Name: measuring synaptic vesicle endocytosis in cultured hippocampal neurons
Uploaded: 2017-09-04T12:30:00.000+00:00
Duration: 7 min 30 s
Description: Watch this Scientific Journal Video about Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons at JoVE.com

我们感谢 Dr. 的 synaptophysin-pHluorin2x 建造, Dr. 提供 VAMP2-phluorin。我们感谢 Dr. 苏珊和弗吉尼亚克罗克的一电子显微镜设施的技术支持和帮助。这项工作得到了美国国家神经疾病研究所和脑卒中内部研究计划的支持, 并得到了 KRIBB 研究计划 (韩国生物医学科学家研究项目) 的资助, 韩国研究所大韩民国生物科学和生物工程。

注意: 下面的协议描述了在培养海马神经元中使用的 pHluorin 成像方法和 em 方法. pHluorin 监测活细胞中突触囊泡蛋白的摄取和 em 检测突触泡的摄取和超微结构的变化. 
 动物护理和程序遵循 nih 的指导方针, 并得到了 nih 动物保育和使用委员会的批准. 
 1. pHluorin 成像 <ol> <li> 海马神经元培养 <ol> <li> 通过组合 4 mm NaHCO 3 和 5 mm HEPES, 并调整到 pH 7.3 与 5 M 氢氧化钠。通过混合 neurobasal 培养基、2% B27、0.5 毫米-l-谷氨酰胺和1% 青霉素-链霉素进行培养基的培养。另外, 准备一个血红蛋白缓冲液与20% 胎牛血清 (HB/20%fbs) 的混合物. 
		注意: 此培养协议基于 Sankaranarayanan, et al. 29 和 Wu , et al. 24 </li> <li> 斩首在产后0天到2天之间的小鼠幼崽变成培养基, 并将大脑提取到4和 #176; HB/20%fbs。切除脑干和丘脑以暴露海马。解剖的海马, 后, 揭露它的脑干和丘脑切除, 并转移到新鲜的4和 #176; HB/20%fbs. 
		注意: 通常, 一个 pup (两个海马) 的收益率大约为 4 x 10 5 单元/mL。用镊子或剪刀清洗附着的膜, 然后转到新鲜的4和 #176; HB/20%fbs。用剪刀切开齿状回, 分离 subiculum, 然后转移到新鲜的4和 #176; C HB/20%fbs. </li>
		<li> 将每个海马体从端部切割到10片, 并将其转移到15毫升的聚丙烯锥形管中. </li>
		<li> 在允许组织沉淀后, 用10毫升的 HB/20%fbs 冲洗, 然后用10毫升的 HB 洗涤三次. </li>
		<li> 准备137毫米氯化钠, 5 毫米氯化钾, 7 毫米 Na2HPO 4 和25毫米 HEPES 的消解溶液, 并调整到 pH 7.2 与5米氢氧化钠。从海马中除去清液。添加10毫克的胰蛋白酶和1毫克的 dnasei 到2毫升的消化液, 并通过无菌0.22 和 #181; m 膜直接在样品颗粒上过滤. </li>
		<li> 在37和 #176 孵育海马5分钟, 然后用10毫升的 HB/20%fbs 冲洗两次, 然后用10毫升的 HB 冲洗一次. </li>
		<li> 将单元格与6毫克的 MgSO 4 & #8729; 7H 2 O 和1毫克的 dnasei 到2毫升 HB 和无菌过滤器通过无菌0.22 和 #181; m 过滤器海马颗粒。小心地研制细胞, 同时注意避免引入气泡。让组织微粒沉淀2分钟, 然后慢慢地将上清液转移到另一根管子上. </li>
		<li> 添加3毫升 HB/20%fbs 到细胞悬浮, 离心机或10分钟在4和 #176; C 和 1000 rpm。在培养基中丢弃上清和重. </li>
		<li> 板6万细胞悬浮在150和 #181; L 培养基上的聚 d-赖氨酸涂层25毫米直径片, 没有溢出的片。在电镀后加入2毫升的5ml 培养基2小时。片是保持在6良好的培养皿或无菌的 Petri 盘. </li>
		<li> 在37和 #176 中维护单元格; C 在 5% CO 2 录制前14-21 天在培养基中加湿培养培养基。在文化成长过程中, 每周更换培养基的上半部两次. </li>
	</ol> </li> <li> 转染 <ol> <li> 电镀后6-7 天, 染突 pHluorin 2X (SpH) 或 VAMP2-pHluorin 进入海马神经元。对于 SpH, 使用巨细胞病毒 (CMV) 启动子, 插入到一个 pcDNA3 矢量 30 。对于 VAMP2-pHluorin, 使用插入到 pCI 向量 31 中的 CMV 启动子. </li>
