依托咪酯激活大鼠骶髓后连合核神经元GABAA门控氯通道电流

目的　 研究依托咪酯 (etomidate ,ET)在急性分离的大鼠骶髓后连合核 (sacraldorsalcommissuralnucleus,SDCN)神经元的药理学特性。方法　采用制霉菌素穿孔膜片钳全细胞记录。结果　在大鼠SDCN神经元 ,ET(10～ 30 0 0 μmol/L)在钳制电位为 - 40mV时 ,可引起内向电流 (IET) ,EC50 为 (33.35± 3.0 7) μmol/L ,该电流可被GABAA受体拮抗剂荷包牡丹碱 (bicuculline)及氯通道阻滞剂印防己毒素 (picrotoxin)以浓度依赖方式所阻断。结论　ET在大鼠SDCN神经元可通过作用于GABAA受体而诱发氯通道电流 ,此作用可能和全麻状态下脊髓水平的麻醉效应有关     

大鼠脑干SP能神经元向腰骶髓后连合核的投射

采用荧光金逆行标记和免疫荧光组织化学技术,观察了大鼠脑干ＳＰ能神经元向腰骶髓后连合核（ＤＣＮ）的投射状况。将ＦＧ压力注入ＤＣＮ后,脑干中缝苍白核、中缝隐核、中缝大核和巨细胞网状核α部腹外侧部的一些ＦＧ逆标神经元呈现ＳＰ样免疫反应阳性。结果表明,脑干的ＳＰ能下行纤维可终止于脊髓ＤＣＮ区。     

大鼠脑-干 SP能神经元向腰骶髓后连合核的投射

采用荧光金逆行标记和免疫荧光组织化学技术,观察了大鼠脑干SP能神经元向腰骶髓后连合核(DCN)的投射状况.将FG压力注入DCN后,脑干中缝苍白核、中缝隐核、中缝大核和巨细胞网状核α部腹外侧部的一些FG逆标神经元呈现SP样免疫反应阳性.结果表明,脑干的SP能下行纤维可终止于脊髓DCN区.     

大鼠腰骶髓后连合核内5—HT样阳性纤维和终末的来源

采用荧光金（ＦＧ）逆行追踪与免疫荧光组织化学染色相结合技术,对大鼠腰骶髓后连合核（ＤＣＮ）内５－羟色胺（５－ＨＴ）能纤维和终末的分布及其来源进行了观察。结果显示ＤＣＮ内分布有大量５－ＨＴ阳性纤维和终末。将ＦＧ注入到ＤＣＮ后,ＦＧ／５－ＨＴ双标细胞出现于延髓中缝核群中的中缝大核、中缝隐核和中缝苍白核以及外侧旁巨细胞核的腹侧部。结果提示５－ＨＴ对ＤＣＮ内盆内脏感觉信息的传递可能具有调控作用。     

大鼠Barrington核内脊髓投射神经元直接接受腰骶髓传入的电镜研究

将结合生物素的葡聚糖胺(BDA)注射到大鼠腰骶髓后,在电镜下观察脑桥Barrington核内腰骶髓投射神经元与来自腰骶髓传入投射纤维间的突触联系.与先前的研究相一致,注射BDA到腰6和骶l节段后,光镜下可见Barrington核内出现大量顺行标记的神经末梢和一定数量的逆行标记细胞.电镜下发现标记的轴突末梢和标记的树突之间存在直接的突触连接.结果表明,Barrington核直接接受腰骶髓的传入投射,提示大鼠脑桥排尿反射的脊髓内上行投射通路中可能存在一条直接通路.     

大鼠Barrington核内脊髓投射神经元直接接受腰骶髓传入的电镜研究

将结合生物素的葡聚糖胺 (BDA)注射到大鼠腰骶髓后 ,在电镜下观察脑桥Barrington核内腰骶髓投射神经元与来自腰骶髓传入投射纤维间的突触联系。与先前的研究相一致 ,注射BDA到腰 6和骶 1节段后 ,光镜下可见Barrington核内出现大量顺行标记的神经末梢和一定数量的逆行标记细胞。电镜下发现标记的轴突末梢和标记的树突之间存在直接的突触连接。结果表明 ,Barrington核直接接受腰骶髓的传入投射 ,提示大鼠脑桥排尿反射的脊髓内上行投射通路中可能存在一条直接通路。     

