Pool Of GABA Research Articles

γ-Aminobutyrate Improves the Postharvest Marketability of Horticultural Commodities: Advances and Prospects.

Postharvest deterioration can result in qualitative and quantitative changes in the marketability of horticultural commodities, as well as considerable economic loss to the industry. Low temperature and controlled atmosphere conditions (low O2 and elevated CO2) are extensively employed to prolong the postharvest life of these commodities. Nevertheless, they may suffer from chilling injury and other physiological disorders, as well as excessive water loss and bacterial/fungal decay. Research on the postharvest physiological, biochemical, and molecular responses of horticultural commodities indicates that low temperature/controlled atmosphere storage is associated with the promotion of γ-aminobutyrate (GABA) pathway activity, with or without the accumulation of GABA, delaying senescence, preserving quality and ameliorating chilling injury. Regardless of whether apple fruits are stored under low temperature/controlled atmosphere conditions or room temperature, elevated endogenous GABA or exogenous GABA maintains their quality by stimulating the activity of the GABA shunt (glutamate GABA succinic semialdehyde succinate) and the synthesis of malate, and delaying fruit ripening. This outcome is associated with changes in the genetic and biochemical regulation of key GABA pathway reactions. Flux estimates suggest that the GABA pool is derived primarily from glutamate, rather than polyamines, and that succinic semialdehyde is converted mainly to succinate, rather than γ-hydroxybutyrate. Exogenous GABA is a promising strategy for promoting the level of endogenous GABA and the activity of the GABA shunt in both intact and fresh-cut commodities, which increases carbon flux through respiratory pathways, restores or partially restores redox and energy levels, and improves postharvest marketability. The precise mechanisms whereby GABA interacts with other signaling molecules such as Ca2+, H2O2, polyamines, salicylic acid, nitric oxide and melatonin, or with phytohormones such as ethylene, abscisic acid and auxin remain unknown. The occurrence of the aluminum-activated malate transporter and the glutamate/aspartate/GABA exchanger in the tonoplast, respectively, offers prospects for reducing transpirational water in cut flowers and immature green fruit, and for altering the development, flavor and biotic resistance of apple fruits.

Slc38a1 Conveys Astroglia-Derived Glutamine into GABAergic Interneurons for Neurotransmitter GABA Synthesis.

GABA signaling is involved in a wide range of neuronal functions, such as synchronization of action potential firing, synaptic plasticity and neuronal development. Sustained GABA signaling requires efficient mechanisms for the replenishment of the neurotransmitter pool of GABA. The prevailing theory is that exocytotically released GABA may be transported into perisynaptic astroglia and converted to glutamine, which is then shuttled back to the neurons for resynthesis of GABA—i.e., the glutamate/GABA-glutamine (GGG) cycle. However, an unequivocal demonstration of astroglia-to-nerve terminal transport of glutamine and the contribution of astroglia-derived glutamine to neurotransmitter GABA synthesis is lacking. By genetic inactivation of the amino acid transporter Solute carrier 38 member a1 (Slc38a1)—which is enriched on parvalbumin+ GABAergic neurons—and by intraperitoneal injection of radiolabeled acetate (which is metabolized to glutamine in astroglial cells), we show that Slc38a1 mediates import of astroglia-derived glutamine into GABAergic neurons for synthesis of GABA. In brain slices, we demonstrate the role of Slc38a1 for the uptake of glutamine specifically into GABAergic nerve terminals for the synthesis of GABA depending on demand and glutamine supply. Thus, while leaving room for other pathways, our study demonstrates a key role of Slc38a1 for newly formed GABA, in harmony with the existence of a GGG cycle.

Augmentation of endogenous GABA pool size induced by Magainin II peptide

BackgroundGamma aminobutyric acid (GABA), an inhibitory neurotransmitter, is produced via decarboxylation of l-glutamate through the glutamic acid decarboxylase (GAD) enzyme. The synchronic action of GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) enzymes convert the GABA metabolite into succinate. Given this background, our research was aimed at probing the effect of Magainin II, on the activity of GABA shunt metabolizing enzymes. MethodsMale NIH mice were administered peripherally by Magainin II (50 μg/kg body weight) and saline solution (%0.9 (w/v)) as the control vehicle. At different time intervals, the mice were sacrificed to evaluate the effect of Magainin II injection on the GABA shunt pathway. The activity of hypothalamic GAD, GABA-T and SSADH enzymes were determined using relevant enzyme assays. ResultsMagainin II effectively enhanced the activity of GAD, by %90, 24 h after injection, while quenching the activities of GABA-T and SSADH by %43 and %71, respectively. In vitro models also revealed the direct but reversible interaction between the peptide and each of the individual enzymes of GABA shunt pathway. ConclusionThis study confirms the probable role of Magainin II in increasing the GABA content of the mouse hypothalamus. This property might candidate the peptide as a novel agent for improving the symptoms of many GABA dependent psychiatric disorders.

