作者要感谢哈特维格·维博尔德的出色技术援助，以及汉堡-埃彭多夫大学医学中心的UKE显微镜成像设施（Umif）提供显微镜和支持。这项研究由DZHK（德国心血管研究中心）[FKZ 81Z4710141]资助。

所有涉及动物的程序都得到汉堡州动物护理和使用委员会（ORG870，959）和北莱茵-威斯特法伦州自然、环境和消费者保护局（LANUV，07/11）的批准，并符合国家卫生研究院的实验室动物护理和使用指南（2011年）。研究使用男性和女性（10-24周）C57BL/6小鼠（库存号000664，杰克逊实验室）和小鼠同源性（db/db）或异质性（db/het:控制）为糖尿病自发突变（Leprdb:Bks.Cg-Dock7m+/+ 勒普db/J，股票号000642，杰克逊实验室）。作者已经使用手头的协议，对60周以上的小鼠没有变化。
1. 木质刺结的定位和解剖
注:尽管描述和图纸大多在较大的物种中可用，但一些出版物以前曾分别使用解剖方法和荧光记者线描述SG在24 只大鼠和25 只小鼠中的位置。
<ol><li>准备50mL的冰冷（3-4°C）肝素化（20单位/mL，见 材料表）磷酸盐缓冲盐水（PBS）。在室温下对 SG 进行解剖 （RT）。</li>
	<li>根据机构和地方指南，通过吸入3%-5%异黄素对鼠标进行深度麻醉。通过失去踏板戒断反射来验证足够的麻醉。斩首小鼠或进行颈椎错位。 注:不正确的颈椎错位可能导致脊椎破裂和胸血管损伤，导致出血，从而阻碍交感链的准备或切除，使SG处于不正确的位置。因此，让有经验的人员在深度消沉中进行颈椎错位或斩首动物是至关重要的。</li>
	<li>用乙醇喷洒皮肤，用梅奥剪刀和窄图案钳子沿着前轴线打开胸腔。切开隔膜，取出肋骨的前部。</li>
	<li>使用伦敦钳子将主动脉和静脉卡瓦抓住隔膜上方，并用斯特拉比斯谟剪刀切割靠近下脊柱的所有血管和结缔组织，从而去除心肺包。</li>
	<li>使用塑料一次性移液器用肝素化 PBS 彻底冲洗胸腔，直到去除所有血液痕迹。</li>
	<li>将躯干置于立体显微镜制备双筒望远镜下，确保胸腔内具有外部光源的良好照明。</li>
	<li>找到第一根肋骨和朗格斯科利肌肉。 注:SG位于双边位置，平行于脊柱在第一根肋骨和脊柱之间的分支，在一个凹槽横向到隆格斯科利肌肉24。SG 的平侧位于朗格斯科利肌肉附近。根据准备情况，交感链的某些部分可能已经可见为与脊柱平行的白色斜纤维。这些可追溯到 SG。</li>
	<li>轻轻地使用杜蒙#5/45钳子的尖端，将结缔组织横向暴露在长腹肌上。</li>
	<li>将钳子转动 180°，然后使用平侧抓住 SG，并以最小的压力将其拉出。</li>
	<li>重复第二个 SG。</li>
	<li>将两个 SG 放在装满冷 PBS 的盘子（直径 6 厘米）中，并以适当的放大倍数进行检查。如有必要，去除多余的血管、脂肪组织和较大的神经。 注:自主结肠被一个结缔组织胶囊包围，由胶原纤维和成纤维细胞26，27组成。这些胶囊的渗透性似乎因物种而异，不同种类的黑帮26岁和28岁。使用 Dumont #5/45 钳子以及如有必要，使用弹簧剪刀去除尽可能多的结缔组织。</li>
	<li>根据实验目标，继续执行本协议的第 2、3 或 5 节。</li>
</ol>2. 全安装免疫造血化学协议
注:此协议是根据心脏整个安装染色4，29改编而成的。在 96 井板的一口井中为每个 SG 执行孵化步骤，并将 100 μL（用于含抗体解决方案）到 200 μL（用于所有其他解决方案）的解决方案进行孵化，以确保完全覆盖。定期用双筒望远镜检查 SG 的覆盖范围并正确浸入。手动取出液体时，配有 200 μL 移液器，在 200 μL 尖端上再加一个 10 μL 尖端。这将防止移液器尖端中 SG 的吸入。使用新制备的溶液和无菌液体来防止细菌生长。
<ol><li>修复 Sg 的组织学 2 小时在 Rt 在 4% 无甲醇副甲醛 （Pfa） / Pbs 。</li>
	<li>尽快处理 SG，但此时 PBS 中的钠酸钠含量为 0.02%（w/v），可在 PBS 的 4-6 °C 下存储 2-4 周。</li>
	<li>准备苏丹黑股解决方案（1%苏丹黑色w/v在100%乙醇），以减少自动荧光和改善信号到背景比30。在 RT 的磁搅拌器上溶解 2-3 小时。 注:使用库存溶液最多 6-8 周，在出现沉降时提前丢弃。</li>
