Name: a method to target and isolate airway-innervating sensory neurons in mice
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支持美国国立卫生研究院授予R01HL105635到SEJ。笔者想感谢迭戈五Bohórquez的技术咨询。我们也感谢R.伊恩·卡明技术援助，并在杜克大学人类疫苗研究所流式细胞仪共享资源基金（北卡罗来纳州达勒姆）进行流式细胞术流。流式细胞仪在杜克区域生物控制实验室，收到来自美国国立卫生研究院国家过敏和传染病研究所（UC6-AI058607）建设部分支持执行。

涉及受试动物的程序已通过杜克大学的机构动物护理和使用委员会（IACUC）。 1.快速的蓝色鼻腔给药对于耐晒蓝，安乐死鼠标前至少2天管理该染料。染料将持续长达八个星期。 <ol><li>麻醉光吸入麻醉（2.5％七氟醚），鼠标，直到呼吸开始放缓。 </li><li>用200μl的移液管与过滤提示，以慢慢灌输的染料溶液40微升（0.4毫坚牢蓝，1％二甲亚砜，二甲基亚砜，在磷酸盐缓冲盐水，PBS）中插入鼻孔，暂停偶尔以确保鼠标抽吸溶液（ 图1A）。 </li><li>按住鼠标垂直，头抬起，轻轻按摩胸部，以确保染料扩散整个肺部。 </li></ol> 2.准备在解剖和分析日<ol><li>准备10毫升0;神经节解离溶液（GADS）由如表1中指定组合的成分。 </li><li>在一个专门的实验室面积进行RNA提取。之前开始的实验中，清洁的表面用70％乙醇，10％的漂白剂，和RNase去污试剂。这包括RNA离心机，任何管架和吸管。确保有只RNA使用专用过滤嘴框。不要使用此设备和区域DNA的准备，因为这会污染样品。 </li><li>混合新鲜的70％和80％的乙醇与无RNase水。制备1毫升，每样品中RNA提取使用。 </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td> 成分 </td><td> 量 </td></tr><tr><td>高级DMEM / F12 </td><td> 9.5毫升</td></tr><tr><td>谷氨酰胺</td><td> 100微升</td></tr><tr><td> HEPES（10毫摩尔） </td><td> 100微升</td></tr><tr><td> N2补充</td><td> 100微升</td></tr><tr><td> B27（无维生素A） </td><td> 200微升</td></tr><tr><td> NGF（50微克/毫升） </td><td> 10微升</td></tr></tbody></table> 表1. 试剂混合物的神经节解离溶液（GADS）。 3.解剖程序<ol><li>填1.5毫升管用500μlGADS，每个实验样品，一个用于正排序控制，一个用于一个负排序控制。在整个解剖刨冰店管。 </li><li>解剖出来的结状神经节，使两个部分削减便于分离，然后把它在实验样品管。请参考下面的朱庇特的协议结状神经节16和17 DRG解剖。 </li><li>解剖和部分切割未标记神经节，如背根神经节（DRG），用于排序控制。使用两个神经节为阳性对照和两个神经节一个阴性对照。 </li><li>为了获得成功分拣足够的结状神经元，从5只小鼠神经节合并到含有GADS 1实验样品管中。 </li></ol> 4.感觉神经节解离<ol><li>吸取了500微升GADS，使神经节在管。通过加入1ml的PBS洗一次神经节，等待神经节沉降到试管底部，然后吸取出来的PBS。 </li><li>加入1 ml GADS和22微升消化酶（胶原酶I / II和蛋白酶混合，2.5毫克/毫升的H 2 O）到每个管中。 </li><li>加入20μl坚牢蓝（5.26毫摩尔）与阳性对照管。 </li><li>把管子中已经设定在37℃水浴或加热块。 注意:神经节消化的时间取决于消化酶的年龄。 1个月内的消化酶溶解，消化30分钟，在2 - 6个月消化45分钟。如果酶是超过6个月大，消化神经节˚F或60分钟。 </li><li>每15分钟摇动管短暂轻弹它们确保神经节被溶液覆盖。 </li><li>的同时，将消化神经节，准备使用粒子溶液（涂有聚乙烯吡咯烷酮的胶体二氧化硅粒子）的密度梯度。 <ol><li>在混合GADS 12％颗粒溶液，每个样品500微升。 </li><li>在混合GADS 28％颗粒溶液，每个样品500微升。 </li><li>慢慢地，小心地加上400微升28％密度的解决方案前400微升12％密度的解决方案在一个新的1.5 ml管每个样品。