GAT-1 regulates both tonic and phasic GABA(A) receptor-mediated inhibition in the cerebral cortex

γ-Aminobutyric acid 1 (GAT-1) is the most copiously expressed GABA transporter; we studied its role in phasic and tonic inhibition in the neocortex using GAT-1 knockout (KO) mice. Immunoblotting and immunocytochemical studies showed that GAT-2 and GAT-3 levels in KOs were unchanged and that GAT-3 was not redistributed in KOs. Moreover, the expression of GAD65/67 was increased, whereas that of GABA or VGAT was unchanged. Microdialysis studies showed that in KOs spontaneous extracellular release of GABA and glutamate was comparable in WT and KO mice, whereas KCl-evoked output of GABA, but not of glutamate, was significantly increased in KOs. Recordings from layer II/III pyramids revealed a significant increase in GABA_AR-mediated tonic conductance in KO mice. The frequency, amplitude and kinetics of spontaneous inhibitory post-synaptic currents (IPSCs) were unchanged, whereas the decay time of evoked IPSCs was significantly prolonged in KO mice. In KO mice, high frequency stimulation of GABAergic terminals induced large GABA_AR-mediated inward currents associated with a reduction in amplitude and decay time of IPSCs evoked immediately after the train. The recovery process was slower in KO than in WT mice. These studies show that in the cerebral cortex of GAT-1 KO mice GAT-3 is not redistributed and GADs are adaptively changed and indicate that GAT-1 has a prominent role in both tonic and phasic GABA_AR-mediated inhibition, in particular during sustained neuronal activity.     

Elevation of Brain GABA Content by Chronic Low-Dosage Administration of Hydrazine, a Metabolite of Isoniazid

Abstract: When γ-aminobutyric acid aminotransferase (GABA-T) activity was measured in vitro in rat brain, neither isoniazid (INH) nor four of its known metabolites (isonicotinic acid, acetylisoniazid, acetylhydrazine, diacetylhydrazine) inhibited the enzyme in concentrations (5 mM) far higher than those likely to be achieved when INH is administered to man. In contrast, hydrazine (5 μM) caused a 50% inhibition of GABA-T without inhibiting glutamic acid decarboxylase (GAD). Rats were injected daily for 109 days with hydrazine (0.08 or 0.16 mmol/kg/day), after which amino acid contents and enzyme activities were measured in their brains. Both hydrazine doses caused significant elevations of whole brain GABA content and reductions of GABA-T activity, but did not affect GAD activity. Chronic administration of hydrazine at thee doses did not reduce weight gain or alter rat behavior, nor did it produce any irreversible pathologic changes in liver or alterations in hepatic aryl hydrocarbon hydroxylase activity. However, hydrazine treatment caused changes in the contents of many brain amino acids besides GABA, and markedly increased concentrations of ornithine, tyrosine, and α-aminoadipic acid in rat plasma. Inhibition of GABA-T activity and the other biochemical alterations observed in patients given high doses of INH probably result from hydrazine formed in the metabolic degradation of INH. Thus administration of hydrazine might be a more direct means of elevating brain GABA content in patients where this seems indicated, and might not entail a greater risk of adverse effects.     

The influence of membrane potential on chloride channels activated by GABA in rat cultured hippocampal neurons

Chloride currents were activated by a low concentration of GABA (0.5 m) in neonatal rat hippocampal neurons cultured for up to 14 days. Currents elicited by 0.5 m GABA in neurons, voltage-clamped using the whole-cell technique with pipettes containing 149 mm Cl^–, reversed close to 0 mV whether pipettes contained 144 mm Na⁺ or 140 mm Cs⁺, and were blocked by 100 m bicuculline. Current-voltage curves showed outward rectification. Single channel currents appeared in cell-attached patches when the pipette tip was perfused with pipette solution containing 0.5 m GABA and disappeared when a solution containing 100 m bicuculline plus 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification and, in some patches, had a much lower probability of opening at hyperpolarized potentials. The average chord conductance in 10 patches hyperpolarized by 80 mV was 7.8±1.6 pS (sem) compared with a chord conductance of 34.1±3.5 pS (sem) in the same patches depolarized by 80 mV. Similar single channel currents were also activated in cell-free, inside-out patches in symmetrical chloride solutions when 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification similar to that seen in cell-attached patches, and some channels had a lower probability of opening at hyperpolarized potentials. The average chord conductance in 13 patches hyperpolarized by 80 mV was 11.8±2.3 pS (sem) compared with 42.1±3.1 pS (sem) in the same patches depolarized by 80 mV.We are grateful to B. McLachlan and M. Robertson for their general assistance, to C. McCulloch and M. Smith for writing computer programs and to W. O'Hare for making the pipette injection device.     

