergic drugs on the resting membrane potential (RMP) and assess the role of cholinergic and GABAergic sensitivity in earthworm muscle electrogenesis. The nicotinic agonists acetylcholine (ACh), carbacholine (CCh) and nicotine depolarize the RMP at concentrations of 5 μM and higher. The nicotinic antagonists (+)tubocurarine, α-bungarotoxin, muscarinic antagonists atropine and hexamethonium do not remove or prevent the CCh-induced depolarization. Verapamil, tetrodotoxin, removal of Cl(-) and Ca(2+) from the solution also cannot prevent the depolarization by CCh. In a Na(+)-free medium, however, CCh lost this depolarization ability and this indicates that the drug opens the sodium permeable pathway. Serotonin, glutamate, glycine, adenosine triphosphate (ATP) and cis-4-aminocrotonic acid (GABA(C) receptor antagonist) had no effect on the RMP. On the other hand, isoguvacin, γ-aminobutyric acid (GABA) and baclofen (GABA(B) receptor agonist) hyperpolarized the RMP. Ouabain, bicucullin (GABA(A) antagonist) and phaclofen (GABA(B) antagonist), as well as the removal of Cl(-), suppressed the effect of GABA and baclofen. CCh did not enhance the depolarization generated by ouabain but, on the other hand, hindered the hyperpolarizing activity of baclofen both in the absence and presence of atropine and (+)tubocurarine. The long-term application of CCh depolarizes the RMP primarily by inhibiting the Na(+)/K(+)-ATPase. The muscle membrane also contains A and B type GABA binding sites, the activation of which increases the RMP at the expense of increasing the action of ouabain- and Cl(-) -sensitive electrogenic pumps.

  5. GABA(B) receptors mediate motility signals for migrating embryonic cortical cells.

    PubMed

    Behar, T N; Smith, S V; Kennedy, R T; McKenzie, J M; Maric, I; Barker, J L

    2001-08-01

    During development, postmitotic neurons migrate from germinal regions into the cortical plate (cp), where lamination occurs. In rats, GABA is transiently expressed in the cp, near target destinations for migrating neurons. In vitro GABA stimulates neuronal motility, suggesting cp cells release GABA, which acts as a chemoattractant during corticogenesis. Pharmacological studies indicate GABA stimulates migration via GABA(B)-receptor (GABA(B)-R) activation. Using immunohistochemistry, RT-PCR and Western blotting, we examined embryonic cortical cell expression of GABA(B)-Rs in vivo. At E17, GABA(B)-R1(+) cells were identified in the ventricular zone (vz) and cp. RT-PCR and Western blotting demonstrated the presence of GABA(B)-R1a and GABA(B)-R1b mRNA and proteins. Using immuno- cytochemistry, GABA(B)-R expression was examined in vz and cp cell dissociates before and after migration to GABA in an in vitro chemotaxis assay. GABA-induced migration resulted in an increase of GABA(B)-R(+) cells in the migrated population. While <20% of each starting dissociate was GABA(B)-R(+), >70% of migrated cells were immunopositive. We used a microchemotaxis assay to analyze cp cell release of diffusible chemotropic factor(s). In vitro, cp dissociates induced vz cell migration in a cell density-dependent manner that was blocked by micromolar saclofen (a GABA(B)-R antagonist). HPLC demonstrated cp cells release micromolar levels of GABA and taurine in several hours. Micromolar levels of both molecules stimulated cell migration that was blocked by micromolar saclofen. Thus, migratory cortical cells express GABA(B)-Rs, cp cells release GABA and taurine, and both molecules stimulate cortical cell movement. Together these findings suggest GABA and/or taurine act as chemoattractants for neurons during rat cortical histogenesis via mechanisms involving GABA(B)-Rs.

  6. Corelease of acetylcholine and GABA from cholinergic forebrain neurons

    PubMed Central

    Saunders, Arpiar; Granger, Adam J; Sabatini, Bernardo L

    2015-01-01

    Neurotransmitter corelease is emerging as a common theme of central neuromodulatory systems. Though corelease of glutamate or GABA with acetylcholine has been reported within the cholinergic system, the full extent is unknown. To explore synaptic signaling of cholinergic forebrain neurons, we activated choline acetyltransferase expressing neurons using channelrhodopsin while recording post-synaptic currents (PSCs) in layer 1 interneurons. Surprisingly, we observed PSCs mediated by GABAA receptors in addition to nicotinic acetylcholine receptors. Based on PSC latency and pharmacological sensitivity, our results suggest monosynaptic release of both GABA and ACh. Anatomical analysis showed that forebrain cholinergic neurons express the GABA synthetic enzyme Gad2 and the vesicular GABA transporter (Slc32a1). We confirmed the direct release of GABA by knocking out Slc32a1 from cholinergic neurons. Our results identify GABA as an overlooked fast neurotransmitter utilized throughout the forebrain cholinergic system. GABA/ACh corelease may have major implications for modulation of cortical function by cholinergic neurons. DOI: http://dx.doi.org/10.7554/eLife.06412.001 PMID:25723967

  7. GABA[subscript A] Receptor Downregulation in Brains of Subjects with Autism

    ERIC Educational Resources Information Center

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Thuras, Paul D.

    2009-01-01

    Gamma-aminobutyric acid A (GABA[subscript A]) receptors are ligand-gated ion channels responsible for mediation of fast inhibitory action of GABA in the brain. Preliminary reports have demonstrated altered expression of GABA receptors in the brains of subjects with autism suggesting GABA/glutamate system dysregulation. We investigated the…

  8. Regulation of GABA-modulin phosphorylation and GABA receptor binding by excitatory amino acids

    SciTech Connect

    Vaccarino, F.; Guidotti, A.

    1987-05-01

    Primary cultures of cerebellar granule cells phosphorylate numerous proteins including GABA-modulin (GM), which is a putative allosteric modulator of GABA receptors. Cell depolarization and treatment with dicarboxylic excitatory amino acids, which activate PI turnover, Ca/sup 2 +/ influx and guanylate cyclase in granule cells increase the phosphorylation of specific proteins. To determine GM phosphorylation by endogenous protein kinases in living granule cell cultures, GM was isolated by immunoprecipitation and reverse-phase HPLC. High K/sup +/, veratridine, glutamate and NMDA treatment stimulated GM phosphorylation over 2-fold. This increase was abolished by the absence of extracellular Ca/sup 2 +/ and was antagonized by Mg/sup 2 +/ ions and by AVP. The excitatory amino acid action was mimicked by phorbol esters but not by forskolin or by cGMP, and thus may be mediated by an activation of protein kinase C (PKC). Moreover, excitatory amino acids increase /sup 3/H-labelled phorbol ester binding sites in granule cell membrane. The same cultures, treated with glutamate or kainate, showed a 50-fold greater efficacy of muscimol for the stimulation of benzodiazepine (BZ) binding. These data-suggest that excitatory amino acid stimulation of neurons triggers PKC translocation and the activated enzyme phosphorylates GM. The extent of GM phosphorylation may regulate the coupling between GABA and BZ binding sites.

  9. Trans-synaptic (GABA-dopamine) modulation of cocaine induced dopamine release: A potential therapeutic strategy for cocaine abuse

    SciTech Connect

    Dewey, S.L.; Straughter-Moore, R.; Chen, R.

    1995-05-01

    We recently developed a new experimental strategy for measuring interactions between functionally-linked neurotransmitter systems in the primate and human brain with PET. As part of this research, we demonstrated that increases in endogenous GABA concentrations significantly reduced striatal dopamine concentrations in the primate brain. We report here the application of the neurotransmitter interaction paradigm with PET and with microdialysis to the investigation of a novel therapeutic strategy for treating cocaine abuse based on the ability of GABA to inhibit cocaine induced increases in striatal dopamine. Using gamma-vinyl GABA (GVG, a suicide inhibitor of GABA transaminase), we performed a series of PET studies where animals received a baseline PET scan with labeled raclopride injection, animals received cocaine (2.0 mg/kg). Normally, a cocaine challenge significantly reduces the striatal binding of {sup 11}C-raclopride. However, in animals pretreated with GVG, {sup 11}C-raclopride binding was less affected by a cocaine challenge compared to control studies. Furthermore, microdialysis studies in freely moving rats demonstrate that GVG (300 mg/kg) significantly inhibited cocaine-induced increases in extracellular dopamine release. GVG also attenuated cocaine-induced increases in locomotor activity. However, at a dose of 100 mg/kg, GVG had no effect. Similar findings were obtained with alcohol. Alcohol pretreatment dose dependantly (1-4 g/kg) inhibited cocaine-induced increases in extracellular dopamine concentrations in freely moving rats. Taken together, these studies suggest that therapeutic strategies targeted at increasing central GABA concentrations may be beneficial for the treatment of cocaine abuse.

  10. An Electrostatic Funnel in the GABA-Binding Pathway

    PubMed Central

    Lightstone, Felice C.

    2016-01-01

    The γ-aminobutyric acid type A receptor (GABAA-R) is a major inhibitory neuroreceptor that is activated by the binding of GABA. The structure of the GABAA-R is well characterized, and many of the binding site residues have been identified. However, most of these residues are obscured behind the C-loop that acts as a cover to the binding site. Thus, the mechanism by which the GABA molecule recognizes the binding site, and the pathway it takes to enter the binding site are both unclear. Through the completion and detailed analysis of 100 short, unbiased, independent molecular dynamics simulations, we have investigated this phenomenon of GABA entering the binding site. In each system, GABA was placed quasi-randomly near the binding site of a GABAA-R homology model, and atomistic simulations were carried out to observe the behavior of the GABA molecules. GABA fully entered the binding site in 19 of the 100 simulations. The pathway taken by these molecules was consistent and non-random; the GABA molecules approach the binding site from below, before passing up behind the C-loop and into the binding site. This binding pathway is driven by long-range electrostatic interactions, whereby the electrostatic field acts as a ‘funnel’ that sweeps the GABA molecules towards the binding site, at which point more specific atomic interactions take over. These findings define a nuanced mechanism whereby the GABAA-R uses the general zwitterionic features of the GABA molecule to identify a potential ligand some 2 nm away from the binding site. PMID:27119953

  11. A Gut Feeling about GABA: Focus on GABAB Receptors

    PubMed Central

    Hyland, Niall P.; Cryan, John F.

    2010-01-01

    γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the body and hence GABA-mediated neurotransmission regulates many physiological functions, including those in the gastrointestinal (GI) tract. GABA is located throughout the GI tract and is found in enteric nerves as well as in endocrine-like cells, implicating GABA as both a neurotransmitter and an endocrine mediator influencing GI function. GABA mediates its effects via GABA receptors which are either ionotropic GABAA or metabotropic GABAB. The latter which respond to the agonist baclofen have been least characterized, however accumulating data suggest that they play a key role in GI function in health and disease. Like GABA, GABAB receptors have been detected throughout the gut of several species in the enteric nervous system, muscle, epithelial layers as well as on endocrine-like cells. Such widespread distribution of this metabotropic GABA receptor is consistent with its significant modulatory role over intestinal motility, gastric emptying, gastric acid secretion, transient lower esophageal sphincter relaxation and visceral sensation of painful colonic stimuli. More intriguing findings, the mechanisms underlying which have yet to be determined, suggest GABAB receptors inhibit GI carcinogenesis and tumor growth. Therefore, the diversity of GI functions regulated by GABAB receptors makes it a potentially useful target in the treatment of several GI disorders. In light of the development of novel compounds such as peripherally acting GABAB receptor agonists, positive allosteric modulators of the GABAB receptor and GABA producing enteric bacteria, we review and summarize current knowledge on the function of GABAB receptors within the GI tract. PMID:21833169

  12. Saturable binding of /sup 35/S-t-butylbicyclophosphorothionate to the sites linked to the GABA receptor and the interaction with gabaergic agents

    SciTech Connect

    Wong, D.T.; Threlkeld, P.G.; Bymaster, F.P.; Squires, R.F.

    1984-02-27

    /sup 35/-S-t-Butylbicyclophosphorothionate (/sup 35/S-TBPS) binds in a concentration-saturable manner to specific sites on membranes from rat cerebral cortex. Using a filtration assay at 25/sup 0/C, in 250 mM NaCl, specific binding of /sup 35/S-TBPS constitutes about 84 to 94 percent of total binding, depending on radioligand concentrations. /sup 35/S-TBPS binding is optimal in the presence of NaCl or NaBr and substantially less in the presence of NaI or NaF. It is sensitive to the treatment with 0.05 percent Triton X-100 but not to repeated freezing and thawing, procedures which increase /sup 3/H-GABA binding. Pharmacological studies show that /sup 35/S-TBPS binding is strongly inhibited by GABA-A receptor agonists (e.g., GABA and muscimol) and by the noncompetitive antagonist, picrotoxin, but not the competitive antagonist, bicuculline. Compounds which enhance binding of radioactive GABA and benzodiazepines, such as the pyrazolopyridines, cartazolate and trazolate, and a diaryl-triazine, LY81067, are also potent inhibitors of /sup 35/S-TBPS binding, with LY81067 being the most effective. The effects of GABA, picrotoxin

  13. The effects of endomorphins on striatal [3H]GABA release induced by electrical stimulation: an in vitro superfusion study in rats.

    PubMed

    Bagosi, Zsolt; Jászberényi, Miklós; Telegdy, Gyula

    2009-05-01

    The endomorphins (EM1 and EM2) are selective endogenous ligands for mu-opioid receptors (MOR1 and MOR2) with neurotransmitter and neuromodulator roles in mammals. In the present study we investigated the potential actions of EMs on striatal GABA release and the implication of different MORs in these processes. Rat striatal slices were preincubated with tritium-labelled GABA ([(3)H]GABA), pretreated with selective MOR1 and MOR2 antagonist beta-funaltrexamine and selective MOR1 antagonist naloxonazine and then superfused with the selective MOR agonists, EM1 and EM2. EM1 significantly decreased the striatal [(3)H]GABA release induced by electrical stimulation. Beta-funaltrexamine antagonized the inhibitory action of EM1, but naloxonazine did not affect it considerably. EM2 was ineffective, even in case of specific enzyme inhibitor diprotin A pretreatment. The results demonstrate that EM1 decreases GABA release in the basal ganglia through MOR2, while EM2 does not influence it.

  14. Agonist pharmacology of two Drosophila GABA receptor splice variants.

    PubMed Central

    Hosie, A. M.; Sattelle, D. B.

    1996-01-01

    1. The Drosophila melanogaster gamma-aminobutyric acid (GABA) receptor subunits, RDLac and DRC 17-1-2, form functional homo-oligomeric receptors when heterologously expressed in Xenopus laevis oocytes. The subunits differ in only 17 amino acids, principally in regions of the N-terminal domain which determine agonist pharmacology in vertebrate ionotropic neurotransmitter receptors. A range of conformationally restricted GABA analogues were tested on the two homo-oligomers and their agonists pharmacology compared with that of insect and vertebrate iontropic GABA receptors. 2. The actions of GABA, isoguvacine and isonipecotic acid on RDLac and DRC 17-1-2 homo-oligomers were compared, by use of two-electrode voltage-clamp. All three compounds were full agonists of both receptors, but were 4-6 fold less potent agonists of DRC 17-1-2 homo-oligomers than of RDLac. However, the relative potencies of these agonists on each receptor were very similar. 3. A more complete agonist profile was established for RDLac homo-oligomers. The most potent agonists of these receptors were GABA, muscimol and trans-aminocrotonic acid (TACA), which were approximately equipotent. RDLac homo-oligomers were fully activated by a range of GABA analogues, with the order of potency: GABA > ZAPA ((Z)-3-[(aminoiminomethyl)thio]prop-2-enoic acid) > isoguvacine > imidazole-4-acetic acid > or = isonipecotic acid > or = cis-aminocrotonic acid (CACA) > beta-alanine. 3-Aminopropane sulphonic acid (3-APS), a partial agonist of RDLac homo-oligomers, was the weakest agonist tested and 100 fold less potent than GABA. 4. SR95531, an antagonist of vertebrate GABAA receptors, competitively inhibited the GABA responses of RDLac homo-oligomers, which have previously been found to insensitive to bicuculline. However, its potency (IC50 500 microM) was much reduced when compared to GABAA receptors. 5. The agonist pharmacology of Drosophila RDLac homo-oligomers exhibits aspects of the characteristic pharmacology of

  15. Dopaminergic neurons modulate GABA neuron migration in the embryonic midbrain.

    PubMed

    Vasudevan, Anju; Won, Chungkil; Li, Suyan; Erdélyi, Ferenc; Szabó, Gábor; Kim, Kwang-Soo

    2012-09-01

    Neuronal migration, a key event during brain development, remains largely unexplored in the mesencephalon, where dopaminergic (DA) and GABA neurons constitute two major neuronal populations. Here we study the migrational trajectories of DA and GABA neurons and show that they occupy ventral mesencephalic territory in a temporally and spatially specific manner. Our results from the Pitx3-deficient aphakia mouse suggest that pre-existing DA neurons modulate GABA neuronal migration to their final destination, providing novel insights and fresh perspectives concerning neuronal migration and connectivity in the mesencephalon in normal as well as diseased brains.

  16. Bioassay-guided fractionation of lemon balm (Melissa officinalis L.) using an in vitro measure of GABA transaminase activity.

    PubMed

    Awad, Rosalie; Muhammad, Asim; Durst, Tony; Trudeau, Vance L; Arnason, John T

    2009-08-01

    A novel pharmacological mechanism of action for the anxiolytic botanical Melissa officinalis L. (lemon balm) is reported. The methanol extract was identified as a potent in vitro inhibitor of rat brain GABA transaminase (GABA-T), an enzyme target in the therapy of anxiety, epilepsy and related neurological disorders. Bioassay-guided fractionation led to the identification and isolation of rosmarinic acid (RA) and the triterpenoids, ursolic acid (UA) and oleanolic acid (OA) as active principles. Phytochemical characterization of the crude extract determined RA as the major compound responsible for activity (40% inhibition at 100 microg/mL) since it represented approximately 1.5% of the dry mass of the leaves. Synergistic effects may also play a role.

  17. GABA transporter 1 tunes GABAergic synaptic transmission at output neurons of the mouse neostriatum

    PubMed Central

    Kirmse, Knut; Dvorzhak, Anton; Kirischuk, Sergei; Grantyn, Rosemarie

    2008-01-01

    GABAergic medium-sized striatal output neurons (SONs) provide the principal output for the neostriatum. In vitro and in vivo data indicate that spike discharge of SONs is tightly controlled by effective synaptic inhibition. Although phasic GABAergic transmission critically depends on ambient GABA levels, the role of GABA transporters (GATs) in neostriatal GABAergic synaptic transmission is largely unknown. In the present study we aimed at elucidating the role of GAT-1 in the developing mouse neostriatum (postnatal day (P) 7–34). We recorded GABAergic postsynaptic currents (PSCs) using the whole-cell patch-clamp technique. Based on the effects of NO-711, a specific GAT-1 blocker, we demonstrate that GAT-1 is operative at this age and influences GABAergic synaptic transmission by presynaptic and postsynaptic mechanisms. Presynaptic GABABR-mediated suppression of GABA release was found to be functional at all ages tested; however, there was no evidence for persistent GABABR activity under control conditions, unless GAT-1 was blocked (P12–34). In addition, whereas no tonic GABAAR-mediated conductances were detected in SONs until P14, application of a specific GABAAR antagonist caused distinct tonic outward currents later in development (P19–34). In the presence of NO-711, tonic GABAAR-mediated currents were also observed at P7–14 and were dramatically increased at more mature stages. Furthermore, GAT-1 block reduced the median amplitude of GABAergic miniature PSCs indicating a decrease in quantal size. We conclude that in the murine neostriatum GAT-1 operates in a net uptake mode. It prevents the persistent activation of presynaptic GABABRs (P12–34) and prevents (P7–14) or reduces (P19–34) tonic postsynaptic GABAAR activity. PMID:18832421

  18. Brain manganese accumulation is inversely related to gamma-amino butyric acid uptake in male and female rats.

    PubMed

    Anderson, Joel G; Cooney, Paula T; Erikson, Keith M

    2007-01-01

    Iron (Fe) is an essential trace metal involved in numerous cellular processes. Iron deficiency (ID) is reported as the most prevalent nutritional problem worldwide. Increasing evidence suggests that ID is associated with altered neurotransmitter metabolism and a risk factor for manganese (Mn) neurotoxicity. Though recent studies have established differences in which the female brain responds to ID-related neurochemical alterations versus the male brain, little is known about the interactions of dietary ID, Mn exposure, and sex on gamma-amino butyric acid (GABA). Male and female Sprague-Dawley rats were randomly divided into four dietary treatment groups: control (CN), control/Mn supplemented, ID, and ID/Mn supplemented. After 6 weeks of treatment, both ID diets caused a highly significant decrease in Fe concentrations across all brain regions compared to CN in both sexes. Both ID and Mn supplementation led to significant accumulation of Mn across all brain regions in both sexes. There was no main effect of sex on Fe or Mn accumulation. Striatal synaptosomes were utilized to examine the effect of dietary intervention on (3)H-GABA uptake. At 4 weeks, there was a significant correlation between Fe concentration and (3)H-GABA uptake in male rats (p < 0.05). At 6 weeks, there was a significant inverse correlation between Mn concentration and (3)H-GABA uptake in male and female rats and a postitive correlation between Fe concentration and (3)H-GABA uptake in female rats (p < 0.05). In conclusion, ID-associated Mn accumulation is similar in both sexes, with Mn levels affecting GABA uptake in both sexes in a comparable fashion.

  19. How and why does tomato accumulate a large amount of GABA in the fruit?

    PubMed Central

    Takayama, Mariko; Ezura, Hiroshi

    2015-01-01

    Gamma-aminobutyric acid (GABA) has received much attention as a health-promoting functional compound, and several GABA-enriched foods have been commercialized. In higher plants, GABA is primarily metabolized via a short pathway called the GABA shunt. The GABA shunt bypasses two steps (the oxidation of α-ketoglutarate to succinate) of the tricarboxylic acid (TCA) cycle via reactions catalyzed by three enzymes: glutamate decarboxylase, GABA transaminase, and succinic semialdehyde dehydrogenase. The GABA shunt plays a major role in primary carbon and nitrogen metabolism and is an integral part of the TCA cycle under stress and non-stress conditions. Tomato is one of the major crops that accumulate a relatively high level of GABA in its fruits. The GABA levels in tomato fruits dramatically change during fruit development; the GABA levels increase from flowering to the mature green stage and then rapidly decrease during the ripening stage. Although GABA constitutes up to 50% of the free amino acids at the mature green stage, the molecular mechanism of GABA accumulation and the physiological function of GABA during tomato fruit development remain unclear. In this review, we summarize recent studies of GABA accumulation in tomato fruits and discuss the potential biological roles of GABA in tomato fruit development. PMID:26322056

  20. P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

    PubMed

    Barros-Barbosa, A R; Lobo, M G; Ferreirinha, F; Correia-de-Sá, P; Cordeiro, J M

    2015-10-15

    Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability.

  1. GABA Metabolism and Transport: Effects on Synaptic Efficacy

    PubMed Central

    Roth, Fabian C.; Draguhn, Andreas

    2012-01-01

    GABAergic inhibition is an important regulator of excitability in neuronal networks. In addition, inhibitory synaptic signals contribute crucially to the organization of spatiotemporal patterns of network activity, especially during coherent oscillations. In order to maintain stable network states, the release of GABA by interneurons must be plastic in timing and amount. This homeostatic regulation is achieved by several pre- and postsynaptic mechanisms and is triggered by various activity-dependent local signals such as excitatory input or ambient levels of neurotransmitters. Here, we review findings on the availability of GABA for release at presynaptic terminals of interneurons. Presynaptic GABA content seems to be an important determinant of inhibitory efficacy and can be differentially regulated by changing synthesis, transport, and degradation of GABA or related molecules. We will discuss the functional impact of such regulations on neuronal network patterns and, finally, point towards pharmacological approaches targeting these processes. PMID:22530158

  2. Dual role of GABA in the neonatal rat hippocampus.

    PubMed

    Khalilov, I; Dzhala, V; Ben-Ari, Y; Khazipov, R

    1999-11-01

    The effects of modulators of GABA-A receptors on neuronal network activity were studied in the neonatal (postnatal days 0-5) rat hippocampus in vitro. Under control conditions, the physiological pattern of activity of the neonatal hippocampal network was characterized by spontaneous network-driven giant depolarizing potentials (GDPs). The GABA-A receptor agonist isoguvacine (1-2 microM) and the allosteric modulator diazepam (2 microM) induced biphasic responses: initially the frequency of GDPs increased 3 to 4 fold followed by blockade of GDPs and desynchronization of the network activity. The GABA-A receptor antagonists bicuculline (10 microM) and picrotoxin (100 microM) blocked GDPs and induced glutamate (AMPA and NMDA)-receptor-mediated interictal- and ictal-like activities in the hippocampal slices and the intact hippocampus. These data suggest that at early postnatal ages GABA can exert a dual - both excitatory and inhibitory - action on the network activity.

  3. Endogenous synthesis of taurine and GABA in rat ocular tissues.

    PubMed

    Heinämäki, A A

    1988-01-01

    The endogenous production of taurine and gamma-aminobutyric acid (GABA) in rat ocular tissues was investigated. The activities of taurine-producing enzyme, cysteine sulfinic acid decarboxylase (CSAD), and GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), were observed in the retina, lens, iris-ciliary body and cornea. The highest specific activity of CSAD was in the cornea and that of GAD in the retina. The discrepancy between CSAD activity and taurine content within the ocular tissues indicates that intra- or extraocular transport processes may regulate the concentration of taurine in the rat eye. The GAD activity and the content of GABA were distributed in parallel within the rat ocular tissues. The quantitative results suggest that the GAD/GABA system has functional significance only in the retina of the rat eye.

  4. [GABA-Receptors in Modulation of Fear Memory Extinction].

    PubMed

    Dubrovina, N I

    2016-01-01

    GABA is the major inhibitory neurotransmitter in the central nervous system determining the efficacy of neuronal interaction. GABA-receptors play a key role in different aspects of fear memory--acquisition and consolidation, retention, reconsolidation and extinction. Extinction is an important behavioural phenomenon which allows organism to adapt its behavior to a changing environment. Extinction of fear memory is a form of new inhibitory learning which interferes with expression of the initial acquired fear conditioning. Resistance to extinction is symptom of depression and posttraumatic stress disorder. The aim of the present review was to summarize own and literary data about GABAergic modulation of fear extinction and pharmacological correction of extinction impairment at influences on GABA(A)- and GABA(B)- receptors.

  5. Benzodiazepines do not potentiate GABA responses in neonatal hippocampal neurons.

    PubMed

    Rovira, C; Ben-Ari, Y

    1991-09-16

    Benzodiazepines (midazolam; flunitrazepam) and pentobarbital increase the response to exogenous gamma-aminobutyric acid (GABA) in adult hippocampal cells. We report in this paper that in contrast pentobarbital but not benzodiazepine potentiate the effects of exogenous (GABA) in neurons recorded from slices of less than two weeks old. This finding suggests that the functional association of benzodiazepine and GABAA receptors is changed during early postnatal life.

  6. Parkinson's Disease and Neurodegeneration: GABA-Collapse Hypothesis

    PubMed Central

    Błaszczyk, Janusz W.

    2016-01-01

    Neurodegenerative diseases constitute a heterogeneous group of age-related disorders that are characterized by a slow but irreversible deterioration of brain functions. Evidence accumulated over more than two decades has implicated calcium-related homeostatic mechanisms, giving rise to the Ca2+ hypothesis of brain aging and, ultimately, cell death. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter within the central (CNS), peripheral and enteric nervous systems. It appears to be involved in a wide variety of physiological functions within and outside the nervous system, that are maintained through a complex interaction between GABA and calcium-dependent neurotransmission and cellular metabolic functions. Within CNS the Ca2+/GABA mechanism stabilizes neuronal activity both at cellular and systemic levels. Decline in the Ca2+/GABA control initiates several cascading processes leading to both weakened protective barriers (in particular the blood-brain barrier) and accumulations of intracellular deposits of calcium and Lewy bodies. Linking such a vital mechanism of synaptic transmission with metabolism (both at cellular and tissue level) by means of a common reciprocal Ca2+/GABA inhibition results in a fragile balance, which is prone to destabilization and auto-destruction. The GABA decline etiology proposed here appears to apply to all human neurodegenerative processes initiated by abnormal intracellular calcium levels. Therefore, the original description of Parkinson's disease (PD) as due to the selective damage of dopaminergic neurons in the mesencephalon should be updated into the concept of a severe multisystemic neurodegenerative disorder of the nervous system, whose clinical symptoms reflect the localization and progression of the most advanced GABA pathology. A future and more complete therapeutic approach to PD should be aimed first at slowing (or stopping) the progression of Ca2+/GABA functional decline. PMID:27375426

  7. GABA Signaling and Neuroactive Steroids in Adrenal Medullary Chromaffin Cells

    PubMed Central

    Harada, Keita; Matsuoka, Hidetada; Fujihara, Hiroaki; Ueta, Yoichi; Yanagawa, Yuchio; Inoue, Masumi

    2016-01-01

    Gamma-aminobutyric acid (GABA) is produced not only in the brain, but also in endocrine cells by the two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67. In rat adrenal medullary chromaffin cells only GAD67 is expressed, and GABA is stored in large dense core vesicles (LDCVs), but not synaptic-like microvesicles (SLMVs). The α3β2/3γ2 complex represents the majority of GABAA receptors expressed in rat and guinea pig chromaffin cells, whereas PC12 cells, an immortalized rat chromaffin cell line, express the α1 subunit as well as the α3. The expression of α3, but not α1, in PC12 cells is enhanced by glucocorticoid activity, which may be mediated by both the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). GABA has two actions mediated by GABAA receptors in chromaffin cells: it induces catecholamine secretion by itself and produces an inhibition of synaptically evoked secretion by a shunt effect. Allopregnanolone, a neuroactive steroid which is secreted from the adrenal cortex, produces a marked facilitation of GABAA receptor channel activity. Since there are no GABAergic nerve fibers in the adrenal medulla, GABA may function as a para/autocrine factor in the chromaffin cells. This function of GABA may be facilitated by expression of the immature isoforms of GAD and GABAA receptors and the lack of expression of plasma membrane GABA transporters (GATs). In this review, we will consider how the para/autocrine function of GABA is achieved, focusing on the structural and molecular mechanisms for GABA signaling. PMID:27147972

  8. Modulation of horizontal cell function by GABA(A) and GABA(C) receptors in dark- and light-adapted tiger salamander retina.

    PubMed

    Yang, X L; Gao, F; Wu, S M

    1999-01-01

    The physiological function of GABA transporters and GABA receptors in retinal horizontal cells (HCs) under dark-and light-adapted conditions were studied by whole-cell voltage clamp and intracellular recording techniques in retinal slices and whole-mounted isolated retinas of the larval tiger salamander. Puff application of GABA in picrotoxin elicited a NO-711 (a potent GABA transporter blocker)-sensitive inward current that did not exhibit a reversal potential in the physiological range, consistent with the idea that these HCs contain electrogenic GABA transporters. Application of GABA in NO-711 elicited a chloride current in HCs; about half of the current was suppressed by bicuculline or I4AA (a GABA(C) receptor antagonist), and the remaining half was suppressed by bicuculline + I4AA or picrotoxin. In whole-mount retinas, NO-711, bicuculline, I4AA, or picrotoxin hyperpolarized the HCs and enhanced the light responses under dark-adapted conditions, and blocked the time-dependent recovery of HC membrane potential and light responses during background illumination. Based on the parallel conductance model, GABA released in darkness mediates a chloride conductance about three times greater than the leak conductance or the glutamate-gated cation conductance. About half of this chloride conductance is mediated by GABA(A) receptors, and the other half is mediated by GABA(C) receptors. These results suggest that GABA released from HCs through the NO-711-sensitive GABA transporters activates GABA(A) and GABA(C) receptors, resulting in chloride conductance increase which leads to a HC depolarization and reduction of the light response. Additionally, GABA transporters also mediate GABA release in background light that is responsible for the recovery of HC membrane potential and light responses.