	<li>准备苏丹黑色工作解决方案，通过离心的库存溶液30分钟全速（13，000 x g）去除碎片和稀释库存70%乙醇到最终浓度0.25%苏丹黑色。</li>
	<li>用登特的漂白剂治疗SG，以改善抗体渗透31。通过混合甲醇 （MeOH）、过氧化氢溶液 30% （w/w） 在 H2O 中和二甲基硫氧化物 （DMSO） 的比例为 4:1:1，新鲜准备登特的漂白剂。每 SG 添加 200 μL，并将板放置在轨道摇床上 1 小时 RT。</li>
	<li>在轨道摇床上孵育 10 分钟，进行降压 MeOH 系列以进行补液:100% MeOH、75% MeOH/PBS、50% MeOH/PBS、25% MeOH/PBS。</li>
	<li>在 RT 的 PBS/1% Triton-X-100 中，通过两次孵育 SG 进行 60 分钟，从而执行渗透。</li>
	<li>从 SG 中删除渗透解决方案，并添加苏丹黑色工作解决方案。在轨道摇床的 RT 上孵育 2 小时。</li>
	<li>同时，通过在 PBS 中加入 5% 受体级牛血清白蛋白 （BSA） 和 0.1% Triton-X-100（15 mL 容器），并让它在滚筒摇床上溶解约 5-10 分钟，从而准备阻塞溶液。通过预褶皱纸过滤器去除碎屑。</li>
	<li>通过倾斜板和小心地从直立侧管道去除苏丹黑色非常小心。 注:不可能看到苏丹的SG是黑色的。使用强光源并缓慢工作。从这一步开始，SG 被染黑，提高了可见度。如果此时在 SG 周围看到任何其他结缔组织，请使用 Dumont #5/45 钳子和弹簧剪刀将其取出。</li>
	<li>加入 200 μL 的 PBS/0.1% 特里顿-X-100 （PBS-T），在轨道摇床的 RT 上洗 5 分钟。</li>
	<li>用移液器吸气，然后重复 2 次，以去除 PBS-T。</li>
	<li>删除 PBS;加入 200 μL 的阻塞溶液，并在轨道摇床上在 4 °C 的夜间孵育。</li>
	<li>第二天，通过在阻塞溶液中添加主要抗体来准备解决方案。从既定协议中调整抗体浓度。 注:包括一个SG作为抗体控制没有原发性抗体（孵化与抗原预吸收抗体或IgG，如果可用，或阻塞缓冲）。</li>
	<li>在轨道摇床上执行 36-48 h 的初级抗体孵化，在 4 °C 下进行。对于细胞大小测量和交感神经元的染色，请使用抗酪氨酸羟基酶的抗体（请参阅抗体建议 材料表 ）。 注意:将 96 井板放在湿室（例如，内衬 ddH2O 湿纸巾的塑料盒），以防止此时蒸发。</li>
	<li>小心地去除抗体溶液，并添加 200 μL 的 PBS-T。将板放在轨道摇床上30分钟。</li>
	<li>移除 PBS-T 并重复洗涤步骤 5 次。</li>
	<li>使用前全速（13，000 x g）离心荧光亚历克萨标签的二级抗体，准备二次抗体工作解决方案1分钟。根据阻塞溶液中的主要抗体 1:500 稀释适当的二级抗体，如果需要核染色，则添加到 SG. 添加 1 μg/mL 的双苯酰胺 H33342 三氯化氢（霍奇斯特染色）。在轨道摇床的 4 °C 下孵育 12-24 小时。</li>
	<li>小心地取下抗体溶液，并添加 200 μL 的 PBS-T。将板放在轨道摇床上30分钟。</li>
	<li>移除 PBS-T 并重复洗涤步骤 5 次。对于最后一步，使用没有特里顿的 PBS。</li>
	<li>要嵌入，请将 50-100 μL 的荧光安装介质（参见 材料表）涂抹在玻璃滑梯上，并放置在准备的双筒望远镜下。使用 Dumont #5/45 钳子从 96 井板中取出 SG，并通过将一端浸入滤纸（例如 Whatman 干燥垫）中去除多余的液体，并将其放在掉落处。</li>
	<li>使用杜蒙#5/45 钳以适当的放大镜校正定位。</li>
	<li>轻轻地将玻璃盖片（20 毫米 x 20 毫米）放在 SG 旁边，然后慢慢下降。 注意:使用过多的安装介质会导致 SG 运动或神经缠绕。如果发生这种情况，请快速取下盖唇并重复步骤 2.9-2.21。</li>
	<li>让滑梯在 RT 的黑暗中干燥过夜。在 4 °C 下，至少可以储存 4-6 周。</li>
</ol>3. 全安装就地杂交
