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 eye-cup. When the retinal tissue is in the dissecting chamber, cold solution should be poured on the tissue every 10 min. The tissue should be sliced quickly without pulling or poking the retinal tissue during the dissection, which could easily damage its structure. Using good dissecting tools (sharp forceps and scissors) is also crucial. A chopper is equally as effective as a vibratome because it allows for speedy maneuvering. Extra-fine forceps should be used to remove vitreous matter as efficiently as possible because residual vitreous damages the retinal structure when slicing the tissue.
Beginners tend to damage the retina when removing the lens. The retina can detach from the eye-cup, or can even be accidentally removed with the lens. To avoid this issue, the sclera can be cut around the Ora Serrata which is located close to the extra-ocular muscle attachment point. An eye-cup with a wide opening makes it easier to remove the lens. Also, it is important not to deform the eye when removing the front portion. Another critical point is fixing the retinal slice onto a coverslip. This can be facilitated by practicing rotating the filter paper without distorting it to prevent the retinal slice from coming off during the experiment. 
A sturdy, well-built rig is crucial for patch clamp recordings. It must be free of electric noise, vibration, and pipette drifting. In addition, light response can be recorded in dark conditions to prevent desensitization. Our cage is made with a metal frame and aluminum panels with black plastic drapes that act as an electric shield while also completely blocking light from entering the cage; computer monitor illumination will not affect the retinal preparation. Use a remote controller for the pipette manipulator and close the cage while setting up the patch clamp configuration, which also avoid illuminating the preparation.
Although we have been tried to simplify the procedures involved, patch clamp study is still a difficult experiment that requires extensive knowledge and training. An automated patch clamp has been developed by some groups20; this system is applicable to dissociated cells and might be an efficient technique for pharmacological screenings. 
In summary, patch clamp recording in dark-adapted retinal slice preparations is a useful technique to investigate visual information processing. Compared to other methods, patch clamp offers better temporal and spatial resolution. Additional technical innovations will make the patch clamp method more user-friendly in the near future.

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 border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:145px;">Company</td>
			<td style="width:203px;">Catalog Number</td>
			<td style="width:323px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mice (28-60 days old, male)</td>
			<td style="width:145px;">Jackson laboratory</td>
			<td style="width:203px;">C57BL/6J strain</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ames&#39; medium powder</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:203px;">A1420</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:145px;">Nikon</td>
			<td style="width:203px;">SMZ745</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_forceps</td>
			<td style="width:145px;">Dumont</td>
			<td style="width:203px;">#4, #5, #55</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_scissors</td>
			<td style="width:145px;">Roboz</td>
			<td style="width:203px;">RS-5605</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_surgery knife</td>
			<td style="width:145px;">Surgistar</td>
			<td style="width:203px;">7514</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">razor blade (for chopper)</td>
			<td style="width:145px;">EMS</td>
			<td style="width:203px;">71970</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chopper</td>
			<td style="width:145px;">handmade</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">infrared viewer</td>
			<td style="width:145px;">Night Owl Optics</td>
			<td style="width:203px;">NOBG1</td>
			<td>It shows bright view.&#160; Focusing small objects is an issue.</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:216px;">infrared pocket scopes</td>
			<td style="width:145px;">B.E. Meyers</td>
			<td style="width:203px;">OWL Gen 3 NV pocketscope</td>
			<td>excellent view</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">puller</td>
			<td style="width:145px;">Sutter</td>
			<td style="width:203px;">P-1000</td>
			<td>excellent.&#160; Make consistent size pipettes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dark box</td>
			<td style="width:145px;">Pelican</td>
			<td style="width:203px;">dark box</td>
			<td>excellent</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:216px;">patch clamp system</td>
			<td style="width:145px;">Scientifica</td>
			<td style="width:203px;">slice scope 2000</td>
			<td style="width:323px;">Excellent setup.&#160; Most key components are included in one package.&#160; Micromanipulators are excellent.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">amplifier</td>
			<td style="width:145px;">Molecular Devices</td>
			<td style="width:203px;">multiclamp 700B</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">acquiring software</td>
			<td style="width:145px;">Molecular Devices</td>
			<td style="width:203px;">pClamp software</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:216px;">light source (LED)</td>
			<td style="width:145px;">Cool LED</td>
			<td style="width:203px;">pE-2 4 channel system</td>
			<td>Excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CCD camera</td>
			<td style="width:145px;">Q-imaging</td>
			<td style="width:203px;">Retiga 2000</td>
			<td>Excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Faraday cage</td>
			<td style="width:145px;">handmade</td>
			<td style="width:203px;"></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（ 图2）。光诱发的兴奋性突触后电位（L-，EPSPS）被诱发响应于步骤光（定时示于黄色）。峰值振幅和取决于细胞类型和子类型而变化的衰减时间。一个神经节细胞响应明亮的刺激（ 图2 D＆E）产生动作电位。在CEL升类型及其形态学亚型是由生理记录（ 图2右侧）后磺酰罗丹明标记显示。 <img alt="图1" src="/files/ftp_upload/52422/52422fig1highres.jpg" width="650" /> 图1.视网膜片的准备工作。（A）视网膜切片准备了10X客观观察。视网膜切片准备（顶部）被附连到一片滤纸（底部）。 （B）中的四图像汇编表示视网膜切片准备用60X物镜观看。每个细胞层清楚地观察到（ONL:外核层，OPL:外丛状层，INL:内核层，彩光:内网状层，GCL:神经节细胞层） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52422/52422fig1highres.jpg" target="_blank">，请点击这里查看此图的放大版本。</a> 图2.光诱发的兴奋性突触后电位（L-，EPSPS）从视网膜神经细胞 。步骤光诱发L-性兴奋（左）。光照强度为30 - 60％韦伯对比。背景光自适应级为4×10 4个光子/微米2 /秒。磺酰罗丹明B膜片钳记录形象化记录的神经元（右）在注射。一种记录吸管仍然附着胞体（A）L-性兴奋和磺酰罗丹明染色从ON锥双极细胞。 （B）的关视锥双极细胞。 （C）无长突细胞。 （ 四）神经节细胞。 （E）OFF神经节细胞。比例尺为2或5 mV的如前所述。光刺激应用于1秒。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52422/52422fig2highres.jpg" target="_blank">请点击这里</a>查看该图的放大版本。

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

Nanyang Technological University

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

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

鼠标采用的空气相Electroolfactogram录制的嗅觉感官神经元响应分析

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

科研

神经科学

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 ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

膜片钳电容测量和Ca 2 +单神经末梢成像在视网膜切片

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

在犁鼻器官的切片准备工作表示一个基因编码的钙传感器成像神经元的反应

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录:神经元表达鉴定的气味受体功能分析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

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 ...

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 ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.