Mechanism by which GABA, through its GABA(A) receptor, modulates glutamate release from rat cortical neurons in culture

In cortical neurons, the GABA(A) agonist, muscimol, increases: (a) basal glutamate release (with a EC50 of 99 +/- 7 microM); (b) intracellular calcium and (c) membrane potential, all of these in a dose-dependent manner. These muscimol effects were specific since they were reversed by bicuculline, a GABA(A) antagonist. When the action of muscimol was measured at different KCl concentrations, an increase or decrease of the glutamate secretion was observed, depending on the KCl concentration in the medium. At low KCl concentration (5.6 mM of KCl), it depolarized, at 20 mM of KCl it had no effect, but at higher KCl concentrations (30-100 microM of KCl), it produced a hyperpolarization in these cells. The mechanism by which the GABA-Cl(-)-channel permits Cl- fluxes, inward or outward, depending on the membrane potential.     

NMDA-mediated release of glutamate and GABA in the subthalamic nucleus is mediated by dopamine: an in vivo microdialysis study in rats

The present study investigated the effects of N-methyl-D-aspartic acid.H2O (NMDA) on the dopamine, glutamate and GABA release in the subthalamic nucleus (STN) by using in vivo microdialysis in rats. NMDA (100 micromol/L) perfused through the microdialysis probe evoked an increase in extracellular dopamine in the STN of the intact rat of about 170%. This coincided with significant increases in both extracellular glutamate (350%) and GABA (250%). The effect of NMDA perfusion on neurotransmitter release at the level of the STN was completely abolished by co-perfusion of the selective NMDA-receptor antagonist MK-801 (10 micromol/L), whereas subthalamic perfusion of MK-801 alone had no effect on extracellular neurotransmitter concentrations. Furthermore, NMDA induced increases in glutamate were abolished by both SCH23390 (8 micromol/L), a selective D1 antagonist, and remoxipride (4 micromol/L), a selective D2 antagonist. The NMDA induced increase in GABA was abolished by remoxipride but not by SCH23390. Perfusion of the STN with SCH23390 or remoxipride alone had no effect on extracellular neurotransmitter concentrations. The observed effects in intact animals depend on the nigral dopaminergic innervation, as dopamine denervation, by means of 6-hydroxydopamine lesioning of the substantia nigra, clearly abolished the effects of NMDA on neurotransmitter release at the level of the STN. Our work points to a complex interaction between dopamine, glutamate and GABA with a crucial role for dopamine at the level of the STN.     

Extrasynaptic release of GABA and dopamine by retinal dopaminergic neurons

In the mouse retina, dopaminergic amacrine (DA) cells synthesize both dopamine and GABA. Both transmitters are released extrasynaptically and act on neighbouring and distant retinal neurons by volume transmission. In simultaneous recordings of dopamine and GABA release from isolated perikarya of DA cells, a proportion of the events of dopamine and GABA exocytosis were simultaneous, suggesting co-release. In addition, DA cells establish GABAergic synapses onto AII amacrine cells, the neurons that transfer rod bipolar signals to cone bipolars. GABA_A but not dopamine receptors are clustered in the postsynaptic membrane. Therefore, dopamine, irrespective of its site of release—synaptic or extrasynaptic—exclusively acts by volume transmission. Dopamine is released upon illumination and sets the gain of retinal neurons for vision in bright light. The GABA released at DA cells'' synapses probably prevents signals from the saturated rods from entering the cone pathway when the dark-adapted retina is exposed to bright illumination. The GABA released extrasynaptically by DA and other amacrine cells may set a ‘GABAergic tone’ in the inner plexiform layer and thus counteract the effects of a spillover of glutamate released at the bipolar cell synapses of adjacent OFF and ON strata, thus preserving segregation of signals between ON and OFF pathways.     

Evidence that nigral GABA mediates behavioural responses elicited by striatal dopamine receptor stimulation

Bilateral intranigral administration of GABA-receptor blockers (picrotoxin or bicuculline) elicits catatonia not surmountable by administration of apomorphine, a dopamine (DA)-receptor agonist. Unilateral intranigral administration of GABA-receptor blockers ipsilateral to a lesion of nigro-striatal DA-neurons induced with 60H-DA, antagonizes the contralateral turning produced by apomorphine. The results indicate that behavioural responses elicited by striatal DA-receptor stimulation are effected through a GABA-mediated inhibition of pars reticulata neurons.     

