图形是用 BioRender.com 创建的。这项工作得到了NIA赠款（R01AG060919）和NSF赠款（2030348）的支持。

注意：实验程序的一般视图如图 1A所示。本研究中对小鼠进行的所有实验均已获得我们机构的动物护理和使用委员会（IACUC）的批准。我们使用3个月大的C57BL6 / J小鼠，这些小鼠在兽医的照顾下饲养在国际实验动物护理协会（AAALAC）批准的动物饲养中。小鼠生活在大笼子里，有12小时的明暗循环，并 随意喂食。
1. 受孕和怀孕验证
<ol><li>放置任何背景和感兴趣的基因型的小鼠进行繁殖。记录女性受孕前的日期和体重。</li>
	<li>6小时后，用探针检查雌性斑块。如果存在斑块，请将雌性与雄性分开。如果斑块不存在，请将雌性留在笼子中直到第二天，然后将小鼠分开。</li>
	<li>在受孕后第 7、10 和 14 天，称女性体重以确认怀孕。</li>
</ol>2. 培养基、24 孔板和材料制备
<ol><li>在细胞分离当天，将即用型聚D-赖氨酸涂层玻璃盖玻片（参见 材料表）置于24孔板中，如下所示：<ol><li>在生物罩下，用70%乙醇对包含15个盖玻片的单个包装进行灭菌并晾干。打开包装并将盖玻片放入 60 毫米板中。水平摇动板以分离盖玻片。然后，倒置板以拾取要放置在 24 孔板孔中的单个盖玻片。</li>
	</ol></li>
	<li>用 1.0 mL 无菌汉克平衡盐溶液 （HBSS） 洗涤盖玻片一次 5 分钟。</li>
	<li>同时，按如下方式制备 20.0 mL 电镀培养基：向 18.31 mL BME（基础培养基鹰，+ 厄尔盐）中，加入 1.0 mL 热灭活胎牛血清 （FBS），加入 200 μL 丙酮酸钠（来自 100 倍原液）、200 μL 谷氨酰胺（来自 200 nM 原液）和 100 μL 青霉素/链霉素（来自 200 倍原液）。</li>
	<li>用1.0mL电镀介质替换孔中的HBSS，并将板置于37°C的培养箱中。</li>
	<li>使用本生燃烧器，对三个直径递减的巴斯德移液器进行火抛光。用一只手握住移液管，将吸头插入火焰中并快速取出。重复该过程，直到尖端变平滑并且直径减小到所需的直径（通过肉眼评估）。</li>
</ol>3. 神经组织解离试剂盒的试剂制备，遵循神经解离试剂盒的说明
<ol><li>在室温下加热消化缓冲液 1 后，通过将 50 μL 酶 1 与 1.91 mL 缓冲液 1 混合来制备酶混合物 1，通过将 15 μL 酶 2 与 30 μL 消化缓冲液 2 混合来制备酶混合物 2。这些混合物足以用于所有胚胎的脑组织。</li>
	<li>在 HBSS 中制备 0.5% 牛血清白蛋白 （BSA），例如，在 50.0 mL HBSS 中制备 0.25 g。</li>
</ol>4. 胚胎提取
<ol><li>高压灭菌两个直细钳，一个弯曲点钳和一对细手术剪刀，并在使用前用70%乙醇消毒。然后，用HBSS填充培养皿。</li>
	<li>在CO2 室中对一个E14-E16孕妇母亲实施安乐死并进行颈椎脱位。 注意：必须在无菌条件下在引擎盖下执行以下步骤：</li>
	<li>用70%乙醇对腹部进行消毒。用手术剪刀和镊子切开从耻骨联合到肋骨剑突的腹腔。</li>
	<li>取出子宫角并将其放入装有冰冷HBSS的100毫米板中并彻底清洗。</li>
	<li>用细镊子从子宫中提取并分离所有胚胎。用精细的手术剪刀和/或镊子快速斩首胚胎。将头部放入装有HBSS的60毫米培养皿中。</li>
</ol>5.下丘脑提取、采集和组织解离
<ol><li>将一个细镊子放在眼腔中以固定大脑。使用其他细镊子，通过剥离去除皮肤和头骨，直到大脑可见。根据其白色外观将大脑与其他组织区分开来。皮肤和头骨是粉红色的，血管系统丰富。</li>
	<li>使用弯曲的点镊子从头骨中取出大脑，然后从嗅球中舀出大脑，将其倒置。</li>
	<li>现在皮层是腹侧的，下丘脑在上表面的背侧可见（图1B）。用弯曲的镊子去除脑膜和血管层，直到大脑看起来白色和清晰。</li>
	<li>用弯曲的镊子将下丘脑区域与大脑的其余部分分开。</li>
	<li>将下丘脑切成 3-4 小块，用移液管将这些碎片转移到 15 mL 管中。</li>
