我们感谢 Broadie 实验室成员对本文的贡献。这项工作得到了 nih R01s MH096832 和 MH084989 K.B. 的支持, nih predoctoral 研究金 F31 MH111144

1. 幼虫胶解剖
<ol><li>从弹性体套件 (材料表) 中彻底混合10部分硅胶弹性体底座与1部分硅胶弹性体固化剂。</li>
	<li>外套 22 x 22 毫米玻璃盖玻片与弹性体和治疗在一个热板在75˚C 几个小时 (直到不再粘到触摸)。</li>
	<li>将一个单一的弹性体涂层玻璃盖玻片到定制的树脂玻璃解剖室(图 1, 底部), 为幼虫解剖作准备。</li>
	<li>用标准微电极拉拔剂从硼硅酸盐玻璃毛细管中制备胶吸管, 以获得所需的锥度和尖端尺寸。</li>
	<li>轻轻折断微尖端, 另一端附加2英尺的柔性塑料管 (1/32 英寸内径, ID; 3/32 "外径, 外径; 1/32" 墙;材料表)配口管 (P2 吸管尖)。</li>
	<li>填充一个小容器 (0.6 毫升离心管帽) 与小容积 (~ 20 µL) 的胶水 (材料表), 为幼虫解剖准备。</li>
	<li>用生理盐水填充房间 (毫米): 128 氯化钠, 2 氯化钾, 4 氯化镁2, 1 CaCl2, 70 蔗糖和 5 2-[4-(2-羟乙基) piperazin-1 基] 磺酸 (HEPES) pH 值7.2。</li>
	<li>添加抗马萝卜过氧化物酶 (HRP) 抗体共轭到 Alexa 647 (anti-HRP:647; 稀释1:10 从1毫克/毫升股票), 以标记 NMJ 突触前终端在解剖21, 22.</li>
	<li>使用精美的画笔 (尺寸 2), 从食品瓶中移除一个流浪的第三个龄, 并放置在弹性体涂层的盖玻璃上。</li>
	<li>用带有附件的嘴产生的负气压填充玻璃微尖, 用少量胶水 (步骤 1.5)。</li>
	<li>将幼虫背侧与镊子一起放置, 将头部粘附在弹性体涂层的盖玻片上, 用一小滴胶水, 用口正面气压。</li>
	<li>重复这个过程与幼虫的后端, 确保动物在两个胶水附件之间绷紧。</li>
	<li>使用剪刀 (刀片3毫米;材料表), 在后部和垂直切口沿背中线进行水平切口 (~ 1 毫米)。</li>
	<li>使用细钳 (#5,材料表), 轻轻地去除背气管, 肠道, 脂肪体和其他内部器官覆盖肌组织。</li>
	<li>重复四体壁襟翼的胶合过程, 确保在水平和纵向上轻轻伸展身体壁。</li>
	<li>用镊子抬起腹神经线 (VNC), 用剪刀小心地切断马达神经, 然后完全删除 VNC。</li>
	<li>用 Ca2 +-游离生理盐水替换解剖盐水 (与上述解剖生理盐水相同, 没有 CaCl2) 以停止 SV 循环。</li>
</ol>2. 备选案文 1: 高 [K+] FM 染料加载
<ol><li>从 FM1-43 的股票溶液 (4 毫米), 增加1µL 到1毫升90毫米氯化钾溶液 (高 [K+] 在解剖盐水) 为最后浓度4µM。</li>
	<li>使用吸管, 用高 [K+] FM 染料溶液替换成像腔中的 Ca2 +无盐盐水, 以刺激 SV 吞染料的吸收。</li>
	<li>立即启动数字计时器, 以确定高 [K+] 去极化型刺激期间的预定时间(e. g, 5 分钟;图 2)。</li>
	<li>要确认一个健康的幼虫制剂, 请注意在高 [K+] 退极化期的持续时间内肌组织的强烈收缩。</li>
	<li>当计时器周期结束时, 快速删除高 [K+] FM 染料解决方案, 并替换为 Ca2 +-免费生理盐水以停止 SV 循环。</li>
	<li>快速连续清洗与 Ca2 +-免费生理盐水 (5x 1 分钟), 以确保高 [K+] FM 染料解决方案被完全删除。</li>
	<li>在新鲜的 Ca2 +-免费生理盐水中保持幼虫的准备, 以便立即用共焦显微镜进行成像。</li>
</ol>3. 成像: 共焦显微术
