一种可用于500μm脑片的高分辨率细胞内标记技术

目的：建立简便高效的、与脑片盲法膜片钳记录相结合的生物胞素细胞内标记染色方法。方法：制备大鼠听皮层脑片（500 μm）,采用盲法脑片膜片钳全细胞记录,泳入生物胞素（0.2％）对记录细胞进行标记,经组织化学显色和甘油封片后,沿显微镜Z轴,每隔30 μm拍摄一帧显微数码图像,利用Adobe Photoshop软件对神经元进行三维重建。结果：标记的神经元层次清楚,可在光镜下分辨出胞体、轴树突分支、棘突等细微结构,而且非特异性背景染色浅;不需要进行厚脑片的二次切片即可对神经元进行三维重建。结论：本方法简便易行,结果可靠,分辨率高,而且对设备要求不高。     

在体大鼠膜片钳全细胞记录技术初探

目的：建立在体大鼠膜片钳全细胞记录方法。方法：固定麻醉大鼠后对其顶叶皮层锥体神经元行全细胞记录。结果：成功记录到顶叶内锥体层神经元电压门控性钠离子通道电流及自发突触活动电流。结论：初步建立了在体大鼠膜片钳全细胞记录方法,但对记录的稳定性仍有待于进一步提高。     

小鼠海马神经元冰冻切片和脑片培养方法的比较

目的:通过冰冻切片和脑片培养方式比较获得更适合脑片实验研究的方法。方法:分别运用急性切片和脑片培养方法,结合全细胞膜片钳技术比较两种脑片处理方法对小鼠海马神经元细胞形态、细胞膜封接难易程度、细胞电生理特性等的差异,获得更适合细胞研究的脑片获取方法。结果:冰冻切片方法切断部分神经纤维,脑片表层出现肿胀或坏死细胞,2-3层细胞可用于膜片钳记录,但不易封接破膜。脑片培养后可使纤维再生,整个脑片细胞形态清晰可见,容易封接破膜,电生理记录波形及基本特性与冰冻切片一致,但脑片培养方法的细胞突触后电流幅度更大、频率更高。结论:脑片培养可修复受损纤维和细胞膜柔韧性,且不改变膜特性,但脑片培养重建了一定数量的细胞间信号连接,使细胞反应性增强,脑片培养方法更适合脑片神经元研究。     

突触前α7烟碱受体对海马神经元兴奋性突触传递的调控

采用盲法膜片钳技术观察突触前烟碱受体(nicotinic acetylcholinel receptors,nAChRs)对海马脑片CAl区锥体神经元兴奋性突触传递的调控作用。结果显示,nAChRs激动剂碘化二甲基苯基哌嗪(dimethylphenyl—piperazinium iodide,DMPP)不能在CAl区锥体神经元上诱发出烟碱电流。DMPP对CAl区锥体神经元自发兴奋性突触后电流(spontaneous excitatory postsynaptic current,sEPSC)具有明显的增频和增幅作用,并呈现明显的浓度依赖关系。DMPP对微小兴奋性突触后电流(miniature excitatory postsynaptic current,mEPSC)具有增频作用,但不具有增幅作用。上述DMPP增强突触传递的作用不能被nAChRs拮抗剂美加明、六烃季铵和双氢-β-刺桐丁所阻断,但可被α-银环蛇毒素阻断。上述结果提示,海马脑片CAl区锥体神经元兴奋性突触前nAChRs含有对α-银环蛇毒素敏感的胡亚单位,其激活可增强海马CAl区锥体神经元突触前递质谷氨酸的释放,从而对兴奋性突触传递发挥调控作用。     

海马脑片锥体神经元钙离子通道的盲法膜片钳研究

目的和方法 :采用大鼠海马脑片盲法膜片钳全细胞记录技术研究CA1区锥体神经元电压门控性Ca2 通道的动力学特征。结果 :大鼠海马脑片CA1区锥体神经元电压门控性Ca2 通道电流具有如下特点 :①激活的阈电位偏低 ,为 (- 4 9.3± 8.6 )mV ,范围为 - 6 5～ - 30mV(n =2 3)。②衰减时间常数τ值较大 ,且变化范围大 (10 0～ 70 0ms) (n =12 ) ,并且衰减具有Ca2 电流幅值的依赖性 ,③稳态失活呈现电压依赖性 ,半失活电压为 (- 5 5 .4± 9.7)mV ,斜率因子为 (5 .3± 0 .9)mV(n =10 )。④当细胞外Ca2 浓度为 2 .5mmol/L时 ,Ca2 通道的反转电位为 (5 5±13)mV(n =10 )。⑤尾电流成分较为单一 ,不表现电压依赖性。另外 ,Ca2 电流对戊脉胺及双氢吡啶类化合物硝苯地平均不敏感。结论 :根据上述Ca2 电流特征 ,海马脑片CA1区锥体神经元上的Ca2 通道主要以N型为主     

