作者要感谢M. Steczkowski语音录音，M. Borczyk图纸，ATrąbczyńska病毒的产生，M.Ziókowska用于基因分型和A. Mirgos与拍摄援助。 KR承认钙调蛋白激酶子从K.戴瑟罗特控制下表达荧光蛋白mCherry重组腺相关病毒（腺相关病毒）的一种恩赐。该项目是在实验室的组织结构和功能，神经生物学，实验生物学Nencki研究所中心的动物模型和实验室的核心功能进行，利用概念的基础设施由欧洲联盟资助 - 在欧洲区域发展基金业务方案"创新经济"2007 - 2013年。这项工作是由国家科学中心的资金支持:索纳塔双2012/05 / E / NZ4 / 0299​​6，异色2013/08 / M / NZ3 / 00861，Symfonia 2013/08 / W / NZ24 / 00691为KR和索纳塔双2014 / 14 / E / NZ4 / 00172 RC

下述所有试验程序都通过实验生物学研究所Nencki，波兰科学院当地伦理委员会的批准。 注意:有些在相关视频的场景被加速。速度因子是在这些场景指示。 1.术前准备<ol><li>消毒在高压釜的液体和棉签所有工具，玻璃容器。使用可有可无的手套。清洁外科表中，立体定位架和所有用70％乙醇的周围区域。用无菌手术垫为所有消毒设备创建的无菌空间。切明胶海绵切成小块并在无菌盐水中浸泡其中。 注:根据指南实验动物的护理和使用，乙醇既不是消毒剂，也不是一个高层次的消毒剂。它应该只被用作灭菌面的清洗/脱脂剂。 </li><li>摆在第动物È感应腔室，并设置异氟烷水平到5％，氧流量为2升/分。这个过程大约需要3分钟。 </li><li>就拿动物出来的感应室。使用尾或脚趾捏以确保动物是完全镇静。 </li><li>使用精确的修剪剃从头部的背面（两耳之间）的毛发到眼睛。 </li><li>放置动物的立体框架，并与耳酒吧稳定的头部。 </li><li>设置麻醉水平1.5-2％异氟烷和0.3升/分钟的氧气。 </li><li>应用眼膏。 </li><li>注射用Tolfedine（4毫克/千克），Butomidor（2毫克/千克）和拜有利（5毫克/千克）动物皮下，以防止炎症，疼痛和感染，分别。 </li><li>注射地塞米松动物肌肉注射（0.2毫克/千克），以防止脑水肿。 注意:可以将注入塞米松皮下或腹膜内防止肌肉损伤。 </li><li>清洁使用S皮肤接着用70％乙醇terile棉签优碘。 </li><li>改变手套和用70％乙醇喷雾它们。 注意:使用一次性手套不要接触无菌区。触摸动物只用无菌手术器械和无菌棉签的提示。 </li></ol> 2.骨窗手术<ol><li>解除用钳子皮肤和使用微剪刀横向切开皮肤沿头部的基部，然后倾斜地眼睛之间的前点。取下皮瓣。 </li><li>应用利多卡因软膏与骨膜无菌棉签，防止出血过多和疼痛。 </li><li>用消毒棉签或手术刀去除骨膜。干用无菌棉签头骨。 </li><li>使用无菌针施加在皮肤上的边缘密氰基丙烯酸酯胶来固定它们，并从用牙科水泥接触，以防止。等待胶水干。 </li><li>躺在无菌3毫米玻璃罩超过Ť他的头骨前方的人字缝。中心RSC盖玻片坐标:AP，前囟门-2.8; ML，前囟门0马克盖玻片边缘用消毒针头刮伤颅骨表面。把玻璃罩回用70％乙醇消毒容器中。 </li><li>使用高速牙钻直径小毛刺勾勒出一个直径为3毫米的圆。清洗从骨粉尘用无菌生理盐水浸渍拭子钻井现场。使用明胶海绵和棉签停止偶尔出血和清洁骨。 </li><li>在钻孔之间通过轻触骨圈并检查其机动性检查骨厚度用细镊子。请记住，骨骼是在缝合的区域厚。停止钻井当骨圆是移动的，只有一个偶数，骨的薄层被留在圆周上。清理所有剩余的骨粉用生理盐水棉签蘸业务领域。 </li><li>滴在钻探领域的无菌生理盐水，覆盖钻CIRCLE。小心地撬起骨圈用细镊子，然后轻轻地但坚定地向上提起，取出骨头。要小心，不要歪斜骨圈，同时提起，以防止硬脑膜可能造成的损害。 </li><li>轻轻涂抹于硬脑膜在无菌盐水浸泡明胶海绵来帮助止血。等到所有的出血完全停止。小心取出明胶海绵不干扰凝血过程。 