我们感谢 Irmgard 维斯的专家技术援助。这项工作得到了 DFG 的支持, 通过显微镜在纳米范围和分子生理学的大脑 (CNMPB, B1-7, T.D.) 的卓越集群。

没有研究与活动物进行。有关安乐死动物获得细胞培养的实验得到了当地动物保护当局 (Tierschutzkommission Universitätsmedizin) 的批准, T10/30 批准号。这些实验是通过批准的协议进行的。
1. 初级海马细胞培养
<ol><li>制备大鼠海马的离解细胞培养在胚胎天 1916,17。将12毫米盖玻片上的细胞涂上聚乙烯亚胺 (裴), 在24井的菜肴中, 密度为 5万-6万细胞/井。使用细胞计数室和相位对比光学检查密度。</li>
	<li>培养神经元3天 (天在体外(DIV) 3) 在24井板材在孵化器在37°c 与 5% CO2。</li>
	<li>使用透射光显微镜 (例如,相位对比光学放大 10-20X) 评估细胞健康指标的盖玻片。检查以下健康状况指标: 清晰的相衬晕, 无串珠结构的突起, 无躯体聚类或突起捆绑。</li>
</ol>2. 转染
注: 以下协议是指对3口井进行双转染。然而, 当准备足够的4口水井时, 该议定书的效果最好。
<ol><li>在锥形瓶中准备500毫升转染缓冲器 (274 毫米氯化钠, 10 毫米氯化钾, 1.4 毫米 Na2HPO4, 15 毫米葡萄糖, 42 毫米 HEPES)。<ol>
<li>溶出8.0 克氯化钠, 0.37 克氯化钾, 0.095 克钠2HPO4, 葡萄糖1.35 克, HEPES 5.0 毫升的锥形瓶中的蒸馏水。</li>
		<li>使用 ph 计将 ph 值调整为6.95 和1米氢氧化钠。</li>
		<li>用蒸馏水将容积调整到500毫升, 用 ph 计检查 ph 值。</li>
		<li>使 20-30 毫升整除数的转染缓冲器具有以下 pH 值由吹打1米氢氧化钠到转染缓冲器: 6.96, 6.97, 6.98, 6.99, 7.00, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06。 注: 转染缓冲液的 pH 值对转染效果至关重要。</li>
		<li>要测试哪个转染缓冲器会导致最大数量的被染细胞, 请从6.96 到7.11 测试每个 pH 值。使用 2.2-2.11 中描述的转染方法和一种表达 GFP 的验证质粒。确定每盖玻片转染的缓冲液 pH 值, 以评估哪个缓冲器工作最好。</li>
		<li>整除最高转染效率为2毫升的缓冲离心管冷冻和存储管在-20 摄氏度。</li>
	</ol>
</li>
	<li>预暖的减少血清培养基, 细胞培养培养基, 蒸馏水到37°c 在水浴。</li>
	<li>在1.5 毫升离心管中准备转染组合。在层流罩下工作, 确保无菌工作条件。<ol>
<li>混合7.5 µL 2 米氯化钙与4µg 每个无内毒素的 DNA (突触素 mOrange 和 mGFP/GFP-Rogdi)。在1.5 毫升的离心管中加入水, 达到60毫升的总容积。</li>
		<li>将60毫升的转染缓冲器添加到混合中。为了获得最好的结果, 添加转染缓冲滴, 同时摇动 DNA 混合轻轻地在漩涡。</li>
		<li>室温 (RT) 孵育20分钟。在孵化时间内, 在层流罩旁边放置管, 避免摇晃孵化管。</li>
	</ol>
</li>
	<li>在层流罩下, 用1000毫升吸管从油井中取出细胞培养介质 ("条件介质"), 并将其存储在孵化器的单独容器中。</li>
	<li>每井加500毫升的血清培养基。孵化细胞在37°c 和 5% CO2 , 直到20分钟潜伏期 (步骤 2.3.3) 结束。</li>
	<li>通过吹打几滴, 加入30毫升的转染混合。丢弃管子底部的残渣。</li>