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

Background

GABA_A receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs [1]. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear [2]. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die [3]–[6]. As many anaesthetics act via GABA_A receptors [7], the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients.

Principal Findings

We demonstrate, using RT-PCR, that monocytes express GABA_A receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA_A receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA_A receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin.

Significance

Our results show that functional GABA_A receptors are present on monocytes with properties similar to CNS GABA_A receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA_A receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA_A receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.     

Excitation by dopamine D-2 receptor agonists, bromocriptine and LY 171555, in caudate nucleus neurons activated by nigral stimulation

Electrophysiological studies using cats anesthetized with alpha-chloralose were carried out to determine whether or not the dopamine D-2 receptor mediates the excitation of the caudate nucleus (CN) neurons activated by stimulation of the substantia nigra (SN). Microiontophoretic application of domperidone (D-2 antagonist) produced a significant inhibition of spikes elicited by SN stimulation in 20 of 27 CN neurons. When bromocriptine and LY 171555 (D-2 agonists) were iontophoretically applied to the CN neurons in which the SN-induced spikes were inhibited by domperidone, an increase in spontaneous firing rate was observed in 18 of 20 neurons and all of 10 neurons tested, respectively. However, no alterations of firing occurred with bromocriptine or LY 171555 in any 7 neurons in which the SN-induced spikes were not affected by domperidone. The increase in firing rate by the D-2 agonists was apparently antagonized during simultaneous application of domperidone and haloperidol, but not affected during application of SCH 23390 (D-1 antagonist). These results strongly suggest that the spike generation of the CN neurons upon SN stimulation is mediated by the dopamine D-2 receptor.     

Disinhibition of nigral GABA output neurons mediates muscular rigidity elicited by striatal opioid receptor stimulation

Unilateral injection of bicuculline (12.5-50 ng) into the substantia nigra pars reticulata (SNR) dose-dependently produced a tonic activity in the electromyogram (EMG), which was antagonized by coadministered muscimol. Bilateral lesions of the caudate nucleus with kainic acid failed to affect the EMG activity produced by unilateral injection of bicuculline into the SNR. Administration of muscimol (12.5 and 25 ng) into the SNR antagonized the tonic activity in the EMG produced by morphine (15 mg/kg i.p.), whereas bicuculline (50 ng) enhanced it. These results suggest that an impairment in the GABAergic neurotransmission in the SNR is relevant for mediating the tonic activity in the EMG produced by morphine and that GABAergic mechanisms in the SNR play an important role in the modulating the opioid-induced alterations in the striatal function.     

GABA(A) receptor function is regulated by lipid bilayer elasticity

Docosahexaenoic acid (DHA) and other polyunsaturated fatty acids (PUFAs) promote GABA(A) receptor [(3)H]-muscimol binding, and DHA increases the rate of GABA(A) receptor desensitization. Triton X-100, a structurally unrelated amphiphile, similarly promotes [(3)H]-muscimol binding. The mechanism(s) underlying these effects are poorly understood. DHA and Triton X-100, at concentrations that affect GABA(A) receptor function, increase the elasticity of lipid bilayers measured as decreased bilayer stiffness using gramicidin channels as molecular force transducers. We have previously shown that membrane protein function can be regulated by amphiphile-induced changes in bilayer elasticity and hypothesized that GABA(A) receptors could be similarly regulated. We therefore studied the effects of four structurally unrelated amphiphiles that decrease bilayer stiffness (Triton X-100, octyl-beta-glucoside, capsaicin, and DHA) on GABA(A) receptor function in mammalian cells. All the compounds promoted GABA(A) receptor [(3)H]-muscimol binding by increasing the binding capacity of high-affinity binding without affecting the associated equilibrium binding constant. A semiquantitative analysis found a similar quantitative relation between the effects on bilayer stiffness and [(3)H]-muscimol binding. Membrane cholesterol depletion, which also decreases bilayer stiffness, similarly promoted [(3)H]-muscimol binding. In whole-cell voltage-clamp experiments, Triton X-100, octyl-beta-glucoside, capsaicin, and DHA all reduced the peak amplitude of the GABA-induced currents and increased the rate of receptor desensitization. The effects of the amphiphiles did not correlate with the expected changes in monolayer spontaneous curvature. We conclude that GABA(A) receptor function is regulated by lipid bilayer elasticity. PUFAs may generally regulate membrane protein function by affecting the elasticity of the host lipid bilayer.     

