现任和前任鲁贝尔的实验室成员。

1。解剖区的制备<ol><li>连续泡沫人工脑脊髓液（学联）的95％O 2 / 5％的CO 2的混合物（pH值在7.2-7.4之间，渗透压295-310 mOsm / L ）。 </li><li>学联是冒泡，清洁的工作区用70％乙醇。同时清洁vibratome和切片刀片。 （DH 2 O），用蒸馏水冲洗刀片。 </li><li>清洁液体吸收垫放在解剖与适当的解剖工具面积。 </li><li>胶琼脂块（琼脂糖40毫克/毫升，即4％，在卫生署2 O）的一个会议厅vibratome切片阶段*。 *提示：事先准备琼脂剥离。行前在冰箱的有盖培养皿内的琼脂。琼脂糖从EMD的化学品或Invitrogen公司购买。 </li></ol> 2。 6孔板用培养液的制备<ol><li>在无菌罩，填补每6孔板的动物1 1毫升培养基孵育35 ° C和5％的CO 2 。 </li><li>许多文化膜插入需要准备和他们在以后使用的防护罩。 </li></ol> 3。鸡脑干隔离<ol><li>将蛋下一个明亮的光源，以确定胚胎（一般鸡蛋大的方面）的空气填充的空间无效。 </li><li>破开的鸡蛋大的一面露出膜囊。在膜囊与手术刀片轻轻取出的鸡胚胎的头部。 </li><li>快速斩首用剪刀头。 </li><li>刀片和眼睛之间略有后的广场一角。通过应用只会有轻微的压力的头骨一个喙到尾鳍正中切口*. *提示：施加的压力是依赖于胚胎的年龄（即，年轻的= =少，年纪较大的更多） </li><li>轻轻推开，暴露颅骨，并确认中线切开皮肤和羽毛。 </li><li>颅骨的延髓部分切片用刀片座。位置刀片后眼睛和整个颅骨和脑组织通过迅速削减* *提示：强大压力，要求彻底清除延髓部分。 </li><li>中线外侧切口，用小剪刀在头骨的尾部地区，稍前头部两侧可见颈部肌肉。拉开头骨和组织暴露小脑和大脑。 </li><li>轻轻地清除颅底脑干，小剪刀切割附着组织。一旦所有组织被切断，脑干应自由移动，远离颅骨* *提示：您可能需要翻转削减附着组织的头骨（大脑下来）。 </li></ol> 4。脑干用于切片的制备<ol><li>一旦从头骨取出，针脑干通过视神经tecta。 </li><li>完全删除轻轻切割小剪刀脚，露出地板第四脑室小脑。 </li><li>从表面用镊子脑干删除任何膜组织和血管，并通过视神经tecta横向削减隔离脑干。 </li><li>将少量的超级胶水，在前面的琼脂块vibratome阶段（对vibratome刀片）。 </li><li>使用镊子，轻轻地，迅速地解除脊髓和地方组织上的超级胶水的喙侧和背侧向vibratome刀片脑干* *提示：吸收多余的胶水应与一个金擦拭，或过滤下一步前纸。 </li><li>轻轻倒入vibratome阶段含氧学联*. *注：本学联可连续冒泡与O 2 / CO 2。然而，在这样一个解决方案，结果体积小（和可能造成的损害）的不必要的运动脑干切片学联冒泡。如果执行迅速，已经含氧从解剖学联是足够的。 </li><li> Vibratome刀片应在20-22 °角。开始vibratome（振荡应在最大振幅）和移动阶段对刀片，使组织的顶部是与刀片平行前切片。 </li><li>快速走向脑干组织的刀片。减慢大大前夕刀片与组织接触。 </li><li>开始与最慢的可能向前运动的组织切片。一旦通过整个组织的冠状切面，轻轻移动从刀片切片，用刷子或碎玻璃，发射抛光的吸管，一端适合用橡胶球。 </li><li>下阶段，在300-500微米的步骤再次切片。重复，直到解剖签名在脑干​​组织中可见。此时，片脑干组织包含在200-1000微米的听觉结构的厚度，取决于组织和实验需要的年龄。 </li><li>仔细切片，即为了保持可用片，第一片代表电路的最尾端的区域（低频率的声音处理），最后切片代表最喙节（高频processiNG）* *注：利用文化片的位置，去第6。 在体外生理存储，请参阅下一节。 </li></ol> 5。 在体外生理切片存储<ol><li>根据年龄的鸡胚胎和切片厚度，每只动物应提供1至6片。 </li><li>轻轻地放置在个别切片室用火抛光的玻璃吸管的一端装有一个橡胶球。商会应编号。将每口井只有一个切片，为了保持一定tonotopic的特殊性（例如，最尾端片室1在过去腔最延髓切片）。商会应充满学联，不断冒泡的95％O 2 / 5％的CO 2（pH值7.4，渗透压295-310 mOsm / L）的混合物。 </li><li>允许切片放入一个温水浴（36℃）约1小时的会议厅，以平衡。 </li><li>删除从温暖的沐浴室，并允许在室温下休息约半小时。 </li><li>片在室温下半小时后，可以从持有室被转移到了〜0.5毫升的录音室，安装了电生理实验显微镜。 </li><li>切片可用于〜6小时后，从温水浴中取出。 </li></ol> 6。器官型切片文化的制备 8 <ol><li>紧随切片过程（见第6章）转让片〜500μL用一个火抛光的玻璃吸管的一端装有一个橡胶球，48孔板冰学联。一个片，每孔* *提示：切片需要至少300微米和微米厚可达1000。 </li><li>已收集切片后，转让的48孔板引擎盖。 </li><li>取出孵化器中的6孔板培养液出，传送到引擎盖。 </li><li>之前把从单一的大脑切片上膜，放置准备膜插入到良好的培养基（见2.2节）。确保无气泡膜目前。 </li><li>使用的玻璃吸管，转移的学联下降到文化膜切片和消除过剩与微学联。重复剩余片（膜片最大的4％）* *提示：确保片不互相接触和地方片膜的中央，让他们不要触摸膜边缘。 </li><li>根据需要尽可能多的脑切片重复* *提示：如果文化是操纵在稍后的时间点，它可能是有利的，限制每6孔板膜量3，以减少时间的文化是从孵化器中删除。 </li><li>改变培养基的3倍引擎盖下一个星期：旧介质膜插入解除以及一双镊子取出一个用无菌提示真空管。加入1毫升的新鲜培养液，而膜插入以及解除。当所有介质改变，放盘进入孵化器。 </li></ol> 7。代表性的成果： <img src="/files/ftp_upload/2527/2527fig1.jpg" alt="图1" /> 图1。鸡听性脑干双耳电路 （一）一个冠状断面示意图（BF）的体外鸡听性脑干切片图像。 （一）中的电路显示了从听觉神经（AN）的传入细胞核magnocellularis（nm）和核angularis（NA）的兴奋性输入。网管项目核laminaris（NL）的双边脑干双方的兴奋性输入。抑制输入NA，新墨西哥州和NL，从上橄榄核（SON）的项目。 在体外片（BF）的序列图像（200微米（二）从尾椎鸡听性脑干延髓（F）的每个），分别代表从低到高频率地区，。电路负责编码时间的声音，主要用于声音定位的属性。比例尺（二）= 500微米，适用于在图像（CF）。 （四）点箭头单细胞NL身体层。 <img src="/files/ftp_upload/2527/2527fig2.jpg" alt="图2" /> 图2。培养的神经元发展正常的生理反应性能 。叠加膜电压变化的响应极化和去极化急性集团（A＆C）和4（B）和7（四）文化日（DIC）的培养组织从当前步骤。在极化电压“凹陷”，在强烈的外向电流减少，以及单个AP发射发展相比，年龄相当于急性组织切片文化的方法同样注意变化。流动注射200毫秒，50-100 Pa的步骤。制冷剂管理计划是-55和-60 mV的之间。 <img src="/files/ftp_upload/2527/2527fig3.jpg" alt="图3" /> 图3。在文化SLI的解剖结构CES E11鸡（DIC）的7天中文化后的听觉脑干的左半边。对微管相关蛋白2（MAP2），胞体和树突中发生的抗体，标记为绿色。通过一个Alexa右旋糖酐染料电核magnocellularis（NM）神经细胞和其轴突群集在红色标记。 NM和NL细胞系是可见的。同侧网管轴突终端可以看到投射到背NL树突（白色箭头）。