<ol><li>使用直立共焦显微镜与40X 水浸泡目标图像 NMJ 染料荧光 (其他显微镜可以使用)。</li>
	<li>图像肌肉 4 NMJ 腹部段 2-4 (其他 NMJs 可以成像) 和收集图像使用适当的软件 (材料表)。</li>
	<li>使用 HeNe 633 nm 激光激发 HRP:647 (带长通滤波器 > 635 nm) 和氩 488 nm 激光器激发 FM1-43 (带通滤波器 530-600 nm)。</li>
	<li>操作确定两个通道的最佳增益和偏移量。 注意: 这些设置在整个实验的其余部分将保持不变。</li>
	<li>采取一个共聚焦 Z 栈通过整个选定的 NMJ 从 HRP 标记的顶端到底部的突触终端。</li>
	<li>仔细注意 NMJ 成像 (段, 侧面和肌肉), 以确保过剩的完全相同的 NMJ 后, 调频染料卸载。</li>
</ol>4. 高 [K+] 刺激: FM 染料卸载
<ol><li>用高 [K+] 生理盐水 (不含 FM1-43 染料) 替换 Ca2 +无盐盐水, 以驱动退极化、SV 胞吐作用和染料释放。</li>
	<li>立即启动数字计时器, 以确定高 [K+] 刺激期间的预定时间 (e. g, 2 分钟;图 2)。</li>
	<li>当计时器周期结束时, 立即删除高 [K+] 生理盐水并替换为 Ca2 +-免费生理盐水以停止 SV 循环。</li>
	<li>用 Ca2 +免费生理盐水 (5x 1 分钟) 快速连续清洗, 以确保完全删除高 [K+] 生理盐水。</li>
	<li>在新鲜的 Ca2 +-免费生理盐水中保持幼虫的准备, 以便立即用共焦显微镜进行成像。</li>
	<li>一定要在相同的 NMJ 上使用相同的共聚焦设置 FM1-43 染料荧光图像。</li>
</ol>5. 备选案文 2: 电刺激调频染料加载
<ol><li>使用微电极拉拔器 (材料表) 准备吸入吸管, 以获得所需的锥度和尖端尺寸。</li>
	<li>用微型锻造将微电极尖端点火, 直到单个马达神经能被紧配合。</li>
	<li>将吸吸管滑入机器人上的电极支架上, 并附着在长柔塑料管和注射器上。</li>
	<li>设置刺激器参数 (e. g, 15 V, 20 Hz 频率, 20 毫秒的持续时间和5分钟 (图 2) 或1分钟 (图 3))。</li>
	<li>在电生理学平台上, 用以上 FM1-43 生理盐水 (4 µM; 1 毫米 CaCl2) 取代 Ca2 +无盐的幼虫制剂。</li>
	<li>将准备工作放在显微镜阶段, 并提高到幼虫和吸吸管的聚焦 (40X 水浸泡目标)。</li>
	<li>吸一圈切断电机神经支配选定的 hemisegment 与负气压的注射器产生的吸入电极。</li>
	<li>在视觉监测所选 hemisegment 的肌肉收缩时, 用短时间刺激来测试吸入电极的功能。</li>
	<li>使用所选参数 (步骤 5.4) 刺激运动神经元, 以驱动 SV 吞和 FM1-43 染料摄取 (图 2)。</li>
	<li>用 Ca2 +无盐 (5x 1 分钟) 快速连续清洗, 以确保 FM1-43 染料溶液完全移除。</li>
	<li>使用上面的共焦成像协议, 在新鲜的 Ca2 +-免费生理盐水中保持幼虫准备。</li>
	<li>仔细注意 NMJ 成像 (段, 侧面和肌肉), 以确保获得完全相同的 NMJ 后, 调频染料卸载。</li>
</ol>6. 电刺激: 调频染料卸料
<ol><li>用常规生理盐水 (无 FM1-43 染料) 替换 Ca2 +无盐生理盐水, 并将制剂放回电生理钻机阶段。</li>
	<li>设置卸载的刺激器参数 (例如、15 V、20 Hz 频率、20毫秒持续时间和2分钟 (图 2) 或二十年代 (图 3))。</li>
	<li>将相同的马达神经吸入到同一个电极上, 然后刺激激活 SV 胞吐作用和 FM1-43 染料释放。</li>
	<li>快速连续洗涤与 Ca2 +-免费生理盐水 (5x 1 分钟), 以确保外部染料完全删除。</li>
