Name: recording light-evoked postsynaptic responses in neurons in dark-adapted, mouse retinal slice preparations using patch clamp techniques
Uploaded: 2015-02-11T14:00:00.000+00:00
Duration: 10 min 30 s
Description: Watch this Scientific Journal Video about Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques at JoVE.com

This work was supported by NIH R01 EY020533, WSU Startup Fund, and RPB grants.

伦理学声明:涉及动物对象的程序批准的机构动物护理和使用委员会（IACUC）韦恩州立大学。 1.准备实验解决方案<ol><li>实际实验之前准备解剖溶液1天到1周。使用的，因为在较低温度下16其较强的缓冲能力视网膜解剖一个HEPES缓冲溶液。混合所有化学物质如下（单位mm）:115氯化钠，氯化钾2.5，2.5 氯化钙 ，氯化镁1.0 2，10 HEPES，28葡萄糖。调节pH至7.4，用NaOH。存放在冰箱中长达1周的解决方案。 </li><li>记录溶液埃姆斯是&#39;中，它是一个人工脑脊髓液（ACSF）设计用于视网膜制剂17。由该实验的当天，称出在管埃姆斯&#39;粉末（4.4克500毫升溶液）。此外，掂量出碳酸氢钠 （0.95克500毫升解决方案），并与混合MES"粉体。 </li><li>由实验当天制备用于膜片钳记录细胞内吸移管溶液。混合所有的化学品如下（以mM计）:111葡萄糖酸钾，1.0氯化钙2，10 HEPES，1.1 EGTA，10的NaCl，1.0的MgCl 2，5 ATP的Mg和1.0 GTP钠。调节pH至7.2，用KOH。过滤与常规注射器过滤的移液管溶液。使〜500微升等分试样，并将其存储在冷冻或深冷冻机。 </li></ol> 2.准备用于实验的当天<ol><li>晚上在实验前，将鼠标在笼子里（C57BL / 6J或相似的背景，4 - 8周龄，雄）在一个黑暗的空间O / N暗适应。 </li><li>解剖准备: <ol><li>填解剖溶液 - 在玻璃烧杯中（100 200毫升）中，将其放置在冰上，并用气泡氧气至少10分钟。 </li><li>启动暗箱视网膜制备存储的氧合。 </li><li>准备的塑料盖玻片重新油脂轨tinal片的准备，并把他们每个人在35毫米的塑料盘。 </li><li>安装一个新的刀片来菜刀（组织切片机）。 </li><li>切断滤膜成两半。这是对视网膜切片。 </li><li>对齐解剖显微镜附近的解剖工具和移液管。工作区必须有良好的组织，使每个工具可以在黑暗中很容易地访问。 </li></ol></li><li>膜片钳录音准备工作: <ol><li>溶解埃姆斯&#39;粉末和的NaHCO 3在蒸馏水（艾姆斯4.4克和的NaHCO 30.95克/500毫升DDH 2 O 的 ）。气泡埃姆斯&#39;中混合氧气（95％O 2和5％CO 2）的至少30分钟，同时加热到记录温度水平（〜35℃）。将pH调节至7.4用NaHCO 3，同时仍然鼓泡。 </li><li>解冻解剖过程中的内部移液器解决方案。它解冻后，细胞内染色增加0.01％磺酰罗丹明B。 </li></ol></li></ol> 3.视网膜解剖<ol><li>在黑暗的房间里，用安乐死二氧化碳和双侧气胸鼠标。经过2 - 使用二氧化碳3分钟，鼠标会失去意识。 </li><li>当鼠标不再响应尾巴捏，迅速剜除眼睛和它们放置在一个塑料盘感冒解剖的解决方案。执行双侧气胸，以确保动物的死亡。快速存储眼睛中在黑暗中的菜。使用红外线观众进行的程序在黑暗中。 注:可替代地，异氟醚，断头，或颈脱位可用于安乐死。安乐死的方法应遵循美国国家卫生研究院（NIH）的和/或机构动物护理和使用委员会的指导方针。 </li><li>在黑暗的房间，调整解剖显微镜和红外照明。在显微镜下，将其从培养皿转眼成10cm的塑料培养皿瓦特第i个冷却解剖的解决方案。连续地通过管道气泡氧放置在塑料培养皿的底部。调节流量，所以它不是太低，而是确保它没有高到足以扰乱解剖。 </li><li>角膜和晶状体摘除: <ol><li>就用微手术刀对角膜的顶部的切口。保持视神经在眼球的另一端防止眼睛移动。延长使用一对手术剪的剪。切出的角膜周围有一些巩膜。 </li><li>抢镜头用细镊子慢慢拉镜头了。如果玻璃体太粘除去透镜，切玻璃体用剪刀。 </li><li>一旦眼杯制成，轻轻倒入冷，含氧解剖溶液进入眼罩用小移液管。 注:眼球快速放气后的切口是在角膜。它维持眼球的形状，因为这有利于在变形眼是非常重要的cedure会损坏视网膜而引起视网膜脱离。 </li></ol></li><li>确定视网膜的背，腹两面: 注意:由于绿色和紫外（UV）不均匀分布的锥体18中 ，识别特定的视网膜区域为光响应录制重要。 <ol><li>识别线横跨视网膜眼罩通过视神经头附近去。此线通过大多横跨视神经腹侧;侧包括视神经是背侧，而另一种是腹侧19。看到这一具有里程碑意义显然在红外观众。 </li><li>根据实验的目的，使得在不必要侧的切口。这将是从眼罩分离视网膜之后是有用的。 </li></ol></li><li>玻璃体切除: <ol><li>任选地，孵育眼杯与透明质酸酶（0.5毫克/毫升）15分钟。 </li><li>删除与特细镊子玻璃体。玻璃体主要是附着在底部和眼杯的外边缘。轻轻拉出，不要触摸或戳视网膜。由于玻璃体可能会被紧紧地贴在眼罩，轻轻取出，避免从眼杯视网膜脱离。继续拉，直到没有张力感觉。 注意:此步骤是为解剖小鼠视网膜尤其重要;然而，这不是对其他物种的视网膜，如大鼠或蝾螈的问题。 </li></ol></li><li>隔离从眼杯视网膜。抓住巩膜并轻轻剥离使用镊子的背侧视网膜。 注:整个安装的视网膜制剂可以从分离的视网膜进行。使三至四个切口处的边缘和扁平视网膜，或切割视网膜成几片。 </li><li>修剪视网膜并丢弃不必要的视网膜的一半（ 步骤3.5）。在相同的塑料盘，切剩下的视网膜成两块。对于每一块的视网膜，切断边缘作视网膜平板，并修剪折叠边缘。 </li><li>转移视网膜板坯上使用大移液管（〜2ml）中的玻璃板。吸起来用小吸管的过量溶液，可使用一片滤纸弄平板坯，然后迅速将一块滤膜上的组织的顶部。 <ol><li>使用小移液管，将一滴解剖上的过滤膜溶液。等待〜15秒，直到溶液散布在膜中。在这段时间内，视网膜组织会粘到过滤膜上。 </li><li>倒在和周围的过滤器越冷解剖的解决方案。与过滤膜的视网膜组织将漂浮。抢用钳子过滤膜，并将其放置在切片室。倾解剖溶液在组织中，并存储该制剂在黑暗中。 注意:这里介绍的方法是至关重要的。以确保它是快速执行这些步骤重要的是视网膜组织附着在过滤器mem的期Brane，不干燥。 </li></ol></li><li>用一把菜刀，切视网膜组织切片。大约200微米厚度的膜片钳研究可行的。 </li><li>将塑料盖玻片，在切片室油脂轨。传输视网膜切片上的油脂轨顶部的塑料盖玻片。旋转片90°，以使横截面是可见的。按过滤膜片向下到盖玻片，然后，覆盖滤纸的侧面与油脂。 <ol><li>后片被固定在塑料盖玻片，抢盖玻片并将其转移到35毫米的塑料培养皿中。倒入冷解剖解决方案到视网膜准备和淹没的准备。储存在黑暗中的每个菜，应连续地氧化。 注:整个过程需要仔细地进行，以使滤膜不​​变形。否则，视网膜切片容易分离的过滤膜。 </li></ol></li></ol><ol><li>录制的准备: <ol><li>总理灌注管与艾姆斯"媒介。让所有的泡沫通过填充管时。 </li><li>使膜片钳记录移液器与车夫。以填充用吸管溶液，浸在吸移管的背侧吸移管，持续1分钟，直到枪头回填。接着，使用微量移液器填料填充吸管〜三分之一。存放在潮湿的吸管盒每个吸管。 </li><li>打开设备的膜片钳记录的;包括计算机，放大器，CCD摄像头，和显微镜。 </li></ol></li><li>关闭房间的灯。将使用红外线观众视网膜切片准备到显微镜阶段室。它被固定后，开始连续灌注。设置灌注温度在33至37℃。 </li><li>查看片表面与CCD相机。专注于目标位置，目标CELL类型驻留。选择膜片钳记录一个看起来健康的细胞（ 即，它应具有光滑的表面寻找和良好的细胞形态）。 </li><li>将在吸管持有人一个记录吸管。推进吸管切片准备。当它接近所述片制剂（〜2毫米以上），以中，找到与显微镜的吸移管的尖端。一旦枪头在显微镜下是可见的，移动的前端向下朝向目标小区。 </li><li>设置放大器。调整吸管在0毫伏/ 0 PA（零），并开始一个连续的电脉冲〜5的MV〜10赫兹。检查吸液管电阻;理想的电阻为5至10兆欧大多数视网膜神经细胞。 </li><li>开始从尖端吹出。使用一个吹口，或使用注射器，施加正压时的吸管蘸入浴溶液中。连续地吹出的内部溶液，直到前端在靶细胞的表面上。 <ol><li>当正压力使得在th小凹坑ê细胞表面，推进尖端轻微并停止吹出来的。检查吸管阻力，这应增加。如果它被连续地增加，离开它并监视电阻，直到达到&gt; 1GΩ（千兆欧密封）。如果电阻不会自发增加，轻轻施加负压，直到它变成一个千兆欧密封。 </li></ol></li><li>在千兆欧密封实现后，改变保持电位-70毫伏。然后，间歇地施加负压破裂枪头内的膜。当进行全细胞构造中，吸液管电阻可以是500兆欧和1GΩ以及电容电流看出之间。有时自发突触后电流可以观察到。 </li><li>记录从-80到+40毫伏的IV关系。不同类型的电压门控通道的激活取决于细胞类型。 </li><li>记录光诱发突触电流或电压。 </li></ol>