Neurotensin speeds inhibition of dopamine neurons through temporal modulation of GABAA and GABAB receptor-mediated synaptic input

Midbrain dopamine neurons play physiological roles in many processes including reward learning and motivated behavior, and are tonically inhibited by γ-aminobutyric acid (GABA)ergic input from multiple brain regions. Neurotensin (NT) is a neuropeptide which acutely modulates midbrain dopamine neuron excitability through multiple mechanisms, one of which is a decrease of GABA-mediated inhibition. However, the mechanisms through which NT depresses GABA signaling are not known. Here we used whole cell patch-clamp electrophysiology of dopamine neurons in mouse brain slices to show that NT acts both presynaptically to increase GABAA and postsynaptically to decrease GABAB receptor-mediated currents in the substantia nigra. The active peptide fragment NT8-13 enhanced GABAA signaling presynaptically by causing an increase in the size of the readily releasable pool of GABA via activation of the NT type-1 receptor and protein kinase A. Conversely, NT8-13 depressed GABAB signaling postsynaptically via the NT type-2 receptor in a process that was modulated by protein kinase C. Both forms of plasticity could be observed simultaneously in single dopamine neurons. Thus, as the kinetics of GABAA signaling are significantly faster than those of GABAB signaling, NT functionally speeds GABAergic input to midbrain dopamine neurons. This finding contributes to our understanding of how neuropeptide-induced plasticity can simultaneously differentiate and integrate signaling by a single neurotransmitter in a single cell and provides a basis for understanding how neuropeptides use temporal shifts in synaptic strength to encode information.

Chronic morphine reduces the readily releasable pool of GABA, a presynaptic mechanism of opioid tolerance.

Chronic treatment with opioids, such as morphine, leads to analgesic tolerance. While postsynaptic opioid tolerance is well documented, the involvement of presynaptic mechanisms remains unclear. We show that chronic morphine reduces the ability of periaqueductal grey (PAG) neurons to maintain GABAergic transmission. This depression of GABAergic transmission was due to a reduction in the effective size of the readily releasable pool. This also led to a reduction in opioid presynaptic inhibition; these presynaptic adaptations need to be considered in the development of strategies to reduce opioid tolerance. The midbrain periaqueductal grey (PAG) plays a critical role in tolerance to the analgesic actions of opioids such as morphine. While numerous studies have identified the postsynaptic adaptations induced by chronic morphine treatment in this and other brain regions, the presence of presynaptic adaptations remains uncertain. We examined GABAergic synaptic transmission within rat PAG brain slices from animals which underwent a low dose morphine treatment protocol which produces tolerance, but not withdrawal. Evoked GABAergic IPSCs (inhibitory postsynaptic currents) were less in morphine compared to control saline treated animals. Postsynaptic GABAA receptor mediated currents and desensitization, presynaptic release probability (Pr ), and inhibition by endogenous neurotransmitters were similar in morphine and saline treated animals. By contrast, the effective size of the readily releasable pool (RRP) was smaller in morphine treated animals. While the μ-opioid agonist DAMGO produced a reduction in Pr and RRP size in saline treated animals, it only reduced Pr in morphine treated animals. Consequently, DAMGO-induced inhibition of evoked IPSCs during short burst stimulation was less in morphine, compared to saline treated animals. These results indicate that low dose chronic morphine treatment reduces presynaptic μ-opioid inhibition by reducing the size of the pool of vesicles available for action potential dependent release. This novel presynaptic adaptation may provide important insights into the development of efficacious pain therapies that can circumvent the development of opioid tolerance.