不要让两层混合。两个不同的层应当是在管可见;其中一个比另一个更暗。 </li></ol></li><li>尽管正在消化神经节，标注新的1.5 ml收集管进行排序（一个耐晒蓝正一耐晒蓝负面每个分拣样品管）。取决于分类设施的要求，这些管必须具有可拆卸的盖子，以便不地干扰Ë与细胞分选仪。 </li><li>合适的消化时间之后，删除与消化酶GADS并用1ml PBS洗涤神经节两次。加入200μl新鲜GADS每管。 </li><li>使用200微升移液器，设置为100微升体积和神经节吸管上下数次以打散细胞。重复，直到没有一块完好的组织能够被看见。避免在溶液产生气泡。 </li><li>通过一个70微米的细胞滤网通过解离的细胞。 </li><li>另外100微升GADS添加到原始解离管拾取留下的任何剩余杂散细胞并穿过细胞过滤的附加的解决方案。使用新的移液管尖，收集已经通过过滤器传递，并紧贴着网状任何剩余的含细胞的液体。悬浮于GADS细胞的最终体积为300微升。 </li><li>仔细层上的以前所作密度梯度的顶部的300微升的细胞。避免气泡和c的混合厄尔与密度层。 </li><li>在在RT 2900 xg离心离心10分钟。 </li><li>而细胞在离心机，如下混合裂解缓冲液（1毫升，每实验样品）:10微升2-巯基乙醇在1毫升缓冲液RLT（从RNA提取试剂盒）。 <ol><li>加入300微升裂解液固蓝积极收集管和600微升至耐晒蓝负面收集管（步骤4.7标记管）。这个量取决于预期要收集的细胞数。对于&lt;2,000个细胞，加300微升，对于&gt; 7000细胞添加600微升。这将确保分拣鞘流体不显著稀释裂解缓冲液。 </li></ol></li><li>一旦离心完成后，小心地取出并丢弃前700微升层。这一层包含的大多数细胞碎片，将与其它细胞分选干涉。 </li><li>加入700μl新鲜GADS包含解决方案和吸管剩余的细胞TE上下几次混合。 </li><li>离心15分钟2,900 xg离心以沉淀细胞。 </li><li>而细胞在离心机，混合加入5微升DNA酶（10毫克/毫升）至1毫升GADS排序GADS（每个样品500微升）。 </li><li>一旦离心完成后，小心地取出弃上清，留​​下细胞沉淀。 </li><li>重悬在200细胞沉淀 - 通过上下吹打与1000微升吸管设定为200微升几次300μl的排序GADS。样品放在冰上。现在，细胞准备好进行排序。 </li></ol> 5.荧光激活细胞（FAC）排序注:本部分要求FACS分拣机的操作知识或技术人员的帮助。 <ol><li>加入1μl碘化丙啶（PI）的储液（50微克/毫升）至每个样品，以测试细胞生存力。拆分阴性对照分为2管，其中一个PI和一个没有。 PI只有在死细胞结合DNA。如果后的前几个种类有标签，不需要使用PI的无死细胞。 </li><li>交通运输细胞流式细胞仪， 例如 ，BD流式细胞仪AriaII运行歌姬8软件，冰上。 </li><li>由于单元尺寸在这一人群中是可变的，使用大喷嘴（100微米）进行排序。以降低应力的单元经验的量和提高细胞生存力使用低压（20磅）。 </li><li>使用FACS分拣机软件来设置分析图，前向散射，侧散射（SSC），坚牢蓝，和PI，如果需要的话。 </li><li>要重新悬浮细胞，加载到分拣机前，然后振荡样品简单。 </li><li>与阴性对照，没有固蓝或PI开始，设置栅极阈值（ 图2A）。这将决定在细胞群的自体荧光的量。可选:在与PI阴性样品，以确定是否有死的细胞群。 </li><li>排序的阳性对照样品（INC与耐晒蓝ubated），设定补偿参数。 </li><li>一旦盖茨和参数设置，开始整理实验样品。样品被分类成含RNA裂解缓冲液制备的收集管。 </li><li>保持冰样本排序，直到细胞分选已完成。 </li><li>开始小区进行排序之后立即提取RNA步骤。 </li></ol> 6. RNA提取<ol><li>在裂解缓冲液涡旋分选的细胞1分钟至均质。 </li><li>移液器向上和向下几次加1体积70％的乙醇和混合。 </li><li>转移到700微升样品，以旋转柱。离心机在8000×g下15秒。丢弃的流量通过。 <ol><li>重复，直到样品的总体积是通过自旋柱。 </li></ol></li><li>添加350微升缓冲液RW1和继续该RNA纯化过程，包括DNA酶处理步骤中，根据制造商的protocol对于细胞。 </li><li>一旦RNA被提取，使用根据制造商的规格的微流体电泳系统检查RNA质量。 </li></ol>

