This work was supported by DFG Schwerpunktprogramm 1392 (project MA 4113/2-2), cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain (project B1-9), and the German Ministry of Research and Education (BMBF; project 1364480).

注：动物的处理和实验进行批准的哥廷根大学委员会，伦理动物实验。 1.准备的仪器和吸管制作<ol><li>确保电穿孔装置由具有大的工作距离在立体显微镜的，并配备了荧光照明和过滤套用于所使用的染料。 </li><li>电穿孔使用一个专用的单个细胞电穿孔仪或附连到示波器的通用方波脉冲发生器。连接所述电穿孔仪输出到移液管保持器和一个浴电极。脉冲发生器的正极端子连接到微管保持器和所述负极端子向浴电极。确保两个吸管保持器和浴电极含有涂有一层薄薄的氯化银的银导线。 </li><li>安装在显微吸管持有人允许精确定位。 </li&gt; <li>从制造硼硅玻璃毛细管内的灯丝电微量。 </li><li>使用水平微量拉马和所描述的贝斯曼等人 13施加改性协议移液器用于膜片钳实验的制造。 </li><li>适应参数，以产生一个较长的刀柄和一个较小的喷嘴开口导致约15-20兆欧姆的单细胞电穿孔较高的吸液管电阻。移液管电阻要低， 例如 ，3-4兆欧，对细胞群体的标签。 </li><li>测量可以直接在吸液管电阻与一个专用的单细胞电穿孔仪或测定用应用程序定义的电压脉冲的后示波器电流流动后用欧姆定律计算出来。 </li></ol> 2.制备电穿孔的解决方案<ol><li>溶于青蛙铃声（98毫米氯化钠，2毫米氯化钾荧光基团偶联葡聚糖，1毫米氯化钙2,2毫米MGC升2，5mM葡萄糖，5mM的丙酮酸钠，10mM的HEPES，pH调至7.8，克分子渗透压浓度为230 mOsmol /升）以3毫摩尔的浓度存在。这些细胞可能不那么明亮，如果较低的染料浓度使用标记。准备大量库存解决方案，并把它分成小等份。冻结，储存（稳定数月）。 注：葡聚糖是在不同尺寸和各种发射/激发光谱可用的（ 例如 ，页面488葡聚糖10KD，Alexa的546葡聚糖10KD，Alexa的568葡聚糖10KD，Alexa的594葡聚糖10KD，TMR-葡聚糖3KD。）。 </li><li>回填具有细长枪头用小体积的葡聚糖溶液（1-5微升）微量。仔细轻弹微量用手指从枪头去除残留的气泡。 </li><li>装在吸管架微量。确保吸液管内的银线是在与染料溶液接触。 </li></ol> 3.选择幼虫X的蟾&lt;/ EM&gt; <ol><li>使用X的白化幼虫蟾用于实验。野生型动物中具有该显示期间共焦/多光子成像的自发荧光发射，并且因此不适合用于所描述的实验，颜料填充的黑素细胞。 </li><li>舞台新科普和法贝尔14后premetamorphotic蝌蚪。选择阶段45-53的实验蝌蚪。 </li></ol> 4.电穿孔荧光基团偶联葡聚糖<ol><li>放置一小块组织培养皿中并覆盖它具有小体积的含0.02％三卡因水（3-氨基苯甲酸乙酯甲磺酸盐，调节至pH 7）。 </li><li>麻醉在水中含0.02％三卡因的蝌蚪。几分钟后，这些动物停止移动。通过触摸蝌蚪确定适当的麻醉。它们应是无响应。 </li><li>小心地从麻醉转移蝌蚪的组织覆盖的培养皿。 </li><li>确保洗浴ELECTR颂关闭电电路。确保电极与湿纸巾接触;直接接触到蝌蚪是没有必要的。 </li><li>定位枪头接近使用显微嗅觉器官。 </li><li>穿透皮肤覆盖的嗅觉器官与枪头和尖小心翼翼地提前进入主嗅上皮，或犁鼻上皮的位于市中心的感觉神经元层。 </li><li>引发正极的方形电压脉冲，以染料转移到的感觉神经元。应用单一电压脉冲（ 如 ，25 V，脉冲长度为25毫秒）或多个脉冲序列（ 例如 ，50 V，脉冲宽度300微秒，400毫秒的火车时间在200赫兹）。 注：确定最佳的电压脉冲参数所需的扩展标签。减小电压脉冲的幅度，持续时间和重复次数，以降低标记的细胞的数目。适用于高电压脉冲的幅度，对期限和数量ulses为更广泛的标签。 </li><li>可视化成功的染料挤压和电利用体视显微镜的荧光照明触发脉冲。成功的电穿孔后的染料迅速扩散到胞体和树突。 </li><li>重复步骤4.5-4.9的蝌蚪的第二个嗅觉器官。 </li><li>转移蝌蚪放入盛有淡水回收的烧杯中。经过约5分钟蝌蚪从麻醉中醒来，并开始正常的游泳动作。 </li><li>后24小时，将电穿孔的染料扩散，在感觉神经元中，并最终到达经由轴突运输嗅球。 </li></ol> 5.安装动物体内可视化细胞及轴突的过程<ol><li>麻醉在水中含0.02％三卡因的蝌蚪。 </li><li>仔细蝌蚪转移到成像室， 比如一个小的硅橡胶盖培养皿蝌蚪大小的凹陷。&lt;/ LI&gt; <li>切一个小矩形变成封口膜的条纹。盖上封口膜的蝌蚪，离开前端脑通过切口窗口暴露出来。修正了上盘针的封口膜，而不伤及蝌蚪。 </li><li>确保蝌蚪被淹没在含有0.02％三卡因足够的水。 </li><li>安装成像室上直立多光子显微镜或共聚焦显微镜的阶段。 </li><li>获得嗅球的图像的三维堆叠。确保该成像程序是尽可能地短，并且不超过10-15分钟。 </li><li>返回蝌蚪变成正常的水在一个单独的油箱，并防止暴露在光线下。 5分钟后，蝌蚪从麻醉中醒来。 </li><li>每天指定的时间间隔后，重复步骤5.1-5.7， 如：，。 </li></ol> 6.安装动物的细胞和轴突的进程离体可视化<ol><li>另外第5该协议的，使用切除大脑准备可视化标记的感觉神经元。 </li><li>麻醉，杀死蝌蚪由大脑的横断处过渡到脊髓。切除组织含有嗅觉器官，嗅神经和端脑前一个块。 </li><li>转移的组织块以蛙振铃溶液和除去结缔组织与细剪至脑的腹侧边露出。 </li><li>转移组织块的成像室，并与铂框架弦用尼龙线固定。 </li><li>装入成像室上的共焦/多光子显微镜的阶段。 </li><li>获得嗅球的图像的三维堆叠。 </li></ol> 7.图像处理和数据评估<ol><li>优化，并应用滤波以提高图像质量和神经元结构的可视化的非本地装置所描述的双门轿跑车，P。等人 15。 注：数据来自于活标本更深的组织层成像实验往往是嘈杂。 </li><li>创建图像栈概述的最大强度的预测。 </li><li>时间推移成像实验筛选数据集的感觉神经元的单明确标识轴突。 </li><li>所描述的鹏等人通过神经元突起的软件辅助重建跟踪细胞形态。16。 </li></ol>

