神经肽Y对海马神经元兴奋性突触活动的影响

目的观察神经肽Y(NPY)对体外培养海马神经元兴奋性突触活动的影响。方法 Wistar大鼠海马神经元体外原代培养,无镁细胞外液处理3h,建立稳定的海马神经元癫痫样放电模型,应用全细胞膜片钳记录技术,观察不同浓度NPY对海马神经元自发兴奋性突触后电流(sEPSCs)的影响。结果 (1)体外培养12d的海马神经元轮廓清晰,折光性良好,彼此间广泛突触联系形成,适于神经元电生理试验。(2)癫痫组神经元sEPSCs发放频率(28.826±1.254HZ)高于对照组(1.296±0.241HZ),差异有统计学意义(P<0.05)。0.1μmol NPY组sEPSCs的频率为0.895±0.146HZ;1μmol NPY组频率为0.461±0.394HZ,与癫痫组(28.826±1.254HZ)相比,均有明显降低,差异有统计学意义(P<0.05)。0.1μmol NPY组的频率降低的程度小于1μmol NPY组神经元,差异有统计学意义(P<0.05)。sEPSCs幅度各组相比无明显差异(P>0.05)。结论 NPY能够抑制癫痫神经元sEPSCs的频率,而不影响幅度,这为NPY基因治疗癫痫提供了电生理学证据。     

不同阿片受体激动剂对海马培养神经元微小兴奋性突触后电流的影响

目的：探讨不同阿片受体激动剂对海马培养神经元微小兴奋性突触后电流（mEPSCs）的影响。方法：原代培养新生sD大鼠海马神经元18d,荧光倒置显微镜下选择健康神经元入组,分别在培养液中加入阿片受体激动剂吗啡（浓度为10μmol·L-1[D—pen2,D—pen5]一enkephalin（DPDPE）和埃托啡（浓度均为1μmol·L-1）处理3d,对照组不加药。全细胞模式记录mEPSCs,用软件MiniAnalysis6．0（Synaptosoft,Inc）对mEPSCs频率及幅度进行分析。结果：吗啡显著降低了海马神经元mEPSCs的频率和幅度,与对照组相比,频率和幅度分别下降了38．5％和38．0％（均P〈0．01）;埃托啡对mEPSCs的幅度无影响（P〉0．05）,但频率增加了26．O％（P〈0．05）;DPDPE对mEPSCs的频率及幅度均无影响（P〉0．05）。结论：不同的阿片受体激动剂由于其引起受体内化的能力不同,对神经元兴奋性突触传递有不同的突触后作用。     

沉默调节蛋白1促进体外神经元的轴突生长

目的探讨沉默调节蛋白1(Sirt1)对神经元轴突生长的影响。方法体外原代分离培养胚胎海马神经元,观察Sirt1在72 h神经元的分布表达;通过RNAi技术下调Sirt1基因,观察其对72 h神经元轴突长度的影响;通过质粒转染过表达Sirt1基因或药物白藜芦醇(RES)激活Sirt1蛋白,检测其对72 h神经元轴突长度的影响。结果免疫荧光染色结果显示Sirt1位于海马神经元的生长圆锥以及胞体和突起,尤其是轴突末端;与正常对照组(Sirt1正常表达组)相比,Sirt1表达下调可显著缩短72 h海马神经元轴突的长度[由(178.3±3.2)μm缩短到(110.2±18.30)μm,P0.01];与正常对照组(Sirt1正常表达组)相比,基因过表达Sirt1可显著增加72 h海马神经元轴突的长度[由(178.3±3.2)μm增长到(310.6±39.5)μm,P0.01]。与药物对照组(DMSO处理组)相比,药物RES激活Sirt1蛋白亦可显著增加72 h海马神经元轴突的长度[由(292.8±11.2)μm增长到(525.1±49.7)μm,P0.01]。结论Sirt1在神经元的轴突生长中起着重要的作用,可作为轴突再生一个潜在的治疗靶点。     

