Name: ex vivo optogenetic dissection of fear circuits in brain slices
Uploaded: 2016-04-05T17:00:00
Description: Watch this Scientific Journal Video about Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices at JoVE.com

We thank Cora H&#252;bner 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.

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<ol><li>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...

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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...

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。

<table>
	<tbody>
		<tr>
			<td colspan="4">Surgery</td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Stoelting, USA</td>
			<td>51670</td>
			<td rowspan="2">can be replaced by other stereotactic frame for mice</td>
		</tr>
		<tr>
			<td>Steretoxic frame mouse adaptor</td>
			<td>Stoelting, USA</td>
			<td>51625</td>
		</tr>
		<tr>
			<td>Gas anesthesia mask for mice</td>
			<td>Stoelting, USA</td>
			<td>50264</td>
			<td>no longer available, replaced by item no. 51609M</td>
		</tr>
		<tr>
			<td>Pressure injection device, Toohey Spritzer</td>
			<td>Toohey Company, USA</td>
			<td>T25-2-900</td>
			<td>other pressure injection devices (e.g. Picospritzer) can be used</td>
		</tr>
		<tr>
			<td>Kwik Fill glass capillaries</td>
			<td>World Precision Instruments, Germany</td>
			<td>1B150F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia machine, IsoFlo</td>
			<td>Eickemeyer, Germany</td>
			<td>213261</td>
			<td></td>
		</tr>
		<tr>
			<td>DC Temperature Controler and heating pad</td>
			<td>FHC, USA</td>
			<td>40-90-8D</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal Micropipette Puller Model P-1000</td>
			<td>Sutter Instruments, USA</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tool sterilizer, Sterilizator 75</td>
			<td>Melag, Germany</td>
			<td>08754200</td>
			<td></td>
		</tr>
		<tr>
			<td>rAAV-hSyn-ChR2(H134R)-eYFP (serotype 2/9)</td>
			<td>Penn Vector Core, USA</td>
			<td>AV-9-26973P</td>
			<td></td>
		</tr>
		<tr>
			<td>rAAV-CAGh-ChR2(H134R)-mCherry (serotype 2/9)&#160;</td>
			<td>Penn Vector Core, USA</td>
			<td>AV-9-20938M</td>
			<td></td>
		</tr>
		<tr>
			<td>rAAV-EF1a-DIOhChR2(H134R)-YFP (serotype 2/1)&#160;</td>
			<td>Penn Vector Core, USA</td>
			<td>AV-1-20298P</td>
			<td></td>
		</tr>
		<tr>
			<td>fast green</td>
			<td>Roth, Germany</td>
			<td>0301.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane Anesthetic, Isofuran CP (1ml/ml)</td>
			<td>CP Pharma, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic, Betadine (providone-iodine)</td>
			<td>Purdure Products, USA</td>
			<td>BSOL32</td>
			<td>can be replaced by other disinfectant</td>
		</tr>
		<tr>
			<td>Analgesic, Metacam Solution (5mg/ml meloxicam)</td>
			<td>Boehringer Ingelheim, Germany</td>
			<td></td>
			<td>can be replaced by other analgesics</td>
		</tr>
		<tr>
			<td>Bepanthen eye ointment</td>
			<td>Bayer, Germany</td>
			<td>0191</td>
			<td>can be replaced by other eye ointment</td>
		</tr>
		<tr>
			<td>Drill NM3000 (SNKG1341 and SNIH1681)</td>
			<td>Nouvag, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sutranox Suture Needle</td>
			<td>Fine Science Tools, Germany</td>
			<td>12050-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Braided Silk Suture</td>
			<td>Fine Science Tools, Germany</td>
			<td>18020-60</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Recordings, light stimulation, and analysis</td>
		</tr>
		<tr>
			<td>artificial cerebrospinal fluid (ACSF)</td>
			<td>for composition see references #16 and #23</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>internal patch solutions</td>
			<td>for composition see references #16 and #23</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MagnesiumSulfate Heptahydrate</td>
			<td>Roth, Germany</td>
			<td>P027.1</td>
			<td>prepare 2M stock solution in purified water</td>
		</tr>
		<tr>
			<td>Slicer, Microm HM650V</td>
			<td>Fisher Scientific, Germany</td>
			<td>920120</td>
			<td></td>
		</tr>
		<tr>
			<td>Cooling unit for tissue slicer, CU65</td>
			<td>Fisher Scientific, Germany</td>
			<td>770180</td>
			<td></td>
		</tr>
		<tr>
			<td>Sapphire blade</td>
			<td>Delaware Diamond Knives</td>
			<td></td>
			<td>custom order, inquire with company</td>
		</tr>
		<tr>
			<td>Stereoscope, SZX2-RFA16</td>
			<td>Olympus, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xcite fluorescent lamp (XI120Q-1492)</td>
			<td>Lumen Dynamics Group, Canada</td>
			<td>2012-12699</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch microscope, BX51WI</td>
			<td>Olympus, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiclamp 700B patch amplifier&#160;</td>
			<td>Molecular Devices, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digitdata 1440A</td>
			<td>Molecular Devices, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PClamp software, Version 10</td>
			<td>Molecular Devices, USA</td>
			<td></td>
