Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABAA receptors by NMDA

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on γ-aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (i) NMDA suppressed GABA-and muscimol (Mus)-activated currents (I_gaba and I_Mus), respectively in the Mg²⁺-free external solution containing 1 μmol/L glycine at a holding potential (V _H ) of −40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 γmol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of I_gaba; (ii) when the neurons were incubated in a Ca²⁺-free bath or pre-loaded with a membrane-permeable Ca²⁺ chelator, BAPTA AM (10 μmol/L), the inhibitory effect of NMDA on I_GAba disappeared. Cd²⁺ (10 μmol/L) or La³⁺ (30 μmol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of I_gaba by NMDA application; (iii) the suppression of I_GAba by NMDA was inhibited by KN-62, a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated that the inhibition of GABA response by NMDA is Ca²⁺-dependent and CaMKII is involved in the process of the Ca²⁺-dependent inhibition.     

催产素对大鼠背根神经节分离细胞GABA激活电流的调制作用

在急性分离的大鼠背根神经节（ｄｏｒｓａｌ ｒｏｏｔ ｇａｎｇｌｉｏｎ,ＤＲＧ）细胞上,应用全细胞膜片箝技术观察了预知催产素（ｏｘｙｔｏｃｉｎ,ＯＴ）对ＧＡＢＡ激活电流的调制作用。结果如下：（１）大多数细胞（４８／５２,９０．５％）对ＧＡＢＡ敏感。（２）ＯＴ可引起５１．３％（２０／３９）的受检细胞出现外向膜电流;４３．６％（１７／３９）无明显膜反应;５．１％（２／３９）出现内向膜电流。（３）预加ＯＴ     

5-HT增强大鼠骶髓后连合核神经元牛磺酸激活的全细胞电流

应用制霉菌素穿孔全细胞记录方法,研究了５－羟色胺对急性分离的大鼠髓后连合核神经元磺酸激活的全细胞电流的调控。     

Neuroprotection of GluR5-containing kainate receptor activation against ischemic brain injury through decreasing tyrosine phosphorylation of N-methyl-D-aspartate receptors mediated by Src kinase

Previous studies indicate that cerebral ischemia breaks the dynamic balance between excitatory and inhibitory inputs. The neural excitotoxicity induced by ionotropic glutamate receptors gain the upper hand during ischemia-reperfusion. In this paper, we investigate whether GluR5 (glutamate receptor 5)-containing kainate receptor activation could lead to a neuroprotective effect against ischemic brain injury and the related mechanism. The results showed that (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a selective GluR5 agonist, could suppress Src tyrosine phosphorylation and interactions among N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A), postsynaptic density protein 95 (PSD-95), and Src and then decrease NMDA receptor activation through attenuating tyrosine phosphorylation of NR2A and NR2B. More importantly, ATPA had a neuroprotective effect against ischemia-reperfusion-induced neuronal cell death in vivo. However, four separate drugs were found to abolish the effects of ATPA. These were selective GluR5 antagonist NS3763; GluR5 antisense oligodeoxynucleotides; CdCl(2), a broad spectrum blocker of voltage-gated calcium channels; and bicuculline, an antagonist of gamma-aminobutyric acid A (GABA(A)) receptor. GABA(A) receptor agonist muscimol could attenuate Src activation and interactions among NR2A, PSD-95 and Src, resulting the suppression of NMDA receptor tyrosine phosphorylation. Moreover, patch clamp recording proved that the activated GABA(A) receptor could inhibit NMDA receptor-mediated whole-cell currents. Taken together, the results suggest that during ischemia-reperfusion, activated GluR5 may facilitate Ca(2+)-dependent GABA release from interneurons. The released GABA can activate postsynaptic GABA(A) receptors, which then attenuates NMDA receptor tyrosine phosphorylation through inhibiting Src activation and disassembling the signaling module NR2A-PSD-95-Src. The final result of this process is that the pyramidal neurons are rescued from hyperexcitability.     