An intracellular motif of P2X(3) receptors is required for functional cross-talk with GABA(A) receptors in nociceptive DRG neurons

Functional cross-talk between structurally unrelated P2X ATP receptors and members of the 'cys-loop' receptor-channel superfamily represents a recently-discovered mechanism for rapid modulation of information processing. The extent and the mechanism of the inhibitory cross-talks between these two classes of ionotropic receptors remain poorly understood, however. Both ionic and molecular coupling were proposed to explain cross-inhibition between P2X subtypes and GABA(A) receptors, suggesting a P2X subunit-dependent mechanism. We show here that cross-inhibition between neuronal P2X(3) or P2X(2+3) and GABA(A) receptors does not depend on chloride and calcium ions. We identified an intracellular QST(386-388) motif in P2X(3) subunits which is required for the functional coupling with GABA(A) receptors. Moreover the cross-inhibition between native P2X(3) and GABA receptors in cultured rat dorsal root ganglia (DRG) neurons is abolished by infusion of a peptide containing the QST motif as well as by viral expression of the main intracellular loop of GABA(A)beta3 subunits. We provide evidence that P2X(3) and GABA(A) receptors are colocalized in the soma and central processes of nociceptive DRG neurons, suggesting that specific intracellular P2X(3)-GABA(A) subunit interactions underlie a pre-synaptic cross-talk that might contribute to the regulation of sensory synaptic transmission in the spinal cord.     

Transmembrane Transport and Release of GABA in the Brain of Rats Subjected to Postnatal Hypoxia

We studied the effects of early postnatal hypoxia on the efficiency of active GABA transport through the plasma membrane of synaptic terminals (synaptosomes) isolated from the cerebral cortex, hippocampus, and thalamus of rats and on non-stimulated and Ca²⁺-stimulated GABA release. The state of hypoxia was induced by exposure of 10- to 12-day-old rats to a respiratory medium with low O₂ content (4% О₂ and 96% N₂) for 12 min (up to the initiation of clonico-tonic seizures). Animals were taken in the experiment 8 to 9 weeks after an episode of hypoxic stress. The intensity of transmembrane transport of GABA was estimated according to accumulation of [³Н]GABA in a coarse synaptosomal fraction. The process was characterized by calculation of the Michaelis constant K _m and also of the initial (within the 1st min) and maximum rates of accumulation of [³Н]GABA. The means of the initial rate of [³Н]GABA accumulation in preparations from the thalamus, cortex, and hippocampus were 205.5 ± 8.8, 266.2 ± 29.6, and 302.3 ± 31.2 pmol/min⋅mg protein, respectively. Hypoxic stress influenced the rates of accumulation of [³Н]GABA in synaptic terminals from the cortex and hippocampus but not in those from the thalamus. According to the characteristics of the response to hypoxic stress, all experimental animals could be classified into two groups. In some rats, accumulation of [³Н]GABA in both cortical and hippocampal synaptosomes decreased insignificantly (by about 15%), while in other animals this parameter increased significantly (by nearly 50%) for the cortex and decreased by 21.5%, on average, for the hippocampus. The affinity of the transporter with respect to [³Н]GABA in the cortex and hippocampus was nearly the same and showed no changes under the influence of hypoxia. The non-stimulated release of [³Н]GABA after the influence of hypoxia increased in all structures, while the depolarization-induced Ca²⁺-dependent release of [³Н]GABA was intensified only in synaptosomes from the cerebral cortex. The mechanisms of development of modifications of GABA-ergic processes under the influence of hypoxic stress in the course of the perinatal period are discussed. Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 293–302, July–August, 2008.     