P.1.g.072 - Striatonigral-nigrotectal circuits regulate the expression of innate fear responses via cannabinoid receptor type 1 on GABAergic neurons

Cannabinoid CB1 receptor (CB1R) activation decreases synaptic GABAergic and glutamatergic transmission and it also controls peripheral metabolism. Here we aimed at testing with 13C NMR isotopomer analysis whether CB1Rs could have a local metabolic role in brain areas having high CB1R density, such as the hippocampus. We labelled hippocampal slices with the tracers [2-13C]acetate, which is oxidized in glial cells, and [U–13C]glucose, which is metabolized both in glia and neurons, to evaluate metabolic compartmentation between glia and neurons. The synthetic CB1R agonist WIN55212-2 (1 μM) significantly decreased the metabolism of both [2-13C]acetate (−11.6 ± 2.0%) and [U–13C]glucose (−11.2 ± 3.4%) in the tricarboxylic acid cycle that contributes to the glutamate pool. WIN55212-2 also significantly decreased the metabolism of [U–13C]glucose (−11.7 ± 4.0%) but not that of [2-13C]acetate contributing to the pool of GABA. These effects of WIN55212-2 were prevented by the CB1R antagonist AM251 (500 nM). These results thus suggest that CB1Rs might be present also in hippocampal astrocytes besides their well-known neuronal localization. Indeed, confocal microscopy analysis revealed the presence of specific CB1R immunoreactivity in astrocytes and pericytes throughout the hippocampus.In conclusion, CB1Rs are able to control hippocampal intermediary metabolism in both neuronal and glial compartments, which suggests new alternative mechanisms by which CB1Rs control cell physiology and afford neuroprotection.

Gad1 mRNA as a reliable indicator of altered GABA release from orexigenic neurons in the hypothalamus.

The strength of γ-aminobutyric acid (GABA)-mediated inhibitory synaptic input is a principle determinant of neuronal activity. However, because of differences in the number of GABA afferent inputs and the sites of synapses, it is difficult to directly assay for altered GABA transmission between specific cells. The present study tested the hypothesis that the level of mRNA for the GABA synthetic enzyme glutamate decarboxylase (GAD) can provide a reliable proxy for GABA release. This was tested in a mouse hypothalamic circuit important in the regulation of energy balance. Fluorescent in situ hybridization results show that the expression of Gad1 mRNA (encoding the GAD67 enzyme) was increased in hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons after an overnight fast, consistent with the ability of GABA from these neurons to stimulate food intake. Optogenetic studies confirmed that the observed increase in Gad1 mRNA correlated with an increase in the probability of GABA release from NPY/AgRP neurons onto downstream proopiomelanocortin neurons. Likewise, there was an increase in the readily releasable pool of GABA in NPY/AgRP neurons. Selective inhibition of GAD activity in NPY/AgRP neurons decreased GABA release, indicating that GAD67 activity, which is largely dictated by expression level, is a key determinant of GABA release. Altogether, it appears that Gad expression may be a reliable proxy of altered GABAergic transmission. Examining changes in Gad mRNA as a proxy for GABA release may be particularly helpful when the downstream targets are not known or when limited tools exist for detecting GABA release at a particular synapse.

CB 1 receptor activation inhibits neuronal and astrocytic intermediary metabolism in the rat hippocampus

Cannabinoid CB 1 receptor (CB 1R) activation decreases synaptic GABAergic and glutamatergic transmission and it also controls peripheral metabolism. Here we aimed at testing with 13C NMR isotopomer analysis whether CB 1Rs could have a local metabolic role in brain areas having high CB 1R density, such as the hippocampus. We labelled hippocampal slices with the tracers [2- 13C]acetate, which is oxidized in glial cells, and [U– 13C]glucose, which is metabolized both in glia and neurons, to evaluate metabolic compartmentation between glia and neurons. The synthetic CB 1R agonist WIN55212-2 (1 μM) significantly decreased the metabolism of both [2- 13C]acetate (−11.6 ± 2.0%) and [U– 13C]glucose (−11.2 ± 3.4%) in the tricarboxylic acid cycle that contributes to the glutamate pool. WIN55212-2 also significantly decreased the metabolism of [U– 13C]glucose (−11.7 ± 4.0%) but not that of [2- 13C]acetate contributing to the pool of GABA. These effects of WIN55212-2 were prevented by the CB 1R antagonist AM251 (500 nM). These results thus suggest that CB 1Rs might be present also in hippocampal astrocytes besides their well-known neuronal localization. Indeed, confocal microscopy analysis revealed the presence of specific CB 1R immunoreactivity in astrocytes and pericytes throughout the hippocampus. In conclusion, CB 1Rs are able to control hippocampal intermediary metabolism in both neuronal and glial compartments, which suggests new alternative mechanisms by which CB 1Rs control cell physiology and afford neuroprotection.

