Name: ex vivo optogenetic dissection of fear circuits in brain slices
Uploaded: 2016-04-05T17:00:00.000+00:00
Duration: 11 min 13 s
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H 2 O 2上的无菌手术盖布。 </li><li>根据制造商的说明在水平微电极拉出器拉出使用3毫米宽框灯丝注射玻璃微（ 图1A，插入图）。 注意:对于深注入，电​​极锥度应足够长达到最腹侧坐标（约5毫米;。为坐标，见1.10）。 </li><li>预混物的病毒溶液1微升和0.2微升在无菌PBS中的0.1％快速绿色溶液（对于在玻璃吸管溶液更好的可视性）。填补玻璃吸管与病毒解决方案和快速的绿色使用带细尖（ 图1A，插图）微升移液管混合物。 注:重组腺相关病毒载体（腺相关病毒）工作被认为是生物安全等级1（BSL 1）工作，并需要在BSL 1实验室进行。在这项研究中（ 图1C）使用rAAVs是:1）mPFC的:重组腺相关病毒-hSyn-的ChR2（H134R）-eYFP（血清型2/9）16; 2）米高梅/ PIN:腺相关病毒CAGh-的ChR2（H134R）-mCherry（血清型2/9）23; 3）mpITC / BLA:腺相关病毒EF1A-DIOhChR2（H134R）-YFP（血清型2/1）23 </li><li>麻醉用小动物麻醉机（异氟烷:在氧诱导3％）鼠标。 注意:在本研究中使用的小鼠为4 -7周龄下列菌株和基因型和:C57BL6 / J野生型（实验在图2A和图3A-D）; GAD67-GFP 24（在图2B和3E-H实验）; TAC2-Cre的25（在图2C和3I-J实验）。 </li><li>耳朵和眼睛之间剃光头。应用的消毒剂（基于providone碘）用棉签剃光头。 </li><li>应用眼药膏，以防止在麻醉过程中眼睛干燥。皮下注入小鼠具有止痛（基于美洛昔康-，0.1毫升5毫克/毫升溶液）。 </li><li>放置在立体定位框架（ 图1A）小鼠和经由气体麻醉面罩维持麻醉（异氟烷:2％的氧气用于维护）。检查麻醉深度使用肢体撤回反射，然后再继续。 注:保持整个外科手术过程中在无菌条件以及可能的;戴一次性口罩，手术去WN和手套。 </li><li>就用剪刀头顶皮肤切口。轻轻拉动皮肤使用钝钳一边，用夹子固定修复，露出颅骨表面，清洁头骨与H 2 O 2。 </li><li>用细尖记号笔和钻孔的两个半球（ 图1B）上骷髅标记注射部位。坐标在这项研究中的注射是（距离前囟（毫米））:mPFC的:前1.9，横向±0.3腹面2.1;米高梅/ PIN:3.0后，横向±1.8，腹3.8; mpITC / BLA:1.45后，横向±3.35和4.75腹。 </li><li>装入填充玻璃吸管上连接到压力注射装置立体定位框架，并把吸移管到前囟的位置。 </li><li>断绝用细直尖镊子玻璃吸管的尖端。轻轻地这样做是为了防止气溶胶的产生的病毒解决方案。确保枪头是开放通过应用一些压力脉冲，并观察维鲁滴挤出的解决。 </li><li>转至所需的注射坐标和使用注射压力注射设备上进行以下设置移液器的内容（约0.5微升）的一半:压力:20磅，平均脉冲宽度:30毫秒，脉冲的平均数:50。 </li><li>离开吸管到位慢（1mm /分钟）之前缩回它〜1分钟。 注意:请不堵塞吸管肯定重复步骤与其他同一半球前吸管。在封锁尖端情况下，清洁或再次折断尖端和零吸管位置前囟门。 </li><li>用PBS（pH值7.4）清洁头盖骨，取出夹子，轻轻地拉在一起的皮肤，缝合个别按钮缝合切口（3 - 4节）。伤口周围施加消毒剂（基于providone碘）。 </li><li>停止麻醉，不要将鼠标无人看管，直到它完全清醒。保持单一一栋或返回到其他动物的公司，只有当完全恢复。术后继续监测健康状况，并adminis之三镇痛药，如果必要的。根据当地的动物护理和使用委员会提出的规则遵循的程序。 </li></ol> 2.急性片的制备<ol><li>准备学联，切削液，工具（剪刀，手术刀，镊子，压舌板，巴斯德吸管），和琼脂块。 注:1升人工脑脊髓液（ACSF）的每个实验是必需的，并且是由双蒸H 2 O作为以前发表16,23溶解化学品制备。切削溶液通过补充200毫升ACSF与0.87毫升2M的硫酸镁原液制备。 </li><li>含氧ACSF和整个用95％ 的 O 2和5％的CO 2在实验切削溶液。 </li><li>使用异氟醚使用小动物麻醉机（氧气3％）深深地麻醉鼠标。检查麻醉深度使用肢体撤回反射，然后再继续。 </li><li>使用大剪刀杀头鼠标，立即冷却头在冰冷的切割溶液。 </li><li>从尾部到延髓和一个中线切口开放性颅骨轻轻推头骨碎片向两侧使用镊子。迅速被轻轻提起它使用一个小的圆形锅铲颅骨取出大脑。切断与小脑手术刀。将脑在冰冷的裁剪解决方案。 </li><li>对于mPFC的注射部位:切断脑部使用解剖刀（含有mPFC的）的前部，并把在冰冷的切割溶液直到切片。 </li><li>除去用滤纸过量切削溶液和脑的后部粘合到vibratome阶段。 </li><li>为倾斜杏仁片，胶上的琼脂块（4％）切大脑在一个35°角（ 图1D，中间）。对于冠状杏仁核切片，MGM / PIN注射部位和注射内侧前额叶皮质部位，直接粘上大脑在舞台上（ 图1D，左和右）。位，仅次于脑额外琼脂块的稳定性，同时切片。 </li><li>的Plac在切割室与使用冷却单元保持在4℃下冰冷含氧切削溶液E的阶段。准备使用蓝宝石刀片杏仁核（320微米）的急性切片。放置切片与在RT含氧ACSF提供的界面室。 </li><li>制备急性扁桃体切片后，放置在接口室在水浴在36℃以回收切片35 - 45分钟。接着，返回界面室至室温。从这些片记录，请继续执行步骤3.2。 </li><li>开始的步骤2.10的过程中，切注射部位切片作为急性扁桃腺切片（步骤2.8 - 2.9）前所述。在水浴片恢复这里不需要。可选:要快速估算注射部位的位置，在装有荧光灯和适当的过滤器集的体视镜观察切片。 </li><li>由两个滤纸和submer夹着他们修复包含事后分析注射部位切片吉宁他们在PBS液O / N 4％多聚甲醛（PFA）。对于注射部位的分析继续执行步骤4.1。 </li></ol>突触前纤维3.可视化和刺激<ol><li>准备纤维和细胞的光遗传学激活补丁显微镜: <ol><li>居中安装的发光二极管（LED）到光传送通路。 </li><li>测量在后焦面并且在每个目标的输出与功率计选择的470纳米的合适的波长的LED的光强度。 </li><li>计算毫瓦/毫米2的光强度，并创建一个校准曲线（LED强度（％）与光输出（毫瓦/毫米2）），用于为470纳米的波长测量值的每一个目标。 </li></ol></li><li>检索来自于安装到配备有荧光灯的直立式显微镜的堰板腔的界面室和地点的急性扁桃体切片。小心定位片使得SLIC在界面室朝上E面也面临着向上的录音室。以1:1的速率灌注以新鲜的含氧ASCF切片 - 2毫升/分钟在〜31℃的温度。 </li><li>观察使用与适当的过滤器集表示的特定荧光蛋白组合荧光灯在切片突触前纤维。使用5倍物镜获取的概要（ 图1E），和60倍的目标为在目标区域内的纤维密度的评估。 注:对于GFP和YFP，使用过滤设置"绿色"（激励二十○分之四百七十二，分束器495，发射490 LP）为mCherry使用过滤器设置"红"（激励四十零分之五百六十，分束器585，发射七十分之六百三十零）作为指定在材料/设备表。 </li><li>打开或根据需要在实验（ 图2D）限制显微镜的光路的孔径。 </li><li>要获取修补程序记录，填补内部解决方案补丁吸管和我装N极持有人。申请正压补丁吸管，慢慢先入沐浴液，然后放低视觉控制到使用显微切片。 <ol><li>接近的从侧面和顶部的膜片吸管感兴趣的神经元。释放正压力时，吸移管是细胞（在细胞表面上的凹坑中可见）的表面上，将获得一个"千兆欧密封"通过施加负压。 </li><li>应用进一步吸破裂膜补片，以获得全细胞记录。接着，使用适当的波长为激活CHR（470纳米），同时记录从细胞电响应刺激与连接的发光二极管标记的纤维。 </li><li>突触刺激开始以低LED强度和增加，直到达到所需的突触电流幅度。触发器由在数据采集软件配置的数字输出控制的定时和脉冲长度（在图示例中的LED3）。 可以使用的其他软件和/或TTL-产生装置到触发LED:音符。 </li></ol></li><li>在根据需要对下一个记录的细胞和/或在特定的药物存在下的显微镜光路打开或限流孔（步骤3.4）重复刺激。 </li><li>录制完毕后，由两个滤纸夹着他们在4％PFA O / N淹没他们的修为事后分析切片。 </li><li>分析电生理数据。 注意:使用适当的软件可视化记录的扫频数据为每个单独的刺激下线。当分析的特效药物，使用峰值检测程序，以获取有关突触电流幅值为所有个人的扫描药物作用时间过程。确定何时药效达到稳定状态。要为数字代表准备响应平均，使用的软件为每个实验条件下平均≥10单独扫描（参见图3B-D，FH，J右图）。 注意:几个备选软件包可用于数据分析。 </li></ol>注射部位4.事后分析<ol><li>洗注射部位（从步骤2.12），并在PBS中的记录位点（如果重新映像是理想的，从步骤3.7）三次的切片。这是通过用新鲜的PBS在旋转摇动器反复更换溶液三次10分钟完成。 </li><li>准备在PBS 2％琼脂溶液中，并让它冷却到〜65℃。切片放置在小直径30毫米的培养皿底部平坦，嵌入琼脂片，让它冷却下来，直到固体。 </li><li>胶嵌入式片或切片的琼脂块到vibratome阶段，并放置在舞台破碎腔用PBS。 </li><li>切除嵌入片70微米厚。可选:切除后，切片可染色， 即 ，具有Neurotrace揭示细胞结构（ 图3A中的C），和/或b为YFP或mCherryŸ免疫荧光染色，以提高从CHR融合蛋白的信号。 </li><li>在幻灯片和盖玻片与安装介质安装片。图像注射部位，如果需要，包含使用荧光显微镜或共焦激光扫描显微镜（ 图2A-C）中的投射区的纤维也图像切片。 </li></ol>