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The authors have nothing to disclose.

这里描述的实验步骤，允许幼虫X的嗅觉器官的标记的感觉神经元蟾通过的荧光团偶联的葡聚糖和随后的感觉轴突生长在活体动物可视电穿孔。通过改变在体内电穿孔的参数，可以控制标记的感觉神经元的数目。由此，能够以标记大基团的感觉上皮，很少或甚至单个细胞的神经元。 以确保所希望的延长神经元标记的是特别谨慎微量特性和电穿孔脉冲是重要的。更高的吸移管的电阻和减小的电压脉冲的幅度，持续时间，重复次数可以减少标记的细胞的量，而降低吸移管的电阻和较高的电压脉冲的幅度，持续时间和脉冲数可以导致更广泛的标签。日E使用荧光葡聚糖的电，如果提供的应用设置相应的即时视觉反馈。但要注意使用的幅度，持续时间和超过协议中提供的值可能会导致细胞损伤，甚至细胞死亡17脉冲数参数。微量的堵塞或破裂的提示也可以阻碍成功的电穿孔。 在X的嗅觉器官活体电穿孔蟾仅限于幼虫期以来postmetamorphotic青蛙的皮肤是强硬的，不能很容易地穿透用微量。 在体内的可视化神经元突起可通过的激发/发射光在更深的脑区域或通过血管的散射受到阻碍。这个问题在更高的幼虫阶段尤为明显，由于较大的大脑，并可能导致使明确的识别细微的轴突过程的更困难的噪声信号。 <p类=“jove_content”&gt;所提出的协议允许以可视化的完整的嗅觉系统的感觉神经元，而不解剖动物，标签的过程中损坏的细胞，制备组织切片或根据需要可替代的方法固定组织，如在全细胞膜片标签-clamp实验18。当组合几个或单个的感觉神经元在体内的时间推移成像的标记，能够以可视化单个成熟的感觉神经元的肾小球连接在较长的时间间隔。这种方式还能够监测未成熟的感觉神经元的轴突突起图形的发展在几个星期。这后一种选择是特别有趣，因为它允许监视单个轴突的生长模式，在活的动物。这将打开以调查控制轴突导向和寻路细胞和分子机制的可能性。几个因素，包括气味受体表达，各种轴突ñ导向分子和感觉神经元的加臭剂诱导的/自发性活动已显示调节的感觉神经元的轴突4,5-目标的发现。 该协议的应用不限于嗅觉的感觉神经元，但也可应用于研究其他类型的细胞， 例如 。，茎发育的大脑或嗅球的僧帽细胞的神经区域的细胞/祖细胞。此外，表现出技术也可以结合使用钙敏感葡聚糖或注入膜透钙染料来获得关于标记的神经元和/或所连接的电路7,19的功能信息。的大范围的荧光团耦合到葡聚糖的可用性允许多个单个细胞或群体具有不同颜色的标签。还质粒DNA溶液，用于荧光蛋白例如编码，适合于电穿孔，并可以进一步提高versatili一节和技术6的有用性。该协议可以进一步增强，使得葡聚糖和DNA或带电吗啉的组合电操纵基因表达13,17。 所描述的方法，当然代表了一种新的工具，调查复杂，但仍不能完全理解的过程，调节脊椎动物的嗅觉系统轴突的指导。

The lifelong turnover of sensory neurons distinguishes the olfactory system from many other sensory and neuronal systems1,2. Newly formed sensory neurons are continuously generated in the basal portion of the olfactory epithelium3 and extend their axons into the olfactory bulb, the first relay station of the olfactory system4. However, the cellular and molecular mechanisms controlling the formation and maintenance of the olfactory map are far from being fully understood4,5.
Here, we describe a protocol for labeling sensory neurons of the olfactory organ of larval X. laevis by in vivo electroporation of fluorophore-coupled dextrans. The presented protocol allows visualization of axonal morphology and connectivity, track axonal development over time and study mechanisms regulating axonal wiring and guidance.
Electroporation is a well established method to introduce charged macromolecules, like dextran-coupled dyes and DNA, into cells6,7. The cell membrane is permeabilized by application of short voltage pulses and the molecules are electrophoretically delivered into the cytosol8. Spatially restricted electroporation using a micropipette permits selective labeling of cells including neurons and has been applied in various neuronal systems including the visual system of X. laevis9,10.
We show how the electroporated animals can be used to study axonal growth patterns and morphology in living animals using confocal laser-scanning or multiphoton microscopy. The described procedure allows identifying the coarse topology of axonal projections of sensory neurons of the main and accessory olfactory system11,12. Using in vivo time lapse imaging, it is also suitable to supervise the glomerular connections of single mature sensory neurons, and to monitor the evolution of the axonal projection patterns of immature sensory neurons12. The described protocol can be applied to investigate the structure and formation of olfactory circuits in the intact animal and can be adapted to other cell types within the olfactory and other neuronal systems.