In vivo neurochemical evidence that newly synthesised GABA activates GABA B, but not GABA A, receptors on dopaminergic nerve endings in the nucleus accumbens of freely moving rats

GABA released from accumbal GABAergic interneurons plays an inhibitory role in the regulation of dopamine efflux through GABA B and GABA A receptors located on accumbal dopaminergic nerve endings. The cytosolic newly synthesised GABA alters vesicular GABA levels and, accordingly, the amount of GABA released from the neuron. Therefore, we hypothesised that glutamic acid decarboxylase (GAD) which generates GABA in accumbal GABAergic neurons, at least partly determines the GABA receptor subtype-mediated GABAergic tonus. To (in)validate this hypothesis, in vivo microdialysis was used to study the effects of an intra-accumbal infusion of the GAD inhibitor l-allylglycine (allylglycine) on the accumbal dopamine efflux of freely moving rats. The intra-accumbal infusion of allylglycine (50.0, 250.0 and 500.0 nmol) dose-dependently increased the accumbal dopamine levels. The co-administration of tetrodotoxin (720 pmol) suppressed the allylglycine (500.0 nmol)-induced dopamine efflux. The intra-accumbal infusion of GABA B receptor agonist baclofen (2.5 and 5.0 nmol) inhibited the allylglycine (500.0 nmol)-induced dopamine efflux. The baclofen’s effects were counteracted by GABA B receptor antagonist saclofen (10.0 nmol). Neither GABA A receptor agonist (muscimol: 25.0 and 250.0 pmol) nor antagonist (bicuculline: 50.0 pmol) altered the allylglycine (250.0 and 500.0 nmol)-induced dopamine efflux. The present study provides in vivo neurochemical evidence that newly synthesised GABA that exerts an inhibitory tonus on the accumbal dopaminergic activity, acts at the level of GABA B receptors, but not GABA A receptors. The present study also shows that there is an allylglycine-insensitive GABA pool that release GABA exerting an inhibitory control of the accumbal dopaminergic activity, at the level of GABA A receptors. This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’.

The role of glia in neuronal recovery following anoxia: In vitro evidence of neuronal adaptation

We investigated the effects of 3 h of anoxia on metabolism of neurons and astrocytes, using a robust cell-based model system that mimics closely the living tissue milieu, i.e., in 3D neural aggregates cultured in bioreactors. Cells were incubated simultaneously with [1- 13C]glucose and [1,2- 13C]acetate; and, the gliotoxin fluorocitrate (FC) was used for glial tricarboxylic acid (TCA) cycle inhibition to assess the role of astrocytes for neuronal metabolism after oxygen deprivation. Results show that culture viability was not compromised by exposure to anoxia with and without FC. Interaction between astrocytes and glutamatergic neurons was altered due to anoxia: labeling in glutamine from [1- 13C]glucose was decreased, whereas that in glutamate from [1,2- 13C]acetate was increased. In contrast, GABA labeling was not affected by anoxia. It was shown that anoxia did not affect astrocytic capacity to synthesize glutamine in the reoxygenation period. The selective action of FC on astrocytes was confirmed. However, the presence of small amounts of glutamate and GABA labeled from acetate indicated residual activity of the glial TCA cycle. Although major metabolic changes were found due to FC-treatment, the intracellular pool of GABA was kept unchanged. Overall, our data clearly confirm that the glutamate–glutamine cycle depends on astrocytic TCA cycle activity and that mitochondrial impairment of astrocytes will ultimately stop metabolic trafficking between astrocytes and glutamatergic neurons. Additionally, our data suggest a metabolic independence of GABAergic neurons from astrocytes even after situations of complete oxygen depletion.