注:SG的全安装原位混合体由corti32 器官和商业RNA荧光原位协议（见 材料表）改编而成。从供应商那里获得感兴趣的基因、缓冲和解决方案的探针。所有孵化步骤均在 RT 执行，如果不提及，则不进行。使用无菌 PBS。如果有兴趣在一口井中染色多个 SG，请使用至少 150 μL 的缓冲器和解决方案。
<ol><li>执行步骤 1.1-1.13 中描述的 SG 解剖。</li>
	<li>固定 SG 1 h 在 200 μL 的 4% 无 MEOH PFA/PBS 在一口井的 96 井板放置在轨道摇床。</li>
	<li>在轨道摇床上以 0.1% Tween-20/PBS 为 30 分钟清洗 SG 三次。</li>
	<li>脱水 SG 在 MeOH/PBS 系列中，通过随后在 50% MeOH/PBS、70% MeOH/PBS 和 100% MeOH 中孵育，每个在轨道摇床上 10 分钟。</li>
	<li>将 SG 存储在 -20 °C 的 100% MeOH 过夜。</li>
	<li>第二天，将孵化器预热至40°C。 使用温度计检查温度。</li>
	<li>在反向 MeOH/PBS 系列（100% MeOH、70% MeOH/PBS、50% MeOH/PBS）中补充水合 SG，每次 10 分钟。</li>
	<li>在 PBS 中，每次清洗 SG 三次，每次 5 分钟。</li>
	<li>同时，在 40 °C 的潜伏期 10 分钟开始预热探测器，然后冷却 10 分钟。</li>
	<li>在 200 μL 的蛋白酶 III 中孵育 SG 15 分钟。</li>
	<li>可选:如果计划进行后续免疫荧光染色，则根据本协议第 2.3、2.4 和 2.8 节执行苏丹黑色处理以淬火自动荧光。</li>
	<li>在轨道摇床上以 0.1% Tween-20/PBS 3x 的 200 μL 清洗 SG，每次 5 分钟。</li>
	<li>可选:如果需要与多个探头共染污，则稀释通道-2 （50 倍） 和第 3 频道探头 （50 倍） 在第 1 频道探头 （1x）中。</li>
	<li>用100微升的探针覆盖SG，寻找感兴趣的基因，并在40°C下在夜间孵育，并轻微激动。将 96 井板放置在 40 °C 的湿室中，用于所有孵化步骤。 注:包括一个SG作为负控制，使用对细菌基因（如二氢二聚二甲酸酯还原酶 ，Dapb）的探针，以检查在以后的步骤中放大试剂的非特异性结合。</li>
	<li>在提供洗涤缓冲器中清洗 SG，每次在轨道摇床上清洗 15 分钟。</li>
	<li>预热 Amp1-3、HRP-C1 和 HRP 拦截器到 RT。如果需要与通道-2和/或通道-2探头共同染色，则需要预热 HRP-C2 和 HRP-C3。</li>
	<li>在轨道摇床上以 4% PFA/PBS 在 RT 中重新修复 SG 10 分钟。</li>
	<li>在 Rt 的轨道摇床上以 200 μL 的供应洗涤缓冲器 3 倍清洗 Sg， 每次 5 分钟。</li>
	<li>为了放大，在轨道摇床上用 100 μL 的 Amp1 孵育 SG，在 40 °C 下进行 35 分钟。</li>
	<li>小心地取出任何液体，在轨道摇床的 RT 上用 200 μL 的供应洗涤缓冲器 3x 清洗 SG，每次 5 分钟。</li>
	<li>在轨道摇床上用 100 μL 的 Amp2 孵化 SG，在 40 °C 下进行 35 分钟。</li>
	<li>重复步骤 3.18。</li>
	<li>在轨道摇床上用 100 μL 的 Amp3 孵化 SG 20 分钟，在 40 °C 下。</li>
	<li>重复步骤 3.18。</li>
	<li>在轨道摇床上以 100 μL 的多路复用 FL v2 HRP-C1 孵育 SG 20 分钟，在 40 °C 处使用。</li>
	<li>重复步骤 3.18。</li>
	<li>准备蛋白石结合的二级抗体 1:1，000 在 200 μL 提供的 TSA 缓冲和孵育 SG 35 分钟在 40 °C 的轨道摇床。在孵化过程中和从这一步开始时，要防止光线照射。</li>
	<li>重复步骤 3.18。</li>
	<li>在轨道摇床上以 100 μL 的多路复用 FL v2 HRP 阻滞器在 40 °C 处孵育 SG 15 分钟。</li>
	<li>重复步骤 3.18。</li>