AmphotericinB在全细胞穿孔膜片钳技术中的应用研究

目的：为研究海德氏突触的电生理特性,建立用amphotericinB穿孔的膜片钳技术。方法：本文应用两性霉素B（amphotericinB）在小鼠脑干的calyx细胞上进行穿孔膜片钳技术的研究。结果：应用amphotericinB进行穿孔后,通道电流衰减现象显著变慢。AmphotericinB的最适浓度为400tc／ml。结论：本研究摸索出了一种稳定的穿孔膜片钳全细胞记录技术,可以更有效,更真实的反应神经元通道电流的电生理特性。为穿孔膜片钳技术在听觉信息传导和调控研究中的应用提供了基础资料。     

皮质酮对体外培养的海马神经元延迟整流钾电流的影响

目的：探讨应激激素皮质酮对海马神经元延迟整流钾电流的影响。方法：膜片钳全细胞记录测量原代培养大鼠海马神经元膜的钾离子电流。结果：在皮质酮的作用下,海马神经元膜的钾离子电流幅度明显下调,激活阈电位升高。结论：过量皮质酮激素可能通过影响延迟整流钾通道损伤海马神经元。     

丙泊酚拮抗戊四氮对大鼠海马CA1区动作电位和突触传递的影响

目的：观察戊四氮对大鼠海马CA1区动作电位（action potential,AP）和兴奋性突触后电流（excitatory postsynaptic current,EPSC）的影响和丙泊酚的拮抗作用。方法：断头法分离Wistar大鼠海马半脑,切片机切出400μm厚度的海马脑片,全细胞电流钳记录CA1区锥体神经元动作电位发放情况,全细胞电压钳记录电刺激Schaeffer侧支／联合纤维诱发的CA1区锥体神经元EPSC的变化。结果：戊四氮使动作电位发放频率增加,EPSC值降低;丙泊酚拮抗戊四氮的作用,使动作电位发放减少甚至消失,EPSC值上升至加入丙泊酚前的2倍左右。结论：丙泊酚拮抗戊四氮对动作电位和EPSC的作用,所以临床上可用于抗癫痫治疗。     

在体膜片钳技术的新进展

在体膜片钳是指在整体动物上直接对其中枢神经元进行全细胞膜片钳记录的技术,在生理学和药理学研究中具有良好的应用前景.常规采用的是盲法记录,最近出现的可视法记录,采用双光子靶向膜片钳（two-photon targeted patching,TPTP）技术,通过基因操作在动物脑内目标神经元中构建特异表达的荧光标志,可以做到对特定神经元亚群的靶向研究.对这两种方法的原理和操作进行了简单的介绍.     

Axon公司膜片钳系统的漏减分析

目的和方法：采用大鼠海马脑片盲法膜片钳的全细胞记录技术,研究美国Axon公司生产的膜片钳系统（Axopatch放大器和pClamp软件）中几种漏减功能的意义和作用机制,重点对定标P／N漏减（Scaled P/N leak subtraction)、膜片钳放大器漏减以及Clampfit处理软件漏减功能的选择与使用进行分析与比较。结果：Clampex采样软件中的定标P／N漏减功能比P／N漏减功能的噪声要小;放大器漏减功能可漏减单一去极化电压幅度所诱发的漏电流,但不能同时对不同电压幅度系列去极化所产生的稳态漏电流进行追踪漏减;Clampfit漏减功能由于其设定只要膜两侧存在电位差就有漏电流产生,因而不适合在记录电压门控性离子通道电流时对稳态漏电流进行漏减。结论：在研究电压门控性离子通道的性质时,可采用P／N漏减功能或定标P／N漏减功能对稳态漏电流进行漏减,而Clampfit漏减功能是不合适的。     

Presynaptic Ca2+ channels compete for channel type-preferring slots in altered neurotransmission arising from Ca2+ channelopathy