基因水平下调PP2A水平可显著抑制海马神经元轴突生长

目的 探讨基因水平下调PP2A对胎鼠海马神经元轴突生成的影响。方法 选取体外培养原代海马神经元为研究模型,首先构建PP2A催化亚基特异性干扰RNA及其对照(si-PP2A/ssi-PP2A)质粒,选择在种植前下调PP2A水平,观察其对海马神经元轴突生成是否有作用; 原代细胞采用Amaxa大鼠神经元核电转试剂盒分别转染EGFP-ssiPP2Ac和EGFP-siPP2Ac,神经元种植培养48 h后固定做免疫荧光双标,分别标记轴突特异性标记物Tau-1和树突特异性标记物MAP-2,观察基因下调PP2A水平对神经元轴突生成的影响。结果 原代海马神经元转染48 h后对照转染组海马神经元神经元轴突和树突均已形成,而干扰RNA转染组神经元轴突生长受到显著抑制,而树突生长未受到明显影响; si-PP2A转染组(干扰转染组)神经元轴突长度仅为对照转染组的38%,单个神经元的平均轴突数目也从正常1.0/neuron下降到0.5/neuron。结论基因下调PP2A水平显著抑制了原代海马神经元轴突生成,结合前期药物下调研究结果提示维持细胞内正常PP2A水平在海马神经元轴突生成中起重要作用。     

Kir 2.3过表达抑制原代海马神经元样放电

目的探究内向整流钾(Kir) 2. 3通道过表达对环噻唑诱导的原代海马神经元样放电的影响。方法分离培养大鼠原代海马神经元,分别使用GFP质粒或Kir 2. 3-GFP质粒转染神经元。通过免疫荧光染色和电压钳技术检测转染效果。使用环噻唑诱导神经元样放电,电流钳记录膜电位钳制在-70 m V条件下的自发放电,膜片钳记录各组神经元的膜电位。结果转染Kir 2. 3-GFP质粒的神经元Kir 2. 3表达水平显著增高(P 0. 05)。环噻唑能够诱导未转染神经元(n=12)及GFP质粒转染神经元(n=6)出现样爆发放电,但是未能诱导Kir 2. 3-GFP质粒转染神经元(n=5)出现样爆发放电。Kir 2. 3-GFP质粒转染组出现样爆发放电神经元的比例显著低于未转染组(P 0. 01)及GFP质粒转染组(P 0. 05)。此外,Kir 2. 3-GFP质粒转染组(n=10)神经元膜电位较未转染组(n=6)及GFP质粒转染组(n=8)显著增高。结论 Kir 2. 3过表达能显著抑制环噻唑诱导的原代海马神经元样放电,其作用机制可能与静息膜电位增高相关。     

Sombati癫癎细胞模型电压依赖性钾电流的变化

目的：采用全细胞膜片钳记录技术研究Sombati癫癎细胞模型电压依赖性K＋电流的变化。方法：取SD新生乳鼠双侧海马,体外原代培养海马神经元,培养至第9天的部分海马神经元经无镁液处理3h制备癫癎细胞模型,采用全细胞膜片钳技术分别记录正常组（未经无镁液处理的海马神经元）和致癎组（经无镁液处理3h后更换为正常维持培养液的海马神经元）海马神经元的电压依赖性外向K＋电流。结果：致癎组外向K＋电流比正常组增大,峰值电流由（596.62±79.23）pA增至（978.68±53.23）pA,两组间比较差异有统计学意义（P=0.000）。与正常组相比,致癎组的I-V曲线明显左移,I-V曲线形态无显著改变。结论：Sombati癫癎细胞模型中,电压依赖性外向K＋电流显著增加可能是癫癎细胞模型癎性放电后为了保持细胞稳态的代偿性保护作用。     

爪蟾视觉发育过程中抑制性GABAa受体功能的变化

目的:阐明从幼年(变态脱尾后2～12周)到成年(变态脱尾后5～10月)爪蟾的视顶盖神经元突触受体变化.方法:盲法脑片全细胞电压钳技术.结果:成年视顶盖神经元的微抑制性突触后电流(mIPSCs)的发放频率(Hz)和平均振幅(pA)分别为2.65±0.69和9.82±1.30,而幼年的分别为0.68±0.23和15.36±2.40,其中成年动物的mIPSCs的频率是幼年频率的3.90倍;乙酰胆碱受体(nAChR)激动剂carbachol均可使幼年及成年的mIPSCs频率增加,增强效率有所不同,幼年增加至对照组的192.0%,而成年组仅为146.2%.结论:随着视顶盖神经元的成熟,其突触GABAa受体功能也相应增强.     