Biphasic action of midazolam on GABAA receptor-mediated responses in rat sacral dorsal commissural neurons

The effect of the benzodiazepine agonist midazolam on gamma-aminobutyric acid(A) (GABA(A)) receptor-mediated currents was investigated in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. Midazolam displayed a biphasic effect on GABA responses. Low concentrations of midazolam (1nM-10 microM) reversibly potentiated GABA (3 microM)-activated Cl(-) currents (I(GABA)) in a bell-shaped manner, with the maximal facilitary effect at 0.1 microM; whereas at higher concentrations (above 10 microM), midazolam had an antagonistic effect on I(GABA). Our further study indicated that midazolam changed GABA(A) receptor affinity to GABA and the effects of midazolam on I(GABA) were voltage-independent. The benzodiazepine receptor antagonist, flumazenil, abolished the facilitary effect of low concentrations of midazolam rather than the antagonism of I(GABA) induced by high doses of midazolam. In addition, activation of protein kinase C prevented the inhibitory effect of midazolam at higher concentrations, but did not influence the effect of midazolam at low concentrations. These results indicate that midazolam interacts with another distinct site other than the central benzodiazepine receptors on GABA(A) receptors as an antagonist at higher concentrations in SDCN neurons.     

Action potential waveforms reveal simultaneous changes in ICa and IK produced by 5-HT in rat dorsal raphe neurons.

Action potentials were recorded from serotonergic dorsal raphe (DR) neurons acutely isolated from the adult rat brain. Action potential waveforms were used as command potentials for whole-cell patch-clamp studies to investigate the Ca2+ and K+ currents underlying action potentials and the modulatory effects of 5-Hydroxytryptamine (5-HT) on them. These data were compared with currents elicited by using rectangular voltage steps of the type commonly used in voltage-clamp experiments. In the same cell, 5-HT simultaneously augmented K+ currents and inhibited Ca2+ currents. Experimental conditions were chosen which allowed us to examine the action of 5-HT on K+ and Ca2+ currents simultaneously or in isolation; 5-HT produced a larger inhibition of calcium current during an action potential waveform compared with that measured by using rectangular steps of voltage. A possible explanation for this finding is that the maximal inhibition is seen immediately after a voltage jump and then decreases with time. Action potentials are, in general, so brief that little time-dependent relief of block is observed. Most of the inhibition of Ca2+ current resulted from a direct effect on Ca2+ channels rather than a shortening of the action potential. The inhibition of Ca2+ current by 5-HT also decreased the Ca(2+)-activated K+ currents. These results suggest that 5-HT reduces DR neuron excitability by the simultaneous activation of K+ channel currents open at the resting potential and the suppression of Ca2+ channel currents.     

Activation of Ca2+-activated Cl- channels by store-operated Ca2+ entry in arterial smooth muscle cells does not require reverse-mode Na+/Ca2+ exchange

The main purpose of this study was to characterize the stimulation of Ca(2+)-activated Cl(-) (Cl(Ca)) by store-operated Ca(2+) entry (SOCE) channels in rabbit pulmonary arterial smooth muscle cells (PASMCs) and determine if this process requires reverse-mode Na(+)/Ca(2+) exchange (NCX). In whole-cell voltage clamped PASMCs incubated with 1 μmol/L nifedipine (Nif) to inhibit Ca(2+) channels, 30 μmol/L cyclopiazonic acid (CPA), a SERCA pump inhibitor, activated a nonselective cation conductance permeable to Na(+) (I(SOC)) during an initial 1-3 s step, ranging from-120 to +60 mV, and Ca(2+)-activated Cl(-) current (I(Cl(Ca))) during a second step to +90 mV that increased with the level of the preceding hyperpolarizing step. Niflumic acid (100 μmol/L), a Cl(Ca) channel blocker, abolished I(Cl(Ca)) but had no effect on I(SOC), whereas the I(SOC) blocker SKF-96365 (50 μmol/L) suppressed both currents. Dual patch clamp and Fluo-4 fluorescence measurements revealed the appearance of CPA-induced Ca(2+) transients of increasing magnitude with increasing hyperpolarizing steps, which correlated with I(Cl(Ca)) amplitude. The absence of Ca(2+) transients at positive potentials following a hyperpolarizing step combined with the observation that SOCE-stimulated I(Cl(Ca)) was unaffected by the NCX blocker KB-R7943 (1 μmol/L) suggest that the SOCE/Cl(Ca) interaction does not require reverse-mode NCX in our conditions.     