注意:缝合区域是高度血管化的，所以在这一点上出血可能被证明是深远的。对于出血完全停止，必须等待足够的时间。它是有用的，以保持通过将它放置在冰上冷却的盐水浸泡的明胶海绵。 </li></ol> 3.病毒注射液<ol><li>连接输液泵的立体定向塔并连接控制器。 </li><li>将35G针头插入注射器。冲洗注射器10次用乙醇消毒，并用无菌生理盐水10次以除去电子商务痕迹thanol。从注射器除去气泡。将注射器进入泵内。 注意:考虑使用其他消毒剂。 </li><li>解冻的rAAV2 / 1 mCherry制剂的单次剂量（建议10 12 PFU），并保持在冰上。填与病毒溶液注射器。 </li><li>中心针上的前囟，然后使用以下坐标轻轻插入海马:AP -2，ML +/- 1.0，DV -1。这些坐标将位于靠近开颅的边缘。等待5分钟，使组织稳定。 </li><li>注入0.7微升的rAAV2 / 1 mCherry溶液以50升/分钟的速率。等待10分钟使病毒充分吸附。轻轻取出针。用明胶海绵吸干如果发生出血。与对侧部位重复。 </li></ol> 4.骨窗植入<ol><li>躺在在钻圆帧硬脑膜顶部的无菌干燥玻璃罩。拿住FORC玻璃罩EPS轻轻压平硬脑膜，并把玻璃罩"边缘靠近颅骨表面。 注意:这是可能的玻璃罩破坏凝块和出血的简历。如果是这样的情况下，解除玻璃罩，将其放置在醇，干燥并返回步骤2.9。 </li><li>用消毒针头敷在玻璃罩边缘茂密的氰基丙烯酸酯胶将其连接到颅骨。等待胶水干。 </li><li>放置在颅骨的前部的​​固定杆（M2螺母或者定制设计）。应用氰基丙烯酸酯胶在条的边缘。等待胶水干。 <ol><li>在广场将成像会议期间使颅窗口的水平定位位置的固定杆。把它作为遥远地从窗口。如果它被置于太靠近窗口，杆和螺钉将其与特制的保持器连接可能对作为在成像过程为目标的一个障碍。 </li></ol></li><l我&gt;准备牙科丙烯酸，并将其应用于玻璃周围的颅骨表面上。它是有用的，以形成在窗口周围的火山口状的形状。这将创造更高版本与水物镜成像应用水腔。 </li><li>创建具有牙科丙烯酸盖，覆盖业务领域，皮肤边缘，固定杆的其余部分，加强各地颅窗口的火山口。等待牙科水泥硬化。 </li><li>从立体框架中取出的动物，并把它放入回收室。 </li><li>等待动物从手术同时观察生理功能恢复。 </li><li> 48小时申请术后镇痛（卡洛芬，10毫克/千克）和抗生素治疗（拜有利，5毫克/千克）。 </li></ol> 5.成像<ol><li>启动钛宝石激光器，功率可达显微镜。在这个实验中使用的系统配备双光子激光，OPO系统和双磷砷化镓PMT。 </li><li>把动物的INDUC化室，诱导麻醉。 </li><li>从在显微镜下气体麻醉面罩感应腔和处取出动物。降低氧气流至0.3升/分和异氟醚浓度为1.5-2％。 </li><li>修复动物用M2螺丝（或其他定制系统）定制的显微镜机架。水平颅窗口。 注意:可以使用显微镜的制造商的头固定系统，虽然具体的定制帧给出更好的结果（改进的头部的稳定性，慢性实验期间在多个会话恒定定位）。 </li><li>上使用retrosplenial皮质的侧面之一的广角镜的设置和低倍率物镜中心视图并将其聚焦盖玻片表面上。 </li><li>适用的水的液滴进入火山口状丙烯酸系好。切换到长距离水浸物镜。移动目标对颅窗口，直到水半月板CONnects试样和目标。 </li><li>切换到双光子设置，并开始使用最低缩放扫描检体从上到下。硬膜的交叉将高达非特异性信号的眩光可见。 </li><li>调整中，根据从荧光细胞以覆盖整个动态范围的信号强度两个通道（GFP和mCherry）显微镜采集设置。 </li><li>找到合适的神经元（与来自其它细胞中分离的树突树）之后仅使用具有最低缩放和5微米的z距离的GFP filterset执行初始扫描。 </li><li>获取扫描栈的最大投影和（使用反色）打印的注释。 </li><li>设置变焦值，使图像所需的形态的细节。使用图像中最大的投影为导向的GFP和mCherry渠道整个树突树。 </li></ol>