胚胎期可卡因暴露对子代大鼠海马CA1锥体神经元mEPSCs 的影响

目的研究胚胎期可卡因暴露(PCE)影响子代空间学习的机制。方法首先从行为学角度分析PCE对子代动物形成可卡因诱导的条件位置偏爱(CPP)能力的影响。进而从电生理角度研究空间记忆受损的原因,通过全细胞膜片钳方式分别记录子代大鼠背侧和腹侧海马兴奋性突触后电流(mEPSC)在PCE影响后的变化。结果 CPP结果显示,PCE明显延长了子代大鼠形成CPP的时间。结合既往研究报道的PCE对子代空间记忆能力的损害,推测PCE对子代CPP的影响是因为空间记忆受损所致。电生理实验结果显示,PCE抑制了子代大鼠经急性可卡因暴露后背侧海马CA1锥体神经元mEPSC振幅增高的变化。结论 PCE造成子代背侧海马CA1锥体神经元功能受损,导致空间记忆受影响,可能是PCE减弱子代形成可卡因诱导CPP能力的基础。     

GABAB受体对大鼠海马CA1区锥体细胞突触传递的作用

目的研究激活GABA_B受体对大鼠海马CA1区锥体细胞突触传递的影响。方法对成年大鼠海马脑片CA1区锥体细胞采用“盲法”全细胞电压钳记录,分别检测和分析巴氯芬(10μmol/L)对自发性的兴奋性突触后电流(EPSCs)和抑制性突触后电流(IPSCs)的影响。结果巴氯芬可显著降低符氨酸能EPSCs和γ-氨基丁酸能IPSCs的频率(P＜0．01),各自达58%±7%(n=17)和42%±10%(n=15),而对它们的幅度无显著性影响。结论巴氯芬对海马CA1区锥体细胞EPSCs和IPSCs的抑制作用属于突触前抑制,推测GABA_B受体所介导的这种抑制作用对CA1区神经元兴奋性的传出具有抑制作用,从而对癫痫的产生有控制作用。     

爪蟾视觉发育过程中抑制性GABAa受体功能的变化

目的 :阐明从幼年 (变态脱尾后 2～ 12周 )到成年 (变态脱尾后 5～ 10月 )爪蟾的视顶盖神经元突触受体变化。 方法 :盲法脑片全细胞电压钳技术。结果 :成年视顶盖神经元的微抑制性突触后电流 (mIPSCs)的发放频率 (Hz)和平均振幅 (pA)分别为 2 .6 5± 0 .6 9和 9.82± 1.30 ,而幼年的分别为 0 .6 8± 0 .2 3和 15 .36± 2 .4 0 ,其中成年动物的mIPSCs的频率是幼年频率的 3.90倍 ;乙酰胆碱受体 (nAChR)激动剂carbachol均可使幼年及成年的mIPSCs频率增加 ,增强效率有所不同 ,幼年增加至对照组的 192 .0 % ,而成年组仅为 14 6 .2 %。 结论 :随着视顶盖神经元的成熟 ,其突触GABAa受体功能也相应增强。     

Fear conditioning enhances spontaneous AMPA receptor-mediated synaptic transmission in mouse hippocampal CA1 area

AMPA receptor-mediated synaptic modifications in the amygdala have been reported to sustain cued fear conditioning. However, the hippocampal formation is also critically involved in fear learning. Therefore, we examined whether fear conditioning is also accompanied by changes in AMPA receptor-mediated synaptic transmission in the hippocampus. We focused on spontaneous miniature excitatory post-synaptic currents (mEPSCs). Young adult mice were trained using tone/footshock pairings and contextual/cued memories were tested 3–4 h and 1 day later. We found that the mEPSC frequency was significantly enhanced when recorded 3 h, but not 24 h, after fear conditioning training. Fear training induced a slight enhancement in the mEPSC amplitude at 3 h after training. The increased mEPSC frequency and amplitude were absent in animals that were only exposed to footshock or novelty or unpaired tone/footshock training. This implies that learning the association between context, tone and footshock transiently enhances hippocampal CA1 spontaneous synaptic transmission, which may contribute to the encoding of the fearful event.     

NT-3 regulates BDNF-induced modulation of synaptic transmission in cultured hippocampal neurons

BDNF and NT-3 can modulate the development and plasticity of central synaptic transmission. Although the expression of NT-3 and BDNF in the rodent hippocampus coincides during perinatal development, little is known about possible functional interactions between both neurotrophins in synaptic development. Here, we have investigated the effects of combined long-term application of NT-3 and BDNF on excitatory glutamatergic (mEPSC) and inhibitory GABAergic miniature synaptic currents (mIPSC) in cultured embryonic hippocampal neurons. Our results show that the BDNF-induced twofold increase in mEPSC frequency is abolished by pre-treatment with NT-3. In addition, the NT-3-induced twofold downregulation of mIPSC frequency is reversed by BDNF. Finally, the BDNF-induced increase in c-fos expression is reduced by 50% after pre-treatment with NT-3. In summary, these data suggest an NT-3 controlled modulation of BDNF signalling in differentiating hippocampal neurons.     

L-phenylalanine selectively depresses currents at glutamatergic excitatory synapses

To explore the hypothesis that L-phenylalanine (L-Phe) depresses glutamatergic synaptic transmission and thus contributes to brain dysfunction in phenylketonuria (PKU), the effects of L-Phe on spontaneous and miniature excitatory postsynaptic currents (s/mEPSCs) in rat and mouse hippocampal and cerebrocortical cultured neurons were studied using the patch-clamp technique. L-Phe depressed the amplitude and frequency of both N-methyl-D-aspartate (NMDA) and non-NMDA components of glutamate receptor (GluR) s/mEPSCs. The IC(50) of L-Phe to inhibit non-NMDAR mEPSC frequency was 0.98 +/- 0.13 mM, a brain concentration seen in classical PKU. In contrast, D-Phe had a significantly smaller effect, whereas L-leucine, an amino acid that competes with L-Phe for brain transporter, had no effect on mEPSCs. Unlike GluR s/mEPSCs, GABA receptor mIPSCs were not attenuated by L-Phe. A high extracellular concentration of glycine prevented the attenuation by L-Phe of NMDAR current, activated by exogenous agonist, and of NMDAR s/mEPSC amplitude, but not of NMDAR s/mEPSC frequency. On the other hand, L-Phe significantly depressed non-NMDAR current activated by low but not high concentrations of exogenous agonists. Glycine-independent attenuation of NMDAR s/mEPSC frequency suggests decreased presynaptic glutamate release caused by L-Phe, whereas decreased amplitudes of NMDAR and non-NMDAR s/mEPSCs are consistent with competition of L-Phe for the glycine- and glutamate-binding sites of NMDARs and non-NMDARs, respectively. The finding that GluR activity is significantly depressed at conditions characteristic of classical PKU indicates a potentially important contribution of impaired GluR function to PKU-related mental retardation and provides important insights into the potential physiological consequences of impaired GluR function.     

Fast excitatory synaptic transmission mediated by nicotinic acetylcholine receptors in Drosophila neurons.