Depolarization-evoked GABA release from myenteric plexus is partially coupled to L-, N-, and P/Q-type calcium channels

1. There are many evidences suggesting that -aminobutyrate (GABA) is an important neurotransmitter and/or neuromodulator in the gut.2. Using the myenteric plexus-longitudinal muscle preparation from the guinea pig ileum, we investigated the evoked release of [³H] GABA from enteric neurons by electrical pulses or high KCl, which occurs in a calcium-dependent and -independent way. In addition, using selective calcium channel blockers, we report the participation of distinct subtypes of calcium channels in the evoked release, showing a minor participation of L- and Q-type calcium channels, while N- and P-type have a participation of approximately 15%, each. However, regardless of the combination of Ca²⁺ channel blockers, we did not observe an inhibition greater than 50% of the calcium-dependent component of [³H] GABA release.3. Thus, while the observed Ca²⁺-independent release mostly probable occur via reversal of the membrane GABA transporter, in our conditions, a considerable portion of the Ca²⁺-dependent evoked release of [³H] GABA is not coupled to L-, N-, or P/Q-type calcium channels, suggesting the involvement of intracellular calcium stores or other ways of getting calcium across the membrane.     

Conductance states activated by glycine and GABA in rat cultured spinal neurones

Summary The conductance properties of single Cl^– channels activated by glycine and gamma-aminobutyric acid (GABA) were examined in rat spinal cord neurones grown in cell culture. The majority (85%) of spinal neurones were sensitive to both glycine and GABA as were most (83%) outside-out patches tested. Glycine and GABA activated multiple conductance state Cl^– channels with linear current-voltage properties when the chloride activities of the solutions bathing both sides of the membrane were similar. Glycine activated six distinct conductance states with conductances of 14, 20, 30, 43, 64 and 93 pS, whereas GABA activated five states with conductances of 13, 20, 29, 39 and 71 pS. The 30 and 43 pS states and the 20 and 29 pS states were observed most frequently with glycine and GABA, respectively. As the values of the glycine- and GABA-activated conductance states form a geometric progression when arranged in ascending order, we concluded that the channels do not consist of a cluster of identical pores. Additional conductance states (50 and 100 pS) were activated by glycine occasionally. The similarity between the conductances of the states activated by the two transmitters is consistent with the proposal that they both activate the same type of Cl^– channel.     

Anticonvulsant effects of the wasp Polybia ignobilis venom on chemically induced seizures and action on GABA and glutamate receptors

Venoms of spiders and wasps are well recognized to present high affinity to the central nervous tissue of many mammalian species. Here we describe the effects of direct exposure of rat (Rattus norvegicus) brains to the crude and denatured venom of the Brazilian social wasp Polybia ignobilis. Lower doses of crude venom injected via intracerebroventricular (i.c.v.) inhibited the exploratory activity of animals, while higher doses provoked severe generalized tonic-clonic seizures, with hind limb extension. The status epilepticus lasted for few minutes leading the animals to respiratory depression and death. In contrast, the denatured venom was anticonvulsant against acute seizures induced by the i.c.v. injection of bicuculline, picrotoxin and kainic acid, but it was ineffective against seizures caused by systemic pentylenetetrazole. Moreover, the [3H]-glutamate binding in membranes from rat brain cortex was inhibited by the denatured venom in lower concentrations than the [3H]-GABA binding. The denatured venom contains free GABA and glutamate (34 and 802 pg/microg of venom, respectively), but they are not the major binding inhibitors. These interactions of venom components with GABA and glutamate receptors could be responsible for the anticonvulsant effects introducing the venom from P. ignobilis as a potential pharmacological source of anticonvulsant drugs.     

In vivo modulation of extracellular hippocampal glutamate and GABA levels and limbic seizures by group I and II metabotropic glutamate receptor ligands

The effects of several metabotropic receptor (mGluR) ligands on baseline hippocampal glutamate and GABA overflow in conscious rats and the modulation of limbic seizure activity by these ligands were investigated. Intrahippocampal mGluR group I agonist perfusion via a microdialysis probe [1 mm (R,S)-3,5-dihydroxyphenylglycine] induced seizures and concomitant augmentations in amino acid dialysate levels. The mGlu1a receptor antagonist LY367385 (1 mm) decreased baseline glutamate but not GABA concentrations, suggesting that mGlu1a receptors, which regulate hippocampal glutamate levels, are tonically activated by endogenous glutamate. This decrease in glutamate may contribute to the reported LY367385-mediated anticonvulsant effect. The mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (50 mg/kg) also clearly abolished pilocarpine-induced seizures. Agonist-mediated actions at mGlu2/3 receptors by LY379268 (100 microm, 10 mg/kg intraperitoneally) decreased basal hippocampal GABA but not glutamate levels. This may partly explain the increased excitation following systemic LY379268 administration and the lack of complete anticonvulsant protection within our epilepsy model with the mGlu2/3 receptor agonist. Group II selective mGluR receptor blockade with LY341495 (1-10 microm) did not alter the rats' behaviour or hippocampal amino acid levels. These data provide a neurochemical basis for the full anticonvulsant effects of mGlu1a and mGlu5 antagonists and the partial effects observed with mGlu2/3 agonists in vivo.     

