Balb/C乳鼠小脑Purkinje细胞原代培养方法

目的建立一种简单、有效的Balb/C乳鼠小脑Purkinje细胞原代培养方法,并初步研究其细胞电生理特性。方法钝性分离生后24h内的乳鼠小脑,采用低浓度胰蛋白酶加机械吹打法获得单细胞悬液,然后用含10%胎牛血清的DMEM/F12培养,24h候更换为1%N2+1%T3+1%谷氨酰胺的DMEM/F12维持培养,3-5d天后进行细胞免疫荧光染色,7-9d天后用全细胞膜片钳单通道法记录钙电流。结果该方法培养的神经元形态典型,细胞学鉴定阳性率高(70%),并能记录到钙通道电流。结论该方法取材容易,操作简便,且培养出的原代Purkinje细胞生长状态较好,神经元的活性较高,可用于电生理研究。     

利用膜片钳技术标记脑片神经元的方法

目的: 介绍一种利用膜片钳技术标记脑片神经元形态的方法。方法: 利用振动切片机切好实验目标部位的脑片,用含有Neurobiotin^TM Tracer的电极内液灌注玻璃微电极,并进行全细胞膜片钳记录;实验结束后将脑片先用4％多聚甲醛固定、漂洗,再用含有Streptavidin-Texas Red和Triton X-100的PB染色,2 h后即可用荧光显微镜观察着色的神经元。结果: 将细胞膜电压钳制在-70 mV,阶跃刺激后神经元表现为逐渐增大的膜电流。电流钳模式记录时,阶跃刺激使神经元去极化,达到阈电位后爆发动作电位。荧光显微镜下可看到胞体和主要突起清晰完整的神经元形态。结论: 本方法适用于在膜片钳实验后观察所记录的神经元的形态特征,操作方便,图像直观清晰。     

银杏内酯B 诱导大鼠骨髓间充质细胞分化为神经元样细胞的电生理研究

目的:研究银杏内酯B(GB)诱导大鼠骨髓间充质细胞(MSCs)分化为神经元样细胞的电生理特性。方法:应用膜片钳技术,采用全细胞记录方式,对由GB诱导的大鼠MSCs进行诱导前后的电生理功能测定。结果:分化后的神经元样细胞较诱导前细胞的膜特性有了显著改变(P<0.05)。结论:大鼠MSCs经过GB诱导能够向功能性神经元方向分化。     

猫体感皮质内脏伤害感受神经元的膜电生理特性

应用细胞内电位记录技术,研究猫ＳＩ中内脏大神经皮质代表区的８５１个神经元膜电生理特性,用胞内注入极化电流的方法,测量和计算出神经元的膜电学参数,并进行内脏痛伤害感受及非相关神经元的电学特性比较,发现二者在膜电阻、时间常数、膜电容、静息电位、细胞活跃程度及神经元深度分布等方面存在差别。注电流引发的频率－电流、动作电位幅值－电流、及Ｉ－Ｖ曲线也有差异,结果提示ＳＩ区内脏伤害感受及非相关神经元在形态及膜结构上可能有不同之处,为痛觉特异学说提供资料。部分神经元用神经生物素进行细胞内电泳标记以显示功能细胞所在层次及形态,从单一皮层感受神经元的反应及形态特点探讨了内脏痛的感受特性。     

Neuritin诱导大鼠骨髓基质细胞分化为神经元样细胞的电生理研究

目的:探讨神经营养因子Neuritin诱导大鼠骨髓基质细胞分化为神经元样细胞的电生理特性.方法:应用膜片钳技术,采用全细胞记录方式,对由Neuritin诱导的大鼠骨髓基质细胞进行诱导前后的电生理功能测定.结果:分化后的神经元样细胞较诱导前细胞的膜特性[静息膜电位(RMP)膜电容(Cm)串联电阻值(RS)]有了显著改变(p＜0.01).分化后细胞记录到K+电流,包括两种成分:外向延迟整流K+电流和内向整流K+电流.结论:骨髓基质细胞经过Neuritin诱导能够向功能性神经元方向分化.     