NMDA-responsible, APV-insensitive receptor in cultured human astrocytes

The present study was conducted to assess N-methyl-D-aspartate (NMDA)-responsible receptors in cultured human astrocytes by monitoring whole-cell membrane currents. NMDA generated currents, that are potentiated by glycine and blocked by Mg2+, with the current/voltage relation showing a reversal potential of +/- 0 mV. The currents were not inhibited by either the selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV), or the non-selective ionotropic glutamate receptor antagonist, kynurenic acid. The currents were inhibited only by 19% in Ca2+-free extracellular solution. Furthermore, GDPbetaS, a broad G-protein inhibitor, inhibited NMDA-induced currents to 82% of original levels. The results of the present study thus suggest that an NMDA-responsible, APV-insensitive receptor with low Ca2+ permeability, distinct from the neuronal NMDA receptors, is expressed in human astrocytes and that the receptor is regulated in part by an unknown G-protein-linked receptor.     

Cu2+ suppresses GABA(A) receptor-mediated responses in rat sacral dorsal commissural neurons

The effect of copper ions (Cu(2+)) on gamma-aminobutyric acid (GABA)-induced responses in acutely dissociated neurons from the rat sacral dorsal commissural nucleus (SDCN) was investigated using a nystatin-perforated patch recording configuration under voltage clamp conditions. The application of Cu(2+) to SDCN neurons reversibly suppressed the GABA (10 microM)-activated Cl(-) current (I(GABA)) in a concentration-dependent manner (1-1000 microM; IC(50) = 24.5 microM). In the presence of Cu(2+) (30 microM), the concentration-response curve of GABA was shifted rightward without reducing I(GABA) recorded under the maximally effective concentration of GABA, thus indicating a dependence of Cu(2+) action on GABA concentration. Inhibition of GABA (10 microM) responses by 30 microM Cu(2+) was essentially voltage independent and was not accompanied by a shift in the reversal potential of the currents. Cu(2+) antagonized the suppressive effect of Zn(2+) in a concentration-dependent manner, suggesting competition between Cu(2+) and Zn(2+) for similar binding sites. These data demonstrate that Cu(2+) is a potent inhibitor of GABA(A) receptor-mediated responses, implying a possible modulatory effect of Cu(2+) on GABAergic synaptic transmission in the mammalian SDCN.     

The phosphorylation state of GluR1 subunits determines the susceptibility of AMPA receptors to calpain cleavage

The alpha-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) is an ionotropic glutamate receptor that governs most of excitatory synaptic transmission in neurons. In vitro biochemical assay has shown that calpain, a Ca2+-activated protease, can cleave AMPAR GluR1 subunits. Our physiological study found that calpain, which was activated by prolonged stimulation of the N-methyl-D-aspartate receptor (100 microM, 10 min), caused a substantial suppression of AMPAR currents in cortical neurons. Since the phosphorylation sites of GluR1 by several protein kinases are located in close proximity to the calpain cleavage sites, we investigated the effect of phosphorylation on the susceptibility of GluR1 to calpain cleavage. Interestingly, we found that the calpain regulation of AMPAR currents was diminished by inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) but was augmented by inhibition of protein phosphatase 1/2A (PP1/2A). In agreement with this, in vitro assay showed that the calpain-induced proteolytic cleavage of GluR1 C-terminal fusion protein was strongly potentiated by adding the purified active CaMKII, and GluR1 phosphorylated at Ser831 by CaMKII is much more sensitive to calpain cleavage. Taken together, our data suggest that calpain activation suppresses AMPA receptor currents via proteolytic cleavage of GluR1 subunits, and the susceptibility of AMPARs to calpain cleavage is determined by the phosphorylation state of GluR1 subunits, which is mediated by CaMKII-PP1/2A activity.     