<ol>
	<li>Grutzendler, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+synaptic+plasticity+and+pathology+in+the+living+mouse+brain.">Two-photon imaging of synaptic plasticity and pathology in the living mouse brain.</a> NeuroRx. 3, 489-496 (2006).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li><li>Trachtenberg, J. T., 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=Long-term+in+vivo+imaging+of+experience-dependent+synaptic+plasticity+in+adult+cortex.">Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex.</a> Nature. 420, 788-794 (2002).</li><li>Holtmaat, 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=Imaging+neocortical+neurons+through+a+chronic+cranial+window.">Imaging neocortical neurons through a chronic cranial window.</a> Cold Spring Harb Protoc. 2012, 694-701 (2012).</li><li>Chow, D. K., 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=Laminar+and+compartmental+regulation+of+dendritic+growth+in+mature+cortex.">Laminar and compartmental regulation of dendritic growth in mature cortex.</a> Nat Neurosci. 12, 116-118 (2009).</li><li>Czajkowski, 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=Encoding+and+storage+of+spatial+information+in+the+retrosplenial+cortex.">Encoding and storage of spatial information in the retrosplenial cortex.</a> Proc Natl Acad Sci U S A. 111, 8661-8666 (2014).</li><li>Czajkowski, 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=Superficially+projecting+principal+neurons+in+layer+V+of+medial+entorhinal+cortex+in+the+rat+receive+excitatory+retrosplenial+input.">Superficially projecting principal neurons in layer V of medial entorhinal cortex in the rat receive excitatory retrosplenial input.</a> J Neurosci. 33, 15779-15792 (2013).</li><li>Feng, G., 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=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Couey, J. J., 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=Recurrent+inhibitory+circuitry+as+a+mechanism+for+grid+formation.">Recurrent inhibitory circuitry as a mechanism for grid formation.</a> Nat Neurosci. 16, 318-324 (2013).</li><li>Miyashita, T., Rockland, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+projections+from+the+hippocampus+to+the+retrosplenial+cortex+in+the+rat.">GABAergic projections from the hippocampus to the retrosplenial cortex in the rat.</a> European Journal of Neuroscience. 26, 1193-1204 (2007).</li></ol>