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这个协议描述为目标气道神经支配的神经元中的迷走神经的节状神经节的方法。一旦标记的神经节轻轻解​​离最佳地保持细胞数量和生存力。这些神经元然后FAC直接分类到裂解缓冲液和RNA提取。此协议的意义是定位，隔离和保持一个特定的感觉细胞群体的质量的能力。基因表达在神经元中的这个小群体描述，以及特定器官的功能和神经网络识别。 在这个协议中的关键步骤包括:通过分离过程，病程进展快随后的细胞分选。它来安排排序时间，使得它离解后，立即开始是很重要的。它同样重要的是所有的RNA提取试剂是新鲜，70％和80％的乙醇溶液制成新鲜，以及用于RNA提取所有管道和设备干净，不含RNase。 这个协议可以被修改，使得细胞被单独地分类成单细胞RNA提取和测序孔板中。它也可以用于分离其它感觉神经节细胞，例如背根神经节（背根节）。不同的身体部位的染料注入追溯的DRG已经先前所述17。该协议也可以被修改，以收集用于功能分析的细胞。代替分拣细胞进入裂解缓冲液，将细胞分类成神经细胞培养基，GADS。这些细胞然后培养并用于功能研究如钙成像，或电。 如果耐晒蓝积极的人口比预期的结果越小，购买新的消化酶。消化酶的年龄相关的消化效率。消化酶需要为1年购买的内使用。在将细胞数高的情况下，RNA提取，品质测试。如果所述RNA被降解，这是一指示该试剂是不新鲜，或已被污染。在这种情况下，一定要彻底清理所有RNA提取方面，移液管，衣架，和任何会与样品管接触。做出新的RNA酶的DNA酶和70免费％和80％的乙醇。用于制造这些溶液中的水和乙醇也应保持从其他实验室用品分开并仅用于RNA提取使用。 FAC分选是用于分离固蓝阳性细胞快速而有效的方法，但是，一个限制是，细胞密度应为每毫升缓冲液1〜2百万个细胞。该协议将电流推细胞分选仪模型的极限。细胞在结状神经节的数目是小的。以获得一个细胞产量足以用于细胞分选，从五只动物的细胞合并。中描述的排序容积为200 - 300微升，具有更少的超过10万悬细胞。 ŧ他转换为每毫升450,000细胞，推荐浓度更低的浓度。一种替代使用细胞分选仪是用荧光显微镜和玻璃吸管24至精选。这种技术是比较慢，并已用于收集30 - 100个细胞一次。因此，与现有方法这个协议提供了更快的高通量方法。 这个协议的未来应用包括确定气道支配神经的特定人群的转录配置文件的能力。使用此协议中收集的高品质的RNA可用作cDNA合成和下一代测序和其他技术的模板来表征所收集的神经元的转录。以这种方式，可以判断哪些基因或通路是特定于这些神经元。这些信息将有助于阐明这些神经元在正常生理条件下发挥的职能作用。一旦基本表达模式已经建立，该系统可以通过物理和化学刺激或病原体的挑战，以确定它们对肺支配神经元基因调控的影响。最后，通过确定哪些基因被上调，我们可以发现新的药理学目标，并开始进行调查的治疗剂的效果与在气道疾病的神经功能的急性和慢性改变干涉。