Increased network activity induces tonic inhibition by emerging a glial GABA pool

Event Abstract Back to Event Increased network activity induces tonic inhibition by emerging a glial GABA pool O. Kékesi1*, L. Héja1, G. Nyitrai1, Á. Dobolyi2, P. Szabó3, M. Palkovits2 and J. Kardos1 1 Hungarian Academy of Sciences, Department of Neurochemistry, Hungary 2 Semmelweis University and Hungarian Academy of Sciences, Hungary 3 Hungarian Academy of Sciences, Department of Pharmacobiochemistry, Hungary Maintenance of the balance between excitation and inhibition is of crucial importance in the brain. Increasing evidence suggest that astroglial glutamate/Na+ symporters induce the reverse action of extrasynaptic astroglial γ-amino-butyric acid/Na+ symporter subtypes GAT-2 and GAT-3, generating tonic inhibition in neurons. The source of the released γ-amino-butyric acid (GABA), however, is uncertain, because GABA is synthesized mainly in neurons from Glu by the enzyme Glu decarboxylase. Here we show that an alternative pathway for the GABA synthetic route exists in astrocytes and this pathway is responsible for the synthesis of the released GABA. We used in vitro hippocampal slices under low-[Mg2+] conditions to measure GABA currents by whole cell patch clamp method in the absence and presence of SNAP-5114, a non-transportable blocker of GAT-2/3. Under control conditions SNAP-5114 application reveals the GAT-2/3 mediated tonic current component. Incubation of the slices with monoamine and diamine oxidase inhibitors (deprenyl and aminoguanidine, respectively) that prevent GABA formation from putrescine eliminated this tonic current component, demonstrating that under low-[Mg2+] conditions astrocytes utilize the polyamine pathway to synthesize GABA for maintaining tonic inhibition. We envision that the discovery of this mechanism will support the development of new pathomechanism-specific treatments for pathological conditions characterized by sustained excitation. Keywords: Neurophysiology, Neuroscience Conference: 13th Conference of the Hungarian Neuroscience Society (MITT), Budapest, Hungary, 20 Jan - 22 Jan, 2011. Presentation Type: Abstract Topic: Neurophysiology Citation: Kékesi O, Héja L, Nyitrai G, Dobolyi Á, Szabó P, Palkovits M and Kardos J (2011). Increased network activity induces tonic inhibition by emerging a glial GABA pool. Front. Neurosci. Conference Abstract: 13th Conference of the Hungarian Neuroscience Society (MITT). doi: 10.3389/conf.fnins.2011.84.00152 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Mar 2011; Published Online: 23 Mar 2011. * Correspondence: Dr. O. Kékesi, Hungarian Academy of Sciences, Department of Neurochemistry, Budapest, Hungary, kekesi.orsolya@chemres.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers O. Kékesi L. Héja G. Nyitrai Á. Dobolyi P. Szabó M. Palkovits J. Kardos Google O. Kékesi L. Héja G. Nyitrai Á. Dobolyi P. Szabó M. Palkovits J. Kardos Google Scholar O. Kékesi L. Héja G. Nyitrai Á. Dobolyi P. Szabó M. Palkovits J. Kardos PubMed O. Kékesi L. Héja G. Nyitrai Á. Dobolyi P. Szabó M. Palkovits J. Kardos Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Presynaptic Actions of General Anesthetics Are Responsible for Frequency-Dependent Modification of Synaptic Transmission in the Rat Hippocampal CA1

In clinical anesthesia, robust surgical stress occasionally causes unintended light anesthesia during operation. To test the hypothesis that neural input condition could modify actions of general anesthetics as a result of presynaptic alteration in the central nervous system, we investigated the mechanisms by which the stimulus frequency modifies synaptic transmission of the rat hippocampus in the presence of general anesthetics. Field population spikes (PSs) of CA1 pyramidal neurons were elicited using orthodromic stimulation of Schaffer collateral-commissural fibers (test-pulse). A second stimulating electrode was placed in the region of the alveus hippocampi to activate recurrent inhibition of area CA1 (pre-pulse). The pre-pulses were applied as train stimuli (100-200 Hz) to activate release and then deplete the neurotransmitter (gamma-aminobutyric acid [GABA]) at presynaptic terminals of inhibitory interneurons. After the activation of inhibitory interneurons with pre-pulses, both IV (thiopental and pentobarbital) and volatile (sevoflurane and isoflurane) anesthetics attenuated the PS amplitudes elicited with test-pulses (test-PS). The IV anesthetics, but not the volatile drugs, produced stimulus frequency- and use-dependent recurrent inhibition of test-PSs. Neither a GABA type A agonist nor a GABA uptake inhibitor produced frequency-dependent modification. The pre-pulse train protocol revealed that IV anesthetics, but not volatile drugs, can enhance GABA release from presynaptic terminals. IV anesthetics, but not volatile drugs, enhance the discharge of a readily releasable pool of GABA vesicles from presynaptic terminals. Depletion of an active pool of GABA after high-frequency stimuli would produce frequency- and use-dependent recurrent inhibition in the presence of IV anesthetics. The stimulus frequency-dependent modification of synaptic transmission might be responsible for the unsuccessful immobilization or hypnosis during general anesthesia after IV anesthetic administration.