	<li>在新鲜的 Ca2 +-免费生理盐水中保持幼虫的准备, 以便立即用共焦显微镜进行成像。</li>
	<li>确保在相同的 NMJ 上使用相同的共焦设置 FM1-43 染料荧光图像。</li>
</ol>7. 选择 3: Channelrhodopsin 刺激 FM 染料装载
<ol><li>在含有 ChR2 因子的全反式视网膜 (溶解于乙醇; 100 µM 最后浓度) 的食物上饲养 ChR2-expressing 幼虫。</li>
	<li>将幼虫制剂放置在有机玻璃室的解剖显微镜阶段, 配备有摄像头端口。</li>
	<li>附上蓝色 LED (470 毫微米;材料表)使用同轴电缆的可编程刺激器, 并将 LED 放到照相机端口。</li>
	<li>利用显微镜变焦功能将蓝色 LED 光束聚焦到解剖幼虫的功能上。</li>
	<li>在 optogenetic 阶段, 用以上 FM1-43 生理盐水 (4 µM; 1 毫米 CaCl2) 取代 Ca2 +无盐的幼虫制剂。</li>
	<li>使用刺激器 (例如、15 V、20 Hz 频率、20毫秒持续时间和5分钟 (图 2)) 设置 LED 参数。</li>
	<li>启动光刺激和跟踪计时器的预先确定的持续时间的 optogenetic 刺激期 (例如, 5 分钟;图 2)。</li>
	<li>当计时器停止时, 请快速删除 FM 染料解决方案, 并替换为 Ca2 +-免费生理盐水以停止 SV 循环。</li>
	<li>用 Ca2 +免费生理盐水 (5x 1 分钟) 快速连续清洗, 以确保 FM 染料溶液完全移除。</li>
	<li>使用上面的成像协议, 在新鲜的 Ca2 +-免费生理盐水中保持幼虫的准备, 以立即成像与共焦显微镜。</li>
	<li>仔细注意 NMJ 成像 (段, 侧面和肌肉), 以确保获得完全相同的 NMJ 后, 调频染料卸载。</li>
</ol>8. Channelrhodopsin 刺激: FM 染料卸载
<ol><li>在解剖显微镜阶段替换 Ca2 +无常规生理盐水 (不含 FM1-43 染料), 并以幼虫为焦点的摄像头端口 LED。</li>
	<li>设置卸载的刺激器参数 (例如, 15 V, 20 Hz 频率, 20 毫秒持续时间和2分钟 (图 2))。</li>
	<li>启动光刺激和跟踪计时器的预先确定的持续时间的 optogenetic 刺激期 (例如, 2 分钟;图 2)。</li>
	<li>当计时器周期结束时, 请快速删除 FM 染料解决方案, 并替换为 Ca2 +-免费生理盐水以停止 SV 循环。</li>
	<li>快速连续洗涤与 Ca2 +-免费生理盐水 (5x 1 分钟), 以确保外部染料完全删除。</li>
	<li>在新鲜的 Ca2 +-免费生理盐水中保持幼虫的准备, 以便立即用共焦显微镜进行成像。</li>
	<li>确保在相同的 NMJ 上使用相同的共焦设置 FM1-43 染料荧光图像。</li>
</ol>9. 荧光定量
<ol><li>加载图像 J (NIH 开源), 并创建一个最大强度投影通过单击图像 |栈 |Z 项目。</li>
	<li>使用 anti-HRP:647 通道, 转到图像 |调整 |阈值和滑动顶部工具栏, 直到只突出显示 NMJ。</li>
	<li>使用魔杖工具, 点击 NMJ。如果 NMJ 不连续, 请按住 Shift 按钮并选择所有部件。</li>
	<li>将图像更改为 FM1-43 染料通道并转到分析 |测量得到荧光测量。</li>
	<li>重复步骤 9.1-9.4 的 "卸载" 图像从相同的 NMJ (确定的部分, 侧面和肌肉)。</li>
	<li>要获得被卸载的染料的百分比, 取被卸载的/被装载的荧光强度的比率。 注意: 可以修改此过程, 以使用 "椭圆形" 或 "徒手" 选择工具, 在溥每溥的基础上进行荧光分析。通过对肌肉荧光的取样可以减去背景荧光。还可以添加代理以减少此背景。</li>
</ol>