	<li>在所有的水井都提供了转染混合, 轻轻摇动24井板, 以确保分配的转染混合在介质中。</li>
	<li>在37摄氏度和 5% CO2上孵化水井60分钟。</li>
	<li>去除并丢弃转染混合, 用细胞培养培养基清洗三次。在每一个井中添加1毫升细胞培养培养基, 并在 RT 中孵化30秒. 除去750毫升培养基, 加入相同数量的新鲜培养基。重复这三次。 注意: 洗涤步骤至关重要。保持每一个井的时间至少没有介质 (i., 拆卸和更换好), 并轻轻地添加洗涤介质。</li>
	<li>去除和丢弃细胞培养培养基, 再加450毫升的条件培养基。</li>
	<li>让神经细胞成熟在孵化器在37°c 和 5% CO2到 DIV 10。</li>
</ol>3. 刺激和 Syt1 吸收
注: 以下协议适用于3口井的吸收。对于任何其他数量的井的退极化, 相应地调整金额。
<ol><li>在锥形瓶中准备50毫升的10x 退极化缓冲器 (640 毫米氯化钠, 700 毫米氯化钾, 10 毫米氯化镁2, 20 毫米 CaCl2, 300 毫米葡萄糖, 200 毫米 HEPES, pH 7.4)。 注意: 退极化缓冲可以保持在4摄氏度, 几个星期。如果还使用非去极化型解决方案, 请准备一个 10x Tyrode 的解决方案, 包括 1290年 mM 氯化钠, 50 毫米氯化钾, 10 毫米氯化镁2, 20 毫米 CaCl2, 300 毫米葡萄糖, 200 毫米 HEPES pH 7.4, 以比较刺激诱导循环与自发回收。稀释至1x 后, 在使用前添加1µM 的河豚毒素以阻止动作电位的产生。<ol>
<li>溶解1.87 克氯化钠, 2.61 克氯化钾, 0.1 克氯化镁2-6H20, 0.15 克 CaCl2-2H2o 和3.0 克 glucose-1 H2o 和 2.38 g HEPES 在50毫升蒸馏水在锥形瓶。用氢氧化钠和无菌过滤溶液来调节 pH 值。稀释1:10 的冷凝水中的缓冲液, 达到1x 浓度。</li>
	</ol>
</li>
	<li>在 1x PBS (pH 7.4) 中准备4% 多聚甲醛 (粉煤灰), 用于刺激后的固定。<ol>
<li>在 1x PBS 中, 500 毫升4% 粉煤灰, 溶解20克多聚甲醛在450毫升蒸馏 H2O。 注: 加热溶液可能加速溶解, 但不加热溶液超过70摄氏度, 因为煤灰可能会解体。 注意: 粉煤灰是有毒的, 潜在的致癌性和致畸。与煤灰一起工作时戴上手套, 在通风罩下干活, 避免进食。</li>
		<li>让解决方案冷却 RT 和添加50毫升的 10x PBS 股票解决方案。使用 ph 计, 用氢氧化钠/HCl 将 ph 值调整为7.4。</li>
	</ol>
</li>
	<li>预热600毫升的1x 退极化缓冲器和10毫升的细胞培养培养基到37°c 在水浴。</li>
	<li>添加1毫升的小鼠 anti-Syt1 抗体 (克隆 604.2) 到1x 退极化缓冲和漩涡10秒。</li>
	<li>从单元格中移除和丢弃单元格培养基。添加200毫升的退极化抗体混合到每个井和孵化5分钟37摄氏度和 5% CO2在孵化器。</li>
	<li>去除和丢弃去极化抗体混合, 用细胞培养培养基清洗三次。在每一个井中加入1毫升细胞培养培养基, 在 RT 中孵育30秒. 除去750毫升培养基, 加入相同数量的新鲜培养基。重复三次。</li>
	<li>去除和丢弃细胞培养基, 在 1x PBS 中加入300毫升4% 的粉煤灰。孵育20分钟, 在4摄氏度。</li>
	<li>每5分钟洗三次, 每个 1x PBS。 注意: 协议可以在这里暂停。</li>
</ol>4. 免疫细胞化学