	<li>可选:用于与通道-2探头共同染色，重复步骤 3.25-3.30 与提供的多路复用 FL v2 HRP-C2。</li>
	<li>可选:用于与通道-3探头共同染色，重复步骤 3.25-3.30 与提供的多路复用 FL v2 HRP-C3。</li>
	<li>如果随后发生免疫荧光染色，执行本协议的第 2.13-2.25 步。</li>
	<li>在轨道摇床上以 1% BSA/PBS 孵育 30 分钟。</li>
	<li>如果需要核染色，在 1 μg/mL 的双苯甲酰胺 H33342 三氯化氢 （霍奇斯特染色） 中孵育 SG 30 分钟，如果需要核染色，并/或在轨道摇床上添加亚历克萨耦合小麦胚芽凝胶素 （WGA， 1:500）。</li>
	<li>重复步骤 3.18。</li>
	<li>按步骤 2.19-2.22 中描述的嵌入。</li>
</ol>4. 木质结肠的成像和分析
<ol><li>在本地成像设备中对嵌入式 SG 进行共聚焦显微镜检查。</li>
	<li>如果需要细胞大小测量，图像SG染色酪氨酸羟基酶（见 材料表）在200倍放大，并采取4-6随机图像从每个SG。</li>
	<li>使用 ImageJ33 软件分析图像以估计单元格大小（例如，使用笔表，参见 材料表）。使用 免费手选择，圈每个单元格，然后单击 分析|测量 获取细胞区域。小心只包括位于 SG 内的完整、完全可见的细胞。</li>
	<li>使用此方法，执行每 SG 约 100 个细胞的测量。</li>
	<li>如果你想比较来自不同老鼠的 SG，请让失明的调查员执行步骤 4.2-4.3。使用统计软件中的频率分布来可视化第 34组之间的大小差异。</li>
</ol>5. 穆林·斯特拉特结帮的分子分析
注意:根据您的实验设计，包括控制。这可能是具有不同基因型和疾病背景和/或其他自主结节的SG，如交感优越的宫颈结节（位于颈部区域，见齐格勒等人的详细描述）或寄生性结节（如颅内结节，见君根等人）。
<ol><li>准备一个2mL管与500微升的酚/瓜尼丁硫氰酸酯溶液（例如，Qiazol）每只动物，并有液氮准备防震霜或考虑商业解决方案，以保护RNA（可选，见材料表）36。快速进行RNA隔离。</li>
	<li>执行步骤 1.1-1.13 中描述的 SG 解剖。</li>
	<li>立即将两个 SG 直接浸入一个管中，用酚/瓜尼丁硫氰酸酯溶液和冲击霜管浸入液氮中。</li>
	<li>在 -80 °C 下存放，直到进一步处理。</li>
	<li>对于组织裂解，让带有 SG 解冻的管子解冻，直到苯酚/瓜尼丁硫氰酸酯溶液液液化，并添加两个 7 mm 不锈钢珠。在 4 °C 的干冰和离心机上冷却组织同质化器（球或搅拌机，例如组织 Lyser II）的金属部件。</li>
	<li>离心管在 500 x g 1 分钟在 4 °C，使 SG 在管的底部。</li>
	<li>将管子放入组织莱瑟的金属部件中，在 20 Hz 处将 1 分钟置于莱塞和莱塞中 1 分钟。</li>
	<li>重复步骤 5.6 和 5.7 最多 5 次，直到无法检测到完整的组织。</li>
	<li>将液体转移到新鲜的 1.5 mL 管中。</li>
	<li>根据制造商的说明，使用基于柱的RNA隔离套件（例如 miRNeasy 迷你套件）执行 RNA 隔离。</li>
	<li>Elute RNA 在 20 μL 的无 RNase 水中，使用光谱光度计测量浓度。 注:为了排除纯RNA与基因组DNA的污染，我们建议用基因组引物和1微升RNA作为模板进行聚合酶链式反应，而不是减去反向转录酶控制。这将节省大量的RNA。如果RNA被污染，请使用外在内子边界引物或内侧引物进行后续定量实时聚合酶链反应。</li>
	<li>使用 250 ng SG RNA 执行 cDNA 合成并使用已建立的协议。在这里，根据制造商的说明使用大容量 cDNA 反向转录套件。</li>
	<li>稀释到 cDNA 的 2.5 ng/μL 的最终浓度，并根据既定协议与适当的探头进行定量实时聚合酶链式反应。在这里，塔克曼检测（见 材料表）使用10 ng cDNA每反应进行。 注:对每个基因执行无模板控制，以排除误报结果。</li>
	<li>使你感兴趣的基因在家养基因上的基因表达正常化（例如 ，Cdkn1b），以比较不同组的SG之间的相对基因表达。</li>
</ol>

