我们感谢霍迈拉·纳瓦比、弗雷德里克·莫雷特和伊莎贝尔·三亚的帮助。这项工作得到了 CNRS、协会特许经营协会（AFM）、ANR YADDLE、拉贝克斯·德维坎、拉贝克斯·皮层、ERC YODA 到 V .C的支持;C.D-B和A.J分别得到拉利格癌症和拉贝克斯·德韦坎奖学金的支持。

1. 克隆策略，以标记普莱辛A1受体与普洛林
<ol><li>选择适当的表达载体作为骨干（例如小鼠受体plexinA1表达载体，安德烈亚斯·普舍尔博士11的一种礼物）。 注意：此普莱辛A1向量设计为实现在等离子膜中高效插入HA-或VSV标记受体。</li>
	<li>通过 PCR 放大黄道 pHluorin 编码序列，使用足够的质粒作为模板（例如，使用 pHluorin 标记的 GABA 受体，Jacob2博士的一种礼物）。如有必要，在引号的 5' 端添加一个限制站点，以方便骨干克隆步骤。</li>
	<li>使用限制位点（如图 1B中描述的 NruI/Kpn2I 限制位点）在信号肽和受体编码序列之间插入 pHluorin 序列。 注意：由于确保受体正确定位的信号肽被切开，因此应将其放置在后。这保证了对信号肽的识别，并防止了普利林从利益受体的。</li>
	<li>对获得的构造进行排序，以确保 PCR 不会引入突变。</li>
</ol>2. 在 COS7 细胞中对 pHluorin 标记的受体 体外 特征
融合蛋白到达血浆膜的能力及其可逆的荧光损失，因为pH度降低，可以使用以下程序得到确认。
<ol><li>第1天。板 1.5 x 105 COS7 细胞在玻璃底 35 毫米盘在 2 毫升完整的杜尔贝科的修改鹰介质 （DMEM - 10% 胎儿牛血清 - 2 毫升 1 mM 丙酸钠 - 25 U/ml 青霉素/链霉素 - 2.5 微克/毫升安非他明 B - pH 7.4）。</li>
	<li>第2天。变异细胞： 注意：细胞应为70-80%的汇流。<ol>
<li>准备 200 μl NaCl 150 mM 并添加 3 μg DNA，即矢量编码 pHluorin 标记受体。轻轻旋转并短暂地旋转。 注：图 1B显示了这些实验中使用的普洛林-普莱辛A1向量的地图。</li>
		<li>添加 10 μl 的转染试剂（或使用的试剂的适当量）。漩涡立即。</li>
		<li>在 Rt 孵化 10 分钟。</li>
		<li>在细胞中加入200微克的转染试剂/DNA混合物。</li>
		<li>轻轻移动板，实现混合的重新分区，并将细胞放回 37 °C 孵化器。</li>
	</ol>
</li>
	<li>第3天。去除转染介质，并更换2毫升的新鲜完整的DMEM。</li>
	<li>第4天。执行 COS7 普洛林 -普莱辛 A1 转感染细胞的活细胞成像：<ol>
<li>准备两个 DMEM 完整介质的语法，并将 pH 分别调整到 3.5 和 9.5。 注意：对于一个 35 mm 板，执行实验需要 1.5 毫升的每个解决方案。</li>
		<li>去除细胞介质，并将其替换为 1 毫升 DMEM 完整中等 pH 7.4。</li>
		<li>用适当类型的管子准备一个 5 毫升注射器，以便在不打开显微镜室的情况下将各种组件直接注入细胞培养介质中。</li>
		<li>使用允许维护 37 °C、5% CO2 潮湿工作气氛的模块。 注意：使用二氧化碳 室的另一种方法是使用 HEPES 缓冲介质（通常根据细胞类型在 10-25 mM 范围内）。</li>
		<li>将细胞放在腔室中，并调整管子和注射器。 注意：显微镜应在录制过程中开始避免机械漂移之前进行平衡。</li>
		<li>打开成像软件并选择多维采集程序。</li>
		<li>查找具有 40 倍目标的转染性 COS7 单元，并在每个单元的软件中标记位置。</li>
		<li>配置 Z 堆栈，以便进行 15μm 深度采集（在板中添加介质时，焦点可能会发生变化）。</li>
		<li>为 GFP 过滤器和相位设置曝光。</li>