		使用1和 #181; 质粒的 g, 通过脂质载体将载体染到靶细胞中。使用培养基从协议步骤 1.1.1, 缺乏血清, 为转染。在转染后改变培养基2小时以降低毒性. 
		注: 在 boutons SpH 低表达时, 将 DNA 浓度提高到2和 #181; g 或与脂质载体的潜伏期, 除非细胞不健康。通常, 4-10 细胞 (0.006-0. 008%) 的神经元转染. </li>
	</ol> </li> <li> 光显微镜 <ol> <li> 配制由119毫米氯化钠组成的生理盐水溶液, 2 毫米 CaCl 2 , 2.5 毫米氯化钾, 25 毫米 HEPES (pH 7.4), 30 毫米葡萄糖, 2 毫米氯化镁 2 , 0.01 毫米 6-氰基-7-nitroquinoxaline-23-酮 (CNQX) 和0.05 毫米 DL-2-amino-5-phosphonovaleric 酸 (AP5)。采取片从培养基板和地方在一个成像室, 允许现场刺激, 使用润滑剂和密封剂, 以避免泄漏。避免让玻璃在片从盘子转移到腔室时, 立即加入750和 #181; 生理盐水溶液. 
		注: CNQX 和 AP5 被用来阻断突触后的活动, 有可能复发的活动。在倒置荧光显微镜上放置一个腔室之前, 使用清洁组织来确认腔室不泄漏。在记录过程中, 由于生理盐水和浸泡油的混合而导致焦点变化, 导致折射率的变化. </li>
		<li> 刺激和记录 <ol> 在倒置的 widefield 显微镜上的 <li> 图像, 带有金属卤化物灯的 60X (1.4 数字孔径) 油浸透镜。可视化 pHluorin 与过滤器集为 480 nm 的励磁峰值, 一个 490 nm 长的通行证镜子, 一个 500-550 nm 发射过滤器, 和一个手动翻转快门。捕获图像每 100 ms, 与 2 x 2 分, 使用电子倍增电荷耦合设备 (EMCCD) 相机. 
			注意: 应考虑几个特性, 以达到适当的条件, 以避免漂白, 包括图像捕获间隔, 和过滤器和分, 这是依赖于设备。在共焦成像的情况下, 应考虑激光功率和曝光时间, 以避免漂白。该设置是在记录至少3分钟没有刺激和记录进行了至少十年代没有刺激检查漂白. </li>
			<li> 选择具有高密度 boutons 的区域, 以便于在每个实验中进行分析。通过其绿色荧光信号来识别转染细胞, 强调溥和 boutons 之间连续表达的圆形或椭圆形的表达模式. </li>
			<li> 利用1毫秒脉冲激发吞, 20 mA 动作电位 (AP) 由脉冲刺激器提供, 并通过刺激隔离单元中的铂电极传递。在刺激过程中和细胞恢复过程中的图像荧光活动. 
			注意: 在死细胞的情况下, 表达比活 boutons 强, 并且显示了一个参差不齐的样式在 boutons 之间. </li>
		</ol> </li> </ol> </li> <li> 图像分析 <ol> <li>为了分析单个溥的荧光强度, 建立了一个 1.5 x 1.5 和 #181 的感兴趣区域; m 平方;溥的大小在大约1.5 和 #181; m. 在刺激之前使用痕迹检查漂白和减去作为背景. </li>
		<li> 规范化荧光变化 (和 #916; f) 与等式: <img alt="Equation" height="11" src="/files/ftp_upload/55862/55862eq1.jpg" width="172"/> 其中 f 最大 和 f 0 是指刺激后的最大增加和基线荧光, 分别。测量吞率的衰变率在前4-10 秒后的最大点 pHluorin 荧光。获得时间常数 (和 #964;) 吞通过拟合 pHluorin 荧光衰变从峰值增加到基线的单指数函数. </li>
	</ol> </li> </ol> 2。电子显微镜 <ol> <li> 在室温下, 用1.5 毫升0.01% 无菌过滤的聚-d-赖氨酸溶液在每个井中为1小时, 然后用灭菌的水冲洗三次, 以制备多聚-d-赖氨酸涂层 6-好的板材。解剖, 培养和维持海马神经元的步骤 1.1.1, 1.1.2, 和1.1.3 分别. </li>
	<li> 准备一个高 K + 的刺激方案, 用 HRP 作为31.5 毫米氯化钠, 2 毫米 CaCl 2 , 90 毫米氯化钾, 25 毫米 HEPES (pH 7.4), 30 毫米葡萄糖, 2 毫米氯化镁 2 , 0.01 毫米 CNQX, 0.05 毫米 AP5, 5 毫克/毫升 HRP, 然后调整到 pH 7.4 与5米氢氧化钠. 