幼年爪蟾视顶盖神经元微兴奋性突触后电流的一些特性

以微兴奋性突触后电流(miniature excitatory postsynaptic currents,mEPSCs)为指标,采用盲法膜片电压钳全细胞记录技术,研究敏感期内爪蟾视顶盖神经元突触前受体调制突触后mEPSCs的作用.结果发现,mEPSCs可以被NMDA受体特异性阻断剂APV和(或)AMPA/KA受体特异性阻断剂CNQX所阻断,NMDA受体激动剂NM-DA可以诱发内向突触后电流.与成年神经元相比,未成年动物神经元的mEPSCs的下降相时间较长,mEPSCs的NMDA成分较多,而AMPA成分较少.尼古丁乙酰胆碱受体激动剂(carbachol,nicotine和cytisine)可使mEPSCs的频率增加,且carbachol使频率增加作用需要Ca2+介导;而乙酰胆碱水解产物choline亦有此作用;尼古丁乙酰胆碱受体的竞争性拮抗剂DH-β-E能够拮抗carbachol诱发的mEPSCs频率增加作用;α3β4亚型尼古丁乙酰胆碱受体拮抗剂mecamyllamine不能拮抗carbachol诱发的mEPSCs频率增加作用,但在低浓度下对含有α7亚单元尼古丁乙酰胆碱受体起特异拮抗作用的MLA却有这种拮抗作用;同时我们还观察到,无Ca2+灌流可以导致巨型PSCs的出现,无镁液可以使mEPSCs的下降相延长.结果表明,mEPSCs是由NMDA受体和AMPA/KA受体共同介导的,尼古丁乙酰胆碱受体以Ca2+依赖的方式参与了mEPSCs的突触前调制,其中可能主要涉及尼古丁乙酰胆碱受体α7亚型.     

幼年爪蟾视顶盖神经元微兴奋性突触后电流的一些特性

以微兴奋性突触后电流 (miniatureexcitatorypostsynapticcurrents,mEPSCs)为指标 ,采用盲法膜片电压钳全细胞记录技术 ,研究敏感期内爪蟾视顶盖神经元突触前受体调制突触后mEPSCs的作用。结果发现 ,mEP SCs可以被NMDA受体特异性阻断剂APV和 (或 )AMPA/KA受体特异性阻断剂CNQX所阻断 ,NMDA受体激动剂NMDA可以诱发内向突触后电流。与成年神经元相比 ,未成年动物神经元的mEPSCs的下降相时间较长 ,mEPSCs的NMDA成分较多 ,而AMPA成分较少。尼古丁乙酰胆碱受体激动剂 (carbachol,nicotine和cytisine)可使mEPSCs的频率增加 ,且carbachol使频率增加作用需要Ca2 介导 ;而乙酰胆碱水解产物choline亦有此作用 ;尼古丁乙酰胆碱受体的竞争性拮抗剂DH β E能够拮抗carbachol诱发的mEPSCs频率增加作用 ;α3β4亚型尼古丁乙酰胆碱受体拮抗剂mecamyllamine不能拮抗carbachol诱发的mEPSCs频率增加作用 ,但在低浓度下对含有α7亚单元尼古丁乙酰胆碱受体起特异拮抗作用的MLA却有这种拮抗作用 ;同时我们还观察到 ,无Ca2 灌流可以导致巨型PSCs的出现 ,无镁液可以使mEPSCs的下降相延长。结果表明 ,mEPSCs是由NMDA受体和AMPA/KA受体共同介导的 ,尼古丁乙酰胆碱受体以Ca2 依赖的方式参与了mEPSCs的突触前调制 ,其中可能主要涉     

海马CA1锥体神经元NMDA及非NMDA受体介导的兴奋性突触后电流的生后变化

应用盲法脑片膜片钳记录方法,研究了幼年大鼠（生后6～21d）及成年大鼠（生后56～70d）离体海马脑片CA1锥体神经元N-甲基-D-门冬氨酸（NMDA）及非NMDA受体介导的兴奋性突触后电流（EPSCs）的生后发育变化。为阻断γ-氨基丁酸（GABA）能抑制性突触活动,灌注液中常规应用GABAA受体拮抗剂荷包牡丹碱（50umol/L）。局部刺激海马辐射层（0.05～0.1Hz）可引起EPSCs。结果显示NMDA受体拮抗剂AP5可减小EPSCs的幅值,减小的程度以幼年大鼠为显著。进一步灌注a-氨基-3羧基-5-甲基异恶唑-4丙酸（AMPA）受体拮抗剂CNQX（20umol/L）可完全阻断残余EPSCs。分析给予AP5前、后EPSCs幅值的大小,可得到NMDA及非NMDA受体介导EPSCs,显示非NMDA受体介导的EPSCs随着发育明显增加,而NMDA万分则降低。上述研究结果表明海马CA1神经元的兴奋性突触活动是由NMDA及非NMDA受体介导的,并且在生后一周内以NMDA成分为主,因此在发育的早期NMDA受体可能更多参与对发育的调节作用。     

