这项工作是支持由美国国立卫生研究院资助EY17606（APS）。

1。准备电极<ol><li>准备记录和参比电极浸泡在1M氯化钠，并推动它们之间的15V〜20秒1 Hz的正弦波</li><li>请使用微量拉马（萨特仪器的P - 97）的记录电极。对于补丁移液器使用硼硅玻璃1.2毫米外径和内径0.69毫米（萨特仪器，CAT＃B120 - 69 - 10）的一个。通过电极尖端测量串联电阻。根据靶细胞的大小选择枪头直径;胞体〜使用直径在20微米〜5MΩ胞体〜在使用直径5微米〜12MΩ。 </li><li>准备地面/参比电极。用注射器填充1毫米氯化钠一小部分聚乙烯管（Intramedic PE205），其中包括3.5％的琼脂（Sigma公司; CAT＃A7002）在1M氯化钠。这超过了Ag / AgCl参比电极放置琼脂桥。 </li></ol> 2。准备解决方案<ol><li>外部液，含有碳酸氢盐（1升）的艾姆斯媒体:1艾姆斯瓶"中等（Sigma公司; CAT＃A1420），1.9克碳酸氢钠 3，7毫升的青霉素，链霉素（Sigma公司; CAT＃P0781），以防止细菌增长。调整渗透压〜280 mOsm。 </li><li>切片的解决方案，艾姆斯介质pH缓冲HEPES（1升）:1瓶Ames的培养基，0.887克的氯化钠，2.38克HEPES，7毫升的青霉素，链霉素。使用1M NaOH和/或1M盐酸调节pH值至7.4〜。调整渗透压〜280 mOsm。 </li><li> 10 -天门冬氨酸钾（K - ASP）移液器内部的解决方案（毫米）:125的K -天门冬氨酸，氯化钾，10 HEPES，5 NMG - HEDTA氯化钙2，1的ATP GTP镁毫克，0.2，0.5， pH值调整到〜7.3 NMG - OH和渗透压调整〜280 mOsm。 </li><li>稳定低胶凝温度琼脂糖包含视网膜（按重量的5％），琼脂7.5克（Sigma公司;猫＃A7002）逆止琼脂融化在150毫升DH 2 O </li></ol> 3。实验日<ol><li>解冻〜1.5毫升的K - ASP内部解决方案，您所选择的荧光团从冰箱。稀释至适当浓度，应凭经验确定的荧光。 </li><li>倒艾姆斯含有碳酸氢钠3（约200毫升）的避光贮存容器，然后将水浴（30-32℃）的地方"媒体和平衡5％的CO 2 / 95％O 2气体</li><li>填充〜110毫升含有HEPES，放置在冰艾姆斯媒体的瓶子和100％O 2的平衡。 </li><li>广场余下的Ames的媒体，包含一个法拉第笼顶部的加热水库碳酸氢钠3，和5％的CO 2 / 95％O 2气体的平衡。封口膜可用于捕获的气体在上述解决方案的空间。 </li><li>准备加入0.75克的低胶凝温度琼脂糖（3％）; HEPES和热量的解决方案〜115 ° C至融化琼脂糖（Sigma公司猫＃A0701）25毫升艾姆斯媒体。搅拌，直至溶液清晰​​，无气泡，并在存储水浴（42℃）。 </li><li>填充的K - ASP的内部解决方案的一个记录的吸液管和检查尖阻力。 </li></ol> 4。开始实验<ol><li>安乐死鼠标，迅速取出眼用弯剪刀。应被用于所有的程序，以防止视觉色素漂白红外护目镜。 </li><li>放在滤纸上，以防止他们从滚动，使用超薄双面刀片眼睛，使角膜缝钳。 </li><li>尽快刺破眼球，转移到与Ames的媒体充满了菜，他们的眼睛。 </li><li>使用角膜为切入点的缝隙，小剪刀剪去其余部分角膜，晶状体和玻璃体后可以去掉。小心视网膜仍然附着眼罩，并存储在黑暗眼罩在32 °艾姆斯"媒体与5％的CO 2 / 95％O 2的平衡彗星。 </li></ol> 5。切片<ol><li> Hemisect一个眼罩，和视网膜脱离的视网膜色素上皮。取出的视网膜边缘，让组织平躺。 </li><li>使用乳胶泡一个小玻璃巴斯德吸管，以填补35毫米的Petri盘与融化的琼脂，并通过琼脂拖过的镊子，以检验其一致性。 </li><li>当你可以开始看到琼脂凝固: <ol class="lalpha"><li>视网膜传送至琼脂顶部。 </li><li>使用一个Kimwipe扭曲，以吸收周围的视网膜多余的溶液。 </li><li>广场2-3滴琼脂直接在视网膜上，并迅速放在一个塑料圆筒视网膜形成了它周围的墙壁。然后往下淌的塑料圆筒两侧额外融化的琼脂，而围绕琼脂内的视网膜。 </li><li>转让的Petri菜吨Ø冰水浴冷却。 </li></ol></li><li> 〜45秒后从冰水浴中删除的组织和使用柱塞挤出琼脂块的塑料圆筒，从视网膜最远的一侧推。 </li><li>使用一个双面刀片切出一个小数据块包含视网膜琼脂和到位，对试样的光盘上的琼脂逆止强力胶块。 </li><li>标本光盘传输振动切片机托盘，倒入托盘允许与视网膜块完全淹没冰冷艾姆斯HEPES。 </li><li>视网膜片〜200微米的厚度，可以切割​​最大的刀片振动速度和向前的刀片速度设定等，它需要4至5秒，穿过视网膜上的每个切片。 </li><li>使用11号手术刀刀片一次删除每个可用的片之一</li><li>含有视网膜的好片放入录制商会和加权对着干的尼龙线片锚。尼龙线按住琼脂周围的视网膜，使视网膜录音通畅。 </li><li>录音室转移到直立显微镜阶段，关闭窗帘，打开红外光源，红外感光摄像头，以查看片。 </li></ol> 6。记录<ol><li>一旦一个良好的视网膜片（完整的感光细胞和适当的切削角度）确定开始灌注视网膜率大于5 mL / min的加热艾姆斯媒体被加热到35 37 ° C和5％的CO 2平衡/ 95％O 2。 </li><li>一些表面的损害可能是由于切割过程明显。这通常是一层薄薄的细胞，可以使用一个破碎的一角吸管去除，并轻轻的吸吮了死细胞机构。这通常会发现下面的活细胞。确定一个健康的细胞体，其位置位于利益阶层。 </li><li>填写与KAsp内部解决方案的一个微量，并申请通过吸管尖正面（向外）的压力，降低进录音室吸管尖，使用显微细胞水平。 </li><li>移动枪头，这样它的酒窝细胞膜，并适用于温柔负（向内）的压力，使高抗密封。全细胞模式，可以实现应用负压电极进入细胞打破。 </li><li>刺激视网膜组织使用的LED就可以唤起反应轻。 </li><li>照片，录音唤起从吸管内部解决的荧光团的荧光的细胞。 </li></ol> 7。代表性的成果<img src="/files/ftp_upload/2107/2107fig1.jpg" alt="图1" /> 图1在这里，一个暗适应视网膜神经元增加强度的光闪烁光诱发的潜力。 <img src="/files/ftp_upload/2107/2107fig2.jpg" alt="图2" /> 图2:从视网膜切片的细胞已被填充的荧光，路西法黄河荧光图像。诱发荧光显示的细胞补丁录音形态。