白藜芦醇抑制大鼠穹隆下器神经元放电

应用细胞外记录单位放电技术,在大鼠穹隆下器脑片上观察了白藜芦醇(resveratrol)对穹隆下器神经元放电的影响。实验结果如下:(1)给予白藜芦醇(1、5、10μmol/L)2min后,大多数穹隆下器神经元(60/65,92.3%)的自发性放电频率呈剂量依赖性降低;(2)预先用0.3mmol/L的L-glutamate灌流穹隆下器脑片,全部放电单位(12/12,100%)放电频率明显增加,表现为癫痫样放电,在此基础上灌流白藜芦醇(5μmol/L)2min,大多数脑片(10/12,83.3%)的癫痫样放电被抑制;(3)预先用L型钙通道开放剂BayK8644灌流,全部(8/8,100%)放电增加,在此基础上灌流白藜芦醇(5μmol/L)2min,其放电全部被抑制;(4)灌流一氧化氮合酶抑制剂NG-nitro-L-argininemethylester(L-NAME)50μmol/L,多数脑片(11/14,78.6%)放电明显增加,在此基础上灌流白藜芦醇(5μmol/L)2min,大部分神经元(9/11,81.8%)放电被抑制;(5)灌流大电导钙激活性钾通道阻断剂tetraethylammoniumchloride(TEA)1mmol/L后,大多数神经元(10/12,83.3%)放电增加,在此基础上灌流白藜芦醇(5μmol/L)2min,(9/10,90%)放电频率明显减低。以上结果提示:白藜芦醇能抑制大鼠穹隆下器神经元自发放电以及由L-glutamate、L-NAME、BayK8644和TEA诱发的放电,可能与白藜芦醇抑制L型钙通道以及促进一氧化氮的释放有关;似乎与大电导钙激活性钾通道无关。     

尼氟灭酸通过钙库钙释放引起豚鼠耳蜗螺旋动脉平滑肌细胞超极化(英文)

本研究旨在观察氯离子通道阻断剂尼氟灭酸（niflumic acid,NFA）引起豚鼠耳蜗螺旋动脉平滑肌细胞产生超极化的机制。以豚鼠为实验动物,运用细胞内微电极和全细胞膜片钳记录技术,观察NFA和其它药物对急性分离的耳蜗螺旋动脉平滑肌细胞的作用。结果显示：NFA、indanyloxyacetic acid94（LAh-94）和diSOdium4,4’-diisothiocyanatostilbene-2,2’-disulfonate（DIDS）可使低静息膜电位的细胞产生超极化,但对高静息膜电位的细胞无明显作用。低静息膜电位细胞的平均静息电位为（-42．47±1．38）mV（n=24）,100μmol／LNFA、10μmol／LIAA-94和200μmol／LDIDS分别使细胞超极化至（13．7±4．3）mV=9,P〈0．01）,（11．4±4．2）mV（n=7,P〈0．01）和（12．3±3．7）mV（n=8,P〈0．01）,这种氯离子通道阻断剂引起细胞超极化反应的效应呈浓度依赖性。NFA引起的超极化和外向电流几乎完全被100nmol／L iberiotoxin、100nmol／L charybdotoxin、10mmol／L tetraethylammonium、50μmol／LBAPTA—AM、10μmol／Lryanodine和0．1-10mmol／Lcaffeine阻断,但不能被100μmol／Lnifedipine、100μmol／LCdCI,和无Ca^2＋灌流外液阻断。结果捉示：氯离_了通道的阻断剂NFA可通过平滑肌细胞内钙库的钙释放增加细胞内钙,进而激活钙依赖的钾通道,产生耳蜗螺旋动脉平滑肌细胞的超极化反应。     

一种改进的适用于膜片钳记录的成年大鼠海马神经元急性分离法

本文建立了一种快速、可靠的急性分离成年大鼠海马神经细胞的方法。此法可将实验大鼠的年龄提高到500d以上,体重300g以上;不损伤神经细胞膜的电学特性：形态上有差异的细胞易于分辨。用膜片钳技术的单通道和全细胞模式证实,在本实验条件下,约95％左右的健康细胞均能形成高阻抗封接,并成功地记录了电压依赖性钾、钠、钙通道,外向整流氯通道和大电导的钙激活钾通道电流。     

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。     

Regulation of neuronal K(+) currents by target-derived factors: opposing actions of two different isoforms of TGFbeta.

The developmental expression of macroscopic Ca(2+)-activated K(+) currents in chick ciliary ganglion neurons is dependent on an avian ortholog of TGFbeta1, known as TGFbeta4, secreted from target tissues in the eye. Here we report that a different isoform, TGFbeta3, is also expressed in a target tissue of ciliary ganglion neurons. Application of TGFbeta3 inhibits the functional expression of whole-cell Ca(2+)-activated K(+) currents evoked by 12 hour treatment with either TGFbeta1 or beta-neuregulin-1 in ciliary ganglion neurons developing in vitro. TGFbeta3 had no effect on voltage-activated Ca(2+) currents. A neutralizing antiserum specific for TGFbeta3 potentiates stimulation of Ca(2+)-activated K(+) currents evoked by a target tissue (iris) extract in cultured ciliary ganglion neurons, indicating that TGFbeta3 is an inhibitory component of these extracts. Intraocular injection of TGFbeta3 causes a modest but significant inhibition of the expression of Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo. Further, intraocular injection of a TGFbeta3-neutralizing antiserum stimulates expression of Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo, indicating that endogenous TGFbeta3 regulates the functional expression of this current. The normal developmental expression of functional Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo is therefore regulated by two different target-derived isoforms of TGFbeta, which produce opposing effects on the electrophysiological differentiation of these neurons.     