作者没有什么可透露的。

对VA发病前交感神经系统神经元和胶质细胞的细胞和分子过程的理解是高度关注的，因为心脏骤停仍然是全世界最常见的死因。因此，在当前的手稿中，我们提供了一系列基本的方法来识别该网络中的穆林 SG - 喃喃元素 ， 并随后对 RNA、蛋白质和细胞水平进行分析。
Murine SG的一个挑战是它的大小和有限的细胞数量39。因此，不同的动物模型?...

心脏自主神经系统是一种受严格控制的交感、寄生和感官成分的平衡，使心脏能够适应环境变化，并具有适当的生理反应1，2。这种平衡的干扰，例如，同情活动的增加，已被确定为发病和维持心室心律失常（VA）3，4的关键驱动因素。因此，自主调制，通过药理学减少与β阻滞剂的交感活性，一直是治疗VA患者几十年的基石5，6。但是，尽管药理学和导管为基础的干预，相关数量的患者仍然患有复发性VA7。
对心脏的交感输入主要通过交感链的双星形结构——神经元细胞体进行中介，这种结构通过无数的胸内神经从脑干传递信息到心脏8、9、10。受伤后从SG发芽的神经与VA和心脏骤死11，12有关，强调SG作为自主调制的目标13，14。通过局部麻醉剂的皮下注射或通过视频辅助胸腔镜检查15、16部分切除SG，可以暂时减少对心脏的交感输入。心脏交感衰竭为治疗难治VA的患者提供了一个选择，结果有希望的结果14，16，17。我们从这些患者的外植SG中了解到，神经元和神经化学改造、神经炎症和胶质活化是同情性重塑的标志，可能有助于或加重自主功能障碍18，19。尽管如此，这些神经元中潜在的细胞和分子过程至今仍然模糊不清，例如，神经元分化成胆碱表型20，21的作用。实验研究提出了治疗VA的新方法，例如，通过光遗传学22减少交感神经活动，但SG的深度表征仍然缺乏许多心脏病理与VA齐头并进。模仿这些病理学的小鼠模型可以研究神经元重塑，这可能先于心律失常12，23的发生。这些可以通过进一步的形态和功能分析来完成，以实现心脏和神经系统的自主特征。在本协议中，我们提供了一系列基本的方法，允许解剖和描述 Murine SG，以增进对 VA 的理解。