作者（KL，MR，KR）拥有专利本条座架待定（PL410001）的权利。

在当前的论文中，我们提出在在RSC突触输入和突触后的目标体内成像同时双光子的协议通过颅窗口。植入程序包括几个关键步骤。首先，将动物深度麻醉并固定在立体定位框架，然后经RSC颅骨变薄沿标出圆形线的钻头和圆形骨除去。出血停止之后，所述的rAAV2 / 1 mCherry注入海马，及玻璃盖被固定到颅骨上钻孔区。最后的固定杆被固定在头部和动物放置在回收室48小时。后所需要的病毒表达大约2-3周时，RSC可以显现。成像协议包括以下步骤的。首先，将动物麻醉并在显微镜下固定。焦点然后使用宽视场显微镜设置，该系统是SWItched进入双光子模式和（GFP和mCherry）感兴趣的信道被可视化。 所提出的技术提供了在先前描述的方案的一个主要的改进。在标准方法中，只有一种类型的标记物可以被检测出来。它可用于图像局部轴突突起和树突树，但没有远距离连接研究是可能的。通过结合两个荧光蛋白，我们启用前和突触后元素的同时进行跟踪。这使得在假定的突触连接体内监测长远。 为了获得在所描述的过程的最佳结果，必须注意几个关键步骤是重要的。在解除了骨圈在步骤2.8硬脑膜任何损坏可能会导致炎症和损害颅窗口的透明度。在步骤2.9，或在过程的任何其他步骤出血停止不足导致血液accumulat离子窗下和显著降低的视场。注射病毒后这是至关重要除去针，为了使病毒注入到仅在注射部位的组织之前等待至少10分钟。它限制了该病毒的皮质无用感染的针移除期间的可能性。病毒转染的面积应与脑组织的事后组织学分析进行检查。应避免在沿着针迹细胞的任何mCherry表达。标准的恢复时间是3周。这一时期是足以使病毒在突触突起达到稳定表达和用于颅窗口完全愈合并稳定。动物应单个容纳在为了防止由轿厢配合除去颅窗口植入物。使用的放大笼的可能，以避免通过敲击金属棒颅窗口的意外损坏被考虑。谅解备忘录的稳定固定在显微镜下本身是必不可少的。任何头部运动，包括呼吸运动，可能会导致在所获得的图像的质量显著降低。这也有利于对颅窗口水平定位到目标，以限制与获得相等焦点为窗口的整个面可能出现的问题。解决刺和终扣明确的标准应适用。一般地，终扣被定义为具有直径比前述光纤7更大的至少3倍轴突肿大。棘被定义为从包含球根状头枝晶轴清晰可辨突起。进一步划分成薄，粗短，蘑菇和支刺种群可能3。由于双光子显微镜的轴向分辨率相对较差，即沿着光轴投射棘分析应避免3。为了明确区分功能终扣和尖刺， 事后免疫标记可以确定前和突触后的标记7进行。 虽然所提出的协议被证明是在我们的实验设计中最有利的，它可以修改它以适合不同的实验的目标。有时是更合适的是使用注射麻醉（如氯胺酮xylasine）代替异氟烷，但它以充分调整剂量，不同品系，年龄和性别的小鼠之间在药物敏感性记差异保持是重要的。它可以使用钻代替球形一个用于钻井环锯，但它可以增至硬脑膜损坏的风险。无菌盐水可与人工脑脊液（ACSF）可以成功地代替，但准备和操作过程中使其保持无菌在任何时候都是重要的。 ACSF是3-4周稳定制备后，如果在此时间之前发生的任何污染，应当立即丢弃。不同的头fixati上的设备可以被应用于，取决于实验设计，包括双光子显微镜下观察清醒小鼠的可能性。 至于其他的技术，这其中也有其局限性。双光子显微镜允许脑组织达500微米深从硬脑膜表面的可视化。对于更深的大脑结构的检查另外的修饰必须应用。我们的协议允许访问最RSC的，但上矢状窦下隐藏结构的部分仍是不可访问。双光子显微镜的分辨率是不够的特定突触的识别以及树突棘详细的形态学分析。额外的技术，如相关电子显微镜必须为了确认疑似结构的存在可以应用。同样重要的是要提到，这是一个较困难的手术技术，它是不RECOmmended为雏运营商。 所呈现的技术可在很宽的范围内的实验被应用。它可以被修改为与双光子显微镜访问不同脑区的调查。它使各种生理和病理状态，包括认知过程和老化或神经和精神疾病的进展过程中轴突和树突的改建同步监测。它允许使用细胞特异性启动子的可视化源自精确限定的神经元亚群的突起。它也可以被调整以适应于清醒和行为的动物实验的协议。此外，钙或pH敏感的蛋白质可以在大脑中表达，以便不仅显现神经元形态，但也改变细胞活性和功能。该方法的另一种可能的修改是利用不同的重组腺相关病毒的血清型为mCherry表达。嵌合2/1血清型公关ovides在注射部位以足够快速起效（2-3周）鲁棒表达。的mCherry水平初始发作后至少12周保持稳定，并在我们的实验中没有检测到逆行标记。为了获得逆行标记，不同的血清型可能被使用，如rAAV9。成像会话可以在任何频率下进行，但至少在24小时的时间间隔，以便允许麻醉后的动物的正确恢复推荐。如果运用得当，这种技术允许通过的几个月中执行相同区域的多个成像会议。对于长期实验（超过6个月），一个的Cre / loxP系统可与AAV载体递送到两侧装接loxP的GFP小鼠系的重组酶被使用。