SAT1, a glutamine transporter, is preferentially expressed in GABAergic neurons

Subsets of GABAergic neurons are able to maintain high frequency discharge patterns, which requires efficient replenishment of the releasable pool of GABA. Although glutamine is considered a preferred precursor of GABA, the identity of transporters involved in glutamine uptake by GABAergic neurons remains elusive. Molecular analyses revealed that SAT1 (Slc38a1) features system A characteristics with a preferential affinity for glutamine, and that SAT1 mRNA expression is associated with GABAergic neurons. By generating specific antibodies against SAT1 we show that this glutamine carrier is particularly enriched in GABAergic neurons. Cellular SAT1 distribution resembles that of GAD67, an essential GABA synthesis enzyme, suggesting that SAT1 can be involved in translocating glutamine into GABAergic neurons to facilitate inhibitory neurotransmitter generation.

Blockade of GABA synthesis only affects neural excitability under activated conditions in rat hippocampal slices

The primary goal of this study was to establish whether inhibition of GABA synthesis was sufficient to induce network hyperexcitability in a rat hippocampal slice model comparable to that seen with GABA receptor blockade. We used field and intracellular recordings from the CA1 region of rat hippocampal slices to determine the physiological effects of blocking GABA synthesis with the convulsant, 3-mercaptoproprionic acid (MPA). We measured the rate of synthesis of GABA and glutamate in slices using 2-13C-glucose as a label source and liquid chromatography–tandem mass spectrometry. There was little effect of 3.5 mM MPA on evoked events under control recording conditions. Tissue excitability was enhanced following a series of stimulus trains; this effect was enhanced when GABA transport was blocked. Evoked inhibitory potentials (IPSPs) failed following repetitive stimulation and MPA. Spontaneous epileptiform activity was seen reliably with elevated extracellular potassium (5 mM). GABA synthesis decreased by 49% with MPA alone and 45% with the combination of MPA and excess potassium; GABA content was not substantially altered. Our data indicate: (1) GABAergic inhibition cannot be significantly compromised by MPA without network activation; (2) GABAergic synaptic inhibition is mediated by newly synthesized GABA; (3) there is a depletable pool of GABA that can sustain GABAergic inhibition when synthesis is impaired under basal, but not activated conditions; (4) overt hyperexcitability is only seen when newly synthesized GABA levels are low.

Glutamine uptake by System A transporters maintains neurotransmitter GABA synthesis and inhibitory synaptic transmission

GABA synthesis is necessary to maintain synaptic vesicle filling, and key proteins in its biosynthetic pathways may play a role in regulating inhibitory synaptic stability and strength. GABAergic neurons require a source of precursor glutamate, possibly from glutamine, although it is controversial whether glutamine contributes to the synaptic pool of GABA. Here we report that inhibition of System A glutamine transporters with alpha-(methyl-amino) isobutyric acid rapidly reduced the amplitude of inhibitory post-synaptic currents and miniature inhibitory post-synaptic currents (mIPSCs) recorded in rat hippocampal area cornu ammonis 1 (CA1) pyramidal neurons, indicating that synaptic vesicle content of GABA was reduced. After inhibiting astrocytic glutamine synthesis by either blocking glutamate transporters or the glutamine synthetic enzyme, the effect of alpha-(methyl-amino) isobutyric acid on mIPSC amplitudes was abolished. Exogenous glutamine did not affect mIPSC amplitudes, suggesting that the neuronal transporters are normally saturated. Our findings demonstrate that a constitutive supply of glutamine is provided by astrocytes to inhibitory neurons to maintain vesicle filling. Therefore, glutamine transporters, like those for glutamate, are potential regulators of inhibitory synaptic strength. However, in contrast to glutamate, extracellular glutamine levels are normally high. Therefore, we propose a supportive role for glutamine, even under resting conditions, to maintain GABA vesicle filling.