<table><tbody><tr><td>96-well plate</td><td>TPP</td><td>92097</td><td>RNAscope</td></tr><tr><td>Adhesion Slides SuperFrost plus&amp;nbsp; 25 x 75 x 1 mm</td><td>R. Langenbrinck</td><td>03-0060</td><td>Microscopy</td></tr><tr><td>Albumin bovine Fraction V receptor grade lyophil.</td><td>Serva</td><td>11924.03</td><td>Whole mount staining</td></tr><tr><td>bisBenzimide H33342 trihydrochloride (Hoechst)</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>B2261</td><td>Whole mount staining</td></tr><tr><td>Chicken anti neurofilament</td><td>EMD Millipore</td><td>AB5539</td><td>Whole mount staining</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Merck, KGA, Darmstadt, Germany</td><td>D8418</td><td>Whole mount staining</td></tr><tr><td>Donkey anti chicken IgY Alexa 647&amp;nbsp;</td><td>Merck, KGA, Darmstadt, Germany</td><td>AP194SA6</td><td>Whole mount staining</td></tr><tr><td>Donkey anti goat IgG Alexa 568&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A11057</td><td>Whole mount staining</td></tr><tr><td>Donkey anti rabbit IgG Alexa 488&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A21206</td><td>Whole mount staining</td></tr><tr><td>Drying block 37-100 mm</td><td>Whatman (Sigma Aldrich)</td><td>WHA10310992&amp;nbsp;</td><td>Whole mount staining</td></tr><tr><td>Eosin Y</td><td>Sigma Aldrich</td><td>E4009</td><td>Whole mount staining</td></tr><tr><td>Ethanol 99 % denatured with MEK, IPA and Bitrex (min. 99,8 %)</td><td>Th.Geyer</td><td>2212.5000</td><td>Whole mount staining</td></tr><tr><td>Eukitt mounting medium</td><td>AppliChem</td><td>253681.0008</td><td>Whole mount staining</td></tr><tr><td>Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td>Whole mount staining</td></tr><tr><td>Fluoromount-G + DAPI</td><td>Southern Biotech</td><td>0100-20</td><td>Whole mount staining</td></tr><tr><td>Goat anti choline acetyltransferase</td><td>EMD Millipore</td><td>AP144P</td><td>Whole mount staining</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; 30% (w/w)</td><td>Merck, KGA, Darmstadt, Germany</td><td>H1009</td><td>Whole mount staining</td></tr><tr><td>Heparin Sodium 25.000 UI / 5ml</td><td>Rotexmedica</td><td>PZN: 3862340</td><td>Preparation SG</td></tr><tr><td>High-capacity cDNA reverse transctiption kit</td><td>Life technologies</td><td>&amp;nbsp;4368813</td><td>RNA isolation</td></tr><tr><td>Isoflurane (Forene)</td><td>Abbott Laboratories</td><td>2594.00.00</td><td>Preparation SG</td></tr><tr><td>Mayer&#039;s hemalum solution</td><td>Merck</td><td>1.09249.0500</td><td>Whole mount staining</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860</td><td>Whole mount staining</td></tr><tr><td>Microscope cover glasses 20x20 mm or smaller</td><td>Marienfeld</td><td>0101040</td><td>Whole mount staining</td></tr><tr><td>miRNeasy Mini Kit</td><td>Qiagen</td><td>217004</td><td>RNA isolation</td></tr><tr><td>NanoDrop 2000c</td><td>Thermo Fisher Scientific</td><td>ND-2000C</td><td>RNA isolation</td></tr><tr><td>Opal 570 Reagent Pack</td><td>Akoya Bioscience</td><td>FP1488001KT</td><td>RNAscope</td></tr><tr><td>Paraformaldehyde, 16% w/v aq. soln., methanol free&amp;nbsp;</td><td>Alfa Aesar</td><td>43368</td><td>Whole mount staining</td></tr><tr><td>Pasteur pipettes, LDPE, unsterile, 3 ml, 154 mm</td><td>Th.Geyer</td><td>7691202</td><td>Whole mount staining</td></tr><tr><td>Phosphate-buffered saline tablets</td><td>Gibco</td><td>18912-014</td><td>Whole mount staining</td></tr><tr><td>Pinzette Dumont SS Forceps</td><td>FineScienceTools</td><td>11203-25</td><td>Preparation SG</td></tr><tr><td>QIAzol Lysis Reagent</td><td>Qiagen&amp;nbsp;</td><td>79306</td><td>RNA isolation</td></tr><tr><td>Rabbit anti tyrosine hydroxylase</td><td>EMD Millipore</td><td>AB152</td><td>Whole mount staining</td></tr><tr><td>RNAlater</td><td>Merck</td><td>R0901-100ML</td><td>RNA isolation (optional)</td></tr><tr><td>RNAscope Multiplex Fluorescent Reagent Kit v2</td><td>biotechne (ACD)</td><td>323100</td><td>RNAscope</td></tr><tr><td>RNAscope Probe-Mm-S100b-C2</td><td>biotechne (ACD)</td><td>431738-C2</td><td>RNAscope</td></tr><tr><td>RNAscope Probe-Mm-Tubb3</td><td>biotechne (ACD)</td><td>423391</td><td>RNAscope</td></tr><tr><td>Stainless steel beads 7 mm&amp;nbsp;</td><td>Qiagen&amp;nbsp;</td><td>69990</td><td>RNA isolation</td></tr><tr><td>Sudan black B</td><td>Roth</td><td>0292.2</td><td>Whole mount staining</td></tr><tr><td>TaqMan Gene Expression Assay Cdkn1b (Mm00438168_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay Choline acetyltransferase (Mm01221880_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay MKi67 (Mm01278617_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay PTPCR (Mm01293577_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay S100b (Mm00485897_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan Gene Expression Assay Tyrosin Hydroxylase (Mm00447557_m1)</td><td>Thermo Fisher Scientific</td><td>4331182</td><td>Gene expression analysis</td></tr><tr><td>TaqMan mastermix</td><td>Applied biosystems</td><td>4370074</td><td>Gene Expression analysis&amp;nbsp;</td></tr><tr><td>Tissue Lyser II</td><td>Qiagen</td><td>85300</td><td>RNA isolation</td></tr><tr><td>Triton X-100 10% solution</td><td>Sigma-Aldrich</td><td>93443-100ml</td><td>Whole mount staining</td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P9416-100ML</td><td>RNAscope</td></tr><tr><td>Wacom bamboo pen</td><td>Wacom</td><td>CTL-460/K</td><td>Cell size measurements</td></tr><tr><td>Whatman prepleated qualitative filter paper, Grade 595 1/2</td><td>Sigma-Aldrich</td><td>WHA10311647</td><td>Whole mount staining</td></tr><tr><td>Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate</td><td>Thermo Fisher Scientific</td><td>W21404</td><td>RNAscope</td></tr></tbody></table>