	<li>当管子在冰上时，对其他胚胎重复这些步骤。</li>
	<li>用 6.0 mL HBSS 填充试管，让组织沉淀，除去上清液，加入酶混合物 1。轻轻混合并搅拌管以防止组织。</li>
	<li>将管在37°C水浴中孵育15分钟，每5分钟搅拌一次组织以重悬组织。</li>
	<li>15 分钟后，加入 30 μL 酶混合物 2。使用最大直径（<1毫米）的移液器巴斯德解离脑组织。上下移液 10 倍，不形成气泡。</li>
	<li>在水浴中于37°C孵育10分钟。轻轻搅拌试管以每5分钟重悬一次组织。</li>
	<li>10 分钟后，加入剩余的 15 μL 酶混合物 2。将组织与其他两个直径减小的火抛光移液器分离 10 倍，上下不形成气泡。</li>
	<li>向具有解离组织的管中，立即加入10.0mL的HBSS-0.5%BSA，并在室温下以300× g 离心10分钟。</li>
	<li>吸出上清液并将细胞沉淀重悬于1.0mL HBS-0.5%BSA中。</li>
</ol>6. 细胞计数
<ol><li>使用HBSS-0.5%BSA以1：5稀释细胞悬液。</li>
	<li>将 10 μL 稀释的细胞悬液放入 Neubauer 计数室中。</li>
	<li>在明场显微镜下，仅计数四个腔室角方块中的细胞。计算平均值并乘以 5 × 104。 注意：确保有>106 个细胞进行细胞分离;最佳细胞数为 107。</li>
</ol>7. 否定选择
注意：阴性选择使用户能够通过分离神经元和非神经元细胞获得纯原代神经元培养物。使用预冷溶液。
<ol><li>将细胞悬液以300× g 离心3分钟（离心可延长至10分钟）。轻轻吸出上清液并将沉淀重悬至80μLHBSS-0.5%BSA中的107 个细胞的浓度。</li>
	<li>加入20μL非神经元细胞生物素抗体混合物，并在4°C下孵育5分钟。</li>
	<li>用 2.0 mL HBSS-0.5% BSA 洗涤细胞以去除游离抗体，并以 300 × g 离心 3 分钟。</li>
	<li>轻轻吸出上清液并将沉淀重悬于80μLHBSS-0.5%BSA中。加入20μL抗生物素微珠，充分混合，并在4°C下孵育10分钟。</li>
	<li>加入 0.5 mL HBSS-0.5% BSA 至多 10 个7 个 细胞，并等待磁柱准备就绪。</li>
</ol>8.磁选，负选择
注意：磁分离是允许将非神经元细胞与神经元细胞分离的关键步骤。含有神经元和非神经元细胞的样品通过磁场，与生物素-抗体-磁珠复合物结合的非神经元细胞被捕获在色谱柱中（图1C）。游离神经元细胞通过色谱柱洗脱并收集在15 mL管中。
<ol><li>使用分离器和MS色谱柱准备支架（包含在套件中），如图 1C所示。</li>
	<li>仅当细胞准备好分离时，才打开色谱柱并设置支架。</li>
	<li>用0.5 mL HBSS-0.5%BSA冲洗色谱柱。等到溶液停止滴落。</li>
	<li>要收集神经元细胞，请将15 mL管放在色谱柱下方，并将0.5 mL的细胞悬液通过色谱柱。将洗脱液收集在管中，直到它停止滴落。为了捕获残留的神经元细胞，洗涤色谱柱3 x 0.5 mL的HBSS-0.5%BSA。</li>
	<li>要收集非神经元细胞，请从磁铁上取下柱并将其放入新的15ml管中。向色谱柱中加入1.0 mL HBSS--0.5%BSA，并使用柱塞收集磁性标记的非神经元细胞。</li>
	<li>将神经元和非神经元细胞以300× g 离心3分钟。轻轻吸出上清液并将细胞重悬于1.0mL的HBSS-0.5%BSA中。按照前面第 6 节中所述对细胞进行计数。</li>
	<li>如果需要，将非神经元细胞接种在 24 孔板中;否则丢弃它们。</li>
</ol>9. 正向选择
注意：一旦获得纯神经元细胞悬液，就会进行正向选择以分离目标细胞。可以通过使用针对表面抗原的特定生物素偶联抗体来分离细胞。该抗体被抗生物素磁珠识别。通过将细胞悬液流过色谱柱，只有感兴趣的细胞被困在磁场中。