Taurine-activated chloride currents in the rat sacral dorsal commissural neurons

The electrophysiological and pharmacological properties of taurine (Tau)-activated Cl⁻ currents (I_Tau) were investigated in the dissociated rat sacral dorsal commissural nucleus (SDCN) neurons using the nystatin perforated patch recording configuration under voltage-clamp conditions. The reversal potential of I_Tau was close to the Cl⁻ equilibrium potential. The I_Tau was not affected by a preceding GABA response but cross-desensitized by a preceding glycine (Gly) response. Strychnine (STR), picrotoxin (PIC), bicuculline (BIC) and Zn²⁺ suppressed the I_Tau in a concentration-dependent manner. The pharmacology of the I_Tau and Gly-induced response (I_Gly) was similar, though Zn²⁺ inhibition on I_Tau differed from that on I_Gly in being much slower in recovery. Serotonin potentiated the I_Tau via protein kinase C. The results indicate that both Tau and Gly act on a strychnine-sensitive site to open the same Cl⁻ channels in the SDCN neurons, and suggest that Tau may act as a functional neurotransmitter in the mammalian SDCN.     

Adenosine suppresses GABAA receptor-mediated responses in rat sacral dorsal commissural neurons

The modulatory effect of adenosine on gamma-aminobutyric acid (GABA)-activated whole-cell currents were investigated in the neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin perforated patch recording configuration under the voltage-clamp conditions. The results showed that: (1) GABA acted on GABAA receptor and elicited inward Cl- currents (IGABA) at a holding potential (VH) of -40 mV; (2) adenosine suppressed GABA-induced Cl- current without affecting the reversal potential of IGABA and the apparent affinity of GABA to its receptor; (3) N6-cyclohexyladenosine mimicked the suppression effect of adenosine on IGABA, whereas 8-cyclopentyl-1,3-dipropylxanthine blocked the suppression effect of adenosine; (4) adenosine fails to suppress IGABA on the neurons that were pretreated with bisindolylmaleimide I (BIM), while after pretreatment with H-89, the inhibitory effect of adenosine on IGABA were not affected; (5) the suppression effect of adenosine on IGABA remained in the presence of BAPTA-AM. The present results indicate that the suppression of adenosine on IGABA is mediated by adenosine A1 receptor and through a Ca2+-independent protein kinase C transduction pathway, and that the interactions between adenosine and GABA might participate in the modulation of nociceptive information transmission at the SDCN.     

Beta-alanine acts on glycine receptors in the rat sacral dorsal commissural neurons

Whole-cell current responses to bath application of beta-alanine (beta-ALA) were investigated in neurons acutely dissociated from the rat sacra! dorsal commissural nucleus (SDCN) using the nystatin perforated patch recording configuration under voltage-clamp conditions. beta-ALA activated inward currents in a concentration-dependent manner over the range of 10-3000 microM with an EC50 of 80.8 microM. The reversal potential of beta-ALA-activated currents (I beta-ALA) was close to the Cl- equilibrium potential. Strychnine and the chloride channel blocker picrotoxin suppressed the I beta-ALA in a concentration-dependent manner with the IC50 values of 0.19 microM and 343.6 microM, respectively. At the concentration of 3 microM, strychnine was sufficient to completely block 100 microM beta-ALA response, whereas it did not show a suppression of GABA response. In contrast, bicuculline, a potent GABAA receptor antagonist, at concentrations up to 10 microM, a dose that could block the GABA response completely, had little or no effect on I beta-ALA. Furthermore, the I beta-ALA was not affected by a preceding GABA response, but rather cross-desensitized with that evoked by glycine. The results indicate that beta-ALA activates the strychnine-sensitive glycine receptors in the SDCN neurons, and suggest that beta-ALA may act as a functional neurotransmitter in the mammalian SDCN.     

Co-release and interaction of two inhibitory co-transmitters in rat sacral dorsal commissural neurons

Conventional whole-cell recording configuration was used to characterize the interaction between two inhibitory neurotransmitters, GABA and glycine, in synaptic bouton preparation obtained from rat sacral dorsal commissural nucleus (SDCN). The co-release of GABA and glycine as well as the interaction between their corresponding receptors was demonstrated. Furthermore, taking advantage of pure glycinergic terminal preparation, the possible interaction between GABA and glycine at synaptic level was studied. The results revealed a novel cross-modulation between the two inhibitory cotransmitters systems. This interaction may contribute to sensory processing such as nociception in the mammalian deep dorsal horn under physiological and/or pathological conditions.     