Several human channelopathies result from mutations in alpha1A, the pore-forming subunit of P/Q-type Ca2+ channels, conduits of presynaptic Ca2+ entry for evoked neurotransmission. We found that wild-type human alpha1A subunits supported transmission between cultured mouse hippocampal neurons equally well as endogenous mouse alpha1A, whereas introduction of impermeant human alpha1A hampered the effect of endogenous subunits. Thus, presynaptic P/Q-type channels may compete for channel type-preferring "slots" that limit their synaptic effectiveness. The existence of slots generates predictions for how neurotransmission might be affected by changes in Ca2+ channel properties, which we tested by studying alpha1A mutations that are associated with familial hemiplegic migraine type 1 (FHM1). Mutant human P/Q-type channels were impaired in contributing to neurotransmission in precise accord with their deficiency in supporting whole-cell Ca2+ channel activity. Expression of mutant channels in wild-type neurons reduced the synaptic contribution of P/Q-type channels, suggesting that competition for type-preferring slots might support the dominant inheritance of FHM1.     

Reversine inhibits spontaneous synaptic transmission in cultured rat hippocampal neurons

The dedifferentiation agent "reversine" [2-(4-morpholinoanilino)-N6-cyclohexyladenine 2], which can induce myogenic lineage-committed cells to become multipotent mesenchymal progenitor cells, was discovered by Shuibing Chen et al. in 2003. But its effects on neurons were unknown. Using patch-clamp technique, we found that reversine inhibits spontaneous synaptic transmission in cultured rat hippocampal neurons without influencing the dynamics function of potassium, sodium and calcium channels. This result suggests that reversine may also act as a dedifferentiation agent in neurons, and inhibiting the synaptic transmission maybe the early step of neuronal dedifferentiation.     

Paired-recordings from synaptically coupled cortical and hippocampal neurons in acute and cultured brain slices

Analysis of synaptic transmission, synaptic plasticity, axonal processing, synaptic timing or electrical coupling requires the simultaneous recording of both the pre- and postsynaptic compartments. Paired-recording technique of monosynaptically connected neurons is also an appropriate technique to probe the function of small molecules (calcium buffers, peptides or small proteins) at presynaptic terminals that are too small to allow direct whole-cell patch-clamp recording. We describe here a protocol for obtaining, in acute and cultured slices, synaptically connected pairs of cortical and hippocampal neurons, with a reasonably high probability. The protocol includes four main stages (acute/cultured slice preparation, visualization, recording and analysis) and can be completed in approximately 4 h.     

Reduction in neuronal L-type calcium channel activity in a double knock-in mouse model of Alzheimer's disease

Increased function of neuronal L-type voltage-sensitive Ca(2+) channels (L-VSCCs) is strongly linked to impaired memory and altered hippocampal synaptic plasticity in aged rats. However, no studies have directly assessed L-VSCC function in any of the common mouse models of Alzheimer's disease where neurologic deficits are typically more robust. Here, we used cell-attached patch-clamp recording techniques to measure L-VSCC activity in CA1 pyramidal neurons of partially dissociated hippocampal "zipper" slices prepared from 14-month-old wild-type mice and memory-impaired APP/PS1 double knock-in mice. Surprisingly, the functional channel density of L-VSCCs was significantly reduced in the APP/PS1 group. No differences in voltage dependency and unitary conductance of L-VSCCs were observed. The results suggest that mechanisms for Ca(2+) dysregulation can differ substantially between animal models of normal aging and models of pathological aging.     

The up-regulation of voltage-gated sodium channels subtypes coincides with an increased sodium current in hippocampal neuronal culture model

Voltage-gated sodium channels (VGSC) have been linked to inherited forms of epilepsy. The expression and biophysical properties of VGSC in the hippocampal neuronal culture model have not been clarified. In order to evaluate mechanisms of epileptogenesis that are related to VGSC, we examined the expression and function of VGSC in the hippocampal neuronal culture model in vitro and spontaneously epileptic rats (SER) in vivo. Our data showed that the peak amplitude of transient, rapidly-inactivating Na⁺ current (I_Na,T) in model neurons was significantly increased compared with control neurons, and the activation curve was shifted to the negative potentials in model neurons in whole cell recording by patch–clamp. In addition, channel activity of persistent, non-inactivating Na⁺ current (I_Na,P) was obviously increased in the hippocampal neuronal culture model as judged by single-channel patch–clamp recording. Furthermore, VGSC subtypes Na_V1.1, Na_V1.2 and Na_V1.3 were up-regulated at the protein expression level in model neurons and SER as assessed by Western blotting. Four subtypes of VGSC proteins in SER were clearly present throughout the hippocampus, including CA1, CA3 and dentate gyrus regions, and neurons expressing VGSC immunoreactivity were also detected in hippocampal neuronal culture model by immunofluorescence. These findings suggested that the up-regulation of voltage-gated sodium channels subtypes in neurons coincided with an increased sodium current in the hippocampal neuronal culture model, providing a possible explanation for the observed seizure discharge and enhanced excitability in epilepsy.     