<ol><li>将纯神经元细胞悬液以300× g 离心3分钟。轻轻吸出上清液并将沉淀重悬于80μLHBSS-0.5%BSA中。按照制造商的说明添加特异性抗体，并在4°C下孵育10分钟。 注意：如果寻求表达LepR的细胞，我们建议使用浓度为0.50μg/ 106细胞的小鼠瘦素R生物素化抗体（参见材料表）。</li>
	<li>用 2.0 mL HBSS-0.5% BSA 洗去多余的抗体，并以 300 × g 离心 3 分钟。</li>
	<li>除去上清液，将沉淀重悬于 80 μL HBSS-0.5% BSA 中，并加入 20 μL 抗生物素微珠。在4°C孵育10分钟。</li>
	<li>对于每 10 个7 个 细胞，加入 0.5 mL HBSS-0.5% BSA，并等待磁柱准备就绪。</li>
</ol>10.磁选，正向选择
<ol><li>准备带有分离器和MS柱的支架。用 0.5 mL HBSS-0.5% BSA 冲洗 MS 色谱柱。等到滴水停止。</li>
	<li>将 15 mL 管置于色谱柱下方，将 0.5 mL 细胞悬液通过色谱柱，并收集包含非特异性神经元细胞的洗脱液。要清洁残留的非特异性神经元细胞柱，请用3 x 0.5 mL HBSS-0.5%BSA洗涤。</li>
	<li>从磁铁上取下色谱柱，将其放入新的 15 mL 管中，然后加入 1.0 mL HBSS-0.5% BSA。使用柱塞冲洗掉目标细胞。</li>
	<li>将两个管以300 ×g 离心3分钟。轻轻除去上清液并将其重悬于0.5mL电镀介质中。</li>
	<li>按照前面第 6 节中所述对细胞进行计数。</li>
	<li>将靶向细胞作为阳性对照，非特异性细胞作为阴性对照，在先前如第2节所述制备的24孔板中以120,000-200,000细胞/mm 3密度接种，并在37°C下在5%CO 2,9%O2和95%湿度下孵育12小时。</li>
</ol>11. 细胞培养维护
<ol><li>用 19.2 mL 神经元培养基、400 μL B27 补充剂（来自 50 倍原液）、200 μL 谷氨酰胺（来自 200 μM 原液）和 100 μL 青霉素/链霉素（来自 200 倍原液）制备 20 mL 培养基。</li>
	<li>更换含有神经元或非神经元细胞的24孔板中的电镀介质。</li>
	<li>用 2 x 1.0 mL HBSS 洗涤。</li>
	<li>加入 1.0 mL 培养基。</li>
	<li>每 2/3 天刷新一次培养基，用 0.5 mL 新培养基替换 0.5 mL 旧培养基。 注意：细胞可以在培养物中保持，并在 体外 使用长达21天（DIV21）。</li>
</ol>12. 神经元免疫荧光染色
<ol><li>染色前12小时，制备由50/50甲醇和丙酮组成的溶液，并在-20°C下冷却过夜。</li>
	<li>用 2 x 1.0 mL 的 1x 磷酸盐缓冲盐水 （PBS） 洗涤 24 孔板中的神经元 5 分钟。</li>
	<li>用 1.0 mL 的 50/50 溶液替换 PBS 溶液，并在冰中孵育 20 分钟。</li>
	<li>用 1x PBS 清洗 3 x 5 分钟。</li>
	<li>在室温下用3%BSA在1x PBS中阻断神经元1小时。</li>
	<li>使用制造商说明中提到的抗体浓度，在 1x PBS 中制备 3% BSA 中的一抗溶液。使用的抗体和浓度列在 材料表中。</li>
	<li>用一抗溶液替换封闭溶液，并在4°C孵育过夜。</li>
	<li>用1x PBS洗涤细胞3 x 10分钟。</li>
	<li>按照制造商的说明使用抗体浓度在1x PBS中用3%BSA制备二抗溶液。使用的二抗列在 材料表中。</li>
	<li>将细胞与二抗溶液在室温下孵育1小时。</li>
	<li>用1x PBS洗涤细胞3 x 10分钟。</li>
	<li>在安装过程中将神经元留在 1x PBS 中。在显微镜载玻片上放置一小滴封片剂（有或没有4'，6-二脒基-2-苯基吲哚用于核鉴定）。用镊子提取一个含有神经元的玻璃盖玻片，并在纸巾上敲击盖玻片的侧面以干燥多余的PBS。翻转封片介质上的盖玻片，确保神经元面向显微镜载玻片;用薄纸轻轻按压并取出多余的封片剂。</li>
	<li>显微镜载玻片已准备好用明场或共聚焦显微镜进行分析。</li>
</ol>