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			<td style="width:100px;">Company</td>
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			<td style="width:167px;">Comments/Description</td>
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			<td height="42" style="height:42px;width:203px;">SZX16</td>
			<td style="width:100px;">Olympus</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">stereomicroscope with fluorescent illumination</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Axon Axoporator 800A</td>
			<td style="width:100px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">single cell electroporator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">ELP-01D</td>
			<td style="width:100px;">npi electronic</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">electroporator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">MMJ</td>
			<td style="width:100px;">M&#228;rzh&#228;user Wetzlar</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">manual micromanipulator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">P-1000</td>
			<td style="width:100px;">Sutter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Horizontal micropipette puller</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:203px;">G150F-4</td>
			<td style="width:100px;">Warner Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">glass capillaries for electroporation pipette fabrication; internal filament makes backfilling easier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Alexa 488-dextran 10kD</td>
			<td style="width:100px;">Life Technologies</td>
			<td style="width:119px;">D22910</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Alexa 546-dextran 10kD</td>
			<td style="width:100px;">Life Technologies</td>
			<td style="width:119px;">D22911</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Alexa 568-dextran 10kD</td>
			<td style="width:100px;">Life Technologies</td>
			<td style="width:119px;">D22912</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Alexa 594-dextran 10kD</td>
			<td style="width:100px;">Life Technologies</td>
			<td style="width:119px;">D22913</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">TMR-dextran 3kD (micro-Ruby)</td>
			<td style="width:100px;">Life Technologies</td>
			<td style="width:119px;">D7162</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">microloader pipette tips</td>
			<td style="width:100px;">eppendorf</td>
			<td style="width:119px;">930001007</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:203px;">tricaine (Ethyl 3-aminobenzoate methanesulfonate)</td>
			<td style="width:100px;">Sigma-Aldrich</td>
			<td style="width:119px;">E10521</td>
			<td style="width:167px;">anesthetic; use gloves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">A1-MP</td>