<ol><li>准备50毫升的阻塞缓冲器。 注: 阻塞缓冲器可保持在-20 摄氏度, 数月。<ol>
<li>将2.5 克蔗糖和1克牛血清白蛋白 (BSA) 溶解在5毫升的 10x PBS 溶液中。添加1.5 毫升10% 洗涤剂库存解决方案。搅拌解决方案, 直到所有的组件已正确溶解, 并添加蒸馏 H2O, 直到达到最终的体积50毫升。整除解决方案并冻结存储整除数。</li>
	</ol>
</li>
	<li>稀释二次, 荧光偶联抗体 (针对主要 Syt1-antibody 种) 在每井200毫升的阻塞缓冲液稀释1:1000。</li>
	<li>卸下并丢弃包含盖玻片的每个井中的 1x PBS。</li>
	<li>在每一个井中加入200毫升的阻断缓冲抗体混合物, 在 RT 中孵化60分钟。 注意: 由于次级抗体是光敏的, 所有前进的步骤必须在黑暗中执行。</li>
	<li>孵化后, 用1毫升 1x PBS 冲洗细胞三次, 5 分钟。</li>
	<li>用嵌入介质将盖玻片嵌入显微镜幻灯片中。<ol>
<li>在显微镜滑梯上添加7毫升的嵌入介质。用注射器把盖玻片从24井板上取出, 然后用镊子抓。 注意: 盖玻片表面的细胞容易受损, 因此必须小心处理镊子。</li>
		<li>将盖玻片浸入蒸馏水中, 去除 PBS, 并通过触摸一个边缘到软组织来仔细干燥。</li>
		<li>将盖玻片翻转到嵌入介质滴上, 使带细胞的表面面对显微镜滑动, 从而将细胞嵌入到嵌入介质中。</li>
	</ol>
</li>
	<li>将幻灯片放在引擎盖下面晾干 1-2 小时 (覆盖它们以避免曝光), 并将它们存储在显微镜下的滑动盒中, 温度为4摄氏度。 注意: 协议可以在这里暂停。</li>
</ol>5. 微观分析
<ol><li>盖玻片干燥后, 将其置于显微镜下, 目标和摄像头。</li>
	<li>调整每个通道的曝光时间, 使少量像素过度曝光, 以确保灰度值的最大分布。 注意: 虽然通道之间的曝光时间可能不同, 但一个通道应保持恒定, 以确保盖玻片之间的可比性。</li>
	<li>获取每盖玻片10个感兴趣区域 (ROIs) 的多通道图像。检查 ROI 是否包含经转染的神经元的轴突过程, 检查 GFP 荧光, 这应该是有点的。</li>
</ol>6. 统计分析
<ol><li>将图像导出为. tif 文件。通过单击 "文件", 将图像加载到 OpenView18中 |加载图像文件。</li>
	<li>选择突触素 mOrange 图像作为通道 1, Rogdi GFP/mGFP 图像作为通道 2, Alexa647 荧光图像作为通道3。</li>
	<li>ROIs 的门槛。<ol>
<li>点击分析|把面积超过 puncta。选择阈值和增量强度值, 以便在目视检查时, 排除漫射荧光, 只留下通道 1 (代表突触素-mOrange 荧光) 图像中的点状信号。对所有图像保持相同的阈值。</li>
	</ol>
</li>
	<li>通过单击 "立即执行", 将这些 ROIs 转移到相应的通道 2 (GFP-Rogdi/mGFP 荧光) 和每个盖玻片区域的 3 (Syt1-fluorescence)。 注意: 只有当单元格被双向转染时, 才应考虑 ROIs。在这种方法中, mOrange 和 GFP 荧光应该是清晰可见的。</li>
	<li>通过单击 "日志数据" 将数据保存到分析日志编辑器中。</li>
	<li>在 " Windows " 选项卡下打开分析日志编辑器, 并复制每个通道的值。将单独通道的值粘贴到电子表格中。</li>
	<li>确定 Syt1-channel 在 ROIs 转染条件下的平均荧光强度 (GFP-Rogdi 和 mGFP)。</li>
	<li>应用适当的统计测试, 如学生的t 检验, 以确定显著的差异。</li>
</ol>