		<li>配置收购时间。 注：对于整个实验的5个感兴趣的领域，每20秒收购10分钟应该足够。</li>
		<li>开始获取，并在 DMEM pH 7.4 介质中拍摄 5 个控制图像。</li>
		<li>暂停收购，注入 1.25 毫升 pH 3.5 完整 DMEM，以实现培养介质中的 pH5.5，标记软件中的事件并恢复获取 5 个时间点。 注意：绿色荧光应逐渐消失。</li>
		<li>暂停收购，注入 1.2 毫升 pH 9.5 完整 DMEM，以实现培养介质中的 pH 7.4，标记软件中的事件并恢复获取 5 个时间点。 注意：绿色荧光应重新出现在等离子膜上。</li>
		<li>分析图像。 图2 显示了通过使用 pHluorin-普莱辛 A1 构造的此类协议获得的具有代表性的图像。
</li>
	</ol>
</li>
</ol>3. 在奥沃电波化的普洛林-普莱辛A1构造
<ol><li>电化前处理鸡蛋：<ol>
<li>在孵化前一周，以 14 °C 的速度将受精卵存放在冰箱中。</li>
		<li>在饱和湿度为 50-52 小时的孵化器中以 38.5 °C （101.3 °F） 孵化卵子，直到胚胎达到 HH1412阶段。 注意：卵子在孵化过程中必须水平放置，以便胚胎正确定位进行电波处理，漂浮在蛋黄顶部。HH14阶段适合在脊髓和多头根结节的分化神经元中获得质粒的表达，具有适当的存活率。</li>
	</ol>
</li>
	<li>电波酸盐胚胎8-10：<ol>
<li>准备电波：<ol>
<li>准备浓度优于2微克/微克的无内毒素DNA质粒，以便能够根据所需的浓度稀释。</li>
			<li>拉足够的玻璃毛细毛，注入不同的DNA溶液。</li>
			<li>准备无菌PBS （-Ca2+; - Mg2+） - 100 U/ml 青霉素/链霉素，在 38.5 °C 下平衡。</li>
			<li>消毒引擎盖、弯曲剪刀和细钳。</li>
			<li>控制电极间距。 注意：一般使用电极之间的 4 mm 空间。</li>
		</ol>
</li>
		<li>窗口鸡蛋13（图3A）：<ol>
<li>使用弯曲的剪刀刺穿鸡蛋钝侧的壳。</li>
			<li>使用 0.9 mm x 25 mm 针头和 5 毫升注射器取出 2 毫升白蛋白。垂直定向针头，以避免损坏蛋黄囊。</li>
			<li>用胶带盖住蛋的顶部，以保持蛋壳的完整性。</li>
			<li>使用弯曲的剪刀，刺穿胶带中间的外壳，以平衡从鸡蛋中取出 2 毫升白蛋白时的压力。然后，切开一个足够大的窗户，使胚胎可视化，并能够在上面工作。</li>
			<li>添加±2毫升无菌温暖 PBS （ - Ca2 +; - Mg2+） - 100 U / ml 青霉素 / 链霉素，以避免胚胎脱水，使其更容易纵者接近。</li>
		</ol>
</li>
		<li>注射DNA并电镀胚胎<ol>
<li>将 PBS 中的质粒稀释 （-Ca2+; - Mg2+），浓度在 0.5-2 μg/μl 之间，并添加快速绿色染料以达到最终 0.025% 的浓度。将DNA混合物加载到毛细毛上。建议使用喷油器。 注意：检查毛细细纸的耐药性是否不是太大（这意味着注射胚胎时可能有困难）也不是太小（这意味着毛细细纸可能太大，可能会损坏胚胎）。此外，高于 2 μg/μl 的核酸浓度可能会导致非特定影响，需要控制。</li>
			<li>用加载的毛细管刺穿蛋黄囊和神经管。以浅角进入神经管，用DNA混合物（图3B）填充从尾部到头部的流明。 注意：快速绿色允许控制注射的准确性。</li>
			<li>快速将 4 mm 铂电极放置在神经管的两侧，达到您希望电波酸盐的水平，并在 31 V 处应用 3 个脉冲，以 500 毫秒间隔 （图 3C）进行 50 毫秒。 注意：避免将电极放在心脏或大型额外的胚胎血管上，以避免损坏发育中的胚胎。气泡应形成在电极上。</li>
			<li>用针头，取出2毫升白蛋白，以降低壳中的水平。</li>
			<li>用胶带密封窗户和钝边。</li>