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作者声明没有相互竞争的利益。

高 [K+] 生理盐水去极化型刺激是迄今为止最容易的三种选择活动相关的 FM 染料循环, 但可能是最小的生理29。这个简单的方法 depolarizes 在整个动物中的每一个可访问的细胞, 所以不允许定向研究。可能在本地应用高 [K+] 生理盐水与微, 但这仍将去极化前/突触细胞和可能突触相关的神经胶质 1.另一个主要关注的问题是高 (K+) 偏极化驱动器?...

用精美的果蝇幼虫神经肌肉结 (NMJ) 谷氨酸突触模型研究了巨大的基因扰动1的突触形成和功能。运动神经元终端由多轴突分支组成, 各有多个突触 boutons。这些宽敞的静脉曲张 (直径达5µm) 包含所有的神经传递机械, 包括均匀谷氨酸突触泡 (SVs; ~ 40 毫微米直径) 在胞浆储备和容易发布池 2.这些囊泡停靠在突触前等离子膜融合点活性区 (AZs), 胞吐作用介导谷氨酸神经递质释放为反式突触通信。随后, SVs 从等离子膜中提取, 通过接吻和运行回收或网格蛋白介导的吞 (CME) 为重复的外周/吞周期。果蝇NMJ 易于访问, 适合于孤立和表征 SV 周期突变体。利用正向基因筛, 新的突变导致了对 SV 周期3的关键基因的鉴定。而且, 从已经已知的基因开始的反向遗传方法, 通过对突变体循环表型4的仔细描述, 导致了新的 SV 循环机制的澄清。果蝇NMJ 几乎是理想的, 作为一个实验性突触准备, 解剖 SV 吞和胞吐作用机制通过方法, 光学跟踪囊泡循环在神经传递。
一系列荧光标记允许视觉跟踪的水泡在循环动力学, 但最多才多艺的是 FM 染料类似物, 首先合成的毛, F., et al。5. 结构上, FM 染料含有亲水头和通过芳香环连接的亲脂尾巴, 其中心区域赋予光谱性质。这些苯乙烯染料在膜中可逆地分解, 不会在膜小叶之间 "翻转", 所以在细胞质中从来没有自由, 而且在膜中的荧光比水的5还要多。可逆插入脂质双层导致荧光6的40倍增加。在神经元突触中, 经典的调频染料标记实验包括在去极化型刺激过程中, 通过 SV 吞, 沐浴与染料的突触准备。然后将外部染料冲走, SV 循环在无钙的响铃解决方案中被捕获, 以图像加载的突触7。第二轮的刺激, 在无染料浴触发 FM 释放通过胞吐作用, 一个过程, 可随后测量荧光强度下降。从单个囊泡到包含数以百计水泡的水池的 SV 种群可以定量地监视6,7。FM 染料被用来解剖活动依赖的动员功能不同的 SV 池, 并比较亲吻和运行与 CME 循环8,9。该方法已被修改, 以单独检测诱发, 自发和微型突触周期活动 (具有高度敏感的设备, 以检测非常小的荧光变化和减少漂白)10。通过将荧光调频信号 photoconverting 为透射电镜11、12、13、14 的电子密标签, 可以将检测范围扩展到超微结构水平..
从历史上看, 高浓度钾的沐浴突触准备 (以下简称 "高 [K+]") 一直是去极化型刺激诱发 SV 循环的选择方法;从青蛙胆碱能 NMJ5到培养啮齿动物大脑海马神经元15, 到果蝇谷氨酸 NMJ 模型16,17。这种高 (K+) 方法简单, 不需要专用设备, 因此大多数实验室都可以访问, 但对应用程序和数据解释都有限制。一个更加生理上适当的方法是使用吸入电极电刺激神经4,5,12。这种方法推动动作电位的传播, 直接刺激突触前神经终端, 结果可以直接与神经传递功能的电生理学分析,13,14, 15, 但需要专用设备, 技术上更具挑战性。随着光遗传学的出现, channelrhodopsin 神经元刺激的使用具有额外的优势, 包括使用二进制 Gal4/UAS 系统20对信道表达进行严密的时空控制.这种方法在技术上比吸力电极刺激更容易, 只需要一个非常便宜的 LED 光源。在这里, 我们使用 FM1-43 的成像 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) 苯乙烯) 吡啶二溴) 来比较和对比这三种不同的刺激方法在果蝇 NMJ: 简单高 [K + ], 挑战电气和新的 channelrhodopsin 方法。