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作者没有什么可透露的。

有三项常规用于研究突触囊泡 (SV) 回收。前两项包括使用 a) 荧光苯乙烯染料, 如 FM1-43 (纳入膜, 在吞期间被纳入细胞器, 并在胞吐作用后释放);和 b) 荧光标记重组 SV 蛋白 (在过度表达后, 纳入蛋白质回收机械)。如果附加的显影根据 pH 值改变荧光, 它们可以用来监测 SV 酸性内部和胞外培养基 ph 值的变化。用 GFP 的 pH 敏感变种标记的重组蛋白称为 Phluorins。所讨论的两种分析方法都有以前...

研究突触囊泡回收在确定突触前的性质如何改变, 无论是在突触可塑性或响应突触功能的扰动。研究 Synaptotagmin-1 (Syt1) 抗体摄取提供了一种测量 SV 循环量的方法。Syt1 是一种 SV 相关蛋白, 充当 Ca2 +传感器, 是 exocytotic 释放神经递质1,2的必要条件。它是一种跨膜蛋白, 具有 C 末端细胞质域外的 sv 和 n-端腔内域的 sv3。在胞吐作用过程中, Syt1 的腔域会暴露于外界介质中。对于这个外在的媒介, 我们增加抗体针对细胞质领域, 在吞期间被内部化。这些抗体可以是预共轭与显影或 immunostained 与二级抗体4,5,6,7。结果 immunosignal 的荧光强度与 SV 回收量成正比。这种方法可用于确定本构和退极化诱发 SV 循环6,8。
Syt1 摄取化验可以执行后, 病毒介导的基因转移到几乎所有的细胞在盘子里或经过稀疏转染少量的细胞。本方法将原发海马神经元的稀疏双转染法与磷酸钙9结合使用。我们使用一种已知的重组标记蛋白积累在 presynapses, 荧光标记突触素, 找到突触前终端和过度表达 tshr 我们的蛋白质的兴趣, Rogdi。这使我们可以测试是否 Rogdi 目标功能突触和影响 SV 回收。基因编码 Rogdi 最初是在屏幕上发现的果蝇突变体的特点是受损的记忆10。在人类中, Rogdi 基因突变导致一种罕见的破坏性疾病, 称为 Kohlschütter-Tönz 综合征。患者患有牙釉质畸形、pharmacoresistant 癫痫、精神运动迟缓;然而, 基因产物的亚细胞定位仍然是难以捉摸的11。因此, Syt1 摄取检测为 GFP 标记的 Rogdi 在功能突触9的定位提供了关键的证据。
这种吸收技术有几个好处。首先, 通过测量 Syt1 荧光标签的荧光强度, 可以实时观测到7、12、6、9的活度成像, 从而实现 SV 循环。此外, 还开发了几种 Syt1 抗体变种。有未加标签的变体, 可以在固定后使用标准染色协议标记为继发抗体, 并附有荧光标签的预共轭变种。最后, 抗体为基础的荧光是有利的, 因为大量选择的商业可用的二级或共轭染料, 可以使用。
当固定和染色神经元时, 也有可能染色额外的蛋白质和进行定位分析。这可以帮助确定它们的位置与回收 SVs 的关系。荧光标签的强度是 SV 回收量的直接度量。此外, 抗体有选择地标记 Syt1-containing 结构, 导致高特异性和小背景荧光4。也可以使用不同的刺激协议, 如退极化缓冲器或电刺激协议9,12,13,14。但是, 在不刺激神经元培养15的情况下, 可以测量基底 SV 循环。
我们的方法专门解决了双转染神经元的 Syt1 抗体摄取与继发抗体 immunolabeling 固定后。但是, 我们在讨论中提到了所有常规使用的方法变体, 使观众有机会根据具体需要调整该协议。