			<li>将鸡蛋放回38.5°C的孵化器中，直到它们达到所需的阶段。</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. 胚胎嵌入和冷冻切除
<ol><li>电波化后48小时，小心收获电波胚胎（HH24阶段）。切掉胶带和一半的胆碱膜。为了防止胚胎沉入蛋黄中，在胚胎下放置一个绞盘，并切开胆碱膜的后半部分。</li>
	<li>将胚胎转移到装满冰冷 PBS 的解剖盘中。</li>
	<li>通过使用荧光立体显微镜在神经管中寻找荧光来检查电扩散效率。 注意：控制质粒编码 RFP 的共电化可能有助于可视化电波区。</li>
	<li>使用微鳞片解剖胚胎，以选择脊髓的电波化区域。</li>
	<li>将解剖胚胎转移到24井板，并固定在pH 7.4 4%甲醛（PFA） - 磷酸盐缓冲盐水（PBS），O/N在4°C。 注意：固定步骤对于使普洛林在其"活"构象中稳定至关重要，因此能够在固定/渗透组织中使用普洛林。虽然固定会大大减缓荧光的pH依赖性变化，但必须考虑到固定后仍可能发生构象/质子变化。因此，以下协议（嵌入、冷冻切除和观察）必须在固定步骤后 3 天内执行，所有缓冲器均位于 pH 7。如果不需要固定，建议对活组织部分进行观察。</li>
	<li>取出 4% PFA 并在 pH 7.4 PBS 中清洗胚胎。</li>
	<li>在PBS-15%蔗糖中孵育胚胎，并保持在4°C，直到胚胎下沉。</li>
	<li>在pH 7.4 7.5%明胶中孵育固定胚胎-15%蔗糖在37°C下45分钟，使胚胎完全嵌入。</li>
	<li>将嵌入模具嵌入冰上，加入 400μl 的 pH 7.4 7.5% 明胶 - 15% 蔗糖，以实现坚实的 2 mm 底座。</li>
	<li>用切口吸气嵌入胚胎，并将胚胎放在固体明胶底座上。</li>
	<li>用pH 7.4 7.5%明胶覆盖-15%蔗糖，并在明胶凝固之前用钳子定位胚胎。</li>
	<li>一旦明胶是固体，准备一个-40°C异丙酚浴（使用干冰或液氮），并冻结明胶块5分钟。</li>
	<li>将冷冻块保持在-80°C。</li>
	<li>将冷冻块置于-20°C，1小时。</li>
	<li>取出模具，用聚乙二醇介质固定夹头上的方块。</li>
	<li>一旦块被紧紧固定，将夹头放在冷冻统计系统中。 注意：使用涂层滑梯避免染色过程中的组织损失。</li>
	<li>执行连续冷冻剖腹产（通常执行 20μm 冷冻切除）。</li>
	<li>让冷冻片在 RT 干燥 15 分钟。 注意：应保护冷冻检查免受不必要的光线照射，以避免 GFP 荧光漂白。</li>
</ol>5. 冷冻剖腹产的微观分析
<ol><li>在RT的pH 7.4 PBS中补充水分，10分钟。 注意：如果需要，核可以沾染为霍赫斯特。</li>
	<li>在 PBS 中使用 0.5 微克/毫升的 Hoechst 溶液，并孵育冷冻切片 15 分钟。</li>
	<li>用pH 7.4 PBS冲洗幻灯片3次，5分钟。</li>
	<li>继续滑动安装。可以使用 pH 7.4（或更基本的）聚乙烯醇安装溶液，使 O/N 变硬：小心定位盖片，以避免滑梯和盖片之间形成气泡。</li>
	<li>让安装介质在黑暗中以 4 °C 变硬 O/N。</li>
	<li>使用倒置的凸显微镜精确可视化 pHluorin-plexinA1 融合蛋白：以最佳针孔和光学分辨率执行 z 堆栈，并使用 20X （NA 0.75） 或 40X （NA 1.3） 镜头。 注意：pHluorin 可检测，其参数与用于检测 GFP 的参数相同（即 排放峰值为 509 nm）。波长激发和检测滤镜设置由成像软件进行最佳定义。在 425-460 nm（激发在 405 nm）之间检测到霍奇斯特，在 485-545 nm 之间检测到 GFP 或 pHluorin（激发在 473 nm 之间），在 575-675 nm 之间检测到 RFP（激发在 559 nm 之间）。</li>
</ol>小鸡胚胎脊髓中普洛林-普莱辛A1和eGFP表达的代表图像显示在 图4中。