	<ol> <li> 在室温下使用 1.5 ml 高 K + 刺激方案刺激海马神经元培养 (称为 K + ), 并在九十年代对每个井加1.5 毫升。在休息条件 (简称 R), 适用于九十年代相同浓度的5毫克/毫升 HRP, 但与生理盐水溶液。对于恢复样本, 应用高 k + 刺激解决方案与 k + 样本, 然后快速清洗和替换生理盐水和孵化为10分钟 </li>
	</ol> </li> <li> 固定和着色 <ol> <li> 使用21.4 克/L na 甲在 pH 值7.4 处准备 0.1 M na 甲缓冲区。在室温下, 用4% 戊二醛在0.1 米甲缓冲中固定细胞, 至少1小时。洗涤三次与 0.1 M Na 甲缓冲为7分钟每个. </li>
		<li> 准备 Diaminobenzidine (民建联) 解决方案, 由0.5 毫克/毫升的民建联与 0.3% H 2 O 2 在 ddH 2 O, 和过滤器与0.22 和 #181; m 过滤器。应用1.5 毫升民建联解决方案为30分钟, 在37和 #176; c. 洗涤三次以 0.1 M Na 甲缓冲为 7 min 每个. 
		警告: 民建联是一种有毒和可疑的致癌物。请使用手套和实验室大衣. 
		注: 与民建联的标签, 是由于其氧化的 H 2 O 2 , 由 HRP 催化。小的增加在组分导致一个增加的信号在样品。增加 HRP 的浓度会加速催化剂的作用。足够大的浓度的 H 2 O 2 允许与 HRP 削弱的副作用, 抑制标记 32 的效果。在这项工作中, 这个标签系统的浓度是根据目前可用的研究选择 33 , 34 。
		<li> 孵育1.5 毫升 1% OsO 4 在 0.1 M Na 甲缓冲区为1和 #176; C 作为后固定。洗涤三次与1.5 毫升 0.1 M Na 甲缓冲区为7分钟每. 
		注意: 由于 OsO 4 的毒性和反应性, 在许多情况下, 将样品放在化学罩的冰上比使用冰箱进行孵化更可取. </li>
		<li> 制备0.1 米醋酸钠缓冲液, 13.61 克/升醋酸钠和11.43 毫升/升冰醋酸在 pH 值5.0。洗涤三次以1.5 毫升 0.1 M 乙酸酯缓冲在 ph 5.0 为7分钟每和孵育与1.5 毫升1% 铀乙酸酯在 0.1 m 醋酸盐缓冲在 ph 5.0 为 1 h 在4和 #176; c. 洗涤三次与1.5 毫升 0.1 m 醋酸盐缓冲7分钟每. </li>
	</ol> </li> <li> 环氧嵌入 <ol> <li> 将神经元培养脱水为50%、70% 和90% 乙醇的单1.5 毫升洗涤, 在每一个7分钟, 然后3毫升1.5 的洗涤在油烟罩中每100% 分钟. </li>
		<li> 混合485毫升/升双酚 A-(epichlorhydrin) 环氧树脂, 160 毫升/升烯丁二酸酐 (DDSA), 340 毫升/升 Methyl-5-Norbornene-2,3-Dicarboxylic 酸酐 (NMA), 和15毫升/升 24, 6-三 (甲基) 苯酚 (DMP-30) 创建环氧树脂树脂.混合彻底, 然后储存在真空中, 以消除气泡. 
		注意: 这是至关重要的, 以消除气泡的树脂, 特别是那些小于肉眼可见, 因为它们可以导致在树脂切片期间的蛀牙. </li>
		<li> 通过将乙醇中的50% 环氧树脂替换为乙醇中的30分钟, 然后在振动筛的室温下将70% 的环氧树脂用于乙醇中, 从而将其渗透到样品中. </li>
		<li> 用100% 环氧树脂开关环氧树脂溶液, 孵育10分钟, 在50和 #176; c. 在50和 #176 进行1小时孵化的两次新的100% 环氧树脂的交换; c. 加入新鲜的100% 环氧树脂, 并允许在50和 #176 硬化; c 过夜, 然后在60和 #176;C 超过36小时硬化. </li>
	</ol> </li> <li> 从具有珠宝商和 #39 的多井板中卸下每个样品; 手锯。选择感兴趣的区域, 密集的细胞浓度, 使用倒置的光显微镜, 然后削减70至 80 nm 块的切片由切片。在切片卡盘中安装剪切区域并加载切片。将卡盘放置在切片中, 然后装上一把与该块表面平行的边缘的金刚石刀。将70至 80 nm 厚度的剖面直接收集到单个网格上. </li>
	<li> 通过重量将醋酸铀溶入水中1% 溶液, 并将柠檬酸铅分解为3% 溶液。Counterstain 1% 水铀醋酸盐浸泡15分钟, 然后3% 水柠檬酸铅5分钟, 以改善样品的对比度. </li>
	<li> EM 成像 <ol> <li> 使用透射电子显微镜检查剖面, 并在 10,000-20,000X 3 的主要放大倍数下记录 CCD 数字照相机的图像. </li>
	</ol> </li> <li> 统计 <ol> <li> 执行 t 测试, 通过比较控制和实验样本之间的测量 (s.e.m.) 的平均值和标准误差来识别显著的差异. </li>
	</ol>
	</li>
</ol>