<table><tbody><tr><td>B27</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A7030-50g</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C3306-100g</td><td></td></tr><tr><td>CoolSNAP HQ2</td><td>Photometrics</td><td></td><td></td></tr><tr><td>dH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Invitrogen</td><td>15230</td><td></td></tr><tr><td>DABCO</td><td>Merck</td><td>8.03456.0100</td><td></td></tr><tr><td>donkey anti mouse Alexa 647</td><td>Jackson-Immunoresearch</td><td>715605151</td><td>antibody</td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>41966</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190</td><td></td></tr><tr><td>Eppendorf tubes</td><td>Eppendorf</td><td>30120094</td><td></td></tr><tr><td>multiwell 24 well</td><td>Fisher Scientific</td><td>087721H</td><td></td></tr><tr><td>tube (50 mL)</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr><tr><td>FBS superior</td><td>BiochromAG</td><td>S0615</td><td></td></tr><tr><td>Glucose</td><td>Merck</td><td>1,083,421,000</td><td></td></tr><tr><td>HBSS</td><td>Invitrogen</td><td>14170</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034-500g</td><td></td></tr><tr><td>Hera Cell 150 (Inkubator)</td><td>ThermoElectron Corporation</td><td></td><td></td></tr><tr><td>KCL</td><td>Sigma-Aldrich</td><td>P9541-500g</td><td></td></tr><tr><td>L-Glutamin</td><td>Gibco</td><td>25030</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Honeywell</td><td>M0250-500g</td><td></td></tr><tr><td>microscope slides</td><td>Fisher Scientific</td><td>10144633CF</td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>Mowiol4-88</td><td>Calbiochem</td><td>475904</td><td></td></tr><tr><td>NaCl</td><td>BioFroxx</td><td>1394KG001</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>BioFroxx</td><td>5155KG001</td><td></td></tr><tr><td>Neurobasal</td><td>Invitrogen</td><td>21103049</td><td></td></tr><tr><td>OpenView Experiment Analysis Application</td><td>Free software, see comments</td><td></td><td>written by Noam E. Ziv, Technion &amp;ndash; Israel Institute of Technology, Haifa, Israel</td></tr><tr><td>PBS (10x)</td><td>Roche</td><td>11666789001</td><td></td></tr><tr><td>Optimem</td><td>Invitrogen</td><td>31985</td><td></td></tr><tr><td>Penstrep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>PFA</td><td>Sigma</td><td>P6148-1kg</td><td></td></tr><tr><td>safety hood</td><td>ThermoElectron</td><td></td><td>Serial No. 40649111</td></tr><tr><td>Sucrose</td><td>neoFroxx</td><td>1104kg001</td><td></td></tr><tr><td>Synaptotagmin1</td><td>Synaptic Systems</td><td>105311</td><td>mouse monoclonal; clone 604.2</td></tr><tr><td>Triton X-100</td><td>Merck</td><td>1,086,031,000</td><td></td></tr><tr><td>Vortex Genius 3&amp;nbsp;</td><td>IKA</td><td>3340001</td><td></td></tr><tr><td>Water bath</td><td>GFL</td><td>1004</td><td></td></tr><tr><td>Zeiss Observer. Z1&amp;nbsp;</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

an optical assay for synaptic vesicle recycling in cultured neurons overexpressing presynaptic proteins

在主动突触前神经终端, 突触囊泡经历了外周和吞的循环。在回收过程中, SV 跨膜蛋白的腔域在细胞表面暴露。其中一种蛋白质是 Synaptotagmin-1 (Syt1)。对 Syt1 的腔域进行的抗体, 一旦添加到培养基中, 就会在 endocytotic 周期内被占用。这种吸收量与 SV 循环的数量成正比, 可以通过免疫荧光进行量化。在这里, 我们结合 Syt1 抗体摄取与双转染培养海马神经元。这使我们能够 (1) 根据重组突触前标记突触素的表达来本地化突触前站点, (2) 使用 Syt1 摄取确定它们的功能, (3) 描述了一种感兴趣的蛋白质的靶向性和作用, GFP-Rogdi。

这种方法的预期结果是每盖玻片大约有50个双转染神经元, 每一个密度为5万个神经元。每个神经元的轴突将显示荧光标记的突触素堆积的多个热点, 表明簇 ofSVs。在功能突触前部位, 重组突触素信号 colocalizes 有点 Syt1 荧光。使用双转染, 无论是 GFP-Rogdi 作为感兴趣的蛋白质 (图 1) 或 mGFP, 因为该控制与重组突触素共同表达。
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Watch this Scientific Journal Video about An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins at JoVE.com

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

我们描述了一种用于培养神经元的突触囊泡 (SV) 循环的光学检测方法。将该协议与双转染相结合, 以表达突触前标记和感兴趣的蛋白质, 使我们能够定位突触前的部位, 它们的突触囊泡再生能力, 并确定感兴趣的蛋白质的作用。

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

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Neuroscience

7.4K 次观看.  University Medical Centre Göttingen. 我们描述了一种用于培养神经元的突触囊泡 (SV) 循环的光学检测方法。将该协议与双转染相结合, 以表达突触前标记和感兴趣的蛋白质, 使我们能够定位突触前的部位, 它们的突触囊泡再生能力, 并确定感兴趣的蛋白质的作用。

视频: An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

突触量化：基于一个免疫细胞化学检测，以量化突触数量

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

科研

神经科学

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

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 ...

