这项研究已经由美国国立卫生研究院资助（PN2EY016525和P41GM103832）的支持。二手烟是由来自WM凯克中心对墨西哥湾沿岸财团（美国国立卫生研究院批准号：T32EB009379）的跨学科生物科学训练的纳米生物学跨学科研究生培养方案的研究奖金支持。 SHS解剖，成长和陶瓷DRG和海马细胞;收集的冷冻电镜和背根神经节和海马轴突冷冻 - 经济数据，重建和颜色标注的​​倾斜系列。 MRG解剖和MNR的实验室提供的海马细胞。资深大律师协助的倾斜系列注释。二手烟是如何解剖和神经生长训练的CW。 SHS，​​WCM和厕所构思的实验。自蔓延高温合成制备的手稿与其他作者的输入。 这部影片的拍摄地在该中心为t的Biozentrum的细胞成像和NanoAnalytics（C-CINA）他巴塞尔大学。 C-CINA集成部的苏黎世联邦理工学院，位于巴塞尔，瑞士的生物系统科学与工程（D-BSSE）。

1。准备菜肴与EM网格的镀初级神经元<ol><li>检查使用光学显微镜在至少25倍的放大倍率黄金EM网格多孔碳的完整性。确保碳孔&gt; 98％不变。 </li><li>电镀初级神经元，用本生灯火焰灭菌的EM网格和并行使其亲水性。使用镊子拿起EM网格的边缘，而不是中心网格区域。注意：切勿将点燃的本生灯无人值守，并且不使用手套或镊子塑料组件，同时执行这些步骤： <ol><li>前点燃本生灯，设置空气和气体调整到最小开启位置。 </li><li>用一个前锋火石或丁烷打火机点燃本生灯。 </li><li>修改空气和煤气的调整来实现一个蓝色的小内内的高，淡蓝色/紫色火焰的火焰。内焰的尖端是火焰的最热的部分。下旋钮照耀在燃烧器调节气体进入燃烧器管的数量，而在燃烧器的桶可以被打开以调节空气进入燃烧器的数量。转动空气调节顺时针方向减小（导致火焰呈紫色）的空气和逆时针方向增加（导致黄色火焰）的空气。 </li><li>使用金​​属镊子（无塑料件）举行了EM网格，通过火焰迅速传递给它的两倍，面对碳的一面朝上。碳侧将出现更多的磨砂（少光泽），并与带有灰色色调的比另一侧。 </li><li>紧接在网格（碳的一面朝上）转移到玻璃底培养皿放置在10厘米本生灯的中心。使用每皿1 EM网格（ 图1）。仅使用玻璃底菜被预消毒， 即通过γ射线照射。 </li></ol></li><li>使用光学显微镜，同时仍保持了EM网格玻璃博特内检查电网的完整性（碳孔完好）OM菜（以避免污染）。当施加任何物质上的网格或任何会与神经元接触，用消毒程序和无菌移液器吸头。 </li><li>在使用过程无菌组织培养罩，慢慢地，仔细地应用于250微升合适的涂布物质在培养皿的中央玻璃面积。确保适当的涂布物质覆盖了整个电磁网格。 <ol><li>对海马神经元，可使用聚-L-赖氨酸（PLL，1毫克/毫升）作为涂层物质，如先前所描述的19制备。请注意，250微升每有EM电网需要，每皿，所以应相应批次。对于背根神经节（DRG）神经元，使用凝胶状蛋白混合物通过Engelbreth - 霍尔姆 - 群（EHS）小鼠肉瘤细胞分泌（见表材料和试剂）作为涂布物质，准备如下。请注意，250微升每有EM电网需要，每皿，所以应相应批次。 <ol><li>解冻股票瓶凝胶状蛋白混合物通过Engelbreth - 霍尔姆 - 群（EHS）小鼠肉瘤细胞在4℃过夜分泌的</li><li>保持冷的凝胶状蛋白混合物通过Engelbreth -霍尔姆-群（EHS）小鼠肉瘤细胞，并用于制造解决方案的所有组件， 即通过保持他们在冰上分泌。 </li><li>而在冰上，在Neurobasal培养基稀释此胶状蛋白质混合物以得到1:20稀释（ 即稀释500μl的胶状蛋白质的混合物到10ml的Neurobasal的）。 </li><li>将稀释后的凝胶状的蛋白混合物为1毫升分装，并立即在-20℃存放任何额外等份℃。 </li><li>每个应用EM电网250微升，每皿，如1.4节所述。 </li></ol></li></ol></li><li>盖培养皿，其顶部和孵化（无论是与锁相环（PLL）海马标本，或用胶状蛋白质混合物DRG标本）1小时在室温下在组织培养罩。 </li><li> ASPI评价所有PLL（海马标本），或从菜品的胶状蛋白质混合物（DRG标本）。要做到这一点，使用真空系统在组织培养罩。使用附连到真空管中的无菌移液管尖端。避免与EM电网直接接触。 </li><li>使用可调节的空气位移移液管小心地应用250微升无菌PBS（磷酸盐缓冲盐水）中的EM网格中的每个培养皿的中心玻璃区域，从而使得EM网格被完全覆盖的PBS中。然后，从每个培养皿中吸出PBS中。重复3次。 </li><li>允许与EM网格的菜组织培养罩15分钟下干燥。确保它们是由光学显微镜的组织培养室检查下完全干燥。请确保在电网无气泡的水分。如果是的话，小心吸旁边的EM网格来消除这种额外的水分。该涂层电网应立即用于电镀的神经元。 </li></ol> 2。准备和排位ATING初级神经元上的EM网格<ol><li>到板原代神经元，第一解剖，胰蛋白酶消化（用2.5％胰蛋白酶）和磨碎以解离的海马（或18日龄的大鼠胚胎背根神经节）成单个细胞，如先前所描述的19。 <ol><li>磨碎是使用神经生物学家描述细胞团块离解成单个细胞，特别是使用了火抛光尖玻璃吸管的一种常见的术语。其结果是通过移液管小心缓慢地拉伸和释放细胞，向上和向下，多次（〜30）来实现。 </li></ol></li><li>遵循如前对背根神经节解剖和细胞分离24所述的方法，注意到使用0.25％胰蛋白酶（在HBSS中）以分离的大鼠DRG神经元，而不是使用提出的酶（木瓜蛋白酶，胶原酶/分散酶），它们更适合于鼠标DRG神经元。 </li><li> （ 即使用血细胞计数）计算分离的神经元的数量并使用该值来计算细胞的适当体积要应用于每个培养皿以达到每个培养皿50000个细胞/ ml的浓度。申请量最大为250微升。请注意，这只是填充中央玻璃面积，而不是整个菜。 </li><li>孵化菜在CO 2培养箱中30分钟，在37℃下让细胞恢复和坚持。 </li><li>慢慢加入1.5毫升回暖媒体到每一道菜，小心不要打扰了EM网格。媒体的类型依赖于细胞类型（海马或DRG）。 <ol><li>准备适当的媒体要么海马神经元19或背根神经节神经元24，这是在使用前37℃水浴中预热。孵育隔夜菜中的CO 2培养箱中培养。 </li><li>对于海马神经元，第二天更换介质。改变一半的媒体，每两天以后的14天。温暖的媒体在使用前37℃水浴。 </LI&gt; <li>对背根神经节神经元中，以下夹层/电镀当天，制备的介质与抗有丝分裂剂（尿苷和5&#39;-氟-2&#39;-脱氧尿苷），使各10mM的储备溶液，单独一个新鲜的库存;使用最终10μM的每种浓度。除去一半的DRG媒体（875微升）中，并加入875微升新鲜培养基与抗有丝分裂剂。 <ol><li>对于DRG神经元，每两天进行的第一周，交替变化介质抗有丝分裂的媒体和标准DRG媒体之间。对于第二个星期，改变标准DRG媒体每两天。 </li></ol></li></ol></li></ol> 3。