双光子显微镜彻底改变了大脑活动的观察生活和行为的动物。自1990年推出它迅速得到普及，现在实现为对体内 1,2大脑活动的许多方面考核最有趣和创新的方法之一。这些应用包括血流测量，神经元激活（ 例如 ，使用钙离子浓度指标或立即早期基因表达）和神经细胞的形态。越来越多的实验室使用双光子显微镜，实现在整个科学界的技术，因为体内脑成像的新标准。 标准方法涉及颅窗口植入在桶或小鼠脑3的视觉皮层（在覆盖有盖玻璃的头盖骨的圆孔）。接着，根据不同的实验方案，在谋本身经历一系列可视化和行为训练会话，允许在一段时间4,5-监视在大脑活动和神经元形态的变化。在两种情况下，开颅仅影响顶骨，而不穿过缝线。它主要是认为该技术的主要缺点是其应用受到限制，以方便皮质诸如桶或视觉皮层。在其他地区颅窗口植入带来了很大的困难，因失血过多和/或空间阻碍。 在本文中，我们提出了retrosplenial皮层（RSC）作为用于体内显微镜6双光子兴趣另一可能的区域上方颅窗口的注入。 RSC负责空间记忆形成脑电路的一个重要因素。解剖学上，RSC是连接皮质，海马，丘脑和区域7神经元网络的一部分。它是积极参与各种行为，如空间学习和灭绝以及空间导航6。 为了可视化神经元的形态学变化，我们使用表达该THY1启动子下的绿色荧光蛋白（GFP）的转基因小鼠品系。在这些小鼠中，绿色荧光蛋白是在用双光子显微镜8在大脑中的神经元，允许皮质轴突和树突的清晰的可视化的大约10％来表示。我们提出另一个创新是重组腺相关病毒血清型2/1（的rAAV2 / 1）特定的神经元的CaMKII子9下编码红色荧光蛋白（mCherry）进入脑的突出到RSC更深结构的注射，如海马。的rAAV2 / 1 mCherry的Thy1肾炎-GFP小鼠海马的表达允许hippocampo-cortica的前和突触后元素的同时进行可视化升突触10。 mCherry的重组腺相关病毒驱动的表达需要两到三周的蛋白质在终末达到足够的水平。这一时期是从开颅手术恢复所需的通常时间是一致的。

<table><tbody><tr><td>&lt;strong&gt;Drug&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>AErrane 8DG9623</td><td>5-2% pre-operative</td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>AErrane 8DG9623</td><td>1.5-2% during surgery</td></tr><tr><td>Dexametasone</td><td>Scan Vet</td><td>Dexasone 2mg/ml</td><td>0.2 mg/kg intramuscular</td></tr><tr><td>Baytril</td><td>Bayer</td><td>2.50%</td><td>5 mg/kg subcutaneously</td></tr><tr><td>Tolfedine</td><td>Vetoquinol</td><td>4%</td><td>4 mg/kg subcutaneously</td></tr><tr><td>Butomidor</td><td>Richter Pharma</td><td>10 mg/ml</td><td>2 mg/kg subcutaneously</td></tr><tr><td>Carprofen</td><td>KRKA-Polska</td><td>Rycarfa 50mg/ml</td><td>10 mg/kg subcutaneously</td></tr><tr><td>Lidocaine</td><td>Jelfa</td><td>Lignocainum</td><td>topically</td></tr><tr><td>Lidocaine</td><td>Jelfa</td><td>20 mg/g</td><td>topically</td></tr><tr><td>&lt;strong&gt;Surgery&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gelfoam</td><td>Ethicon</td><td>Spongostan dental; REF MS0005</td><td></td></tr><tr><td>Eye ointment</td><td>Dedra</td><td>Lubrithal</td><td>topically</td></tr><tr><td>CA glue</td><td>Pelikan Daniel</td><td>20G Huste</td><td></td></tr><tr><td>Dental acrylic</td><td>SpofaDental</td><td>Duracryl Plus</td><td></td></tr><tr><td>Stereotaxic frame</td><td>Stoelting</td><td>51500D</td><td></td></tr><tr><td>&lt;strong&gt;Tool&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Coverglass</td><td>Harvard Apparatus</td><td>HSE-64-0720</td><td>3 mm diameter</td></tr><tr><td>Dental drill</td><td>Sigmed</td><td>Keystone KVet</td><td></td></tr><tr><td>Fixation bar</td><td>Custom made</td><td>N/A</td><td>M2 or M3 screw nuts could be used</td></tr><tr><td>Forceps</td><td>Renex</td><td>PN-7B-SA</td><td></td></tr><tr><td>Micro scissors</td><td>Falcon</td><td>BM.183.180</td><td></td></tr><tr><td>Dissection microscope</td><td>KOZO</td><td>XTL6445T</td><td></td></tr><tr><td>&lt;strong&gt;Imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Holder frame</td><td>Custom made</td><td>N/A</td><td></td></tr><tr><td>Two-photon microscope</td><td>Zeiss</td><td>Upright Axio Examiner Z1</td><td>Laser unit: Coherent Chameleon 690-1040nm with Optical Parametric Oscillator 1050-1300nm. Objectives: EC-PLAN-NEUFLUAR 10x/0.1 and LD Plan-APOCHROMAT 20x/1.0. Detection: Zeiss bandpass filters BP 500-550 (GFP) and BP 570-610 (mCherry) separated by beam splitter at 560nm and coupled to two GaAsP photodetectors.&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Virus</td><td>gift from K. Deisseroth</td><td></td><td>Recombinant adeno-associated virus (rAAV) expressing fluorescent protein mCherry under the control of CaMK promoter</td></tr></tbody></table>