			<td>used to control data acquisition and stimulation</td>
		</tr>
		<tr>
			<td>Bath temperature controler, TC05</td>
			<td>Luigs &#38; Neumann, Germany</td>
			<td>200-100 500 0145</td>
			<td></td>
		</tr>
		<tr>
			<td>Three axis micromanipulator Mini 25</td>
			<td>Luigs &#38; Neumann, Germany</td>
			<td>210-100 000 0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator controller SM7</td>
			<td>Luigs &#38; Neumann, Germany</td>
			<td>200-100 900 7311</td>
			<td></td>
		</tr>
		<tr>
			<td>glass capillaries for patch pipettes</td>
			<td>World Precision Instruments, Germany</td>
			<td>GB150F-8P</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose nitrate filterpaper for interface chamber&#160;</td>
			<td>Satorius Stedim Biotech, Germany</td>
			<td>13006--50----ACN</td>
			<td></td>
		</tr>
		<tr>
			<td>LED unit, CoolLED pE</td>
			<td>CoolLED, UK</td>
			<td>244-1400</td>
			<td rowspan="4">CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation</td>
		</tr>
		<tr>
			<td>CoolLED 100 Dual Adapt</td>
			<td>CoolLED, UK</td>
			<td>pE-ADAPTOR-50E</td>
		</tr>
		<tr>
			<td>LED unit, USL 70/470</td>
			<td>Rapp Optoelectronic</td>
			<td>L70-000</td>
		</tr>
		<tr>
			<td>Dual port adapter</td>
			<td>Rapp Optoelectronic</td>
			<td>inquire with company</td>
		</tr>
		<tr>
			<td>Filter set red (excitation)</td>
			<td>AHF, Germany</td>
			<td>F49-560</td>
			<td rowspan="3">Filters can be bought as set F46-008</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (beamsplitter)</td>
			<td>AHF, Germany</td>
			<td>F48-585</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (emission)</td>
			<td>AHF, Germany</td>
			<td>F47-630</td>
		</tr>
		<tr>
			<td>Filter set green (excitation)</td>
			<td>AHF, Germany</td>
			<td>F39-472</td>
			<td rowspan="3">Alternatives: filterset F36-149 or F46-002 (with bandpass emission)</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (beamsplitter)</td>
			<td>AHF, Germany</td>
			<td>F43-495W</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (emission)</td>
			<td>AHF, Germany</td>
			<td>F76-490</td>
		</tr>
		<tr>
			<td>LaserCheck, handheld power meter</td>
			<td>Coherent, USA</td>
			<td><a>1098293</a></td>
			<td></td>
		</tr>
		<tr>
			<td>IgorPro Software, Version 6</td>
			<td>Wavemetrics, USA</td>
			<td></td>
			<td rowspan="2">for electrophysiology data analysis, other alternative software packages can also be used&#160;</td>
		</tr>
		<tr>
			<td>Neuromatic suite of macros for IgorPro</td>
			<td><a href="http://www.neuromatic.thinkrandom.com/">http://www.neuromatic.thinkrandom.com</a></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Post hoc analysis of injections and projections</td>
		</tr>
		<tr>
			<td>Paraformaldehyde powder (PFA)</td>
			<td>Roth, Germany</td>
			<td>0335.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurotrace 435/455 blue fluorescent Nissl stain</td>
			<td>Invitrogen</td>
			<td>N-21479</td>
			<td></td>
		</tr>
		<tr>
			<td>agar-agar for embedding and resectioning</td>
			<td>Roth, Germany</td>
			<td>5210.3</td>
			<td></td>
		</tr>
		<tr>
			<td>30 x 10 mm petri dishes for embedding</td>
			<td>SPL Life Sciences</td>
			<td></td>
			<td>alternatives can be used</td>
		</tr>
		<tr>
			<td>Slides, Super Frost</td>
			<td>R. Langenbrinck, Germany</td>
			<td>61303802</td>
			<td>alternatives can be used</td>
		</tr>
		<tr>
			<td>cover slips</td>
			<td>R. Langenbrinck, Germany</td>
			<td>3000302</td>
			<td>alternatives can be used</td>
		</tr>
		<tr>
			<td>Vecta Shield mounting medium</td>
			<td>Vector Laboratories, USA</td>
			<td>H-1000</td>
			<td>alternative mounting media can be used</td>
		</tr>
		<tr>
			<td>cellulose nitrate filter for flattening slices for fixation</td>
			<td>Satorius Stedim Biotech, Germany</td>
			<td>11406--25------N</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope LSM 710</td>
			<td>Zeiss, Germany</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo optogenetic dissection of fear circuits in brain slices

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。

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...

Watch this Scientific Journal Video about Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices at JoVE.com

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

在脑片恐惧电路体外光遗传学解剖

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。

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

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Neuroscience

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

科研

神经科学

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

大脑切片生物素化：一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

从成年斑马鱼中制备新鲜视网膜切片用于前体成像实验

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Optogenetic 在行为小鼠海马θ振荡中的作用

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

利用体外大鼠脑切片研究变性主要事件的一种替代方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

监测小鼠睡眠/觉醒状态期间神经回路的光遗传学操作

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...