调频染料循环在突触: 高钾偏极化, 电和 Channelrhodopsin 刺激的比较
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 幼虫胶解剖 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。 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)。 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。 用标准微电极拉拔剂从硼硅酸盐玻璃毛细管…
Representative Results
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?…
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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
Materials
| SylGard 184 Silicone Elastomer Kit | Fisher Scientific | NC9644388 | To put on cover glass for dissections |
| Microscope Cover Glass 22×22-1 | Fisherbrand | 12-542-B | To put SylGard on for dissections |
| Aluminum Top Hot Plate Type 2200 | Thermolyne | HPA2235M | To cure the SylGard |
| Plexi glass dissection chamber | N/A | N/A | Handmade |
| Borosilicate Glass Capillaries | WPI | 1B100F-4 | To make suction and glue micropipettes |
| Laser-Based Micropipette Puller | Sutter Instrument | P-2000 | To make suction and glue micropipettes |
| Tygon E-3603 Laboratory Tubing | Component Supply Co. | TET-031A | For mouth and suction pipette |
| P2 pipette tip | USA Scientific | 1111-3700 | For mouth pipette |
| 0.6-mL Eppendorf tube cap | Fisher Scientific | 05-408-120 | Used to put glue in for dissection |
| Vetbond 3M | WPI | vetbond | Glue used for dissections |
| Potassium Chloride | Fisher Scientific | P-217 | Forsaline |
| Sodium Chloride | Millipore Sigma | S5886 | For saline |
| Magnesium Chloride | Fisher Scientific | M35-500 | For saline |
| Calcium Chloride Dihydrate | Millipore Sigma | C7902 | For saline |
| Sucrose | Fisher Scientific | S5-3 | For saline |
| HEPES | Millipore Sigma | H3375 | For saline |
| HRP:Alexa Fluor 647 | Jackson ImmunoResearch | 123-605-021 | To label neuronal membranes |
| Paintbrush | Winsor & Newton | 94376864793 | To manipulate the larvae |
| Dumont Dumostar Tweezers #5 | WPI | 500233 | Used during dissection |
| 7 cm McPherson-Vannas Microscissors (blades 3 mm) | WPI | 14177 | Used during dissection |
| FM1-43 | Fisher Scientific | T35356 | Fluorescent styryl dye |
| Digital Timer | VWR | 62344-641 | For timing FM dye load/unload |
| LSM 510 META laser-scanning confocal microscope | Zeiss | For imaging the fluorescent markers | |
| Zen 2009 SP2 version 6.0 | Zeiss | Software for imaging on confocal | |
| HeNe 633nm laser | Lasos | To excite HRP:647 during imaging | |
| Argon 488nm laser | Lasos | To excite the FM dye during imaging | |
| Micro-Forge | WPI | MF200 | To fire polish glass micropipettes |
| 20mL Syringe Slip Tip | BD | 301625 | To suck up the axon for electrical stimulation. |
| Micro Manipulator (magnetic base) | Narishige | MMN-9 | To control the suction electrode for electrical stimulation |
| Stimulator | Grass | S48 | To control the LED and electrical stimulation |
| Zeiss Axioskop Microscope | Zeiss | Used during electrical stimulation. | |
| 40X Achroplan Water Immersion Objective | Zeiss | Used during electrical stimulation and confocal imaging | |
| All-trans Retinal | Millipore Sigma | R2500 | Essential co-factor for ChR2 |
| Zeiss Stemi Microscope with camera port | Zeiss | 2000-C | Used during channelrhodopsin stimulation |
| LED 470nm | ThorLabs | M470L2 | Used for ChR activation |
| T-Cube LED Driver | ThorLabs | LEDD1B | To control the LED |
| LED Power Supply | Cincon Electronics Co. | TR15RA150 | To power the LED |
| Optical Power and Energy Meter | ThorLabs | PM100D | To measure LED intensity |
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Citar este artigo
Kopke, D. L., Broadie, K. FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation. J. Vis. Exp. (135), e57765, doi:10.3791/57765 (2018).