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The authors have nothing to disclose.

Good recordings can only be achieved with good retinal preparations and well-designed patch clamp setups. Although all the steps described above are important, the discussion highlights some critical steps both for the dissection and recordings. 
For dissection, two things are especially important: cooling and oxygenation. After enucleating the eye, quickly remove the front part of the eye in a dissecting chamber with oxygen-bubbled, cooled dissecting solution, and pour cold solution into the ...

The retina is one of the unique parts of the nervous system. As an accessible part of the brain, its synaptic architecture has been well characterized. In addition, the functions of this neural network can be examined with a physiological stimulus: light. If the retinal tissue is isolated in a dark room with appropriate procedures, neurons in the tissue will respond to light. This preparation has been used to study visual signal processing and elucidate various synaptic mechanisms and neural network functions, as well as disease mechanisms. 
Light responses in retinal neurons have been recorded for decades. Early studies used sharp electrodes to make intracellular recordings from mudpuppy retinal neurons1. In the 1980s, the patch clamp technique was invented2, and soon became a popular method among vision researchers3,4. Single cell recordings from lower vertebrates, including mudpuppy and fish retinal neurons, were popular methods that contributed to the elucidation of visual signal processing mechanisms5,6. 
After genetic mutation techniques were developed, the mouse retina became a more popular model for vision researchers7-9. The mammalian retina is more attractive than that of lower vertebrates because it is evolutionarily closer to the human retina, and there is an opportunity to use disease models. However, mouse retinal cells are small and fragile10, and making retinal preparations and conducting patch clamp recordings in a dark room is challenging. As technology has improved, diverse approaches have become available to study visual signaling mechanisms such as imaging studies11 and the electroretinogram (ERG)12. Nevertheless, single cell recording with the patch clamp method is still important because it is highly temporally and spatially sensitive compared to other methods. Therefore, we have continuously conducted patch clamp recordings and improved our methods to investigate visual signal processing in mouse retinal slice preparations13-15.
In this video tutorial, the protocols are presented with important tips. Good recordings can only be achieved with good preparation. Practicing animal dissection and building a sturdy patch clamp rig will enable most researchers to achieve successful recordings.

<table><tbody><tr><td>Mice (28-60 days old, male)</td><td>Jackson laboratory</td><td>C57BL/6J strain</td><td></td></tr><tr><td>Ames&#039; medium powder</td><td>Sigma</td><td>A1420</td><td>excellent</td></tr><tr><td>Stereo microscope</td><td>Nikon</td><td>SMZ745</td><td>excellent</td></tr><tr><td>Dissecting tool: forceps</td><td>Dumont</td><td>#4, #5, #55</td><td>excellent</td></tr><tr><td>Dissecting tool: scissors</td><td>Roboz</td><td>RS-5605</td><td>excellent</td></tr><tr><td>Dissecting tool: surgery knife</td><td>Surgistar</td><td>7514</td><td>excellent</td></tr><tr><td>Razor blade (for chopper)</td><td>EMS</td><td>71970</td><td>excellent</td></tr><tr><td>Chopper</td><td>handmade</td><td></td><td></td></tr><tr><td>Infrared viewer</td><td>Night Owl Optics</td><td>NOBG1</td><td>It shows bright view. Focusing small objects is an issue.</td></tr><tr><td>Puller</td><td>Sutter</td><td>P-1000</td><td>excellent; makes consistent size pipettes.</td></tr><tr><td>Dark box</td><td>Pelican</td><td>dark box</td><td>excellent</td></tr><tr><td>Patch clamp system</td><td>Scientifica</td><td>slice scope 2000</td><td>Excellent setup. Most key components are included in one package. Micromanipulators are excellent.</td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>multiclamp 700B</td><td>Excellent and easy control.</td></tr><tr><td>Acquiring software</td><td>Molecular Devices</td><td>pClamp software</td><td>Excellent and easy control.</td></tr><tr><td>Light source (LED)</td><td>Cool LED</td><td>pE-2 4 channel system</td><td>Excellent</td></tr><tr><td>CCD camera</td><td>Q-imaging</td><td>Retiga 2000</td><td>Excellent</td></tr><tr><td>Faraday cage</td><td>handmade</td><td></td><td></td></tr></tbody></table>