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Structural characterization and preliminary application.</a> Biochemistry. 41, 15477-15488 (2002).</li><li>Rose, T., Schoenenberger, P., Jezek, K., Oertner, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+refinement+of+vesicle+cycling+at+schaffer+collateral+synapses.">Developmental refinement of vesicle cycling at schaffer collateral synapses.</a> Neuron. 77, 1109-1121 (2013).</li><li>Li, Y., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pHTomato,+a+red,+genetically+encoded+indicator+that+enables+multiplex+interrogation+of+synaptic+activity.">pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity.</a> Nat. Neurosci. 15, 1047-1053 (2012).</li><li>de Wit, J., Toonen, R. 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作者没有什么可透露的。

该协议提供了一个分步程序，以遵循轴向导受体在细胞培养和小鸡胚胎脊髓发育环境中的动态。
要设计一种 诺沃 普洛林标记蛋白，需要考虑两点关于克隆策略。首先，pHluorin 标签应暴露在酸性内生体的流明中，因此应暴露在细胞外隔间，以便可视化等离子膜受体池。因此，pHluorin序列对受体序列的正确定位直接取决于所研究的受体类型（即受体的N端或C端部分?...

在导航过程中，轴突集成了多个环境提示，引导他们朝着目标前进。这些线索激活轴龙终端表面的制导受体，即生长锥体，进而启动适当的信号通路。因此，受体细胞表面分布的时间和空间调节对于确定生长锥体1的灵敏度至关重要。在此背景下，中线交叉通过通信轴突是研究受体细胞表面水平调节的极好模型。在正在发育的脊髓中，小卖部轴突最初被吸引到穿过中线的腹腔底板上。穿越后，他们失去了对地板吸引器的响应，并获得对地板排斥反应，以便他们能够退出地板，导航到他们的最终目的地在神经系统的对立面2，3。调节生长锥表面的受体可用性是响应性切换到中线提示4，5的机制之一。因此，选择性地监测生长锥体等离子膜中的受体至关重要。我们在这里描述了一种基于绿色荧光蛋白（GFP）变种的pH依赖荧光特性的方法，以具体可视化在 体外 和 体外血浆膜的轴向导受体，在发育中的小鸡脊髓中。
罗斯曼和他的同事设计了点突变pH敏感变种的GFP，包括黄道pHluorin6。黄道磷脂素在接触酸性pH（<6）时具有无荧光特性，同时在中性pH处具有荧光。这允许区分细胞内酸性隔间（即内分泌体，贩运卵泡）与荧光受体中加入等离子膜的无荧光受体，从而暴露在细胞外中性pH7中。我们利用这一点来监测普莱辛A1的等离子膜本地化，这是一种轴向制导受体，对中线驱虫剂血红素3B5（图1A）的生长锥反应进行调停。我们在这里描述了pHluorin-plexinA1构造的体外特征，以及在发育中的小鸡脊髓中这种构造的ovo电波8-10，然后是低温剖腹产的微观分析，从而能够以空间和时间分辨率在体内跟随轴向受体动力学。