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R., Perez-Carrera, D., Crespo-Ramirez, M., Fuxe, K., Perez de la Mora, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intercalated+paracapsular+islands+as+a+module+for+integration+of+signals+regulating+anxiety+in+the+amygdala.">The intercalated paracapsular islands as a module for integration of signals regulating anxiety in the amygdala.</a> Brain Res. 1476, 211-234 (2012).</li><li>Asede, D., Bosch, D., Luthi, A., Ferraguti, F., Ehrlich, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+inputs+to+intercalated+cells+provide+fear-learning+modulated+inhibition+to+the+basolateral+amygdala.">Sensory inputs to intercalated cells provide fear-learning modulated inhibition to the basolateral amygdala.</a> Neuron. 86 (2), 541-554 (2015).</li><li>Tamamaki, N., Yanagawa, Y., Tomioka, R., Miyazaki, J., Obata, K., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+expression+and+colocalization+with+calretinin,+parvalbumin,+and+somatostatin+in+the+GAD67-GFP+knock-in+mouse.">Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse.</a> J Comp Neurol. 467 (1), 60-79 (2003).</li><li>Mar, L., Yang, F. C., Ma, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+marking+and+characterization+of+Tac2-expressing+neurons+in+the+central+and+peripheral+nervous+system.">Genetic marking and characterization of Tac2-expressing neurons in the central and peripheral nervous system.</a> Mol Brain. 5, (2012).</li><li>Jackman, S. L., Beneduce, B. M., Drew, I. R., Regehr, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achieving+high-frequency+optical+control+of+synaptic+transmission.">Achieving high-frequency optical control of synaptic transmission.</a> J Neurosci. 34 (22), 7704-7714 (2014).</li><li>Li, H., Penzo, M. A., Taniguchi, H., Kopec, C. D., Huang, Z. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+induction+of+synaptic+plasticity+using+a+light-sensitive+channel.">Optical induction of synaptic plasticity using a light-sensitive channel.</a> Nat Methods. 4 (2), 139-141 (2007).</li><li>Britt, J. P., Benaliouad, F., McDevitt, R. A., Stuber, G. D., Wise, R. A., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+and+behavioral+profile+of+multiple+glutamatergic+inputs+to+the+nucleus+accumbens.">Synaptic and behavioral profile of multiple glutamatergic inputs to the nucleus accumbens.</a> Neuron. 76 (4), 790-803 (2012).</li><li>Kohl, M. M., Shipton, O. A., Deacon, R. M., Rawlins, J. N., Deisseroth, K., Paulsen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemisphere-specific+optogenetic+stimulation+reveals+left-right+asymmetry+of+hippocampal+plasticity.">Hemisphere-specific optogenetic stimulation reveals left-right asymmetry of hippocampal plasticity.</a> Nat Neurosci. 14 (11), 1413-1415 (2011).</li><li>Morozov, A., Sukato, D., Ito, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+suppression+of+plasticity+in+amygdala+inputs+from+temporal+association+cortex+by+the+external+capsule.">Selective suppression of plasticity in amygdala inputs from temporal association cortex by the external capsule.</a> J Neurosci. 31 (1), 339-345 (2011).</li><li>Davidson, B. L., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+gene+delivery+to+the+nervous+system.">Viral vectors for gene delivery to the nervous system.</a> Nat Rev Neurosci. 4 (5), 353-364 (2003).</li><li>Aschauer, D. F., Kreuz, S., Rumpel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+transduction+efficiency,+tropism+and+axonal+transport+of+AAV+serotypes+1,+2,+5,+6,+8,+and+9+in+the+mouse+brain.">Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8, and 9 in the mouse brain.</a> PLoS One. 8 (9), (2013).</li><li>Salegio, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+of+adeno-associated+viral+vectors+is+serotype-dependent.">Axonal transport of adeno-associated viral vectors is serotype-dependent.</a> Gene Ther. 20 (3), 348-352 (2013).</li><li>Holehonnur, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+viral+serotypes+produce+differing+titers+and+differentially+transduce+neurons+within+the+rat+basal+and+lateral+amygdala.">Adeno-associated viral serotypes produce differing titers and differentially transduce neurons within the rat basal and lateral amygdala.</a> BMC Neurosci. 15, (2014).</li><li>McFarland, N. R., Lee, J. S., Hyman, B. T., McLean, P. 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The authors declare that they have no competing financial interests.