小鼠胚胎心肌细胞的分离及电生理特性

本文旨在探索小鼠胚胎心肌细胞的分离方法并观察其电生理特性。应用胶原酶B消化法获得不同时期单个小鼠胚胎心肌细胞;利用全细胞膜片钳技术,记录胚胎心肌细胞的超极化激活的非选择性内向阳离子电流(If)和L-钙电流(ICa-L),并用电流钳记录其自发性动作电位。胚胎心肌细胞通过相差显微镜依据其形态和自发性收缩进行鉴定。本法分离所获得的胚胎心肌细胞容易进行全细胞膜片钳记录,可用于记录If,ICa-L．电流和自发性动作电位,己证实胚胎心肌细胞If和Ica-L的电生理特性与成年起搏细胞或心肌细胞相似。本实验建立的分离方法简单、稳定、有效、可靠,最早可获得8．5d的胚胎心肌细胞。胚胎心肌细胞的电生理记录为探索胚胎心肌细胞的电生理特性提供了一个可用的模型,并可能为某些心脏疾病产生的机制提供实验依据。     

大鼠,豚鼠心肌细胞的简单,快速分离

本文介绍了一种简单、快速分离成年大鼠、豚鼠心肌细胞的方法。所分得的细胞形态结构完整,具有良好的钙耐受性,膜片钳上易于形成高阻抗接封,因而适于作各种电生理记录。     

可视膜片钳法记录初级传入纤维介导的脊髓背角神经元突触后电流

本文描述了用明胶半包埋法制备带背根脊髓薄片的实验步骤,和在脊髓背角记录由初级传入纤维介导的突触后电流的可视膜片钳法。手术制备一段带背根的脊髓标本,并用20％的明胶包埋在琼脂块上,再用振动切片机切片获得带背根的脊髓薄片。通过红外线可视的引导,在脊髓背角神经元上建立全细胞封接模式。在钳制电压为-70mV条件下,记录自发的和背根刺激引起的兴奋性突触后电流。以传入纤维的传导速度与刺激阈值为指标,可以区分A样纤维与C样纤维兴奋性突触后电流。在钳制电压为0mV条件下,记录自发的和背根刺激引起的抑制性突触后电流。用5μmol／L的士宁或20μmol／L的荷包牡丹碱分离出γ-氨基丁酸能或甘氨酸能的抑制性突触后电流。用可视膜片钳方法可以准确测量脊髓背角神经元的突触后电流,从而研究初级传入突触的传递过程。更重要的是,在红外线可视观察的帮助下,建立膜片钳封接的成功率显著提高,同时也使记录研究脊髓背角深层神经元变得更加容易。本研究为探索初级传入突触传递过程提供了一个有效的方法。     

全细胞膜片钳研究离体小鼠下丘亚核神经元电生理特性

下丘(inferior colliculus,IC)是听觉通路的重要中继站。本实验采用红外可视脑片全细胞膜片钳技术对昆明小鼠IC神经元的电生理特性进行研究。共记录获得88个神经元,其中背侧核(dorsal cortex of IC,ICd)神经元21个,中央核(central nucleus of IC,ICc)神经元43个,外侧核(external cortex of IC,ICx)神经元24个。根据神经元对注入正电流的发放模式,将其分为起始型(6.8%,n=6),适应型(39.8%,n=35)和持续型(53.4%,n=47)三种类型。约一半的神经元(49/88)具有超极化电流激活的内向电流(hyperpolarization-activated inward current,Ih)。ICd和ICc神经元主要表现为持续型(61.9%和67.4%),ICx主要为适应型(75%)。比较发现,ICd、ICc及ICx神经元之间在动作电位发放阈值和膜时间常数等参数上存在显著差异。以上结果表明IC亚核神经元的电生理特性存在差异,这些差异可能与神经元的类型、它们所接受的投射以及在声信息处理中作用不同相关。     

猫扣带回前部内脏伤害感受神经元的膜电学特性

目的：研究猫扣带回前部内脏大神经刺激相关神经元的膜电生理特性,以便从神经元水平进一步了解大脑皮质内脏伤害感受的特性及机制,为痛觉理论“特异性学说”提供新的实验依据。方法：应用在体玻璃微电极细胞内电位记录技术及细胞内注入极化电流的方法,测量和计算神经元的膜电学参数。结果：将20只猫扣带回前部176个内脏大神经刺激相关神经元,分为内脏伤害(148个)和非伤害(28个)感受神经元。发现它们在膜电阻、时间常数、膜电容及I—V曲线等方面存在差异。注入去极化电流引发的放电幅值及频率也存在差异。结论：扣带回前部内脏伤害与非伤害感受神经元可能在细胞膜结构、细胞大小等形态学方面存有差别。     