Difficulty in recording from single neurons in vivo has precluded functional analyses of transmission at central synapses in Drosophila, where the neurotransmitters and receptors mediating fast synaptic transmission have yet to be identified. Here we demonstrate that spontaneously active synaptic connections form between cultured neurons prepared from wild-type embryos and provide the first direct evidence that both acetylcholine and GABA mediate fast interneuronal synaptic transmission in Drosophila. The predominant type of fast excitatory transmission between cultured neurons is mediated by nicotinic acetylcholine receptors (nAChRs). Detailed analysis of cholinergic transmission reveals that spontaneous EPSCs (sEPSCs) are composed of both evoked and action potential-independent [miniature EPSC (mEPSC)] components. The mEPSCs are characterized by a broad, positively skewed amplitude histogram in which the variance is likely to reflect differences in the currents induced by single quanta. Biophysical characteristics of the cholinergic mEPSCs include a rapid rise time (0.6 msec) and decay (tau = 2 msec). Regulation of mEPSC frequency by external calcium and cobalt suggests that calcium influx through voltage-gated channels influences the probability of ACh release. In addition, brief depolarization of the cultures with KCl can induce a calcium-dependent increase in sEPSC frequency that persists for up to 3 hr after termination of the stimulus, illustrating one form of plasticity at these cholinergic synapses. These data demonstrate that cultured embryonic neurons, amenable to both genetic and biochemical manipulations, present a unique opportunity to define genes/signal transduction cascades involved in functional regulation of fast excitatory transmission at interneuronal cholinergic synapses in Drosophila.     

神经肽Y对海马神经元“癫痫样”动作电位的影响

目的探讨神经肽Y(neuropeptide Y,NPY)对海马神经元"癫痫样"动作电位的影响。方法用无镁细胞外液处理原代培养12 d的海马神经元3 h,诱导海马神经元癫痫样放电,建立海马神经元癫痫样放电模型;用全细胞膜片钳电流钳模式检测神经元动作电位,分别给予0.1μmol/L和1μmol/L NPY各1μL,给药时间10 s,观察其对神经元动作电位频率及波幅的影响。结果无镁细胞外液处理神经元3 h,可以形成稳定的海马神经元癫痫样放电模型,频率16～23 Hz,波幅75～96 mV。模型组神经元动作电位频率为(18.00±2.32)Hz,而0.1μmol/L和1μmol/L NPY组分别为(4.75±1.04)Hz和(1.50±0.75)Hz。与模型组相比较,两种浓度NPY组均降低了动作电位发放的频率(P<0.05)。模型组神经元动作电位波幅为(82.25±5.17)mV,而0.1μmol/L和1μmol/L NPY组分别为(49.75±2.49)mV和(40.00±2.20)mV。与模型组相比较,两种浓度NPY组均降低了动作电位发放的波幅(P<0.05)。两种浓度NPY之间相比较,也有统计学差异(P<0.05)。1μmol/LNPY明显抑制了动作电位发放的频率和波幅。结论 NPY能够抑制无镁细胞外液诱发的神经元癫痫样电活动,为应用NPY抑制癫痫发作提供了细胞电生理学证据。     

Neuroprotective effect of amantadine on corticosterone‐induced abnormal glutamatergic synaptic transmission of CA3‐CA1 pathway in rat's hippocampal slices

Depression is a psychiatric disorder and chronic stress, leading to altered glucocorticoid secretion patterns, is one of the factors that induce depression. Our previous study showed that amantadine significantly attenuated the impairments of synaptic plasticity and cognitive function a rat model of CUS. However, little is known regarding the underlying mechanism. In the present study, the whole‐cell patch‐clamp technique was applied to examine the protection effect of amantadine on the hippocampus CA3‐CA1 pathway. Evoked excitatory postsynaptic currents (eEPSCs), miniature excitatory postsynaptic currents (mEPSCs), paired‐pulse ratio (PPR) and the action potentials of CA3 neurons were recorded. Our data showed that corticosterone increased the amplitude of eEPSCs and decreased the value of paired‐pulse ratio (PPR), but both of them were significantly reversed by amantadine. In addition, the frequency of mEPSC was considerably increased by corticosterone, but it was reduced by amantadine. Moreover, we used the Fluo‐3/AM image to detect the Ca²⁺ influx in primary cultured hippocampal neurons. The results showed that the intracellular calcium levels were significantly decreased by amantadine in the corticosterone treated neurons. Additionally, the superoxide dismutase (SOD) and catalase (CAT) activities were reduced by corticosterone, while they were enhanced by either amantadine or low‐calcium artificial cerebral spinal fluid (ACSF). These results suggest that amantadine significantly improves corticosterone‐induced abnormal glutamatergic synaptic transmission of CA3‐CA1 synapses presynaptically and alleviates the activities of antioxidant enzymes via regulating the calcium influx.     

