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Introduction
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신경과학

在脑片恐惧电路体外光遗传学解剖

Published: April 05, 2016
doi:
10.3791/53628
, ,
1Hertie Institute for Clinical Brain Research and Werner Reichardt Centre for Integrative Neuroscience,University of Tuebingen, 2Max Planck Florida Institute for Neuroscience

Summary

光遗传学方法被广泛地用于操纵神经活动和评估大脑功能的后果。在这里，技术概述了在光激活Channelrhodopsin 在体内表达，允许在恐惧相关的电路体外特定的远程及本地的神经连接的突触性质的分析。

Abstract

光遗传学方法现在广泛使用的通过光组合光活化的蛋白和神经活动的后续处理的靶向表达研究神经种群和电路的功能。 Channelrhodopsins（CHRS）是光门控阳离子通道，当融合于荧光蛋白的表达可用于可视化和特定细胞类型的并行激活和在脑的限定的区域其轴突突起。通过病毒载体的立体定向注射，CHR融合蛋白可被组成性或有条件地限定的脑区域的特定细胞中表达，并且它们的轴突突起随后可以通过在脑切片的体外光遗传学活化解剖和功能的研究。旨在明白无法与传统的电刺激的方法来解决的连接突触性质时，这是特别重要的，或在确定新颖AFFE租金而被理解很差先前传出的连接。这里，有几个实施例说明此技术如何可以应用于调查这些问题，在杏仁核阐明恐惧相关的电路。杏仁核是恐惧和情绪记忆获取和恐惧的表情，和存储的重点地区。证据许多线表明，内侧前额叶皮质（内侧前额叶皮质）参与恐惧采集和灭绝的不同方面，但其与杏仁核精确连通刚刚开始被理解。首先，示出如何体外光遗传学激活可以用来研究mPFC的传入和靶细胞间的突触通信的各方面在基底外侧杏仁核（BLA）。此外，它示出本体外光遗传学方法如何可以适用于评估使用一组GABA能神经元中的杏仁核改进的连接图案，所述paracapsular闰细胞簇（mpITC），作为一个例子。

Introduction

可视化和脑区和特定类型的神经元之间的特定连接的同时激活精密工具正在成为了解功能连接基本健康的大脑功能和疾病状态更重要。理想情况下，这需要与鉴定神经元沟通精确突触性生理研究。这是不能在一个单一的急性脑切片保存脑区之间的连接，尤其如此。在过去，这已经在很大程度上在单独的实验取得。一方面，神经示踪剂注入体内已采用结合随后的光或前和突触后伙伴的电子显微镜分析。另一方面，当从原点的区域中纤维束被保留，并在切片准备访问的，电刺激已经被用于评估在所述目标区域的细胞突触通信机制。

与光遗传学的出现，的光门控阳离子通道，诸如Channelrhodopsins（CHRS）融合于荧光蛋白的靶向表达，现在使神经元和它们的轴突轨迹的活化，同时允许其可视化和事后解剖分析1- 4。因为从父胞体5切断时CHR表达轴突甚至可以刺激，有可能在脑切片于：1）评估从那些不与传统的电刺激可访问的大脑区域的输入，因为纤维束是不可分离或特定的轨迹不知道; 2）明确标识产地为进行推测，但不完全了解具体投入的区域; 3）调查定义的细胞类型之间的功能连接，包括本地和长期预测。因为许多优点的，电路的脑切片这个光遗传学映射已成为广LY在过去几年中使用，以及各种用于荧光标记CHRS表达病毒载体的可容易地从商业供应商获得。比传统的电刺激光遗传学激活一些关键的优势是对组织无损伤，由于刺激电极，纤维刺激特异性的位置，因为电刺激也可以招募通道或其他附近的细胞，和同样快速，时间精确刺激纤维。此外，病毒载体的立体定向注射可以很容易地针对特定脑区6和条件或细胞类型特异性表达可以通过依赖于Cre的表达和/或特异性启动子7来实现。这里，这种技术被应用于远距离映射和局部电路中的恐惧系统。

杏仁核是恐惧和情绪记忆8,9购置和恐惧的表情，和存储的重点地区。除了来回m为杏仁核，内侧前额叶皮质（mPFC的）和海马（HC）被相互连接到杏仁核的结构，有牵连的恐惧和消光存储器10,11采集，固结和检索的各个方面。在内侧前额叶皮质的活动细分出现在控制高和低的恐惧发挥双重作用状态12,13。这可以部分由内侧前额叶皮质，以将控制杏仁核活动和产出的杏仁核的直接连接介导的。因此，在过去的几年中，一些研究离体切片实验开始调查在杏仁核14-17 mPFC的传入和特定的靶细胞间的突触相互作用。