<table><tbody><tr><td>SylGard 184 Silicone Elastomer Kit</td><td>Fisher Scientific</td><td>NC9644388</td><td>To put on cover glass for dissections</td></tr><tr><td>Microscope Cover Glass 22x22-1</td><td>Fisherbrand</td><td>12-542-B</td><td>To put SylGard on for dissections</td></tr><tr><td>Aluminum Top Hot Plate Type 2200</td><td>Thermolyne</td><td>HPA2235M</td><td>To cure the SylGard</td></tr><tr><td>Plexi glass dissection chamber</td><td>N/A</td><td>N/A</td><td>Handmade</td></tr><tr><td>Borosilicate Glass Capillaries</td><td>WPI</td><td>1B100F-4</td><td>To make suction and glue micropipettes</td></tr><tr><td>Laser-Based Micropipette Puller</td><td>Sutter Instrument</td><td>P-2000</td><td>To make suction and glue micropipettes</td></tr><tr><td>Tygon E-3603 Laboratory Tubing</td><td>Component Supply Co.</td><td>TET-031A</td><td>For mouth and suction pipette</td></tr><tr><td>P2 pipette tip</td><td>USA Scientific</td><td>1111-3700</td><td>For mouth pipette</td></tr><tr><td>0.6-mL Eppendorf tube cap</td><td>Fisher Scientific</td><td>05-408-120</td><td>Used to put glue in for dissection</td></tr><tr><td>Vetbond 3M</td><td>WPI</td><td>vetbond</td><td>Glue used for dissections</td></tr><tr><td>Potassium Chloride</td><td>Fisher Scientific</td><td>P-217</td><td>Forsaline</td></tr><tr><td>Sodium Chloride</td><td>Millipore Sigma</td><td>S5886</td><td>For saline</td></tr><tr><td>Magnesium Chloride</td><td>Fisher Scientific</td><td>M35-500</td><td>For saline</td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Millipore Sigma</td><td>C7902</td><td>For saline</td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-3</td><td>For saline</td></tr><tr><td>HEPES</td><td>Millipore Sigma</td><td>H3375</td><td>For saline</td></tr><tr><td>HRP:Alexa Fluor 647</td><td>Jackson ImmunoResearch</td><td>123-605-021</td><td>To label neuronal membranes</td></tr><tr><td>Paintbrush</td><td>Winsor &amp;amp; Newton</td><td>94376864793</td><td>To manipulate the larvae</td></tr><tr><td>Dumont Dumostar Tweezers #5</td><td>WPI</td><td>500233</td><td>Used during dissection</td></tr><tr><td>7 cm McPherson-Vannas Microscissors (blades 3 mm)</td><td>WPI</td><td>14177</td><td>Used during dissection&amp;nbsp;</td></tr><tr><td>FM1-43</td><td>Fisher Scientific</td><td>T35356</td><td>Fluorescent styryl dye</td></tr><tr><td>Digital Timer</td><td>VWR</td><td>62344-641</td><td>For timing FM dye load/unload&amp;nbsp;</td></tr><tr><td>LSM 510 META laser-scanning confocal microscope</td><td>Zeiss</td><td></td><td>For imaging the fluorescent markers</td></tr><tr><td>Zen 2009 SP2 version 6.0</td><td>Zeiss</td><td></td><td>Software for imaging on confocal</td></tr><tr><td>HeNe 633nm laser</td><td>Lasos</td><td></td><td>To excite HRP:647 during imaging</td></tr><tr><td>Argon 488nm laser</td><td>Lasos</td><td></td><td>To excite the FM dye during imaging</td></tr><tr><td>Micro-Forge</td><td>WPI</td><td>MF200</td><td>To fire polish glass micropipettes</td></tr><tr><td>20mL Syringe Slip Tip</td><td>BD</td><td>301625</td><td>To suck up the axon for electrical stimulation.</td></tr><tr><td>Micro Manipulator (magnetic base)</td><td>Narishige</td><td>MMN-9</td><td>To control the suction electrode for electrical stimulation</td></tr><tr><td>Stimulator</td><td>Grass</td><td>S48</td><td>To control the LED and electrical stimulation</td></tr><tr><td>Zeiss Axioskop Microscope</td><td>Zeiss</td><td></td><td>Used during electrical stimulation.</td></tr><tr><td>40X Achroplan Water Immersion Objective</td><td>Zeiss</td><td></td><td>Used during electrical stimulation and confocal imaging</td></tr><tr><td>All-trans Retinal</td><td>Millipore Sigma</td><td>R2500</td><td>Essential co-factor for ChR2</td></tr><tr><td>Zeiss Stemi Microscope with camera port</td><td>Zeiss</td><td>2000-C</td><td>Used during channelrhodopsin stimulation</td></tr><tr><td>LED 470nm</td><td>ThorLabs</td><td>M470L2</td><td>Used for ChR activation</td></tr><tr><td>T-Cube LED Driver</td><td>ThorLabs</td><td>LEDD1B</td><td>To control the LED</td></tr><tr><td>LED Power Supply</td><td>Cincon Electronics Co.</td><td>TR15RA150</td><td>To power the LED</td></tr><tr><td>Optical Power and Energy Meter</td><td>ThorLabs</td><td>PM100D</td><td>To measure LED intensity</td></tr></tbody></table>