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作者没有什么可透露的。

这里我们展示了两种监测突触囊泡吞的方法。在第一种方法中, 我们监测 pHluorin 融合的突触泡蛋白在转染神经元和随后电刺激。其次, 我们使用的 EM 成像的 HRP 吸收诱导氯化钾。我们使用不同的刺激有两个原因。首先, 高钾的应用诱导了培养中所有神经元的去极化。这有助于 em 检查, 因为我们的 em 方法不能区分陶土和受激神经元。其次, 微的形态学变化更可靠地观察后, 强烈的刺激, 如高钾刺激, 而...

神经递质储存在突触泡中, 由胞释放。突触囊泡膜和蛋白质被吞化, 并在下一轮胞中重复使用。突触囊泡的吞对维持突触囊泡池和从细胞膜上去除突起的泡囊很重要。ph 敏感的绿色荧光蛋白 pHluorin, 这是在酸性情况下淬火和 dequenched 在中性 ph 值, 已被用来测量吞时间课程在活细胞1,2,3。pHluorin 蛋白通常附着在突触囊泡蛋白的腔侧, 如突或 VAMP2/synaptobrevin。在休息, pHluorin 是淬火在 5.5 pH 流明的突触泡。囊泡融合到等离子体膜暴露水泡腔的胞外溶液的 pH 值是〜 7.3, 导致增加 pHluorin 荧光。胞后, 增加的荧光衰变, 由于吞的突触囊泡蛋白, 其次是囊泡 re-acidification 在这些回收泡。虽然衰变反映了吞和水泡 re-acidification, 它主要反映吞, 因为 re-acidification 在大多数情况下比吞快1,4。re-acidification 的时间常数为 3-4 s 或更少的5,6, 它通常比十年代或更多的泡吞4,5所需的速度快。如果需要进行实验来区分吞和 re-acidification, 使用 4-Morpholineethanesulfonic 酸 (MES) 溶液 (25 mM) 和 pH 值为5.5 的酸性淬火实验可以用来确定是否检索突触囊泡蛋白从等离子膜通过吞1,3,4。因此, pHluorin 荧光强度的增加反映了外、吞的平衡, 神经刺激后的减少具体反映了吞。
pHluorin 成像不仅可以用来测量吞的时间过程, 也可用于突触囊泡池的大小7,8, 以及诱发释放和自发释放9的概率。许多因素和蛋白质参与调节吞, 如钙, 可溶性 NSF-附着蛋白受体 (诱捕) 蛋白, 脑源性神经营养因子 (BDNF) 和磷酸已被确定使用 pHluorin 成像1,2,10,11,12,13,14,15,16. 此外, 神经递质的释放不仅可在原发神经元中检测, 而且可以在显微镜17的神经母细胞瘤中发现。最近, pHluorin 变体、dsRed、mOrange 和 pHTomato 被开发用于在单个突触1819中监视多个因素的同时录制。例如, pHTomato 已与突融合, 并与基因编码的钙指示剂 (GCaMP5K) 一起使用, 以监测前囊泡融合, 并在突触后室20中的 Ca2 +流入。因此, pHluorin 附在突触蛋白提供了一个有用的方法来分析吞和胞的关系。
EM 是另一种方法, 通常用于研究吞, 由于高空间分辨率, 显示超微结构的变化, 在吞。两个一般的领域是能够可视化的病理改变神经元细胞21和跟踪囊泡蛋白22。特别是, 观察突触囊泡摄取, 膜曲率涂覆格在 periactive 区, 和内涵结构是可能的 EM3,23,24,25 ,26,27,28。虽然 EM 涉及潜在的文物, 如固定诱发的畸形, 可能会影响吞, 和数据分析是劳动密集型, 该决议提供了一个诱人的机会, 可视化细胞结构。在吞27期间, 可能存在的定影问题和 EM 时间分辨率的限制可以通过高压冻结来克服, 提供一种快速和非化学的方法来稳定当前的微妙结构。