location, dissection, and analysis of the murine stellate ganglion

自主神经系统是心脏电生理学的一大驱动力。特别是其同情分支的作用是心室心律失常（VA）病理生理学中正在进行的研究。交感链的双子星形结构（SG）   中的神经元   是交感基础设施的重要组成部分。SG 是治疗难治 VA 患者通过心脏交感衰减而公认的治疗目标。虽然在SG的神经元重塑和胶质激活已经描述了VA患者，潜在的潜在的细胞和分子过程，潜在的心律失常开始之前，只是不够了解，应该阐明，以改善自主调节。小鼠模型允许我们研究交感神经元改造，但对缺乏经验的研究者来说，识别穆林SG是具有挑战性的。因此，许多常见的心脏病缺乏对穆林SG的深入细胞和分子生物学研究。在这里，我们描述了解剖和研究成年小鼠SG的基本剧目，用于RNA水平分析（基因表达分析的RNA分离，原位杂交）、蛋白质水平（免疫荧光全安装染色）和细胞水平（基本形态、细胞大小测量）。我们提出潜在的解决方案，以克服制备技术中的挑战，以及如何通过淬火自流来改善染色。这允许通过既定标记对神经元和胶质细胞进行可视化，以确定细胞组成和重塑过程。这里介绍的方法允许SG的特征获得关于容易发生VA的小鼠自主功能障碍的进一步信息，并可以辅之以额外的技术来研究心脏自主神经系统的神经元和胶质成分。

图 1可视化如何识别和解剖 SG.图 1A显示了位置的示意图图，而图 1B在切除心肺包后将视图呈现到胸腔中。左和右长部的科利肌肉从 SG 和肋骨笼是方向的重要地标。解剖是沿着肌肉和第一根肋骨之间的虚线进行的。SG 和交感链变得可见为白色结构（图 1C）。图 1D显示了左长腹肌和第一根...

Watch this Scientific Journal Video about Location, Dissection, and Analysis of the Murine Stellate Ganglion at JoVE.com

Location, Dissection, and Analysis of the Murine Stellate Ganglion

心脏自主神经系统的病理生理变化，特别是其交感分支的病理生理变化，有助于心室心律失常的发病和维持。在本协议中，我们展示了如何描述粘膜结节，以增进对基础分子和细胞过程的理解。

穆林·斯特拉特·甘利翁的位置、解剖和分析

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

location-dissection-and-analysis-of-the-murine-stellate-ganglion

Research

JoVE Journal

Medicine

9.9K 次观看.  EVK Düsseldorf, cNEP, cardiac Neuro- and Electrophysiology Research Consortium. 心脏自主神经系统的病理生理变化，特别是其交感分支的病理生理变化，有助于心室心律失常的发病和维持。在本协议中，我们展示了如何描述粘膜结节，以增进对基础分子和细胞过程的理解。