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">&lt;strong&gt;Surgery&lt;/strong&gt;</td></tr><tr><td>Stereotactic frame</td><td>Stoelting, USA</td><td>51670</td><td>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><td>can be replaced by other stereotactic frame for mice</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)&amp;nbsp;</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)&amp;nbsp;</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 colspan="4">&lt;strong&gt;Recordings, light stimulation, and analysis&lt;/strong&gt;</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&amp;nbsp;</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 &amp;amp; Neumann, Germany</td><td>200-100 500 0145</td><td></td></tr><tr><td>Three axis micromanipulator Mini 25</td><td>Luigs &amp;amp; Neumann, Germany</td><td>210-100 000 0010</td><td></td></tr><tr><td>Micromanipulator controller SM7</td><td>Luigs &amp;amp; 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&amp;nbsp;</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>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><td>CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation</td></tr><tr><td>LED unit, USL 70/470</td><td>Rapp Optoelectronic</td><td>L70-000</td><td>CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation</td></tr><tr><td>Dual port adapter</td><td>Rapp Optoelectronic</td><td>inquire with company</td><td>CoolLED or USL 70/470 and appropriate adapters are two alternative choices for LED stimulation</td></tr><tr><td>Filter set red (excitation)</td><td>AHF, Germany</td><td>F49-560</td><td>Filters can be bought as set F46-008</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (beamsplitter)</td><td>AHF, Germany</td><td>F48-585</td><td>Filters can be bought as set F46-008</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (emission)</td><td>AHF, Germany</td><td>F47-630</td><td>Filters can be bought as set F46-008</td></tr><tr><td>Filter set green (excitation)</td><td>AHF, Germany</td><td>F39-472</td><td>Alternatives: filterset F36-149 or F46-002 (with bandpass emission)</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (beamsplitter)</td><td>AHF, Germany</td><td>F43-495W</td><td>Alternatives: filterset F36-149 or F46-002 (with bandpass emission)</td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (emission)</td><td>AHF, Germany</td><td>F76-490</td><td>Alternatives: filterset F36-149 or F46-002 (with bandpass emission)</td></tr><tr><td>LaserCheck, handheld power meter</td><td>Coherent, USA</td><td>1098293</td><td></td></tr><tr><td>IgorPro Software, Version 6</td><td>Wavemetrics, USA</td><td></td><td>for electrophysiology data analysis, other alternative software packages can also be used&amp;nbsp;</td></tr><tr><td>Neuromatic suite of macros for IgorPro</td><td>http://www.neuromatic.thinkrandom.com</td><td></td><td>for electrophysiology data analysis, other alternative software packages can also be used&amp;nbsp;</td></tr><tr><td colspan="4">&lt;strong&gt;Post hoc analysis of injections and projections&lt;/strong&gt;</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 ...