<table><tbody><tr><td>Lipofectamine LTX with Plus</td><td>Thermo Fisher</td><td>15338-100</td><td>Transfection of plasmid DNA including synaptophysin or VAMP2-pHluorin</td></tr><tr><td>neurobasal medium</td><td>Thermo Fisher</td><td>21103-049</td><td>Growth medium for neuron, Warm up to 37&amp;deg;C before use</td></tr><tr><td>B27</td><td>Thermo Fisher</td><td>17504-044</td><td>Gradient for neuronal differentiation</td></tr><tr><td>Glutamax</td><td>Thermo Fisher</td><td>35050-061</td><td>Gradient for neuronal culture</td></tr><tr><td>Poly-D-Lysine coated coverslip</td><td>Neuvitro</td><td>GG-25-pdl</td><td>Substrate for neuronal growth and imaging of pHluorin</td></tr><tr><td>Trypsin XI from bovine pancrease</td><td>Sigma</td><td>T1005</td><td>Neuronal culture-digest hippocampal tissues</td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma</td><td>D5025</td><td>Neuronal culture-inhibits viscous cell suspension</td></tr><tr><td>pulse stimulator</td><td>A-M systems</td><td>model 2100</td><td>Apply electrical stimulation</td></tr><tr><td>Slotted bath with field stimulation</td><td>Warner Instruments</td><td>RC-21BRFS</td><td>Apply electrical stimulation</td></tr><tr><td>stimulus isolation unit</td><td>Warner Instruments</td><td>SIU102</td><td>Apply electrical stimulation</td></tr><tr><td>lubricant</td><td>Dow corning</td><td>111</td><td>pHluorin imaging-seal with coverslip and imaging chamber, avoid leak from chamber</td></tr><tr><td>AP5</td><td>Tocris</td><td>3693</td><td>Gradient for normal saline, selective NMDA receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity</td></tr><tr><td>CNQX</td><td>Tocris</td><td>190</td><td>Gradient for normal saline, competitive AMPA/kainate receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity</td></tr><tr><td>Illuminator</td><td>Nikon</td><td>C-HGFI</td><td>Metal halide light source for pHluorin</td></tr><tr><td>EMCCD camera</td><td>Andor</td><td>iXon3</td><td>pHluorin imaging, detect pHluorin fluorescence intensity</td></tr><tr><td>Inverted microscopy</td><td>Nikon</td><td>Ti-E</td><td>Imaging for synaptophysin or VAMP2 pHluorin transfected cells</td></tr><tr><td>NIS-Elements AR</td><td>Nikon</td><td>NIS-Elements Advanced Research</td><td>Software for imaging acquisition and analysis</td></tr><tr><td>Igor Pro</td><td>WaveMetrics</td><td>Igor pro</td><td>Software for imaging analysis and data presentation</td></tr><tr><td>imaging chamber</td><td>Warner Instruments</td><td>RC21B</td><td>pHluorin imaging, apply field stimulation on living cells</td></tr><tr><td>poly-l-lysine</td><td>Sigma</td><td>P4832</td><td>Electron microscopy, substrate for neuronal growth, apply on multiwell plate for 1 h at room temperature then wash with sterilized water 3 times</td></tr><tr><td>Horseradish peroxidase(HRP)</td><td>Sigma</td><td>P6782</td><td>Electron microscopy, labeling of endocytosed synaptic vesicles by catalyzing DAB in presence hydrogen peroxide, final concentration is 5 mg/mL in normal saline, make fresh before use</td></tr><tr><td>Na cacodylate</td><td>Electron Microscopy Sciences</td><td>12300</td><td>Electron microscopy, buffer for fixatives and washing, final concentration is 0.1 N</td></tr><tr><td>3,3&amp;prime;-Diaminobenzidine(DAB)</td><td>Sigma</td><td>D8001</td><td>Electron microscopy, labeling of endocytosed synaptic vesicles, substrate for HRP, final concentration is 0.5 mg/mL in DDW and filtered, make fresh before use</td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma</td><td>H1009</td><td>Electron microscopy, labeling of endocytosed synaptic vesicles by inducing HRP-DAB reaction, final concentration is 0.3% in DDW, make fresh before use</td></tr><tr><td>glutaraldehyde</td><td>Electron Microscopy Sciences</td><td>16365</td><td>Electron microscopy, fixatives, final concentration is 4% in Na-cacodylate buffer, make fresh before use, shake well before to use</td></tr><tr><td>TEM</td><td>JEOL</td><td>200CX</td><td>Electron microscopy, imaging of endocytosed vesicles and ultrastructural changes</td></tr><tr><td>CCD digital camera</td><td>AMT</td><td>XR-100</td><td>Electron microscopy, capturing images</td></tr><tr><td>Lead citrate</td><td>Leica microsystems</td><td>16707235</td><td>Electron microscopy, grid staining</td></tr></tbody></table>