Two pools of Triton X‐100‐insoluble GABAA receptors are present in the brain, one associated to lipid rafts and another one to the post‐synaptic GABAergic complex

Rat forebrain synaptosomes were extracted with Triton X-100 at 4 degrees C and the insoluble material, which is enriched in post-synaptic densities (PSDs), was subjected to sedimentation on a continuous sucrose gradient. Two pools of Triton X-100-insoluble gamma-aminobutyric acid type-A receptors (GABA(A)Rs) were identified: (i) a higher-density pool (rho = 1.10-1.15 mg/mL) of GABA(A)Rs that contains the gamma2 subunit (plus alpha and beta subunits) and that is associated to gephyrin and the GABAergic post-synaptic complex and (ii) a lower-density pool (rho = 1.06-1.09 mg/mL) of GABA(A)Rs associated to detergent-resistant membranes (DRMs) that contain alpha and beta subunits but not the gamma2 subunit. Some of these GABA(A)Rs contain the delta subunit. Two pools of GABA(A)Rs insoluble in Triton X-100 at 4 degrees C were also identified in cultured hippocampal neurons: (i) a GABA(A)R pool that forms clusters that co-localize with gephyrin and remains Triton X-100-insoluble after cholesterol depletion and (ii) a GABA(A)R pool that is diffusely distributed at the neuronal surface that can be induced to form GABA(A)R clusters by capping with an anti-alpha1 GABA(A)R subunit antibody and that becomes solubilized in Triton X-100 at 4 degrees C after cholesterol depletion. Thus, there is a pool of GABA(A)Rs associated to lipid rafts that is non-synaptic and that has a subunit composition different from that of the synaptic GABA(A)Rs. Some of the lipid raft-associated GABA(A)Rs might be involved in tonic inhibition.

Differential subcellular and subsynaptic distribution of GABA A and GABA B receptors in the monkey subthalamic nucleus

The activation of GABA receptor subtype A (GABA A) and GABA receptor subtype B (GABA B) receptors mediates differential effects on GABAergic and non-GABAergic transmission in the basal ganglia. To further characterize the anatomical substrate that underlies these functions, we used immunogold labeling to compare the subcellular and subsynaptic localization of GABA A and GABA B receptors in the subthalamic nucleus (STN). Our findings demonstrate major differences and some similarities in the distribution of GABA A and GABA B receptors in the monkey STN. The immunoreactivity for GABA A receptor α1 subunits is mostly bound to the plasma membrane, whereas GABA B R1 subunit α1 immunoreactivity is largely expressed intracellularly. Plasma membrane-bound GABA A α1 subunit aggregate in the main body of putative GABAergic synapses, while GABA B R1 receptors are found at the edges of putative glutamatergic or GABAergic synapses. A large pool of plasma membrane-bound GABA A and GABA B receptors is extrasynaptic. In conclusion, these findings demonstrate a significant degree of heterogeneity between the distributions of the two major GABA receptor subtypes in the monkey STN. Their pattern of synaptic localization puts forward interesting questions regarding their mechanisms of activation and functions at GABAergic and non-GABAergic synapses.

Serotonergic regulation of the GABAergic transmission in the rat basal ganglia

The GABAergic neurons represent a major neuronal population in the basal ganglia. Although alterations in serotonin (5-HT) transmission are associated with neurodegenerative diseases involving these regions, the influence exerted by 5-HT afferents on GABAergic populations remains poorly understood. Here, we examined the consequences of 5,7-dihydroxytryptamine-induced lesion of 5-HT neurons on glutamic acid decarboxylase (GAD) activity, mRNA expression of the two isoforms of the enzyme, GAD65 and GAD67, GABA uptake, and extracellular GABA levels in the striatum. The 5-HT depletion produced an increase in GAD activity without modifying GAD65 and GAD67 mRNA levels, suggesting that 5-HT acts at the posttranscriptional level to regulate striatal GABA synthesis. No change in GAD activity was measured in the main striatal target structures, the globus pallidus and substantia nigra. Striatal GABA uptake and extracellular levels of GABA measured under basal conditions in freely moving rats were maintained in a normal range following 5-HT deprivation. By contrast, depolarization-induced increases in extracellular levels of GABA were larger in the striatum of 5-HT-deprived rats than in controls, which may be accounted for by an increase in a releasable pool of GABA due to increased synthesis rate. Together, these results suggest that 5-HT afferents may exert a phasic inhibitory control on striatal GABA transmission. Therefore, a decrease in striatal 5-HT transmission in disease states, such as Parkinson's disease, may contribute to pathological changes in striatal GABA neuron activity by increasing their reactivity to depolarizing stimuli.