Adenosine A2A receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1

Neurotransmitter transporters are regulated by phosphorylation but little is known about endogenous substances and receptors that regulate this process. Adenosine is an ubiquitous neuromodulator operating G-protein coupled receptors, which affect the activity of several kinases. We therefore evaluated the influence of adenosine upon the GABA transporter 1 (GAT-1) mediated GABA uptake into hippocampal synaptosomes. Removal of endogenous adenosine (adenosine deaminase, 1 U/mL) decreased GABA uptake, an effect mimicked by blockade of A2A receptors (2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine, 50 nM) but not A1 or A2B receptors. A2A receptor activation (4-[2-[[6-amino-9-( N -ethyl-β- d -ribofuranuronamidosyl)-9H-purin-yl]amino]ethyl]benzenepropanoic acid hydrochloride, 3–100 nM) enhanced GABA uptake by increasing the transporter Vmax without change of K_M. This was mimicked by adenylate cyclase activation (forskolin, 10 μM) and prevented by protein kinase A (PKA) inhibition ( N -[2-( p -bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, 1 μM), which per se did not influence GABA transport. Blockade of protein kinase C (PKC) (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide, 1 μM) facilitated GABA transport whereas PKC activation (4-β-phorbol-didecanoate, 250 nM) inhibited it. PKA blockade did not affect the facilitatory action of the PKC inhibitor or the inhibitory action of the PKC activator. However, when adenylate cyclase was activated neither activation nor inhibition of PKC affected GABA uptake. It is concluded that A2A receptors, through activation of the adenylate cyclase/cAMP/PKA transducing pathway facilitate GAT-1 mediated GABA transport into nerve endings by restraining tonic PKC-mediated inhibition.     

SKF38393抑制大鼠DRG分离神经元GABA-激活电流

在大鼠新鲜分离ＤＲＧ神经元标本上应用全细胞膜片箝记录,观察了多巴胺Ｄ１受体的选择性激动剂ＳＫＦ３８３９３ＨＣＩ对ＧＡＢＡ－激活电流的作用。大部分受检细胞对ＧＡＢＡ敏感,１０＾－６－１０＾－３－ｍｏｌ／Ｌ ＧＡＢＡ可于引起呈剂量依赖性的明显去敏感作用的内向电流。     

GABA介导杏仁外侧核对皮层A Ⅰ区神经元声反应的抑制:在体微电泳研究

在SD大鼠上应用多顺利完成微电极方法,观察微电泳CABA及其受体的拮抗剂或激动剂对杏仁外侧核（LA）抑制皮层AⅠ神经元声反应效应的影响。结果显示,电泳GABA能抑制皮层AⅠ区神经元的电活动,电泳GABAA受体拮抗剂bicuculline（BIC）则能易化其反应;电刺激LA能抑制皮层AⅠ区听神经元声反应,电泳GABA产生类拟于刺激LA的抑制效应;LA对皮层AⅠ区神经的抑制效应能被BIC所翻转,而不能被什氨酸受体拮抗剂strychnine所翻转,电泳GABAB型受体例激动剂baclofen对神经元声反应无影响。上术结果表明,GABA可能是介民LA抑制皮层AⅠ区神经元声反应的最终递质,并且是通过GABAA受体作用的。     

Inhibition of [3H]GABA Binding to Postsynaptic Receptors in Human Cerebellar Synaptic Membranes by Carboxyl and Amino Derivatives of GABA

Fifty synthetic analogues of GABA were tested for their ability ot interact with GABA receptors, using [3H]GABA binding to human cerebellar membranes as an in vitro model. The most active compounds were found to be aliphatic and heterocyclic aminosulphonic acids. Compounds with highly substituted nitrogen atoms were only weakly active unless a long alkyl chain, which can interact with the postsynaptic membrane, was present. It was concluded that a pyramidal nitrogen atom is favoured fro binding of GABA analogues to human cerebellar membranes.     

