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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E. <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+cells+of+the+amygdala.">The intercalated cells of the amygdala.</a> J Comp Neurol. 247 (2), 246-271 (1986).</li><li>Busti, D., 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=Different+fear+states+engage+distinct+networks+within+the+intercalated+cell+clusters+of+the+amygdala.">Different fear states engage distinct networks within the intercalated cell clusters of the amygdala.</a> J Neurosci. 31 (13), 5131-5144 (2011).</li><li>Palomares-Castillo, E., Hernandez-Perez, O. 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. J., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+modification+of+a+central+amygdala+fear+circuit.">Experience-dependent modification of a central amygdala fear circuit.</a> Nat Neurosci. 16 (3), 332-339 (2013).</li><li>Petreanu, L., Mao, T., Sternson, S. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+subcellular+organization+of+neocortical+excitatory+connections.">The subcellular organization of neocortical excitatory connections.</a> Nature. 457 (7233), 1142-1145 (2009).</li><li>Felix-Ortiz, A. C., Beyeler, A., Seo, C., Leppla, C. A., Wildes, C. P., Tye, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLA+to+vHPC+inputs+modulate+anxiety-related+behaviors.">BLA to vHPC inputs modulate anxiety-related behaviors.</a> Neuron. 79 (4), 658-664 (2013).</li><li>Chu, H. Y., Ito, W., Li, J., Morozov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target-specific+suppression+of+GABA+release+from+parvalbumin+interneurons+in+the+basolateral+amygdala+by+dopamine.">Target-specific suppression of GABA release from parvalbumin interneurons in the basolateral amygdala by dopamine.</a> J Neurosci. 32 (42), 14815-14820 (2012).</li><li>Zhang, Y. P., Oertner, T. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transduction+efficiency+of+recombinant+AAV+serotypes+1,+2,+5,+and+8+in+the+rat+nigrostriatal+system.">Comparison of transduction efficiency of recombinant AAV serotypes 1, 2, 5, and 8 in the rat nigrostriatal system.</a> J Neurochem. 109 (3), 838-845 (2009).</li><li>Miyashita, T., Shao, Y. R., Chung, J., Pourzia, O., Feldman, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+channelrhodopsin-2+(ChR2)+expression+can+induce+abnormal+axonal+morphology+and+targeting+in+cerebral+cortex.">Long-term channelrhodopsin-2 (ChR2) expression can induce abnormal axonal morphology and targeting in cerebral cortex.</a> Front Neural Circuits. 7, (2013).</li></ol>

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">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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

15.8K 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

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神经元形态的研究方法:<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

科研

神经科学

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

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

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 ...

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

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

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

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

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

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

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 ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

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

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

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

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

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

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 diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

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

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

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

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

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

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, ...

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

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

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 ...

在脑片恐惧电路体外光遗传学解剖

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神经元形态的研究方法: