Insulin reduces neuronal excitability by turning on GABA(A) channels that generate tonic current

Insulin signaling to the brain is important not only for metabolic homeostasis but also for higher brain functions such as cognition. GABA (γ-aminobutyric acid) decreases neuronal excitability by activating GABA(A) channels that generate phasic and tonic currents. The level of tonic inhibition in neurons varies. In the hippocampus, interneurons and dentate gyrus granule cells normally have significant tonic currents under basal conditions in contrast to the CA1 pyramidal neurons where it is minimal. Here we show in acute rat hippocampal slices that insulin (1 nM) "turns on" new extrasynaptic GABA(A) channels in CA1 pyramidal neurons resulting in decreased frequency of action potential firing. The channels are activated by more than million times lower GABA concentrations than synaptic channels, generate tonic currents and show outward rectification. The single-channel current amplitude is related to the GABA concentration resulting in a single-channel GABA affinity (EC(50)) in intact CA1 neurons of 17 pM with the maximal current amplitude reached with 1 nM GABA. They are inhibited by GABA(A) antagonists but have novel pharmacology as the benzodiazepine flumazenil and zolpidem are inverse agonists. The results show that tonic rather than synaptic conductances regulate basal neuronal excitability when significant tonic conductance is expressed and demonstrate an unexpected hormonal control of the inhibitory channel subtypes and excitability of hippocampal neurons. The insulin-induced new channels provide a specific target for rescuing cognition in health and disease.     

Protein kinase C phosphorylation regulates membrane insertion of GABAA receptor subtypes that mediate tonic inhibition

Tonic inhibition in the brain is mediated largely by specialized populations of extrasynaptic receptors, γ-aminobutyric acid receptors (GABA(A)Rs). In the dentate gyrus region of the hippocampus, tonic inhibition is mediated primarily by GABA(A)R subtypes assembled from α4β2/3 with or without the δ subunit. Although the gating of these receptors is subject to dynamic modulation by agents such as anesthetics, barbiturates, and neurosteroids, the cellular mechanisms neurons use to regulate their accumulation on the neuronal plasma membrane remain to be determined. Using immunoprecipitation coupled with metabolic labeling, we demonstrate that the α4 subunit is phosphorylated at Ser(443) by protein kinase C (PKC) in expression systems and hippocampal slices. In addition, the β3 subunit is phosphorylated on serine residues 408/409 by PKC activity, whereas the δ subunit did not appear to be a PKC substrate. We further demonstrate that the PKC-dependent increase of the cell surface expression of α4 subunit-containing GABA(A)Rs is dependent on Ser(443). Mechanistically, phosphorylation of Ser(443) acts to increase the stability of the α4 subunit within the endoplasmic reticulum, thereby increasing the rate of receptor insertion into the plasma membrane. Finally, we show that phosphorylation of Ser(443) increases the activity of α4 subunit-containing GABA(A)Rs by preventing current run-down. These results suggest that PKC-dependent phosphorylation of the α4 subunit plays a significant role in enhancing the cell surface stability and activity of GABA(A)R subtypes that mediate tonic inhibition.     

Elevation of Basal Protein Kinase C Activity Increases Ethanol Sensitivity of GABAA Receptors in Rat Hippocampal CA1 Pyramidal Neurons

Abstract: The ability of ethanol to enhance GABA_A receptor function remains controversial; conflicting observations have been made even in the same brain region, and when using apparently similar methodologies. In this study we characterized a single protocol variable, the initial incubation temperature of brain slices, that had dramatic effects on the ethanol sensitivity of GABA_A inhibitory postsynaptic currents (IPSCs) recorded from rat hippocampal CA1 pyramidal neurons. Incubation of hippocampal slices at relatively low temperatures (11–15°C) immediately after slice preparation significantly affected a number of physiological and biochemical parameters. Such slices showed a decrease in extracellular inhibitory postsynaptic potential amplitude, a significant increase in the ethanol sensitivity of GABA_A IPSCs in CA1 pyramidal neurons, no change in pentobarbital or flunitrazepam potentiation of IPSCs, and an increase in basal protein kinase C (PKC) activity relative to slices incubated at 31–33°C. In addition, the increase in ethanol sensitivity of GABA_A IPSCs was blocked by chelerythrine, a selective inhibitor of PKC. These results suggest that differences in hippocampal slice incubation protocols may have contributed to the disparate results of previous investigations of ethanol modulation of GABA_A receptor-mediated synaptic transmission in the rat hippocampus. In addition, these findings provide further evidence that PKC activity positively modulates the interaction between ethanol and GABA_A receptors in the mammalian brain.     

Graded response to GABA by native extrasynaptic GABA receptors

GABA is the main inhibitory neurotransmitter in the mammalian CNS. GABA in the brain is commonly associated with a fast, point-to-point form of signalling called synaptic transmission (phasic inhibition), but there is growing evidence that GABA participates in another, slower and more diffuse form of signalling often referred to as tonic inhibition. Unresolved questions regarding tonic neuronal inhibition concern activation and functional properties of extrasynaptic GABAA receptors (GABARex) present on neurones. Extrasynaptic receptors are exposed to submicromolar GABA concentrations and may modulate the overall excitability of neurones and neuronal networks. Here, we examined GABA-activated single-channel currents in dentate gyrus granule neurones in rat hippocampal slices. We activated three types (I, II, III) of GABARex channels by nanomolar GABA concentrations (EC50 I: 27 +/- 12; II: 4 +/- 3; III: 43 +/- 19 nm). The channels opened after a delay and the single-channel conductance was graded (gammamax I: 61 +/- 3; II: 85 +/- 8, III: 40 +/- 3 pS). The channels were differentially modulated by 1 microm diazepam, 200 nm zolpidem, 1 microm flumazenil and 50 nm THDOC (3alpha, 21-dihydroxy-5alpha-pregnan-20-one), consistent with the following minimal subunit composition of GABARex I alpha1betagamma2, GABARex II alpha4betagamma2 and GABARex III alphabetadelta channels.     

Cholecystokinin-2 receptors couple to cAMP-protein kinase A to depress excitatory synaptic currents in rat nucleus accumbens in vitro

We recently reported that the activation of cholecystokinin-2 receptors depress evoked excitatory postsynaptic currents (EPSCs) in nucleus accumbens (NAc) indirectly through gamma-aminobutyric acid (GABA) acting on gamma-aminobutyric acid-B (GABA(B)) receptors. Here, we determined the second messenger system that couples cholecystokinin-2 receptors to the observed synaptic depression. Using in vitro forebrain slices of rats and whole-cell patch recording, we tested the hypothesis that cholecystokinin-2 receptors are coupled to cAMP and protein kinase A signaling pathway. Cholecystokinin-8S induced inward currents and depressed evoked EPSCs. Forskolin, an activator of adenylyl cyclase and rolipram that is an inhibitor of phosphodiesterase type IV, independently increased EPSC amplitude and blocked the inward current and synaptic depression induced by cholecystokinin-8S. Furthermore, the membrane-permeable cAMP analog, 8-bromo-cAMP, blocked the cholecystokinin-8S effects. H89, a protein kinase A inhibitor, also blocked cholecystokinin-8S effects. However, depression of the evoked EPSC by baclofen, a GABA(B) receptor agonist, was not blocked by H89 or forskolin. These findings indicate that cholecystokinin-2, but not GABA(B), receptors are coupled to the adenylyl cyclase-cAMP-protein kinase A signaling pathway in the NAc to induce inward currents and cause synaptic depression.     

8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors

Nicotinic acetylcholine (ACh) receptors, such as alpha7, alpha3beta4 and alpha4beta2 receptors in the hippocampus, are suggested to modulate neurotransmitter release. 8-[2-(2-Pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) (100 nM), a linoleic acid derivative, potentiated responses of alpha7, alpha3beta4 and alpha4beta2 ACh receptors expressed in Xenopus oocytes that are blocked by 3-(1-[dimethylaminopropyl] indol-3-yl)-4-[indol-3-yl] maleimide (GF109203X), a selective inhibitor of protein kinase C (PKC), except for alpha3beta4 ACh receptors. DCP-LA enhanced the nicotine-triggered release of GABA from rat hippocampal slices in the presence of tetrodotoxin in a bell-shaped dose-dependent manner at concentrations ranging from 10 nM to 10 microM, although DCP-LA by itself had no effect on GABA release. The DCP-LA action was inhibited by GF109203X or alpha-bungarotoxin, an inhibitor of alpha7 ACh receptors, but not by mecamylamine or dihydro-beta-erithroidine, an inhibitor of alpha3beta4 and alpha4beta2 ACh receptors. A similar effect on GABA release was obtained with 12-O-tetradecanoylphorbol 13-acetate, a PKC activator. DCP-LA (100 nM) also enhanced GABA release triggered by choline, an agonist of alpha7 ACh receptors, but not 3-[2(s)-azetidinylmethoxy] pyridine, an agonist of alpha4beta2 ACh receptors. In addition, DCP-LA (100 nM) increased the rate of nicotine-triggered GABA(A) receptor-mediated miniature inhibitory post-synaptic currents, monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was also inhibited by GF109203X or alpha-bungarotoxin but not by mecamylamine. Thus, the results of the present study indicate that DCP-LA stimulates GABA release by enhancing activity of pre-synaptic alpha7 ACh receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via a PKC pathway.     

Clustering of extrasynaptic GABA(A) receptors modulates tonic inhibition in cultured hippocampal neurons

Tonic inhibition plays a crucial role in regulating neuronal excitability because it sets the threshold for action potential generation and integrates excitatory signals. Tonic currents are known to be largely mediated by extrasynaptic gamma-aminobutyric acid type A (GABA(A)) receptors that are persistently activated by submicromolar concentrations of ambient GABA. We recently reported that, in cultured hippocampal neurons, the clustering of synaptic GABA(A) receptors significantly affects synaptic transmission. In this work, we demonstrated that the clustering of extrasynaptic GABA(A) receptors modulated tonic inhibition. Depolymerization of the cytoskeleton with nocodazole promoted the disassembly of extrasynaptic clusters of delta and gamma(2) subunit-containing GABA(A) receptors. This effect was associated with a reduction in the amplitude of tonic currents and diminished shunting inhibition. Moreover, diffuse GABA(A) receptors were less sensitive to the GAT-1 inhibitor NO-711 and to flurazepam. Quantitative analysis of GABA-evoked currents after prolonged exposure to submicromolar concentrations of GABA and model simulations suggest that clustering affects the gating properties of extrasynaptic GABA(A) receptors. In particular, a larger occupancy of the singly and doubly bound desensitized states can account for the modulation of tonic inhibition recorded after nocodazole treatment. Moreover, comparison of tonic currents recorded during spontaneous activity and those elicited by exogenously applied low agonist concentrations allows estimation of the concentration of ambient GABA. In conclusion, receptor clustering appears to be an additional regulating factor for tonic inhibition.     

Pharmacological analysis of the activation and receptor properties of the tonic GABA(C)R current in retinal bipolar cell terminals

GABAergic inhibition in the central nervous system (CNS) can occur via rapid, transient postsynaptic currents and via a tonic increase in membrane conductance, mediated by synaptic and extrasynaptic GABA(A) receptors (GABA(A)Rs) respectively. Retinal bipolar cells (BCs) exhibit a tonic current mediated by GABA(C)Rs in their axon terminal, in addition to synaptic GABA(A)R and GABA(C)R currents, which strongly regulate BC output. The tonic GABA(C)R current in BC terminals (BCTs) is not dependent on vesicular GABA release, but properties such as the alternative source of GABA and the identity of the GABA(C)Rs remain unknown. Following a recent report that tonic GABA release from cerebellar glial cells is mediated by Bestrophin 1 anion channels, we have investigated their role in non-vesicular GABA release in the retina. Using patch-clamp recordings from BCTs in goldfish retinal slices, we find that the tonic GABA(C)R current is not reduced by the anion channel inhibitors NPPB or flufenamic acid but is reduced by DIDS, which decreases the tonic current without directly affecting GABA(C)Rs. All three drugs also exhibit non-specific effects including inhibition of GABA transporters. GABA(C)R ρ subunits can form homomeric and heteromeric receptors that differ in their properties, but BC GABA(C)Rs are thought to be ρ1-ρ2 heteromers. To investigate whether GABA(C)Rs mediating tonic and synaptic currents may differ in their subunit composition, as is the case for GABA(A)Rs, we have examined the effects of two antagonists that show partial ρ subunit selectivity: picrotoxin and cyclothiazide. Tonic and synaptic GABA(C)R currents were differentially affected by both drugs, suggesting that a population of homomeric ρ1 receptors contributes to the tonic current. These results extend our understanding of the multiple forms of GABAergic inhibition that exist in the CNS and contribute to visual signal processing in the retina.     

P物质抑制培养大鼠海马大锥体细胞GABA—激活电流

研究主要探讨P物质（SP）对GABA－激活电流的调制。实验在培养的新生大鼠海马大锥体细胞上进行。应用全细胞膜片箝技术记录GABA激活的内向电流。在被检的大锥体细胞中,有72％（66／92）的神经元对GABA和SP同时敏感,预后SP后,GABA激活电流明显地被抑制,此抑制作用是呈剂量依赖性的。在预加10^-8,10^-7,10^-6,10^-5mol/LSP后,GABA的激活电流分别降低18％,24．8％,25．9％和28％,用SP的拮抗剂 spantide能阻断此种抑制作用,在电极中灌注H7（PKC抑制剂）能取消此抑制作用,上述结果提示：SP对GABA激活电流的抑制作用是SP作用于SP受体,通过胞内第二信使,使GABAA受体通道复合体胞内磷酸化所致。     

Amygdala Kindling Alters Protein Kinase C Activity in Dentate Gyrus

Kindling is a use-dependent form of synaptic plasticity and a widely used model of epilepsy. Although kindling has been widely studied, the molecular mechanisms underlying induction of this phenomenon are not well understood. We determined the effect of amygdala kindling on protein kinase C (PKC) activity in various regions of rat brain. Kindling stimulation markedly elevated basal (Ca(2+)-independent) and Ca(2+)-stimulated phosphorylation of an endogenous PKC substrate (which we have termed P17) in homogenates of dentate gyrus, assayed 2 h after kindling stimulation. The increase in P17 phosphorylation appeared to be due at least in part to persistent PKC activation, as basal PKC activity assayed in vitro using an exogenous peptide substrate was increased in kindled dentate gyrus 2 h after the last kindling stimulation. A similar increase in basal PKC activity was observed in dentate gyrus 2 h after the first kindling stimulation. These results document a kindling-associated persistent PKC activation and suggest that the increased activity of PKC could play a role in the induction of the kindling effect.     

GABAA receptor phosphorylation and functional modulation in cortical neurons by a protein kinase C-dependent pathway

GABA(A) receptors are critical mediators of fast synaptic inhibition in the brain, and the predominant receptor subtype in the central nervous system is believed to be a pentamer composed of alpha, beta, and gamma subunits. Previous studies on recombinant receptors have shown that protein kinase C (PKC) and PKA directly phosphorylate intracellular serine residues within the receptor beta subunit and modulate receptor function. However, the relevance of this regulation for neuronal receptors remains poorly characterized. To address this critical issue, we have studied phosphorylation and functional modulation of GABA(A) receptors in cultured cortical neurons. Here we show that the neuronal beta3 subunit is basally phosphorylated on serine residues by a PKC-dependent pathway. PKC inhibitors abolish basal phosphorylation, increasing receptor activity, whereas activators of PKC enhance beta3 phosphorylation with a concomitant decrease in receptor activity. PKA activators were shown to increase the phosphorylation of the beta3 subunit only in the presence of PKC inhibitors. We also show that the main sites of phosphorylation within the neuronal beta3 subunit are likely to include Ser-408 and Ser-409, residues that are important for the functional modulation of beta3-containing recombinant receptors. Furthermore, PKC activation did not change the total number of GABA(A) receptors in the plasma membrane, suggesting that the effects of PKC activation are on the gating or conductance of the channel. Together, these results illustrate that cell-signaling pathways that activate PKC may have profound effects on the efficacy of synaptic inhibition by directly modulating GABA(A) receptor function.     

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

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

A-type GABA receptor as a central target of TRPM8 agonist menthol

Menthol is a widely-used cooling and flavoring agent derived from mint leaves. In the peripheral nervous system, menthol regulates sensory transduction by activating TRPM8 channels residing specifically in primary sensory neurons. Although behavioral studies have implicated menthol actions in the brain, no direct central target of menthol has been identified. Here we show that menthol reduces the excitation of rat hippocampal neurons in culture and suppresses the epileptic activity induced by pentylenetetrazole injection and electrical kindling in vivo. We found menthol not only enhanced the currents induced by low concentrations of GABA but also directly activated GABA(A) receptor (GABA(A)R) in hippocampal neurons in culture. Furthermore, in the CA1 region of rat hippocampal slices, menthol enhanced tonic GABAergic inhibition although phasic GABAergic inhibition was unaffected. Finally, the structure-effect relationship of menthol indicated that hydroxyl plays a critical role in menthol enhancement of tonic GABA(A)R. Our results thus reveal a novel cellular mechanism that may underlie the ambivalent perception and psychophysical effects of menthol and underscore the importance of tonic inhibition by GABA(A)Rs in regulating neuronal activity.     

Kainate receptor activation enhances both tonic and phasic GABA-ergic inhibition in CA1 hippocampal interneurons

Kainate receptor agonists are powerful convulsants and excitotoxins. It has been a lot of controversy around functions of these receptors in the brain. It is shown in this article that kainate enhances evoked GABAergic IPSC (phasic currents) in CA1 interneurons in concentration-dependent manner. The phenomenon is likely to be due to kainate-mediated lowering of the threshold for action potential generation in interneuron axons and increased number of terminals responding to the same stimulus strength. Kainate application also induced an enhancement in tonic GABAergic conductance. This phenomenon can be attributed to massive extracellular GABA accumulation caused by interneuron firing in the presence of kainate. Extracellular GABA also shunts synaptic currents by activating tonic conductance as well as desensitizing synaptic GABAA receptors. Thus, the enhancement of the evoked IPSCs by 1 microM kainate was complicated by early and transient decrease. The kainate receptor-mediated enhancement of GABAergic tonic and phasic signalling to interneurons can contribute to the depression of GABAergic transmission to pyramidal neurons. The consequence of this phenomenon may play a major role in the epileptogenic action of this agent.     

Mechanisms of homomeric alpha1 glycine receptor endocytosis

Little is known regarding the mechanism(s) by which glycine receptors are endocytosed. Here we examined the endocytosis of homomeric alpha1 glycine receptors expressed in HEK 293 cells using immunofluorescence/confocal microscopy and whole-cell patch-clamp recordings. Our studies demonstrate that constitutive endocytosis of glycine receptors is blocked by the dominant negative dynamin construct K44A and that intracellular dialysis with peptide P4, a dynamin/amphiphysin-disrupting peptide, increased whole-cell glycine-gated chloride currents. To examine whether receptor endocytosis could be regulated by PKC, experiments with the PKC activator PMA (phorbol 12-myristate 13-acetate) were performed. PMA, but not its inactive analogue PMM (phorbol 12-monomyristate), stimulated receptor endocytosis and inhibited glycine-gated chloride currents. Similar to constitutive endocytosis, PKC-stimulated endocytosis was blocked by dynamin K44A. Mutation of a putative AP2 adaptin dileucine motif (L314A, L315A) present in the receptor cytoplasmic loop blocked PMA-stimulated receptor endocytosis and also prevented PMA inhibition of glycine receptor currents. In patch-clamp experiments, intracellular dialysis of a 12-amino acid peptide corresponding to the region of the receptor containing the dileucine motif prevented PKC modulation of wild-type glycine receptors. Unlike PKC modulation of the receptor, constitutive endocytosis was not affected by mutation of this dileucine motif. These results demonstrate that PKC activation stimulates glycine receptor endocytosis, that both constitutive endocytosis and PKC-stimulated endocytosis are dynamin-dependent, and that PKC-stimulated endocytosis, but not constitutive endocytosis, occurs via the dileucine motif (L314A, L315A) within the cytoplasmic loop of the receptor.     

Stimulation of A(2A) adenosine receptor phosphorylation by protein kinase C activation: evidence for regulation by multiple protein kinase C isoforms.

Activation of the A(2A) adenosine receptor (A(2A)AR) contributes to the neuromodulatory and neuroprotective effects of adenosine in the central nervous system. Here we demonstrate that, in rat C6 glioma cells stably expressing an epitope-tagged canine A(2A)AR, receptor phosphorylation on serine and threonine residues can be increased by pretreatment with either the synthetic protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or endothelin 1, which increases PKC activity via binding to endogenous endothelin(A) receptors. Under conditions in which PMA was maximally effective, activation of other second messenger-regulated kinases was without effect. While basal and PMA-stimulated phosphorylation were unaffected by the A(2A)AR-selective antagonist ZM241385, they were both blocked by GF109203X (a selective inhibitor of conventional and novel PKC isoforms) and rottlerin (a PKCdelta-selective inhibitor) but not Go6976 (selective for conventional PKC isoforms). However, coexpression of the A(2A)AR with each of the alpha, betaI, and betaII isoforms of PKC increased basal and PMA-stimulated phosphorylation. Mutation of the three consensus PKC phosphorylation sites within the receptor (Thr298, Ser320, and Ser335) to Ala failed to inhibit either basal or PMA-stimulated phosphorylation. In addition, phosphorylation of the receptor was not associated with detectable changes in either its signaling capacity or cell surface expression. These observations suggest that multiple PKC isoforms can stimulate A(2A)AR phosphorylation via activation of one or more downstream kinases which then phosphorylate the receptor directly. In addition, it is likely that phosphorylation controls interactions with regulatory proteins distinct from those involved in the classical cAMP signaling pathway utilized by this receptor.     

Modulation of Tonic GABA Currents by Anion Channel and Connexin Hemichannel Antagonists

Anion channels and connexin hemichannels are permeable to amino acid neurotransmitters. It is hypothesized that these conductive pathways release GABA, thereby influencing ambient GABA levels and tonic GABAergic inhibition. To investigate this, we measured the effects of anion channel/hemichannel antagonists on tonic GABA currents of rat hippocampal neurons. In contrast to predictions, blockade of anion channels and hemichannels with NPPB potentiated tonic GABA currents of neurons in culture and acute hippocampal slices. In contrast, the anion channel/hemichannel antagonist carbenoxolone (CBX) inhibited tonic currents. These findings could result from alterations of ambient GABA concentration or direct effects on GABA_A receptors. To test for effects on GABA_A receptors, we measured currents evoked by exogenous GABA. Coapplication of NPPB with GABA potentiated GABA-evoked currents. CBX dose-dependently inhibited GABA-evoked currents. These results are consistent with direct effects of NPPB and CBX on GABA_A receptors. GABA release from hippocampal cell cultures was directly measured using HPLC. Inhibition of anion channels with NPPB or CBX did not affect GABA release from cultured hippocampal neurons. NPPB reduced GABA release from pure astrocytic cultures by 21%, but the total GABA release from astrocytes was small compared to that of mixed cultures. These data indicate that drugs commonly used to antagonize anion channels and connexin hemichannels may affect tonic currents via direct effects on GABA_A receptors and have negligible effects on ambient GABA concentrations. Interpretation of experiments using NPPB or CBX should include consideration of their effects on tonic GABA currents.     

Activation of muscarinic receptors inhibits beta-amyloid peptide-induced signaling in cortical slices

Deposition of fibrillar aggregates of the beta-amyloid peptide (Abeta) is a key pathologic feature during the early stage of Alzheimer's disease. The initial neuronal responses to Abeta in cortical circuits and the regulation of Abeta-induced signaling remain unclear. In this study, we found that exposure of cortical slices to Abeta(1-42) or Abeta(25-35) induced a marked increase in the activation of protein kinase C (PKC) and Ca(2+)/calmodulin-dependent kinase II (CaMKII), two enzymes critically involved in a variety of cellular functions. Activation of M1 muscarinic receptors, but not nicotinic receptors, significantly inhibited the Abeta activation of PKC and CaMKII. Increasing inhibitory transmission mimicked the M1 effect on Abeta, whereas blocking GABA(A) receptors eliminated the M1 action. Moreover, electrophysiological evidence shows that application of Abeta to cortical slices induced action potential firing and enhanced excitatory postsynaptic currents, whereas muscarinic agonists potently increased inhibitory postsynaptic currents. These results suggest that Abeta activates PKC and CaMKII through enhancing excitatory activity in glutamatergic synaptic networks. Activation of M1 receptors inhibits Abeta signaling by enhancing the counteracting GABA(ergic) inhibitory transmission. Thus the muscarinic reversal of the Abeta-induced biochemical and physiological changes provides a potential mechanism for the treatment of Alzheimer's disease with cholinergic enhancers.     

Plasticity of pre- and postsynaptic GABAB receptor function in the paraventricular nucleus in spontaneously hypertensive rats

GABA(B) receptor function is upregulated in the paraventricular nucleus (PVN) of the hypothalamus in spontaneously hypertensive rats (SHR), but it is unclear whether this upregulation occurs pre- or postsynaptically. We therefore determined pre- and postsynaptic GABA(B) receptor function in retrogradely labeled spinally projecting PVN neurons using whole cell patch-clamp recording in brain slices in SHR and Wistar-Kyoto (WKY) rats. Bath application of the GABA(B) receptor agonist baclofen significantly decreased the spontaneous firing activity of labeled PVN neurons in both SHR and WKY rats. However, the magnitude of reduction in the firing rate was significantly greater in SHR than in WKY rats. Furthermore, baclofen produced larger membrane hyperpolarization and outward currents in labeled PVN neurons in SHR than in WKY rats. The baclofen-induced current was abolished by either including G protein inhibitor GDPbetaS in the pipette solution or bath application of the GABA(B) receptor antagonist in both SHR and WKY rats. Blocking N-methyl-d-aspartic acid receptors had no significant effect on baclofen-elicited outward currents in SHR. In addition, baclofen caused significantly greater inhibition of glutamatergic excitatory postsynaptic currents (EPSCs) in labeled PVN neurons in brain slices from SHR than WKY rats. By contrast, baclofen produced significantly less inhibition of GABAergic inhibitory postsynaptic currents (IPSCs) in labeled PVN neurons in SHR than in WKY rats. Although microinjection of the GABA(B) antagonist into the PVN increases sympathetic vasomotor tone in SHR, the GABA(B) antagonist did not affect EPSCs and IPSCs of the PVN neurons in vitro. These findings suggest that postsynaptic GABA(B) receptor function is upregulated in PVN presympathetic neurons in SHR. Whereas presynaptic GABA(B) receptor control of glutamatergic synaptic inputs is enhanced, presynaptic GABA(B) receptor control of GABAergic inputs in the PVN is attenuated in SHR. Changes in both pre- and postsynaptic GABA(B) receptors in the PVN may contribute to the control of sympathetic outflow in hypertension.     

Protein kinase C activation stimulates the phosphorylation and internalization of the sst2A somatostatin receptor

The sst2A receptor is expressed in the endocrine, gastrointestinal, and neuronal systems as well as in many hormone-sensitive tumors. This receptor is rapidly internalized and phosphorylated in growth hormone-R2 pituitary cells following somatostatin binding (Hipkin, R. W., Friedman, J., Clark, R. B., Eppler, C. M., and Schonbrunn, A. (1997) J. Biol. Chem. 272, 13869-13876). The protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), also stimulates sst2A phosphorylation. Here we examine the mechanisms and consequences of PMA and agonist-induced sst2A phosphorylation. Like somatostatin, both PMA and bombesin increased sst2A receptor phosphorylation within 2 min. The PKC inhibitor GF109203X blocked PMA- and bombesin- stimulated sst2A phosphorylation, whereas stimulation by the somatostatin analog SMS 201-995 was unaffected. Agonist and PMA each stimulated phosphorylation in two receptor domains, the third intracellular loop and the C-terminal tail. Functionally, PMA dramatically increased the internalization of the sst2A receptor-ligand complex. This PMA stimulation was blocked by GF109203X, whereas basal internalization was unaffected. However, neither basal nor PMA-stimulated internalization was altered by pertussis toxin, whereas both were blocked by hypertonic sucrose. Therefore PKC activation and agonist binding stimulate sst2A phosphorylation by distinct mechanisms, and PKC potentiates internalization of the sst2A receptor via clathrin-coated pits. Thus, hormonal stimulation of PKC-coupled receptors may provide a mechanism for regulating the inhibitory actions of somatostatin in target tissue.