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Abstract
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Protocol
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Neuroscience

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

Published: March 31, 2014
doi:
10.3791/50557
, , , ,
1Department of Molecular Physiology & Biophysics,University of Iowa Carver College of Medicine, 2Department of Biology & Biochemistry,University of Bath

Summary

我们描述了使用的苯乙烯调频染料图像突触囊泡循环的功能神经末梢。这个协议可以不仅诱发，也自发和微型突触活动的应用。该协议扩展了多种可有效评估的突触事件。

Abstract

在功能神经末梢突触囊泡的胞吐接受和内吞作用。这种突触囊泡循环可以使用苯乙烯调频染料，它揭示了膜有效的营业额分析。对于使用调频染料的传统协议的设计分析神经元以下的刺激（诱发）突触活动。最近，协议，成为可用于分析伴随弱突触活动，如自发或微型突触活动的调频信号。在FM信号的这些小的变化分析，需要对成像系统是足够敏感以检测强度的微小变化，但振幅较大的那个伪迹变化被抑制。在这里，我们描述了可应用于诱发的，自发的和微型的突触活动，并使用培养的海马神经元作为一个例子的协议。这个协议还包含评估调频染料的光漂白速率的一种手段，因为这是一个显著源成像强度的微小变化，当文物。

Introduction

突触小泡的功能是突触传递的重要决定因素。这些囊泡释放神经递质，当他们与突触前质膜（胞吐作用）熔化，并且它们成为准备发布的另一周期从质膜（内吞作用）进行再生和重新装载神经递质之后。研究的动态及相关突触囊泡循环机制已被引入苯乙烯调频染料1大大加快。这些两亲性分子，其具有带正电荷的亲水性头部基团和疏水性尾（多种染料在图1A中，FM1-43的图1B中stereoview），能可逆地进入和退出脂膜不渗透其中。调频染料组共享影响的光，它们发出的一系列类似的功能。例如，FM2-10，FM1-43，和FM1-84有两个环状化合物和升之间的一个双键瓦特绿色发光。它们之间的区别是疏水尾，这决定了其疏水性，因此，出口处的从膜的速率（departitioning）的长度。在FM5-95和FM4-64的情况下，三个双键连接的环状化合物，它们显示红色发射。这些染料差异相对于它们的亲水部分。在所有的FM染料，当它们被插入到生物膜，由于增加量子产率在相对于所述亲水性环境的疏水性环境中的荧光强度增加。因此，改变FM强度代表了改变膜的营业额。不同的颜色（发射光谱）和疏水性使调频染料一种多用途的研究工具，在突触囊泡循环。

基于这些特性，调频染料大多根据在分析突触小泡循环利用下面的方案（ 图2）中使用。神经元S是沐浴在包含FM染料的细胞外液，使其能够被带进突触小泡（SVS），因为它们通过内吞作用（染色）形成。的染料，然后洗出通过应用染料-自由胞外溶液，这表明功能神经末梢， 即只有那些积极进行内吞作用将包含突触小泡被装入染料（ 图2底部）的群集。随后的胞吐作用导致的调频染料的损失到细胞外空间以及随之而来的损失荧光（脱色;由于两者departitioning到亲水环境和扩散远离胞吐作用的位点）。因此，改变FM的荧光强度是突触囊泡外切和内吞作用的指标。

调频染料已被用于染色和脱色的各种生物和制剂2,3突触囊泡。例子包括哺乳动物neurONAL培养4-9，哺乳动物的脑片10,11，神经肌肉接头12,13，视网膜双极神经细胞14,15和16耳蜗毛细胞。

通常，在这样的实验中，无论染色和脱色是通过广泛刺激神经元（诱发活性）触发。然而，最近，突触小泡循环响应于弱刺激也被分析，因为在没有外部刺激（自发和微型突触活动）9,17-19具有回收利用。自发和微型突触活动被定义为那些发生在没有外界刺激的，前者涉及动作电位（ 图3）的自发发射。这些微弱的突触活动与调频信号比由粗放刺激引发的较小的变化相关联。测量要求的变化，调频fluorescENCE强度准确反映突触囊泡的胞吐或胞吞作用，但强度不是伪迹的变化。伪像的原因之一是通过调频染料质膜的非特异性染色的存在。此组件的逐步洗脱将导致一个逐渐变化的测得的荧光强度，这将被错误地归因于突触活动。这个因子可以通过适当的方法来减少（见协议）。神器的最显着的原因是调频染料突触小泡内保留的漂白。在调频强度的光漂白相关的变更必须在比较所测得的生物（突触）的变化很小。敏感的摄像机的最新发展， 例如在电子倍增电荷耦合器件（EMCCD）相机，使得它可以最小化，通过缩短曝光时间，削弱用于激发荧光团的光的强度的光漂白。神器的另一个原因是我的漂移n个光显微镜的聚焦水平。成像会话期间的焦点漂移可以通过机械或热效应引起，并且将错误导致所测量的荧光强度的变化。