  9. Expression of GABA receptor rho subunits in rat brain.

    PubMed

    Boue-Grabot, E; Roudbaraki, M; Bascles, L; Tramu, G; Bloch, B; Garret, M

    1998-03-01

    The GABA receptor rho1, rho2, and rho3 subunits are expressed in the retina where they form bicuculline-insensitive GABA(C) receptors. We used northern blot, in situ hybridization, and RT-PCR analysis to study the expression of rho subunits in rat brains. In situ hybridization allowed us to detect rho-subunit expression in the superficial gray layer of the superior colliculus and in the cerebellar Purkinje cells. RT-PCR experiments indicated that (a) in retina and in domains that may contain functional GABA(C) receptors, rho2 and rho1 subunits are expressed at similar levels; and (b) in domains and in tissues that are unlikely to contain GABA(C) receptors, rho2 mRNA is enriched relative to rho1 mRNA. These results suggest that both rho1 and rho2 subunits are necessary to form a functional GABA(C) receptor. The use of RT-PCR also showed that, except in the superior colliculus, rho3 is expressed along with rho1 and rho2 subunits. We also raised an antibody against a peptide sequence unique to the rho1 subunit. The use of this antibody on cerebellum revealed the rat rho1 subunit in the soma and dendrites of Purkinje neurons. The allocation of GABA(C) receptor subunits to identified neurons paves the way for future electrophysiological studies.

  10. Control of cortical neuronal migration by glutamate and GABA.

    PubMed

    Luhmann, Heiko J; Fukuda, A; Kilb, W

    2015-01-01

    Neuronal migration in the cortex is controlled by the paracrine action of the classical neurotransmitters glutamate and GABA. Glutamate controls radial migration of pyramidal neurons by acting primarily on NMDA receptors and regulates tangential migration of inhibitory interneurons by activating non-NMDA and NMDA receptors. GABA, acting on ionotropic GABAA-rho and GABAA receptors, has a dichotomic action on radially migrating neurons by acting as a GO signal in lower layers and as a STOP signal in upper cortical plate (CP), respectively. Metabotropic GABAB receptors promote radial migration into the CP and tangential migration of interneurons. Besides GABA, the endogenous GABAergic agonist taurine is a relevant agonist controlling radial migration. To a smaller extent glycine receptor activation can also influence radial and tangential migration. Activation of glutamate and GABA receptors causes increases in intracellular Ca(2+) transients, which promote neuronal migration by acting on the cytoskeleton. Pharmacological or genetic manipulation of glutamate or GABA receptors during early corticogenesis induce heterotopic cell clusters in upper layers and loss of cortical lamination, i.e., neuronal migration disorders which can be associated with neurological or neuropsychiatric diseases. The pivotal role of NMDA and ionotropic GABA receptors in cortical neuronal migration is of major clinical relevance, since a number of drugs acting on these receptors (e.g., anti-epileptics, anesthetics, alcohol) may disturb the normal migration pattern when present during early corticogenesis.

  11. Regionally specific human GABA concentration correlates with tactile discrimination thresholds.

    PubMed

    Puts, Nicolaas A J; Edden, Richard A E; Evans, C John; McGlone, Francis; McGonigle, David J

    2011-11-16

    The neural mechanisms underlying variability in human sensory perception remain incompletely understood. In particular, few studies have attempted to investigate the relationship between in vivo measurements of neurochemistry and individuals' behavioral performance. Our previous work found a relationship between GABA concentration in the visual cortex and orientation discrimination thresholds (Edden et al., 2009). In the present study, we used magnetic resonance spectroscopy of GABA and psychophysical testing of vibrotactile frequency thresholds to investigate whether individual differences in tactile frequency discrimination performance are correlated with GABA concentration in sensorimotor cortex. Behaviorally, individuals showed a wide range of discrimination thresholds ranging from 3 to 7.6 Hz around the 25 Hz standard. These frequency discrimination thresholds were significantly correlated with GABA concentration (r = -0.58; p < 0.05) in individuals' sensorimotor cortex, but not with GABA concentration in an occipital control region (r = -0.04). These results demonstrate a link between GABA concentration and frequency discrimination in vivo, and support the hypothesis that GABAergic mechanisms have an important role to play in sensory discrimination.

  12. GABA-independent GABAA Receptor Openings Maintain Tonic Currents

    PubMed Central

    Wlodarczyk, Agnieszka I.; Sylantyev, Sergiy; Herd, Murray B.; Kersanté, Flavie; Lambert, Jeremy J.; Rusakov, Dmitri A.; Linthorst, Astrid C.E.; Semyanov, Alexey; Belelli, Delia; Pavlov, Ivan; Walker, Matthew C.

    2013-01-01

    Activation of GABAA receptors (GABAARs) produces two forms of inhibition: ‘phasic’ inhibition generated by the rapid, transient activation of synaptic GABAARs by presynaptic GABA release, and tonic inhibition generated by the persistent activation of peri- or extrasynaptic GABAARs which can detect extracellular GABA. Such tonic GABAAR-mediated currents are particularly evident in dentate granule cells in which they play a major role in regulating cell excitability. Here we show that in rat dentate granule cells in ex-vivo hippocampal slices, tonic currents are predominantly generated by GABA-independent GABAA receptor openings. This tonic GABAAR conductance is resistant to the competitive GABAAR antagonist SR95531, which at high concentrations acts as a partial agonist, but can be blocked by an open channel blocker picrotoxin. When slices are perfused with 200 nM GABA, a concentration that is comparable to cerebrospinal fluid concentrations but is twice that measured by us in the hippocampus in vivo using zero-net-flux microdialysis, negligible GABA is detected by dentate granule cells. Spontaneously opening GABAARs, therefore, maintain dentate granule cell tonic currents in the face of low extracellular GABA concentrations. PMID:23447601

  13. Control of cortical neuronal migration by glutamate and GABA

    PubMed Central

    Luhmann, Heiko J.; Fukuda, A.; Kilb, W.

    2015-01-01

    Neuronal migration in the cortex is controlled by the paracrine action of the classical neurotransmitters glutamate and GABA. Glutamate controls radial migration of pyramidal neurons by acting primarily on NMDA receptors and regulates tangential migration of inhibitory interneurons by activating non-NMDA and NMDA receptors. GABA, acting on ionotropic GABAA-rho and GABAA receptors, has a dichotomic action on radially migrating neurons by acting as a GO signal in lower layers and as a STOP signal in upper cortical plate (CP), respectively. Metabotropic GABAB receptors promote radial migration into the CP and tangential migration of interneurons. Besides GABA, the endogenous GABAergic agonist taurine is a relevant agonist controlling radial migration. To a smaller extent glycine receptor activation can also influence radial and tangential migration. Activation of glutamate and GABA receptors causes increases in intracellular Ca2+ transients, which promote neuronal migration by acting on the cytoskeleton. Pharmacological or genetic manipulation of glutamate or GABA receptors during early corticogenesis induce heterotopic cell clusters in upper layers and loss of cortical lamination, i.e., neuronal migration disorders which can be associated with neurological or neuropsychiatric diseases. The pivotal role of NMDA and ionotropic GABA receptors in cortical neuronal migration is of major clinical relevance, since a number of drugs acting on these receptors (e.g., anti-epileptics, anesthetics, alcohol) may disturb the normal migration pattern when present during early corticogenesis. PMID:25688185

  14. Pharmacological characterisation of a cell line expressing GABA B1b and GABA B2 receptor subunits.

    PubMed

    Hirst, Warren D; Babbs, Adam J; Green, Andrew; Minton, Jayne A L; Shaw, Tracy E; Wise, Alan; Rice, Simon Q; Pangalos, Menelas N; Price, Gary W

    2003-04-01

    The gamma-aminobutyric acid (GABA(B)) receptor has been shown to be a heterodimer consisting of two receptor subunits, GABA(B1) and GABA(B2). We have stably co-expressed these two subunits in a CHO cell line, characterised its pharmacology and compared it to the native receptor in rat brain membranes. Radioligand binding using [3H]CGP54626A demonstrated a similar rank order of potency between recombinant and native receptors: CGP62349>CGP54626A>SCH 50911>3-aminopropylphosphinicacid(3-APPA)>GABA>baclofen>saclofen>phaclofen. However, differences were observed in the affinity of agonists, which were higher at the native receptor, suggesting that in the recombinant system a large number of the receptors were in the low agonist affinity state. In contrast, [35S]GTPgammaS binding studies did not show any differences between recombinant and native receptors with the full agonists GABA and 3-APPA. Measurement of cAMP accumulation in the cells revealed a degree of endogenous coupling of the receptors to G-proteins. This is most likely to be due to the high expression levels of receptors (B(max)=22.5+/-2.5pmol/mg protein) in this experimental system. There was no evidence of GABA(B2) receptors, when expressed alone, binding [3H]CGP54626A, [3H]GABA, [3H]3-APPA nor of GABA having any effect on basal [35S]GTPgammaS binding or cAMP levels.

  15. GABA receptors, alcohol dependence and criminal behavior.

    PubMed

    Terranova, Claudio; Tucci, Marianna; Sartore, Daniela; Cavarzeran, Fabiano; Di Pietra, Laura; Barzon, Luisa; Palù, Giorgio; Ferrara, Santo D

    2013-09-01

    The aim of this study was to analyze the connection between alcohol dependence and criminal behavior by an integrated genetic-environmental approach. The research, structured as a case-control study, examined 186 alcohol-dependent males; group 1 (N = 47 convicted subjects) was compared with group 2 (N = 139 no previous criminal records). Genetic results were innovative, highlighting differences in genotype distribution (p = 0.0067) in group 1 for single-nucleotide polymorphism rs 3780428, located in the intronic region of subunit 2 of the GABA B receptor gene (GABBR2). Some environmental factors (e.g., grade repetition) were associated with criminal behavior; others (e.g., attendance at Alcoholics Anonymous) were inversely related to convictions. The concomitant presence of the genetic and environmental factors found to be associated with the condition of alcohol-dependent inmate showed a 4-fold increase in the risk of antisocial behavior. The results need to be replicated on a larger population to develop new preventive and therapeutic proposals.

  16. Involvement of noradrenaline in potentiation of the head-twitch response by GABA-related drugs.

    PubMed

    Singh, L; Heaton, J C; Rea, P J; Handley, S L

    1986-01-01

    The involvement of noradrenaline in the potentiation of head-twitching by drugs acting at GABA and related receptors has been examined in mice. The direct 5-HT agonist 5-methoxy-N,N-dimethyltryptamine was used to induce the head-twitch. The dopamine beta-oxidase inhibitor FLA-63 depleted whole brain noradrenaline by 90% and potentiated head-twitching when this was measured 4 h after injection. The GABAA-receptor agonists muscimol and 3-aminopropanesulphonic acid (3-APS), a low dose of pentobarbitone, diazepam and picrotoxin (PTX) potentiated control head-twitch rates but had no further effect when head-twitching had been potentiated by FLA-63. The potentiating action of FLA-63 was prevented by the beta-adrenoceptor antagonist metoprolol; the latter having no effect on control head-twitch rates. Muscimol and diazepam potentiation was examined and found to be blocked by metoprolol. The GABAB-receptor agonist baclofen reduced head-twitching. This was also prevented by FLA-63. The role of beta-adrenoceptors in modulating these actions of GABA-related drugs is discussed.

  17. Role of GABA-active neurosteroids in the efficacy of metyrapone against cocaine addiction.

    PubMed

    Schmoutz, Christopher D; Guerin, Glenn F; Goeders, Nicholas E

    2014-09-01

    Previous research has demonstrated a complicated role for stress and HPA axis activation in potentiating various cocaine-related behaviors in preclinical models of drug dependence. However, the investigation of several antiglucocorticoid therapies has yielded equivocal results in reducing cocaine-related behaviors, possibly because of varying mechanisms of actions. Specifically, research suggests that metyrapone (a corticosterone synthesis inhibitor) may reduce cocaine self-administration in rats via a nongenomic, extra-adrenal mechanism without altering plasma corticosterone. In the current experiments, male rats were trained to self-administer cocaine infusions and food pellets in a multiple, alternating schedule of reinforcement. Metyrapone pretreatment dose-dependently decreased cocaine self-administration as demonstrated previously. Pharmacological inhibition of neurosteroid production by finasteride had significant effects on cocaine self-administration, regardless of metyrapone pretreatment. However, metyrapone's effects on cocaine self-administration were significantly attenuated with bicuculline pretreatment, suggesting a role for GABA-active neurosteroids in cocaine-reinforced behaviors. In vitro binding data also confirmed that metyrapone does not selectively bind to GABA-related proteins. The results of these experiments support the hypothesis that metyrapone may increase neurosteroidogenesis to produce effects on cocaine-related behaviors.

  18. Gamma-vinyl GABA inhibits methamphetamine, heroin, or ethanol-induced increases in nucleus accumbens dopamine.

    PubMed

    Gerasimov, M R; Ashby, C R; Gardner, E L; Mills, M J; Brodie, J D; Dewey, S L

    1999-10-01

    We examined the acute effect of the irreversible GABA-transaminase inhibitor, gamma-vinyl GABA (GVG, Sabril((R)), Vigabatrin((R))) on increases in nucleus accumbens (NAc) dopamine (DA) following acute administration of methamphetamine, heroin, or ethanol. Methamphetamine (2.5 mg/kg) produced a dose-dependent increase (2, 700%) in NAc DA. GVG preadministration (300 or 600 mg/kg), however, inhibited this response by approximately 39 and 61%, respectively. The lower dose of methamphetamine (1.25 mg/kg), increased DA by 1, 700%. This response was inhibited to a similar extent (44%) regardless of the GVG dose preadministered (300 or 600 mg/kg). In addition, heroin-induced increases in NAc DA (0.5 mg/kg, 170%) were inhibited or completely abolished by GVG (150 or 300 mg/kg, 65 and 100%, respectively). Finally, at half the dose necessary for heroin, GVG (150 mg/kg) also completely abolished ethanol-induced increases in NAc DA following a 0.25 g/kg challenge dose (140%). Taken with our previous findings using nicotine or cocaine as the challenge drug, these results indicate that GVG attenuates increases in NAc DA by a mechanism common to many drugs of abuse. However, it appears unlikely that an acute dose of GVG can completely inhibit increases in NAc DA following challenges with a drug whose mechanism of action is mediated primarily through the DA reuptake site.

  19. Saturable binding of /sup 35/S-t-butylbicyclophosphorothionate to the sites linked to the GABA receptor and the interaction with gabaergic agents

    SciTech Connect

    Wong, D.T.; Threlkeld, P.G.; Bymaster, F.P.; Squires, R.F.

    1984-02-27

    /sup 35/S-t-Butylbicyclophosphorothionate (/sup 35/S-TBPS) binds in a concentration-saturable manner to specific sites on membranes from rat cerebral cortex. Using a filtration assay at 25/sup 0/C, in 250 mM NaCl, specific binding of /sup 35/S-TBPS constitutes about 84 to 94 percent of total binding, depending on radioligand concentrations. /sup 35/S-TBPS binding is optimal in the presence of NaCl or NaBr and substantially less in the presence of NaI or NaF. It is sensitive to the treatment with 0.05 percent Triton X-100 but not to repeated freezing and thawing, procedures which increase /sup 3/H-GABA binding. Pharmacological studies show that /sup 35/S-TBPS binding is strongly inhibited by GABA-A receptor agonists (e.g., GABA and muscimol) and by the noncompetitive antagonist, picrotoxin, but not the competitive antagonist, bicuculline. Compounds which enhance binding of radioactive GABA and benzodiazepines, such as the pyrazolopyridines, cartazolate and tracazolate, and a diaryltriazine, LY81067, are also potent inhibitors of /sup 35/S-TBPS binding, with LY81067 being the most effective. The effects of GABA, picrotoxin and LY81067 on the saturable binding of /sup 35/S-TBPS in cortical membranes are compared. The present findings are consistent with the interpretation that /sup 35/S-TBPS bind at or near the picrotoxin-sensitive anion recognition sites of the GABA/benzodiazepine/picrotoxin receptor complex.

  20. Potentiation of the ionotropic GABA receptor response by whiskey fragrance.

    PubMed

    Hossain, Sheikh Julfikar; Aoshima, Hitoshi; Koda, Hirofumi; Kiso, Yoshinobu

    2002-11-06

    It is well-known that the target of most mood-defining compounds is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activity in the human brain. To study the effects of whiskey fragrance on the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting rat whole brain mRNA or cRNA prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors. Most whiskey components such as phenol, ethoxy, and lactone derivatives potentiated the electrical responses of GABA(A) receptors, especially ethyl phenylpropanoate (EPP), which strongly potentiated the response. When this compound was applied to mice through respiration, the convulsions induced by pentetrazole were delayed, suggesting that EPP was absorbed by the brain, where it could potentiate the GABA(A) receptor responses. The extract of other alcoholic drinks such as wine, sake, brandy, and shochu also potentiated the responses to varying degrees. Although these fragrant components are present in alcoholic drinks at low concentrations (extremely small quantities compared with ethanol), they may also modulate the mood or consciousness of the human through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic fragrant compounds are easily absorbed into the brain through the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response.

  1. Analysis of GABA(A)- and GABA(B)-receptor mediated effects on intracellular Ca(2+) in DRG hybrid neurones.

    PubMed

    Yokogawa, T; Kim, S U; Krieger, C; Puil, E

    2001-09-01

    1. Using pharmacological analysis and fura-2 spectrofluorimetry, we examined the effects of gamma-aminobutyric acid (GABA) and related substances on intracellular Ca(2+) concentration ([Ca(2+)]i) of hybrid neurones, called MD3 cells. The cell line was produced by fusion between a mouse neuroblastoma cell and a mouse dorsal root ganglion (DRG) neurone. 2. MD3 cells exhibited DRG neurone-like properties, such as immunoreactivity to microtubule-associated protein-2 and neurofilament proteins. Bath applications of capsaicin and alpha, beta-methylene adenosine triphosphate reversibly increased [Ca(2+)]i. However, repeated applications of capsaicin were much less effective. 3. Pressure applications of GABA (100 microM), (Z)-3-[(aminoiminomethyl) thio] prop-2-enoic acid sulphate (ZAPA; 100 microM), an agonist at low affinity GABA(A)-receptors, or KCl (25 mM), transiently increased [Ca(2+)]i. 4. Bath application of bicuculline (100 nM - 100 microM), but not picrotoxinin (10 - 25 microM), antagonized GABA-induced increases in [Ca(2+)]i in a concentration-dependent manner (IC(50)=9.3 microM). 5. Ca(2+)-free perfusion reversibly abolished GABA-evoked increases in [Ca(2+)]i. Nifedipine and nimodipine eliminated GABA-evoked increases in [Ca(2+)]i. These results imply GABA response dependence on extracellular Ca(2+). 6. Baclofen (500 nM - 100 microM) activation of GABA(B)-receptors reversibly attenuated KCl-induced increases in [Ca(2+)]i in a concentration-dependent manner (EC(50)=1.8 microM). 2-hydroxy-saclofen (1 - 20 microM) antagonized the baclofen-depression of the KCl-induced increase in [Ca(2+)]i. 7. In conclusion, GABA(A)-receptor activation had effects similar to depolarization by high external K(+), initiating Ca(2+) influx through high voltage-activated channels, thereby transiently elevating [Ca(2+)]i. GABA(B)-receptor activation reduced Ca(2+) influx evoked by depolarization, possibly at Ca(2+)-channel sites in MD3 cells.

  2. Snake neurotoxin α-bungarotoxin is an antagonist at native GABA(A) receptors.

    PubMed

    Hannan, Saad; Mortensen, Martin; Smart, Trevor G

    2015-06-01

    The snake neurotoxin α-bungarotoxin (α-Bgtx) is a competitive antagonist at nicotinic acetylcholine receptors (nAChRs) and is widely used to study their function and cell-surface expression. Increasingly, α-Bgtx is also used as an imaging tool for fluorophore-labelling studies, and given the structural conservation within the pentameric ligand-gated ion channel family, we assessed whether α-Bgtx could bind to recombinant and native γ-aminobutyric type-A receptors (GABAARs). Applying fluorophore-linked α-Bgtx to recombinant αxβ1/2γ2 GABAARs expressed in HEK-293 cells enabled clear cell-surface labelling of α2β1/2γ2 contrasting with the weaker staining of α1/4β1/2γ2, and no labelling for α3/5/6β1/2γ2. The labelling of α2β2γ2 was abolished by bicuculline, a competitive antagonist at GABAARs, and by d-tubocurarine (d-Tc), which acts in a similar manner at nAChRs and GABAARs. Labelling by α-Bgtx was also reduced by GABA, suggesting that the GABA binding site at the receptor β-α subunit interface forms part of the α-Bgtx binding site. Using whole-cell recording, high concentrations of α-Bgtx (20 μM) inhibited GABA-activated currents at all αxβ2γ2 receptors examined, but at lower concentrations (5 μM), α-Bgtx was selective for α2β2γ2. Using α-Bgtx, at low concentrations, permitted the selective inhibition of α2 subunit-containing GABAARs in hippocampal dentate gyrus granule cells, reducing synaptic current amplitudes without affecting the GABA-mediated tonic current. In conclusion, α-Bgtx can act as an inhibitor at recombinant and native GABAARs and may be used as a selective tool to inhibit phasic but not tonic currents in the hippocampus.

  3. Ethanol and acetaldehyde induce similar changes in extracellular levels of glutamate, taurine and GABA in rat anterior cingulate cortex.

    PubMed

    Zuo, Gong Cheng; Yang, Jing Yu; Hao, Yue; Dong, Ying Xu; Wu, Chun Fu

    2007-03-30

    It is controversial regarding to the roles of acetaldehyde and ethanol in the central nervous system. In the present study, the effects of acetaldehyde and ethanol on extracellular levels of glutamate, taurine and GABA in the anterior cingulate cortex (ACC) of freely moving rats were investigated by using the microdialysis technique coupled to high performance liquid chromatography (HPLC) with fluorescent detection. The result showed that glutamate levels were significantly decreased after acute administration of acetaldehyde (AcH, 20 and 100 mg/kg, i.p.), while taurine levels were significantly increased after the higher dose of acetaldehyde (100 mg/kg, i.p.). GABA levels had no changes at any doses of acetaldehyde tested. Interestingly, similar changes of these amino acids were induced by ethanol (EtOH, 3 g/kg, i.p.) when sodium azide (NaN3, 10 mg/kg, i.p.), a catalase inhibitor that can reduce brain ethanol metabolism, was used simultaneously. These findings suggest that acetaldehyde and ethanol have the similar effects on the extracellular output of glutamate, taurine and GABA in the ACC.

  4. Production of gaba (γ – Aminobutyric acid) by microorganisms: a review

    PubMed Central

    Dhakal, Radhika; Bajpai, Vivek K.; Baek, Kwang-Hyun

    2012-01-01

    GABA (γ-aminobutyric acid) is a four carbon non-protein amino acid that is widely distributed in plants, animals and microorganisms. As a metabolic product of plants and microorganisms produced by the decarboxylation of glutamic acid, GABA functions as an inhibitory neurotransmitter in the brain that directly affects the personality and the stress management. A wide range of traditional foods produced by microbial fermentation contain GABA, in which GABA is safe and eco-friendly, and also has the possibility of providing new health-benefited products enriched with GABA. Synthesis of GABA is catalyzed by glutamate decarboxylase, therefore, the optimal fermentation condition is mainly based on the biochemical properties of the enzyme. Major GABA producing microorganisms are lactic acid bacteria (LAB), which make food spoilage pathogens unable to grow and act as probiotics in the gastrointestinal tract. The major factors affecting the production of GABA by microbial fermentation are temperature, pH, fermentation time and different media additives, therefore, these factors are summarized to provide the most up-dated information for effective GABA synthesis. There has been a huge accumulation of knowledge on GABA application for human health accompanying with a demand on natural GABA supply. Only the GABA production by microorganisms can fulfill the demand with GABA-enriched health beneficial foods. PMID:24031948

  5. Investigating GABA and its function in platelets as compared to neurons.

    PubMed

    Kaneez, Fatima Shad; Saeed, Sheikh Arshad

    2009-08-01

    We have recently suggested that platelets could be used as a model for neuronal receptors. In this paper we have investigated gamma-aminobutyric acid (GABA) metabolism and GABA receptors in platelets and in cultured neurons to see whether platelets' GABA mimics neuronal GABA receptor activities. We used the ELISA technique for detecting the GABA concentration in platelet rich plasma and cultured neurons. The functional effects of GABA and its receptor ligands on platelets were determined using an aggregometer. We found that the GABA concentration is 30% lower in platelets than in neurons and in both preparations GABA was metabolized by GABA transaminase (GABA-T). GABA potentiated calcium dependent platelet aggregation with a higher value in washed platelets suspension (WPS) then in platelet rich plasma (PRP). This effect was inhibited by benzodiazepines, calcium channel blockers and the selective phosphoinositide 3-kinase antagonist Wortmannin. GABA neurotransmission is involved in most aspects of normal brain function and can be perturbed in many neuropathologic conditions. We concluded that platelets could be further developed to be used as a peripheral model to study neuronal GABAergic function and its abnormality in diseases such as epilepsy and schizophrenia. Furthermore our results indicated that PI3-kinase is involved in calcium dependent GABA induced platelet aggregation as this synergistic effect is inhibited by Wortmannin in dose dependent manner.

  6. Marlin-1, a novel RNA-binding protein associates with GABA receptors.

    PubMed

    Couve, Andrés; Restituito, Sophie; Brandon, Julia M; Charles, Kelly J; Bawagan, Hinayana; Freeman, Katie B; Pangalos, Menelas N; Calver, Andrew R; Moss, Stephen J

    2004-04-02

    GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Whereas heterodimerization between GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits is essential for functional expression, how neurons coordinate the assembly of these critical receptors remains to be established. Here we have identified Marlin-1, a novel GABA(B) receptor-binding protein that associates specifically with the GABA(B)R1 subunit in yeast, tissue culture cells, and neurons. Marlin-1 is expressed in the brain and exhibits a granular distribution in cultured hippocampal neurons. Marlin-1 binds different RNA species including the 3'-untranslated regions of both the GABA(B)R1 and GABA(B)R2 mRNAs in vitro and also associates with RNA in cultured neurons. Inhibition of Marlin-1 expression via small RNA interference technology results in enhanced intracellular levels of the GABA(B)R2 receptor subunit without affecting the level of GABA(B)R1. Together our results suggest that Marlin-1 functions to regulate the cellular levels of GABA(B) R2 subunits, which may have significant effects on the production of functional GABA(B) receptor heterodimers. Therefore, our observations provide an added level of regulation for the control of GABA(B) receptor expression and for the efficacy of inhibitory synaptic transmission.

  7. Nicotinamide ribosyl uptake mutants in Haemophilus influenzae.

    PubMed

    Herbert, Mark; Sauer, Elizabeta; Smethurst, Graeme; Kraiss, Anita; Hilpert, Anna-Karina; Reidl, Joachim

    2003-09-01

    The gene for the nicotinamide riboside (NR) transporter (pnuC) was identified in Haemophilus influenzae. A pnuC mutant had only residual NR uptake and could survive in vitro with high concentrations of NR, but could not survive in vivo. PnuC may represent a target for the development of inhibitors for preventing H. influenzae disease.

  8. Ethanol increases GABA release in the embryonic avian retina.

    PubMed

    Pohl-Guimarães, Fernanda; Calaza, Karin da Costa; Yamasaki, Edna Nanami; Kubrusly, Regina Célia Cussa; Reis, Ricardo Augusto de Melo

    2010-04-01

    Several mechanisms underlying ethanol action in GABAergic synapses have been proposed, one of these mechanisms is on GABA release. Here, we report that in ovo exposure to ethanol induces an increase on GABA release in the embryonic chick retina. Eleven-day-old chick embryos (E11) received an injection of either phosphate buffer saline (PBS) or ethanol (10%, v/v, diluted in PBS), and were allowed to develop until E16. A single glutamate stimulus (2 mM) showed approximately a 40% increase on GABA release in E16 retinas when compared to controls. The effect was dependent on NMDA receptors and GAD65 mRNA levels, which were increased following the ethanol treatment. However, the numbers of GABA-, GAD-, and NR1-immunoreactive cells, and the expression levels of these proteins, were not affected. We conclude that ethanol treatment at a time point when synapses are being formed during development selectively increases GABA release in the retina via a NMDA receptor-dependent process.

  9. Spinal GABA-B receptor modulates neutrophil recruitment to the knee joint in zymosan-induced arthritis.

    PubMed

    Bassi, Gabriel S; do C Malvar, David; Cunha, Thiago M; Cunha, Fernando Q; Kanashiro, Alexandre

    2016-08-01

    Recent studies have demonstrated that the central nervous system controls inflammatory responses by activating complex efferent neuroimmune pathways. The present study was designed to evaluate the role that central gamma-aminobutyric acid type B (GABA-B) receptor plays in neutrophil migration in a murine model of zymosan-induced arthritis by using different pharmacological tools. We observed that intrathecal administration of baclofen, a selective GABA-B agonist, exacerbated the inflammatory response in the knee after zymosan administration characterized by an increase in the neutrophil recruitment and knee joint edema, whereas saclofen, a GABA-B antagonist, exerted the opposite effect. Intrathecal pretreatment of the animals with SB203580 (an inhibitor of p38 mitogen-activated protein kinase) blocked the pro-inflammatory effect of baclofen. On the other hand, systemic administration of guanethidine, a sympatholytic drug that inhibits catecholamine release, and nadolol, a beta-adrenergic receptor antagonist, reversed the effect of saclofen. Moreover, saclofen suppressed the release of the pro-inflammatory cytokines into the knee joint (ELISA) and pain-related behaviors (open field test). Since the anti-inflammatory effect of saclofen depends on the sympathetic nervous system integrity, we observed that isoproterenol, a beta-adrenergic receptor agonist, mimics the central GABA-B blockade decreasing knee joint neutrophil recruitment. Together, these results demonstrate that the pharmacological manipulation of spinal GABAergic transmission aids control of neutrophil migration to the inflamed joint by modulating the activation of the knee joint-innervating sympathetic terminal fibers through a mechanism dependent on peripheral beta-adrenergic receptors and central components, such as p38 MAPK.

  10. GABA depolarizes immature neurons and inhibits network activity in the neonatal neocortex in vivo.

    PubMed

    Kirmse, Knut; Kummer, Michael; Kovalchuk, Yury; Witte, Otto W; Garaschuk, Olga; Holthoff, Knut

    2015-07-16

    A large body of evidence from in vitro studies suggests that GABA is depolarizing during early postnatal development. However, the mode of GABA action in the intact developing brain is unknown. Here we examine the in vivo effects of GABA in cells of the upper cortical plate using a combination of electrophysiological and Ca(2+)-imaging techniques. We report that at postnatal days (P) 3-4, GABA depolarizes the majority of immature neurons in the occipital cortex of anaesthetized mice. At the same time, GABA does not efficiently activate voltage-gated Ca(2+) channels and fails to induce action potential firing. Blocking GABA(A) receptors disinhibits spontaneous network activity, whereas allosteric activation of GABA(A) receptors has the opposite effect. In summary, our data provide evidence that in vivo GABA acts as a depolarizing neurotransmitter imposing an inhibitory control on network activity in the neonatal (P3-4) neocortex.