<ol>
	<li>Howard, M. A., Burger, R. M., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+switch+to+GABAergic+inhibition+dependent+on+increases+in+Kv1-type+K++currents.">A developmental switch to GABAergic inhibition dependent on increases in Kv1-type K+ currents.</a> J Neurosci. 27, 2112-2123 (2007).</li><li>Kuba, H., Koyano, K., Ohmori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+membrane+conductance+improves+coincidence+detection+in+the+nucleus+laminaris+of+the+chicken.">Development of membrane conductance improves coincidence detection in the nucleus laminaris of the chicken.</a> J Physiol. 540, 529-542 (2002).</li><li>Gao, H., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+of+intrinsic+and+synaptic+properties+of+chicken+nucleus+laminaris+neurons.">Early development of intrinsic and synaptic properties of chicken nucleus laminaris neurons.</a> Neuroscience. 153, 131-143 (2008).</li><li>Sanchez, J. T., Wang, Y., Rubel, E. W., Barria, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+glutamatergic+synaptic+transmission+in+binaural+auditory+neurons.">Development of glutamatergic synaptic transmission in binaural auditory neurons.</a> J Neurophysiol. 104, 1774-1789 (2010).</li><li>Jackson, H., Hackett, J. T., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+development+of+brain+stem+auditory+nuclei+in+the+chick:+ontogeny+of+postsynaptic+responses.">Organization and development of brain stem auditory nuclei in the chick: ontogeny of postsynaptic responses.</a> J Comp Neurol. 210, 80-86 (1982).</li><li>Rubel, E. W., Parks, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+development+of+brain+stem+auditory+nuclei+of+the+chicken:+tonotopic+organization+of+n.+magnocellularis+and+n.+laminaris.">Organization and development of brain stem auditory nuclei of the chicken: tonotopic organization of n. magnocellularis and n. laminaris.</a> J Comp Neurol. 164, 411-433 (1975).</li><li>Rubel, E. W., Smith, D. J., Miller, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+development+of+brain+stem+auditory+nuclei+of+the+chicken:+ontogeny+of+n.+magnocellularis+and+n.+laminaris.">Organization and development of brain stem auditory nuclei of the chicken: ontogeny of n. magnocellularis and n. laminaris.</a> J Comp Neurol. 166, 469-489 (1976).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37, 173-182 (1991).</li><li>Reyes, A. D., Rubel, E. W., Spain, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+properties+underlying+the+firing+of+neurons+in+the+avian+cochlear+nucleus.">Membrane properties underlying the firing of neurons in the avian cochlear nucleus.</a> J Neurosci. 14, 5352-5364 (1994).</li><li>Monsivais, P., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accommodation+enhances+depolarizing+inhibition+in+central+neurons.">Accommodation enhances depolarizing inhibition in central neurons.</a> J Neurosci. 21, 7823-7830 (2001).</li><li>Lu, T., Trussell, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+elimination+of+endbulb+synapses+in+the+chick+cochlear+nucleus.">Development and elimination of endbulb synapses in the chick cochlear nucleus.</a> J Neurosci. 27, 808-8017 (2007).</li><li>Kuba, H., Yamada, R., Fukui, I., Ohmori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tonotopic+specialization+of+auditory+coincidence+detection+in+nucleus+laminaris+of+the+chick.">Tonotopic specialization of auditory coincidence detection in nucleus laminaris of the chick.</a> J Neurosci. 25, 1924-1934 (2005).</li><li>Trussell, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+for+preservation+of+timing+in+central+auditory+pathways.">Cellular mechanisms for preservation of timing in central auditory pathways.</a> Curr Opin Neurobiol. 7, 487-492 (1997).</li><li>Trussell, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+mechanisms+for+coding+timing+in+auditory+neurons.">Synaptic mechanisms for coding timing in auditory neurons.</a> Annu Rev Physiol. 61, 477-496 (1999).</li><li>Sorensen, S. A., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+level+and+integrity+of+synaptic+input+regulates+dendrite+structure.">The level and integrity of synaptic input regulates dendrite structure.</a> J Neurosci. 26, 1539-1550 (2006).</li></ol>