Calcium permeability and modulation of nicotinic acetylcholine receptor-channels in rat parasympathetic neurons.

Neuronal nicotinic acetylcholine (ACh)-activated currents in rat parasympathetic ganglion cells were examined using whole-cell and single-channel patch clamp recording techniques. The whole-cell current-voltage (I-V) relationship exhibited strong inward rectification and a reversal (zero current) potential of -3.9 mV in nearly symmetrical Na+ solutions (external 140 mM Na+/internal 160 mM Na+). Isosmotic replacement of extracellular Na+ with either Ca2+ or Mg2+ yielded the permeability (Px/PNa) sequence Mg2+ (1.1) > Na+ (1.0) > Ca2+ (0.65). Whole-cell ACh-induced current amplitude decreased as [Ca2+]0 was raised from 2.5 mM to 20 mM, and remained constant at higher [Ca2+]0. Unitary ACh-activated currents recorded in excised outside-out patches had conductances ranging from 15-35 pS with at least three distinct conductance levels (33 pS, 26 pS, 19 pS) observed in most patches. The neuronal nicotinic ACh receptor-channel had a slope conductance of 30 pS in Na+ external solution, which decreased to 20 pS in isotonic Ca2+ and was unchanged by isosmotic replacement of Na+ with Mg2+. ACh-activated single channel currents had an apparent mean open time (tau 0) of 1.15 +/- 0.16 ms and a mean burst length (tau b) of 6.83 +/- 1.76 ms at -60 mV in Na+ external solution. Ca(2+)-free external solutions, or raising [Ca2+]0 to 50-100 mM decreased both the tau 0 and tau b of the nAChR channel. Varying [Ca2+]0 produced a marked decrease in NP0, while substitution of Mg2+ for Na+ increased NP0. These data suggest that activation of the neuronal nAChR channel permits a substantial Ca2+ influx which may modulate Ca(2+)-dependent ion channels and second messenger pathways to affect neuronal excitability in parasympathetic ganglia.     

Cross-talk between NMDA and GABA<Subscript>A</Subscript> receptors in cultured neurons of the rat inferior colliculus

Neuronal ion channels of different types often do not function independently but will inhibit or potentiate the activity of other types of channels, a process called cross-talk. The N-methyl-D-aspartate receptor (NMDA receptor) and the γ-aminobutyric acid type A receptor (GABA_A receptor) are important excitatory and inhibitory receptors in the central nervous system, respectively. Currently, cross-talk between the NMDA receptor and the GABA_A receptor, particularly in the central auditory system, is not well understood. In the present study, we investigated functional interactions between the NMDA receptor and the GABA_A receptor using whole-cell patch-clamp techniques in cultured neurons from the inferior colliculus, which is an important nucleus in the central auditory system. We found that the currents induced by aspartate at 100 μmol L⁻¹ were suppressed by the pre-perfusion of GABA at 100 μmol L⁻¹, indicating cross-inhibition of NMDA receptors by activation of GABA_A receptors. Moreover, we found that the currents induced by GABA at 100 μmol L⁻¹ (I _GABA) were not suppressed by the pre-perfusion of 100 μmol L⁻¹ aspartate, but those induced by GABA at 3 μmol L⁻¹ were suppressed, indicating concentration-dependent cross-inhibition of GABA_A receptors by activation of NMDA receptors. In addition, inhibition of IGABA by aspartate was not affected by blockade of voltage-dependent Ca²⁺ channels with CdCl₂ in a solution that contained Ca²⁺, however, CdCl₂ effectively attenuated the inhibition of I _GABA by aspartate when it was perfused in a solution that contained Ba²⁺ instead of Ca²⁺ or a solution that contained Ca²⁺ and 10 mmol L⁻¹ BAPTA, a membrane-permeable Ca²⁺ chelator, suggesting that this inhibition is mediated by Ca²⁺ influx through NMDA receptors, rather than voltage-dependent Ca²⁺ channels. Finally, KN-62, a potent inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), reduced the inhibition of I _GABA by aspartate, indicating the involvement of CaMKII in this cross-inhibition. Our study demonstrates a functional interaction between NMDA and GABA_A receptors in the inferior colliculus of rats. The presence of cross-talk between these receptors suggests that the mechanisms underlying information processing in the central auditory system may be more complex than previously believed.     