Interleukin-1beta enhances NMDA receptor-mediated current but inhibits excitatory synaptic transmission

Interleukin (IL)-1beta is often characterized as the prototypic proinflammatory cytokine but is involved in various pathophysiological conditions in the central nervous system (CNS). A whole-cell recording technique was used to observe its effect on N-methyl-D-aspartate (NMDA)-evoked currents and spontaneous synaptic activity in cultured rat hippocampal neurons. The results showed that the frequencies but not the amplitudes of spontaneous excitatory postsynaptic currents (sEPSC) and miniature excitatory postsynaptic currents (mEPSC) were decreased by 10 or 100 ng/ml IL-1beta. IL-1beta at these concentrations also increased the NMDA receptor-mediated current. In addition, 10 ng/ml IL-1beta significantly increased the amplitude of the voltage-dependent Ca2+ current (I(Ca)). The increase in I(Ca) following treatment of cultures with IL-1beta resulted mainly from an increase in L-type current. These data suggest that IL-1beta modulates hippocampus-related functions via its effect on synaptic activity and Ca2+ signaling in neurons.     

Modulation of excitatory synaptic transmission by endogenous glutamate acting on presynaptic group II mGluRs in rat substantia nigra compacta

Excitatory synaptic inputs from the subthalamic nucleus (STN) have been proposed to underlie burst firing of substantia nigra pars compacta (SNc) dopamine (DA) neurons in Parkinson's disease. Given the potential importance of the STN-SNc synapse in health and disease, our goal was to study how transmission at this synapse is regulated. We tested the hypothesis that neurotransmission at STN-SNc synapses is tonically inhibited by endogenous glutamate acting on presynaptic group II metabotropic glutamate receptors (mGluRs). By using whole-cell recording techniques in brain slices, we examined the effect of LY341495, a mGluR antagonist that is most potent at group II mGluRs, on excitatory postsynaptic currents (EPSCs) that either were evoked in SNc DA neurons by stimulation of the STN or were spontaneously occurred in the presence of tetrodotoxin (miniature EPSCs; mEPSCs). LY341495 increased the evoked EPSC amplitude and mEPSC frequency without changing mEPSC amplitude. In contrast, the group III mGluR antagonist UBP1112 failed to increase the evoked EPSC amplitude. An elevation of extracellular glutamate concentration by a glutamate transporter inhibitor, TBOA, suppressed the evoked EPSCs. LY341495, but not UBP1112, partially reversed the TBOA action. The modulations of EPSCs by TBOA and LY341495 were associated with changes in paired-pulse facilitation ratio. Furthermore, TBOA decreased mEPSC frequency, which was partially reversed by LY341495, without affecting mEPSC amplitude. The results indicate that presynaptic group II mGluRs at STN-SNc synapses appear to be partially activated by a basal level of extracellular glutamate and able to sense the change in extracellular glutamate concentration, subsequently modulating synaptic glutamate release.     

Impaired hippocampal synaptic transmission and plasticity in mice lacking fibroblast growth factor 14

Humans with an autosomal dominant missense mutation in fibroblast growth factor 14 (FGF14) have impaired cognitive abilities and slowly progressive spinocerebellar ataxia. To explore the mechanisms that may account for this phenotype, we show that synaptic transmission at hippocampal Schaffer collateral-CA1 synapses and short- and long-term potentiation are impaired in Fgf14-/- mice, indicating abnormalities in synaptic plasticity. Examination of CA1 synapses in Fgf14-/- mice show a significant reduction in the number of synaptic vesicles docked at presynaptic active zones and a significant synaptic fatigue/depression during high/low-frequency stimulation. In addition, mEPSC frequency, but not amplitude, is decreased in hippocampal neurons derived from Fgf14-/- mice. Furthermore, expression of selective synaptic proteins in Fgf14-/- mice was decreased. These findings suggest a novel role for FGF14 in regulating synaptic plasticity via presynaptic mechanisms by affecting the mobilization, trafficking, or docking of synaptic vesicles to presynaptic active zones.