N-methyl-D-aspartate enhancement of the glycine response in the rat sacral dorsal commissural neurons

The effect of N-methyl-D-aspartate (NMDA) on the glycine (Gly) response was examined in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. The application of 100 microM NMDA to SDCN neurons reversibly potentiated Gly-activated Cl- currents (IGly) without affecting the Gly binding affinity and the reversal potential of IGly. A selective NMDA receptor antagonist, APV (100 microM), blocked the NMDA-induced potentiation of IGly, whereas 50 microM CNQX, a non-NMDA receptor antagonist, did not. The potentiation effect was reduced when NMDA was applied in a Ca2+-free extracellular solution or in the presence of BAPTA AM, and was independent of the activation of voltage-dependent Ca2+ channels. Pretreatment with KN-62, a selective Ca2+-calmodulin-dependent protein kinase II (CaMKII) inhibitor, abolished the NMDA action. Inhibition of calcineurin (CaN) further enhanced the NMDA-induced potentiation of IGly. In addition, the GABAA receptor-mediated currents were suppressed by NMDA receptor activation in the SDCN neurons. The present results show that Ca2+ entry through NMDA receptors modulates the Gly receptor function via coactivation of CaMKII and CaN in the rat SDCN neurons. This interaction may represent one of the important regulatory mechanisms of spinal nociception. The results also suggest that GABAA and Gly receptors may be subject to different intracellular modulatory pathways.     

Gamma-aminobutyric acid-induced responses in acutely dissociated neurons from the rat sacral dorsal commissural nucleus

The electrophysiological and pharmacological properties of GABA-activated Cl- currents (IGABA) were investigated in enzymatically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons using the nystatin perforated patch recording configuration under voltage-clamp conditions. Exogenous application of GABA to SDCN neurons induced Cl- currents which increased in a concentration-dependent manner. Bicuculline (BIC) and strychnine (STR) antagonized the IGABA in a concentration-dependent manner. Zn2+ suppressed the IGABA with an IC50 of 2.8 X 10(-5) M. Muscimol mimicked the IGABA, while baclofen evoked no response. Pentobarbital (PB) and 5beta-pregnan-3alpha-ol-20-one (pregnanolone, PGN) also induced GABAA-mimic Cl- currents. Diazepam (DZP), PB and PGN all enhanced the IGABA by increasing the apparent affinity of the GABAA receptors to GABA. Moreover, spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) were observed in mechanically dissociated SDCN neurons attached with synaptic boutons, so called 'synaptic bouton preparation'. These results indicate that SDCN neurons express GABAA receptors with relatively low sensitivity to Zn2+ inhibition, and that GABA may have a functional role as an inhibitory transmitter in the SDCN regulating nociceptive, analgesic, and autonomic functions.     

Mechanism underlying inhibition of the capsaicin receptor-mediated current by pregnenolone sulfate in rat dorsal root ganglion neurons

The effect of pregnenolone sulfate (PS) on the capsaicin receptor-mediated current was studied in acutely dissociated rat dorsal root ganglion neurons using the whole-cell voltage-clamp technique. PS acted extracellularly to inhibit the capsaicin-induced current in a not competitive but concentration-dependent manner, with an EC50 of 13 microM and a maximal inhibition of 65%. Furthermore, antagonism of the capsaicin response by PS is neither voltage- nor agonist-dependent, indicating that PS did not act as an open-channel blocker. The inhibitory action of PS on the capsaicin-induced current may provide a basis for reducing capsaicin receptor-mediated nociception.     

Block of sodium currents in rat dorsal root ganglion neurons by diphenhydramine

To elucidate the local anesthetic mechanism of diphenhydramine, its effects on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion (DRG) neurons were examined by the whole-cell voltage clamp method. Diphenhydramine blocked TTX-S and TTX-R sodium currents with K(d) values of 48 and 86 microM, respectively, at a holding potential of -80 mV. It shifted the conductance-voltage curve for TTX-S sodium currents in the depolarizing direction but had little effect on that for TTX-R sodium currents. Diphenhydramine caused a shift of the steady-state inactivation curve for both types of sodium currents in the hyperpolarizing direction. The time-dependent inactivation became faster and the recovery from the inactivation was slowed by diphenhydramine in both types of sodium currents. Diphenhydramine produced a profound use-dependent block when the cells were repeatedly stimulated with high-frequency depolarizing pulses. The use-dependent block was more pronounced in TTX-R sodium currents. The results show that diphenhydramine blocks sodium channels of sensory neurons similarly to local anesthetics.