  11. Refuting the challenges of the developmental shift of polarity of GABA actions: GABA more exciting than ever!

    PubMed Central

    Ben-Ari, Yehezkel; Woodin, Melanie A.; Sernagor, Evelyne; Cancedda, Laura; Vinay, Laurent; Rivera, Claudio; Legendre, Pascal; Luhmann, Heiko J.; Bordey, Angelique; Wenner, Peter; Fukuda, Atsuo; van den Pol, Anthony N.; Gaiarsa, Jean-Luc; Cherubini, Enrico

    2012-01-01

    During brain development, there is a progressive reduction of intracellular chloride associated with a shift in GABA polarity: GABA depolarizes and occasionally excites immature neurons, subsequently hyperpolarizing them at later stages of development. This sequence, which has been observed in a wide range of animal species, brain structures and preparations, is thought to play an important role in activity-dependent formation and modulation of functional circuits. This sequence has also been considerably reinforced recently with new data pointing to an evolutionary preserved rule. In a recent “Hypothesis and Theory Article,” the excitatory action of GABA in early brain development is suggested to be “an experimental artefact” (Bregestovski and Bernard, 2012). The authors suggest that the excitatory action of GABA is due to an inadequate/insufficient energy supply in glucose-perfused slices and/or to the damage produced by the slicing procedure. However, these observations have been repeatedly contradicted by many groups and are inconsistent with a large body of evidence including the fact that the developmental shift is neither restricted to slices nor to rodents. We summarize the overwhelming evidence in support of both excitatory GABA during development, and the implications this has in developmental neurobiology. PMID:22973192

  12. Refuting the challenges of the developmental shift of polarity of GABA actions: GABA more exciting than ever!

    PubMed

    Ben-Ari, Yehezkel; Woodin, Melanie A; Sernagor, Evelyne; Cancedda, Laura; Vinay, Laurent; Rivera, Claudio; Legendre, Pascal; Luhmann, Heiko J; Bordey, Angelique; Wenner, Peter; Fukuda, Atsuo; van den Pol, Anthony N; Gaiarsa, Jean-Luc; Cherubini, Enrico

    2012-01-01

    DURING BRAIN DEVELOPMENT, THERE IS A PROGRESSIVE REDUCTION OF INTRACELLULAR CHLORIDE ASSOCIATED WITH A SHIFT IN GABA POLARITY: GABA depolarizes and occasionally excites immature neurons, subsequently hyperpolarizing them at later stages of development. This sequence, which has been observed in a wide range of animal species, brain structures and preparations, is thought to play an important role in activity-dependent formation and modulation of functional circuits. This sequence has also been considerably reinforced recently with new data pointing to an evolutionary preserved rule. In a recent "Hypothesis and Theory Article," the excitatory action of GABA in early brain development is suggested to be "an experimental artefact" (Bregestovski and Bernard, 2012). The authors suggest that the excitatory action of GABA is due to an inadequate/insufficient energy supply in glucose-perfused slices and/or to the damage produced by the slicing procedure. However, these observations have been repeatedly contradicted by many groups and are inconsistent with a large body of evidence including the fact that the developmental shift is neither restricted to slices nor to rodents. We summarize the overwhelming evidence in support of both excitatory GABA during development, and the implications this has in developmental neurobiology.

  13. Regulation of Local Ambient GABA Levels via Transporter-Mediated GABA Import and Export for Subliminal Learning.

    PubMed

    Hoshino, Osamu

    2015-06-01

    Perception of supraliminal stimuli might in general be reflected in bursts of action potentials (spikes), and their memory traces could be formed through spike-timing-dependent plasticity (STDP). Memory traces for subliminal stimuli might be formed in a different manner, because subliminal stimulation evokes a fraction (but not a burst) of spikes. Simulations of a cortical neural network model showed that a subliminal stimulus that was too brief (10 msec) to perceive transiently (more than about 500 msec) depolarized stimulus-relevant principal cells and hyperpolarized stimulus-irrelevant principal cells in a subthreshold manner. This led to a small increase or decrease in ongoing-spontaneous spiking activity frequency (less than 1 Hz). Synaptic modification based on STDP during this period effectively enhanced relevant synaptic weights, by which subliminal learning was improved. GABA transporters on GABAergic interneurons modulated local levels of ambient GABA. Ambient GABA molecules acted on extrasynaptic receptors, provided principal cells with tonic inhibitory currents, and contributed to achieving the subthreshold neuronal state. We suggest that ongoing-spontaneous synaptic alteration through STDP following subliminal stimulation may be a possible neuronal mechanism for leaving its memory trace in cortical circuitry. Regulation of local ambient GABA levels by transporter-mediated GABA import and export may be crucial for subliminal learning.

  14. GABA-B receptor activation and conflict behavior

    SciTech Connect

    Ketelaars, C.E.J.; Bollen, E.L.; Rigter, H.; Bruinvels, J.

    1988-01-01

    Baclofen and oxazepam enhance extinction of conflict behavior in the Geller-Seifter test while baclofen and diazepam release punished behavior in Vogel's conflict test. In order to investigate the possibility that the effect of the selective GABA-B receptor agonist baclofen is mediated indirectly via the GABA-A/benzodiazepine receptor complex, the effect of pretreatment of rats with baclofen on (/sup 3/H)-diazepam binding to washed and unwashed cortical and cerebellar membranes of rats has been studied. Baclofen pretreatment increase Bmax in washed cerebellar membranes when bicuculline was present in the incubation mixture. No effect was seen in cortical membranes. The present results render it unlikely that the effect of baclofen on extinction of conflict behavior and punished drinking is mediated via the GABA-A/benzodiazepine receptor complex. 50 references, 1 figure, 4 tables.

  15. Facilitation of amphetamine-induced hypothermia in mice by GABA agonists and CCK-8.

    PubMed

    Boschi, G; Launay, N; Rips, R

    1991-04-01

    1. Amphetamine-induced hypothermia in mice is facilitated by dopaminergic stimulation and 5-hydroxytryptaminergic inhibition. The present study was designed to investigate: (a) the involvement of other neuronal systems, such as the gamma-aminobutyric acid (GABA), the opioid and the cholecystokinin (CCK-8) systems; (b) the possible contribution of hydroxylated metabolites of amphetamine to the hypothermia; (c) the capacity of dopamine itself to induce hypothermia and its mechanisms, in order to clarify the resistance of amphetamine-induced hypothermia to certain neuroleptics. 2. Pretreatment with the GABA antagonists, bicuculline and picrotoxin, did not inhibit amphetamine-induced hypothermia. The GABAB agonist, baclofen (2.5 mg kg-1, i.p.) potentiated this hypothermia, whereas the GABAA agonist, muscimol, did not. gamma-Butyrolactone (GBL) (40 mg kg-1, i.p.) and the neuropeptide CCK-8 (0.04 mg kg-1, i.p.) also induced potentiation. The opioid antagonist, naloxone, was without effect. 3. Dopamine itself (3, 9, 16 and 27 micrograms, i.c.v.) induced less hypothermia than the same doses of amphetamine. Sulpiride did not block dopamine-induced hypothermia, but pimozide (4 mg kg-1, i.p.), cis(z)flupentixol (0.25 mg kg-1, i.p.) and haloperidol (5 micrograms, i.c.v.) did. The direct dopamine receptor agonist, apomorphine, did not alter the hypothermia. Neither the 5-hydroxytryptamine (5-HT) receptor blocker, cyproheptadine, nor the inhibitor of 5-HT synthesis, p-chlorophenylalanine (PCPA), modified dopamine-induced hypothermia. Fluoxetine, an inhibitor of 5-HT reuptake, had no effect, whereas quipazine (6 mg kg-1, i.p.), a 5-HT agonist, totally prevented the hypothermia. Hypothermia was unaffected by pretreatment with CCK-8. 4. These data indicate that the hypothermia induced by amphetamine involves not only dopaminergic and 5-hydroxytryptaminergic systems which are functionally antagonistic, but is also facilitated by direct or indirect GABA and CCK-8 receptor stimulation

  16. GABA excitation in mouse hilar neuropeptide Y neurons

    PubMed Central

    Fu, Li-Ying; van den Pol, Anthony N

    2007-01-01

    Neuropeptide Y-containing interneurons in the dentate hilar area play an important role in inhibiting the activity of hippocampal circuitry. Hilar cells are often among the first lost in hippocampal epilepsy. As many types of neurons are found in the hilus, we used a new transgenic mouse expressing green fluorescent protein (GFP) in a subset of neurons that colocalized neuropeptide Y (NPY), somatostatin (SST), and GABA for whole-cell, perforated, and cell-attached recording in 240 neurons. As these neurons have not previously been identifiable in live slices, they have not been the focus of physiological analysis. Hilar NPY neurons showed modest spike frequency adaptation, a large 15.6 ± 1.0 mV afterhyperpolarization, a mean input resistance of 335 ± 26 mΩ, and were capable of fast-firing. Muscimol-mediated excitatory actions were found in a nominally Ca2+-free/high-Mg2+ bath solution using cell-attached recording. GABAA receptor antagonists inhibited half the recorded neurons and blocked burst firing. Gramicidin perforated-patch recording revealed a GABA reversal potential positive to both the resting membrane potential and spike threshold. Together, these data suggest GABA is excitatory to many NPY cells. NPY and SST consistently hyperpolarized and reduced spike frequency in these neurons. No hyperpolarization of NPY on membrane potential was detected in the presence of tetrodotoxin, AP5, CNQX and bicuculline, supporting an indirect effect. Under similar conditions, SST hyperpolarized the cells, suggesting a direct postsynaptic action. Depolarizing actions of GABA and GABA-dependent burst-firing may synchronize a rapid release of GABA, NPY, and SST, leading to pre- and postsynaptic inhibition of excitatory hippocampal circuits. PMID:17204505

  17. GABA-receptor complex in monkeys treated with MPTP

    SciTech Connect

    Huffman, R.D.; Ticku, M.K.

    1986-03-01

    Tissue samples from the brains of monkeys made parkinsonian by the depletion of dopamine (DA) with dopaminergic neurotoxin (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1.4-3.4 mg/kg, i.v.) were assayed for changed in GABA ((/sup 3/H)-GABA), benzodiazepine ((/sup 3/H)-flunitrazepam) and picrotoxin ((/sup 35/S)-TBPS) binding sites. One point binding assays were performed on globus pallidus (GP), substantia nigra reticulata (SN/sub R/) and VA-VL thalamic samples. GABA binding was markedly increased in the SN/sub R/ (129 +/- 12%, n = 2) and GP (108 +/- 33%, n = 4) and not altered in the striatum or thalamus. However, benzodiazepine binding was increased in the striatum (170%; 257 fm/mg, control; 692 fm/mg, treated) and GP (28%; 317 fm/mg, control, 405 fm/mg, treated) and (/sup 35/S)-TBPS binding was also increased in GP (100%; 32.5 fm/mg, control; 65.5 fm/mg, treated). atScatchard analysis of (/sup 3/H)-GABA binding was also performed on tissue samples of motor cortex, cerebellar vermis and striatum pooled from half brains of 4 parkinsonian and 2 control monkeys. Depletion of DA (92 +/- 5%) in the striatum of these monkeys was not associated with any change in the K/sub D/ or B/sub max/ for the high or low affinity GABA binding sites in the striatum, motor cortex or cerebellum. Thus, in the basal ganglia, DA depletion is associated with an increase in GABA binding sites in GP and SN/sub R/, an increase in picrotoxin binding sites in GP and an increase in benzodiazepine binding sites in the striatum.

  18. Posttraumatic GABA(A)-mediated [Ca2+]i increase is essential for the induction of brain-derived neurotrophic factor-dependent survival of mature central neurons.

    PubMed

    Shulga, Anastasia; Thomas-Crusells, Judith; Sigl, Thomas; Blaesse, Anne; Mestres, Pedro; Meyer, Michael; Yan, Qiao; Kaila, Kai; Saarma, Mart; Rivera, Claudio; Giehl, Klaus M

    2008-07-02

    A shift of GABA(A)-mediated responses from hyperpolarizing to depolarizing after neuronal injury leads to GABA(A)-mediated increase in [Ca2+](i). In addition, central neurons become dependent on BDNF for survival. Whether these two mechanisms are causally interrelated is an open question. Here, we show in lesioned CA3 hippocampal neurons in vitro and in axotomized corticospinal neurons in vivo that posttraumatic downregulation of the neuron-specific K-Cl cotransporter KCC2 leads to intracellular chloride accumulation by the Na-K-2Cl cotransporter NKCC1, resulting in GABA-induced [Ca2+](i) transients. This mechanism is required by a population of neurons to survive in a BDNF-dependent manner after injury, because blocking GABA(A)-depolarization with the NKCC1 inhibitor bumetanide prevents the loss of neurons on BDNF withdrawal. The resurgence of KCC2 expression during recovery coincides with loss of BDNF dependency for survival. This is likely mediated through BDNF itself, because injured neurons reverse their response to this neurotrophin by switching the BDNF-induced downregulation of KCC2 to upregulation.

  19. Attenuation of γ-aminobutyric acid (GABA) transaminase activity contributes to GABA increase in the cerebral cortex of mice exposed to β-cypermethrin.

    PubMed

    Han, Y; Cao, D; Li, X; Zhang, R; Yu, F; Ren, Y; An, L

    2014-03-01

    The current study investigated the γ-aminobutyric acid (GABA) levels and GABA metabolic enzymes (GABA transaminase (GABA(T)) and glutamate decarboxylase (GAD)) activities at 2 and 4 h after treatment, using a high-performance liquid chromatography with ultraviolet detectors and colorimetric assay, in the cerebral cortex of mice treated with 20, 40 or 80 mg/kg β-cypermethrin by a single oral gavage, with corn oil as vehicle control. In addition, GABA protein (4 h after treatment), GABA(T) protein (2 h after treatment) and GABA receptors messenger RNA (mRNA) expression were detected by immunohistochemistry, Western blot and real-time quantitative reverse transcriptase polymerase chain reaction, respectively. β-Cypermethrin (80 mg/kg) significantly increased GABA levels in the cerebral cortex of mice, at both 2 and 4 h after treatment, compared with the control. Also, GABA immunohistochemistry results suggested that the number of positive granules was increased in the cerebral cortex of mice 4 h after exposure to 80 mg/kg β-cypermethrin when compared with the control. Furthermore, the results also showed that GABA(T) activity detected was significantly decreased in the cerebral cortex of mice 2 h after β-cypermethrin administration (40 or 80 mg/kg). No significant changes were found in GAD activity, or the expression of GABA(T) protein and GABAB receptors mRNA, in the cerebral cortex of mice, except that 80 mg/kg β-cypermethrin caused a significant decrease, compared with the vehicle control, in GABAA receptors mRNA expression 4 h after administration. These results suggested that attenuated GABA(T) activity induced by β-cypermethrin contributed to increased GABA levels in the mouse brain. The downregulated GABAA receptors mRNA expression is most likely a downstream event.

  20. [GABA, a key transmitter for fetal brain maturation].

    PubMed

    Ben-Ari, Yehezkel

    2007-01-01

    GABA, the principal inhibitory transmitter excites immature neurons in all animal species studied. This is due to the higher intracellular concentration of chloride at early developmental stages. Excitatory actions of GABA play an important action in brain maturation. Recent observations also suggest an abrupt shift during delivery that exerts a neuro-protective action contributing to reduce the sequels of trauma and anoxic episodes. These observations have important clinical implications in relation to delivery associated insults but also preterm delivery and more generally consumption of agents during gestation.

  1. Induction of rotational behaviour by intranigral baclofen suggests possible GABA-agonist activity.

    PubMed

    Waddington, J L

    1977-10-15

    In rats, unilateral injections of the GABA-derivative baclofen into the zona reticulata of the substantia nigra produced a contralateral rotation that was translated to ipsilateral rotation under the influence of amphetamine. These results mimic those following unilateral elevation of GABA levels in the substantia nigra and suggest that baclofen may have some GABA agonist activity following intracerebral injection.

  2. Edited magnetic resonance spectroscopy detects an age-related decline in brain GABA levels.

    PubMed

    Gao, Fei; Edden, Richard A E; Li, Muwei; Puts, Nicolaas A J; Wang, Guangbin; Liu, Cheng; Zhao, Bin; Wang, Huiquan; Bai, Xue; Zhao, Chen; Wang, Xin; Barker, Peter B

    2013-09-01

    Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. Although measurements of GABA levels in vivo in the human brain using edited proton magnetic resonance spectroscopy ((1)H-MRS) have been established for some time, it is has not been established how regional GABA levels vary with age in the normal human brain. In this study, 49 healthy men and 51 healthy women aged between 20 and 76 years were recruited and J-difference edited spectra were recorded at 3T to determine the effect of age on GABA levels, and to investigate whether there are regional and gender differences in GABA in mesial frontal and parietal regions. Because the signal detected at 3.02 ppm using these experimental parameters is also expected to contain contributions from both macromolecules (MM) and homocarnosine, in this study the signal is labeled GABA+ rather than GABA. Significant negative correlations were observed between age and GABA+ in both regions studied (GABA+/Cr: frontal region, r=-0.68, p<0.001, parietal region, r=-0.54, p<0.001; GABA+/NAA: frontal region, r=-0.58, p<0.001, parietal region, r=-0.49, p<0.001). The decrease in GABA+ with age in the frontal region was more rapid in women than men. Evidence of a measureable decline in GABA is important in considering the neurochemical basis of the cognitive decline that is associated with normal aging.

  3. Comparative Mapping of GABA-Immunoreactive Neurons in the Buccal Ganglia of Nudipleura Molluscs.

    PubMed

    Gunaratne, Charuni A; Katz, Paul S

    2016-04-15

    Phylogenetic comparisons of neurotransmitter distribution are important for understanding the ground plan organization of nervous systems. This study describes the γ-aminobutyric acid (GABA)-immunoreactive (GABA-ir) neurons in the buccal ganglia of six sea slug species (Mollusca, Gastropoda, Euthyneura, Nudipleura). In the nudibranch species, Hermissenda crassicornis, Tritonia diomedea, Tochuina tetraquetra, and Dendronotus iris, the number of GABA-ir neurons was highly consistent. Another nudibranch, Melibe leonina, however, contained approximately half the number of GABA-ir neurons. This may relate to its loss of a radula and its unique feeding behavior. The GABA immunoreactivity in a sister group to the nudibranchs, Pleurobranchaea californica, differed drastically from that of the nudibranchs. Not only did it have significantly more GABA-ir neurons but it also had a unique GABA distribution pattern. Furthermore, unlike the nudibranchs, the Pleurobranchaea GABA distribution was also different from that of other, more distantly related, euopisthobranch and panpulmonate snails and slugs. This suggests that the Pleurobranchaea GABA distribution may be a derived feature, unique to this lineage. The majority of GABA-ir axons and neuropil in the Nudipleura were restricted to the buccal ganglia, commissures, and connectives. However, in Tritonia and Pleurobranchaea, we detected a few GABA-ir fibers in buccal nerves that innervate feeding muscles. Although the specific functions of the GABA-ir neurons in the species in this study are not known, the innervation pattern suggests these neurons may play an integrative or regulatory role in bilaterally coordinated behaviors in the Nudipleura.

  4. Systematic analysis of γ-aminobutyric acid (GABA) metabolism and function in the social amoeba Dictyostelium discoideum.

    PubMed

    Wu, Yuantai; Janetopoulos, Chris

    2013-05-24

    While GABA has been suggested to regulate spore encapsulation in the social amoeba Dictyostelium discoideum, the metabolic profile and other potential functions of GABA during development remain unclear. In this study, we investigated the homeostasis of GABA metabolism by disrupting genes related to GABA metabolism and signaling. Extracellular levels of GABA are tightly regulated during early development, and GABA is generated by the glutamate decarboxylase, GadB, during growth and in early development. However, overexpression of the prespore-specific homologue, GadA, in the presence of GadB reduces production of extracellular GABA. Perturbation of extracellular GABA levels delays the process of aggregation. Cytosolic GABA is degraded by the GABA transaminase, GabT, in the mitochondria. Disruption of a putative vesicular GABA transporter (vGAT) homologue DdvGAT reduces secreted GABA. We identified the GABAB receptor-like family member GrlB as the major GABA receptor during early development, and either disruption or overexpression of GrlB delays aggregation. This delay is likely the result of an abolished pre-starvation response and late expression of several "early" developmental genes. Distinct genes are employed for GABA generation during sporulation. During sporulation, GadA alone is required for generating GABA and DdvGAT is likely responsible for GABA secretion. GrlE but not GrlB is the GABA receptor during late development.

  5. Antihistamine effect on synaptosomal uptake of serotonin, norepinephrine and dopamine

    NASA Technical Reports Server (NTRS)

    Brown, P. A.; Vernikos, J.

    1980-01-01

    A study on the effects of five H1 and H2 antihistamines on the synaptosomal uptake of serotonin (5HT), norepinephrine (NE), and dopamine (DA) is presented. Brain homogenates from female rats were incubated in Krebs-Ringer phosphate buffer solution in the presence of one of three radioactive neurotransmitters, and one of the five antihistamines. Low concentrations of pyrilamine competitively inhibited 5HT uptake, had little effect on NE uptake, and no effect on DA uptake. Promethazine, diphenhydramine, metiamide, and cimetidine had no effect on 5HT or DA uptake at the same concentration. Diphenhydramine had a small inhibitory effect on NE uptake. It is concluded that pyrilamine is a selective and potent competitive inhibitor of 5HT uptake at concentrations between .05 and .5 micromolars.

  6. Dual effect of GABA on the contractile activity of the guinea-pig isolated urinary bladder.

    PubMed

    Maggi, C A; Santicioli, P; Meli, A

    1985-06-01

    The effects of GABA and related substances were examined in isolated detrusor strips from the dome of the guinea-pig urinary bladder. GABA (0.01-1 mM) produced concentration-related phasic contractions of isolated strips from the guinea-pig urinary bladder dome. This effect of GABA was mimicked by homotaurine and muscimol, selective GABAA receptor agonists but not by (+/-)-baclofen, a selective GABAB receptor agonist. A specific cross desensitization was observed between GABA, homotaurine and muscimol but not between (+/-)-baclofen and GABA. GABA (1 mM)-induced contractions were antagonized by picrotoxin, a selective GABAA receptor antagonist. GABA-induced contractions were almost abolished by tetrodotoxin (0.5 microM, TTX) thus indicating their neurogenic origin. In addition GABA-induced contractions were partially antagonized by atropine (to about the same extent as those produced by dimethylphenylpiperazinium (DMPP), a ganglionic stimulant), but were unaffected by hexamethonium (10 microM), phentolamine (0.2 microM) or indomethacin (5 microM). In the presence of GABA the contractile effect of both DMPP (TTX-sensitive) and acetylcholine (ACh, TTX-insensitive) were significantly reduced. Similar findings were obtained with DMPP, i.e. in preparations exposed to this ganglionic stimulant both GABA- and ACh-induced contractions were depressed. Homotaurine but not (+/-)-baclofen mimicked the depressant effect of GABA on DMPP-induced contractions. The depressant effect of GABA on ACh-induced contractions of the guinea-pig urinary bladder was neurogenic in origin, i.e., was not observed in preparations exposed to TTX. These experiments indicate that GABA has a dual effect on the contractile behaviour of the guinea-pig isolated urinary bladder. Recently it has been proposed that endogenous GABA plays a neuromodulatory role in this organ. Our data suggest that in the early phase of neurogenic activation of detrusor muscle (micturition reflex) GABA might transiently

  7. Benzodiazepine-dependent stabilization of GABA(A) receptors at synapses.

    PubMed

    Gouzer, Géraldine; Specht, Christian G; Allain, Laure; Shinoe, Toru; Triller, Antoine

    2014-11-01

    GABA(A) receptors constitutively enter and exit synapses by lateral diffusion in the plane of the neuronal membrane. They are trapped at synapses through their interactions with gephyrin, the main scaffolding protein at inhibitory post-synaptic densities. Previous work has shown that the synaptic accumulation and diffusion dynamics of GABA(A)Rs are controlled via excitatory synaptic activity. However, it remains unknown whether GABA(A)R activity can itself impact the surface trafficking of the receptors. Here we report the effects of GABA(A)R agonists, antagonists and allosteric modulators on the receptor's surface dynamics. Using immunocytochemistry and single particle tracking experiments on mouse hippocampal neurons, we show that the agonist muscimol decreases GABA(A)R and gephyrin levels at synapses and accelerates the receptor's lateral diffusion within 30–120 min of treatment. In contrast, the GABA(A)R antagonist gabazine increased GABA(A)R amounts and slowed down GABA(A)R diffusion at synapses. The response to GABA(A)R activation or inhibition appears to be an adaptative regulation of GABAergic synapses. Surprisingly, the positive allosteric modulator diazepam abolished the regulation induced by muscimol, and this effect was observed on α1, α2, α5 and γ2 GABA(A)R subunits. Altogether these results indicate that diazepam stabilizes synaptic GABA(A)Rs and thus prevents the agonist-induced regulation of GABA(A)R levels at synapses. This occurred independently of neuronal activity and intracellular calcium and involved GABA(A)R–gephyrin interactions, suggesting that the changes in GABA(A)R diffusion depend on conformational changes of the receptor. Our study provides a new molecular mechanism involved in the adaptative response to changes in GABA(A)R activity and benzodiazepine treatments.

  8. Inhibition of GABA-gated chloride channels by 12,14-dichlorodehydroabietic acid in mammalian brain

    PubMed Central

    Nicholson, Russell A; Lees, George; Zheng, Jian; Verdon, Bernard

    1999-01-01

    12,14-dichlorodehydroabietic acid (12,14-Cl2DHA) reduced GABA-stimulated uptake of 36Cl− into mouse brain synaptoneurosomes suggesting inhibition of mammalian GABAA receptor function. 12,14-Cl2DHA did not affect the binding of [3H]-muscimol to brain membranes but displaced specifically bound [3H]-EBOB. The inhibitory effect on [3H]-EBOB binding was not reversible. 12,14-Cl2DHA reduced the availability of [3H]-EBOB binding sites (Bmax) without changing the KD of the radioligand for remaining sites. 12,14-Cl2DHA did not affect the rate of association of [3H]-EBOB with its chloride channel receptor, but increased the initial rate of [3H]-EBOB dissociation. 12,14-Cl2DHA enhanced the incidence of EPSCs when rapidly applied to cultured rat cortical neurones. Longer exposures produced block of IPSCs with marked increases in the frequency of EPSCs and min EPSCs. 12,14-Cl2DHA also irreversibly suppressed chloride currents evoked by pulses of exogenous GABA in these cells. Ultimately, 12,14-Cl2DHA inhibited all synaptic traffic and action currents in current clamped cells indicating that, in contrast to picrotoxinin (which causes paroxysmal bursting), it is not fully selective for the GABAA receptor-chloride channel complex. The depolarizing block seen with 12,14-Cl2DHA in amphotericin-perforated preparations implicates loss of Ca2+ buffering in the polarity change and this may account for inhibition of spontaneous action potentials. Our investigation demonstrates that 12,14-Cl2DHA blocks GABA-dependent chloride entry in mammalian brain and operates as a non-competitive insurmountable GABAA antagonist. The mechanism likely involves either irreversible binding of 12,14-Cl2DHA to the trioxabicyclooctane recognition site or a site that is allosterically coupled to it. We cannot exclude, however, the possibility that 12,14-Cl2DHA causes localized proteolysis or more extensive conformational change within a critical subunit of the chloride channel. PMID:10204999

  9. Role of proline and GABA in sexual reproduction of angiosperms

    PubMed Central

    Biancucci, Marco; Mattioli, Roberto; Forlani, Giuseppe; Funck, Dietmar; Costantino, Paolo; Trovato, Maurizio

    2015-01-01

    Two glutamate derivatives, proline and γ-aminobutyric acid (GABA), appear to play pivotal roles in different aspects of sexual reproduction in angiosperms, although their precise function in plant reproduction and the molecular basis of their action are not yet fully understood. Proline and GABA have long been regarded as pivotal amino acids in pollen vitality and fertility. Proline may constitute up to 70% of the free amino acid pool in pollen grains and it has been recently shown that Arabidopsis mutants affected in the first and rate-limiting step in proline synthesis produce aberrant and infertile pollen grains, indicating that proline synthesis is required for pollen development and fertility. Concerning GABA, a large body of evidence points to this glutamate derivative as a key determinant of post-pollination fertilization. Intriguingly, proline has also been associated with pollination, another aspect of sexual reproduction, since honeybees were reported to show a strong preference for proline-enriched nectars. In this review, we survey current knowledge on the roles of proline and GABA in plant fertility, and discuss future perspectives potentially capable to improve our understanding on the functions of these amino acids in pollen development, pollination, and pollen tube guidance. PMID:26388884

  10. Novel functions of GABA signaling in adult neurogenesis

    PubMed Central

    PONTES, Adalto; ZHANG, Yonggang; HU, Wenhui

    2013-01-01

    Neurotransmitter gamma-aminobutiric acid (GABA) through ionotropic GABAA and metabotropic GABAB receptors plays key roles in modulating the development, plasticity and function of neuronal networks. GABA is inhibitory in mature neurons but excitatory in immature neurons, neuroblasts and neural stem/progenitor cells (NSCs/NPCs). The switch from excitatory to inhibitory occurs following the development of glutamatergic synaptic input and results from the dynamic changes in the expression of Na+/K+/2Cl− co-transporter NKCC1 driving Cl− influx and neuron-specific K+/Cl− co-transporter KCC2 driving Cl− efflux. The developmental transition of KCC2 expression is regulated by Disrupted-in-Schizophrenia 1 (DISC1) and brain-derived neurotrophic factor (BDNF) signaling. The excitatory GABA signaling during early neurogenesis is important to the activity/experience-induced regulation of NSC quiescence, NPC proliferation, neuroblast migration and newborn neuronal maturation/functional integration. The inhibitory GABA signaling allows for the sparse and static functional networking essential for learning/memory development and maintenance. PMID:24285940

  11. Glutamate and GABA modulate dopamine in the pedunculopontine tegmental nucleus.

    PubMed

    Steiniger, Björn; Kretschmer, Beate D

    2003-04-01

    The pedunculopontine tegmental nucleus (PPTg) has an important anatomical position connecting basal ganglia and limbic systems with motor execution structures in the pons and spinal cord. It receives glutamatergic and GABAergic input and has additional reciprocal connections with mesencephalic dopaminergic neurons, suggesting that the PPTg plays a key role in frontostriatal information processing. In vivo microdialysis in freely moving rats, in combination with behavioral analysis, was used in this study to investigate whether the dopaminergic input can be modulated at the level of the PPTg via N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) or GABA(B) receptors. Stimulation of the GABA(B) receptor decreased dopamine release in the PPTg while that of the AMPA and NMDA receptors increased it. A time-related comparison of the effects of NMDA (0.75 and 1 mM) and AMPA (50 and 25 microM) revealed a more long-lasting effect after AMPA stimulation than after NMDA. However, only the infusion of the GABA(B) receptor agonist baclofen (100 and 200 microM) stimulated stereotyped behavior (e.g. sniffing, digging or head movements) and contralateral circling. This study clearly demonstrates that GABAergic as well as glutamatergic terminals in the PPTg are critically involved in the modulation of the dopamine system. Moreover, a decrease in PPTg dopamine via GABA(B) receptor stimulation seems to be behaviorally relevant.