在EM网格玻璃化神经元<ol><li>准备设备和材料全部冻结和存储黄金的EM电网在低温下：配有湿度室17玻璃化设备，细点专业镊子液氮的玻璃化机，长平点镊子，杜瓦（S）（LN 2），Coolant容器，EM格收纳盒，无钙滤纸。 </li><li>开始玻璃化装置。湿度设定为100％和温度为32℃。在“控制台”部分，设置印迹时间为零秒。这允许手动穿过玻璃化机器的湿度腔室的侧面窗口印迹。 </li><li>处理好与手套的无钙滤纸，分层他们这样三个文件堆叠。切堆栈成宽约0.5厘米的条是〜2厘米。弯曲它们以90°角，使得纸张的一面具有0.5厘米×0.5厘米面（ 图2）。使用镊子，取出中间纸并将其放置在另一个无钙滤纸直到使用。 </li><li>把网格存储按钮，在按钮持有人的玻璃化冷冻室中。充满液氮的冷却剂容器的内室中，并等待直至完全蒸发。填写日的外腔用液氮冷却剂ë容器，直到达到稳定的温度下进行到下一个步骤。用高纯度的气态乙烷，将冷凝成液体状态的冷却腔中填充内腔。 </li><li>从孵化器移动盘（ES）到大100毫米聚苯乙烯菜运到玻璃化冷冻室，如果不是在附近。使用专业的玻璃化镊子从菜仔细挑选了EM网格。注意哪个方面的神经元生长的EM网格;的位置将重要的一个步骤。请使用镊子黑色滑锁牢固地锁定在EM网格镊子。 </li><li>插入镊子进入玻璃化机对EM网格使得侧上的神经元被附着面的左侧，远离玻璃化机器的侧面开口的孔。缩回专业镊子放入玻璃化机。 </li><li>放置冷却剂容器中的相应夹持器玻璃化机。它应该充满2足够LN和液态乙烷。使用玻璃化机器的适当屏幕的命令，提高冷却剂腔室向上，直到它的刷新的湿度室的底部。 </li><li>与扁平点镊子，把握滤纸，使得短边（0.5厘米×0.5厘米面）垂直于所述镊子的一个边缘。这张脸会接触到电磁电网直接接触印迹（ 图2B）。小心地将滤纸放入侧孔的玻璃化冷冻机的恒温恒湿试验箱（ 图2A）。稳定地保持对EM网格纸（面朝远离试样）为10秒。丢弃的文件之后，立即使用玻璃化冷冻机的自动化暴跌，冻结试样在液体乙烷。 </li><li>仔细的1的冷冻水合EM网格转移到时隙中的一个网格存储按钮。重复此过程，以获得更多的EM网格的菜肴。在这些实验中使用了EM网格存储按钮可以存储多个冷冻水合EM网格。 </li></ol> 4。图像采集，处理和注释<ol><li>进行收集的2-D型电子显微镜照片和/或使用低剂量条件下的低温电子显微镜的神经突的三维倾斜系列5。的2-D图像的目的是要评估网格的质量在冰厚度和可能的领域而言是对3-D倾斜系列有用。 <ol><li>在这种情况下，使用连接到配备有一个倾斜液氮冷冻传输座200千伏电子显微镜，在20K显微​​镜的放大倍率在目标underfocus的7微米和4.4采样一个集成的4K x 4K CCD摄像头采集的所有图像/像素。 </li><li>取得的样品的三维断层图像，采取了一系列的投影图像的同时逐步倾斜的样品人透射电子显微镜（TEM）的翁一根轴。由于倾斜角度等实验设置，有可用的自动采集倾斜系列5几种不同的软件。这里显示的3-D倾斜系列采用半自动化的倾斜系列采集软件26在一个范围-60°〜5°增量60°，〜60 E / A 2的累积剂量收集在同一个显微镜。 </li></ol></li><li>重建倾斜系列使用先前用于其他样品5,29描述的图像处理软件21的突起的。 </li><li>由第一分割的断层图像，并使用3-D图像处理软件，如前所述5创造表面模型的颜色标注的突起的三维特征。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

