This work is supported by a National Science Foundation award (1145581) to LWG.

注意：整个过程是根据美国国立卫生研究院的指导方针进行的，经批准由伊利诺伊大学芝加哥分校的动物护理和使用委员会。 1，解决方案和文化传媒准备<ol><li>保留所有的解决方案在-20°C至六个月。保持培养基在4℃下进行长达3个月。 </li><li>通过混合1毫升ITSX（胰岛素 - 转硒补充）的青霉素 - 链霉素溶液和1毫升到100毫升的DMEM制备100ml培养基。 </li><li>通过混合250毫升DMEM中，加入2.5ml的100mM的CaCl 2，和2.5毫升的50mM EDTA制备的酶溶液。 </li><li>通过混合225毫升DMEM，25毫升胎牛血清，625毫克的白蛋白，和625毫克的胰蛋白酶抑制剂的制备失活的解决方案。 </li><li>准备了154毫米的NaCl，5.6 mM的氯化钾，5.0毫米肝素钠，3.6毫米的碳酸氢钠，5.6毫米的葡萄糖洛克的解决方案。将pH调节至7.3，用NaOH。 </li><li>制备50毫克/毫升的聚-D-赖氨酸（PDL）溶液。用1毫克/毫升的最终浓度大衣盖玻片。 </li><li>制备140 mM氯化钠，5mM的氯化钾，2mM的氯化钙 ，1mM的MgCl 2的，10毫米的HEPES-NaOH和10mM葡萄糖的细胞外溶液。将pH调节至7.3，用NaOH为〜310纳摩尔/ kg的渗透压。 </li><li>准备了50毫米的NaCl，100毫米TEACl，5 mM的氯化钾，2毫米氯化钙2，1毫米氯化镁2，和10毫米的HEPES吸管的解决方案。调节pH值至7.3，用NaOH与〜290纳摩尔/ kg的渗透压。 </li></ol> 2，肾上腺隔离<ol><li>高压釜，对大型和小型清扫剪刀和两对镊子。在整个过程中戴上手套，然后将解剖托盘上一个冰桶。 </li><li>在使用0-3天取小鼠。安乐死的动物用加压的二氧化碳罐后斩首。 </li><li>利用小剪刀来剪下来的小狗的背下从宫颈癌放疗脊椎<html> CAL尾鳍地区。可以肯定表面的皮肤下的切割和未刺穿进入体腔。 </li><li>其次，剥离皮肤远离脊柱，使肌肉暴露在有脊柱的一个明确的说法。从这里出发，让在颈椎脊髓transectional切割，然后进行沿脊柱两侧的两个平行的切口。 </li><li>与一对保持动物的主体钳子，使用其他钳子抓住脊柱和剥离背部脊柱直到肾脏被暴露。 </li><li>此时，可视化对肾脏顶部的肾上腺。然后小心地从每个动物的两个腺体成锥形套管内填充洛克的解决方案。 注：有时腺体会坚持镊子。如果是这样的话，轻轻用另一对镊子在去除从钳子和进洛克溶液的腺体中提供帮助。腺体可保持在该状态下长达2小时。 </li></ol>E“&gt; 3。组织消化<ol><li>在此期间，气泡，用5％CO 2 +95％O 2的酶溶液15分钟，在使用之前。确保平衡的溶液变成从一个明亮的粉红色到粉红色/橙色。 </li><li>木瓜蛋白酶加入到新鲜鼓泡酶溶液在20〜25单位/ ml的最终浓度。在用木瓜蛋白酶的酶溶液孵育每对接头的每个动物为40-60分钟，在37℃。 </li><li>加失活溶液的75％至每个管并在37℃进行另外10分钟。这个孵育灭活木瓜蛋白酶的酶活性。 </li><li>在温育10min，灭活液后，轻轻腺体转移到新标管，然后清洗腺体3X文化传媒。 </li></ol> 4，细胞分离和电镀<ol><li>洗涤后，放置两个腺体成200μL培养基，然后轻轻地滴定腺体和上下贯通的移液管尖与200μl的移液管，直到不再有一大块组织在该溶液中。 注意：当进行滴定，维持腺体在管与来自200μl的移液管尖端向下的力底部和接合组织轻缓地。务必使用缓慢，持续的招，绝不吹打快速或突然。 </li><li>增加一个额外的250微升培养基，使培养液的总体积为450微升。 </li><li>板60微升此含细胞溶液到每七个12毫米PDL-预涂覆的盖玻片，它被放置在一个60×15毫米培养皿中。 </li><li>一次镀，将盘插入，并设定为37℃，并保持5％的CO 2流的稳定/供应30分钟，以确保细胞存活的环境中孵化。孵育后，轻轻地加入5 ml预先温热的培养基放入培养皿中，并返回盘放回培养箱中放置24小时前，细胞公文包ð录音。 注意：通常情况下，可以使用的细胞电生理记录长达4天。 </li></ol> 5，细胞识别和千兆欧密封形成<ol><li>将12毫米盖玻片进入细胞外液。由于整个肾上腺用于细胞制剂，拌入培养与皮质细胞（小和比铬细胞）的调光器的嗜铬细胞（通常是非常明亮的带褐/米色着色和近乎完美的圆形（ 图1）。 </li><li>拉使用可编程的P-97牵引补丁移液器四个阶段。浸枪头到融化的蜡，这将有助于降低玻璃的电容再火波兰尖端“井喷走”这起蜡枪头内。 注意：当装满补丁吸管的解决方案，补丁移液器通常有〜2MΩ电阻。 </li><li>在数米接近补丁吸管靠近电池icrons。实现了小区连接的配置，补丁吸管更靠近的小区的方法中查找任何轻微的细胞变形。轻轻推吸，直到GΩ的密封来实现的。 </li></ol> 6，细胞贴附电容录音<ol><li>一旦GΩ的印章则透露，通过使用EPC-7加膜片钳放大器使细胞贴附记录和两相模拟锁相放大器（见设备附表）。申请为50毫伏（有效值）的振幅和20千赫频率的锁定放大器的正弦波。 </li><li>在1毫秒的时间常数和24分贝设置的锁定放大器的输出滤波器。设置锁定放大器，使得由温和抽吸脉冲产生的瞬态电容变化只在贴片电容（Im）的痕迹，没有投影到补丁电导（Re）中的跟踪显示（ 图2）的相位。 注：请参阅详细的系统重新校准ference 19。 </li><li>识别典型的电容变化由“向下”的步骤（ 图3）。 注：固体录音将被视为一个“阶梯”式的相似与许多向下的步骤，这表明内在单囊泡。打补丁的过程作为一个机械刺激，因为动作电流通常可以从大多数细胞记录。 </li><li>名的文件作为单独的文件夹，包括日期：该文件中/日/年，每个细胞贴附记录作为自己的个人编号（XX）。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