<table border="0" cellpadding="0" cellspacing="0" width="1007">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">COS7 cells</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:219px;">CRL-1651</td>
			<td style="width:379px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">DMEM GlutaMAX</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">61965-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sodium pyruvate</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">11360-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Amphotericin B</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">A2942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fetal bovine serum</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Penicillin/Streptomycin</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:264px;">Exgen500 reagent</td>
			<td style="width:147px;">Euromedex Fermentas</td>
			<td style="width:219px;">ET0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">PBS -Ca2+ -Mg2+</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fast green dye</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">F7252</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">32% Paraformaldehyde aqueous solution</td>
			<td style="width:147px;">Electron Microscopy</td>
			<td style="width:219px;">15714-S</td>
			<td>Dilute extemporaneously in PBS to achieve a 4% solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Gelatin from cold water fish skin</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">G7041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sucrose</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryomount</td>
			<td style="width:147px;">Histolab</td>
			<td style="width:219px;">00890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Hoechst 34580</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:219px;">H21486</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Mowiol 4-88</td>
			<td style="width:147px;">Fluka</td>
			<td style="width:219px;">81381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Consumables</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Bottom-glass 35 mm dish</td>
			<td style="width:147px;">MatTek</td>
			<td style="width:219px;">P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">5 ml Syringe</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:219px;">SS-05S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Needles 0.9 mm x 25 mm</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:219px;">NN-2025R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Capillaries</td>
			<td style="width:147px;">CML</td>
			<td style="width:219px;">PP230PO</td>
			<td>capillaries are stretched manually in the flame</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Superfrost Plus Slides</td>
			<td style="width:147px;">Thermo Scientific</td>
			<td>4951PLUS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Curved scissors</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">129-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Microscalpel</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">10316-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Forceps</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">Dumont #5 REF#11254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Equipment/software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Time lapse microscope</td>
			<td style="width:147px;">Zeiss</td>
			<td style="width:219px;">Observer 1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">Temp module S</td>
			<td style="width:147px;">PECON for Zeiss</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">CO2 module S</td>
			<td style="width:147px;">PECON for Zeiss</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Metamorph software</td>
			<td style="width:147px;">Metamorph</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">Eggs incubator</td>
			<td style="width:147px;">Sanyo</td>
			<td style="width:219px;">MIR154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Electroporator apparatus</td>
			<td style="width:147px;">Nepa Gene CO., LTD</td>
			<td style="width:219px;">CUY21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Electrodes</td>
			<td style="width:147px;">Nepa Gene CO., LTD</td>
			<td style="width:219px;">CUY611P7-4</td>
			<td>4 mm platinum electrodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fluorescence stereomicroscope</td>
			<td style="width:147px;">LEICA</td>
			<td style="width:219px;">MZ10F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryostat</td>
			<td style="width:147px;">MICROM</td>
			<td style="width:219px;">HM550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Confocal microscope</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:219px;">FV1000, X81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fluoview software</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CLC Main Workbench software</td>
			<td style="width:147px;">CLC Bio</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of phluorin to assess the dynamics of axon guidance receptors in cell culture and in the chick embryo

在开发过程中，轴突制导受体在调节轴突对吸引力和排斥性暗示的敏感性方面起着至关重要的作用。事实上，激活制导受体是信号机制的第一步，允许轴尖，生长锥，对配体作出反应。因此，在细胞表面调节它们的可用性是参与设置生长锥敏感性的机制之一。我们在这里描述了一种方法，以精确可视化在体外和体内发育的小鸡脊髓的轴向导受体的间子-时间细胞表面动力学。我们利用绿色荧光蛋白 （GFP） 变种的 pH 依赖荧光特性，专门检测用于等离子膜的轴向制导受体的分数。我们首先描述了这种pH依赖结构的体外验证，我们进一步详细说明了它们在体内的使用，在小鸡脊髓和弦，以评估轴向导受体的兴趣的间断-时间动力学。

<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50883/50883fig1highres.jpg" src="/files/ftp_upload/50883/50883fig1.jpg"> 图1。A. 在细胞环境中的普洛林-普莱辛A1荧光特性的方案。PHluorin 在 pH 是酸性 （<6） 的细胞内隔间中是无荧光的，例如在贩运静脉或内分泌体中，当暴露在 pH 是中性的细胞外介质中时，它是荧光的。这只能可视化普洛林-普莱辛A1受体?...

Watch this Scientific Journal Video about Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo at JoVE.com

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

我们在这里描述了使用pH敏感的绿色荧光蛋白变种，pHluorin，来研究在细胞表面贩运的轴向制导受体的间歇性时间动力学。pHluorin标记的受体在细胞培养和 体内都表达，使用小鸡胚胎的电波。

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

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JoVE Journal

Neuroscience

8.2K 次观看.  University of Lyon. 我们在这里描述了使用pH敏感的绿色荧光蛋白变种，pHluorin，来研究在细胞表面贩运的轴向制导受体的间歇性时间动力学。pHluorin标记的受体在细胞培养和 体内都表达，使用小鸡胚胎的电波。

视频: Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

科研

神经科学

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

小鼠胚胎感觉轴突预测整个安装成像

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

使用的跟踪经典条件反射，评估胎儿酒精谱系障碍大鼠模型海马功能障碍

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

使用初级培养的希波坎帕神经元来研究Axon初始片段的组装

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Isolation and Culture of Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

小鸡西里神经神经元的分离与培养

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the ...