离体下丘脑神经元的激素、代谢和电调节

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H., Oster, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mouse+primary+hypothalamic+neuronal+cultures+for+circadian+bioluminescence+assays.">Generation of mouse primary hypothalamic neuronal cultures for circadian bioluminescence assays.</a> Bio-protocol. 11 (5), e3944 (2021).</li><li>Foo, 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=.">.</a> Purification of rat and mouse astrocytes by immunopanning. 2013 (5), 421-432 (2013).</li><li>Emery, B., Dugas, J. 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研究下丘脑神经元的生化和电学特性是了解新陈代谢、体温调节、情绪管理、喂养行为等分子基础的关键。然而，下丘脑的神经元异质性使这项工作具有挑战性，需要分离和研究特定下丘脑亚群的方法。
体内 技术采用CRE重组酶、光遗传学、纤维光度法和钙成像。这些方法主要用于研究下丘脑神经元的电特性，目前很少有方法可用于研究它们的非电属性。本研究中开发的MACS技术可以提供一种适合 于体外分离 特定下丘脑神经元亚群的技术，从而提供有针对性的治疗和分析。与不同神经元群体的共培养相比，神经元培养更易于管理。此外，纯培养物可避免因神经胶质细胞和小胶质细胞的存在而产生的混杂效应。因此，可以研究来自相同下丘脑区域和类型的神经元，以响应特定的代谢和激素输入。
在该协议中，我们选择了表达LepR的下丘脑神经元。培养分离的LepR +细胞以研究其难以在体内研究的细胞，形态和分子特征 。 培养物的纯度为99%，支持该方法的准确性。此外，LepR +细胞在DIV7直到DIV21都是健康和可行的。
但是，这种技术有一些局限性。E18 或更早的纯神经元培养物难以维持。因此，提取窗口仅限于E14-E16。这意味着E16之后发生的细胞变化被遗漏了。例如，ARC神经元中瘦素受体的表达在出生后早期增加22。必须尽快进行分离程序，以减少细胞应激和死亡并提高产量。该过程可能需要长达5小时;因此，必须保持无菌条件并将操作减少到最低限度。由于可用组织量低，正向选择可能导致低产量，从而限制了单次制备可以进行的实验数量。观察到神经元死亡升高，可能是由于细胞密度低，神经元连接和神经元内支持减少。
此外，靶向目标抗原的抗体必须与细胞表面结合以保证正确分离;通常，用于流式细胞术的抗体适用于MACS技术。如果以前未在细胞分离方法中使用过抗体，则需要验证和滴定实验来确定理想的用途和浓度。靶细胞的提取需要细胞表面标记物。在这里，我们使用了生物素化抗体，但原则上也可以使用与其他分子偶联的抗体，例如FITC（异硫氰酸荧光素）和PE（纯化的抗藻红蛋白）。MACS技术也可以应用于表达荧光团的神经元，如GFP或其他Tag蛋白，潜在地提高特异性和产量。如果不使用荧光团，另一种方法是在进行活细胞实验之前通过免疫荧光确认目标分子的表达。未来的研究将测试这些替代方案的有效性。
这项研究没有解决的一个重要方面涉及亚神经元群体的“保真度”。我们确定培养的LepR +神经元表达POMC，这是天然ARC POMC 神经元的特征。然而，需要更多的测试来得出结论，LepR +神经元培养物概括了它们的 体内 天然对应物。总体而言，这里介绍的MACS神经元分离方案可能为研究 体外 下丘脑机制提供了一种有效且有效的方法，否则很难 在体内进行研究。