在恐惧学习，关于空调，无条件刺激的感觉信息到达通过特定丘脑和皮层区域的预测杏仁核。这些投入可塑性在basol外侧部（LA）神经元ateral杏仁核（BLA）是恐惧条件9,18潜在的重要机制。越来越多的证据表明，在杏仁核并行塑料方法涉及抑制元件以控制恐惧存储器19。 A组集群抑制性神经元是GABA能内侧paracapsular闰细胞（mpITCs），但其确切的连通性和功能还没有完全搞清楚20-22。这里，光遗传学电路映射用于评估这些细胞的传入和传出连接以及它们在杏仁核上目标神经元的影响，这表明mpITCs接收来自丘脑和大脑皮层的中继站23直接感觉输入。在mpITCs或BLA神经元CHR的特异性表达使得局部相互作用的映射，揭示mpITCs抑制，但也相互通过，BLA主要的神经元​​激活，将它们放置在该有效控制BLA活性的新的前馈和反馈抑制电路23。

Protocol

伦理学声明：所有实验程序均符合有关研究使用动物的欧盟指令和当地动物护理和使用委员会批准（Regierungspräsidium蒂宾​​根，巴登 – 符腾堡州，德国的状态）负责图宾根大学。 1.立体定向注射过程准备使用消毒器无菌工具（剪刀，手术刀，夹具，钻头，针，缝合材料）。安排无菌工具和其他所需的解决方案和手术用品如无菌棉互换，消毒，无菌磷酸盐缓冲盐水（PBS，pH7.4）中，和…

Representative Results

这部分显示了体外光遗传学方法，并从不同的实验策略代表结果的工作流程进行调查感觉和调节的长期预测到BLA和mpITC神经元以及mpITC和BLA之间的本地连接属性的生理特性。 在所需的坐标到小鼠脑所选病毒载体的立体定向注射后（ 图1A-C中 ，病毒表达时间：2 – 6周，这取决于实验），在杏仁核注射部位和投射…

Discussion

本协议描述了体外 ，可以很容易地在大多数，如果不是全部实行神经回路和本地连接的光遗传学研究的方法，通过用〜470纳米的表面荧光光端口LED装备他们直立片膜片钳记录设置。在切片轴突突起的光遗传学刺激的主要优点是，它允许为特定的激活和不与传统的电刺激可访问连接的性质的调查，因为相应的纤维束没有公知的，没有明确的定义，或在不保留一个单一的脑片。作为相关的恐惧…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Cora Hübner and Andrea Gall for help in acquiring some of the representative results. This work was supported by the Werner Reichardt Centre for Integrative Neuroscience (CIN) at the University of Tuebingen, an Excellence Initiative funded by the Deutsche Forschungsgemeinschaft (DFG) within the framework of the Excellence Initiative (EXC 307), and by funds from the Charitable Hertie Foundation.