measuring synaptic vesicle endocytosis in cultured hippocampal neurons

在吞, 融合突触泡在神经末端被检索, 允许囊泡再循环和因而维持突触传输在重复的神经生火期间。损伤的吞在病理条件下导致突触强度和脑功能下降。在这里, 我们描述的方法用于测量突触囊泡吞在哺乳动物海马突触在神经元培养。我们监测突触囊泡蛋白吞融合的突触囊膜蛋白, 包括突和 VAMP2/synaptobrevin, 在水泡腔侧, 与 pHluorin, pH 敏感的绿色荧光蛋白, 增加其荧光强度随着 pH 值的增加而增大。在胞, 水泡流明 ph 增加, 而在吞水泡流明 ph 值是 re-acidified。因此, pHluorin 荧光强度的增加表明融合, 而减少表明吞的标签突触泡蛋白。除了使用 pHluorin 成像方法记录吞, 我们还通过电子显微镜 (EM) 测量了辣根过氧化物酶 (HRP) 对囊泡的摄取量来监测水泡膜的吞。最后, 在高钾诱导去极化后的不同时间, 我们监测了神经末端膜凹坑的形成。HRP 吸收和膜坑形成的时间过程表明吞的时间过程。

使用脂质载体法, SpH 表达了海马神经元, 允许识别 boutons (图 1a)。电刺激细胞诱导胞, 并相应增加荧光强度。荧光 (δ) 的增加是通过终止刺激 (图 1b) 停止的。随着吞的增加, 荧光量也随之缓慢下降。在 VAMP2-pHluorin 的情况下, VAMP2 扩散沿轴突从一个溥后刺激4。原始数据使用任意单位, 并被规范化到基线以获得衰变?...

Watch this Scientific Journal Video about Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons at JoVE.com

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

突触囊泡吞是通过光学显微镜检测 pHluorin 融合与突触泡蛋白和电子显微镜的囊泡摄取。

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 ...

measuring-synaptic-vesicle-endocytosis-in-cultured-hippocampal-neurons

Research

JoVE Journal

Neuroscience

9.8K 次观看.  National Institute of Neurological Disorders and Stroke. 突触囊泡吞是通过光学显微镜检测 pHluorin 融合与突触泡蛋白和电子显微镜的囊泡摄取。

视频: Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

测量中使用FM标签的神经元的胞外分泌

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

科研

生物学

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 ...

神经科学

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 ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

在原代培养海马神经元的密集核心囊泡的实时成像

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

嵌入后突触蛋白的免疫标记海马切片培养

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

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 ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

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

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

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 ...

Transmission Electron Microscopic Imaging of Synaptic Vesicle Endocytosis in Mouse Hippocampal Neurons 

<a href='https://app.jove.com/v/55862/measuring-synaptic-vesicle-endocytosis-in-cultured-hippocampal-neurons'>Source: Villarreal, S., et. al., Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons. J. Vis. Exp. (2017)</a>This video demonstrates the analysis of synaptic vesicle endocytosis in cultured hippocampal neurons through horseradish peroxidase labeling and electron microscopy.

Source: Villarreal, S., et. al., Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons. J. Vis. Exp. (2017)This video demonstrates the ...