下丘脑是大脑的一个多管齐下的区域，介导内分泌、自主神经、内脏和行为功能，包括进食、新陈代谢、睡眠、体温、社会行为和 1,2,3,4,5。功能异质性是通过生化和电机制的协同组合实现的：下丘脑神经元激发动作电位，分泌和释放激素和神经肽，以调节大脑区域和身体器官。最后，下丘脑神经元翻译来自身体的稳态信息，以长期和短期反馈和前馈调节做出反应6。
下丘脑的复杂神经元环境包括大细胞内分泌神经元，释放催产素和加压素;细小细胞神经元，主要参与全身激素调节，例如向垂体释放促甲状腺激素释放激素 （TRH） 和促肾上腺激素释放激素 （CRH）;大的肽能投射神经元，释放食欲素和黑色素浓缩激素（MCH）;以及弓形核（ARC）的细小细胞肽能神经元释放POMC（原黑皮质素）和AgRP（刺鼠相关蛋白），分别命名为ARCPOMC和ARC AgRP。与分泌细胞一起，其他兴奋性和抑制性神经元，包括多巴胺能、谷氨酰胺能和 GABA 能神经元 7，参与形成下丘脑内和下丘脑外回路，从而创建具有相当细胞异质性的大规模协调网络8。
下丘脑多样性一直是研究人员在过去50年中一直试图克服的挑战。为了研究发育，成熟和衰老的下丘脑的这种异质性，研究人员一方面一直在使用单细胞RNA测序来探索神经元组织以及分子和转录组学特征。这项工作为下丘脑神经元的各种作用提供了深刻的见解，并解决了细胞身份与其在生理系统中的可能作用之间的联系8,9,10。另一方面，已经通过光遗传学操作和纤维光度法行为方法研究了神经元功能，从而可以仔细观察电路结构。在过去的二十年中，Cre-重组酶技术使研究人员能够在个体遗传学上刺激或抑制目标神经元组，同时观察行为和身体反应的变化6,11,12。
然而，这些方法从一般角度检查下丘脑功能，而没有深入研究特定的细胞机制或其在复杂的下丘脑环境中作用的生物学基础。为了解决这个问题，很少有研究专注于利用异质原代下丘脑培养物研究分子、生化和电学特性。这些研究试图在复杂的环境中剖析特定的神经元过程，并生成生理机制的综合模型13,14,15。然而，非特定文化带来了重大挑战。例如，神经元的生理连接和解剖分布被来自不同下丘脑区域的神经元电镀破坏，这些神经元通常不会相互作用，从而产生混杂效应。此外，每个区域都有不同的角色和杂色的神经元群体，这使得研究简单的生物过程变得困难。
为了应对这些挑战，在过去十年中，已经实施了新方法来分离感兴趣的神经元，例如免疫淘选，荧光激活细胞分选（FACS）和磁性激活细胞分选（MACS）。免疫淘选是一种用于纯化靶细胞的策略，使用抗体包被的培养皿进行一系列非神经元（阴性）和神经元（阳性）选择。虽然该技术原则上可以产生高产量的纯化细胞培养物，但在实践中，主要用于星形胶质细胞和少突胶质细胞，因为这些细胞可以抵抗数小时的操纵16,17。FACS技术是一种强大的工具，可以使用流式细胞术18,19,20根据荧光标记物和细胞特征对细胞进行分选。然而，很少有研究使用这种方法分离细胞进行细胞培养。该技术价格昂贵，需要高技能人员使用和维护;此外，在分选程序结束时保持活细胞和无菌细胞具有挑战性21。总体而言，MACS似乎是一种简单，不昂贵的技术，可以获得下丘脑原代神经元的高纯度和活力培养物。该方法利用通过抗体与细胞相连的磁珠。这允许使用色谱柱的磁场隔离细胞。
在这里，我们描述了一种基于MACS技术的方法，该方法通常用于皮质神经元。该协议原则上允许分离可行且高度纯的下丘脑神经元。在这项研究中，我们制备表达瘦素受体（LepR）的神经元的原代培养物，例如ARCPOMC和ARC AgRP 神经元，它们仅存在于弓形核中。这些神经元以生化和电学方式对瘦素（一种由脂肪组织分泌的厌食激素）做出反应。因此，培养物中这组神经元的分离允许 在体外研究它们的激素，代谢和电学特性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Embryo extraction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 curved point forceps</td>
			<td>Fine Science Tools</td>
			<td>11270-20</td>
			<td>Dumont</td>
		</tr>
		<tr>
			<td>1 fine surgical scissor</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td>Dumont</td>
		</tr>
		<tr>
			<td>100 mm Petri dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>2 straight fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>Dumont</td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>Corning</td>
			<td>430196</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Decon Laboratories, INC.</td>
			<td>2801</td>
			<td>Ethanol 190 Proof</td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads 1mL</td>
			<td>Miltenyi Biotec</td>
			<td>130-115-390</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MAP2 antibody</td>
			<td>Abcam</td>
			<td>ab5392</td>
			<td>1 : 800</td>
		</tr>
		<tr>
			<td>Bench pads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9418-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Y</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Z</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads 1mL</td>
			<td>Miltenyi Biotec</td>
			<td>130-115-390</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9418-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Y</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer Z</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme A</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme P</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>GG-12-1.5, 12 mm dia.#1.5 thick 100 pc cell culture tested German coverglasses</td>
			<td>Neuvitro Corporation</td>
			<td>GG-12-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco B-27 Supplement 10 mL</td>
			<td>ThermoFisher&#160;</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Basal Medium Eagle (BME) 500 mL&#160;</td>
			<td>ThermoFisher</td>
			<td>21010046</td>