  12. Action of tremorgenic mycotoxins on GABA/sub A/ receptor

    SciTech Connect

    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 (GABA/sub A/) receptor binding and function in rat brain and on binding of a voltage-operated Cl/sup -/ channel in Torpedo electric organ. None of the mycotoxins had significant effect on (/sup 3/H)muscimol or (/sup 3/H)flunitrazepam binding to the GAMA/sup A/ receptor. However, only the four tremorgenic mycotoxins inhibited GABA-induced /sup 36/Cl/sup -/ influx and (/sup 35/S)t-butylbicyclophosphorothionate ((/sup 35/S)TBPS) binding in rate brain membranes, while the nontremorgenic verruculotoxin had no effect. Inhibition of (/sup 35/S)TBPS binding by paspalinine was non-competitive. This suggests that tremorgenic mycotoxins inhibit GABA/sub A/ receptor function by binding close to the receptor's Cl/sup -/ channel. On the voltage-operated Cl/sup -/ channel, only high concentrations of verruculogen and verruculotoxin caused significant inhibition of the channel's binding of (/sup 35/S)TBPS. The data suggest that the tremorgenic action of these mycotoxins may be due in part to their inhibition of GABA/sub A/ receptor function. 21 references, 4 figures, 2 tables.

  13. Cocaine inhibition of GABA(A) current: role of dephosphorylation.

    PubMed

    Ye, Jiang-Hong; Ren, Jun

    2006-01-01

    Acute cocaine toxicity is frequently associated with seizures. The mechanisms underlying the convulsant effect of cocaine are not well understood. Previously, we have shown that cocaine depresses whole-cell current evoked by gamma-aminobutyric acid (GABA) in hippocampal neurons freshly isolated from rats. Cocaine's effect was voltage-independent and concentration-dependent. In the present study, using whole-cell patch-clamp recording on rat neurons freshly isolated from hippocampus, we examined the intracellular mechanisms involved in cocaine's action. Increasing intracellular Ca(2+) concentration ([Ca]i) from 0.01 to 5 microM strongly increased the depressant effect of cocaine. By contrast, 1-[N, O-bis (5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific antagonist of Ca/calmodulin-dependent protein kinase (CaMKII), attenuated or enhanced cocaine's action in different neurons: in three out of nine neurons dialysed with 5 microM KN-62,1 mM cocaine depressed GABA current by only 33%, but in another three out of nine neurons, cocaine depressed GABA current by as much as 83%. Chelerythrine (a specific CaCa(2+)/phospholipid-dependent protein kinase C [PKC] antagonist) had minimal effect on cocaine's action. We suggest that cocaine induces an increase in [Ca]i, which stimulates phosphatase activity and thus leads to dephosphorylation of GABA receptors. This dephosphorylation-mediated disinhibitory action may play a role in cocaine-induced convulsant states.

  14. Ionotropic GABA Receptors and Distal Retinal ON and OFF Responses

    PubMed Central

    Popova, E.

    2014-01-01

    In the vertebrate retina, visual signals are segregated into parallel ON and OFF pathways, which provide information for light increments and decrements. The segregation is first evident at the level of the ON and OFF bipolar cells in distal retina. The activity of large populations of ON and OFF bipolar cells is reflected in the b- and d-waves of the diffuse electroretinogram (ERG). The role of gamma-aminobutyric acid (GABA), acting through ionotropic GABA receptors in shaping the ON and OFF responses in distal retina, is a matter of debate. This review summarized current knowledge about the types of the GABAergic neurons and ionotropic GABA receptors in the retina as well as the effects of GABA and specific GABAA and GABAC receptor antagonists on the activity of the ON and OFF bipolar cells in both nonmammalian and mammalian retina. Special emphasis is put on the effects on b- and d-waves of the ERG as a useful tool for assessment of the overall function of distal retinal ON and OFF channels. The role of GABAergic system in establishing the ON-OFF asymmetry concerning the time course and absolute and relative sensitivity of the ERG responses under different conditions of light adaptation in amphibian retina is also discussed. PMID:25143858

  15. GABA mediated excitation in immature rat CA3 hippocampal neurons.

    PubMed

    Cherubini, E; Rovira, C; Gaiarsa, J L; Corradetti, R; Ben Ari, Y

    1990-01-01

    Intracellular recordings from rat hippocampal neurons in vitro during the first postnatal week revealed the presence of spontaneous giant depolarizing potentials (GDPs). These were generated by the synchronous discharge of a population of neurons. GDPs reversed polarity at -27 and -51 mV when recorded with KCl or K-methylsulphate filled electrodes, respectively. GDPs were blocked by the GABAA receptor antagonist bicuculline (10 microM). Iontophoretic or bath applications of GABA (10-300 microM) in the presence of tetrodotoxin (1 microM), induced a membrane depolarization or in voltage clamp experiments an inward current which reversed polarity at the same potential as GDPs. The response to GABA was blocked in a non-competitive manner by bicuculline (10 microM) and did not desensitize. GABA mediated GDPs were presynaptically modulated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Their frequency was reduced or blocked by NMDA receptor antagonists and by the rather specific non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The frequency of GDPs was enhanced by glycine and D-serine (10-30 microM) in a strychnine insensitive manner. This effect was blocked by AP-5, suggesting that it was mediated by the allosteric modulatory site of the NMDA receptor. These observations suggest that most of the 'excitatory' drive in immature neurons is mediated by GABA acting on GABAA receptors; furthermore excitatory amino acids modulate the release of GABA by a presynaptic action on GABAergic interneurons.

  16. Unsaturated phosphinic analogues of gamma-aminobutyric acid as GABA(C) receptor antagonists.

    PubMed

    Chebib, M; Vandenberg, R J; Froestl, W; Johnston, G A

    1997-06-25

    The phosphinic and methylphosphinic analogues of gamma-aminobutyric acid (GABA) are potent GABA(C) receptor antagonists but are even more potent as GABA(B) receptor agonists. Conformationally restricted unsaturated phosphinic and methylphosphinic analogues of GABA and some potent GABA(B) receptor phosphonoamino acid antagonists were tested on GABA(C) receptors in Xenopus oocytes expressing human retinal rho1 mRNA. 3-Aminopropyl-n-butyl-phosphinic acid (CGP36742), an orally active GABA(B) receptor antagonist, was found to be a moderately potent GABA(C) receptor antagonist (IC50 = 62 microM). The unsaturated methylphosphinic and phosphinic analogues of GABA were competitive antagonists of the GABA(C) receptors, the order of potency being [(E)-3-aminopropen-1-yl]methylphosphinic acid (CGP44530, IC50 = 5.53 microM) > [(E)-3-aminopropen-1-yl]phosphinic acid (CGP38593, IC50 = 7.68 microM) > [(Z)-3-aminopropen-1-yl]methylphosphinic acid (CGP70523, IC50 = 38.94 microM) > [(Z)-3-aminopropen-1-yl]phosphinic acid (CGP70522, IC50 > 100 microM). This order of potency differs from that reported for these compounds as GABA(B) receptor agonists, where the phosphinic acids are more potent than the corresponding methylphosphinic acids.

  17. GAD67-mediated GABA synthesis and signaling regulate inhibitory synaptic innervation in the visual cortex.

    PubMed

    Chattopadhyaya, Bidisha; Di Cristo, Graziella; Wu, Cai Zhi; Knott, Graham; Kuhlman, Sandra; Fu, Yu; Palmiter, Richard D; Huang, Z Josh

    2007-06-21

    The development of GABAergic inhibitory circuits is shaped by neural activity, but the underlying mechanisms are unclear. Here, we demonstrate a novel function of GABA in regulating GABAergic innervation in the adolescent brain, when GABA is mainly known as an inhibitory transmitter. Conditional knockdown of the rate-limiting synthetic enzyme GAD67 in basket interneurons in adolescent visual cortex resulted in cell autonomous deficits in axon branching, perisomatic synapse formation around pyramidal neurons, and complexity of the innervation fields; the same manipulation had little influence on the subsequent maintenance of perisomatic synapses. These effects of GABA deficiency were rescued by suppressing GABA reuptake and by GABA receptor agonists. Germline knockdown of GAD67 but not GAD65 showed similar deficits, suggesting a specific role of GAD67 in the maturation of perisomatic innervation. Since intracellular GABA levels are modulated by neuronal activity, our results implicate GAD67-mediated GABA synthesis in activity-dependent regulation of inhibitory innervation patterns.

  18. The endogenous GABA bioactivity of camel, bovine, goat and human milks.

    PubMed

    Limon, Agenor; Gallegos-Perez, Jose-Luis; Reyes-Ruiz, Jorge M; Aljohi, Mohammad A; Alshanqeeti, Ali S; Miledi, Ricardo

    2014-02-15

    GABA orally administered has several beneficial effects on health, including the regulation of hyperglycaemic states in humans. Those effects are similar to the effects reported for camel milk (CMk); however, it is not known whether compounds with GABAergic activity are present in milk from camels or other species. We determined CMk free-GABA concentration by LS/MS and its bioactivity on human GABA receptors. We found that camel and goat milks have significantly more bioavailable GABA than cow and human milks and are able to activate GABAρ receptors. The relationship between GABA and taurine concentrations suggests that whole camel milk may be more efficient to activate GABAρ1 receptors than goat milk. Because GABAρ receptors are normally found in enteroendocrine cells in the lumen of the digestive tract, these results suggest that GABA in camel and goat milk may participate in GABA-modulated functions of enteroendocrine cells in the GI lumen.

  19. GABA signalling modulates plant growth by directly regulating the activity of plant-specific anion transporters.

    PubMed

    Ramesh, Sunita A; Tyerman, Stephen D; Xu, Bo; Bose, Jayakumar; Kaur, Satwinder; Conn, Vanessa; Domingos, Patricia; Ullah, Sana; Wege, Stefanie; Shabala, Sergey; Feijó, José A; Ryan, Peter R; Gilliham, Matthew; Gillham, Matthew

    2015-07-29

    The non-protein amino acid, gamma-aminobutyric acid (GABA) rapidly accumulates in plant tissues in response to biotic and abiotic stress, and regulates plant growth. Until now it was not known whether GABA exerts its effects in plants through the regulation of carbon metabolism or via an unidentified signalling pathway. Here, we demonstrate that anion flux through plant aluminium-activated malate transporter (ALMT) proteins is activated by anions and negatively regulated by GABA. Site-directed mutagenesis of selected amino acids within ALMT proteins abolishes GABA efficacy but does not alter other transport properties. GABA modulation of ALMT activity results in altered root growth and altered root tolerance to alkaline pH, acid pH and aluminium ions. We propose that GABA exerts its multiple physiological effects in plants via ALMT, including the regulation of pollen tube and root growth, and that GABA can finally be considered a legitimate signalling molecule in both the plant and animal kingdoms.

  20. Local GABA Concentration Predicts Perceptual Improvements After Repetitive Sensory Stimulation in Humans.

    PubMed

    Heba, Stefanie; Puts, Nicolaas A J; Kalisch, Tobias; Glaubitz, Benjamin; Haag, Lauren M; Lenz, Melanie; Dinse, Hubert R; Edden, Richard A E; Tegenthoff, Martin; Schmidt-Wilcke, Tobias

    2016-03-01

    Learning mechanisms are based on synaptic plasticity processes. Numerous studies on synaptic plasticity suggest that the regulation of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) plays a central role maintaining the delicate balance of inhibition and excitation. However, in humans, a link between learning outcome and GABA levels has not been shown so far. Using magnetic resonance spectroscopy of GABA prior to and after repetitive tactile stimulation, we show here that baseline GABA+ levels predict changes in perceptual outcome. Although no net changes in GABA+ are observed, the GABA+ concentration prior to intervention explains almost 60% of the variance in learning outcome. Our data suggest that behavioral effects can be predicted by baseline GABA+ levels, which provide new insights into the role of inhibitory mechanisms during perceptual learning.

  1. Photorelease of GABA with Visible Light Using an Inorganic Caging Group

    PubMed Central

    Rial Verde, Emiliano M.; Zayat, Leonardo; Etchenique, Roberto; Yuste, Rafael

    2008-01-01

    We describe the selective photorelease of γ-amino butyric acid (GABA) with a novel caged-GABA compound that uses a ruthenium complex as photosensor. This compound (“RuBi-GABA”) can be excited with visible wavelengths, providing greater tissue penetration, less photo-toxicity, and faster photorelease kinetics than currently used UV light-sensitive caged compounds. Using pyramidal neurons from neocortical brain slices, we show that RuBi-GABA uncaging induces GABA-A receptor-mediated responses, has no detectable side effects on endogenous GABAergic and glutamatergic receptors and generates responses with kinetics and spatial resolution comparable to the best caged GABA compounds presently available. Finally, we illustrate two potential applications of RuBi-GABA uncaging: GABA receptor mapping, and optical silencing of neuronal firing. PMID:18946542

  2. Selected Gamma Aminobutyric Acid (GABA) Esters may Provide Analgesia for Some Central Pain Conditions

    PubMed Central

    Goldberg, Joel S.

    2010-01-01

    Central pain is an enigmatic, intractable condition, related to destruction of thalamic areas, resulting in likely loss of inhibitory synaptic transmission mediated by GABA. It is proposed that treatment of central pain, a localized process, may be treated by GABA supplementation, like Parkinson’s disease and depression. At physiologic pH, GABA exists as a zwitterion that is poorly permeable to the blood brain barrier (BBB). Because the pH of the cerebral spinal fluid (CSF) is acidic relative to the plasma, ion trapping may allow a GABA ester prodrug to accumulate and be hydrolyzed within the CSF. Previous investigations with ester local anesthetics may be applicable to some GABA esters since they are weak bases, hydrolyzed by esterases and cross the BBB. Potential non-toxic GABA esters are discussed. Many GABA esters were investigated in the 1980s and it is hoped that this paper may spark renewed interest in their development. PMID:20703328

  3. The role of GABA in the regulation of GnRH neurons

    PubMed Central

    Watanabe, Miho; Fukuda, Atsuo; Nabekura, Junichi

    2014-01-01

    Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction. Gamma-amino butyric acid (GABA) has long been implicated as one of the major players in the regulation of GnRH neurons. Although GABA is typically an inhibitory neurotransmitter in the mature adult central nervous system, most mature GnRH neurons show the unusual characteristic of being excited by GABA. While many reports have provided much insight into the contribution of GABA to the activity of GnRH neurons, the precise physiological role of the excitatory action of GABA on GnRH neurons remains elusive. This brief review presents the current knowledge of the role of GABA signaling in GnRH neuronal activity. We also discuss the modulation of GABA signaling by neurotransmitters and neuromodulators and the functional consequence of GABAergic inputs to GnRH neurons in both the physiology and pathology of reproduction. PMID:25506316

  4. Iron uptake and translocation by macrocystis pyrifera

    SciTech Connect

    Manley, S.L.

    1981-10-01

    Parameters of iron uptake have been determined for blade tissue of Macrocystis pyrifera (L.) C. Ag. These include the effects of iron concentration, light, various inhibitors, and blade type. All experiments were conducted in the defined artificial seawater Aquil. Iron uptake is light independent, energy dependent, and dependent on the reduction from Fe/sup 3+/ to Fe/sup 2+/. Iron is concentrated in the sieve tube exudate; exudate analysis revealed the presence of other micronutrients. Iron and other micronutrient translocation is discussed.

  5. Prejunctional GABA-B inhibition of cholinergic, neurally-mediated airway contractions in guinea-pigs.

    PubMed

    Chapman, R W; Danko, G; Rizzo, C; Egan, R W; Mauser, P J; Kreutner, W

    1991-01-01

    GABA is a known inhibitory neurotransmitter in the CNS. Recent studies have also demonstrated the presence of GABA in peripheral tissue, including lung. To delineate a role for GABA in lung, the effect of GABA and selective GABA agonists and antagonists on neuronally-induced airway contractions in guinea pigs were studied. In vitro, tracheal contractions induced by electrical field stimulation (EFS) were inhibited by tetrodotoxin and atropine indicating that the contractions were mediated by neuronal release of acetylcholine. The contractions caused by EFS, but not those by exogenous acetylcholine, were inhibited by GABA (EC50 = 4.5 microM) and the selective GABA-B agonist baclofen (EC50 = 9 microM), but not by the GABA-A agonist, muscimol. The inhibitory effect of baclofen was not affected by the GABA-A antagonist, bicuculline, but was significantly reversed with the GABA-B antagonists, 3-aminopropylphosphonic acid (3-APPA) (pA2 = 4.5) and 2-hydroxysaclofen (pA2 = 4.1). In vivo, vagal nerve stimulation (5 V, 20 Hz, 0.5 ms, 5 s) in anesthetized, mechanically ventilated guinea-pigs caused cholinergic-dependent bronchospasms that were inhibited by intravenous GABA (3 and 10 mg/kg) and baclofen (1-10 mg/kg), but not by muscimol. The inhibitory effects of GABA and baclofen against vagal bronchospasm were blocked by 3-APPA (5 mg/kg, i.v.), but not by bicuculline. Responses to the GABA-B agonists were unaltered after the treatment of animals with phentolamine or propranolol to block alpha-adrenergic and beta-adrenergic receptors, respectively. Bronchospasm due to intravenous methacholine was also unchanged by GABA and baclofen.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Effect of various GABA-receptor agonists and antagonists on anaphylactic histamine release in the guinea-pig ileum.

    PubMed

    Luzzi, S; Franchi-Micheli, S; Ciuffi, M; Rosi, E; Zilletti, L

    1987-04-01

    In this paper we confirm the previously reported inhibition by GABA of anaphylactic histamine release from isolated guinea-pig ileum longitudinal muscle. Moreover we report that: GABA-inhibition of anaphylactic histamine release is mimicked both by GABA-A and GABA-B agonists; both GABA-A and GABA-B antagonists are effective in reversing GABA's inhibitory effect; the effect is exerted specifically by GABA-ergic drugs: taurine and beta-alanine are ineffective; the GABA-ergic effect seems not to involve cholinergic and adrenergic transmission. It is concluded that it might be interesting to assess the clinical value of GABA-ergic drugs in allergic gut disorders.

  7. An improved asymmetric synthetic route to a novel triple uptake inhibitor antidepressant (2S,4R,5R)-2-benzhydryl-5-((4-methoxybenzyl)amino)tetrahydro-2H-pyran-4-ol (D-142)

    PubMed Central

    Gopishetty, Bhaskar; Gogoi, Sanjib; Dutta, Aloke

    2011-01-01

    Triple monoamine reuptake inhibitors have been implicated in the development of a new generation of antidepressants with higher efficacy than the currently existing therapies. In this paper, we have developed an alternative efficient synthetic route for triple monoamine reuptake inhibitor D-142 in 18.5% overall yield in 11 steps starting from diphenylmethane. D-142 was developed by us recently. The key step of the present synthetic strategy is the preferential formation of a bromohydrin from olefin via a cis-bromoinum intermediate, which introduced significant efficiency in the overall synthesis. Furthermore, we have developed an efficient way to recycle the optically active intermediate diol back to the desired chiral epoxide. PMID:21927543

  8. Regulation of GABA release by nicotinic acetylcholine receptors in the neonatal rat hippocampus

    PubMed Central

    Maggi, Laura; Sher, Emanuele; Cherubini, Enrico

    2001-01-01

    The whole-cell configuration of the patch-clamp technique was used to study the modulation of giant depolarizing potentials (GDPs) by nicotinic acetylcholine receptors (nAChRs) in CA3 hippocampal neurons in slices from postnatal day (P) 2–6 rats.Bath application of nicotine increased GDP frequency in a concentration-dependent manner. For example, nicotine (0.5–1 μm) enhanced GDP frequency from 0.05 ± 0.04 to 0.17 ± 0.04 Hz. This effect was prevented by the broad-spectrum nicotinic receptor antagonist dihydro-β-erythtroidine (DHβE, 50 μm) and partially antagonized by methyllycaconitine (MLA, 50 nm) a competitive antagonist of α7 nAChRs. GDP frequency was also enhanced by AR-17779 (100 μm), a selective agonist of α7 nAChRs.The GABAA receptor antagonist bicuculline (10 μm) and the non-NMDA glutamate receptor antagonist DNQX (20 μm) blocked GDPs and prevented the effects of nicotine on GDPs. In the presence of DNQX, nicotine increased GABA-mediated synaptic noise, indicating that this drug may have a direct effect on GABAergic interneurons.Bath application of edrophonium (20 μm), a cholinesterase inhibitor, in the presence of atropine (1 μm), increased GDP frequency, indicating that nAChRs can be activated by ACh released from the septo-hippocampal fibres. This effect was prevented by DHβE (50 μm).In the majority of neurons tested, MLA (50 nm) and DHβE (50 μm) reduced the frequency of GDPs with different efficacy: a reduction of 98 ± 11 and 61 ± 29 % was observed with DHβE and MLA, respectively. In a subset of cells (40 % in the case of MLA and 17 % in the case of DHβE) these drugs induced a twofold increase in GDP frequency.It is suggested that, during development, nAChRs modulate the release of GABA, assessed as GDPs, through distinct nAChRs. The rise of intracellular calcium via nAChRs would further strengthen GABA-mediated oscillatory activity. This can be crucial for consolidation of synaptic contacts and for the fine-tuning of the

  9. Muscarinic receptor modulation of GABA-mediated giant depolarizing potentials in the neonatal rat hippocampus

    PubMed Central

    Avignone, Elena; Cherubini, Enrico

    1999-01-01

    The whole-cell patch clamp technique was used to study the role of muscarinic receptors in regulating the frequency of giant depolarizing potentials (GDPs) in CA3 hippocampal neurones in slices from postnatal (P) P1-P8 rats. Atropine (1 μM) reduced the frequency of GDPs by 64·2 ± 2·9%. The acetylcholinesterase inhibitor edrophonium (20 μM) increased the frequency of GDPs in a developmentally regulated way. This effect was antagonized by the M1 muscarinic receptor antagonist pirenzepine. In the presence of edrophonium, tetanic stimulation of cholinergic fibres induced either an enhancement of GDP frequency (179 ± 79%) or a membrane depolarization (27 ± 16 mV) associated with an increase in synaptic noise. These effects were prevented by atropine. Application of carbachol (3 μM) produced an increase in GDP frequency that at P5-P6 was associated with a membrane depolarization and an increase in synaptic noise. These effects were prevented by atropine, pirenzepine (3 μM) and bicuculline (10 μM). In the presence of pirenzepine, carbachol reduced GDP frequency by 50 ± 4%. Conversely, in the presence of methoctramine (3 μM), carbachol enhanced GDP frequency by 117 ± 4%. It is concluded that endogenous acetylcholine, through the activation of M1 receptors, enhances the release of γ-aminobutyric acid (GABA), in a developmentally regulated way. On the other hand, carbachol exerts both an up- and downregulation of GABA release through the activation of M1 and M2 receptors, respectively. PMID:10373692

  10. ALPHA-1 ADRENORECEPTORS MODULATE GABA RELEASE ONTO VENTRAL TEGMENTAL AREA DOPAMINE NEURONS

    PubMed Central

    Velásquez-Martínez, M.C.; Vázquez-Torres, R.; Rojas, L.V.; Sanabria, P.; Jiménez-Rivera, C.A.

    2014-01-01

    The ventral tegmental area (VTA) plays an important role in reward and motivational processes involved in drug addiction. Previous studies have shown that alpha1-adrenoreceptors (α1-AR) are primarily found presynaptically at this area. We hypothesized that GABA released onto VTA-dopamine (DA) cells is modulated by presynaptic α1-AR. Recordings were obtained from putative VTA-DA cells of male Sprague-Dawley rats (28–50 days postnatal) using whole-cell voltage clamp technique. Phenylephrine (10µM; α1-AR agonist) decreased the amplitude of GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) evoked by electrical stimulation of afferent fibers (n=7; p<0.05). Prazosin (1µM, α1-AR antagonist), blocked this effect. Paired-pulse ratios were increased by phenylephrine application (n=13; p<0.05) indicating a presynaptic site of action. Spontaneous IPSCs frequency but not amplitude, were decreased in the presence of phenylephrine (n=7; p<0.05). However, frequency or amplitude of miniature IPSCs were not changed (n=9; p>0.05). Phenylephrine in low Ca2+ (1mM) medium decreased IPSC amplitude (n=7; p<0.05). Chelerythrine (a protein kinase C inhibitor) blocked the α1-AR action on IPSC amplitude (n=6; p<0.05). Phenylephrine failed to decrease IPSCs amplitude in the presence of paxilline, a BK channel blocker (n=7; p<0.05). Taken together, these results demonstrate that α1-ARs at presynaptic terminals can modulate GABA release onto VTA-DA cells. Drug-induced changes in α1-AR could contribute to the modifications occurring in the VTA during the addiction process. PMID:25261018

  11. At immature mossy fibers-CA3 connections, activation of presynaptic GABA(B) receptors by endogenously released GABA contributes to synapses silencing.

    PubMed

    Safiulina, Victoria F; Cherubini, Enrico

    2009-01-01

    Early in postnatal life correlated GABAergic activity in the hippocampus is thought to play a crucial role in synaptogenesis and in the development of adult neuronal networks. Unlike adulthood, at this developmental stage, mossy fibers (MF) which are the axons of granule cells, release GABA into CA3 principal cells and interneurons. Here, we tested the hypothesis that at MF-CA3 connections, tonic activation of GABA(B) autoreceptors by GABA is responsible for the low probability of release and synapse silencing. Blocking GABA(B) receptors with CGP55845 enhanced the probability of GABA release and switched on silent synapses while the opposite was observed with baclofen. Both these effects were presynaptic and were associated with changes in paired-pulse ratio and coefficient of variation. In addition, enhancing the extracellular GABA concentration by repetitive stimulation of MF or by blocking the GABA transporter GAT-1, switched off active synapses, an effect that was prevented by CGP55845. In the presence of CGP55845, stimulation of MF-induced synaptic potentiation. The shift of E(GABA) from the depolarizing to the hyperpolarizing direction with bumetanide, a blocker of the cation-chloride co-transporter NKCC1, prevented synaptic potentiation and caused synaptic depression, suggesting that the depolarizing action of GABA observed in the presence of CGP55845 is responsible for the potentiating effect. It is proposed that, activation of GABA(B) receptors by spillover of GABA from MF terminals reduces the probability of release and contributes to synapses silencing. This would act as a filter to prevent excessive activation of the auto-associative CA3 network and the emergence of seizures.

  12. Statins impair glucose uptake in tumor cells.

    PubMed

    Malenda, Agata; Skrobanska, Anna; Issat, Tadeusz; Winiarska, Magdalena; Bil, Jacek; Oleszczak, Bozenna; Sinski, Maciej; Firczuk, Małgorzata; Bujnicki, Janusz M; Chlebowska, Justyna; Staruch, Adam D; Glodkowska-Mrowka, Eliza; Kunikowska, Jolanta; Krolicki, Leszek; Szablewski, Leszek; Gaciong, Zbigniew; Koziak, Katarzyna; Jakobisiak, Marek; Golab, Jakub; Nowis, Dominika A

    2012-04-01

    Statins, HMG-CoA reductase inhibitors, are used in the prevention and treatment of cardiovascular diseases owing to their lipid-lowering effects. Previous studies revealed that, by modulating membrane cholesterol content, statins could induce conformational changes in cluster of differentiation 20 (CD20) tetraspanin. The aim of the presented study was to investigate the influence of statins on glucose transporter 1 (GLUT1)-mediated glucose uptake in tumor cells. We observed a significant concentration- and time-dependent decrease in glucose analogs' uptake in several tumor cell lines incubated with statins. This effect was reversible with restitution of cholesterol synthesis pathway with mevalonic acid as well as with supplementation of plasma membrane with exogenous cholesterol. Statins did not change overall GLUT1 expression at neither transcriptional nor protein levels. An exploratory clinical trial revealed that statin treatment decreased glucose uptake in peripheral blood leukocytes and lowered (18)F-fluorodeoxyglucose ((18)F-FDG) uptake by tumor masses in a mantle cell lymphoma patient. A bioinformatics analysis was used to predict the structure of human GLUT1 and to identify putative cholesterol-binding motifs in its juxtamembrane fragment. Altogether, the influence of statins on glucose uptake seems to be of clinical significance. By inhibiting (18)F-FDG uptake, statins can negatively affect the sensitivity of positron emission tomography, a diagnostic procedure frequently used in oncology.

  13. Synchronization by Food Access Modifies the Daily Variations in Expression and Activity of Liver GABA Transaminase

    PubMed Central

    De Ita-Pérez, Dalia; Vázquez-Martínez, Olivia; Villalobos-Leal, Mónica

    2014-01-01

    Daytime restricted feeding (DRF) is an experimental protocol that influences the circadian timing system and underlies the expression of a biological clock known as the food entrained oscillator (FEO). Liver is the organ that reacts most rapidly to food restriction by adjusting the functional relationship between the molecular circadian clock and the metabolic networks. γ-Aminobutyric acid (GABA) is a signaling molecule in the liver, and able to modulate the cell cycle and apoptosis. This study was aimed at characterizing the expression and activity of the mostly mitochondrial enzyme GABA transaminase (GABA-T) during DRF/FEO expression. We found that DRF promotes a sustained increase of GABA-T in the liver homogenate and mitochondrial fraction throughout the entire day-night cycle. The higher amount of GABA-T promoted by DRF was not associated to changes in GABA-T mRNA or GABA-T activity. The GABA-T activity in the mitochondrial fraction even tended to decrease during the light period. We concluded that DRF influences the daily variations of GABA-T mRNA levels, stability, and catalytic activity of GABA-T. These data suggest that the liver GABAergic system responds to a metabolic challenge such as DRF and the concomitant appearance of the FEO. PMID:24809054

  14. The action of GABA receptor agonists and antagonists on muscle membrane conductance in Schistocerca gregaria.

    PubMed Central

    Murphy, V. F.; Wann, K. T.

    1988-01-01

    1. The properties of postsynaptic gamma-aminobutyric acid (GABA) receptors in the extensor tibiae muscle of Schistocerca gregaria were studied by conventional electrophysiological recording techniques. 2. GABA and other active GABA receptor agonists produced rapid, dose-dependent, reversible increases in membrane conductance. 3. In two microelectrode experiments the ED50 for GABA was approximately 1 mM. In three microelectrode experiments (assuming short cable theory conditions) the ED50 for GABA was 2.3 mM. The Hill coefficient for GABA estimated from the latter experiments was 1.4. 4. The relative potency of muscimol/GABA at the ED50 for GABA was 1.36. 3-Aminopropane sulphonic acid (3-APS) and isonipecotic acid were weakly active, baclofen and piperidine-4-sulphonic acid (P4S) were inactive. Isoguvacine produced depolarizations and increases in conductance in preparations which hyperpolarized in response to GABA. These depolarizations were enhanced by both picrotoxin and pitrazepin although the increases in input conductance were depressed. 5. Picrotoxin (20 microM), (+)-bicuculline (20-100 microM) and pitrazepin (1-10 microM) all reversibly antagonized GABA-induced responses. Such antagonism was not competitive in the case of picrotoxin and (+)-bicuculline but was competitive for pitrazepin. Schild plot analysis gave an average pA2 value of 5.5 for pitrazepin. 6. The significance of these results is briefly discussed. PMID:2850061

  15. Relaxation and immunity enhancement effects of gamma-aminobutyric acid (GABA) administration in humans.

    PubMed

    Abdou, Adham M; Higashiguchi, S; Horie, K; Kim, Mujo; Hatta, H; Yokogoshi, H

    2006-01-01

    The effect of orally administrated gamma-aminobutyric acid (GABA) on relaxation and immunity during stress has been investigated in humans. Two studies were conducted. The first evaluated the effect of GABA intake by 13 subjects on their brain waves. Electroencephalograms (EEG) were obtained after 3 tests on each volunteer as follows: intake only water, GABA, or L-theanine. After 60 minutes of administration, GABA significantly increases alpha waves and decreases beta waves compared to water or L-theanine. These findings denote that GABA not only induces relaxation but also reduces anxiety. The second study was conducted to see the role of relaxant and anxiolytic effects of GABA intake on immunity in stressed volunteers. Eight acrophobic subjects were divided into 2 groups (placebo and GABA). All subjects were crossing a suspended bridge as a stressful stimulus. Immunoglobulin A (IgA) levels in their saliva were monitored during bridge crossing. Placebo group showed marked decrease of their IgA levels, while GABA group showed significantly higher levels. In conclusion, GABA could work effectively as a natural relaxant and its effects could be seen within 1 hour of its administration to induce relaxation and diminish anxiety. Moreover, GABA administration could enhance immunity under stress conditions.