			<td style="width:100px;">Nikon</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiphoton microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">LSM 780</td>
			<td style="width:100px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">confocal microscope</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Imaris</td>
			<td style="width:100px;">Bitplane</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">alternative software for neuronal tracing</td>
		</tr>
	</tbody>
</table>

the olfactory system as a model to study axonal growth patterns and morphology in vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

所述协议可以成功地应用于电穿孔和麻醉X的嗅觉系统的感觉神经元的轴突过程的体内可视化蟾 ，使用共聚焦激光扫描或者多光子显微术（ 图1）。 电穿孔使荧光团偶联的葡聚糖进入并迅速传播内的嗅觉器官的细胞。这是有帮助的应用荧光照明的触发电压脉冲后验证成功的标志。根据电穿孔参数， 例如 。，吸液管电阻，将细胞（ 图2A，B）或嗅觉器官的单细胞（ 图2C）的基团标记。标记的感觉神经元的轴突可以显现在嗅神经（ 图2D）和轴突的过程也可以被观察到，在嗅球，成功的电穿孔后，通常24小时（ 图3）。感觉神经元群体的电穿孔允许可视化在嗅球（ 图3A）的粗配线图案。单个细胞电穿孔可以用于调查个体轴索突起图形，轴突分叉并连接到肾小球的结构在嗅球（ 图3C-E）。 十，透明的白化蝌蚪蟾许可证活体共焦或麻醉蝌蚪的完整的大脑标记的感觉神经元的多光子成像。的轴突生长模式的发展，可以跟踪在数天/周反复在同一标记的感觉神经元（ 图4）的体内可视化。根据不同的感觉神经元的电穿孔的时间点，在嗅球轴突图案可以有很大的不同的成熟度。成熟的神经元已经拥有精心设计的轴突分支功能绺连接到肾小球编辑区域。成熟神经元的轴突生长模式是相当稳定的，但轴突分支和肾小球簇改进的伸长率可观察（ 图4A-E）。感觉神经元可以在体内观察到的更长的时间内， 例如 。，大于三个周（ 图4F-1）。另一方面，未成熟的神经元的轴突仍处于初期生长的过程中，不具有簇绒arborizations和尚未连接到它们的最终肾小球目标。实验协议允许成熟过程中追踪这些神经元的发育， 如：，轴突伸长，分叉和建立簇绒arborizations（ 图4J-L）的。更多的例子也看到Hassenklöver和曼齐尼12。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/52143/52143fig1highres.jpg" width="500"/&gt; 麻醉X的嗅觉器官图1.原理的实验方案概述（一）主嗅上皮感觉神经元（MOE）或犁鼻器（VNO） 蟾幼虫通过电穿孔，使用玻璃移液管填充有荧光葡聚糖溶液进行标记。标记的神经元的轴突可以通过嗅觉神经（ON），随后，最终达到嗅球（OB）。（B）中的蝌蚪被麻醉并标记的神经元是使用在特定的时间间隔的共焦或多光子显微镜反复研究。 （C）的标记细胞的轴突生长模式的渐进发展，可以在后面几天的时间跨度为两周。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/52143/52143fig2highres.jpg" width="500" /> 图2. ELECtroporation在嗅觉器官的感觉神经元中。（A）电穿孔与低电阻移液器导致多种细胞在嗅觉器官的标签。在这个典型的例子多个嗅觉受体神经元（ORNs，充满箭头）和两个柱状支持细胞（旺，星号）进行染色。虚线划分的嗅觉上皮（OE）。（B）的磨浆电穿孔参数， 例如 ，边界，增加了吸液管电阻，限制了标记细胞的数目。在这个例子中，一个单一的感觉神经元（实心箭头）和一个相邻的支持细胞（星号）电穿孔后进行染色。注意单个轴突离开嗅觉上皮（空心箭头）。（C）的成功单细胞电穿孔导致的个体感觉神经元（实心箭头）并在嗅觉上皮与其连接的轴突（空心箭头）的专属标记。 <s仲&gt;（D）中的单个标记的轴突（空心箭头）可以通过嗅神经（ON），进入嗅球被遵循。 <img alt="图3" fo:content-width="4in" src="/files/ftp_upload/52143/52143fig3highres.jpg" width="400" /> 图3.可视化在切除嗅球轴突突起。（A）中 ，在嗅球的感觉神经元的轴突突起的粗拓扑可以用较低的电阻电吸移管被可视化。一嗅球半球和其相关联的嗅神经（ON）的描绘。四葡聚糖耦合到不同的荧光团进行了电穿孔在嗅觉器官的4远的地方：横向（绿），中间（黄色），内侧弹性模量（红色）和虚拟网络运营商（橙色）。这允许可视化嗅球（AOB）和主嗅球（MOB）的三个主凸域。 <str翁&gt;（B）的叠加在嗅球的结构单嗅觉感觉神经元的不同的轴突生长型态。示出被合并的来自不同幼虫标本来自多个单细胞荧光染色的三维重建。单嗅觉轴突伸入嗅球和球面肾小球（实心箭头）形成簇绒arborizations /突触（CE）的例子。另外，在X中蟾嗅神经轴突连接到一个，两个或多个肾小球（实心箭头，也看到Hassenklöver和曼齐尼12）之前经常分叉（空心箭头）。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/52143/52143fig4highres.jpg" width="500" /> 图4 中的单个嗅觉神经元轴突体内延时成像。（AE）成功的单个细胞电穿孔后的标记的轴突可反复可视化在嗅球（OB）。这个例子说明，研究了一个星期的个人轴突。整体的形态不发生变化很大，因此两个主要分支点可被识别（开放箭头）。需要注意的是在时间过程中，1肾小球的绒头经历连续减少（实心箭头），而另外两个肾小球簇保持稳定（星号）。（FI）此代表性示例示出的长期观测的可行性，因为这特定轴突进行了研究三个多星期。可以检测它的生长模式没有明显的变化。（JL）在生长过程中的未成熟的感觉轴突的一个例子被描述。它尚未连接到肾小球和肾小球特征簇的丢失。经过5天的轴突分支被拉长，轴突分叉多次和细arborizations是确立。

