作者想感谢L. Belluscio对手术的摄影器材，D.权进行拍摄的援助，K.，用于视频编辑的援助，和K.麦克劳德所有的背景音乐。 KW承认院内研究项目的镍氢电池事业部和基因，认知和精神病计划的慷慨支持。这项工作是支持的镍氢电池院内研究计划（VC，YY，SMKW），和NIAAA部院内临床和生物研究发展计划（VC，RMC，DML）。

下面描述的实验过程的心理健康动物管理和使用委员会由国家批准，并按照国家研究院实验动物护理和使用健康指南 。 1。手术前准备<ol><li>在炎热的珠灭菌器无菌手术前清洗所有的工具，清洁手术部位用70％的乙醇，放下干净的罩布。戴无菌手套。与新鲜制备的1.2％阿佛丁解决方案，由于在0.02毫升/克，腹腔注射麻醉动物。或者，麻醉用异氟醚气体通过一个鼻锥体，用于诱导的5％，1.5％以进行维护，与一个被动清道夫电路。检查用尾巴或脚趾捏，以确保全面镇静麻醉水平。 </li><li>量的动物的眼睛用无菌的眼用软膏，并注入新鲜制备的地塞米松（0.2毫克/千克），卡洛芬（5毫克/公斤）subcutaneously以防止脑水肿及炎症11。刮头发，两耳之间的头骨，并与聚维酮碘擦洗和三个交70％的酒精棉签消毒皮肤。 </li><li>挂载该动物在立体定位手术与earbars阶段，在37℃下与下面的动物组的水环流加热垫定期检查动物的麻醉水平，并根据需要补充原有的麻醉剂量。 </li><li>注入200μL0.5％的疼痛控制下的头皮麻木的区域。在头骨切开皮肤及去除皮瓣。删除骨膜，用干净的棉签和干燥的地区。 </li></ol> 2。慢性颅窗手术<ol><li>使用一个高速牙钻一个0.5毫米毛刺，轻轻地勾勒出一个3-5毫米直径的圆过的大脑区域的利益。定期湿钻井现场，用无菌0.9％生理盐水和清除骨用干净的棉签上的灰尘。如果骨出血，用gelfoam浸种用无菌生理盐水涂抹出血，并等待其停止。 </li><li>当最后的骨层，细尖镊子提起并取下骨岛。杜拉附件的底架上的骨头，可能会遇到，如果窗口越过骨缝。这些应轻轻取出的骨瓣解除。 </li><li>辊蘸明胶海绵轻轻地覆盖在外露的硬脑膜清洁其表面，并等待硬脑膜出血停止。 关键的步骤：请不要继续，直到所有的硬脑膜出血已停止。血液变得被困在里面的颅骨窗口通常会导致一个不透明的窗口。 </li><li>如果感兴趣的区域是硬脑膜的位置是特别厚，除去它上面的硬脑膜下可能是必要的。如果是这样的情况下，轻轻分开硬脑膜从软以下，非常细的钳子，使一个小切口，在解除硬膜与另一对细钳子，然后抓住切割边缘，并轻轻分裂硬脑膜。硬膜薄，但强大的，所以要小心不要切的大脑完整的硬脑膜边缘。 </li><li> ，用无菌的ACSF或无菌生理盐水冲洗区。 </li><li>躺在无菌的盖玻片（3-5毫米）以上的持续时间或软。 关键的步骤：如果周围的头骨曲线显着，使用Kwiksil 12胶粘剂填补的空间硬脑膜或软将不紧密的接触盖玻片地区将不被成像。 </li><li>使用氰基丙烯酸酯凝胶覆盖的弹性体粘接剂和玻璃盖玻片的边缘。覆盖整个暴露颅骨氰基丙烯酸酯胶，或krazyglue和牙科水泥混合物。 关键的一步 ：不要让任何氰基丙烯酸酯凝胶接触到大脑表面。 </li><li>嵌入一​​个特制的金属头固定杆的另一端的头骨。 </li><li>动物返回到温暖的回收室，一个intraperitonial酮洛芬，注射5毫克/公斤，对于后ŕ疼痛管理。持续两天手术后的镇痛。 </li><li>经过两个星期的术后恢复，动物麻醉与异氟醚（归纳为5％，1.5％，为维护），将其安装在一个特制的显微镜载物台头固定架，并检查颅窗的透光与蓝色光的照射下。脑表面血管清晰颅窗质量的高度指示。如果血管边缘分明，窗口是可能是可用的。两个星期的手术后的恢复时间，这样就可以有足够的时间从手术中恢复过来的动物。颅窗口通常清除和改善，在这段时间内，并保持光学透明额外的几周到几个月，直到再生的颅骨或硬脑膜增厚窗口质量下降。 </li></ol> 3。行为协议和激光扫描双光子显微镜<ol><li>动物有明确的Çranial窗口将被暴露在环境刺激或行为训练会话，然后成像后在最大弧GFP表达的时间。动物开始行为协议之前，应保持在一个一致的饲养笼的环境，以尽量减少一天到天基线电弧GFP表达水平的变化。 </li><li>行为训练或环境刺激范式开始，根据对大脑的地区的利益。例如，动物可以接触到不同的视觉环境，在连续数天，和视觉上的刺激，以识别特定刺激的反应在视觉皮层的10后拍摄的每一天。 </li><li> ARC-GFP荧光的神经元通常两个小时后，刺激达到了登峰造极的地步。实验时间轴可以优化，以便于检测电弧在神经元中的GFP表达的荧光峰值水平。 </li><li>后一个行为的训练或environmenta的升刺激，麻醉动物，异氟醚（归纳为5％，1.5％，为维护）和双光子显微镜安装在为客户定制的显微镜载物台头固定框架下的动物。 </li><li>动物的头位置是固定的，直接连接到舞台上，使用植入的金属条上的骷髅头固定架。异氟醚和氧气连续地供给到鼠标通过鼻锥体。使用加热垫，保持体温。 </li><li>确保在一个黑暗的房间中进行双光子激光扫描显微镜，环境光探测器的保护。使用20倍或25倍的水（1.05数值孔径）浸没透镜的成像。首先，外延荧光照明下，获得的图像在大脑区域感兴趣的用CCD照相机未来图像对齐表面的血管图案。 </li><li>启动双光子激光扫描获得3-D图像堆栈。使用Olympus FV1000MPE多光子显微镜被用来在我们的设置。的双光子激光的激发波长设定为920纳米，和从目标发射的激光的功率被设定为约50毫瓦。同时检测在绿色和红色通道中（在570 nm处的分色镜，在495-540 nm和570-625纳米的屏障过滤器），使用一个外部的双通道的photomutiplier检测系统放置在靠近试样发出的荧光。 ARC-GFP荧光只出现在组织的自体荧光的绿色通道，而出现两个通道中的13，14。 </li><li>典型的图像栈具有约320x320x100微米的尺寸（宽度×长度×深度），水平分辨率为0.5μm/像素​​，和垂直分辨率为3μm/像素​​。 </li><li>获取图像的堆栈后，返回到其家笼子里的动物。请勿打扰的动物，直到下一次的行为和成像会议。重复的行为和成像整个天DESIR程序编辑。使用先前获得的脑表面的血管图像的方向返回到相同的成像位置。 </li></ol>