Store Ca2+ depletion enhances NMDA responses in cultured human astrocytes.

NMDA produced whole-cell membrane currents in cultured human astrocytes. The currents were not inhibited by the selective NMDA receptor antagonist, APV, while they were partially inhibited by the broad G-protein inhibitor, GDPbetaS. NMDA-induced currents were enhanced by either the microsomal Ca2+/ATPase inhibitors, thapsigargin and cyclopiazonic acid, or the ATP-uncoupler, dinitrophenol (DNP). In the Ca2+ assay, NMDA increased intracellular calcium concentration. The increase was inhibited by 26% in Ca2+-free extracellular solution, and it was not inhibited by APV. The results of the present study suggest that NMDA responses in human astrocytes are regulated by store Ca2+ depletion-associated signal.     

Allopregnanolone enhancement of GABAergic transmission in rat medial preoptic area neurons

Gamma-aminobutyric acid (GABA)-mediated transmission in the medial preoptic area (MPOA) of the hypothalamus plays an important role in functions such as sex steroid hormone dynamics and control of body temperature. The action of allopregnanolone, the primary metabolite of progesterone, on GABAergic transmission was investigated by employing patch clamp whole cell recording on acutely dissociated rat MPOA neurons with the functional connection of presynaptic terminals. Allopregnanolone enhanced spontaneous GABA release on the MPOA neurons and induced prolonged decay of miniature GABAergic-inhibitory postsynaptic currents (mIPSCs). The facilitation of GABA release from the presynaptic terminals by allopregnanolone disappeared in Ca2+-free extracellular solution. The presynaptic action of this neurosteroid was also blocked by bumetanide, a blocker of cation-Cl- cotransporters, and by removal of extracellular Na+. The results suggest that allopregnanolone enhances GABAergic transmission at the MPOA neurons by pre- and postsynaptic mechanisms. The enhancement of GABA release by allopregnanolone might require a high Cl- concentration in the presynaptic terminal maintained by Na+-dependent, bumetanide-sensitive mechanisms (e.g., Na+-K+-Cl- cotransporter) and might be mediated by Ca2+ influx into presynaptic terminal.     

Inositol trisphosphate isomers, but not inositol 1,3,4,5-tetrakisphosphate, induce calcium influx in Xenopus laevis oocytes

To investigate the mechanisms by which inositol phosphates regulate cytosolic free Ca2+ concentration ([Ca2+]c), we injected Xenopus oocytes with inositol phosphates and measured Ca2+-activated Cl- currents as an assay of [Ca2+]c. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) injection (0.1-10.0 pmol) induced an initial transient Cl- current (I1) followed by a second more prolonged Cl- current (I2). Both currents were Ca2+-dependent, but the source of Ca2+ was different. Release of intracellular Ca2+ stores produced I1, whereas influx of extracellular Ca2+ produced I2; Ca2+-free bathing media and inorganic calcium channel blockers (Mn2+, Co2+) did not alter I1 but completely and reversibly inhibited I2. Injection of the Ins(1,4,5)P3 metabolite, inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (0.2-10.0 pmol) generated a Ca2+-dependent Cl- current with superimposed current oscillations that resulted from release of intracellular Ca2+, not Ca2+ influx. Injection of the Ins(1,3,4,5)P4 metabolite, inositol 1,3,4-trisphosphate (10.0 pmol), or the synthetic inositol trisphosphate isomer, inositol 2,4,5-trisphosphate (1.0-10.0 pmol), mimicked the effect of Ins(1,4,5)P3, stimulating an I1 resulting from release of intracellular Ca2+ and an I2 resulting from influx of extracellular Ca2+. The results indicate that several inositol trisphosphate isomers stimulate both release of intracellular Ca2+ and influx of extracellular Ca2+. Ins(1,3,4,5)P4 also stimulated release of intracellular Ca2+, but it was neither sufficient nor required for Ca2+ influx.