  16. Endogenous GABA levels in the pontine reticular formation are greater during wakefulness than during REM sleep

    PubMed Central

    Vanini, Giancarlo; Wathen, Bradley L.; Lydic, Ralph; Baghdoyan, Helen A.

    2011-01-01

    Studies using drugs that increase or decrease GABAergic transmission suggest that GABA in the pontine reticular formation (PRF) promotes wakefulness and inhibits rapid eye movement (REM) sleep. Cholinergic transmission in the PRF promotes REM sleep, and levels of endogenous acetylcholine (ACh) in the PRF are significantly greater during REM sleep than during wakefulness or non-REM (NREM) sleep. No previous studies have determined whether levels of endogenous GABA in the PRF vary as a function of sleep and wakefulness. This study tested the hypothesis that GABA levels in cat PRF are greatest during wakefulness and lowest during REM sleep. Extracellular GABA levels were measured during wakefulness, NREM sleep, REM sleep, and the REM sleep-like state (REMNeo) caused by microinjecting neostigmine into the PRF. GABA levels varied significantly as a function of sleep and wakefulness, and decreased significantly below waking levels during REM sleep (−42%) and REMNeo (−63%). The decrease in GABA levels during NREM sleep (22% below waking levels) was not statistically significant. Compared to NREM sleep, GABA levels decreased significantly during REM sleep (−27%) and REMNeo (−52%). Comparisons of REM sleep and REMNeo revealed no differences in GABA levels or cortical EEG power. GABA levels did not vary significantly as a function of dialysis site within the PRF. The inverse relationship between changes in PRF levels of GABA and ACh during REM sleep indicates that low GABAergic tone combined with high cholinergic tone in the PRF contributes to the generation of REM sleep. PMID:21325533

  17. Effect of antioxidant treatment on spinal GABA neurons in a neuropathic pain model in the mouse.

    PubMed

    Yowtak, June; Wang, Jigong; Kim, Hee Young; Lu, Ying; Chung, Kyungsoon; Chung, Jin Mo

    2013-11-01

    One feature of neuropathic pain is a reduced spinal gamma-aminobutyric acid (GABA)-ergic inhibitory function. However, the mechanisms behind this attenuation remain to be elucidated. This study investigated the involvement of reactive oxygen species in the spinal GABA neuron loss and reduced GABA neuron excitability in spinal nerve ligation (SNL) model of neuropathic pain in mice. The importance of spinal GABAergic inhibition in neuropathic pain was tested by examining the effects of intrathecally administered GABA receptor agonists and antagonists in SNL and naïve mice, respectively. The effects of SNL and antioxidant treatment on GABA neuron loss and functional changes were examined in transgenic GAD67-enhanced green fluorescent protein positive (EGFP+) mice. GABA receptor agonists transiently reversed mechanical hypersensitivity of the hind paw in SNL mice. On the other hand, GABA receptor antagonists made naïve mice mechanically hypersensitive. Stereological analysis showed that the numbers of enhanced green fluorescent protein positive (EGFP+) GABA neurons were significantly decreased in the lateral superficial laminae (I-II) on the ipsilateral L5 spinal cord after SNL. Repeated antioxidant treatments significantly reduced the pain behaviors and prevented the reduction in EGFP+ GABA neurons. The response rate of the tonic firing GABA neurons recorded from SNL mice increased with antioxidant treatment, whereas no change was seen in those recorded from naïve mice, which suggested that oxidative stress impaired some spinal GABA neuron activity in the neuropathic pain condition. Together the data suggest that neuropathic pain, at least partially, is attributed to oxidative stress, which induces both a GABA neuron loss and dysfunction of surviving GABA neurons.

  18. Glutamate, GABA, and glutamine are synchronously upregulated in the mouse lateral septum during the postpartum period.

    PubMed

    Zhao, Changjiu; Gammie, Stephen C

    2014-12-03

    Dramatic structural and functional remodeling occurs in the postpartum brain for the establishment of maternal care, which is essential for the growth and development of young offspring. Glutamate and GABA signaling are critically important in modulating multiple behavioral performances. Large scale signaling changes occur in the postpartum brain, but it is still not clear to what extent the neurotransmitters glutamate and GABA change and whether the ratio of glutamate/GABA remains balanced. In this study, we examined the glutamate/GABA-glutamine cycle in the lateral septum (LS) of postpartum female mice. In postpartum females (relative to virgins), tissue levels of glutamate and GABA were elevated in LS and increased mRNA was found for the respective enzymes producing glutamate and GABA, glutaminase (Gls) and glutamate decarboxylase 1 and 2 (Gad1 and Gad2). The common precursor, glutamine, was elevated as was the enzyme that produces it, glutamate-ammonia ligase (Glul). Additionally, glutamate, GABA, and glutamine were positively correlated and the glutamate/GABA ratio was almost identical in the postpartum and virgin females. Collectively, these findings indicate that glutamate and GABA signaling are increased and that the ratio of glutamate/GABA is well balanced in the maternal LS. The postpartum brain may provide a useful model system for understanding how glutamate and GABA are linked despite large signaling changes. Given that some mental health disorders, including depression and schizophrenia display dysregulated glutamate/GABA ratio, and there is increased vulnerability to mental disorders in mothers, it is possible that these postpartum disorders emerge when glutamate and GABA changes are not properly coordinated.

  19. The reciprocal regulation of stress hormones and GABA(A) receptors.

    PubMed

    Mody, Istvan; Maguire, Jamie

    2011-01-01

    Stress-derived steroid hormones regulate the expression and function of GABA(A) receptors (GABA(A)Rs). Changes in GABA(A)R 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 GABA(A)R subunit expression, stress hormones can also be metabolized to neuroactive derivatives which can alter the function of GABA(A)Rs. Neurosteroids allosterically modulate GABA(A)Rs at concentrations comparable to those during stress. In addition to the actions of stress-derived steroid hormones on GABA(A)Rs, GABA(A)Rs 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, GABA(A)R δ subunit-containing receptors representing a novel feedback mechanism onto the HPA axis. Further, it has been demonstrated that neurosteroidogenesis and neurosteroid actions on GABA(A)R 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 GABA(A)Rs as well as the importance of GABA(A)Rs in regulating the production of steroid hormones. This review incorporates what we currently know about changes in GABA(A)Rs following stress and the role in HPA axis regulation.

  20. UWA-121, a mixed dopamine and serotonin re-uptake inhibitor, enhances L-DOPA anti-parkinsonian action without worsening dyskinesia or psychosis-like behaviours in the MPTP-lesioned common marmoset.

    PubMed

    Huot, Philippe; Johnston, Tom H; Lewis, Katie D; Koprich, James B; Reyes, M Gabriela; Fox, Susan H; Piggott, Matthew J; Brotchie, Jonathan M

    2014-07-01

    L-3,4-Dihydroxyphenylalanine (L-DOPA) is the most effective treatment for Parkinson's disease (PD), but its long-term administration is complicated by wearing-off and dyskinesia. UWA-101, a dual, equipotent inhibitor of dopamine (DAT) and serotonin (SERT) transporters, has previously been shown to successfully extend duration of anti-parkinsonian benefit of L-DOPA (ON-time), without exacerbating dyskinesia, in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned marmoset. However, UWA-101 is racemic and it is unclear whether one or both enantiomers contribute to its actions, and whether a better therapeutic effect might be attained by using a single antipode. In the current study, we synthesised the two enantiomers of UWA-101, R-101 (UWA-121) and S-101 (UWA-122), characterised their pharmacological profiles and administered them to MPTP-lesioned marmosets. Parkinsonism, dyskinesia, psychosis-like behaviours and duration of ON-time were evaluated. UWA-121 is a dual DAT > SERT inhibitor, with an approximate 10:1 DAT:SERT affinity ratio (inhibitory constants (Ki) of 307 and 3830 nM, respectively). In combination with L-DOPA, UWA-121 extended duration of ON-time when compared to L-DOPA/vehicle treatment (by 40%, P < 0.01). UWA-121 also extended duration of ON-time without dyskinesia (by 215%, P < 0.05) and ON-time without psychosis-like behaviours when compared to L-DOPA/vehicle treatment (by 345%, P < 0.01). UWA-121 did not worsen the severity of dyskinesia or psychosis-like behaviours (P > 0.05). UWA-122 is a selective SERT inhibitor (Ki 120 nM, Ki at DAT > 50 μM) and, in combination with L-DOPA, had no effect on ON-time, dyskinesia or psychosis-like behaviours (P > 0.05). These data indicate that dual DAT and SERT inhibitors effectively enhance L-DOPA anti-parkinsonian action without worsening dyskinesia and that compounds with such a pharmacological profile represent promising agents against wearing-off in PD.

  1. [Autoimmune encephalitis induced by antibodies against GABA-A receptor].

    PubMed

    González R, Pablo; Hudson A, Lorena; Basáez M, Esteban; Miranda C, Marcelo

    2016-11-01

    Among autoimmune encephalitides, a prevalent group are those associated with antibodies against the N-Methyl-D-aspartate receptor, which present with behavior abnormalities, psychosis, seizures and abnormal movements. A new variant, mediated by antibodies against the GABA-A receptor, was recen-tly described. We report a 66-years-old female with this form of encephalitis whose main manifestation was the presence of severe seizures leading to status epilepticus. The patient had a good response to immunomodulatory therapy with intravenous methylprednisolone, azathioprine and anticonvulsants. The laboratory tests initially detected anti-thyroid peroxidase antibodies which lead to the misdiagnosis of Hashimoto Encephalitis, which was ruled out after the detection of antibodies against GABA-A receptor. No malignancy was detected.

  2. Ketosis and brain handling of glutamate, glutamine, and GABA.

    PubMed

    Yudkoff, Marc; Daikhin, Yevgeny; Horyn, Oksana; Nissim, Ilana; Nissim, Itzhak

    2008-11-01

    We hypothesize that one mechanism of the anti-epileptic effect of the ketogenic diet is to alter brain handling of glutamate. According to this formulation, in ketotic brain astrocyte metabolism is more active, resulting in enhanced conversion of glutamate to glutamine. This allows for: (a) more efficient removal of glutamate, the most important excitatory neurotransmitter; and (b) more efficient conversion of glutamine to GABA, the major inhibitory neurotransmitter.

  3. Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases

    PubMed Central

    Yuan, Hongjie; Low, Chian-Ming; Moody, Olivia A.; Jenkins, Andrew

    2015-01-01

    The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-d-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases. PMID:25904555

  4. Role of gamma-aminobutyric acid (GABA) and metabotropic glutamate receptors in nicotine reinforcement: potential pharmacotherapies for smoking cessation.

    PubMed

    Markou, Athina; Paterson, Neil E; Semenova, Svetlana

    2004-10-01

    Previous work indicated a role for GABA and glutamate in the reinforcing effects of drugs of abuse. The present studies assessed the effects of GABAergic and glutamatergic manipulations on the reinforcing effects of nicotine as assessed by intravenous nicotine self-administration. Male Wistar rats were allowed to self-administer either of two nicotine doses under a fixed ratio or a progressive ratio schedule of reinforcement. The effects of a glutamatergic compound on nicotine self-administration in male DBA/2J mice were also explored. Finally, to assess for nonspecific effects of the drug manipulations, the effects of all test compounds on responding maintained by a food reinforcer were investigated. The pharmacological manipulations used were: gamma-vinyl-GABA (vigabatrin or GVG), an irreversible inhibitor of GABA transaminase, the GABAB receptor agonists (-)baclofen and CGP44532, and the metabotropic glutamate receptor 5 (mGluR5) antagonist MPEP. GVG, CGP44532, and (-)baclofen dose-dependently decreased nicotine self-administration on the fixed-ratio schedule, but also decreased food-maintained responding. Furthermore, CGP44532 decreased breakpoints for nicotine and food at identical doses under the progressive-ratio schedule. MPEP dose-dependently decreased nicotine self-administration with no effect on food-maintained responding in rats. MPEP also decreased nicotine self-administration in the mice. These results demonstrate that activation of GABAB receptors or blockade of mGluR5 decreased nicotine self-administration. Although there was some selectivity for the effects of the GABAergic manipulations, there was clear selectivity of the effects of MPEP on nicotine- versus food-maintained responding. Thus, compounds that increase GABAergic neurotransmission and antagonists at mGluR5 have potential as anti-smoking medications for humans.

  5. The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling.

    PubMed

    Calver, A R; Robbins, M J; Cosio, C; Rice, S Q; Babbs, A J; Hirst, W D; Boyfield, I; Wood, M D; Russell, R B; Price, G W; Couve, A; Moss, S J; Pangalos, M N

    2001-02-15

    GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.

  6. Flavonoid nutraceuticals and ionotropic receptors for the inhibitory neurotransmitter GABA.

    PubMed

    Johnston, Graham A R

    2015-10-01

    Flavonoids that are found in nutraceuticals have many and varied effects on the activation of ionotropic receptors for GABA, the major inhibitory neurotransmitter in our brains. They can act as positive or negative modulators enhancing or reducing the effect of GABA. They can act as allosteric agonists. They can act to modulate the action of other modulators. There is considerable evidence that these flavonoids are able to enter the brain to influence brain function. They may have a range of effects including relief of anxiety, improvement in cognition, acting as neuroprotectants and as sedatives. All of these effects are sought after in nutraceuticals. A number of studies have likened flavonoids to the widely prescribed benzodiazepines as 'a new family of benzodiazepine receptor ligands'. They are much more than that with many flavonoid actions on ionotropic GABA receptors being insensitive to the classic benzodiazepine antagonist flumazenil and thus independent of the classic benzodiazepine actions. It is time to consider flavonoids in their own right as important modulators of these vital receptors in brain function. Flavonoids are rarely consumed as a single flavonoid except as dietary supplements. The effects of mixtures of flavonoids and other modulators on GABAA receptors need to be more thoroughly investigated.

  7. GABA Not Only a Neurotransmitter: Osmotic Regulation by GABAAR Signaling

    PubMed Central

    Cesetti, Tiziana; Ciccolini, Francesca; Li, Yuting

    2012-01-01

    Mature macroglia and almost all neural progenitor types express γ-aminobutyric (GABA) A receptors (GABAARs), whose activation by ambient or synaptic GABA, leads to influx or efflux of chloride (Cl−) depending on its electro-chemical gradient (ECl). Since the flux of Cl− is indissolubly associated to that of osmotically obliged water, GABAARs regulate water movements by modulating ion gradients. In addition, since water movements also occur through specialized water channels and transporters, GABAAR signaling could affect the movement of water by regulating the function of the channels and transporters involved, thereby affecting not only the direction of the water fluxes but also their dynamics. We will here review recent observations indicating that in neural cells GABAAR-mediated osmotic regulation affects the cellular volume thereby activating multiple intracellular signaling mechanisms important for cell proliferation, maturation, and survival. In addition, we will discuss evidence that the osmotic regulation exerted by GABA may contribute to brain water homeostasis in physiological and in pathological conditions causing brain edema, in which the GABAergic transmission is often altered. PMID:22319472

  8. Phenibut (beta-phenyl-GABA): a tranquilizer and nootropic drug.

    PubMed

    Lapin, I

    2001-01-01

    Phenibut (beta-phenyl-gamma-aminobutyric acid HCl) is a neuropsychotropic drug that was discovered and introduced into clinical practice in Russia in the 1960s. It has anxiolytic and nootropic (cognition enhancing) effects. It acts as a GABA-mimetic, primarily at GABA(B) and, to some extent, at GABA(A) receptors. It also stimulates dopamine receptors and antagonizes beta-phenethylamine (PEA), a putative endogenous anxiogenic. The psychopharmacological activity of phenibut is similar to that of baclofen, a p-Cl-derivative of phenibut. This article reviews the structure-activity relationship of phenibut and its derivatives. Emphasis is placed on the importance of the position of the phenyl ring, the role of the carboxyl group, and the activity of optical isomers. Comparison of phenibut with piracetam and diazepam reveals similarities and differences in their pharmacological and clinical effects. Phenibut is widely used in Russia to relieve tension, anxiety, and fear, to improve sleep in psychosomatic or neurotic patients; as well as a pre- or post-operative medication. It is also used in the therapy of disorders characterized by asthenia and depression, as well as in post-traumatic stress, stuttering and vestibular disorders.

  9. Wakefulness Is Governed by GABA and Histamine Cotransmission

    PubMed Central

    Yu, Xiao; Ye, Zhiwen; Houston, Catriona M.; Zecharia, Anna Y.; Ma, Ying; Zhang, Zhe; Uygun, David S.; Parker, Susan; Vyssotski, Alexei L.; Yustos, Raquel; Franks, Nicholas P.; Brickley, Stephen G.; Wisden, William

    2015-01-01

    Summary Histaminergic neurons in the tuberomammilary nucleus (TMN) of the hypothalamus form a widely projecting, wake-active network that sustains arousal. Yet most histaminergic neurons contain GABA. Selective siRNA knockdown of the vesicular GABA transporter (vgat, SLC32A1) in histaminergic neurons produced hyperactive mice with an exceptional amount of sustained wakefulness. Ablation of the vgat gene throughout the TMN further sharpened this phenotype. Optogenetic stimulation in the caudate-putamen and neocortex of “histaminergic” axonal projections from the TMN evoked tonic (extrasynaptic) GABAA receptor Cl− currents onto medium spiny neurons and pyramidal neurons. These currents were abolished following vgat gene removal from the TMN area. Thus wake-active histaminergic neurons generate a paracrine GABAergic signal that serves to provide a brake on overactivation from histamine, but could also increase the precision of neocortical processing. The long range of histamine-GABA axonal projections suggests that extrasynaptic inhibition will be coordinated over large neocortical and striatal areas. PMID:26094607

  10. Distribution of GABA-like immunoreactivity in the rat amygdaloid complex.

    PubMed

    Nitecka, L; Ben-Ari, Y

    1987-12-01

    The distribution of GABA-like (GABA-Li) immunoreactivity in the rat amygdaloid complex was studied by using an anti-GABA antibody. GABA-Li positive neurons and processes were present in every nucleus of the complex. Three patterns of immunoreactivity were revealed: (1) the intercalated masses and the lateral olfactory tract nucleus exhibited the most intense staining of the neuropil, and virtually every neuron was labeled, (2) the central and medial nuclei contained intensely labeled neuropil and moderately labeled neurons, and (3) in the remaining nuclei, the neuropil was weakly labeled, and relatively numerous GABA-Li neurons were present. Our results suggest that: (1) the intercalated masses and lateral olfactory tract nucleus consist of large aggregates of GABA-Li immunoreactive neurons, and (2) the lateral, basal dorsal, and the posterior cortical nuclei may constitute a significant source of GABAergic connections to other amygdaloid nuclei, in particular to the medial and central nuclei.

  11. The dynamics of GABA signaling: Revelations from the circadian pacemaker in the suprachiasmatic nucleus.

    PubMed

    Albers, H Elliott; Walton, James C; Gamble, Karen L; McNeill, John K; Hummer, Daniel L

    2017-01-01

    Virtually every neuron within the suprachiasmatic nucleus (SCN) communicates via GABAergic signaling. The extracellular levels of GABA within the SCN are determined by a complex interaction of synthesis and transport, as well as synaptic and non-synaptic release. The response to GABA is mediated by GABAA receptors that respond to both phasic and tonic GABA release and that can produce excitatory as well as inhibitory cellular responses. GABA also influences circadian control through the exclusively inhibitory effects of GABAB receptors. Both GABA and neuropeptide signaling occur within the SCN, although the functional consequences of the interactions of these signals are not well understood. This review considers the role of GABA in the circadian pacemaker, in the mechanisms responsible for the generation of circadian rhythms, in the ability of non-photic stimuli to reset the phase of the pacemaker, and in the ability of the day-night cycle to entrain the pacemaker.

  12. Allosteric modulation by benzodiazepines of GABA-gated chloride channels of an identified insect motor neurone.

    PubMed

    Buckingham, Steven D; Higashino, Yoshiaki; Sattelle, David B

    2009-11-01

    The actions of benzodiazepines were studied on the responses to GABA of the fast coxal depressor (D(f)) motor neurone of the cockroach, Periplaneta americana. Ro5-4864, diazepam and clonazepam were investigated. Responses to GABA receptors were enhanced by both Ro5-4864 and diazepam, whereas clonazepam, a potent-positive allosteric modulator of human GABA(A) receptors, was ineffective on the native insect GABA receptors of the D(f) motor neurone. Thus, clear pharmacological differences exist between insect and mammalian native GABA-gated chloride channels with respect to the actions of benzodiazepines. The results enhance our understanding of invertebrate GABA-gated chloride channels which have recently proved important in (a) comparative studies aimed at identifying human allosteric drug-binding sites and (b) understanding the actions of compounds used to control ectoparasites and insect crop pests.

  13. Effect of experimental diabetes on GABA-mediated inhibition of neurally induced contractions in rat isolated trachea.

    PubMed

    Ozdem, S S; Sadan, G; Usta, C; Taşatargil, A

    2000-04-01

    1. In the present study, we investigated the effect of GABA and selective GABA agonists and antagonists on neurally induced tracheal contractions in streptozotocin (STZ) diabetic rats. 2. Contractile responses to electrical field stimulation (EFS) in rat tracheal rings were completely abolished by atropine and tetrodotoxin, but were unaffected by the ganglion blocker hexamethonium, indicating that they were mediated via neuronal release of acetylcholine (ACh). 3. Contractions induced by EFS, but not by exogenous ACh, were inhibited by GABA and the selective GABA(B) receptor agonist baclofen, but not by the selective GABA(A) receptor agonist 3-aminopropane sulphonic acid. The inhibitory effects of GABA or baclofen were not affected by the GABA(A) antagonist bicuculline, but were significantly reversed by the GABA(B) antagonist phaclofen. 4. The inhibitory effects of both GABA and baclofen were found to be significantly greater in trachea from control rats compared with tissues from diabetic rats. 5. Non-adrenergic, non-cholinergic relaxation responses elicited by EFS in precontracted tracheal rings from diabetic and control rats were similar in magnitude and were unaffected by GABA or GABA analogues. 6. These results suggest that GABA decreases the response to EFS by directly inhibiting the evoked release of ACh through GABA(B) receptors in rat trachea and that STZ-induced diabetes causes an impairment in the inhibitory effect of GABA on neurally induced contractions in this tissue.

  14. Association of GABA(B) receptors and members of the 14-3-3 family of signaling proteins.

    PubMed

    Couve, A; Kittler, J T; Uren, J M; Calver, A R; Pangalos, M N; Walsh, F S; Moss, S J

    2001-02-01

    Two GABA(B) receptors, GABA(B)R1 and GABA(B)R2, have been cloned recently. Unlike other G protein-coupled receptors, the formation of a heterodimer between GABA(B)R1 and GABA(B)R2 is required for functional expression. We have used the yeast two hybrid system to identify proteins that interact with the C-terminus of GABA(B)R1. We report a direct association between GABA(B) receptors and two members of the 14-3-3 protein family, 14-3-3eta and 14-3-3zeta. We demonstrate that the C-terminus of GABA(B)R1 associates with 14-3-3zeta in rat brain preparations and tissue cultured cells, that they codistribute after rat brain fractionation, colocalize in neurons, and that the binding site overlaps partially with the coiled-coil domain of GABA(B)R1. Furthermore we show a reduced interaction between the C-terminal domains of GABA(B)R1 and GABA(B)R2 in the presence of 14-3-3. The results strongly suggest that GABA(B)R1 and 14-3-3 associate in the nervous system and begin to reveal the signaling complexities of the GABA(B)R1/GABA(B)R2 receptor heterodimer.

  15. Status epilepticus enhances tonic GABA currents and depolarizes GABA reversal potential in dentate fast-spiking basket cells

    PubMed Central

    Yu, Jiandong; Proddutur, Archana; Elgammal, Fatima S.; Ito, Takahiro

    2013-01-01

    Temporal lobe epilepsy is associated with loss of interneurons and inhibitory dysfunction in the dentate gyrus. While status epilepticus (SE) leads to changes in granule cell inhibition, whether dentate basket cells critical for regulating granule cell feedforward and feedback inhibition express tonic GABA currents (IGABA) and undergo changes in inhibition after SE is not known. We find that interneurons immunoreactive for parvalbumin in the hilar-subgranular region express GABAA receptor (GABAAR) δ-subunits, which are known to underlie tonic IGABA. Dentate fast-spiking basket cells (FS-BCs) demonstrate baseline tonic IGABA blocked by GABAAR antagonists. In morphologically and physiologically identified FS-BCs, tonic IGABA is enhanced 1 wk after pilocarpine-induced SE, despite simultaneous reduction in spontaneous inhibitory postsynaptic current (sIPSC) frequency. Amplitude of tonic IGABA in control and post-SE FS-BCs is enhanced by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), demonstrating the contribution of GABAAR δ-subunits. Whereas FS-BC resting membrane potential is unchanged after SE, perforated-patch recordings from FS-BCs show that the reversal potential for GABA currents (EGABA) is depolarized after SE. In model FS-BCs, increasing tonic GABA conductance decreased excitability when EGABA was shunting and increased excitability when EGABA was depolarizing. Although simulated focal afferent activation evoked seizurelike activity in model dentate networks with FS-BC tonic GABA conductance and shunting EGABA, excitability of identical networks with depolarizing FS-BC EGABA showed lower activity levels. Thus, together, post-SE changes in tonic IGABA and EGABA maintain homeostasis of FS-BC activity and limit increases in dentate excitability. These findings have implications for normal FS-BC function and can inform studies examining comorbidities and therapeutics following SE. PMID:23324316

  16. Nitric oxide and GABA mediate bi-directional cardiovascular effects of orexin in the nucleus tractus solitarii of rats.

    PubMed

    Shih, C-D; Chuang, Y-C

    2007-11-09

    The present study investigated the cardiovascular effects of orexin (OX)-A and OX-B in the nucleus tractus solitarii (NTS) and delineated the engagement of nitric oxide (NO) and GABA in OX-induced cardiovascular responses. In adult male Sprague-Dawley rats maintained under propofol anesthesia, microinjection bilaterally into the NTS of OX-A or OX-B evoked bi-directional cardiovascular effects in a dose-dependent manner. At a lower dose (5 pmol), OX-A or OX-B decreased systemic arterial pressure (SAP), heart rate (HR), and power density of the vasomotor components of SAP signals, our experimental index for sympathetic neurogenic vasomotor tone. At higher doses (>20 pmol), these two compounds elicited cardiovascular excitatory responses. These bi-directional cardiovascular effects of OX were abolished by co-injection of an OX(1) receptor antagonist, 1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl-urea hydrochloride (SB-334867, 0.75 nmol) or the OX(2) receptor antiserum (1:20). In addition, the vasodepressor effects of low dose (5 pmol) OX-A or OX-B in the NTS were attenuated by a nitric oxide synthase (NOS) inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME, 5 nmol), a neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (2.5 pmol) or the soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazole[4,3-alpha]quinoxalin-1-one (250 pmol). The vasopressor effects of high dose (200 pmol) OX were reversed by co-administration with GABA(A) or GABA(B) receptor antagonist, bicuculline methiodine (10 pmol) or 2-hydroxy saclofen (100 pmol), or l-NAME (5 nmol). Our results indicate that OX-A or OX-B elicited bi-directional cardiovascular effects via OX receptor-dependent mechanisms. The vasodepressor effects of OX were induced by the nNOS-derived NO and activation of sGC-associated signaling pathway, whereas the vasopressor effects were mediated by interaction with GABAergic or nitrergic neurotransmission in the NTS.

  17. Hyperpolarizing inhibition develops without trophic support by GABA in cultured rat midbrain neurons.

    PubMed

    Titz, Stefan; Hans, Michael; Kelsch, Wolfgang; Lewen, Andrea; Swandulla, Dieter; Misgeld, Ulrich

    2003-08-01

    During a limited period of early neuronal development, GABA is depolarizing and elevates [Ca2+]i, which mediates the trophic action of GABA in neuronal maturation. We tested the attractive hypothesis that GABA itself promotes the developmental change of its response from depolarizing to hyperpolarizing (Ganguly et al. 2001). In cultured midbrain neurons we found that the GABA response changed from depolarizing to hyperpolarizing, although GABAA receptors had been blocked throughout development. In immature neurons prolonged exposure of the cells to nanomolar concentrations of GABA or brief repetitive applications of GABA strongly diminished the elevation of [Ca+]i by GABA. As revealed by gramicidin perforated-patch recording, reduced [Ca2+]i responses were due to a diminished driving force for Cl-. This suggests that immature neurons do not have an efficient inward transport that can compensate the loss of cytosolic Cl-resulting from sustained GABAA receptor activation by ambient GABA. Transient increases in external K+, which can induce voltage-dependent Cl- entry, restored GABA-induced [Ca2+]i elevations. In mature neurons, GABA reduced [Ca2+]i provided that background [Ca2+]i was elevated by the application of an L-type Ca2+ channel agonist. This was probably due to a hyperpolarization of the membrane by Cl- currents. K(+)-Cl- cotransport maintained the gradient for hyperpolarizing Cl-currents. We conclude that in immature midbrain neurons an inward Cl- transport is not effective although the GABA response is depolarizing. Further, GABA itself is not required for the developmental switch of GABAergic responses from depolarizing to hyperpolarizing in cultured midbrain neurons.