几十年来，鸡的脑干急性切片准备工作已用于研究听觉处理9，10 。这种方法提供了大量的体外生理双耳处理的数据，从发展和成熟的状态4，11 ，12。被知道关于这个高度专业化的电路和每个细胞核中的作用时间处理的声音13，14次。事实上，短期的实验操作和特点电路的结构和功能的影响已被证明有利 15 。但是从长远发展的角度，这样的操作不可能和技术?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Chicken ACSF</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>M-11624</td>
<td>130 mM</td>
</tr>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>M-10576</td>
<td>26 mM</td>
</tr>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-9333</td>
<td>3 mM</td>
</tr>
<tr>
<td>NaH2PO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S-8282</td>
<td>1.25 mM</td>
</tr>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G-7528</td>
<td>10 mM</td>
</tr>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>M33-500</td>
<td>1 mM</td>
</tr>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>423525000</td>
<td>2 mM</td>
</tr>
<tr>
<td>Chicken culture medium (store at 4&deg;C)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>advanced minimum essential medium (with NEAA, sodium pyruvate at 110 mg/l, without L-glutamate)</td>
<td>&nbsp;</td>
<td>Gibco Invitrogen</td>
<td>12492-013</td>
<td>48.00%</td>
</tr>
<tr>
<td>Earl's balanced salt solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E-2888</td>
<td>24.00%</td>
</tr>
<tr>
<td>L-glutamine (200 mM)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G-7513</td>
<td>1.00%</td>
</tr>
<tr>
<td>Glucose solution (200 g/l, sterile filtered)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G-7528</td>
<td>2.75%</td>
</tr>
<tr>
<td>horse serum (heat inactivated, sterile filtered)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H-1138</td>
<td>24.00%</td>
</tr>
<tr>
<td>Penicillin-streptomycin*</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-0781</td>
<td>1.00 ml 
* = Add to 100 ml culture medium if needed to prevent contamination</td>
</tr>
<tr>
<td>Specific equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Millicell-CM cell culture inserts</td>
<td>PICMORG50</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6-well plate</td>
<td>6 Well Cell Culture Cluster</td>
<td>Corning Incorporated</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>Forma Scientific CO2 Water Jacketed Incubator</td>
<td>Forma Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