这里我们描述的协议和设备，使得有可能使用调频染料来分析突触小泡循环，即使在弱的或无刺激的情况下，特别是在微型突触活动。我们发现囊泡的染色和脱色的例子中诱发和自发突触活动，利用培养的啮齿动物的海马神经元，并且成像脱色阶段。我们还说明了如何评估的调频染料光漂白的程度，在没有任何调频染料损失是由于突触活动。

Protocol

1。神经元从哺乳动物脑原代培养在这项研究中进行的所有动物的程序是由爱荷华大学的机构动物护理和使用委员会批准。 海马准备从小鼠海马CA3-CA1区或大鼠分离的细胞培养在出生后0-1天19,20。镀上12毫米的盖玻片（厚度数0）预置与大鼠神经胶质饲养层，在24孔培养皿中，并以12,000个细胞/孔的密度将海马细胞。 培养海马神经元至少8天为功能性神经末梢?…

Representative Results

作为一个例子，我们展示代表结果的突触小泡的脱色时间过程（ 图4）。培养海马神经元采用自发突触活动（步骤2.3）沾上FM4-64，用无染料溶液（冲洗液2）。成像显示了使用自发活动（步骤5.3）（连续线的起始部分， 图4A）初始脱色时间过程。这后面是脱色时间过程使用三轮诱发活性与10赫兹场刺激为120秒每个（步骤5.1）。测量诱发脱色量的一种方法是说明与对应于囊泡?…

Discussion

我们已经描述了协议的染色和脱色突触小泡响应引起的，自发的和微型突触活动，并为在脱色相位成像。除了现有的协议，我们已观察的基础上微型突触活动的调频脱色的一个新的协议。使用这些协议，我们先前确定的运动障碍肌张力障碍的小鼠模型中培养的神经元异常。相较于他们的同行在野生型小鼠中，这些神经元后行加速在Ca 2 +的依赖性突触囊泡的胞吐的时候用高活性20刺?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢原田实验室的成员在整个这项工作的执行有益的讨论。这项工作是由美国心脏协会，肌张力障碍医学研究基金会，爱德华马林克罗特，小基金会，美国国家科学基金会和白厅基金，以资助NCH

Materials

Pulse generator AMPI Master-8
Isolated stimulator Digitimer DS3
Inverted microscope Nikon Eclipse TS100 This is used for assessing the cell morphology at low magnification.
Inverted microscope Nikon Eclipse TiE This is used for high-resolution fluorescence and transmitted light imaging, with minimal focus drift.
Objective lens Nikon Water-immersion lens is recommended. Oil-immersion lens is usable unless an imaged structure is deep from the coverslip surface (e.g. >10 μm).
Filter cube Nikon 77032509 490/20-nm ex, 510-nm dclp, 520-nm-LP em for FM1-43
Filter cube Nikon 77032809 490/20-nm ex, 510-nm dclp, 650-nm-LP em for FM4-64. 
EMCCD camera Andor Technology iXon EM+ DU-860 This EMCCD camera is used for high-sensitivity detection of fluorescence.
Liquid recirculating chiller Solid State Cooling Systems Oasis 160 This is used for continuously perfusing the camera with chilled water for maintaining a temperature of -80°C, and thereby reducing noise.
LED CoolLED-Custom Interconnect 490 nm  This light source is used for rapid on/off control of fluorescence excitation.
Image acquisition software Andor Technology Solis
Imaging chamber Warner Instruments RC-21BRFS
Fast perfusion system Warner Instruments SF-77B
CNQX Tocris Bioscience 1045
D,L-AP5 Tocris Bioscience 0106
Tetrodotoxin Tocris Bioscience 1069 Caution: toxic reagent. Handle with care.
FM1-43 Invitrogen T35356
Aldehyde-fixable FM1-43 (FM1-43FX) Invitrogen F35355
FM4-64 Invitrogen T13320
Aldehyde-fixable FM4-64 (FM4-64FX) Invitrogen F34653
Ionomycin Sigma-Aldrich I0634
Hanks’ balanced salt Sigma-Aldrich H2387
Minimum Essential Medium Invitrogen 51200-038 This solution does not contain phenol red that will interfere with fluorescence imaging.
Paraformaldehyde Electron Microscopy Sciences 15710 Caution: toxic reagent. Handle with care.
Sucrose Sigma-Aldrich S7903
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Tags

Synaptic Vesicle RecyclingFM DyesEvoked Synaptic ActivitySpontaneous Synaptic ActivityMiniature Synaptic ActivityMembrane TurnoverHippocampal NeuronsPhotobleaching
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Iwabuchi, S., Kakazu, Y., Koh, J., Goodman, K. M., Harata, N. C. Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities. J. Vis. Exp. (85), e50557, doi:10.3791/50557 (2014).
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