Slice culture of the olfactory bulb of Xenopus laevis tadpoles

We report on the development of a slice culture of amphibian brain tissue. In particular, we cultured slices from Xenopus laevis tadpoles that contain the olfactory mucosae, the olfactory nerves, the olfactory bulb and the telencephalon. During 6 days in roller tubes the slices flattened, starting from 250 microm and decreasing to approximately 40 microm, corresponding to about three cell layers. Dendritic processes could be followed over distances as long as 200 microm. Neurons in the cultured slice could be recorded using the patch clamp technique and simultaneously imaged using an inverted laser scanning microscope. We characterized the main neuron types of the olfactory bulb, i.e. mitral cells and granule cells, by correlating their typical morphological features in the acute slice with the electrophysiological properties in both the acute slice and slice culture. This correlation allowed unambiguous identification of mitral cells and granule cells in the slice culture.     

Blind patch clamp recordings in embryonic and adult mammalian brain slices

To obtain electrophysiological recordings in brain slices, sophisticated and expensive pieces of equipment can be used. However, costly microscope equipment with infrared differential interference contrast optics is not always necessary or even desirable. For instance, obtaining a randomized unbiased sample in a given preparation would be better accomplished if cells were not directly visualized before recording. In addition, some preparations require thick slices, and direct visualization is not possible. Here we describe a protocol for the 'blind patch clamp method' that we developed several years ago to perform electrophysiological recordings in mammalian brain slices using a standard patch clamp amplifier, dissecting microscope and recording chamber. Overall, it takes approximately 3-4 h to set up this procedure.     

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。     

A novel silicon patch‐clamp chip permits high‐fidelity recording of ion channel activity from functionally defined neurons

We report on a simple and high‐yield manufacturing process for silicon planar patch‐clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high‐quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high‐impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high‐fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole‐cell current recordings obtained from a voltage‐clamp stimulation protocol, and in current‐clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch‐clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high‐information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity. Biotechnol. Bioeng. 2010;107:593–600. © 2010 Wiley Periodicals, Inc.     

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.     

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents. The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.Download video file.^{(217M, mp4)}     

Electrophysiological Properties of Motor Neurons in a Mouse Model of Severe Spinal Muscular Atrophy: In Vitro versus In Vivo Development

We examined the electrophysiological activity of motor neurons from the mouse model of severe spinal muscular atrophy (SMA) using two different methods: whole cell patch clamp of neurons cultured from day 13 embryos; and multi-electrode recording of ventral horns in spinal cord slices from pups on post-natal days 5 and 6. We used the MED64 multi-electrode array to record electrophysiological activity from motor neurons in slices from the lumbar spinal cord of SMA pups and their unaffected littermates. Recording simultaneously from up to 32 sites across the ventral horn, we observed a significant decrease in the number of active neurons in 5–6 day-old SMA pups compared to littermates. Ventral horn activity in control pups is significantly activated by serotonin and depressed by GABA, while these agents had much less effect on SMA slices. In contrast to the large differences observed in spinal cord, neurons cultured from SMA embryos for up to 21 days showed no significant differences in electrophysiological activity compared to littermates. No differences were observed in membrane potential, frequency of spiking and synaptic activity in cells from SMA embryos compared to controls. In addition, we observed no difference in cell survival between cells from SMA embryos and their unaffected littermates. Our results represent the first report on the electrophysiology of SMN-deficient motor neurons, and suggest that motor neuron development in vitro follows a different path than in vivo development, a path in which loss of SMN expression has little effect on motor neuron function and survival.     

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as α4 L9′A mice ¹ and α6 L9′S mice ², allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.     

Electrophysiological recording from brain slices.

This article discusses several of the currently used methodologies for recording from brain slices. Aspects of slice preparation as well as appropriate uses for the various slice models (i.e., thin or thick slices) are considered. The merits of extracellular and intracellular electrophysiological recording and their uses are discussed. In addition, mechanisms of neuronal circuit activation and stimulation are presented.