preparation and culture of chicken auditory brainstem slices

鸡听性脑干是一个完善的模型系统，已广泛应用于研究谨慎时期的发展1-4以及5-7在中枢神经系统的时空编码机制的解剖和生理听觉处理。 在这里，我们提出了一个方法来准备鸡听性脑干片可用于急性实验程序或长期的实验操作的器官切片文化。 鸡听性脑干是由原子核angularis，magnocellularis，laminaris和优越的橄榄油。这些原子核负责双耳进行无害化处理和单一的冠状片的准备工作，保持整个电路。最终，器官切片文化可以提供机会，如突触活动，前和突触后成分的表达，表达方面控制的兴奋性和差异表达基因操纵一些发展参数这种方法可用于扩大对神经回路的发展，完善和成熟的一般知识。

Watch this Scientific Journal Video about Preparation and Culture of Chicken Auditory Brainstem Slices at JoVE.com

Preparation and Culture of Chicken Auditory Brainstem Slices

鸡听性脑干是由原子核负责双耳进行无害化处理。一个单一的冠状切片制剂保持整个电路，而培养的方法，在分子，细胞和网络水平研究神经细胞结构和听觉功能的发展提供了一个独特的准备。

鸡脑干切片的制备及文化

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

preparation-and-culture-of-chicken-auditory-brainstem-slices

Research

JoVE Journal

Neuroscience

11.0K 次观看.  University of Washington. 鸡听性脑干是由原子核负责双耳进行无害化处理。一个单一的冠状切片制剂保持整个电路，而培养的方法，在分子，细胞和网络水平研究神经细胞结构和听觉功能的发展提供了一个独特的准备。

视频: Preparation and Culture of Chicken Auditory Brainstem Slices

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

低聚β-淀粉样蛋白的制备 1-42和突触可塑性损伤诱导海马脑片

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

科研

神经科学

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

鸡胚胎脊髓片培养协议

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

长时程增强一个非常稳定的和可重复的记录和保存海马鼠标片制备工艺的改进

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

斜脊髓切片用于神经前根刺激的制备

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

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

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

成年小鼠视网膜电生理研究水平切片的制备

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

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

产后大鼠听觉脑干反应及外毛细胞全细胞贴片钳记录

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

c57bl6 小鼠耳蜗植入术与电唤起脑干反应记录

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

头脊髓和薄片体外制备心律失常活性

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Methods Article

鸡脑干切片的制备及文化