<ol>
	<li>Weblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+along+and+between+rods+in+the+tiger+salamander+retina.">Transmission along and between rods in the tiger salamander retina.</a> J Physiol. 280, 449-470 (1978).</li><li>Wu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+connections+among+retina+neurons+in+living+slices.">Synaptic connections among retina neurons in living slices.</a> J Neurosci Methods. 20, 139-149 (1987).</li><li>Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+contrast+adaptation+in+salamander+bipolar+cell.">Temporal contrast adaptation in salamander bipolar cell.</a> J Neurosci. 21, 9445-9454 (2001).</li><li>Armstrong-Gold, C., &amp;amp, R. i. e. k. e., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bandpass+filtering+at+the+rod+to+second-order+cell+synapse+in+salamander+(Ambystoma+tigrinum)+retina.">Bandpass filtering at the rod to second-order cell synapse in salamander (Ambystoma tigrinum) retina.</a> J Neurosci. 23, 3796-3806 (2003).</li><li>Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+plan+of+the+retina.">The fundamental plan of the retina.</a> Nat Neurosci. 4, 877-886 (2001).</li><li>Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+the+mammalian+retina.">Parallel processing in the mammalian retina.</a> Nat Rev Neurosci. 5, 747-757 (2004).</li></ol>

片脊椎动物视网膜的录音已经几十年 1,2，已经相当成功，提供了一个如何视网膜电路编码入射光更深入的了解。振动切片，而不是直接用刀片切割的优点是视网膜切片，在表面产生较少的损害。具有吸吸管，这是容易消除这种损害健康细胞和目标，只是在表面之下，他们的连接保留在视网膜的电路，其中大部分的细胞类型已经确定了5,6，轻，显示强劲的反应。因此，从暗适应视?...

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<td>Micropipette Puller</td>
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<td>Sutter Instruments</td>
<td>P-97</td>
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<td>Borosilicate glass</td>
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<td>Sutter Instruments</td>
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<td>Intramedic</td>
<td>PE205</td>
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<td>Agar</td>
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<td>Sigma</td>
<td>A7002</td>
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<td>Low gelling temp agarose</td>
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patch clamp recordings from mouse retinal neurons in a dark-adapted slice preparation

启动时，在我们的视网膜感光细胞的视色素吸收光能量的光子，并启动一个封闭的环核苷酸门控通道在细胞膜，导致细胞内事件级联我们的视觉体验。膜电位的变化，导致神经递质的释放杆和锥突触终端数量的减少反过来。为了测量在感光膜电位光诱发的变化如何导致在视网膜上下游活动，科学家们从几十年 1,2的视网膜切片准备的电生理记录。在过去，这些片被切断与视网膜组织，是连接到滤纸上的刀片手动，近年来已开发的另一种方法的切片，使视网膜组织中嵌入胶凝温度琼脂低和在阴凉的解决方案与切片振动切片3,4。这种准备产生表面损伤少和非常强大的光诱发反应的视网膜切片。在这里，我们的文件，此过程中如何可以根据红外光，以避免视觉色素漂白。

Watch this Scientific Journal Video about Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation at JoVE.com

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

在这里，我们描述了产生暗适应对小鼠视网膜电生理记录片的过程。

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

patch-clamp-recordings-from-mouse-retinal-neurons-dark-adapted-slice

Research

JoVE Journal

Neuroscience

14.4K 次观看.  University of Southern California. 在这里，我们描述了产生暗适应对小鼠视网膜电生理记录片的过程。

视频: Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

科研

神经科学

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

膜片钳记录在内耳毛细胞的分离斑马鱼

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</subATP酶微囊的制备和表演膜片钳记录的Submitochondrial泡膜技术

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

同时光感受器和二阶神经元在两栖类动物的视网膜切片制备全细胞记录

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

隔离的感觉神经元中<em&gt;海兔丫</em谷氨酸能电流膜片钳记录

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

从胚胎斑马鱼的毛特纳细胞膜片钳记录

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...