Control of Na+ spike backpropagation by intracellular signaling in the pyramidal neuron dendrites

The integrative function of neurons depends on the somato-dendritic distribution and properties of voltage-gated ion channels. Sodium, potassium, calcium, and hyperpolarization-activated cyclic nucleotide-gated K+ (HCN) channels expressed in the dendrites can be modulated by a number of neurotransmitters and second-messenger systems. For example, activation of protein kinases leads to an increase in dendritic excitability by removing a slow inactivation of Na+ channels and decreasing the activity of transient K+ channels in the apical dendrites of hippocampal pyramidal neurons. Consequently, action potentials propagating along the dendrites can be modified significantly by a variety of neuromodulatory synaptic inputs.     

CoCl2预处理对大鼠海马神经元急性低氧后Na+、K+电流的影响

目的：观察氯化钴(COCl2)预处理对急性低氧后海马神经元电压门控性Na^ 、K^ 电流的影响。方法：原代培养大鼠海马神经元,分为COCl2预处理和非处理组,采用膜片钳全细胞记录技术,检测急性低氧后海马神经元钠电流(INa)、钾电流(Ik)的变化。结果：急性低氧后,海马神经元INa、Ik电流幅度明显降低,INa阈值右移,而经CoCl2预处理的海马神经元INa、Ik电流的降低幅度明显减轻。结论：COCl2预处理减轻急性低氧所致的INa、Ik电流变化,对神经元有明显的保护作用。     

Modulatory effects of diadenosine polyphosphates on different types of calcium channels in the rat central neurons

Modulatory effects of diadenosine tetraphosphate (Ap₄A) and diadenosine pentaphosphate (Ap₅A) on Ca²⁺ channels were studied on isolated hippocampal neurons and synaptosomes taken from the rat midbrain. In experiments on synaptosomes obtained from the whole brain, Ap₅A applied at a concentration of 100 µM increased the intrasynaptosomal calcium level (measured by means of spectrofluorometry) for 26±1.8 nM, i.e., by 24±2%. Nifedipine failed to block this effect in synaptosomes and in hippocampal neurons. The high voltage-activated Ca²⁺ currents were identified by recording from freshly isolatedCA3 neurons using a whole-cell patch-clamp technique. Current-voltage relationships were measured in control and after incubation with 5 µM Ap₅A. In the majority of tested pyramidal neurons, the latter procedure resulted in a reversible increase in the high voltage-activated currents through Ca²⁺ channels measured at a holding potential of –100 mV, but not of –40 mV. Potentiation of the currents through Ca²⁺ channels in hippocampal neurons as well as an increase in intrasynaptosomal [Ca²⁺] could be irreversibly blocked by 5 µM -conotoxin, but not by 200 nM -Aga-IVA. These data indicate that diadenosine polyphosphates enhance the activity of N-type but not of L-type or P-type Ca²⁺ channels in many central neurons of the rat brain.Neirofiziologiya/Neurophysiology, Vol. 26, No. 6, pp. 409–416, November–December, 1994.     

Calcium-activated chloride channels (CaCCs) regulate action potential and synaptic response in hippocampal neurons

Central neurons respond to synaptic inputs from other neurons by generating synaptic potentials. Once the summated synaptic potentials reach threshold for action potential firing, the signal propagates leading to transmitter release at the synapse. The calcium influx accompanying such signaling opens calcium-activated ion channels for feedback regulation. Here, we report a mechanism for modulating hippocampal neuronal signaling that involves calcium-activated chloride channels (CaCCs). We present evidence that CaCCs reside in hippocampal neurons and are in close proximity of calcium channels and NMDA receptors to shorten action potential duration, dampen excitatory synaptic potentials, impede temporal summation, and raise the threshold for action potential generation by synaptic potential. Having recently identified TMEM16A and TMEM16B as CaCCs, we further show that TMEM16B but not TMEM16A is important for hippocampal CaCC, laying the groundwork for deciphering the dynamic CaCC modulation of neuronal signaling in neurons important for learning and memory.