体感神经转导热，机械，化学，和引起的内源和环境因素有害刺激。这些传入神经的细胞体存在于感觉神经节，如背根，三叉神经痛，或节状神经节。每个感觉神经节支配的身体的特定区域，并包含支配该区域内的单独的器官和组织的细胞。例如，背根神经节（DRG）位于脊柱和整个身体和四肢延伸过程，而三叉神经节位于头骨，包含支配脸，眼睛，脑膜或上气道1的神经元， 2。迷走神经的节状神经节位于颅骨下颈部和包含在整个胃肠道，心脏扩大的神经纤维细胞体，和下呼吸道和肺部3。在人类的结状神经节是独立的，但是，在小鼠它所稠合与颈神经节，这也支配肺部4。此稠合神经节通常被称为颈/结状复杂，迷走神经的神经节，或简单地结状神经节5。这里，它被称为结状神经节。 结状的传入纤维从内脏信息传递到脑干的孤束（NTS）的核心。感觉输入到这个独特神经节控制的功能，例如肠能动性6，心脏速率7，呼吸8,9，和刺激性激活呼吸道反应-10,11-一个多样化。同的功能和神经支配的器官这种多样性，这是定位和分离结状神经节的器官特异性亚群为了研究个体神经通路的关键。然而，鉴于结状的小尺寸和神经元的有限数量的包含这不是一个简单的任务。每只小鼠结状神经节中含有大约5000元12除了支持卫星细胞的广泛的人口。 5％支配气管 - 5000结状神经元中，只有3。因此，气道神经支配的神经元内由于呼吸刺激或病状的任何功能，形态或分子变化，将在密集结状神经节丢失。 为了解决这个问题，一种方法的开发是为了鉴定和分离那支配气道的神经元。呼吸道暴露于荧光示踪染料，以确定在随后支配结状神经元。耐晒蓝是由神经元拾起并迅速传播到它保留长达八周的13元胞体- 15。一旦确定，温和的，但有效的，解离的协议被用来保存染料标记和细胞生存力荧光活化的细胞（FAC）排序。分选的细胞用于提取高质量核糖核酸（RNA），以确定基因表达或f或其他下游分子分析。这个协议提供了用于分离该支配的关心组织的感觉神经元的有用和健壮的技术。

<table><tbody><tr><td>Fast Blue</td><td>Polysciences, Inc.</td><td>17740-2</td><td>stock 2 mg/ml in water</td></tr><tr><td>NeuroTrace 530/615 red Nissle stain</td><td>Life Technologies</td><td>N21482</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>D128-500</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS) Ca and Mg free</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Advanced DMEM/F12</td><td>Gibco</td><td>12634-010</td><td></td></tr><tr><td>glutamine (Glutamax)</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>N2</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>B27 (no vitamin A)</td><td>Gibco</td><td>12587-010</td><td></td></tr><tr><td>Nerve Growth Factor (NGF)</td><td>Sigma</td><td>N6009</td><td>stock 50 &amp;micro;g/ml in PBS/10% FBS</td></tr><tr><td>digestion enzyme, Liberase DH Research Grade</td><td>Roche</td><td>5401054001</td><td>stock 2.5 mg/ml in water</td></tr><tr><td>particle solution (Percoll)</td><td>Sigma</td><td>P1644-25ML</td><td></td></tr><tr><td>Heating block</td><td>LabNet</td><td></td><td></td></tr><tr><td>70 &amp;micro;m cell strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>Absolute Ethanol (200 proof)</td><td>Fisher Scientific</td><td>BP2818-500</td><td></td></tr><tr><td>RNase free water</td><td>Fisher Scientific</td><td>BP2484-100</td><td></td></tr><tr><td>RNase decontamination reagent, RNase AWAY</td><td>invitrogen</td><td>10328-011</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>VWR</td><td>EM-6010</td><td></td></tr><tr><td>RNA extraction kit, RNeasy Plus Micro Kit</td><td>Qiagen</td><td>74034</td><td></td></tr><tr><td>DNase kit, RNase-Free DNase Set</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>DNase</td><td>Sigma</td><td>D5025-15KU</td><td>stock 10 mg/ml in 0.15 M NaCl</td></tr><tr><td>Propidium Iodide</td><td>Sigma</td><td>P4170-10MG</td><td>stock 10 &amp;micro;g/ml in PBS</td></tr><tr><td>Microfluidic electrophoresis system (TapeStation 2200)</td><td>Agilent</td><td></td><td></td></tr></tbody></table>