ex-vivo-optogenetic-dissection-of-fear-circuits-in-brain-slices

Research

JoVE Journal

Neuroscience

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

15.9K 次观看.  University of Tuebingen. 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。 

视频: Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

Optogenetically有针对性的前脑电路的激光扫描光刺激

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

科研

神经科学

Assessing Rodent Empathy: Observational Fear and Vicarious Freezing Method

Observational Fear as a Model of Affective Empathy in Mice

Empathy, characterized by the ability to recognize and share the emotions of others, plays a fundamental role in shaping social interactions. It allows individuals to respond to the emotional states of others, promoting prosocial behaviors and social bonding. Observational fear is a fundamental aspect of affective empathy, where an observer witnesses a demonstrator undergoing aversive experiences and subsequently exhibits fear behaviors. This socially induced vicarious freezing response in observers, known as emotional contagion or affect sharing, is regarded as an indicator of empathy-like traits in rodents. Here, we present a protocol that delineates the assessment of vicarious freezing responses in observers exposed to conspecific demonstrators receiving aversive foot shocks. The utilization of observational fear assays in rodents has become a widely adopted method for studying the neural mechanisms underlying affective empathy. Given the universality of observational fear across mammals, this methodology further contributes to advancing our understanding of the neural substrates of empathy in humans.

The observational fear paradigm assesses vicarious freezing in rodents as a model for affective empathy. This procedure entails exposing observer rodents to conspecific demonstrators receiving aversive foot shocks to elicit empathic freezing responses. By employing observational fear assays, researchers can investigate the neural mechanisms underlying affective empathy.

Empathy, characterized by the ability to recognize and share the emotions of others, plays a fundamental role in shaping social interactions. It allows ...

行为学

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

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

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

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

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, ...

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

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo 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 ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

脑切片中传入通路的光遗传学激活和挥发性麻醉剂对反应的调节

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. Traditional in vitro electrophysiological techniques use electrical stimulation to evoke synaptic transmission. A major drawback of this method is its nonspecific nature; all axons in the region of the stimulating electrode will be activated, making it difficult to attribute an effect to a particular afferent connection. This issue can be overcome by replacing electrical stimulation with optogenetic-based stimulation. We describe a method for combining optogenetics with in vitro patch-clamp recordings. This is a powerful tool for the study of both basal synaptic transmission and synaptic plasticity of precise anatomically defined synaptic connections and is applicable to almost any pathway in the brain. Here, we describe the preparation and handling of a viral vector encoding channelrhodopsin protein for surgical injection into a pre-synaptic region of interest (medial prefrontal cortex) in the rodent brain and making of acute slices of downstream target regions (lateral entorhinal cortex). A detailed procedure for combining patch-clamp recordings with synaptic activation by light stimulation to study short- and long-term synaptic plasticity is also presented. We discuss examples of experiments that achieve pathway- and cell-specificity by combining optogenetics and Cre-dependent cell labeling. Finally, histological confirmation of the pre-synaptic region of interest is described along with biocytin labeling of the post-synaptic cell, to allow further identification of the precise location and cell type.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

离体 从内侧前额叶皮层到外嗅皮层的远距离突触传递和可塑性的光遗传学询问

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. ...