Watch this Scientific Journal Video about The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo at JoVE.com

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

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Neuroscience

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

10.8K 次观看.  University of Göttingen. We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

视频: The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

一<em&gt;线虫</em&gt;研究Amylopathy模型系统

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

科研

神经科学

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

A Free-breathing fMRI Method to Study Human Olfactory Function

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, evaluation of the functions of the human central olfactory system with functional magnetic resonance imaging (fMRI) is still a challenge because of several technical difficulties. There are some significant variables to take into account when considering an effective method for mapping the function of the central olfactory system using fMRI, including proper odorant selection, the interaction between odor presentation and respiration, and potential anticipation of or habituation to odorants. An event-related, respiration-triggered olfactory fMRI technique can accurately administer odorants to stimulate the olfactory system while minimizing potential interference. It can effectively capture the precise onsets of fMRI signals in the primary olfactory cortex using our data post-processing method. The technique presented here provides an efficient and practical means for generating reliable olfactory fMRI results. Such a technique can ultimately be applied in the clinical realm as a diagnostic tool for diseases associated with olfactory degeneration, including Alzheimer&#39;s and Parkinson&#39;s disease, as we begin to further understand the complexities of the human olfactory system.

We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

一种免费呼吸功能磁共振成像方法研究人类嗅觉功能

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

大鼠模型中的面部神经外科研究同氧抑制与再生

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

与嗅觉恩希廷格利亚合体培养的 Axotom 化大鼠视网膜甘利翁神经元，作为成人轴再生体外模型

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures&#160;throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

利用 体内 出生后电穿孔研究小脑颗粒神经元形态和突触发育

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

优化离 体 视网膜电图的设置和条件，以研究小眼睛和大眼睛的视网膜功能

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...