			<td>(+) Earle&#39;s Salts, (-) L-Glutamine&#160;</td>
		</tr>
		<tr>
			<td>Gibco HBBS (1x) Hanks&#39; Balanced Salt Solution&#160; 500 mL</td>
			<td>ThermoFisher</td>
			<td>14025092</td>
			<td>Calcium, Magnesium, No phenol red</td>
		</tr>
		<tr>
			<td>Gibco HI FBS 100 mL</td>
			<td>ThermoFisher</td>
			<td>16140-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco L-Glutamine 200 mM (100x)</td>
			<td>ThermoFisher</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Penicilline/Streptomicine&#160;</td>
			<td>ThermoFisher</td>
			<td>15140-122</td>
			<td>10,000 U/mL</td>
		</tr>
		<tr>
			<td>Gibco Sodium Pyruvate (100 mM) 100 mL</td>
			<td>ThermoFisher</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator and Starting Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Leptin R Biotinylated Antibody</td>
			<td>R&#38;D Systems&#160;</td>
			<td>ABAF497</td>
			<td>0.25 &#956;g/106 cells</td>
		</tr>
		<tr>
			<td>MS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Neaubeaur-Improved Brightline 100 &#181;m Chamber</td>
			<td>Hausser Scientific&#160;</td>
			<td>3120</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural Tissue Dissociation Kit - Postnatal Neurons</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuronal Culture Medium 500 mL&#160;</td>
			<td>ThermoFisher</td>
			<td>88283</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Neuronal Cell Biotin-Antibody Cocktail mouse 1 mL</td>
			<td>Miltenyi Biotec</td>
			<td>130-115-389</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus SZ61 Zoom Stereomicroscope&#160;</td>
			<td>Olympus Life Science</td>
			<td>SZ61/SZ51&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Primary Neuron Isolation Kit</td>
			<td>ThermoFisher</td>
			<td>88280Y</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MAP2 antibody</td>
			<td>Abcam</td>
			<td>ab5392</td>
			<td>1 : 800</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488</td>
			<td>ThermoFisher</td>
			<td>A32766</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488</td>
			<td>ThermoFisher</td>
			<td>A32790</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS)&#160;</td>
			<td>Sigma Aldrich&#160;</td>
			<td>MFCD00131855</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647</td>
			<td>ThemoFisher</td>
			<td>A32933</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>ThermoFisher</td>
			<td>A11037</td>
			<td>1 : 200</td>
		</tr>
		<tr>
			<td>Invitrogen Leptin Receptor Recombinant Rabbit Monoclonal Antibody (JA73-01)</td>
			<td>ThermoFisher</td>
			<td>MA5-32685</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Mouse Leptin R Biotinylated Antibody</td>
			<td>R&#38;D Systems&#160;</td>
			<td>ABAF497</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>POMC Rabbit mAb&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>D3R1U</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>PSD95 (D74D3) XP Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>D74D3#3409</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Streptavidin, Alexa Fluor 594 conjugate</td>
			<td>ThermoFisher</td>
			<td>S11227</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Synapsin 1 Monoclonal Antibody (7H10G6)</td>
			<td>ThermoFisher</td>
			<td>MA5-31919</td>
			<td>1 : 500</td>
		</tr>
		<tr>
			<td>Vectashield Plus Antifade Mountina Medium with DAPI 10 mL</td>
			<td>Vector Laboratories&#160;</td>
			<td>H-2000</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of targeted hypothalamic neurons for studies of hormonal, metabolic, and electrical regulation