Modulation of glutamate release from parallel fibers by mGlu4 and pre-synaptic GABA(A) receptors

The regulation of pre-synaptic glutamate release is important in the maintenance and fidelity of excitatory transmission in the nervous system. In this study, we report a novel interaction between a ligand-gated ion channel and a G-protein coupled receptor which regulates glutamate release from parallel fiber axon terminals. Immunocytochemical analysis revealed that GABA(A) receptors and the high affinity group III metabotropic glutamate receptor subtype 4 (mGlu4) are co-localized on glutamatergic parallel fiber axon terminals in the cerebellum. GABA(A) and mGlu4 receptors were also found to co-immunoprecipitate from cerebellar membranes. Independently, these two receptors have opposing roles on glutamate release: pre-synaptic GABA(A) receptors promote, while mGlu4 receptors inhibit, glutamate release. However, coincident activation of GABA(A) receptors with muscimol and mGlu4 with the agonist (2S)-S-2-amino-4-phosphonobutanoic acid , increased glutamate release from [(3) H]glutamate-loaded cerebellar synaptosomes above that observed with muscimol alone. Further support for an interaction between GABA(A) and mGlu4 receptors was obtained in the mGlu4 knockout mouse which displayed reduced binding of the GABA(A) ligand [(35) S]tert-butylbicyclophosphorothionate, and decreased expression of the α1, α6, β2 GABA(A) receptor subunits in the cerebellum. Taken together, our data suggest a new role for mGlu4 whereby simultaneous activation with GABA(A) receptors acts to amplify glutamate release at parallel fiber-Purkinje cell synapses.     

Nerve growth factor-induced glutamate release is via p75 receptor,ceramide, and Ca(2+) from ryanodine receptor in developing cerebellar neurons

Very little is known about the contribution of a low affinity neurotrophin receptor, p75, to neurotransmitter release. Here we show that nerve growth factor (NGF) induced a rapid release of glutamate and an increase of Ca2+ in cerebellar neurons through a p75-dependent pathway. The NGF-induced release occurred even in the presence of the Trk inhibitor K252a. The release caused by NGF but not brain-derived neurotrophic factor was enhanced in neurons overexpressing p75. Further, after transfection of p75-small interfering RNA, which down-regulated the endogenous p75 expression, the NGF-induced release was inhibited, suggesting that the NGF-induced glutamate release was through p75. We found that the NGF-increased Ca2+ was derived from the ryanodine-sensitive Ca2+ receptor and that the NGF-increased Ca2+ was essential for the NGF-induced glutamate release. Furthermore, scyphostatin, a sphingomyelinase inhibitor, blocked the NGF-dependent Ca2+ increase and glutamate release, suggesting that a ceramide produced by sphingomyelinase was required for the NGF-stimulated Ca2+ increase and glutamate release. This action of NGF only occurred in developing neurons whereas the brain-derived neurotrophic factor-mediated Ca2+ increase and glutamate release was observed at the mature neuronal stage. Thus, we demonstrate that NGF-mediated neurotransmitter release via the p75-dependent pathway has an important role in developing neurons.     

Regulated secretion of neurotrophins by metabotropic glutamate group I (mGluRI) and Trk receptor activation is mediated via phospholipase C signalling pathways

Neurotrophins (NTs) play an essential role in modulating activity-dependent neuronal plasticity. In this context, the site and extent of NT secretion are of crucial importance. Here, we demonstrate that the activation of phospolipase C (PLC) and the subsequent mobilization of Ca(2+) from intracellular stores are essential for NT secretion initiated by both Trk and glutamate receptor activation. Mutational analysis of tyrosine residues, highly conserved in the cytoplasmic domain of all Trk receptors, revealed that the activation of PLC-gamma in cultured hippocampal neurons and nnr5 cells is necessary to mobilize Ca(2+) from intracellular stores, the key mechanism for regulated NT secretion. A similar signalling mechanism has been identified for glutamate-mediated NT secretion-which in part depends on the activation of PLC via metabotropic receptors-leading to the mobilization of Ca(2+) from internal stores by inositol trisphosphate. Thus, PLC-mediated signal transduction pathways are the common mechanisms for both Trk- and mGluRI-mediated NT secretion.     