Phenylarsine oxide inhibits α-latrotoxin-stimulated [ [formula omitted]]GABA release from rat brain synaptosomes

Phosphatidylinositol 4,5-biphosphate has been implicated in a variety of membrane-trafficking processes, including exocytosis of neurotransmitters. However, there are contradictory findings concerned ability of phenylarsine oxide (PAO), an inhibitor of phosphatidylinositol 4-kinase, to affect exocytotic release of different types of neurotransmitters. We bent our efforts to a detailed analysis of action of PAO on Ca 2+-dependent and Ca 2+-independent [ 3 H ]GABA release produced by exposure of rat brain synaptosomes to different concentrations of α-latrotoxin. We also compared PAO action on α-latrotoxin- and 4-aminopyridine (4-AP)-evoked [ 3 H ]GABA release. The experiments have shown that release of [ 3 H ]GABA evoked by the depolarization with 4-AP was decreased by 80% as a result of action of 3 μM PAO and the complete inhibition of release was observed with 10 μM PAO. When α-latrotoxin as a stimulant was applied, release of [ 3 H ]GABA was increased as toxin concentration used was elevated from 0.5 to 3.0 nM, however, concomitantly, the response of the toxin-induced [ 3 H ]GABA release to PAO became attenuated: 10 μM PAO led to almost complete inhibition of the effect of 0.5 nM α-latrotoxin and only partly decreased (by 40%) the response to 3.0 nM α-latrotoxin. To test whether the efficacy of PAO depended on the toxin-induced outflow of cytosolic [ 3 H ]GABA, synaptosomes with depleted cytosolic [ 3 H ]GABA pool were also exploited. Depletion was performed by means of heteroexchange of cytosolic [ 3 H ]GABA with nipecotic acid. The experiments have shown that treatment of loaded synaptosomes with nipecotic acid resulted in some increase of [ 3 H ]GABA release evoked by 0.5 nM α-latrotoxin, but in the two-fold decrease of the response to 3.0 nM α-latrotoxin. PAO essentially inhibited [ 3 H ]GABA release from depleted synaptosomes irrespective of α-latrotoxin concentration used. Therefore, the amount of [ 3 H ]GABA released from cytosolic pool determined, in considerable degree, the insensitivity of α-latrotoxin action to PAO. Thus, our data show that subnanomolar concentrations of α-latrotoxin may be used for stimulation of exocytotic release of [ 3 H ]GABA. Exposure of synaptosomes with nanomolar toxin concentrations leads not only to stimulation of exocytosis, but also to leakage of [ 3 H ]GABA from cytosolic pool. PAO potently inhibits exocytotic release of [ 3 H ]GABA and its inhibitory effectiveness is diminished as far as the outflow of [ 3 H ]GABA is elevated.

Does extracellular calcium determine what pool of GABA is the target for α-latrotoxin?

Presynaptic neurotoxin α-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 α-latrotoxin to Ca 2+-dependent (neurexin 1α) 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 α-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 α-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 α-latrotoxin was greatly augmented with respect to the control response. Another way to assay what GABA pool was involved in α-latrotoxin-induced release lays in an analysis of the effects of depolarisation and α-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 α-latrotoxin effect was revealed. Employing digitonin-permeated synaptosomes, we have shown that α-latrotoxin could stimulate [ 3 H ]GABA release in the medium with 1 mM EGTA, this effect of the toxin was blocked by concanavalin A and was ATP-dependent. The latter suggests that α-latrotoxin-released neurotransmitter has the vesicular nature. We assume that the type of the toxin membrane receptor does not determine the mechanisms of [ 3 H ]GABA release evoked by α-latrotoxin.