simultaneous two-photon in vivo imaging of synaptic inputs and postsynaptic targets in the mouse retrosplenial cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

GFP的神经元在大鼠Thy1-GFP报道小鼠的子集的表达允许在皮质树突和在RSC局部轴突突起的体内成像。 图1A示出了用可见光多个GFP阳性树突图像的堆叠的最大投影。细胞体通过动脉遮蔽。 图1B示出了在图1A所示的树枝状分支的单一平面的缩放图像（数字变焦3×）。树突形态（棘，丝状伪足）的细节都清晰可见。 GFP的通道是通过使用带通发射滤波器500-550毫微米获得。 在的rAAV2 / 1 mCherry到背侧海马的注入使得海马轴突和突触终扣在RSC终端的可视化。这些终端可以在mCherry通道（带通发射滤波器570-610纳米）来检测。 图1。双通道 RSC神经元和海马预测到RSC 体内双光子成像。（ 一 ）表达GFP的细胞在RSC的片段（以反色显示的图像）的概述。在低倍率（0.7倍数码变焦）拍摄的100微米厚叠显示最大投影。 （B）在（A）所表示的片段的单一光学平面在GFP的通道高倍率（3倍数码变焦）收购。使用mCherry采集设置（C）在（A）表示单光学平面片段。请参阅协议用 ​​于检测过滤器的详细信息。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53528/53528fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex at JoVE.com

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

simultaneous-two-photon-vivo-imaging-synaptic-inputs-postsynaptic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

11.4K 次观看.  Nencki Institute of Experimental Biology. This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

视频: Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

有针对性的特定功能的微域的新皮质相结合的内源信号和双光子成像标记的神经元

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

科研

神经科学

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

行为学

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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 ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

<meta charset="utf8"></head><body>小鼠感觉皮层发育中的神经元活动映射

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following the discovery of numerous genes contributing to these developmental changes, it is now known that neuronal activity also substantially modulates these processes. In the developing cerebral cortex, neurons exhibit synchronized activity patterns that are specialized to each primary sensory area. These patterns markedly differ from those observed in the mature cortex, emphasizing their role in regulating area-specific developmental processes. Deficiencies in neuronal activity during development can lead to various brain diseases. These findings highlight the need to examine the regulatory mechanisms underlying activity patterns in neuronal development. This paper summarizes a series of protocols to visualize primary sensory areas and neuronal activity in neonatal mice, to image the activity of individual neurons within the cortical subfields using two-photon microscopy in vivo, and to analyze subfield-related activity correlations. We show representative results of patchwork-like synchronous activity within individual barrels in the somatosensory cortex. We also discuss various potential applications and some limitations of this protocol.

<meta charset="utf8"></head><body>作者聚焦：从双光子显微镜和单神经元成像破译神经回路形成

Primary sensory areas in the neocortex exhibit unique spontaneous activities during development. This article describes how to visualize individual neuron activities and primary sensory areas to analyze area-specific synchronous activities in neonatal mice in vivo.

在单神经元分辨率下新生小鼠感觉皮层自发活动的体内可视化

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following ...

同时双光子

摘要

探索更多视频

此视频中的章节

有针对性的特定功能的微域的新皮质相结合的内源信号和双光子成像标记的神经元

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

双光子

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

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

小鼠视网膜的经瞳双光子体内成像

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

在单神经元分辨率下新生小鼠感觉皮层自发活动的体内可视化

在单神经元分辨率下新生小鼠感觉皮层自发活动的体内可视化