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.

使用调频染料诱发过程中，自发的，微型突触活动检查突触囊泡循环的

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

海马神经元中突触囊泡吞的测量

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

一种用于监测 ampa 受体贩运的高含量检测方法

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

Presynapse Formation Assay Using Presynapse Organizer Beads and &ldquo;Neuron Ball&rdquo; Culture

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in appropriate order by transport from cell bodies and/or local translation in immature synapses. However, it is not fully understood how synaptic proteins accumulate in synapses in proper order. Here, we present a novel method to analyze presynaptic formation by using the combination of neuron ball culture with beads to induce presynapse formation. Neuron balls that is neuronal cell aggregates provide axonal sheets far from cell bodies and dendrites, so that weak fluorescent signals of presynapses can be detected by avoiding overwhelming signals of cell bodies. As beads to trigger presynapse formation, we use beads conjugated with leucine-rich repeat transmembrane neuronal 2 (LRRTM2), an excitatory presynaptic organizer. Using this method, we demonstrated that vesicular glutamate transporter 1 (vGlut1), a synaptic vesicle protein, accumulated in presynapses faster than Munc18-1, an active zone protein. Munc18-1 accumulated translation-dependently in presynapse even after removing cell bodies. This finding indicates the Munc18-1 accumulation by local translation in axons, not transport from cell bodies. In conclusion, this method is suitable to analyze accumulation of synaptic proteins in presynapses and source of synaptic proteins. As neuron ball culture is simple and it is not necessary to use special apparatus, this method could be applicable to other experimental platforms.

Presynapse formation is a dynamic process including accumulation of synaptic proteins in proper order. In this method, presynapse formation is triggered by beads conjugated with a presynapse organizer protein on axonal sheets of &#8220;neuron ball&#8221; culture, so that accumulation of synaptic proteins is easy to be analyzed during presynapse formation.

使用预突子形成测定使用预突子组织者珠和"神经元球"文化

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

肌萎缩性侧索硬化症模型中突触功能的实时荧光测量

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Live Imaging of Synaptic Vesicle Recycling in the Neuromuscular Junction of Dissected Larval Zebrafish

Neuronal communication is mediated by synaptic transmission, which depends primarily on the release of neurotransmitters stored in synaptic vesicles (SVs) in response to an action potential (AP). Since SVs are recycled locally at the presynaptic terminal, coordination of SV exocytosis and endocytosis is important for sustained synaptic transmission. A pH-sensitive green fluorescent protein, called pHluorin, provides a powerful tool to monitor SV exo/endocytosis by targeting it to the SV lumen. However, tracking AP-driven SV recycling with the pHluorin-based probes is still largely limited to in vitro culture preparations because the introduction of genetically encoded probes and subsequent optical imaging is technically challenging in general for in vivo animal models or tissue preparations. Zebrafish is a model system offering valuable features, including ease of genetic manipulation, optical clarity, and rapid external development. We recently generated a transgenic zebrafish that highly expresses a pHluorin-labeled probe at motor neuron terminals and developed a protocol to monitor AP-driven SV exo/endocytosis at the neuromuscular junction (NMJ), a well-established synapse model that forms in vivo. In this article, we show how to prepare larval zebrafish NMJ preparation suitable for pHluorin imaging. We also show that the preparation allows time-lapse imaging under conventional upright epifluorescence microscope, providing a cost-effective platform for analyzing NMJ function.

The zebrafish larval neuromuscular junction is an attractive model for studying synaptic physiology. It is amenable to many experimental techniques, including electrophysiology and optical imaging. Here, we describe a protocol for imaging synaptic transmission using a pHluorin-based probe under an upright epifluorescence microscope.

解剖斑马鱼幼鱼神经肌肉接头突触囊泡循环的实时成像

Neuronal communication is mediated by synaptic transmission, which depends primarily on the release of neurotransmitters stored in synaptic vesicles (SVs) ...