  18. Excitatory actions of GABA in the intact neonatal rodent hippocampus in vitro

    PubMed Central

    Valeeva, Guzel; Valiullina, Fliza; Khazipov, Roustem

    2013-01-01

    The excitatory action of gamma-aminobutyric acid (GABA) is considered to be a hallmark of the developing nervous system. However, in immature brain slices, excitatory GABA actions may be secondary to neuronal injury during slice preparation. Here, we explored GABA actions in the rodent intact hippocampal preparations and at different depths of hippocampal slices during the early post-natal period [post-natal days (P) 1–7]. We found that in the intact hippocampus at P1–3: (i) GABA exerts depolarizing action as seen in cell-attached single GABA(A) channel recordings; (ii) GABA(A) receptor (GABA(A)-R) agonist isoguvacine and synaptic activation of the GABA(A)-Rs increase the frequency of multiple unit activity and the frequency of the network-driven giant depolarizing potentials (GDPs); and that (iii) Na+–K+–2Cl- cotransporter (NKCC1) antagonist bumetanide suppresses GDPs and the excitatory actions of isoguvacine. In the hippocampal slices at P2–5, isoguvacine and synaptic activation of GABA(A)-Rs-evoked excitatory responses at all slice depths, including surface and core. Thus, GABA exerts excitatory actions in the intact hippocampus (P1–3) and at all depths of hippocampal slices (P2–5). Therefore, the excitatory actions of GABA in hippocampal slices during the first post-natal days are not due to neuronal injury during slice preparation, and the trauma-related excitatory GABA actions at the slice surface are a fundamentally different phenomenon observed during the second post-natal week. PMID:23467988

  19. Hyperpolarizing Inhibition Develops without Trophic support by GABA in Cultured Rat Midbrain Neurons

    PubMed Central

    Titz, Stefan; Hans, Michael; Kelsch, Wolfgang; Lewen, Andrea; Swandulla, Dieter; Misgeld, Ulrich

    2003-01-01

    During a limited period of early neuronal development, GABA is depolarizing and elevates [Ca2+]i, which mediates the trophic action of GABA in neuronal maturation. We tested the attractive hypothesis that GABA itself promotes the developmental change of its response from depolarizing to hyperpolarizing (Ganguly et al. 2001). In cultured midbrain neurons we found that the GABA response changed from depolarizing to hyperpolarizing, although GABAA receptors had been blocked throughout development. In immature neurons prolonged exposure of the cells to nanomolar concentrations of GABA or brief repetitive applications of GABA strongly diminished the elevation of [Ca2+]i by GABA. As revealed by gramicidin perforated-patch recording, reduced [Ca2+]i responses were due to a diminished driving force for Cl−. This suggests that immature neurons do not have an efficient inward transport that can compensate the loss of cytosolic Cl− resulting from sustained GABAA receptor activation by ambient GABA. Transient increases in external K+, which can induce voltage-dependent Cl− entry, restored GABA-induced [Ca2+]i elevations. In mature neurons, GABA reduced [Ca2+]i provided that background [Ca2+]i was elevated by the application of an L-type Ca2+ channel agonist. This was probably due to a hyperpolarization of the membrane by Cl− currents. K+-Cl− cotransport maintained the gradient for hyperpolarizing Cl− currents. We conclude that in immature midbrain neurons an inward Cl− transport is not effective although the GABA response is depolarizing. Further, GABA itself is not required for the developmental switch of GABAergic responses from depolarizing to hyperpolarizing in cultured midbrain neurons. PMID:12938674

  20. Pressure-Dependent Changes in the Release of GABA by Cerebrocortical Synaptosomes

    DTIC Science & Technology

    1989-01-01

    DTICL9? Undersea Biomedical Research, Vol. 16, No. 3. 1989 N V918f, NOV09198 B U Pressure-dependent changes in the release of GABA 1 by...Center, Bothesda. Maryland 20814-5055 Gilman SC, Colton JS, Dutka AJ. Pressure-dependent changes in the release of GABA by cerebrocortical synaptosomes...evoked [3H]--aminobutyric acid ( GABA ) release from isolated presynaptic terminals (synaptosomes) (15, 16). The current study was designed to evaluate

  1. Modulatory action of taurine on the release of GABA in cerebellar slices of the guinea pig.

    PubMed

    Namima, M; Okamoto, K; Sakai, Y

    1983-01-01

    For the purpose of demonstrating the action of taurine as a neuromodulator in addition to its suggested neurotransmitter function, the effects of taurine and muscimol on the depolarization-induced Ca-dependent release of [3H] gamma-aminobutyric acid ([3H]GABA) and L-[3H]glutamate in cerebellar slices from guinea pigs were investigated. The release of [3H]GABA was found to be greatly decreased by a GABA agonist, muscimol, and by taurine, but not by glycine. The release of L-[3H]glutamate was little affected by taurine. The release of [3H]GABA, was enhanced by bicuculline and strychnine, but not by picrotoxin, and the suppressive action of muscimol on the GABA release was antagonized by bicuculline, picrotoxin, and strychnine, suggesting the possible existence of presynaptic autoreceptors for GABA in the cerebellum. The suppressive action of taurine on the release of [3H]GABA, on the other hand, was blocked only by bicuculline. These results suggest that taurine reduced the release of [3H]GABA from cerebellar slices by acting on the GABA autoreceptors or, more likely, on other types of receptors that are sensitive to bicuculline. As a possible mechanism for this modulatory action of taurine, the blockade by this amino acid of the influx of Ca2+ into cerebellar tissues was tentatively suggested.

  2. Enhanced phasic GABA inhibition during the repair phase of stroke: a novel therapeutic target

    PubMed Central

    Paz, Jeanne T.; Wang, Eric Hou Jen; Badgely, Corrine; Olson, Andrew; Micheva, Kristina D.; Wang, Gordon; Lemmens, Robin; Tran, Kevin V.; Nishiyama, Yasuhiro; Liang, Xibin; Hamilton, Scott A.; O’Rourke, Nancy; Smith, Stephen J.; Huguenard, John R.; Bliss, Tonya M.

    2016-01-01

    Ischaemic stroke is the leading cause of severe long-term disability yet lacks drug therapies that promote the repair phase of recovery. This repair phase of stroke occurs days to months after stroke onset and involves brain remapping and plasticity within the peri-infarct zone. Elucidating mechanisms that promote this plasticity is critical for the development of new therapeutics with a broad treatment window. Inhibiting tonic (extrasynaptic) GABA signalling during the repair phase was reported to enhance functional recovery in mice suggesting that GABA plays an important function in modulating brain repair. While tonic GABA appears to suppress brain repair after stroke, less is known about the role of phasic (synaptic) GABA during the repair phase. We observed an increase in postsynaptic phasic GABA signalling in mice within the peri-infarct cortex specific to layer 5; we found increased numbers of α1 receptor subunit-containing GABAergic synapses detected using array tomography, and an associated increased efficacy of spontaneous and miniature inhibitory postsynaptic currents in pyramidal neurons. Furthermore, we demonstrate that enhancing phasic GABA signalling using zolpidem, a Food and Drug Administration (FDA)-approved GABA-positive allosteric modulator, during the repair phase improved behavioural recovery. These data identify potentiation of phasic GABA signalling as a novel therapeutic strategy, indicate zolpidem’s potential to improve recovery, and underscore the necessity to distinguish the role of tonic and phasic GABA signalling in stroke recovery. PMID:26685158

  3. Excitatory GABA: How a Correct Observation May Turn Out to be an Experimental Artifact

    PubMed Central

    Bregestovski, Piotr; Bernard, Christophe

    2012-01-01

    The concept of the excitatory action of GABA during early development is based on data obtained mainly in brain slice recordings. However, in vivo measurements as well as observations made in intact hippocampal preparations indicate that GABA is in fact inhibitory in rodents at early neonatal stages. The apparent excitatory action of GABA seems to stem from cellular injury due to the slicing procedure, which leads to accumulation of intracellular Cl− in injured neurons. This procedural artifact was shown to be attenuated through various manipulations such as addition of energy substrates more relevant to the in vivo situation. These observations question the very concept of excitatory GABA in immature neuronal networks. PMID:22529813

  4. [GABA--the basic mediator of excitation in the early stages of hippocampal development].

    PubMed

    Khazipov, R N; Zefirov, A L; Ben-Ari, E

    1998-01-01

    GABA is the principal neurotransmitter of inhibition in the adult mammalian brain. However, at early stages of development, including embryonic period and first week of postnatal life, GABA plays the role of main neurotransmitter of excitation. The paradoxical excitatory effect of GABA is due to an inversed chloride gradient and therefore a depolarizing direction of GABA-A receptor mediated responses. In addition, another type of GABAergic inhibition mediated by postsynaptic GABA-B receptors is not functional at early stage of life. In the neonatal rat hippocampus, GABA, acting via GABA-A receptors, activates voltage gated sodium and calcium channels and potentiates the activity of NMDA receptors by reducing their voltage dependent Mg2+ block. The temporal window when GABA exerts excitatory actions coincides with a particular pattern of activity of hippocampal neuronal network that is characterized by periodical giant depolarizing potentials (GDPs) reminiscent of interictal-like epileptiform discharges. Recent studies have shown that GDPs result from the synchronous discharge of GABAergic interneurons and principal glutamatergic pyramidal cells and are mediated by the synergistic excitatory actions of GABA-A and glutamate receptors. GDPs provide synchronous intracellular Ca2+ oscillations and may therefore be implicated in hebbian modulation of developing synapses and activity-dependent formation of the hippocampal network.

  5. GABA signaling promotes synapse elimination and axon pruning in developing cortical inhibitory interneurons.

    PubMed

    Wu, Xiaoyun; Fu, Yu; Knott, Graham; Lu, Jiangteng; Di Cristo, Graziella; Huang, Z Josh

    2012-01-04

    Accumulating evidence indicates that GABA acts beyond inhibitory synaptic transmission and regulates the development of inhibitory synapses in the vertebrate brain, but the underlying cellular mechanism is not well understood. We have combined live imaging of cortical GABAergic axons across time scales from minutes to days with single-cell genetic manipulation of GABA release to examine its role in distinct steps of inhibitory synapse formation in the mouse neocortex. We have shown previously, by genetic knockdown of GABA synthesis in developing interneurons, that GABA signaling promotes the maturation of inhibitory synapses and axons. Here we found that a complete blockade of GABA release in basket interneurons resulted in an opposite effect, a cell-autonomous increase in axon and bouton density with apparently normal synapse structures. These results not only demonstrate that GABA is unnecessary for synapse formation per se but also uncover a novel facet of GABA in regulating synapse elimination and axon pruning. Live imaging revealed that developing GABAergic axons form a large number of transient boutons, but only a subset was stabilized. Release blockade led to significantly increased bouton stability and filopodia density, increased axon branch extension, and decreased branch retraction. Our results suggest that a major component of GABA function in synapse development is transmission-mediated elimination of subsets of nascent contacts. Therefore, GABA may regulate activity-dependent inhibitory synapse formation by coordinately eliminating certain nascent contacts while promoting the maturation of other nascent synapses.

  6. A study on quality components and sleep-promoting effects of GABA black tea.

    PubMed

    Zhao, Wenfang; Li, Yun; Ma, William; Ge, Yazhong; Huang, Yahui

    2015-10-01

    The aims of this study were to analyze the changes in quality components of gamma (γ)-aminobutyric acid (GABA) black tea during processing, and to investigate the effect of three dosages of GABA black tea on sleep improvement. The results showed that the GABA content was increased significantly up to 2.70 mg g(-1) after vacuum anaerobic and aerobic treatment. In addition, the content of GABA after drying reached 2.34 mg g(-1), which achieved the standard of GABA tea. During the entire processing of GABA black tea, the contents of tea polyphenols, caffeine and total catechins displayed a gradually descending trend, while the contents of free amino acids and GABA were firstly increased, and then reduced. The GABA black tea had significant effects on prolonging the sleeping time with sodium pentobarbital (P < 0.05) and significantly enhancing the sleeping rate induced by sodium pentobarbital at a sub-threshold dose (P < 0.05). But its effect on shortening the sleeping latency period induced by sodium barbital was not significant (P > 0.05). It had no effect on directly inducing sleep and the mouse body weight. The extract of GABA black tea improved the sleeping quality of mice to extend with an optimal effect being found in the high dose-treated mice.

  7. Edited Magnetic Resonance Spectroscopy Detects an Age-Related Decline in Nonhuman Primate Brain GABA Levels

    PubMed Central

    Killiany, Ronald J.

    2016-01-01

    Recent research had shown a correlation between aging and decreasing Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the brain. However, how GABA level varies with age in the medial portion of the brain has not yet been studied. The purpose of this study was to investigate the GABA level variation with age focusing on the posterior cingulate cortex, which is the “core hub” of the default mode network. In this study, 14 monkeys between 4 and 21 years were recruited, and MEGA-PRESS MRS was performed to measure GABA levels, in order to explore a potential link between aging and GABA. Our results showed that a correlation between age and GABA+/Creatine ratio was at the edge of significance (r = −0.523, p = 0.081). There was also a near-significant trend between gray matter/white matter ratio and the GABA+/Creatine ratio (r = −0.518, p = 0.0848). Meanwhile, the correlation between age and grey matter showed no significance (r = −0.028, p = 0.93). Therefore, age and gray matter/white matter ratio account for different part of R-squared (adjusted R-squared = 0.5187) as independent variables for predicting GABA levels. Adjusted R-squared is about 0.5 for two independent variables. These findings suggest that there is internal neurochemical variation of GABA levels in the nonhuman primates associated with normal aging and structural brain decline. PMID:27660760

  8. THIP and isoguvacine are partial agonists of GABA-stimulated benzodiazepine receptor binding.

    PubMed

    Karobath, M; Lippitsch, M

    1979-10-15

    The effects of THIP and isoguvacine on 3H-flunitrazepam binding to washed membranes prepared from the cerebral cortex of adult rats have been examined. THIP, which has only minimal stimulatory effects on benzodiazepine (BZ) receptor binding, has been found to inhibit the stimulation induced by small concentrations (2 microM) of exogenous GABA. While isoguvacine stimulates BZ receptor binding, although to a smaller extent than GABA, it also antagonizes the stimulation of BZ receptor binding induced by GABA. Thus THIP and isoguvacine exhibit the properties of a partial agonist of GABA-stimulated BZ receptor binding.

  9. Effect of three anorectic drugs on brain GABA levels and synthesis in the Zucker rat.

    PubMed

    Orosco, M; Bremond, J; Jacquot, C; Cohen, Y

    1983-01-01

    1. Genetically obese Zucker rats and their lean littermates were submitted to a subchronic treatment with fenfluramine, mazindol and amphetamine. GABA levels and synthesis index were measured in different brain areas. 2. GABA levels, similar in obese and lean controls, were not changed after the three treatments. 3. A higher synthesis index of GABA was found in lean rats, in the striatum after mazindol and in the hypothalamus after amphetamine. 4. The three drugs increased the synthesis index of GABA in the remainder of the brain of both obese and lean rats.

  10. Neurotransmitters as food supplements: the effects of GABA on brain and behavior.

    PubMed

    Boonstra, Evert; de Kleijn, Roy; Colzato, Lorenza S; Alkemade, Anneke; Forstmann, Birte U; Nieuwenhuis, Sander

    2015-01-01

    Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the human cortex. The food supplement version of GABA is widely available online. Although many consumers claim that they experience benefits from the use of these products, it is unclear whether these supplements confer benefits beyond a placebo effect. Currently, the mechanism of action behind these products is unknown. It has long been thought that GABA is unable to cross the blood-brain barrier (BBB), but the studies that have assessed this issue are often contradictory and range widely in their employed methods. Accordingly, future research needs to establish the effects of oral GABA administration on GABA levels in the human brain, for example using magnetic resonance spectroscopy. There is some evidence in favor of a calming effect of GABA food supplements, but most of this evidence was reported by researchers with a potential conflict of interest. We suggest that any veridical effects of GABA food supplements on brain and cognition might be exerted through BBB passage or, more indirectly, via an effect on the enteric nervous system. We conclude that the mechanism of action of GABA food supplements is far from clear, and that further work is needed to establish the behavioral effects of GABA.

  11. Comparing GABA-dependent physiological measures of inhibition with proton magnetic resonance spectroscopy measurement of GABA using ultra-high-field MRI.

    PubMed

    Dyke, Katherine; Pépés, Sophia E; Chen, Chen; Kim, Soyoung; Sigurdsson, Hilmar P; Draper, Amelia; Husain, Masud; Nachev, Parashkev; Gowland, Penelope A; Morris, Peter G; Jackson, Stephen R

    2017-03-09

    Imbalances in glutamatergic (excitatory) and GABA (inhibitory) signalling within key brain networks are thought to underlie many brain and mental health disorders, and for this reason there is considerable interest in investigating how individual variability in localised concentrations of these molecules relate to brain disorders. Magnetic resonance spectroscopy (MRS) provides a reliable means of measuring, in vivo, concentrations of neurometabolites such as GABA, glutamate and glutamine that can be correlated with brain function and dysfunction. However, an issue of much debate is whether the GABA observed and measured using MRS represents the entire pool of GABA available for measurement (i.e., metabolic, intracellular, and extracellular) or is instead limited to only some portion of it. GABA function can also be investigated indirectly in humans through the use of non-invasive transcranial magnetic stimulation (TMS) techniques that can be used to measure cortical excitability and GABA-mediated physiological inhibition. To investigate this issue further we collected in a single session both types of measurement, i.e., TMS measures of cortical excitability and physiological inhibition and ultra-high-field (7 T) MRS measures of GABA, glutamate and glutamine, from the left sensorimotor cortex of the same group of right-handed individuals. We found that TMS and MRS measures were largely uncorrelated with one another, save for the plateau of the TMS IO curve that was negatively correlated with MRS-Glutamate (Glu) and intra-cortical facilitation (10ms ISI) that was positively associated with MRS-Glutamate concentration. These findings are consistent with the view that the GABA concentrations measured using the MRS largely represent pools of GABA that are linked to tonic rather than phasic inhibition and thus contribute to the inhibitory tone of a brain area rather than GABAergic synaptic transmission.

  12. Expression of brain derived neurotrophic factor, activity-regulated cytoskeleton protein mRNA, and enhancement of adult hippocampal neurogenesis in rats after sub-chronic and chronic treatment with the triple monoamine re-uptake inhibitor tesofensine.

    PubMed

    Larsen, Marianne H; Rosenbrock, Holger; Sams-Dodd, Frank; Mikkelsen, Jens D

    2007-01-26

    The changes of gene expression resulting from long-term exposure to monoamine antidepressant drugs in experimental animals are key to understanding the mechanisms of action of this class of drugs in man. Many of these genes and their products are either relevant biomarkers or directly involved in structural changes that are perhaps necessary for the antidepressant effect. Tesofensine is a novel triple monoamine reuptake inhibitor that acts to increase noradrenaline, serotonin, and dopamine neurotransmission. This study was undertaken to examine the effect of sub-chronic (5 days) and chronic (14 days) administration of Tesofensine on the expression of brain derived neurotrophic factor (BDNF) and activity-regulated cytoskeleton protein (Arc) in the rat hippocampus. Furthermore, hippocampi from the same animals were used to investigate the effect on cell proliferation by means of Ki-67- and NeuroD-immunoreactivity. We find that chronic, but not sub-chronic treatment with Tesofensine increases BDNF mRNA in the CA3 region of the hippocampus (35%), and Arc mRNA in the CA1 of the hippocampus (65%). Furthermore, the number of Ki-67- and neuroD-positive cells increased after chronic, but not sub-chronic treatment. This study shows that Tesofensine enhances hippocampal gene expression and new cell formation indicative for an antidepressant potential of this novel drug substance.

  13. Multiple Forms of Endocannabinoid and Endovanilloid Signaling Regulate the Tonic Control of GABA Release

    PubMed Central

    Lee, Sang-Hun; Ledri, Marco; Tóth, Blanka; Marchionni, Ivan; Henstridge, Christopher M.; Dudok, Barna; Kenesei, Kata; Barna, László; Szabó, Szilárd I.; Renkecz, Tibor; Oberoi, Michelle; Watanabe, Masahiko; Limoli, Charles L.; Horvai, George; Soltesz, Ivan

    2015-01-01

    Persistent CB1 cannabinoid receptor activity limits neurotransmitter release at various synapses throughout the brain. However, it is not fully understood how constitutively active CB1 receptors, tonic endocannabinoid signaling, and its regulation by multiple serine hydrolases contribute to the synapse-specific calibration of neurotransmitter release probability. To address this question at perisomatic and dendritic GABAergic synapses in the mouse hippocampus, we used a combination of paired whole-cell patch-clamp recording, liquid chromatography/tandem mass spectrometry, stochastic optical reconstruction microscopy super-resolution imaging, and immunogold electron microscopy. Unexpectedly, application of the CB1 antagonist and inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide], but not the neutral antagonist NESS0327 [8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-5,6-dihydro-4H-benzo[2,3]cyclohepta[2,4-b]pyrazole-3-carboxamine], significantly increased synaptic transmission between CB1-positive perisomatic interneurons and CA1 pyramidal neurons. JZL184 (4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate), a selective inhibitor of monoacylglycerol lipase (MGL), the presynaptic degrading enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), elicited a robust increase in 2-AG levels and concomitantly decreased GABAergic transmission. In contrast, inhibition of fatty acid amide hydrolase (FAAH) by PF3845 (N-pyridin-3-yl-4-[[3-[5-(trifluoromethyl)pyridin-2-yl]oxyphenyl]methyl]piperidine-1-carboxamide) elevated endocannabinoid/endovanilloid anandamide levels but did not change GABAergic synaptic activity. However, FAAH inhibitors attenuated tonic 2-AG increase and also decreased its synaptic effects. This antagonistic interaction required the activation of the transient receptor potential vanilloid receptor TRPV1, which was concentrated on postsynaptic

  14. The supramammillary nucleus mediates primary reinforcement via GABA(A) receptors.

    PubMed

    Ikemoto, Satoshi

    2005-06-01

    The supramammillary nucleus (SUM), a dorsal layer of the mammillary body, has recently been implicated in positive reinforcement. The present study examined whether GABA(A) receptors in the SUM or adjacent regions are involved in primary reinforcement using intracranial self-administration procedures. Rats learned quickly to lever-press for infusions of the GABA(A) antagonist picrotoxin into the SUM. Although picrotoxin was also self-administered into the posterior hypothalamic nuclei and anterior ventral tegmental area, these regions were less responsive to lower doses of picrotoxin than the SUM. The finding that rats learned to respond selectively on the lever triggering drug infusions is consistent with picrotoxin's reinforcing effect. Coadministration of the GABA(A) agonist muscimol disrupted picrotoxin self-administration, and another GABA(A) antagonist, bicuculline, was also self-administered into the SUM; thus, the reinforcing effect of picrotoxin is mediated by GABA(A) receptors. Since rats did not self-administer the GABA(B) antagonist 2-hydroxysaclofen into the SUM, the role of GABA(B) receptors may be distinct from that of GABA(A) receptors. Pretreatment with the dopamine receptor antagonist SCH 23390 (0.05 mg/kg, i.p.) extinguished picrotoxin self-administration into the SUM, suggesting that the reinforcing effects of GABA(A) receptor blockade depend on normal dopamine transmission. In conclusion, the blockade of GABA(A) receptors in the SUM is reinforcing, and the brain 'reward' circuitry appears to be tonically inhibited via supramammillary GABA(A) receptors and more extensive than the meso-limbic dopamine system.

  15. Hepatic uptake of gamma-butyrobetaine, a precursor of carnitine biosynthesis, in rats.

    PubMed

    Fujita, Masaharu; Nakanishi, Takeo; Shibue, Yuta; Kobayashi, Daisuke; Moseley, Richard H; Shirasaka, Yoshiyuki; Tamai, Ikumi

    2009-10-01

    Gamma-butyrobetaine (GBB) is a precursor in the biosynthesis of carnitine, which plays an important role in the beta-oxidation of fatty acids, and is converted to carnitine by gamma-butyrobetaine dioxygenase (BBD) predominantly in liver. We investigated the molecular mechanism of hepatic uptake of GBB in rat hepatocytes. Cellular localization of rat Octn2 (rOctn2:Slc22A5) was studied by Western blot analysis. Uptake of deuterated GBB (d(3)-GBB) was examined in HEK293 cells expressing rOctn2 (HEK293/rOctn2) and freshly isolated rat hepatocytes. d(3)-GBB was quantified by use of liquid chromatography-tandem mass spectrometry. Western blot analysis demonstrated an expression of OCTN2 protein in hepatic basolateral membrane but not in bile canalicular membrane fraction. Furthermore, we found that d(3)-GBB was taken up by rOctn2 in an Na(+)-dependent manner with K(m) value of 13 microM. The apparent K(m) value for d(3)-GBB transport in freshly isolated rat hepatocytes was 9 microM. d(3)-GBB uptake by the rat hepatocytes was inhibited by gamma-aminobutyric acid (GABA) to 30% of the control, whereas it was inhibited by carnitine to 62% of the control, even at 500 microM. Furthermore, d(3)-GBB uptake by rat hepatocytes was decreased by 45% with rat Gat2 (Slc6A13, a major liver GABA transporter) silenced by the microRNA method. Accordingly, the present study clearly demonstrates that GBB is taken up by hepatocytes for carnitine biosynthesis not only via Octn2 but also via the GABA transporter, possibly Gat2.

  16. Clarithromycin in GABA-related Hypersomnolence: A Randomized, Crossover Trial

    PubMed Central

    Trotti, Lynn Marie; Saini, Prabhjyot; Bliwise, Donald L.; Freeman, Amanda A.; Jenkins, Andrew; Rye, David B.

    2016-01-01

    Objective Some central hypersomnolence syndromes are associated with a positive allosteric modulator of GABA-A receptors in cerebrospinal fluid. Negative allosteric modulators of GABA-A receptors, including clarithromycin, have been reported to reduce sleepiness in these patients. We sought to systematically assess the effects of clarithromycin on objective vigilance and subjective sleepiness. Methods This was a five-week, randomized, placebo-controlled, double-blind, crossover trial of clarithromycin 500 mg with breakfast and lunch, in patients with hypersomnolence syndromes (excluding narcolepsy with cataplexy) and evidence for abnormal cerebrospinal fluid potentiation of GABA-A receptors. The study occurred at a university-affiliated medical center. The primary outcome measure was median reaction time on the psychomotor vigilance task (PVT) at week 2 in each condition. Secondary outcomes included the Epworth Sleepiness Scale, Stanford Sleepiness Scale, Functional Outcomes of Sleep, Pittsburgh Sleep Quality Index, the SF-36, and additional PVT measures. Results Twenty-three patients began treatment. Three patients dropped out, and final analyses were performed on twenty complete cases. Median reaction time was not significantly different between clarithromycin and placebo. Subjective measures of sleepiness were significantly improved on clarithromycin versus placebo. Altered taste perception occurred, but was the only side effect more common on clarithromycin than placebo. No serious adverse events occurred. Interpretation Subjective sleepiness, but not psychomotor vigilance, improved during a two-week course of clarithromycin. Although additional studies are needed, this suggests that clarithromycin may be a reasonable treatment option in patients with treatment-refractory hypersomnolence. This trial was registered at clinicaltrials.gov (NCT01146600) and supported by the American Sleep Medicine Foundation. PMID:26094838

  17. Glutamate and GABA in Vestibulo-Sympathetic Pathway Neurons

    PubMed Central

    Holstein, Gay R.; Friedrich, Victor L. Jr.; Martinelli, Giorgio P.

    2016-01-01

    The vestibulo-sympathetic reflex (VSR) actively modulates blood pressure during changes in posture. This reflex allows humans to stand up and quadrupeds to rear or climb without a precipitous decline in cerebral perfusion. The VSR pathway conveys signals from the vestibular end organs to the caudal vestibular nuclei. These cells, in turn, project to pre-sympathetic neurons in the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively). The present study assessed glutamate- and GABA-related immunofluorescence associated with central vestibular neurons of the VSR pathway in rats. Retrograde FluoroGold tract tracing was used to label vestibular neurons with projections to RVLM or CVLM, and sinusoidal galvanic vestibular stimulation (GVS) was employed to activate these pathways. Central vestibular neurons of the VSR were identified by co-localization of FluoroGold and cFos protein, which accumulates in some vestibular neurons following galvanic stimulation. Triple-label immunofluorescence was used to co-localize glutamate- or GABA- labeling in the identified VSR pathway neurons. Most activated projection neurons displayed intense glutamate immunofluorescence, suggestive of glutamatergic neurotransmission. To support this, anterograde tracer was injected into the caudal vestibular nuclei. Vestibular axons and terminals in RVLM and CVLM co-localized the anterograde tracer and vesicular glutamate transporter-2 signals. Other retrogradely-labeled cFos-positive neurons displayed intense GABA immunofluorescence. VSR pathway neurons of both phenotypes were present in the caudal medial and spinal vestibular nuclei, and projected to both RVLM and CVLM. As a group, however, triple-labeled vestibular cells with intense glutamate immunofluorescence were located more rostrally in the vestibular nuclei than the GABAergic neurons. Only the GABAergic VSR pathway neurons showed a target preference, projecting predominantly to CVLM. These data provide the first

  18. Chronic stress shifts the GABA reversal potential in the hippocampus and increases seizure susceptibility

    PubMed Central

    MacKenzie, Georgina; Maguire, Jamie

    2014-01-01

    The most commonly reported precipitating factor for seizures is stress. However, the underlying mechanisms whereby stress triggers seizures are not yet fully understood. Here we demonstrate a potential mechanism underlying changes in neuronal excitability in the hippocampus following chronic stress, involving a shift in the reversal potential for GABA (EGABA) associated with a dephosphorylation of the potassium chloride co-transporter, KCC2. Mice subjected to chronic restraint stress (30 mins/day for 14 consecutive days) exhibit an increase in serum corticosterone levels which is associated with increased susceptibility to seizures induced with kainic acid (20 mg/kg). Following chronic stress, but not acute stress, we observe a dephosphorylation of KCC2 residue S940, which regulates KCC2 cell surface expression and function, in the hippocampus. To determine the impact of alterations in KCC2 expression following chronic stress, we performed gramicidin perforated patch recordings to measure changes in EGABA and neuronal excitability of principal hippocampal neurons. We observe a depolarizing shift in EGABA in hippocampal CA1 pyramidal neurons after chronic stress. In addition, there is an increase in the intrinsic excitability of CA1 pyramidal neurons, evident by a shift in the input-output curve which could be reversed with the NKCC1 inhibitor, bumetanide. These data uncover a potential mechanism involving chronic stress-induced plasticity in chloride homeostasis which may contribute to stress-induced seizure susceptibility. PMID:25524838

  19. [Effect of the GABA derivative phenibut on learning].

    PubMed

    Shul'gina, G I; Ziablitseva, E A

    2005-01-01

    The learning effect of phenibut, a beta-phenyl derivative of the inhibitory neuromediator GABA, used in a subcutaneous dose of 40 mg/kg 2 hours before each experiment, was studied in rabbits. The injection of phenibut was shown to enhance not only the inhibitory, but also excitatory components of cerebral cortical neuronal responses to all applied stimuli. The findings support the concept that the GABAergic neuromediator system is involved in the elaboration of internal inhibition and explain the sense of intracerebral processes that ensure both the sedative properties of phenibut and its ability to improve patients' systemic tone and health status when brain dysfunctions are treated.

  20. [Cardioprotective effect of GABA derivatives in acute alcohol intoxication].

    PubMed

    Perfilova, V N; Tiurenkov, I N; Berestovitskaia, V M; Vasil'eva, O S

    2006-01-01

    Cardioprotective properties of GABA analogs under conditions of acute alcoholic intoxication have been studied using the following functional tests: volume loads, tests for adrenoreactivity, and maximum isometric load. The experiments showed that a 32% aqueous ethanol solution intraperitoneally injected in a dose of 8 g/kg produces a cardiotoxic action, which is manifested by a decrease in the inotropic reserve in load tests. Citrocard (50 mg/kg), phenibut (50 mg/kg), and piracetam (200 mg/kg) prevent the alcohol-induced myocardium injury, as shown by the heart contractility retained on a higher level in the test group than in the control group.

  1. Thyroid Scan and Uptake

    MedlinePlus

    ... procedures within the last two months that used iodine-based contrast material. Your doctor will instruct you ... a type of nuclear medicine imaging. The radioactive iodine uptake test (RAIU) is also known as a ...

  2. Thyroid Scan and Uptake

    MedlinePlus Videos and Cool Tools

    ... A thyroid scan is a type of nuclear medicine imaging. The radioactive iodine uptake test (RAIU) is ... thyroid function, but does not involve imaging. Nuclear medicine is a branch of medical imaging that uses ...

  3. Modulation of GABA-A receptors of astrocytes and STC-1 cells by taurine structural analogs.

    PubMed

    Reyes-Haro, Daniel; Cabrera-Ruíz, Elizabeth; Estrada-Mondragón, Argel; Miledi, Ricardo; Martínez-Torres, Ataúlfo

    2014-11-01

    Taurine activates and modulates GABA receptors in vivo as well as those expressed in heterologous systems. This study aimed to determine whether the structural analogs of taurine: homotaurine and hypotaurine, have the ability to activate GABA-A receptors that include GABAρ subunits. The expression of GABA-A receptors containing GABAρ has been reported in the STC-1 cells and astrocytes. In both cell types, taurine, homo-, and hypotaurine gated with low efficiency a picrotoxin-sensitive GABA-A receptor. The known bimodal modulatory effect of taurine on GABAρ receptors was not observed; however, differences between the activation and deactivation rates were detected when they were perfused together with GABA. In silico docking simulations suggested that taurine, hypo-, and homotaurine do not form a cation-π interaction such as that generated by GABA in the agonist-binding site of GABAρ. This observation complements the electrophysiological data suggesting that taurine and its analogs act as partial agonists of GABA-A receptors. All the observations above suggest that the structural analogs of taurine are partial agonists of GABA-A receptors that occupy the agonist-binding site, but their structures do not allow the proper interaction with the receptor to fully gate its Cl(-) channel.