下丘脑通过控制食物摄入量、体温和激素释放等功能来调节基本的代谢过程。由于下丘脑的功能由神经元群的特定亚群控制，因此分离它们的能力为研究代谢机制提供了主要工具。在这方面，下丘脑的神经元复杂性带来了特殊的挑战。 
由于这些原因，已经探索了新技术，例如磁性激活细胞分选（MACS）。本文描述了磁激活细胞分选（MACS）的新应用，使用微珠技术从产前小鼠大脑中分离目标神经元群。该技术简单，保证了具有高可重复性的高度纯度和活性的原代下丘脑神经元培养。轻轻解离下丘脑，选择性分离神经元并与神经胶质细胞分离，最后，使用细胞表面标志物的特异性抗体，选择感兴趣的群体。 
一旦分离出来，靶向神经元可用于研究它们的形态、电学和内分泌特征及其在正常或病理条件下的反应。此外，鉴于下丘脑在调节喂养、新陈代谢、压力、睡眠和动机方面的多种作用，仔细观察目标和区域特异性神经元可能会深入了解它们在这个复杂环境中的任务。

本文描述了一种分离下丘脑靶向神经元的方案 （图1）。该方法的范围是在受控和孤立的背景下研究特定的神经元特征。因此，在E14-E16从怀孕的母亲中提取小鼠胚胎。脑膜被移除，下丘脑与大脑的其余部分分离。将组织与使用参考解离试剂盒新鲜制备的两种酶混合物轻轻解离。首先，将非神经元细胞与神经元细胞分离——神经胶质细胞、小胶质细胞和神经元收集在同一单细胞悬液中。为此，使用识别非神经元表面表位的抗体混合物标记非神经元细胞。孵育后，抗体-细胞复合物与磁性微珠偶联，随后通过磁柱捕获非神经元细胞。
该步骤产生了两种细胞悬液，一种包含非神经元，另一种含有神经元细胞。两种悬浮液都可以立即镀层。或者，可以使用相同的策略进一步操纵神经元细胞悬液以将神经元亚群（靶向悬浮液）与其他亚群分离。根据感兴趣的实验，神经元细胞可以接种125,000至200,000个细胞/ mm3。密度较低的培养物可用于以单细胞分辨率分析神经元：从轴突发育、突触形成和传递到电生理学。密度较大的培养物可用于生化分析，包括 DNA 和 RNA 提取、蛋白质印迹、南部印迹、北部印迹、实时荧光定量 PCR 和 RNA 测序。
在这项研究中，LepR的目标是分离参与黑皮质素系统的神经元，如ARCPOMC和ARC AgRP神经元。细胞的接种密度范围从LepR +神经元的120,000个细胞/ mm 3到通用神经元群的200,000个细胞/ mm3。48小时后，LepR +神经元开始形成神经突（图2）。在DIV4，轴突延伸显示出进展，而树突过程开始出现。在DIV6，神经元充分发育，因此可以进行分析。LepR +神经元的免疫荧光实验显示LepR的表达率为99%（绿色，图3A）。未观察到神经胶质细胞或其他非神经元细胞，证实了原代神经元培养物的纯度。通过微管相关蛋白2（MAP2）染色确认细胞的神经元性质，鉴定轴突和树突突起（图3B）。在DIV10时，30%的LepR +细胞表达POMC（红色）。这是意料之中的，因为大多数LepR +细胞表达POMC或AgRP。图3C，D说明了POMC和LepR信号之间的共定位。请注意，正如预期的那样，共定位在核及其周围很突出。
使用含有异质性下丘脑神经元群的一般培养物进行对照。免疫荧光显示突触连接和功能，通过Synapsin-1（绿色）和PSD 95（红色）共染色评估（图4A，B）。一般培养物中存在的LepR +神经元数量为~5%，这一百分比与在磁分离过程中选择了大多数表达LepR的神经元的概念一致（代表性LepR +细胞如图4C，D所示）。本研究期间生成或分析的所有数据均可在 https://doi.org/10.5061/dryad.cnp5hqc9c 获得。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65674/65674fig01.jpg"> 图1：实验流程图和设置 。 （A）实验程序的图形表示。去-不去：≥106 个细胞是进行细胞分离所必需的;最佳细胞数为 107。（B）E16胚胎大脑的代表性图像。指示下丘脑和脑膜。 （C）用于目标细胞分离和分离的MACS装置。标明了磁性支架、磁选机和色谱柱。 <a href="https://www.jove.com/files/ftp_upload/65674/65674fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65674/65674fig02.jpg"> 图2：DIV2和DIV6之间的神经元培养 。 （A）从阳性选择中获得的LepR +细胞显示细胞密度和连通性降低，但神经突（红色箭头），轴突（蓝色箭头）和树突（绿色箭头）正常发育。细胞以120,000个细胞/mm 3的密度接种。比例尺 = 100 μm。（B）通用下丘脑神经元，以200,000个细胞/ mm3的密度接种，显示出正常的发育和生长特征以及连接性（黑色箭头）。比例尺 = 100 μm。缩写：DIV = 体外天数;LepR = 瘦素受体。为构建此数字而生成或分析的所有数据均可在 https://doi.org/10.5061/dryad.cnp5hqc9c 获得。 <a href="https://www.jove.com/files/ftp_upload/65674/65674fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65674/65674fig03.jpg"> 图 3： 体内 神经元由培养的 LepR+ 神经元概括。 （A）表达LepR的神经元培养物的代表性图像（绿色;DAPI 是蓝色的）。百分之九十九的细胞表达LepR。神经元以120,000个细胞/ mm3 密度接种。在DIV7，神经元呈现细长的轴突，树突成熟和神经元连接（箭头）。比例尺 = 40 μm。（B）使用抗MAP2免疫荧光来确认细胞的神经元性质。展示了神经元特异性形态，例如轴突、树突和突起（箭头）。比例尺 = 40 μm。（C）LepR +细胞的放大。LepR为绿色，DAPI为蓝色。比例尺 = 10 μm。（D）与LepR（绿色）和POMC（红色）共染色。大约30%的LepR +神经元对POMC具有共免疫反应性，POMC在细胞核水平（红色箭头）中检测到。比例尺 = 10 μm。缩写：DAPI = 4'，6-二脒基-2-苯基吲哚;MAP2 = 微管相关蛋白 2;POMC = 原黑皮质素;LepR = 瘦素受体。为构建此数字而生成或分析的所有数据均可在 https://doi.org/10.5061/dryad.cnp5hqc9c 获得。<a href="https://www.jove.com/files/ftp_upload/65674/65674fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65674/65674fig04.jpg"> 图 4：体内细胞由培养的通用下丘脑神经元重述。 （A）用Synapsin 1（绿色），PSD95（红色）和DAPI（蓝色）染色的通用下丘脑神经元培养物的代表性图像。神经元表现出发达的连接性和突触功能（箭头）。比例尺 = 40 μm。（B）（A）中框的放大倍数，显示突触1（绿色）和PSD95（红色，箭头）的共定位。比例尺 = 20 μm。（中，四）代表性图像显示一般培养物中的LepR +细胞。LepR +细胞（绿色）占总数的~5%。代表性的LepR +细胞用箭头表示。比例尺 = 40 μm。（中、女）（C，D）中盒子的放大倍数显示位于躯体处的绿色点状瘦素受体。比例尺 = 20 μm。为构建此数字而生成或分析的所有数据均可在 https://doi.org/10.5061/dryad.cnp5hqc9c 获得。<a href="https://www.jove.com/files/ftp_upload/65674/65674fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Hypothalamic Neural Mechanism Insights at JoVE.com