Materials

Surgery
Stereotactic frame Stoelting, USA 51670 can be replaced by other stereotactic frame for mice
Steretoxic frame mouse adaptor Stoelting, USA 51625
Gas anesthesia mask for mice Stoelting, USA 50264 no longer available, replaced by item no. 51609M
Pressure injection device, Toohey Spritzer Toohey Company, USA T25-2-900 other pressure injection devices (e.g. Picospritzer) can be used
Kwik Fill glass capillaries World Precision Instruments, Germany 1B150F-4
Anesthesia machine, IsoFlo Eickemeyer, Germany 213261
DC Temperature Controler and heating pad FHC, USA 40-90-8D
Horizontal Micropipette Puller Model P-1000 Sutter Instruments, USA P-1000
Surgical tool sterilizer, Sterilizator 75 Melag, Germany 08754200
rAAV-hSyn-ChR2(H134R)-eYFP (serotype 2/9) Penn Vector Core, USA AV-9-26973P
rAAV-CAGh-ChR2(H134R)-mCherry (serotype 2/9)  Penn Vector Core, USA AV-9-20938M
rAAV-EF1a-DIOhChR2(H134R)-YFP (serotype 2/1)  Penn Vector Core, USA AV-1-20298P
fast green Roth, Germany 0301.1
Isoflurane Anesthetic, Isofuran CP (1ml/ml) CP Pharma, Germany
Antiseptic, Betadine (providone-iodine) Purdure Products, USA BSOL32 can be replaced by other disinfectant
Analgesic, Metacam Solution (5mg/ml meloxicam) Boehringer Ingelheim, Germany can be replaced by other analgesics
Bepanthen eye ointment Bayer, Germany 0191 can be replaced by other eye ointment
Drill NM3000 (SNKG1341 and SNIH1681) Nouvag, Switzerland
Sutranox Suture Needle Fine Science Tools, Germany 12050-01
Braided Silk Suture Fine Science Tools, Germany 18020-60
Recordings, light stimulation, and analysis
artificial cerebrospinal fluid (ACSF) for composition see references #16 and #23
internal patch solutions for composition see references #16 and #23
MagnesiumSulfate Heptahydrate Roth, Germany P027.1 prepare 2M stock solution in purified water
Slicer, Microm HM650V Fisher Scientific, Germany 920120
Cooling unit for tissue slicer, CU65 Fisher Scientific, Germany 770180
Sapphire blade Delaware Diamond Knives custom order, inquire with company
Stereoscope, SZX2-RFA16 Olympus, Japan
Xcite fluorescent lamp (XI120Q-1492) Lumen Dynamics Group, Canada 2012-12699
Patch microscope, BX51WI Olympus, Japan
Multiclamp 700B patch amplifier  Molecular Devices, USA
Digitdata 1440A Molecular Devices, USA
PClamp software, Version 10 Molecular Devices, USA used to control data acquisition and stimulation
Bath temperature controler, TC05 Luigs & Neumann, Germany 200-100 500 0145
Three axis micromanipulator Mini 25 Luigs & Neumann, Germany 210-100 000 0010
Micromanipulator controller SM7 Luigs & Neumann, Germany 200-100 900 7311
glass capillaries for patch pipettes World Precision Instruments, Germany GB150F-8P
Cellulose nitrate filterpaper for interface chamber  Satorius Stedim Biotech, Germany 13006–50—-ACN
LED unit, CoolLED pE CoolLED, UK 244-1400 CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation
CoolLED 100 Dual Adapt CoolLED, UK pE-ADAPTOR-50E
LED unit, USL 70/470 Rapp Optoelectronic L70-000
Dual port adapter Rapp Optoelectronic inquire with company
Filter set red (excitation) AHF, Germany F49-560 Filters can be bought as set F46-008
                     (beamsplitter) AHF, Germany F48-585
                     (emission) AHF, Germany F47-630
Filter set green (excitation) AHF, Germany F39-472 Alternatives: filterset F36-149 or F46-002 (with bandpass emission)
                         (beamsplitter) AHF, Germany F43-495W
                         (emission) AHF, Germany F76-490
LaserCheck, handheld power meter Coherent, USA 1098293
IgorPro Software, Version 6 Wavemetrics, USA for electrophysiology data analysis, other alternative software packages can also be used 
Neuromatic suite of macros for IgorPro http://www.neuromatic.thinkrandom.com
Post hoc analysis of injections and projections
Paraformaldehyde powder (PFA) Roth, Germany 0335.2
Neurotrace 435/455 blue fluorescent Nissl stain Invitrogen N-21479
agar-agar for embedding and resectioning Roth, Germany 5210.3
30 x 10 mm petri dishes for embedding SPL Life Sciences alternatives can be used
Slides, Super Frost R. Langenbrinck, Germany 61303802 alternatives can be used
cover slips R. Langenbrinck, Germany 3000302 alternatives can be used
Vecta Shield mounting medium Vector Laboratories, USA H-1000 alternative mounting media can be used
cellulose nitrate filter for flattening slices for fixation Satorius Stedim Biotech, Germany 11406–25——N
Confocal Laser Scanning Microscope LSM 710 Zeiss, Germany
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Tags

Ex Vivo OptogeneticsFear CircuitsBrain SlicesNeural ProjectionsMicro-circuitsChannel RhodopsinPatch-clamp RecordingsSynaptic PropertiesLocal ConnectivityLong-range ConnectivityBrain AreasConventional Electrical Stimulation TechniquesViral InjectionsExpression TimeBrain Slices PreparationLight Activation And RecordingsRecombinant Adeno-associated Viral VectorMedial Pre-frontal CortexVibratome
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Bosch, D., Asede, D., Ehrlich, I. Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices. J. Vis. Exp. (110), e53628, doi:10.3791/53628 (2016).
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