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没有利益冲突的声明。

这里描述的体内成像方法，使弧基因表达的变化在相同的一组神经元在活的动物多天的反复检查。这是一个高效，灵活的方法来获取信息在单个神经元的神经可塑性相关的分子动力学响应不同的行为体验。标准组织化学方法，如原位杂交和免疫组织化学可以实现单细胞的第3号决议，但缺乏能够跟踪多天在同一个神经元的基因表达的变化。通过适当记者15生物发光，磁...

<table border="1"><tbody><tr><td> 设备名称： </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> FV1000多光子激光扫描显微镜</td><td>奥林巴斯</td><td> FV1000MPE </td><td>成像</td></tr><tr><td>解剖显微镜</td><td> Omano </td><td> 555V107 </td><td>手术</td></tr><tr><td>立体定向手术对小鼠的阶段</td><td>哈佛设备</td><td> 726335 </td><td>手术</td></tr><tr><td> 20X或25X水浸物镜</td><td>奥林巴斯</td><td> XLPL25XWMP </td><td>成像</td></tr><tr><td>显微镜载物台头固定架</td><td>订制</td><td> N / A </td><td>成像</td></tr><tr><td>精细镊子</td><td>精细科学工具</td><td> 11251-20 </td><td>手术</td></tr><tr><td>钻牙毛刺</td><td>精细科学工具</td><td> 19007-05 </td><td>手术</td></tr><tr><td> CCD相机</td><td> QImaging </td><td> QICAM 12位</td><td>成像</td></tr></tbody></table>

in vivo two-photon imaging of experience-dependent molecular changes in cortical neurons

大脑的能力来改变响应经验健康的大脑功能是必不可少的，并且在这个过程中的异常导致的脑功能障碍的各种1,2。为了更好地理解大脑回路的反应机制，动物的经验，需要能够监控的经验依赖的分子在一个给定的神经元的变化，在相当长的一段时间内，在活的动物。虽然经验和相关联的神经活动被称为触发1,2在神经元中的基因表达的变化，大多数的方法来检测这样的变化不允许重复观察多天相同的神经元的或不具有足够的分辨率来观察单个神经元的3 ，4。在这里，我们描述了在体内双光子显微镜相结合的方法，经验依赖的基因表达的改变在个别大脑皮质神经元的基因编码的荧光记者跟踪<html>当然，每天的日常经验。 一个行之有效的经验依赖的基因活动调节细胞骨架相关蛋白（ARC）5,6。转录的弧是迅速和高度诱导的神经元的活动加剧3，其蛋白产物调控谷氨酸受体的内吞作用和长时程突触可塑性7。电弧中的表达已被广泛使用作为分子标记映射涉及在特定的行为3的神经回路。在这些研究中，电弧的表达在固定的脑组织切片的原位杂交或免疫组化检测。虽然这些方法发现的表达定位于弧后行为的经验，如何弧的表达模式可能会改变的细胞反复多次发作或独特的经历，天兴奋性神经元的一个子集。 ntent“ 在体内双光子显微镜提供了一个强大的方法来考察经验依赖的细胞变化的活脑8,9。为了使双光子显微镜检查弧在现场神经元中的表达，我们以前产生了连锁反应鼠标线中，在其中的GFP记者弧的内源性启动子10的控制下被放置。此协议描述了外科手术准备和跟踪的经验依赖性电弧GFP表达模式在神经元中的活的动物的合奏的成像程序，在该方法中，慢性头颅窗弧-GFP小鼠的皮质区植入这些动物，然后反复后几天的过程中所需的行为范式的双光子显微镜拍摄，这种方法一般适用于动物携带其他的荧光记者的经验依赖的分子变化4。

<html>该协议描述了一个方法来跟踪个人皮层神经元在活的动物的经验依赖的分子变化。一种慢性颅窗口第一次创建了一个带有荧光的记者在小鼠基因表达的兴趣皮层区域。双光子显微镜然后可以加上各种行为范式来观察行为诱导的分子变化，个别神经元和跟踪多天（ 图1）在相同的一组神经元中的这种变化。 弧-GFP小鼠，可以可靠地成像依赖于经验在Arc基因的表达...

Watch this Scientific Journal Video about In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons at JoVE.com

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

经验依赖的分子在神经元的变化是大脑的能力相适应，行为问题的关键。一个<em在体内</em&gt;双光子成像方法的描述，在这里，可以追踪个人皮层神经元的分子变化，通过基因编码的记者。

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 ...

in-vivo-two-photon-imaging-experience-dependent-molecular-changes

Research

JoVE Journal

Neuroscience

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

21.5K 次观看.  National Institute of Mental Health. 经验依赖的分子在神经元的变化是大脑的能力相适应，行为问题的关键。一个

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

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

一个慢性双光子薄颅骨窗口技术在体内 Neuroinflammation模型小鼠小胶质细胞成像

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

科研

神经科学

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

在皮肤中的单神经末梢体内双光子显微镜

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

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 ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

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.

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 ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

大鼠皮层神经元丝状肌动蛋白（F-肌动蛋白）泪点的量化

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

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 ...