recording light-evoked postsynaptic responses in neurons in dark-adapted, mouse retinal slice preparations using patch clamp techniques

视网膜是通往视觉系统。要了解视觉信号处理机制，我们研究视网膜神经网络的功能。网络中的视网膜神经元包括许多亚型。超过10种亚型双极细胞，神经节细胞，和无长突细胞已经鉴定了形态学研究。视网膜神经元的多个亚型被认为编码可视信令的显着特征，例如运动和颜色，并形成多个神经通路。然而，在视频信号处理的每个神经元的功能角色尚未完全了解。膜片钳的方法是有用的，解决这一根本问题。在这里，一个协议来记录使用膜片钳记录在暗适应条件鼠标视网膜神经元光诱发突触反应提供。鼠标眼睛的暗适应O / N，和视网膜切片准备使用红外照明和解剖观众在黑暗的房间。红外灯不激活鼠标光感受器，从而保留了它们的光响应。膜片钳是用来记录在视网膜神经元的光诱发反应。在录音表征神经元形态亚型荧光染料注入。此过程使我们能够确定在小鼠视网膜的每个神经元的生理功能。

代表性的切片制备示于图1的片制剂是在一条直线的位置，示出在一个平面上的光感受器向神经节细胞，并没有从滤纸分离。如果一个切片是倾斜的，制剂的仅有部分为焦点，这使得它很难确定适当的细胞用于膜片钳。对于录音，它选择一个良好的索玛，通常有一个光亮的表面，均匀的圆形，并没有明显的暗斑是很重要的。被做了全细胞构造后，将切片暴露于背景光，然后到步骤光（ ...

Watch this Scientific Journal Video about Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques at JoVE.com

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

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. ...

recording-light-evoked-postsynaptic-responses-neurons-dark-adapted

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.4K 次观看.  Wayne State University School of Medicine. 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.

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

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

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

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 ...

科研

神经科学

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

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.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

全细胞记录的光在视网膜切片诱发兴奋性突触电流

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 ...

生物学

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 ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

星爆无长突细胞的膜片钳在Deafferentated小鼠视网膜的平板安装准备

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

成年小鼠视网膜电生理研究水平切片的制备

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Light-Evoked Postsynaptic Responses in Neurons of Mouse Retinal Slices

Source: Hellmer, C. B., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52422">Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques.</a> J. Vis. Exp. (2015).
This video demonstrates the recording of light-evoked post-synaptic responses in a dark-adapted mouse retinal tissue slice preparation. The retinal slice is placed in a recording chamber, and a recording pipette is used to establish electrical continuity with a retinal ganglion cell. Light pulses are applied to generate excitatory post-synaptic potentials in the ganglion cells, which are recorded by the pipette.

小鼠视网膜切片神经元中的光诱发突触后反应

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Experimental ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

Photostimulation and Whole-Cell Patch Clamp Recordings of Neurons in Mouse Hippocampal Slices

Source: Richevaux, L., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59534">In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices.</a> J. Vis. Exp. (2019)
The video demonstrates a protocol to study synaptic interactions and neuronal responses in a transfected mouse hippocampal brain slice. It involves illuminating long-range sensory neurons to trigger action potentials and recording the resulting excitatory post-synaptic potentials in the recorded neuron using the whole-cell patch-clamp technique.

小鼠海马切片中神经元的光刺激和全细胞膜片钳记录

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Solutions for acute slice ...

Electrophysiology Techniques

Optogenetic Stimulation and Electrophysiological Recording of Synaptic Events in Rat Brain Slices

Source: Kinnavane, L., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/63077">Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure for studying synaptic efficiency in the lateral entorhinal cortex (LEC) by optogenetically stimulating medial prefrontal cortex (mPFC) axons and recording the responses of pyramidal neurons in the LEC. This method combines patch-clamp recordings with synaptic activation by light stimulation to study synaptic plasticity.

大鼠脑切片中突触事件的光遗传学刺激和电生理记录

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Electrophysiology and ...