fm dye cycling at the synapse: comparing high potassium depolarization, electrical and channelrhodopsin stimulation

FM 染料用于研究突触囊泡 (SV) 周期。这些双亲探头有亲水性的头和疏水的尾巴, 使他们水溶性的能力, 可逆进入和退出膜脂双层。这些苯乙烯染料在水介质中相对非荧光, 但插入到等离子膜的外小叶中会导致荧光 > 40X 的增加。在神经元突触中, FM 染料在 sv 吞的内部和之间被内部内化, 并与 sv 胞吐作用一起释放, 提供了一个强有力的工具来可视化神经传递突触前阶段。谷氨酸突触发育和功能的一个主要遗传模型是果蝇神经肌肉结 (NMJ), 其中 FM 染料成像已广泛用于量化 SV 动力学在广泛的突变条件。NMJ 突触终端是容易接近的, 与一个美丽的阵列大突触 boutons 理想的成像应用。在这里, 我们比较和对比三种方法来刺激果蝇NMJ 驱动活动依赖 FM1-43 染料摄取/释放: 1) 浴应用高 [K+] 去极化神经肌肉组织, 2) 吸入电极马达神经刺激去极化的突触前神经终端和 3) 靶向转基因表达 channelrhodopsin 变种为光刺激, 空间控制的退极化。这些方法中的每一个都对研究果蝇NMJ 的 SV 周期的遗传突变效应有好处和坏处。我们将讨论这些利弊, 以协助选择刺激方法, 以及每个战略的具体方法。除了荧光成像, FM 染料可以 photoconverted 电子密集信号可视化使用透射电镜 (TEM) 研究 SV 周期机制的超微结构水平。我们提供的比较共聚焦和电子显微成像的不同方法的果蝇NMJ 刺激, 以帮助指导选择的未来实验范式。