视频: Location, Dissection, and Analysis of the Murine Stellate Ganglion

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

蟹电压敏感染料Stomatogastric神经节神经元的光学成像

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

科研

神经科学

Homarus Americanus Stomatogastric Nervous System Dissection

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the activity of interest and are accessible and amenable to experimental methods. The stomatogastric nervous system (STNS) of the American lobster (Homarus americanus; also know was the Atlantic or Maine lobster) has been established as a model system for studying rhythm generating networks and neuromodulation of networks. The STNS consists of 3 anterior ganglia (2 commissural ganglia and an oesophageal ganglion), containing modulatory neurons that project centrally to the stomatogastric ganglion (STG). The STG contains approximately 30 neurons that comprise two central pattern generating networks, the pyloric and gastric networks that underlie feeding behaviors in crustaceans1,2. While it is possible to study this system in vivo3, the STNS continues to produce its rhythmic activity when isolated in vitro. Physical isolation of the STNS in a dish allows for easy access to the somata in the ganglia for intracellular electrophysiological recordings and to the nerves of the STNS for extracellular recordings. Isolating the STNS is a two-part process. The first part, dissecting the stomach from the animal, is described in an accompanying video article4. In this video article, fine dissection techniques are used to isolate the STNS from the stomach. This procedure results in a nervous system preparation that is available for electrophysiological recordings.

Homarus Americanus Stomatogastric Nervous System Dissection

We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).

Homarus Americanus Stomatogastric神经系统解剖

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the ...

生物学

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of cells (approximately 25 in C. borealis) which are well characterized. The cells in the STG exhibit a broad range of outputs and are responsible for the motor actions of the stomach. The stomach contains the gastric mill which breaks down food with three internal teeth, and the pylorus which filters the food before it reaches the midgut. The STG produces two rhythmic outputs to control the gastric mill and pylorus known as central pattern generators (CPGs). Each cell in the STG can participate in one or both of these rhythms. These CPGs allow for the study of neuromodulation, homeostasis, cellular and network variability, network development, and network recovery.
The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric nervous system (STNS) of the Jonah crab (C. borealis) can be used for electrophysiology, immunohistochemistry, and cell culture studies. The STNS extraction is done in two parts: the gross and fine dissection.

癌症北欧化工 Stomatogastric神经系统解剖

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

分析小鼠沿着轴突生长的雪旺氏细胞发展

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

Surgical Technique for Superior Cervical Ganglionectomy in a Murine Model

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary gland secretion, circadian rhythm, macular degeneration, immune function, and cardiac physiology. Murine surgical sympathectomy is a method for studying the effects of adrenergic innervation by allowing for complete, unilateral adrenergic ablation while avoiding the need for repeated pharmacologic intervention and the associated side effects. However, surgical sympathectomy in mice is technically challenging because of the small size of the superior cervical ganglion. This study describes a surgical technique for reliably identifying and resecting the superior cervical ganglion to ablate the sympathetic nervous system. The successful identification and removal of the ganglion are validated by imaging the fluorescent sympathetic ganglia using a transgenic mouse, identifying post-resection Horner's syndrome, staining for adrenergic markers in the resected ganglia, and observing diminished adrenergic immunofluorescence in the target organs following sympathectomy. This model enables future studies of cancer progression as well as other physiological processes regulated by the sympathetic nervous system.

The present protocol describes a mouse model of the ablation of adrenergic innervation by identifying and resecting the superior cervical ganglion.

小鼠模型中上颈神经节切除术的手术技术

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary ...

癌症研究

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.

佩尔维奇自主性甘利亚与雄性大鼠和雌性大鼠相关神经的解剖

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons ...

Whole Central and Peripheral Nervous System Mice Dissection

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent nervous system, nor is there a complete schematic representing it that is freely available. Only methods to harvest the brain, the spinal cord, a specific dorsal root ganglion, and the sciatic nerve (separately) are available. Here, we provide detailed pictures and a schematic of the central and peripheral murine nervous system. More importantly, we outline a robust procedure to perform its dissection. The 30 min pre-dissection step allows isolating the intact nervous system within the vertebra with muscles free of viscera and skin. A 2-4 h dissection follows it under a micro-dissection microscope to expose the spinal cord and the thoracic nerves, and finally peel the whole central and peripheral nervous system off the carcass. This protocol represents a significant step forward in studying the anatomy and pathophysiology of the nervous system globally. For example, the dissected dorsal root ganglions from a neurofibromatosis type I mice model can be further processed for histology to unravel changes in tumor progression.

Here, we present a protocol to complete the dissection of central and peripheral nervous systems of mice. The dissected tissues are further analyzed in downstream applications, such as taking gross pictures or histology.

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent ...

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome.
Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice.
This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

This protocol describes the detailed, low-input sample preparation for single-nucleus sequencing, including the dissection of mouse superior cervical and stellate ganglia, cell dissociation, cryopreservation, nucleus isolation, and hashtag barcoding.

低输入细胞核分离和多路复用小鼠交感神经节条形码抗体进行单核RNA测序

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into ...

医学

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 ...