  4. Inhibitory effect of GABA on sympathetic neurotransmission in rabbit ear artery.

    PubMed

    Manzini, S; Maggi, C A; Meli, A

    1985-01-01

    GABA 100 microM inhibited neurogenic vasoconstrictor responses elicited in rabbit ear artery by field stimulation at various frequencies. GABA was ineffective both on resting tone and on noradrenaline (0.05-5 microM)- and high-K+ (24-54 mM)-induced tonic contraction. GABA effectiveness against field stimulation-induced vasoconstriction was inversely related to the frequency of stimulation. This action of GABA was mimicked by a selective GABAB agonist, such as baclofen (100 microM), but not by selective GABAA agonists as muscimol (100 microM) and homotaurine (100 microM). The selective GABAB antagonists 5-aminovaleric acid (1 mM) and homotaurine (100 microM) completely suppressed the inhibitory effect of GABA on field stimulation-induced vasoconstrictions. GABA action was partially reversed also by the selective GABAA antagonist picrotoxin (100 microM); however, this drug "per se" determined an increase in amplitude of field stimulation-induced contractions which in turn could have determined the minor effect of GABA. As a whole these data suggest that GABA can inhibit sympathetic neurotransmission in rabbit ear artery through the stimulation of a prejunctional receptor of the GABAB subtype.

  5. Immature Responses to GABA in Fragile X Neurons Derived from Human Embryonic Stem Cells

    PubMed Central

    Telias, Michael; Segal, Menahem; Ben-Yosef, Dalit

    2016-01-01

    Fragile X Syndrome (FXS) is the most common form of inherited cognitive disability. However, functional deficiencies in FX neurons have been described so far almost exclusively in animal models. In a recent study we found several functional deficits in FX neurons differentiated in-vitro from human embryonic stem cells (hESCs), including their inability to fire repetitive action potentials, and their lack of synaptic activity. Here, we investigated the responses of such neurons to pulse application of the neurotransmitter GABA. We found two distinct types of responses to GABA and sensitivity to the GABA-A receptor antagonist bicuculline; type 1 (mature) characterized by non-desensitized responses to GABA as well as a high sensitivity to bicuculline, and type 2 (immature) which are desensitized to GABA and insensitive to bicuculline. Type 1 responses were age-dependent and dominant in mature WT neurons. In contrast, FX neurons expressed primarily type 2 phenotype. Expression analysis of GABA-A receptor subunits demonstrated that this bias in human FX neurons was associated with a significant alteration in the expression pattern of the GABA-A receptor subunits α2 and β2. Our results indicate that FMRP may play a role in the development of the GABAergic synapse during neurogenesis. This is the first demonstration of the lack of a mature response to GABA in human FX neurons and may explain the inappropriate synaptic functions in FXS. PMID:27242433

  6. Phasic, Nonsynaptic GABA-A Receptor-Mediated Inhibition Entrains Thalamocortical Oscillations

    PubMed Central

    Rovó, Zita; Mátyás, Ferenc; Barthó, Péter; Slézia, Andrea; Lecci, Sandro; Pellegrini, Chiara; Astori, Simone; Dávid, Csaba; Hangya, Balázs

    2014-01-01

    GABA-A receptors (GABA-ARs) are typically expressed at synaptic or nonsynaptic sites mediating phasic and tonic inhibition, respectively. These two forms of inhibition conjointly control various network oscillations. To disentangle their roles in thalamocortical rhythms, we focally deleted synaptic, γ2 subunit-containing GABA-ARs in the thalamus using viral intervention in mice. After successful removal of γ2 subunit clusters, spontaneous and evoked GABAergic synaptic currents disappeared in thalamocortical cells when the presynaptic, reticular thalamic (nRT) neurons fired in tonic mode. However, when nRT cells fired in burst mode, slow phasic GABA-AR-mediated events persisted, indicating a dynamic, burst-specific recruitment of nonsynaptic GABA-ARs. In vivo, removal of synaptic GABA-ARs reduced the firing of individual thalamocortical cells but did not abolish slow oscillations or sleep spindles. We conclude that nonsynaptic GABA-ARs are recruited in a phasic manner specifically during burst firing of nRT cells and provide sufficient GABA-AR activation to control major thalamocortical oscillations. PMID:24849349

  7. Effect of GABA, a Bacterial Metabolite, on Pseudomonas fluorescens Surface Properties and Cytotoxicity

    PubMed Central

    Dagorn, Audrey; Chapalain, Annelise; Mijouin, Lily; Hillion, Mélanie; Duclairoir-Poc, Cécile; Chevalier, Sylvie; Taupin, Laure; Orange, Nicole; Feuilloley, Marc G. J.

    2013-01-01

    Different bacterial species and, particularly Pseudomonas fluorescens, can produce gamma-aminobutyric acid (GABA) and express GABA-binding proteins. In this study, we investigated the effect of GABA on the virulence and biofilm formation activity of different strains of P. fluorescens. Exposure of a psychotropic strain of P. fluorescens (MF37) to GABA (10−5 M) increased its necrotic-like activity on eukaryotic (glial) cells, but reduced its apoptotic effect. Conversely, muscimol and bicuculline, the selective agonist and antagonist of eukaryote GABAA receptors, respectively, were ineffective. P. fluorescens MF37 did not produce biosurfactants, and its caseinase, esterase, amylase, hemolytic activity or pyoverdine productions were unchanged. In contrast, the effect of GABA was associated to rearrangements of the lipopolysaccharide (LPS) structure, particularly in the lipid A region. The surface hydrophobicity of MF37 was marginally modified, and GABA reduced its biofilm formation activity on PVC, but not on glass, although the initial adhesion was increased. Five other P. fluorescens strains were studied, and only one, MFP05, a strain isolated from human skin, showed structural differences of biofilm maturation after exposure to GABA. These results reveal that GABA can regulate the LPS structure and cytotoxicity of P. fluorescens, but that this property is specific to some strains. PMID:23743829

  8. Beta Peak Frequencies at Rest Correlate with Endogenous GABA+/Cr Concentrations in Sensorimotor Cortex Areas

    PubMed Central

    Baumgarten, Thomas J.; Oeltzschner, Georg; Hoogenboom, Nienke; Wittsack, Hans-Jörg; Schnitzler, Alfons; Lange, Joachim

    2016-01-01

    Neuronal oscillatory activity in the beta band (15–30 Hz) is a prominent signal within the human sensorimotor cortex. Computational modeling and pharmacological modulation studies suggest an influence of GABAergic interneurons on the generation of beta band oscillations. Accordingly, studies in humans have demonstrated a correlation between GABA concentrations and power of beta band oscillations. It remains unclear, however, if GABA concentrations also influence beta peak frequencies and whether this influence is present in the sensorimotor cortex at rest and without pharmacological modulation. In the present study, we investigated the relation between endogenous GABA concentration (measured by magnetic resonance spectroscopy) and beta oscillations (measured by magnetoencephalography) at rest in humans. GABA concentrations and beta band oscillations were measured for left and right sensorimotor and occipital cortex areas. A significant positive linear correlation between GABA concentration and beta peak frequency was found for the left sensorimotor cortex, whereas no significant correlations were found for the right sensorimotor and the occipital cortex. The results show a novel connection between endogenous GABA concentration and beta peak frequency at rest. This finding supports previous results that demonstrated a connection between oscillatory beta activity and pharmacologically modulated GABA concentration in the sensorimotor cortex. Furthermore, the results demonstrate that for a predominantly right-handed sample, the correlation between beta band oscillations and endogenous GABA concentrations is evident only in the left sensorimotor cortex. PMID:27258089

  9. Temperature dependence and GABA modulation of (TH)triazolam binding in the rat brain

    SciTech Connect

    Earle, M.E.; Concas, A.; Wamsley, J.K.; Yamamura, H.I.

    1987-07-27

    The hypnotic triazolam (TZ), a triazolobenzodiazepine displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. The authors major objectives were the direct measurement of the temperature dependence and the gamma-aminobutyric acid (GABA) effect of (TH)TZ binding in the rat brain. Saturation studies showed a shift to lower affinity with increasing temperatures (K/sub d/ = 0.27 +/- 08 nM at 0C; K/sub d/ = 1.96 +/- 0.85 nM at 37C) while the B/sub max/ values remained unchanged (1220 +/- 176 fmoles/mg protein at 0C and 1160 +/- 383 fmoles/mg protein at 37C). Saturation studies of (TH)TZ binding in the presence or absence of GABA (100 M) showed a GABA-shift. At 0C the K/sub d/ values were (K/sub d/ = 0.24 +/- 0.03 nM/-GABA; K/sub d/ = 0.16 +/- 0.04/+GABA) and at 37C the K/sub d/ values were (K/sub d/ = 1.84 +/- 0.44 nM/-GABA; K/sub d/ = 0.95 +/- 0.29 nM/+GABA). In contrast to reported literature, the authors findings show that TZ interacts with benzodiazepine receptors with a temperature dependence and GABA-shift consistent with predicted behavior for benzodiazepine agonists. 20 references, 3 tables.

  10. Cyclic AMP-dependent protein kinase phosphorylation facilitates GABA(B) receptor-effector coupling.

    PubMed

    Couve, A; Thomas, P; Calver, A R; Hirst, W D; Pangalos, M N; Walsh, F S; Smart, T G; Moss, S J

    2002-05-01

    GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.

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

    PubMed

    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.

  12. Elevated levels of GABA+ in migraine detected using (1) H-MRS.

    PubMed

    Aguila, Maria-Eliza R; Lagopoulos, Jim; Leaver, Andrew M; Rebbeck, Trudy; Hübscher, Markus; Brennan, Patrick C; Refshauge, Kathryn M

    2015-07-01

    γ-Aminobutyric acid (GABA) has been implicated in several pain conditions, yet no study has systematically evaluated GABA levels in migraine using (1) H-MRS. The accurate detection, separation and quantification of GABA in individuals with migraine could elucidate the role of this neurotransmitter in migraine pathophysiology. Such information may eventually be useful in the diagnosis and development of more effective treatments for migraine. The aims of this study were therefore to compare the concentration of GABA+ in individuals with migraine with that in asymptomatic individuals, and to determine the diagnostic potential of GABA+ in the classification of those with or without migraine. In this case-control study, GABA+ levels in the brain were determined in 19 participants with migraine and 19 matched controls by (1) H-MRS using Mescher-Garwood point-resolved spectroscopy (MEGA-PRESS) sequence. The diagnostic accuracy of GABA+ for the detection of migraine and the optimal cut-off value were determined by receiver operating characteristic analysis. GABA+ levels were significantly higher (p = 0.002) in those with migraine [median, 1.41 institutional units (IU); interquartile range, 1.31-1.50 IU] than in controls (median, 1.18 IU; interquartile range, 1.12-1.35 IU). The GABA+ concentration appears to have good accuracy for the classification of individuals with or without migraine [area under the curve (95% confidence interval), 0.837 (0.71-0.96); p < 0.001]. The optimal GABA+ cut-off value for migraine was 1.30 IU, with a sensitivity of 84.2%, specificity of 68.4% and positive likelihood ratio of +2.67. The outcomes of this study suggest altered GABA metabolism in migraine. These results add to the scarce evidence on the putative role of GABA in migraine and provide a basis to further explore the causal relationship between GABA+ and the pathophysiology of migraine. This study also demonstrates that GABA+ concentration has good diagnostic accuracy for migraine

  13. Influence of GABA and GABA-producing Lactobacillus brevis DPC 6108 on the development of diabetes in a streptozotocin rat model.

    PubMed

    Marques, T M; Patterson, E; Wall, R; O'Sullivan, O; Fitzgerald, G F; Cotter, P D; Dinan, T G; Cryan, J F; Ross, R P; Stanton, C

    2016-06-01

    The aim of this study was to investigate if dietary administration of γ-aminobutyric acid (GABA)-producing Lactobacillus brevis DPC 6108 and pure GABA exert protective effects against the development of diabetes in streptozotocin (STZ)-induced diabetic Sprague Dawley rats. In a first experiment, healthy rats were divided in 3 groups (n=10/group) receiving placebo, 2.6 mg/kg body weight (bw) pure GABA or L. brevis DPC 6108 (~10(9)microorganisms). In a second experiment, rats (n=15/group) were randomised to five groups and four of these received an injection of STZ to induce type 1 diabetes. Diabetic and non-diabetic controls received placebo [4% (w/v) yeast extract in dH2O], while the other three diabetic groups received one of the following dietary supplements: 2.6 mg/kg bw GABA (low GABA), 200 mg/kg bw GABA (high GABA) or ~10(9) L. brevis DPC 6108. L. brevis DPC 6108 supplementation was associated with increased serum insulin levels (P<0.05), but did not alter other metabolic markers in healthy rats. Diabetes induced by STZ injection decreased body weight (P<0.05), increased intestinal length (P<0.05) and stimulated water and food intake. Insulin was decreased (P<0.05), whereas glucose was increased (P<0.001) in all diabetic groups, compared with non-diabetic controls. A decrease (P<0.01) in glucose levels was observed in diabetic rats receiving L. brevis DPC 6108, compared with diabetic-controls. Both the composition and diversity of the intestinal microbiota were affected by diabetes. Microbial diversity in diabetic rats supplemented with low GABA was not reduced (P>0.05), compared with non-diabetic controls while all other diabetic groups displayed reduced diversity (P<0.05). L. brevis DPC 6108 attenuated hyperglycaemia induced by diabetes but additional studies are needed to understand the mechanisms involved in this reduction.

  14. Activation of metabotropic GABA receptors increases the energy barrier for vesicle fusion.

    PubMed

    Rost, Benjamin R; Nicholson, Patrick; Ahnert-Hilger, Gudrun; Rummel, Andreas; Rosenmund, Christian; Breustedt, Joerg; Schmitz, Dietmar

    2011-09-15

    Neurotransmitter release from presynaptic terminals is under the tight control of various metabotropic receptors. We report here that in addition to the regulation of Ca(2+) channel activity, metabotropic GABA(B) receptors (GABA(B)Rs) at murine hippocampal glutamatergic synapses utilize an inhibitory pathway that directly targets the synaptic vesicle release machinery. Acute application of the GABA(B)R agonist baclofen rapidly and reversibly inhibits vesicle fusion, which occurs independently of the SNAP-25 C-terminus. Using applications of hypertonic sucrose solutions, we find that the size of the readily releasable pool remains unchanged by GABA(B)R activation, but the sensitivity of primed vesicles to hypertonic stimuli appears lowered as the response amplitudes at intermediate sucrose concentrations are smaller and release kinetics are slowed. These data show that presynaptic GABA(B)Rs can inhibit neurotransmitter release directly by increasing the energy barrier for vesicle fusion.

  15. Unsaturated Analogues of the Neurotransmitter GABA: trans-4-Aminocrotonic, cis-4-Aminocrotonic and 4-Aminotetrolic Acids.

    PubMed

    Johnston, Graham A R

    2016-03-01

    Analogues of the neurotransmitter GABA containing unsaturated bonds are restricted in the conformations they can attain. This review traces three such analogues from their synthesis to their use as neurochemicals. trans-4-Aminocrotonic acid was the first conformationally restricted analogue to be extensively studied. It acts like GABA across a range of macromolecules from receptors to transporters. It acts similarly to GABA on ionotropic receptors. cis-4-Aminocrotonic acid selectively activates bicuculline-insensitive GABAC receptors. 4-Aminotetrolic acid, containing a triple bond, activates bicuculline-sensitive GABAA receptors. These findings indicate that GABA activates GABAA receptors in extended conformations and GABAC receptors in folded conformations. These and related analogues are important for the molecular modelling of ionotropic GABA receptors and to the development of new agents acting selectively on these receptors.

  16. Pioneer GABA cells comprise a subpopulation of hub neurons in the developing hippocampus

    PubMed Central

    Picardo, M.A.; Guigue, P.; Bonifazi, P.; Batista-Brito, R.; Allene, C.; Ribas, A.; Fishell, G.; Baude, A.; Cossart, R.

    2011-01-01

    Summary Connectivity in the developing hippocampus displays a functional organization particularly effective in supporting network synchronization, as it includes superconnected hub neurons. We have previously shown that hub network function is supported by a subpopulation of GABA neurons. However it is unclear whether hub cells are only transiently present or later develop into distinctive subclasses of interneurons. These questions are difficult to assess given the heterogeneity of the GABA neurons and the poor early expression of markers. To circumvent this conundrum we used “genetic fate mapping” that allows for the selective labelling of GABA neurons based on their place and time of origin. We show that early generated GABA cells form a subpopulation of hub neurons, characterized by an exceptionally widespread axonal arborisation and the ability to single-handedly impact network dynamics when stimulated. Pioneer hub neurons remain into adulthood where they acquire the classical markers of long-range projecting GABA neurons. PMID:21867885

  17. Excitatory Synaptic Responses Mediated by GABA_A Receptors in the Hippocampus

    NASA Astrophysics Data System (ADS)

    Michelson, Hillary B.; Wong, Robert K. S.

    1991-09-01

    Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the cortex. Activation of postsynaptic GABA_A receptors hyperpolarizes cells and inhibits neuronal activity. Synaptic responses mediated by GABA_A receptors also strongly excited hippocampal neurons. This excitatory response was recorded in morphologically identified interneurons in the presence of 4-aminopyridine or after elevation of extracellular potassium concentrations. The synaptic excitation sustained by GABA_A receptors synchronized the activity of inhibitory interneurons. This synchronized discharge of interneurons in turn elicited large-amplitude inhibitory postsynaptic potentials in pyramidal and granule cells. Excitatory synaptic responses mediated by GABA_A receptors may thus provide a mechanism for the recruitment of GABAergic interneurons through their recurrent connections.

  18. Cloning of the. gamma. -aminobutyric acid (GABA). rho. sub 1 cDNA: A GABA receptor subunit highly expressed in the retina

    SciTech Connect

    Cutting, G.R.; Lu, Luo; Kasch, L.M.; Montrose-Rafizadeh, C.; Antonarakis, S.E.; Guggino, W.B.; Kazazian, H.H. Jr. ); O'Hara, B.F.; Donovan, D.M.; Shimada, Shoichi ); Uhl, G.R. Johns Hopkins Univ. School of Medicine, Baltimore, MD )

    1991-04-01

    Type A {gamma}-aminobutyric acid (GABA{sub A}) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. The authors have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence is 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABA{sub A} subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA {rho}{sub 1}, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family.

  19. Presynaptic Control of Corticostriatal Synapses by Endogenous GABA

    PubMed Central

    Logie, Christopher; Bagetta, Vincenza

    2013-01-01

    Corticostriatal terminals have presynaptic GABAB receptors that limit glutamate release, but how these receptors are activated by endogenous GABA released by different types of striatal neurons is still unknown. To address this issue, we used single and paired whole-cell recordings combined with stimulation of corticostriatal fibers in rats and mice. In the presence of opioid, GABAA, and NK1 receptor antagonists, antidromic stimulation of a population of striatal projection neurons caused suppression of subsequently evoked EPSPs in projection neurons. These effects were larger at intervals of 500 ms than 1 or 2 s, and were fully blocked by the selective GABAB receptor antagonist CGP 52432. Bursts of spikes in individual projection neurons were not able to inhibit evoked EPSPs. Similarly, spikes in fast spiking interneurons and low-threshold spike interneurons failed to elicit detectable effects mediated by GABAB receptors. Conversely, spikes in individual neurogliaform interneurons suppressed evoked EPSPs, and these effects were blocked by CGP 52432. These results provide the first demonstration of how GABAB receptors are activated by endogenous GABA released by striatal neuronal types. PMID:24068811

  20. GABA System in Schizophrenia and Mood Disorders: A Mini Review on Third-Generation Imaging Studies

    PubMed Central

    Chiapponi, Chiara; Piras, Federica; Piras, Fabrizio; Caltagirone, Carlo; Spalletta, Gianfranco

    2016-01-01

    Third-generation neuroimaging research has been enriched by advances in magnetic resonance spectroscopy (MRS) measuring the concentration of important neurotrasmitters, such as the inhibitory amino acid GABA. Here, we performed a systematic mini-review on brain MRS studies measuring GABA concentration in patients affected by schizophrenia (SZ), bipolar disorder (BD), and major depressive disorder (MDD). We wondered whether multimodal investigations could overcome intrinsic technical limits of MRS giving a broader view of mental disorders pathogenesis. In SZ, unimodal studies gave mixed results, as increased, decreased, or unaltered GABA levels were reported depending on region, disease phase, and treatment. Conversely, multimodal results showed reduced level of glutamate, but not of GABA, in patients mirrored by in vitro biochemical findings revealing hippocampal reduction in glutamate signaling in SZ, and no deficits in GABA synthesis. Moreover, a mouse model confirmed the unique pathological characteristic of glutamate function in SZ. Unimodal studies in BD revealed again, inconsistent results, while no multimodal investigations including MRS on GABA exist. In MDD, unimodal studies could not differentiate patients from controls nor characterize high-risk subjects and remitted patients. However, a multimodal study combining functional magnetic resonance imaging and MRS revealed that cingulate cortex activity is related to glutamate, N-acetylaspartate levels and anhedonia in patients, and to GABA concentration in healthy subjects, improving the distinction between MDD and physiology. Overall, our results show that unimodal studies do not indicate GABA as a biomarker for the psychiatric disorders considered. Conversely, multimodal studies can widen the understanding of the link between psychopathology, genetics, neuroanatomy, and functional–biochemical brain activity in mental disorders. Although scarce, multimodal approaches seem promising for moving from GABA

  1. Depolarizing actions of GABA in immature neurons depend neither on ketone bodies nor on pyruvate.

    PubMed

    Tyzio, Roman; Allene, Camille; Nardou, Romain; Picardo, Michel A; Yamamoto, Sumii; Sivakumaran, Sudhir; Caiati, Maddalena D; Rheims, Sylvain; Minlebaev, Marat; Milh, Mathieu; Ferré, Pascal; Khazipov, Rustem; Romette, Jean-Louis; Lorquin, Jean; Cossart, Rosa; Khalilov, Ilgam; Nehlig, Astrid; Cherubini, Enrico; Ben-Ari, Yehezkel

    2011-01-05

    GABA depolarizes immature neurons because of a high [Cl(-)](i) and orchestrates giant depolarizing potential (GDP) generation. Zilberter and coworkers (Rheims et al., 2009; Holmgren et al., 2010) showed recently that the ketone body metabolite DL-3-hydroxybutyrate (DL-BHB) (4 mM), lactate (4 mM), or pyruvate (5 mM) shifted GABA actions to hyperpolarizing, suggesting that the depolarizing effects of GABA are attributable to inadequate energy supply when glucose is the sole energy source. We now report that, in rat pups (postnatal days 4-7), plasma D-BHB, lactate, and pyruvate levels are 0.9, 1.5, and 0.12 mM, respectively. Then, we show that DL-BHB (4 mM) and pyruvate (200 μM) do not affect (i) the driving force for GABA(A) receptor-mediated currents (DF(GABA)) in cell-attached single-channel recordings, (2) the resting membrane potential and reversal potential of synaptic GABA(A) receptor-mediated responses in perforated patch recordings, (3) the action potentials triggered by focal GABA applications, or (4) the GDPs determined with electrophysiological recordings and dynamic two-photon calcium imaging. Only very high nonphysiological concentrations of pyruvate (5 mM) reduced DF(GABA) and blocked GDPs. Therefore, DL-BHB does not alter GABA signals even at the high concentrations used by Zilberter and colleagues, whereas pyruvate requires exceedingly high nonphysiological concentrations to exert an effect. There is no need to alter conventional glucose enriched artificial CSF to investigate GABA signals in the developing brain.

  2. Comparative density of CCK- and PV-GABA cells within the cortex and hippocampus

    PubMed Central

    Whissell, Paul D.; Cajanding, Janine D.; Fogel, Nicole; Kim, Jun Chul

    2015-01-01

    Cholecystokinin (CCK)- and parvalbumin (PV)-expressing neurons constitute the two major populations of perisomatic GABAergic neurons in the cortex and the hippocampus. As CCK- and PV-GABA neurons differ in an array of morphological, biochemical and electrophysiological features, it has been proposed that they form distinct inhibitory ensembles which differentially contribute to network oscillations and behavior. However, the relationship and balance between CCK- and PV-GABA neurons in the inhibitory networks of the brain is currently unclear as the distribution of these cells has never been compared on a large scale. Here, we systemically investigated the distribution of CCK- and PV-GABA cells across a wide number of discrete forebrain regions using an intersectional genetic approach. Our analysis revealed several novel trends in the distribution of these cells. While PV-GABA cells were more abundant overall, CCK-GABA cells outnumbered PV-GABA cells in several subregions of the hippocampus, medial prefrontal cortex and ventrolateral temporal cortex. Interestingly, CCK-GABA cells were relatively more abundant in secondary/association areas of the cortex (V2, S2, M2, and AudD/AudV) than they were in corresponding primary areas (V1, S1, M1, and Aud1). The reverse trend was observed for PV-GABA cells. Our findings suggest that the balance between CCK- and PV-GABA cells in a given cortical region is related to the type of processing that area performs; inhibitory networks in the secondary cortex tend to favor the inclusion of CCK-GABA cells more than networks in the primary cortex. The intersectional genetic labeling approach employed in the current study expands upon the ability to study molecularly defined subsets of GABAergic neurons. This technique can be applied to the investigation of neuropathologies which involve disruptions to the GABAergic system, including schizophrenia, stress, maternal immune activation and autism. PMID:26441554

  3. Daily changes of GABA and taurine concentrations in various hypothalamic areas are affected by chronic hyperprolactinemia.

    PubMed

    Duvilanski, Beatriz H; Alvarez, M Pilar; Castrillón, Patricia O; Cano, Pilar; Esquifino, Ana I

    2003-03-01

    This study was designed to characterize, in anterior, mediobasal, and posterior hypothalamic and median eminence, the 24h changes of gamma aminobutyric acid (GABA) and taurine (TAU) contents in adult male rats and to analyze whether chronic hyperprolactinemia may affect these patterns. Rats were turned hyperprolactinemic by a pituitary graft. Plasma prolactin (PRL) levels increased after pituitary grafting at all time points examined. A disruption of the circadian rhythm was observed in pituitary-grafted rats, whereas GABA and TAU content followed daily rhythms in all areas studied in controls. In the mediobasal hypothalamus, two peaks for each amino acid were found at midnight and midday. In the anterior hypothalamus, GABA and TAU showed only one peak of concentration at midnight. In the posterior hypothalamus, the values of both GABA and TAU were higher during the light as compared to the dark phase of the photoperiod. In the median eminence GABA content peaked at 20:00h, the time when TAU exhibited the lowest values. Hyperprolactinemia abolished the 24h changes of GABA in the mediobasal hypothalamus and reduced its content as compared to controls. Hyperprolactinemia advanced the diurnal peak of TAU to 12:00h in the mediobasal hypothalamus and did not modify the 24:00h peak. In the anterior hypothalamus, hyperprolactinemia increased GABA and TAU contents during the light phase while it decreased them during the dark phase of the photoperiod. In the posterior hypothalamus hyperprolactinemia did not modify GABA or TAU patterns as compared to controls. In the median eminence hyperprolactinemia increased the 20:00h peak of GABA and shift advanced the decrease in TAU content at 20:00h and its maximum at 24:00h as compared to controls. These data show that GABA and TAU content exhibit specific daily patterns in each hypothalamic region studied. PRL differentially affects the daily pattern of these amino acids in each hypothalamic region analyzed.

  4. Excitatory action of gamma-aminobutyric acid (GABA) on crustacean neurosecretory cells.

    PubMed

    García, U; Onetti, C; Valdiosera, R; Aréchiga, H

    1994-02-01

    1. Intracellular and voltage-clamp recordings were obtained from a selected population of neurosecretory (ns) cells in the X organ of the crayfish isolated eyestalk. Pulses of gamma-aminobutyric acid (GABA) elicited depolarizing responses and bursts of action potentials in a dose-dependent manner. These effects were blocked by picrotoxin (50 microM) but not by bicuculline. Picrotoxin also suppressed spontaneous synaptic activity. 2. The responses to GABA were abolished by severing the neurite of X organ cells, at about 150 microns from the cell body. Responses were larger when the application was made at the neuropil level. 3. Topical application of Cd2+ (2 mM), while suppressing synaptic activity, was incapable of affecting the responses to GABA. 4. Under whole-cell voltage-clamp, GABA elicited an inward current with a reversal potential dependent on the chloride equilibrium potential. The GABA effect was accompanied by an input resistance reduction up to 33% at a -50 mV holding potential. No effect of GABA was detected on potassium, calcium, and sodium currents present in X organ cells. 5. The effect of GABA on steady-state currents was dependent on the intracellular calcium concentration. At 10(-6) M [Ca2+]i, GABA (50 microM) increased the membrane conductance more than threefold and shifted the zero-current potential from -25 to -10 mV. At 10(-9) M [Ca2+]i, GABA induced only a 1.3-fold increase in membrane conductance, without shifting the zero-current potential. 6. These results support the notion that in the population of X organ cells sampled in this study, GABA acts as an excitatory neurotransmitter, opening chloride channels.

  5. 2-Aminoethyl Methylphosphonate, a Potent and Rapidly Acting Antagonist of GABAA-ρ1 Receptors

    SciTech Connect

    Xie, A.; Yan, J.; Yue, L.; Feng, F.; Mir, F.; Abdel-Halim, H.; Chebib, M.; Le Breton, G. C.; Standaert, R. F.; Qian, H.; Pepperberg, D. R.

    2011-08-02

    All three classes of receptors for the inhibitory neurotransmitter GABA (GABAR) are expressed in the retina. This study investigated roles of GABAR, especially GABA(C)R (GABA(A)-rho), in retinal signaling in vivo by studying effects on the mouse electroretinogram (ERG) of genetic deletion of GABA(C)R versus pharmacological blockade using receptor antagonists. Brief full-field flash ERGs were recorded from anesthetized GABA(C)R(-/-) mice, and WT C57BL/6 (B6) mice, before and after intravitreal injection of GABA(C)R antagonists, TPMPA, 3-APMPA, or the more recently developed 2-AEMP; GABA(A)R antagonist, SR95531; GABA(B)R antagonist, CGP, and agonist, baclofen. Intravitreal injections of TPMPA and SR95531 were also made in Brown Norway rats. The effect of 2-AEMP on GABA-induced current was tested directly in isolated rat rod bipolar cells, and 2-AEMP was found to preferentially block GABA(C)R in those cells. Maximum amplitudes of dark (DA) and light-adapted (LA) ERG b-waves were reduced in GABA(C)R(-/-) mice, compared to B6 mice, by 30-60%; a-waves were unaltered and oscillatory potential amplitudes were increased. In B6 mice, after injection of TPMPA (also in rats), 3-APMPA or 2-AEMP, ERGs became similar to ERGs of GABA(C)R(-/-) mice. Blockade of GABA(A)Rs and GABA(B)Rs, or agonism of GABA(B)Rs did not alter B6 DA b-wave amplitude. The negative scotopic threshold response (nSTR) was slightly less sensitive in GABA(C)R(-/-) than in B6 mice, and unaltered by 2-AEMP. However, amplitudes of nSTR and photopic negative response (PhNR), both of which originate from inner retina, were enhanced by TPMPA and 3-APMPA, each of which has GABA(B) agonist properties, and further increased by baclofen. The finding that genetic deletion of GABA(C)R, the GABA(C)R antagonist 2-AEMP, and other antagonists all reduced ERG b-wave amplitude, supports a role for CABA(C)R in determining the maximum response amplitude of bipolar cells contributing to the b-wave. GABA(C)R antagonists differed

  6. In vivo electroretinographic studies of the role of GABAC receptors in retinal signal processing

    SciTech Connect

    Wang, Jing; Mojumder, Deb Kumar; Yan, Jun; Xie, An; Standaert, Robert F.; Qian, Haohua; Pepperberg, David R.; Frishman, Laura J.