我们证明了大鼠胚胎神经元（背根神经节和海马）可以生长在金电子显微镜（EM）的网格，并冻结在玻璃冰足够薄，其轴突使用2-D冷冻电镜和3-D冷冻待成像ET。虽然海马神经元突起先前已使用冷冻ET 8,10,18,23成像，协议使用市售的设备一直缺乏足够详细的成功复制。此外，虽然他们的研究已经率先使用冷冻ET的可视化海马突起，我们的研究探讨低温ET的适用性，以不止一种类型的神经元标本?...

神经元建立复杂的电路必需的中枢和周围神经系统的通过阐述树突接收信息和轴突，往往是相当冗长的，与下游神经元进行通信的功能。神经突增生起到胚胎发育和神经细胞的分化和维持轴突的过程中的基础性作用支持神经系统的审慎的功能。神经炎过程还具有批判性的神经损伤和再生，以及神经系统疾患。神经元结构的研究是至关重要的了解双方的正常和病变的大脑。幸运的是，生理有关的神经元细胞培养系统存在，可以概括复杂和异构的细胞结构。基于坚实的实验平台，有效的可视化策略，使神经元形态的定性和定量分析的澄清是必要的。特别有用会是一个详细的方法，提供了用于可视化的神经突，既轴突和树突在纳米尺度一致的平台。 传统的电子显微镜需要脱水和树脂包埋过程中使用有机溶剂，可以从自己的真实状态引起扭曲的标本。迄今为止，在纳米尺度结构最大表征是基于较大的细胞或组织所承受这样苛刻的化学物质-从而限制的结果4,9,25解释。此外，对于电子束的穿透，切片所需的生物体或细胞的突起呈现的厚度大于1微米的12。最后，切片或磨碎的组织结果集合中的离散量特定的数据集，使突起的细长的特点繁琐的定义。即使冷冻电镜，其中切片冷冻水合的样品是可能的，该方法引入了压缩ARTIF使徒行传1。 近年来，研究人员已经学会了如何直接在EM电网和速冻它们在液态乙烷采用低温ET 8,10,18,23随后可视化轴突生长的海马神经元。然而，这些研究要么使用一个特制的装置10,23，或对产生足够薄玻璃冰常规可视化8,18的印迹步缺乏细节。例如，一项研究建议使用30-40秒，吸干了EM网格10，但是，这个值进行了优化不能用于一般用途，但具体是对于定做暴跌冷冻设备。使用一个特制的设备，而不是市售17保持湿度暴跌冻结的样品前可能构成障碍的广泛的可重复性。 虽然这些研究已经破土动工了冷冻ET可视化突起，我们已经迈出了一步，探讨AP冷冻的ET plicability各种神经元标本（海马及背根神经节神经元）的。此外，我们将讨论这两种最优和次优的结果，以及人们可以利用冷冻ET此类标本遇到潜在的文物。 定义一个详细的技术维护和在纳米尺度可视化整个轴突在接近本机的状态将加强开展超微结构研究的能力，更多的研究者。为此，我们使用市售设备来制备未定影的，未染色的神经元来可视化突起描述一种有效和详细的协议。这是迈向健康的详细轴突的超微结构，并奠定基础，了解什么是结构上的差异存在于神经系统疾病模型的重要的第一步。因为冷冻的ET能在纳米尺度解决在3-D未定影的，未染色的神经突的特点，该方法将使得能够作为永远脱颖而出定义神经炎架构12。

<table border="0" cellpadding="0" cellspacing="0" width="970">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Dumont #7 Tweezers</td>
			<td style="width:196px;">Electron Microscopy Sciences</td>
			<td style="width:309px;">72803-01</td>
			<td style="width:171px;">For handling EM grids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottom dishes</td>
			<td>MatTek Corp.</td>
			<td>P35G-1.5-10C</td>
			<td>For growing sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electron microscopy grids</td>
			<td>Quantifoil</td>
			<td>Holey Carbon, Au 200, R 2/2</td>
			<td>For growing sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium-free filter paper</td>
			<td>Whatman</td>
			<td>1541-055</td>
			<td>For blotting sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large flat point long tweezers</td>
			<td>Excelta Corporation</td>
			<td>E003-000590, 25-SA</td>
			<td>For blotting sample </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vitrification device and tweezers</td>
			<td>FEI</td>
			<td>Vitrobot Mark III</td>
			<td>For freezing sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini grid storage boxes</td>
			<td>Ted Pella, Inc.</td>
			<td>160-40</td>
			<td>For storing EM grids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryo transfer holder</td>
			<td>Gatan</td>
			<td>626 Single Tilt Liquid Nitrogen Cryo Transfer Holder</td>
			<td>For imaging samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Semi-automated tilt series acquisition software</td>
			<td>SerialEM</td>
			<td>http://bio3d.colorado.edu/SerialEM/</td>
			<td>For imaging samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image processing software</td>
			<td>IMOD eTomo</td>
			<td>http://bio3d.colorado.edu/imod/</td>
			<td>For image processing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Transmission electron microscope for cryoEM</td>
			<td>JEOL, Tokyo</td>
			<td>200-kV JEM2100 LaB6 electron microscope</td>
			<td>For imaging samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4k x 4k CCD camera</td>
			<td>Gatan</td>
			<td>N/A</td>
			<td>For imaging samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-D annotation software</td>
			<td>Visage Imaging GmbH</td>
			<td>Amira/Avizo</td>
			<td>For processing 3-D data</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software for digitally stitching 2-D images</td>
			<td>Adobe</td>
			<td>Adobe Photoshop</td>
			<td>For processing 2-D data</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, High Glucose</td>
			<td>Invitrogen</td>
			<td>11965-118</td>
			<td>For hippocampal culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boric acid</td>
			<td>Sigma Aldrich</td>
			<td>B-0252</td>
			<td>For hippocampal culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium tetraborate</td>
			<td>Sigma Aldrich</td>
			<td>B-9876</td>
			<td>For hippocampal culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine </td>
			<td>Sigma Aldrich</td>
			<td>P2636-500MG</td>
			<td>For hippocampal culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter system</td>
			<td>Corning</td>
			<td>430758</td>
			<td>For hippocampal culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal medium</td>
			<td>Invitrogen</td>
			<td>21103-049</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27 supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant rat b-NGF</td>
			<td>R&#38;D Systems</td>
			<td>556-NG</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uridine</td>
			<td>Sigma</td>
			<td>U3003-5G</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5&#39;-Fluoro-2&#39;-deoxyuridine</td>
			<td>Sigma</td>
			<td>F0503-100MG</td>
			<td>For DRG culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>For DRG culture</td>
		</tr>
	</tbody>
</table>