a method to target and isolate airway-innervating sensory neurons in mice

体感神经转导热，机械，化学，和引起的内源和环境因素有害刺激。这些传入神经元的细胞体位于感觉神经节内。感觉神经节支配身体的特定器官或部分。例如，背根神经节（DRG）位于脊柱和整个身体和四肢延伸工艺。三叉神经节位于头盖骨和支配面，和上呼吸道。结状神经节的迷走神经传入整个肠道，心脏和肺延长。结状神经元控制的功能，例如一个多样化:呼吸率，气道发炎，和咳嗽反射。因此，要理解和操纵它们的功能，这是至关重要的，以鉴定和分离气道特定的神经元亚群。在小鼠中，气道暴露于荧光示踪剂染料，坚牢蓝，对特定气道结状神经元逆行示踪秒。结状神经节解离和荧光激活细胞（FAC）排序是用来收集染料阳性细胞。接着，高品质的核糖核酸（RNA）从用于下一代测序染料阳性细胞中提取。使用特定的神经元基因表达被确定这种方法的气道。

使用这种方法，气道神经支配的神经元通过鼻内滴注坚牢蓝（ 图1A）标记。两天后，固蓝标记的细胞出现在节状神经节（ 图1C）。这些细胞构成了3 - 结状神经节的神经元总人口的5％。已用于此目的的其他逆行染料包括的DiI（1,1&#39;-双十八烷基-3,3,3&#39;，3&#39;- tetramethylindocarbocyanine高氯酸盐）和荧光金。肺暴露的DiI标签结状神经元。但是，的DiI花费更长的时间到达神经节，7 -滴注18,19后14天，使用因此一个主动转运染料。荧光金是也被用于标记的气道神经元14,20的主动转运染料。根据我们的经验，但是，这种染料一旦在结状神经节它很快就被卫星细胞而不是神经元拾起达到的细胞体（数据未显示）。快速蓝色是一个备用主动运输荧光染料，成功地，迅速 ​​，标签大鼠21及气道结节支配神经13,14 DRG。该耐晒蓝的方法，因此，会出现更强大的和可重复的。 的结状神经节的解剖以前已经描述16和它的位置在图1B中简要地示出。消化开始节状神经节被切除之后。为了防止聚集在一起的神经元，并允许一个更清洁的排序，使用温和的消化酶，清除任何碎片，并添加RNA酶的神经节排序的解决方案。多消化酶混合料有效性测试。温和的消化酶混合物22被审判，但是，对于成人节消化它不是强大到足以掰开细胞及时。可替换地，以前使用的木瓜蛋白酶的23组合，蛋白酶，和胶原酶的酶混合物，被证明是过于激进和引起不希望的细胞死亡（数据未显示）。概述的消化协议，使用指定的酶混合物，被认为是理想的具有高细胞活力及时消化。 以前的方法已经使用手册细胞用玻璃吸管标记的细胞与未标记的细胞24分离采摘。这种技术更适合于少数细胞的单细胞分析。然而，48个细胞/ hr的最大速率这种技术是不切合实际的数百或数千的细胞的整个人群需要被收集。当目标是分离高质量的RNA，时间成为关键变量。以尽快开始RNA提取，以防止降解是重要的。 FAC分选允许整个标记的人口的集合中〜45分钟和RNA提取后立即开始。 ○:保together.within页="1"&gt;当排序的神经元它从破碎神经元突起清除尽可能多的碎片是很重要的。密度梯度用来从离解的样品清除碎屑。它在足够高的力到离心机以确保C-纤维神经元，其中有一个小的半径，〜5微米24，通过梯度被迫是重要的。 图2A和B显示用于设置分配门的阴性和阳性对照。如在图2C中可以看出，解离的固蓝标记结状细胞分类成两个群体。从该样品所收集的细胞数列于图2D。分拣效率此方法是73 - 85％。当神经元进行排序前向散射和侧向散射参数没有添加特异性。因为神经元不是球形这些参数均无法可靠地报告小区大小。 ENT"FO:保持-together.within页="1"&gt;对于高质量的RNA产量，细胞需要直接分类到裂解缓冲液和RNA应立即分拣的微流体电泳系统用于测试之后被提取。的RNA（ 图3）的质量。的18S和28S峰通过用于计算的RNA完整性数（RIN）凝胶电泳来确定。对于RNA测序样品RIN应大于7。这RNA样品现在准备待测序。 <img alt="图1" src="/files/ftp_upload/53917/53917fig1.jpg" /> 图1.气道元支配神经元的固蓝标记在迷走神经的节状神经节。 A）耐晒蓝滴鼻的表示进入肺部。下光七氟醚麻醉，吸管固蓝的PBS的40微升含有1％的DMSO到小鼠的鼻孔，确保鼠标在流体抽吸。 <s仲&gt; B）表示将结状神经节所在的图。它显示为迷走神经，从而沿颈动脉。C同时运行的一个增厚）至少2天后，固蓝出现在节状神经节，标记气道的神经元。结状的复染用红色荧光染色尼氏，这些污渍所有神经元。比例尺为100微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53917/53917fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53917/53917fig2.jpg" /> 图2.荧光激活细胞（FAC）排序盖茨为固蓝阳性细胞。要设置分配门使用负（A）和阳性（B）的控制，以建立固蓝阳性群体。阴性对照组（A）的分离DRG细胞届在没有支配肺部。阳性对照（B）的解离了在体外用固蓝孵育，它们已被解剖后的DRG细胞。由于游离神经元的非球形形状，前向散射和侧向散射参数不会帮助定义细胞群。C）代表的排序的解离结状小鼠的气道已暴露于固蓝。 的D节细胞）的细胞计数为（C）。收集超过1000耐晒蓝（FB）细胞。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53917/53917fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53917/53917fig3.jpg" /> 图3. RNA质量和RNA完整性（RIN）的分配使用微流体电泳系统的评估。使用选rophoresis系统RNA的质量，从描绘凝胶图像上的18S和28S的峰进行计算。较低的峰代表降解RNA。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53917/53917fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice at JoVE.com