    2015-07-08

    The retina expresses all three classes of receptors for the inhibitory neurotransmitter GABA (GABAR). Our study investigated roles of GABAR, especially GABA(C)R (GABA(A)-rho), in retinal signaling in vivo by studying effects on the mouse electroretinogram (ERG) of genetic deletion of GABA(C)R versus pharmacological blockade using receptor antagonists. Brief full-field flash ERGs were recorded from anesthetized GABA(C)R(-/-) mice, and WT C57BL/6 (B6) mice, before and after intravitreal injection of GABA(C)R antagonists, TPMPA, 3-APMPA, or the more recently developed 2-AEMP; GABA(A)R antagonist, SR95531; GABA(B)R antagonist, CGP, and agonist, baclofen. Intravitreal injections of TPMPA and SR95531 were also made in Brown Norway rats. The effect of 2-AEMP on GABA-induced current was tested directly in isolated rat rod bipolar cells, and 2-AEMP was found to preferentially block GABA(C)R in those cells. Maximum amplitudes of dark (DA) and light-adapted (LA) ERG b-waves were reduced in GABA(C)R(-/-) mice, compared to B6 mice, by 30-60%; a-waves were unaltered and oscillatory potential amplitudes were increased. In B6 mice, after injection of TPMPA (also in rats), 3-APMPA or 2-AEMP, ERGs became similar to ERGs of GABA(C)R(-/-) mice. Blockade of GABA(A)Rs and GABA(B)Rs, or agonism of GABA(B)Rs did not alter B6 DA b-wave amplitude. Furthermore, the negative scotopic threshold response (nSTR) was slightly less sensitive in GABA(C)R(-/-) than in B6 mice, and unaltered by 2-AEMP. However, amplitudes of nSTR and photopic negative response (PhNR), both of which originate from inner retina, were enhanced by TPMPA and 3-APMPA, each of which has GABA(B) agonist properties, and further increased by baclofen. The finding that genetic deletion of GABA(C)R, the GABA(C)R antagonist 2-AEMP, and other antagonists all reduced ERG b-wave amplitude, supports a role for CABA(C)R in determining the maximum response amplitude of bipolar cells contributing to the b-wave. GABA(C)R antagonists

  7. Endocytotic uptake of nutrients in carnivorous plants.

    PubMed

    Adlassnig, Wolfram; Koller-Peroutka, Marianne; Bauer, Sonja; Koshkin, Edith; Lendl, Thomas; Lichtscheidl, Irene K

    2012-07-01

    Carnivorous plants trap, digest and absorb animals in order to supplement their mineral nutrition. Nutrients absorbed by the plant include different nitrogen species, phosphate, potassium, trace elements and small organic compounds. Uptake is usually thought to be performed via specific channels, but this study provides evidence that endocytosis is involved as well. Traps of the carnivorous plants Nepenthes coccinea, Nepenthes ventrata, Cephalotus follicularis, Drosophyllum lusitanicum, Drosera capensis, Dionaea muscipula, Aldrovanda vesiculosa, Genlisea violacea × lobata, Sarracenia psittacina and Sarracenia purpurea were stained with methylene blue in order to identify possible sites of uptake. The permeable parts of the traps were incubated with fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) and other fluorescent endocytosis markers, combined with the soluble protein BSA or respiratory inhibitors. Uptake was studied by confocal microscopy. In Nepenthes, small fluorescent vesicles became visible 1 h after incubation with FITC-BSA. These vesicles fused to larger compartments within 30 h. A similar behaviour was found in the related genera Drosera, Dionaea, Aldrovanda and Drosophyllum but also in Cephalotus with glands of different evolutionary origin. In Genlisea and Sarracenia, no evidence for endocytosis was found. We propose that in many carnivorous plants, nutrient uptake by carriers is supplemented by endocytosis, which enables absorption and intracellular digestion of whole proteins. The advantage for the plant of reducing secretion of enzymes for extracellular digestion is evident.

  8. Characterization of iron uptake from transferrin by murine endothelial cells.

    PubMed

    Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

    2000-01-01

    Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB.

  9. The glutamate transporters EAAT2 and EAAT3 mediate cysteine uptake in cortical neuron cultures.

    PubMed

    Chen, Yongmei; Swanson, Raymond A

    2003-03-01

    Cysteine availability is normally the rate-limiting factor in glutathione synthesis. How neurons obtain cysteine from extracellular space is not well established. Here we used mouse cortical neuron cultures to examine the role of the excitatory amino acid transporters (EAATs) in neuronal cysteine uptake. The cultured neurons expressed both EAAT2 and EAAT3. Cysteine uptake was predominantly (> 85%) Na+-dependent, with an apparent Km of 37 microm. Cysteine uptake was reduced by the EAAT substrates l-glutamate and l-aspartate and by synthetic EAAT inhibitors. The non-selective EAAT inhibitor threo-beta-hydroxyaspartate had a significantly greater maximal inhibitory effect than did the EAAT2-selective inhibitor, dihydrokainate, indicating uptake by both EAAT2 and EAAT3. Serine, a substrate of ASC uptake system, had negligible effects on cysteine uptake at 10-fold excess concentrations. To assess the functional importance of EAAT-mediated cysteine uptake in neuronal glutathione synthesis, cultures were treated with diethylmaleate to deplete glutathione, then incubated with cysteine in the presence or absence of EAAT inhibitors. Threo-beta-benzyloxyaspartate and the non-transportable inhibitor threo-beta-hydroxyaspartate both inhibited the cysteine-dependent glutathione synthesis. The findings suggest that neuronal EAAT activity can be a rate-limiting step for neuronal glutathione synthesis and that the primary function of EAATs expressed by neurons in vivo may be to transport cysteine.

  10. Modulation of GABA release from the thalamic reticular nucleus by cocaine and caffeine: role of serotonin receptors.

    PubMed

    Goitia, Belén; Rivero-Echeto, María Celeste; Weisstaub, Noelia V; Gingrich, Jay A; Garcia-Rill, Edgar; Bisagno, Verónica; Urbano, Francisco J

    2016-02-01

    Serotonin receptors are targets of drug therapies for a variety of neuropsychiatric and neurodegenerative disorders. Cocaine inhibits the re-uptake of serotonin (5-HT), dopamine, and noradrenaline, whereas caffeine blocks adenosine receptors and opens ryanodine receptors in the endoplasmic reticulum. We studied how 5-HT and adenosine affected spontaneous GABAergic transmission from thalamic reticular nucleus. We combined whole-cell patch clamp recordings of miniature inhibitory post-synaptic currents (mIPSCs) in ventrobasal thalamic neurons during local (puff) application of 5-HT in wild type (WT) or knockout mice lacking 5-HT2A receptors (5-HT2A -/-). Inhibition of mIPSCs frequency by low (10 μM) and high (100 μM) 5-HT concentrations was observed in ventrobasal neurons from 5-HT2A -/- mice. In WT mice, only 100 μM 5-HT significantly reduced mIPSCs frequency. In 5-HT2A -/- mice, NAN-190, a specific 5-HT1A antagonist, prevented the 100 μM 5-HT inhibition while blocking H-currents that prolonged inhibition during post-puff periods. The inhibitory effects of 100 μM 5-HT were enhanced in cocaine binge-treated 5-HT2A -/- mice. Caffeine binge treatment did not affect 5-HT-mediated inhibition. Our findings suggest that both 5-HT1A and 5-HT2A receptors are present in pre-synaptic thalamic reticular nucleus terminals. Serotonergic-mediated inhibition of GABA release could underlie aberrant thalamocortical physiology described after repetitive consumption of cocaine. Our findings suggest that both 5-HT1A , 5-HT2A and A1 receptors are present in pre-synaptic TRN terminals. 5-HT1A and A1 receptors would down-regulate adenylate cyclase, whereas 5-HT1A would also increase the probability of the opening of G-protein-activated inwardly rectifying K(+) channels (GIRK). Sustained opening of GIRK channels would hyperpolarize pre-synaptic terminals activating H-currents, resulting in less GABA release. 5-HT2A -would activate PLC and IP3 , increasing intracellular [Ca(2+) ] and

  11. A Rice Mutant Defective in Si Uptake1

    PubMed Central

    Ma, Jian Feng; Tamai, Kazunori; Ichii, Masahiko; Wu, Guo Feng

    2002-01-01

    Rice (Oryza sativa) accumulates silicon (Si) in the tops to levels up to 10.0% of shoot dry weight, but the mechanism responsible for high Si uptake by rice roots is not understood. We isolated a rice mutant (GR1) that is defective in active Si uptake by screening M2 seeds (64,000) of rice cv Oochikara that were treated with 10−3 m sodium azide for 6 h at 25oC. There were no phenotypic differences between wild type (WT) and GR1 except that the leaf blade of GR1 remained droopy when Si was supplied. Uptake experiments showed that Si uptake by GR1 was significantly lower than that by WT at both low and high Si concentrations. However, there was no difference in the uptake of other nutrients such as phosphorus and potassium. Si concentration in the xylem sap of WT was 33-fold that of the external solution, but that of GR1 was 3-fold higher than the external solution at 0.15 mm Si. Si uptake by WT was inhibited by metabolic inhibitors including NaCN and 2,4-dinitrophenol and by low temperature, whereas Si uptake by GR1 was not inhibited by these agents. These results suggest that an active transport system for Si uptake is disrupted in GR1. Analysis of F2 populations between GR1 and WT showed that roots with high Si uptake and roots with low Si uptake segregated at a 3:1 ratio, suggesting that GR1 is a recessive mutant of Si uptake. PMID:12481095

  12. Role of neuronal glutamate transporter in the cysteine uptake and intracellular glutathione levels in cultured cortical neurons.

    PubMed

    Himi, T; Ikeda, M; Yasuhara, T; Nishida, M; Morita, I

    2003-12-01

    Cysteine uptake is the rate-limiting process in glutathione synthesis. Previously we have shown that the inhibitors of excitatory amino acid transporters (EAATs) significantly enhance glutamate toxicity via depletion of intracellular glutathione. In this study we show evidence that the neuronal glutamate transporter EAAT3 is directly enrolled in cysteine uptake in cultured neurons. Neuronal cysteine uptake was dependent on the extracellular sodium, and was suppressed by EAAT inhibitors. Cysteine uptake was suppressed by extracellular glutamate and aspartate, substrates of EAATs, and not by substrates of cysteine transporters. Intracellular glutathione levels were reduced by EAAT inhibitors, and not by inhibitors of cysteine transporters. Knock down of EAAT3 expression using antisense oligonucleotide significantly reduced cysteine uptake, intracellular glutathione level, and neuronal viability against oxidative stress. These facts indicate that EAAT3 functions as a cysteine transporter, and this function seems to be unique and distinct from cysteine transporters that have been reported.

  13. Contents of Neo-flavored Tea (GABA Kintaro) Containing γ-Aminobutyric Acid

    NASA Astrophysics Data System (ADS)

    Shiraki, Yoshiya

    The contents of γ-aminobutyric acid (GABA), catechins, theaflavins, caffeine and pheophorbide-a in neo-flavored tea (GABA Kintaro tea) were analyzed. 1)The amounts of GABA were increased over 1.5mg/g by means of infrared ray irradiation with agitation treatment. 2)There was a tendency for the amount of catechins to be decreased by this treatment, whereas the amount of theaflavins tended to increase with the same treatment. The composition of these contents in this GABA Kintaro tea was almost the same as that of black tea. 3)There was a tendency for the amount of caffeine to be decreased by this treatment. 4)There was a tendency for the amount of pheophorbide-a to be increased by this treatment. 5)The result of this study showed that the amounts of GABA and theaflavins in this GABA Kintaro tea were higher than ordinary green tea but contained few catechins.It became clear that the amount of pheophorbide-a in this GABA Kintaro tea was less than the standard value established in processed chlorella.

  14. Prevention of GABA reduction during dough fermentation using a baker's yeast dal81 mutant.

    PubMed

    Ando, Akira; Nakamura, Toshihide

    2016-10-01

    γ-Aminobutyric acid (GABA) is consumed by yeasts during fermentation. To prevent GABA reduction in bread dough, a baker's yeast mutant AY77 deficient in GABA assimilation was characterized and utilized for wheat dough fermentation. An amber mutation in the DAL81 gene, which codes for a positive regulator of multiple nitrogen degradation pathways, was found in the AY77 strain. The qPCR analyses of genes involved in nitrogen utilization showed that transcriptional levels of the UGA1 and DUR3 genes encoding GABA transaminase and urea transporter, respectively, are severely decreased in the AY77 cells. The AY77 strain cultivated by fed-batch culture using cane molasses exhibited inferior gas production during dough fermentation compared to that of wild-type strain AY13. However, when fed with molasses containing 0.5% ammonium sulfate, the mutant strain exhibited gas production comparable to that of the AY13 strain. In contrast to the AY13 strain, which completely consumed GABA in dough within 5 h, the AY77 strain consumed no GABA under either culture condition. Dough fermentation with the dal81 mutant strain should be useful for suppression of GABA reduction in breads.

  15. Regulation of (/sup 3/H)GABA release from strips of guinea pig urinary bladder

    SciTech Connect

    Shirakawa, J.; Taniyama, K.; Iwai, S.; Tanaka, C.

    1988-12-01

    The presence of receptors that regulate the release of gamma-aminobutyric acid (GABA) was studied in strips of the guinea pig urinary bladder. GABA (10(-8)-10(-5) M) and muscimol (10(-8)-10(-5) M), but not baclofen (10(-5) M), reduced the Ca2+-dependent, tetrodotoxin-resistant release of (/sup 3/H)GABA evoked by high K+ from the urinary bladder strips preloaded with (/sup 3/H)GABA. The inhibitory effect of muscimol was antagonized by bicuculline and potentiated by diazepam, clonazepam, and pentobarbital sodium. The potentiating effect of clonazepam was antagonized by Ro 15-1788. Acetylcholine (ACh) inhibited the high K+-evoked release of (/sup 3/H)GABA. The inhibitory effect of ACh was antagonized by atropine sulfate and pirenzepine but not by hexamethonium. Norepinephrine (NE) inhibited the evoked release of (/sup 3/H)GABA. The inhibitory effect of NE was mimicked by clonidine, but not by phenylephrine, and was antagonized by yohimbine but not by prazosin. These results provide evidence that the release of GABA from strips of guinea pig urinary bladder is regulated via the bicuculline-sensitive GABAA receptor, M1-muscarinic, and alpha 2-adrenergic receptors.

  16. GABA is the principal fast-acting excitatory transmitter in the neonatal brain.

    PubMed

    Leinekugel, X; Khalilov, I; McLean, H; Caillard, O; Gaiarsa, J L; Ben-Ari, Y; Khazipov, R

    1999-01-01

    gamma-aminobutyric acid (GABA) is the principal neurotransmitter of inhibition in the adult mammalian brain. However, at early stages of development, including the embryonic period and first week of postnatal life, GABA plays the role of main neurotransmitter of excitation. The paradoxical excitatory effect of GABA is caused by an inverted chloride gradient and, therefore, a depolarizing direction of GABA type A (GABAA) receptor mediated responses. In addition, another type of GABAergic inhibition mediated by postsynaptic GABA type B (GABAB) receptors is not functional at early stage of life. In the neonatal rat hippocampus, GABA, acting via GABAA receptors, activates voltage-gated sodium and calcium channels and potentiates the activity of N-methyl-D-aspartate (NMDA) receptors by reducing their voltage-dependent Mg2+ block. The temporal window when GABA exerts excitatory actions coincides with a particular pattern of activity of hippocampal neuronal network that is characterized by periodical giant depolarizing potentials (GDPs) reminiscent of interictal-like epileptiform discharges. Recent studies have shown that GDPs result from the synchronous discharge of GABAergic interneurons and principal glutamatergic pyramidal cells, and they are mediated by the synergistic excitatory actions of GABAA and glutamate receptors. GDPs provide synchronous intracellular Ca2+ oscillations and may, therefore, be implicated in hebbian modulation of developing synapses and activity-dependent formation of the hippocampal network.

  17. Cadmium exposure disrupts GABA and taurine regulation of prolactin secretion in adult male rats.

    PubMed

    Caride, A; Fernández-Pérez, B; Cabaleiro, T; Esquifino, A I; Lafuente, A

    2009-03-28

    This work was undertaken to evaluate the possible effects of cadmium exposure on 24 h changes of gamma-aminobutyric acid (GABA) and taurine median eminence and pituitary contents. Also the possible alterations of the regulatory mechanisms of GABA and taurine on prolactin secretion were evaluated. Adult male rats were given cadmium at a dose of 25 mg/l of cadmium chloride in the drinking water for 30 days. Control age-matched rats received cadmium free water. Metal exposure induced the appearance of a maximal value of prolactin at 08:00 h. In median eminence, cadmium abolished the GABA and taurine maximal values and decreased GABA and taurine mean levels. In the anterior pituitary, cadmium treatment phase advanced 12 h the peak observed in controls at 00:00 h for both amino acids. There was a positive correlation between GABA and taurine contents in median eminence and the anterior pituitary in both control and cadmium-exposed animals. However, the correlation between GABA or/and taurine with prolactin levels disappeared in cadmium-exposed animals. These results suggest that cadmium exposure affects GABA and taurine daily pattern in the median eminence and anterior pituitary, and those changes explain, at least in part, the modification in the regulatory pattern of prolactin secretion.

  18. Hypothesis/review: contribution of putrescine to 4-aminobutyrate (GABA) production in response to abiotic stress.

    PubMed

    Shelp, Barry J; Bozzo, Gale G; Trobacher, Christopher P; Zarei, Adel; Deyman, Kristen L; Brikis, Carolyne J

    2012-09-01

    4-Aminobutyrate (GABA) accumulates in various plant parts, including bulky fruits such as apples, in response to abiotic stress. It is generally believed that the GABA is derived from glutamate, although a contribution from polyamines is possible. Putrescine, but not spermidine and spermine, generally accumulates in response to the genetic manipulation of polyamine biosynthetic enzymes and abiotic stress. However, the GABA levels in stressed plants are influenced by processes other than putrescine availability. It is hypothesized that the catabolism of putrescine to GABA is regulated by a combination of gene-dependent and -independent processes. The expression of several putative diamine oxidase genes is weak, but highly stress-inducible in certain tissues of Arabidopsis. In contrast, candidate genes that encode 4-aminobutyraldehyde dehydrogenase are highly constitutive, but not stress inducible. Changes in O(2) availability and cellular redox balance due to stress may directly influence the activities of diamine oxidase and 4-aminobutyraldehyde dehydrogenase, thereby restricting GABA formation. Apple fruit is known to accumulate GABA under controlled atmosphere storage and therefore could serve as a model system for investigating the relative contribution of putrescine and glutamate to GABA production.

  19. A tonoplast Glu/Asp/GABA exchanger that affects tomato fruit amino acid composition.

    PubMed

    Snowden, Christopher J; Thomas, Benjamin; Baxter, Charles J; Smith, J Andrew C; Sweetlove, Lee J

    2015-03-01

    Vacuolar accumulation of acidic metabolites is an important aspect of tomato fruit flavour and nutritional quality. The amino acids Asp and Glu accumulate to high concentrations during ripening, while γ-aminobutyrate (GABA) shows an approximately stoichiometric decline. Given that GABA can be catabolised to form Glu and subsequently Asp, and the requirement for the fruit to maintain osmotic homeostasis during ripening, we hypothesised the existence of a tonoplast transporter that exports GABA from the vacuole in exchange for import of either Asp or Glu. We show here that the tomato vacuolar membrane possesses such a transport property: transport of Glu across isolated tonoplast vesicle membranes was trans-stimulated in counterexchange mode by GABA, Glu and Asp. We identified SlCAT9 as a candidate protein for this exchanger using quantitative proteomics of a tonoplast-enriched membrane fraction. Transient expression of a SlCAT9-YFP fusion in tobacco confirmed a tonoplast localisation. The function of the protein was examined by overexpression of SlCAT9 in transgenic tomato plants. Tonoplast vesicles isolated from transgenic plants showed higher rates of Glu and GABA transport than wild-type (WT) only when assayed in counterexchange mode with Glu, Asp, or GABA. Moreover, there were substantial increases in the content of all three cognate amino acids in ripe fruit from the transgenic plants. We conclude that SlCAT9 is a tonoplast Glu/Asp/GABA exchanger that strongly influences the accumulation of these amino acids during fruit development.

  20. Contribution of metabotropic GABA(B) receptors to neuronal network construction.

    PubMed

    Gaiarsa, Jean-Luc; Kuczewski, Nicola; Porcher, Christophe

    2011-11-01

    In the 1980s, Bowery and colleagues discovered the presence of a novel, bicuculline-resistant and baclofen-sensitive type of GABA receptor on peripheral nerve terminals, the GABA(B) receptor. Since this pioneering work, GABA(B) receptors have been identified in the Central Nervous System (CNS), where they provide an important inhibitory control of postsynaptic excitability and presynaptic transmitter release. GABA(B) receptors have been implicated in a number of important processes in the adult brain such as the regulation of synaptic plasticity and modulation of rhythmic activity. As a result of these studies, several potential therapeutic applications of GABA(B) receptor ligands have been identified. Recent advances have further shown that GABA(B) receptors play more than a classical inhibitory role in adult neurotransmission, and can in fact function as an important developmental signal early in life. Here we summarize current knowledge on the contribution of GABA(B) receptors to the construction and function of developing neuronal networks.

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

    PubMed Central

    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

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

    PubMed

    Snell, Heather D; Gonzales, Eric B

    2015-06-01

    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.

  3. Focal uncaging of GABA reveals a temporally defined role for GABAergic inhibition during appetitive associative olfactory conditioning in honeybees.

    PubMed

    Raccuglia, Davide; Mueller, Uli

    2013-07-16

    Throughout the animal kingdom, the inhibitory neurotransmitter γ-aminobutyric acid (GABA) is a key modulator of physiological processes including learning. With respect to associative learning, the exact time in which GABA interferes with the molecular events of learning has not yet been clearly defined. To address this issue, we used two different approaches to activate GABA receptors during appetitive olfactory conditioning in the honeybee. Injection of GABA-A receptor agonist muscimol 20 min before but not 20 min after associative conditioning affects memory performance. These memory deficits were attenuated by additional training sessions. Muscimol has no effect on sensory perception, odor generalization, and nonassociative learning, indicating a specific role of GABA during associative conditioning. We used photolytic uncaging of GABA to identify the GABA-sensitive time window during the short pairing of the conditioned stimulus (CS) and the unconditioned stimulus (US) that lasts only seconds. Either uncaging of GABA in the antennal lobes or the mushroom bodies during the CS presentation of the CS-US pairing impairs memory formation, while uncaging GABA during the US phase has no effect on memory. Uncaging GABA during the CS presentation in memory retrieval also has no effect. Thus, in honeybee appetitive olfactory learning GABA specifically interferes with the integration of CS and US during associative conditioning and exerts a modulatory role in memory formation depending on the training strength.

  4. Aging of whiskey increases the potentiation of GABA(A) receptor response.

    PubMed

    Koda, Hirofumi; Hossain, Sheikh Julfikar; Kiso, Yoshinobu; Aoshima, Hitoshi

    2003-08-27

    It is known that the target of most mood-defining compounds such as ethanol is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activities in the human brain. Because both extracts of whiskey by pentane and fragrant components in whiskey potentiate the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting cRNAs prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors in order to study the effects of whiskey itself on the GABA(A) receptor-mediated response. Whiskey itself also potentiated the electrical responses of GABA(A) receptors generally more than ethanol at the same concentration as that of the whiskey. The potentiation of the GABA(A) receptor-mediated response increased with the aging period of the whiskey. Inhalation of whiskey to mice increased the sleeping time induced by pentobarbital more than that of the same concentration of ethanol as the whiskey. These results suggest that not only ethanol but also minor components in whiskey play an important role in the potentiation of GABA(A) receptor-mediated response and possibly the sedative effect of whiskey. Although the minor components are present in extremely small quantities compared with ethanol in alcoholic beverages, they may modulate the mood or consciousness of humans through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic compounds are easily absorbed into the brain across the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response.

  5. GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations.

    PubMed

    Ben-Ari, Yehezkel; Gaiarsa, Jean-Luc; Tyzio, Roman; Khazipov, Rustem

    2007-10-01

    Developing networks follow common rules to shift from silent cells to coactive networks that operate via thousands of synapses. This review deals with some of these rules and in particular those concerning the crucial role of the neurotransmitter gamma-aminobuytric acid (GABA), which operates primarily via chloride-permeable GABA(A) receptor channels. In all developing animal species and brain structures investigated, neurons have a higher intracellular chloride concentration at an early stage leading to an efflux of chloride and excitatory actions of GABA in immature neurons. This triggers sodium spikes, activates voltage-gated calcium channels, and acts in synergy with NMDA channels by removing the voltage-dependent magnesium block. GABA signaling is also established before glutamatergic transmission, suggesting that GABA is the principal excitatory transmitter during early development. In fact, even before synapse formation, GABA signaling can modulate the cell cycle and migration. The consequence of these rules is that developing networks generate primitive patterns of network activity, notably the giant depolarizing potentials (GDPs), largely through the excitatory actions of GABA and its synergistic interactions with glutamate signaling. These early types of network activity are likely required for neurons to fire together and thus to "wire together" so that functional units within cortical networks are formed. In addition, depolarizing GABA has a strong impact on synaptic plasticity and pathological insults, notably seizures of the immature brain. In conclusion, it is suggested that an evolutionary preserved role for excitatory GABA in immature cells provides an important mechanism in the formation of synapses and activity in neuronal networks.

  6. Microtransplantation of cellular membranes from squid stellate ganglion reveals ionotropic GABA receptors.

    PubMed

    Conti, Luca; Limon, Agenor; Palma, Eleonora; Miledi, Ricardo

    2013-02-01

    The squid has been the most studied cephalopod, and it has served as a very useful model for investigating the events associated with nerve impulse generation and synaptic transmission. While the physiology of squid giant axons has been extensively studied, very little is known about the distribution and function of the neurotransmitters and receptors that mediate inhibitory transmission at the synapses. In this study we investigated whether γ-aminobutyric acid (GABA) activates neurotransmitter receptors in stellate ganglia membranes. To overcome the low abundance of GABA-like mRNAs in invertebrates and the low expression of GABA in cephalopods, we used a two-electrode voltage clamp technique to determine if Xenopus laevis oocytes injected with cell membranes from squid stellate ganglia responded to GABA. Using this method, membrane patches containing proteins and ion channels from the squid's stellate ganglion were incorporated into the surface of oocytes. We demonstrated that GABA activates membrane receptors in cellular membranes isolated from squid stellate ganglia. Using the same approach, we were able to record native glutamate-evoked currents. The squid's GABA receptors showed an EC(50) of 98 μmol l(-1) to GABA and were inhibited by zinc (IC(50) = 356 μmol l(-1)). Interestingly, GABA receptors from the squid were only partially blocked by bicuculline. These results indicate that the microtransplantation of native cell membranes is useful to identify and characterize scarce membrane proteins. Moreover, our data also support the role of GABA as an ionotropic neurotransmitter in cephalopods, acting through chloride-permeable membrane receptors.

  7. GABA-B receptor activation inhibits the in vitro migration of malignant hepatocytes.

    PubMed

    Lodewyks, Carly; Rodriguez, Jose; Yan, Jing; Lerner, Betty; Lipschitz, Jeremy; Nfon, Charles; Rempel, Julia Darlene; Uhanova, Julia; Minuk, Gerald Yosel

    2011-06-01

    There are conflicting data regarding whether activation of γ-aminobutyric acid-B (GABA-B) receptors results in inhibition of tumor growth and invasion. The objectives of this study were to document the effects of the GABA-B receptor agonist baclofen on malignant hepatocyte proliferation and migration. We also sought to determine whether any effects on cell migration were mediated by changes in cyclic adenosine monophosphate (cAMP) signaling or matrix metalloproteinase (MMP) expression. Finally, GABA-B(1) and -B(2) receptor expression was documented in 2 malignant hepatocyte cell lines (PLC/PRF/5 and Huh-7) and 12 sets of human hepatocellular carcinoma and adjacent nontumor tissues. Cell proliferative activity was documented by WST-1 absorbance, migration by wound healing assays, cAMP levels by enzyme-linked immunoassay (ELISA), MMP by immunohistochemistry and ELISA, and GABA-B receptor expression by flow cytometry and reverse transcriptase - polymerase chain reaction. Although baclofen had no effect on cell proliferation, wound healing was delayed, an effect that was reversed by the GABA-B receptor antagonist CGP. cAMP levels were decreased in Huh-7 but not PLC cells exposed to baclofen. MMP expression remained unaltered in both cell lines. Finally, GABA-B(1) receptor expression was present and consistently expressed, but GABA-B(2) expression was limited and varied with the number of cell passages and (or) duration of culture. In conclusion, activation of GABA-B receptors has no effect on malignant hepatocyte proliferation but does decrease cell migration. This inhibitory effect may involve cAMP signaling but not MMP expression. GABA-B(2) receptor expression is limited and variable, which may help to explain discrepancies with previously published results.

  8. Loss of functional GABA(A) receptors in the Alzheimer diseased brain.

    PubMed

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

    2012-06-19

    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.

  9. The TM2 6' position of GABA(A) receptors mediates alcohol inhibition.

    PubMed

    Johnson, W David; Howard, Rebecca J; Trudell, James R; Harris, R Adron

    2012-02-01

    Ionotropic GABA(A) receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABA(A)Rs. We used Xenopus laevis oocytes to investigate the 6' position in the second transmembrane region of GABA(A)Rs as a site influencing alcohol inhibition. We asked whether modification of the 6' position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6' position in either α2 or β2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6' position. We verified a role for the 6' position in previously tested α2β2 as well as more physiologically relevant α2β2γ2s GABA(A)Rs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABA(A)R homolog, revealing that the 6' position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6' positions in both α2 and β2 GABA(A)R subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels.

  10. Semisynthetic preparation of amentoflavone: A negative modulator at GABA(A) receptors.

    PubMed

    Hanrahan, Jane R; Chebib, Mary; Davucheron, Neil L M; Hall, Belinda J; Johnston, Graham A R

    2003-07-21

    Amentoflavone is found in a number of plants with medicinal properties, including Ginkgo biloba and Hypericum perforatum (St. John's Wort). We have developed a rapid and economic semi-synthetic preparation of amentoflavone from biflavones isolated from autumnal Ginkgo biloba leaves. Several studies have shown that amentoflavone binds to benzodiazepine receptors. Using two electrode voltage-clamp methodology, amentoflavone has been shown to be a negative modulator of GABA at GABA(A) alpha(1)beta(2)gamma(2L) receptors expressed in Xenopus laevis oocytes This action appears to be independent of the flumazenil-sensitive benzodiazepine modulatory sites on the GABA(A) receptor.

  11. Molecular diversity of GABA-gated chloride channels in the rat anterior pituitary.