作者聚焦：下丘脑神经机制洞察 

在这里，我们提出了一种在培养中培养特定下丘脑细胞亚型的方案。可以根据机会/独特的膜标志物选择细胞，并用于许多应用，包括免疫荧光、电生理和生化测定。

分离靶向下丘脑神经元，用于激素、代谢和电调节研究

The hypothalamus regulates fundamental metabolic processes by controlling functions as varied as food intake, body temperature, and hormone release. As ...

我们的研究范围是研究与下丘脑功能障碍有关的代谢紊乱。我们有兴趣研究靶向哺乳动物下丘脑神经元的生化和细胞机制，特别是表达瘦素受体的神经元。下丘脑是一个复杂的大脑区域，涉及维持进食、睡眠、动机和新陈代谢。这意味着一个非常多样化的神经社区。只有少数人群被彻底研究过，其他大多数需要进一步研究并进一步解释它们的细胞机制。下丘脑神经元及其功能主要通过电生理学、化学遗传学和光遗传学进行研究。这些技术在理解动物行为和大脑回路方面非常有用。然而，对驱动这种行为的细胞和分子机制知之甚少。只有通过更深入地观察细胞水平，我们才能解决这一差距。

isolation-targeted-hypothalamic-neurons-for-studies-hormonal

Research

JoVE Journal

Neuroscience

Isolation of Targeted Hypothalamic Neurons for Studies of Hormonal, Metabolic, and Electrical Regulation

3.3K 次观看.  Rutgers University. 我们的研究范围是研究与下丘脑功能障碍有关的代谢紊乱。我们有兴趣研究靶向哺乳动物下丘脑神经元的生化和细胞机制，特别是表达瘦素受体的神经元。下丘脑是一个复杂的大脑区域，涉及维持进食、睡眠、动机和新陈代谢。这意味着一个非常多样化的神经社区。只有少数人群被彻底研究过，其他大多数需要进一步研究并进一步解释它们的细胞机制。下丘脑神经元及?...

视频: 作者聚焦：下丘脑神经机制洞察 

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

闪光光解笼状化合物在嗅感觉神经元的纤毛

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

科研

神经科学

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

隔离的感觉神经元中<em&gt;海兔丫</em谷氨酸能电流膜片钳记录

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

成人下丘脑神经发生局灶性放射性抑制功能审讯

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

使用电动和一维和二维文化磁场神经元的外部激励

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

微粒相关α synuclein 聚合体对原代神经元的外源性管理--纤维形成的强大细胞模型

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...