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

一种方法为Target和隔离气道支配感觉神经元的小鼠

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

a-method-to-target-isolate-airway-innervating-sensory-neurons

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

13.1K 次观看.  Yale University. Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

视频: A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

科研

生物学

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

面神经干切断小鼠:模型，研究运动神经元损伤反应

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

医学

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

在鼠背耳蜗核的听觉通路的光遗传学刺激直接可视化

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

神经科学

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

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

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

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

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

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

在小鼠犁鼻急性组织切片定义细胞群的深入生理学分析

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

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 ...

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

靶向小鼠交感神经元：一种腰交感神经切除术方法

Surgical Lumbar Sympathectomy in Mice

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays many critical roles in maintaining bodily homeostasis, but it has rarely been studied in the context of peripheral nerve injury. The extent of postganglionic sympathetic neuronal functions in distal targets in the periphery is currently unclear. To better explore the role of sympathetic innervation of peripheral targets, a surgical &#34;knock-out&#34; model provides an alternative approach. Although this can be achieved chemically, chemical destruction of postganglionic sympathetic neurons can be nonspecific and dose-dependent. The use of a surgical lumbar sympathectomy in mice, once thought to be &#34;virtually not practicable&#34; in small animals, allows for specific targeting of postganglionic sympathetic neurons that innervate the hind limbs. This manuscript describes how to surgically remove the L2-L5 lumbar sympathetic ganglia from a mouse as a survival surgery, which reliably decreases the hind paw sweat response and the number of sympathetic axons in the sciatic nerve.

作者聚焦：探索交感神经元的可塑性

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue targets.

小鼠手术腰交感神经切除术

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays ...

一种方法为Target和隔离气道支配感觉神经元的小鼠

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此视频中的章节

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

面神经干切断小鼠:模型，研究运动神经元损伤反应

在鼠背耳蜗核的听觉通路的光遗传学刺激直接可视化

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

在小鼠犁鼻急性组织切片定义细胞群的深入生理学分析

斜脊髓切片用于神经前根刺激的制备

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

小鼠手术腰交感神经切除术

小鼠手术腰交感神经切除术

小鼠手术腰交感神经切除术