细胞贴附电容测量要求，以顺利取得录音提供高品质的几个关键步骤：1）肾上腺编制可行的，健康的细胞; 2）客运专线涂层的盖玻片; 3）千兆欧密封的形成; 4）该系统的噪声电平;和5）的相位校正。 动物手术，修饰可以潜在地使是调整手术方法，以最适合的灵巧和防止剥离时发生损坏。此外，关于定位，除去肾上腺足够的做法是必要的，这将被用于电生理记录的细胞全部来?...

突触传递是由含有神经递质的突触小泡的胞吐作用介导的，而这些囊泡必须经过神经末梢内本地吞再循环以维持长期的神经元沟通。由于突触传递在大脑中的重要作用，了解构成突触小泡循环是实现在整个蜂窝通信更好的理解的重要基础的分子机器。在细胞模型系统，肾上腺嗜铬细胞提供了一些最明确的洞察分子机制基础突触囊泡循环。胞吐作用，在神经递质释放的最后步骤，已经极大地研究并通过使用肾上腺嗜铬细胞1,2的检查。事实上，大多数的分子玩家编排的形成，目标，对接和融合分泌颗粒已经确定，由于应用程序股利ERSE技术，嗜铬细胞1。此外，通过提供一个机会以允许参与胞吐作用的蛋白质机械的单泡的分辨率，嗜铬细胞仍然是一个功能强大的模型，以解决囊泡融合3的问题。 细胞贴附电容测量首先在胞吐3中解决单囊泡融合利用。以通过细胞膜导纳测量与小区连接的配置4-7膜片钳技术来检测囊泡一样小〜中直径为60nm已证明的胞吐作用。导纳的定义是多么容易的电路或设备将允许电流流动的措施;它是阻抗的倒数。因此，导纳测量提供的膜电容的理解。这是由小泡膜进入细胞膜的结合来实​​现;这表明掺入改变表面面积8。每个囊泡融合导致膜电容9,10逐步增加。此外，该导纳测量提供了膜电导和胞吐事件3中 ​​的融合孔导。由于这种技术提供了在胞吐过程中识别单囊泡动力学的独特工具，我们的实验室最近应用这个概念，检测单囊泡11,12的内吞作用。 我们特别感兴趣是网格蛋白介导的内吞作用（CME），它已被认为是一个基本的看家成分在许多细胞13和作为突触小泡胞吞作用在神经端子14,15的主通路。 CME是已知的生物学上重要的，但是，它的动力学仍不能很好地理解，由于在监测单数内吞事件的技术限制。考虑到嗜铬细胞和神经元之间的1胞吐机制的相似性，这是PLausible在嗜铬细胞分裂机制可能会适用于突触小泡内吞作用的神经元。细胞贴附电容测量已用于监测个体的内吞事件并分析裂变动力学，其中大多数方法都未能解决。在我们的细胞附着的录音，在20kHz的正弦波叠加在保持电势，并且输出电流是从两相分离成薄膜电导的一个信道和膜电容，在其它信道锁定放大器16 18。从变化的膜电导和电容，可以计算裂变孔隙，这可能对应于管状薄膜颈部的内化泡连接到前囊夹断质膜的动力学。总的来说，这项技术使我们有机会研究囊泡分裂的调控机制CME的过程中。