图 1显示了与活动相关的 FM 染料成像协议的工作流。无论后来使用何种刺激方法, 实验总是以相同的幼虫胶解剖开始。图 1a是解剖幼虫的示意图, 显示腹神经线 (VNC)、辐射神经和重复 hemisegmental 肌肉模式。VNC 被删除, 准备沐浴在 FM1-43 的4µM 解决方案中 (图 1b, 粉红色)。然后, 在 FM 染料存在的情况下, 使用三?...

Watch this Scientific Journal Video about FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation at JoVE.com

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

调频染料循环在突触: 高钾偏极化, 电和 Channelrhodopsin 刺激的比较

突触囊泡 (SV) 循环是神经元突触中细胞间通讯的核心机制。调频染料的摄取和释放是定量测定 SV 内胞吐作用的主要手段。在这里, 我们比较所有的刺激方法, 以驱动 FM1-43 循环在果蝇神经肌肉连接 (NMJ) 模型突触。

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them ...

fm-dye-cycling-at-synapse-comparing-high-potassium-depolarization

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Cell Biology

Neuroscience

Unit 1

Channels and the Electrical Properties of Membranes

SCIENTISTS IN ACTION

8.4K 次观看.  Vanderbilt University. 突触囊泡 (SV) 循环是神经元突触中细胞间通讯的核心机制。调频染料的摄取和释放是定量测定 SV 内胞吐作用的主要手段。在这里, 我们比较所有的刺激方法, 以驱动 FM1-43 循环在果蝇神经肌肉连接 (NMJ) 模型突触。

视频: FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

突触小泡池，利用苯乙烯染料的光转化的研究

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

科研

神经科学

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

生物学

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 ...

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 ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

通过神经树桩将钙染料加载到青蛙神经末梢:青蛙神经肌肉接头中的钙瞬态登记

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

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) ...

FM Dyes in Vesicle Recycling

FM dyes are a class of fluorescent molecules that has found important use in studying the vesicle recycling process. By virtue of a chemical structure, these molecules can insert themselves into the outer leaflet of phospholipid bilayer membranes. After membrane insertion, they are internalized into the cell via endocytosed vesicles, and released when these vesicles recycle back to the membrane. Since, these dyes fluoresce strongly in the hydrophobic environment within membranes and weakly in the extracellular compartment, FM fluorescence levels can be used to track vesicular activity throughout the recycling process.This video provides an introduction to the use of FM dyes in experiments aimed to examine vesicle recycling. We first review the biochemistry of FM dyes and how their properties permit their use in these experiments. We then go through a general protocol for using FM dyes in such studies, and finally, discuss some recent research that makes use of these unique molecules.

FM 染料在囊泡循环再造

FM dyes are a class of fluorescent molecules that has found important use in studying the vesicle recycling process. By virtue of a chemical structure, ...

教育

JoVE Science Education

高级生物学

细胞生物学

Microelectrode-Mediated FM Dye Loading and Unloading in Drosophila Larvae Nerve Cells

Source: Kopke, D. L., et al., <a href="https://app.jove.com/v/57765">FM dye cycling at the synapse: comparing high potassium depolarization, electrical and channelrhodopsin stimulation.</a> J. Vis. Exp. (2018)
In this video, Drosophila larvae nerve fibers are immersed in FM dye, which binds to cell membrane lipids. Suction microelectrode and electrical pulses stimulate the endocytosis process, incorporating the FM dye into newly formed endocytic vesicles. This method, which visualizes presynaptic stages of neurotransmission, is useful for studying synaptic function and neurotransmitter release.

微电极介导的果蝇幼虫神经细胞中的 FM 染料加载和卸载

1. Larval Glue Dissection

	Thoroughly mix 10 parts of silicone elastomer base with 1 part of silicone elastomer curing agent from the elastomer kit.
	...