Glutamate-induced swelling of cultured astrocytes is mediated by metabotropic glutamate receptor

The effects of glutamate and its agonists and antagonists on the swelling of cultured astrocytes were studied. Swelling of astrocytes was measured by [3H]-O-methyl-D-glucose uptake. Glutamate at 0.5, 1 and 10mmol/L and irons-l-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD), a metabotropic glutamate receptor (mGluR) agonist, at 1 mmol/L caused a significant increase in astrocytic volume, whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) was not effective. L-2-amino-3-phosphonopropionic acid (L-AP3), an antagonist of mGluR, blocked the astrocytic swelling induced by trans-ACPD or glutamate. In Ca2+-free condition, glutamate was no longer effective. Swelling of astrocytes induced by glutamate was not blocked by CdCl2 at 20 μmol/L, but significantly reduced by CdCl2 at 300 μmol/L and dantrolene at 30 μmol/L. These findings indicate that mGluR activation results in astrocytic swelling and both extracellular calcium and internal calcium stores play important roles in the genes     

Discovery of a novel potent GABA(B) receptor agonist

Structure-activity studies have led to a discovery of 3-(4-pyridyl)methyl ether derivative 9d that has 25- to 50-fold greater functional potency than R-baclofen at human and rodent GABA(B) receptors in vitro. Mouse hypothermia studies confirm that this compound crosses the blood-brain barrier and is approximately 50-fold more potent after systemic administration.     

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

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.     

Adenosine A2A receptor activation is determinant for BDNF actions upon GABA and glutamate release from rat hippocampal synaptosomes

Adenosine, through A_2A receptor (A_2AR) activation, can act as a metamodulator, controlling the actions of other modulators, as brain-derived neurotrophic factor (BDNF). Most of the metamodulatory actions of adenosine in the hippocampus have been evaluated in excitatory synapses. However, adenosine and BDNF can also influence GABAergic transmission. We thus evaluated the role of A_2AR on the modulatory effect of BDNF upon glutamate and GABA release from isolated hippocampal nerve terminals (synaptosomes). BDNF (30 ng/ml) enhanced K⁺-evoked [³H]glutamate release and inhibited the K⁺-evoked [³H]GABA release from synaptosomes. The effect of BDNF on both glutamate and GABA release requires tonic activation of adenosine A_2AR since for both neurotransmitters, the BDNF action was blocked by the A_2AR antagonist SCH 58261 (50 nM). In the presence of the A_2AR agonist, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 (30 nM), the effect of BDNF on either glutamate or GABA release was, however, not potentiated. It is concluded that both the inhibitory actions of BDNF on GABA release as well as the facilitatory action of the neurotrophin on glutamate release are dependent on the activation of adenosine A_2AR by endogenous adenosine. However, these actions could not be further enhanced by exogenous activation of A_2AR.     

Nicotine induced excitation of locus coeruleus neurons is mediated via release of excitatory amino acids

Previous electrophysiological studies have shown that systemically administered nicotine in low doses increases the firing rate of rat locus coeruleus (LC) neurons. In the present study, this action of nicotine was found to be prevented by pretreatment with kynurenic acid (1 mumol; i.c.v.). In addition, pretreatment with MK 801 effectively blocked the nicotine induced LC excitation in most neurons tested (60%) whereas the rest were left unaffected by this treatment. It is suggested that excitatory amino acids (EAA), e.g. released from nerve terminals from nucleus paragigantocellularis (PGi), indirectly mediated the effect of nicotine on LC neurons.     

Transporter mediated GABA release in the retina: role of excitatory amino acids and dopamine

In general, the release of neurotransmitters in the central nervous system is accomplished by a calcium-dependent process which constitutes a common feature of exocytosis, a conserved mechanism for transmitter release in all species. However, neurotransmitters can also be released by the reversal of their transporters. In the retina, a large portion of GABA is released by this mechanism, which is under the control of neuroactive agents, such as excitatory amino acids and dopamine. In this review, we will focus on the transporter mediated GABA release and the role played by excitatory amino acids and dopamine in this process. First, we will discuss the works that used radiolabeled GABA to study the outflow of the neurotransmitter and then the works that took into consideration the endogenous pool of GABA and the topography of GABAergic circuits influenced by excitatory amino acids and dopamine.     