<table border="0" cellpadding="0" cellspacing="0" width="352">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Name of Material/ Equipment</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:88px;">Poly-D-Lysine</td>
			<td rowspan="2" style="width:88px;">Sigma</td>
			<td rowspan="2" style="width:88px;">P0899</td>
			<td rowspan="2" style="width:88px;"></td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:88px;">DMEM&#160;&#160;</td>
			<td style="width:88px;"></td>
			<td rowspan="2" style="width:88px;">15066024</td>
			<td rowspan="2" style="width:88px;">Keep out of UV</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:88px;">Dulbecco&#8217;s Modified Eagle Medium</td>
			<td style="width:88px;">Life Technologies</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Cover Glass</td>
			<td style="width:88px;">Carolina Biological</td>
			<td style="width:88px;">633029</td>
			<td style="width:88px;">12mm</td>
		</tr>
		<tr height="34">
			<td height="56" rowspan="2" style="height:57px;width:88px;">Penicillin Streptomycin</td>
			<td rowspan="2" style="width:88px;">Life Technologies</td>
			<td rowspan="2" style="width:88px;">15140122</td>
			<td rowspan="2" style="width:88px;">100mL</td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Insulin-Trans-Sel-X</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">51500056</td>
			<td style="width:88px;">Only thaw on ICE!</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Papain</td>
			<td style="width:88px;">Worthington</td>
			<td style="width:88px;">39S11614</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="56" rowspan="2" style="height:57px;width:88px;">EPC-7 plus patch amplifier</td>
			<td rowspan="2" style="width:88px;">HEKA</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BNC-2090 data acquisition board</td>
			<td rowspan="2" style="width:88px;">National Instruments</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Igor data acquisition software</td>
			<td style="width:88px;">Wavemetrics</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">P-97 pipette puller</td>
			<td style="width:88px;">Sutter Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Microforge</td>
			<td style="width:88px;">Scientific Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="60">
			<td height="161" rowspan="3" style="height:161px;width:88px;">Borosilicate glass capillaries</td>
			<td rowspan="3" style="width:88px;">Sutter Instruments</td>
			<td rowspan="3" style="width:88px;">B150-110-10</td>
			<td style="width:88px;">Outer diameter-1.5 mm</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Inner diameter 1.10 mm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Length- 10 cm</td>
		</tr>
	</tbody>
</table>

methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

细胞存活率和千兆欧姆密封的质量是确定细胞贴附电容记录的质量是至关重要的。因此，为促使有效和高效的细胞培养物之前，电生理记录，和典型的活细胞被示出在图1中是至关重要的。实践和时间将是在实现千兆欧姆密封高品质很有帮助。如果人们可以清楚地看到，当在协议5.3步中描述的补丁吸管的临近细胞的细胞变形，有获得高品质的密封更好的机会。 图3显示膜电...

Watch this Scientific Journal Video about Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell at JoVE.com

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

方法在小鼠肾上腺嗜铬细胞细胞贴附电容测量

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

methods-for-cell-attached-capacitance-measurements-mouse-adrenal

Research

JoVE Journal

Neuroscience

8.7K 次观看.  University of Illinois at Chicago. After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

视频: Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

超声引导下显微注射到小鼠前脑在子宫内在E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

科研

神经科学

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

在小鼠模型中静脉自我管理的方法

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

方法对NF-κB依赖的成年神经发生的调制与分析

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

在对小鼠坐骨神经体内的电测量

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The SCN1A-associated genetic epilepsies comprise a wide spectrum of seizure disorders with incomplete penetrance and clinical variability. SCN1A mutations can result in a large variety of seizure phenotype ranging from simple, self-limited fever-associated febrile seizures (FS), moderate-level genetic epilepsy with febrile seizures plus (GEFS+) to more severe Dravet Syndrome (DS). Although FS are commonly seen in children below 6-7 years of age who do not have genetic epilepsy, FS in GEFS+ patients continue to occur into adulthood. Traditionally, experimental FS have been induced in mice by exposing the animal to a stream of dry air or heating lamps, and the rate of change in body temperature is often not well controlled. Here, we describe a custom-built heating chamber, with a plexiglass front, that is fitted with a digital temperature controller and a heater-equipped electric fan, which can send heated forced air into the test arena in a temperature-controlled manner. The body temperature of a mouse placed in the chamber, monitored through a rectal probe, can be increased to 40-42 &#176;C in a reproducible manner by increasing the temperature inside the chamber. Continual visual monitoring of the animals during the heating period demonstrates induction of heat-induced seizures in mice carrying an FS mutation at a body temperature that does not elicit behavioral seizures in wild-type litter mates. Animals can be easily removed from the chamber and placed on a cooling pad to rapidly return body temperature to normal. This method provides for a simple, rapid, and reproducible screening protocol for the occurrence of heat-induced seizures in epilepsy mouse models.

The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.

癫痫小鼠模型中热诱发癫痫发作的行为筛查

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

两种剥离方法，用于分离小鼠视网膜中的光感受器细胞区室以进行蛋白质分析

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...