preparation of primary neurons for visualizing neurites in a frozen-hydrated state using cryo-electron tomography

突起，无论是树突和轴突，是神经元细胞过程，使电脉冲的神经元之间的传导。定义突起的结构是如何理解这些过程动议，支持突触宣传材料和信号的关键。电子显微镜（EM）传统上被用来评估突起内的超微结构特征，但是，接触有机溶剂的脱水和树脂的嵌入过程中，可能会扭曲结构。一个重要的未满足的目标是有关程序，以便不被这种假象影响结构性评估的制定。在这里，我们已经建立了种植和速冻对整个电子显微镜网格其次是他们检查与低温电子断层扫描（冷冻ET）不同的主神经元的轴突详细的和可重复的协议。这种技术允许冷冻，水合突起在纳米级分辨率的三维可视化，便于ASSEssment他们的形态差异。我们的协议产生背根神经节（DRG）突起，和海马神经突在他们接近天然状态的可视化前所未有的看法。因此，这些方法为未来在正常的神经元和那些由神经系统疾病影响的突起研究奠定了基础。

在此之前通过低温冷冻ET和成像，光学显微镜图像应采取的EM网格上的神经元生长。突起应清晰可见，但不与另一个显著重叠。在图3A中的着色方块表示被放大的显示在图3B中，其中，突起在网格的格子延伸更高倍率的区域。每个网格正方形是由支撑的神经元及其突起一个多孔碳片。这些漏洞是显而易见的在4K放大，这表明神经元的身体作为一个比较大的，黑暗的，电子致?...

Watch this Scientific Journal Video about Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography at JoVE.com

Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

以保留神经流程超微结构分析，我们描述了一种协议，用于在电子显微镜网格后用快速冷冻原代神经元的电镀，得到的样品悬浮在一层玻璃的冰。这些样本可以检查与一个低温电子显微镜可视化结构在纳米尺度。

用于可视化神经突在冷冻水合状态下使用低温电子断层扫描准备初级神经元

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the ...

preparation-primary-neurons-for-visualizing-neurites-frozen-hydrated

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Neuroscience

79.0K 次观看.  Baylor College of Medicine. 以保留神经流程超微结构分析，我们描述了一种协议，用于在电子显微镜网格后用快速冷冻原代神经元的电镀，得到的样品悬浮在一层玻璃的冰。这些样本可以检查与一个低温电子显微镜可视化结构在纳米尺度。 

视频: Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

科研

神经科学

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

制备<em&gt;果蝇</em&gt;中央神经元<em在原位</em&gt;膜片钳

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

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 ...

Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a commercially available pre-assembled plastic chip for compartmentalizing cultured primary rat hippocampal neurons. These plastic chips, contained within the footprint of a standard microscope slide, are compatible with high-resolution, live, and fluorescence imaging. This protocol demonstrates how to retrograde label neurons via isolated axons using a modified rabies virus encoding a fluorescent protein, create isolated microenvironments within one compartment, and perform axotomy and immunocytochemistry on-chip. Neurons are cultured for &gt;3 weeks within the plastic chips, illustrating the compatibility of these chips for long-term neuronal cultures.

This protocol describes the use of plastic chips to culture and compartmentalize primary murine neurons. These chips are preassembled, user-friendly, and compatible with high-resolution, live, and fluorescence imaging. This protocol describes how to plate rat hippocampal neurons within these chips and perform fluidic isolation, axotomy and immunostaining.

预组装塑料微流体芯片在原生小鼠神经元分割中的应用

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

定量自发 Ca2+ 通量及其在原发性小鼠中脑神经元中的下游效应

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

使用比率指标对原代神经元中的线粒体谷胱甘肽氧化还原状态进行实时成像

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...