Visualization and Mapping of Neuronal Pathways in an Amygdala Brain Slice

Source: Bosch, D., et al. <a href="https://app.jove.com/v/53628">Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices.</a> J. Vis. Exp. (2016).
This video demonstrates a procedure for analyzing synaptic connectivity between medial prefrontal cortex (mPFC) and amygdala neurons using acute amygdala brain slices from mice. The mPFC neurons in these mice express channelrhodopsin fused to a fluorescent protein. The channelrhodopsins facilitate ion influx and action potential generation in the mPFC neurons upon light activation. Consequently, mPFC neurons release neurotransmitters that bind to postsynaptic amygdala neurons, triggering postsynaptic currents that are recorded to visualize mPFC-amygdala connectivity.

杏仁核脑切片中神经元通路的可视化和映射

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Visualization and ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

An Electrophysiological Study of Retinogeniculate and Corticogeniculate Synapses in Mouse Brain Slices

Source: Chen, X., et al. <a href="https://app.jove.com/v/59680">Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function.</a> J. Vis. Exp. (2019).
The video describes the electrophysiological recording of retinogeniculate and corticogeniculate synapses in mouse brain slices. The slices containing the dorsal lateral geniculate nucleus (dLGN) are placed in a recording chamber. A stimulating pipette is positioned on either the optic tract to stimulate retinal ganglion cells (RGCs) and investigate retinogeniculate synapses or the nucleus reticularis thalami to stimulate cortical neurons and investigate corticogeniculate synapses. Upon stimulation, the RGCs and cortical neurons release excitatory neurotransmitters, which produce synaptic currents in the LGN relay neurons. These currents are recorded using a patch-clamp recording pipette.

小鼠脑切片中视网膜生殖突触和皮质生殖突触的电生理学研究

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Solutions

	Dissection solution
	
...

Electrophysiology Techniques

Generation of Tilted Amygdala Slices from a Mouse Brain

Source: Bosch, D., et al. <a href="https://app.jove.com/v/53628">Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices.</a> J. Vis. Exp. (2016)
The video demonstrates the generation of tilted amygdala slices from a mouse brain. The brain is positioned at an angle on the vibratome specimen stage to obtain slices containing the amygdala and axons from the medial prefrontal cortex that form synaptic connections with the amygdala. The brain slices are maintained at the physiological temperature to preserve the tissue for further experiments.

从小鼠大脑中生成倾斜的杏仁核切片

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Acute Slices

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在脑片恐惧电路体外光遗传学解剖

摘要

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此视频中的章节

Optogenetically有针对性的前脑电路的激光扫描光刺激

Observational Fear as a Model of Affective Empathy in Mice

利用体外大鼠脑切片研究变性主要事件的一种替代方法

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

脑切片中传入通路的光遗传学激活和挥发性麻醉剂对反应的调节

原位急性离体胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

离体从内侧前额叶皮层到外嗅皮层的远距离突触传递和可塑性的光遗传学询问

杏仁核脑切片中神经元通路的可视化和映射

小鼠脑切片中视网膜生殖突触和皮质生殖突触的电生理学研究

从小鼠大脑中生成倾斜的杏仁核切片

在脑片恐惧电路体外光遗传学解剖

摘要

探索更多视频

此视频中的章节

Optogenetically有针对性的前脑电路的激光扫描光刺激

Observational Fear as a Model of Affective Empathy in Mice

利用体外大鼠脑切片研究变性主要事件的一种替代方法

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

脑切片中传入通路的光遗传学激活和挥发性麻醉剂对反应的调节

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

离体 从内侧前额叶皮层到外嗅皮层的远距离突触传递和可塑性的光遗传学询问

杏仁核脑切片中神经元通路的可视化和映射

小鼠脑切片中视网膜生殖突触和皮质生殖突触的电生理学研究

从小鼠大脑中生成倾斜的杏仁核切片

原位急性离体胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

离体从内侧前额叶皮层到外嗅皮层的远距离突触传递和可塑性的光遗传学询问