Transporter mediated GABA release in the retina: Role of excitatory amino acids and dopamine

In general, the release of neurotransmitters in the central nervous system is accomplished by a calcium-dependent process which constitutes a common feature of exocytosis, a conserved mechanism for transmitter release in all species. However, neurotransmitters can also be released by the reversal of their transporters. In the retina, a large portion of GABA is released by this mechanism, which is under the control of neuroactive agents, such as excitatory amino acids and dopamine. In this review, we will focus on the transporter mediated GABA release and the role played by excitatory amino acids and dopamine in this process. First, we will discuss the works that used radiolabeled GABA to study the outflow of the neurotransmitter and then the works that took into consideration the endogenous pool of GABA and the topography of GABAergic circuits influenced by excitatory amino acids and dopamine.     

Endogenous phosphorylation of the GABA(A) receptor protein is counteracted by a membrane-associated phosphatase

Incubation of bovine brain membranes with [gamma-33P]ATP phosphorylated mainly a 51-kDa band. Electrophoretic co-migration was observed for 33P- and [3H]flunitrazepam-labeled bands in both membrane fractions and in affinity-purified GABA(A) receptor (GABAA-R) preparations. An alpha-subunit monoclonal antibody adsorbed most of the radiolabeled-band, suggesting that the labeled-membrane polypeptide corresponds to the GABA(A)-R alpha1-subunit, which is the only GABA(A)-R subunit with a molecular weight of 51 kDa. The phosphorylation rate was much faster in membranes than in purified receptor. Dephosphorylation was detected in membranes only. The membrane-bound phosphatase was potently inhibited by vanadate and Zn2+>Mn2+ , but was insensitive to okadaic acid (a phosphatase 1, 2 and 2B inhibitor), cyclosporin (specific calcineurin inhibitor) and phosphatase-1 inhibitor. Endogenous kinase was activated by divalent cations including calcium (Mg2- > Mn2+ > Ca2+), whilst dephosphorylation did not require the presence of Ca2+ ions. This suggests that at least one membrane-bound phosphatase counteracts the endogenous phosphorylation of the GABA(A)-R: the lack of dephosphorylation in the purified receptor preparation indicates that, in contrast to the endogenous kinase, no phosphatase is closely associated with the receptor protein complex.     

Alcohol-induced blood-brain barrier dysfunction is mediated via inositol 1,4,5-triphosphate receptor (IP3R)-gated intracellular calcium release

The blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC), pericytes and astrocytes controls the transport of ions, peptides and leukocytes in and out of the brain. Tight junctions (TJ) composed of TJ proteins (occludin, claudins and zonula occludens) ensure the structural integrity of the BMVEC monolayer. Neuropathologic studies indicated that the BBB was impaired in alcohol abusers; however, the underlying mechanism of BBB dysfunction remains elusive. Using primary human BMVEC, we previously demonstrated that oxidative stress induced by ethanol (EtOH) metabolism in BMVEC activated myosin light chain kinase (MLCK), resulting in the enhanced phosphorylation of either cytoskeletal or TJ proteins, and in BBB impairment. We proposed that EtOH metabolites stimulated inositol 1,4,5-triphosphate receptor (IP(3)R)-operated intracellular calcium (Ca(2+)) release, thereby causing the activation of MLCK in BMVEC. Indeed, treatment of primary human BMVEC with EtOH or its metabolites resulted in the increased expression of IP(3)R protein and IP(3)R-gated intracellular Ca(2+) release. These functional changes paralleled MLCK activation, phosphorylation of cytoskeletal/TJ proteins, loss of BBB integrity, and enhanced leukocyte migration across BMVEC monolayers. Inhibition of either EtOH metabolism or IP(3)R activation prevented BBB impairment. These findings suggest that EtOH metabolites act as signaling molecules for the activation of MLCK via the stimulation of IP(3)R-gated intracellular Ca(2+) release in BMVEC. These putative events can lead to BBB dysfunction in the setting of alcoholism, and to neuro-inflammatory disorders promoting leukocyte migration across the BBB.