Modulation of cell surface GABA(B) receptors by desensitization, trafficking and regulated degradation

Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity.-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system,and mediates its effects via two classes of receptors:the GABA A and GABA B receptors.GABA A receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission.GABA B receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission.The extent of inhibitory neurotransmission is determined by a variety of factors,such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g.,phosphorylation),as well as by the number of receptors present in the plasma membrane available for signal transduction.The level of GABA B receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation.In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA B receptors in the plasma membrane,and thereby signaling strength.     

Inhibition of Striatal GABA Release by the Adenosine A2a Receptor Is Not Mediated by Increases in Cyclic AMP

Abstract: The adenosine A_2a receptor inhibition of potassium (15 m M )-evoked GABA release from striatal nerve terminals has been examined. High extracellular calcium concentrations (4 m M ) reduced the effect of the A_2a receptor agonist CGS-21680 (1 n M ). CGS-21680 inhibited GABA release in the presence of the L-type calcium channel blocker nifedipine, which itself inhibited evoked GABA release (by 16 ± 4%). ω-Conotoxin inhibited the evoked release by 45 ± 4% and prevented the action of CGS-21680. Forskolin and 8-bromo cyclic AMP both stimulated evoked GABA release at low concentrations, but at higher concentrations they abolished the inhibition by CGS-21680 without affecting the evoked release. The nonselective protein kinase inhibitor H-7 inhibited both the evoked release and the inhibition by CGS-21680, whereas the selective protein kinase A and G inhibitor HA-1004 had no effect on either evoked release or the action of CGS-21680. Pretreatment with pertussis toxin did not affect the A_2a receptor-mediated inhibition. Therefore, the effect of A_2a receptor stimulation was not mediated by protein kinases A or G but was inhibited by elevated cyclic AMP levels and mimicked by inhibitors of the N-type calcium channel and protein kinase C.     

硫酸镁对大鼠海马CA1区神经元钠电流的抑制作用

利用全细胞膜片钳技术研究了硫酸镁 (MgSO4 )对大鼠海马CA1区神经元钠电流的影响。结果表明 ,MgSO4 可浓度依赖和电压依赖地抑制钠电流 ,半数抑制浓度为 4 0 5mmol/L。这一抑制作用与刺激频率无关。结果还表明 ,4mmol/LMgSO4 不影响钠电流的失活过程 ,却使半数激活电压由 - 5 5 8± 6 8mV变为 - 3 4 2± 6 2mV (n =8,P <0 0 1) ,而激活曲线的斜率因子不变。结果提示 ,MgSO4 抑制大鼠海马CA1区神经元的钠电流可能是其抗缺血缺氧造成的中枢神经系统损伤的机制之一     

Activity- and sleep-dependent regulation of tonic inhibition by Shisa7

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Functional asymmetry of the conserved cystine loops in alphabetagamma GABA A receptors revealed by the response to GABA activation and drug potentiation

Ligand-gated ion channels respond to specific neurotransmitters by transiently opening an integral membrane ion-selective pore, allowing ions to move down their electrochemical gradient. A distinguishing feature of all members of the ligand-gated ion channel superfamily is the presence of a 13-amino acid disulfide loop (Cys-loop) in the extracellular ligand-binding domain. Structural data derived from the acetylcholine receptor place this loop at the interface between the ligand-binding domain and the transmembrane pore-forming domain where it is ideally located to participate in coupling ligand binding to channel opening. We have introduced specific mutations into a conserved motif at the mid-point of the Cys-loop of the GABA A receptor subunits alpha1, beta2 and gamma2S where the sequence reads aromatic, proline, aliphatic (ArProAl motif). Receptors carrying a mutation in the Cys-loop of one of their subunits were expressed in L929 cells and responses to both GABA and drugs were assessed using the whole-cell patch clamp technique. Drug potentiation and direct activation were significantly enhanced by mutations in this Cys-loop but these effects were subunit-dependent. Currents in response to agonists were larger when mutations were carried in the alpha and beta subunits but not in the gamma subunit. In contrast, potentiation of current responses by diazepam, etomidate and pentobarbital were all enhanced when mutations were carried in the alpha and gamma subunits, but not the beta subunit. Since the disruption of interactions mediated through the ArProAl motif enhances the mutant receptor's response to both agonist and drugs we suggest that this motif in the Cys-loop of the wild-type receptor participates in interactions that create activation barriers to conformational changes during channel gating.