0920060 NSF资助，NIH资助DC004395 NHMRC赠款1009482，科学和医学研究的新南威尔士州办事处，加内特过时和罗德尼·威廉斯纪念基金会的支持。

1。头部约束概述<ol><li>组装一个定制的头后 ，插入1/16"不锈钢辊针直刺入钻了一个洞在3/32"不锈钢杆形成一个十字架。头后的垂直一块应该是约20毫米和跨一块约15毫米的水平。点击垂直杆的一端接受＃1-72螺钉。 （ 图1）。 </li><li>在手术过程中及录音，头后固定在一个定制的黄铜或铝安装酒吧 ＃1-72螺丝（ 图2）。安装杆的长片，应该是约90毫米长和宽5-10毫米。适合对开长条，角度为45度15毫米扩展成槽切头后。为了防止头部转动，头部后紧贴横梁上安装杆底部的小槽。 </li><li>越来越多的酒吧，然后连接到一个特制的铝安装集团K（ 图3）。安装块大约为30毫米×30毫米×25毫米。这种安装块附着到微操作机器人，使头后的位置，可以准确地沿中线放置于前囟门。两件一起安装用螺丝安装块。一个块中切出允许安装杆滑动，并用螺钉固定。安装酒吧台面平行。在录制过程中，争取在一个定制的立体框架（ 图4）酒吧。设计stereotax是安装在相同的位置在手术过程中在安装块吧。这样的安排，确保鼠标将始终对准跨越实验会话立体框架。 </li><li>钨棒（直径0.010"），以削减约5毫米的长度，约1毫米弯曲45°。这杆为接地脚（ 图1）。 </li></ol> 2。立体铝ignment为开颅手术 注意:下面列出的所有程序按照标准的无菌手术技术已经由华盛顿州立大学动物护理和使用委员会和加尔文研究所动物伦理委员会批准。 <ol><li>在感应室内放置5％异氟醚麻醉的小鼠。尾巴和/或脚趾掐反应缺乏确保该动物是完全麻醉。 </li><li>将鼠标鼠标适配器（Stoelting）的装备在啮齿类动物立体框架。鼠标的方向正确，将里面的牙齿咬栏孔，并拧紧，所以它是紧贴鼻夹。将鼠标作为直咬酒吧和鼻钳可能。将制成的乳胶手套，一块鼠标的鼻子，口罩，并保持在5％的异氟醚，直到鼠标的呼吸率约为1呼吸/秒。关闭异氟醚1.5 - 2.0％。固定头，采用t他耳酒吧小心不要刺破耳膜。酒吧应该是贴身但不渗入耳道太深。 </li><li>鼠标下方放置一个加热垫。 </li><li>申请眼药膏，以防止眼睛干燥。 </li><li>剃的头皮之间的耳朵小剃须刀或细点的剪刀眼睛。 </li><li>清洗和消毒氯碱hexidine（或优碘），然后用酒精擦洗冲洗3次重复擦拭头皮。 </li><li>来打开颅骨顶部的皮肤做一个切口，沿中线从后脑勺眼睛。 </li><li>用手术刀刮去骨膜切口边缘。 （根据实验目标）如果需要的话，仔细地收回镊子颈部背面的肌肉，使肌肉沿束撕裂，以减少出血。适用于头皮组织下面的明胶海绵，以减少水分，防止皮肤从头骨。 </li><li>干颅骨表面用棉签蘸异丙醇，以提高可视化颅骨地标。 </li><li>将头骨标准立体坐标13。罚款点探针插入电极支架和重视它的立体定向显微操作。一个尖锐的金属棒工作以及为此目的（ 图5）。 </li><li>使用点，以确定头骨上的囟门（ 图5A）和Lambda（ 图5B）。这些点之间来回移动探头，检查每个位置的内侧外侧不超过50微米的坐标不同。需要仔细松动耳酒吧，横向转移的头部和耳朵酒吧，并重新拧紧耳酒吧定位头部。 </li><li>确认在每个位置的背腹的高度不超过50微米的不同。调整鼻钳的高度，这将旋转轴耳酒吧周围的头骨，平的头骨，如果有必要的间距。耳杆的高度，可能还需要进行调整。 </li><li>使用地图集13日确定利益的目标（ 图5C）相对囟坐标。上述目标的头骨（例如，一个"X"标记，或用墨汁点四方形的角落）将开颅手术（ 图5D）。 </li></ol> 3。头后和接地引脚安装<ol><li>使用蚀刻凝胶清洁头骨。使用撒施擦洗和遍布表面蚀刻胶（10秒）。用清水洗净，完全晾干。适用底漆牙科水泥颅骨表面采用撒施。传播新喷头的粘合剂。固化胶UV光枪（1周期）。 </li><li>选择一个远端目标站点的接地引脚的位置。孔在地面p的短端使用一个＃65 miniblade手术刀的一角，也足够宽头骨仔细一小孔在进入和休息后，脑膜。接地引脚应为导向，以便它指向前囟门和未来的头后（ 图6A）。 </li><li>头后固定安装的酒吧和封锁和附加的微操作机器人。移动的微操作机器人在三个层面上，定位在头后，前囟门刚刚接触的头骨（ 图6B）。基地的头后应该趴在颅骨平。 </li><li>使用解剖探头，适用于头后和接地引脚（ 图6C），周围有少量的牙科水泥。按水泥，以确保良好的接触与头骨。注意:不要使用过多的水泥或不会变硬均匀。防治紫外光枪（3-4周期从多个角度）的水泥。 </li><li>适用于水泥的第二阶段和周围的头部后建立的基础和覆盖的头后的沟槽（ 图6D）。 </li></ol> 4。开颅手术<ol><li>找到了开颅手术的参考标志，这将部分底漆掩盖。使用65＃手术刀，以纪念3毫米×3平方毫米围绕这个站点和应用利多卡因下降，降低灵敏度，以减少出血。 </li><li>使每个广场的一侧（多达10个）多得分，小心不要通过骨骼和削减到大脑（ 图7A）。 </li><li>一旦骨瓣松动，使用手术刀的一角撬起来，离开硬脑膜完好（ 图7B）。 </li><li>用骨蜡覆盖。 </li></ol> 5。手术后<ol><li>关闭气体麻醉和从鼠标的立体框架。 </li><li>用棉签申请周围裸露的皮肤利多卡因软膏，以缓和伤口周围的轰动。使用另一种棉签应用抗生素软膏裸露的皮肤。注入酮洛芬（5毫克/公斤的IM或IP基于个人IACUC兽医偏好的）手术后镇痛。鼠标一般会和天然气停止分钟内移动。 </li><li>将鼠标在一个温暖的笼子和显示器直到它准备返回到动物的住房。 </li><li>动物应每天检查至少一次后，它已经从手术中恢复。观看不良的饮食和/或饮酒和无精打采行为的迹象。如果怀疑疼痛，直到缓解酮洛芬每24小时（在手术后相同剂量）。利多卡因局部可应用的伤口，如果动物被刮伤或显示不适的迹象。动物一般恢复手术，无并发症或疼痛。 </li></ol> 6。录音和染料注射液<ol><li>等待后，头部手术后至少一天前使用鼠标录音。 </li><li>塑造地方在泡沫束缚设备的鼠标鼠标的身体。鼠标已经被改编到该设备，从而减少其焦虑。典型的适应断奶后大约一个星期前，手术和实验小鼠每天处理，放置在发泡设备鼠标，时间为1-5分钟。暂停在立体框架（ 图4）的设备。 </li><li>安全的头后安装吧，这是现在固定的立体框架（ 图4）。 </li><li>用生硬的22号注射器针头尖端终止线连接到接地引脚。 </li><li>在开颅取出骨蜡。取出硬脑膜如果有必要使用一个miniblade手术刀尖端。 </li><li>驱动电极，以适当的立体位置，并开始录制（ 图8）。只有电生理记录，各种电极都可以使用。电极的选择是需要录音的位置和类型的依​​赖。清醒鼠标录音电极的选择将是什么研究员在麻醉鼠标使用相同。对于合并的电生理记录染料/示踪剂注射，微量电极使用10。微量充满染料/示踪剂的选择，以便它可以被微电泳注入的电生理实验结束。电极也可用于多管，这样可以药理操纵。多管电极安装在一个单一的录音微量（ 图8）。这些技术标准文献和麻醉与清醒的录音是不是不同。 </li><li>录音可为4-5个小时，连续超过3-4天。 </li><li>在录制会议结束时，染料/示踪剂可以注射。因为有没有在大脑疼痛受体，离子导入或进入大脑的染料示踪注射是不可能造成的不适。然而，一次注射是该地区的电反应特性的特点，动物，可能是麻醉异氟醚f或注射。这将消除任何潜在的不必要的感觉。因为染料/示踪已经在微管，可以主动和地线接通的电生理记录设置的电流发生器，微电泳注入染料/示踪。使用适当的协议，在电极的染料/示踪。删除从headpost克制和身体克制的动物，并返回到其家乡笼。 </li><li>在实验结束后，按照适当的协议安乐死和组织处理，以恢复注射部位和标签。 </li></ol> 7。代表结果成功安装的头后，允许实验者记录单，多单元的响应，从麻醉的，清醒的鼠标多天。约束系统可以稳定在大脑中单个神经元的电生理记录。极好的隔离单的单位和强烈反响ţØ刺激，可记录多天从相同的大脑结构，例如在小鼠下丘（ 图9A，日图9B，一天两）。带了一个好头安装后，将始终对准每个鼠标的立体定位仪和鼠标立体定向图谱，13，特别是脑细胞的可靠定位。这个可靠的定位，使染料和示踪剂注入后多个录音天的具体结构。例如，评估降预测从听觉中脑听觉脑干，生物素葡聚糖胺（可视作为chromagen使用的二氨基联苯胺）后，在注射部位的神经元的反应特性电生理识别，可以注射到小鼠下丘（ 图10A） （ 图10A，一个神经元与最佳频率为51千赫），组织处理后，对侧耳蜗核背侧nterograde标签可以被可视化和策划（ 图10B）。显微照片也可以得到更高的分辨率，查看顺标轴突和终端（ 图10C）。 <img alt="图1" src="/files/ftp_upload/3755/3755fig1.jpg" /> 图1。 <img alt="图2" src="/files/ftp_upload/3755/3755fig2.jpg" /> 图2。 <img alt="图3" src="/files/ftp_upload/3755/3755fig3.jpg" /> 图3。 <img alt="图4" src="/files/ftp_upload/3755/3755fig4.jpg" /> 图4。 <img alt="图5" src="/files/ftp_upload/3755/3755fig5.jpg" /> 图5。 <img alt="图6" src="/files/ftp_upload/3755/3755fig6.jpg" /> <s阮富仲&gt;图6。 <img alt="图7" src="/files/ftp_upload/3755/3755fig7.jpg" /> 图7。 <img alt="图8" src="/files/ftp_upload/3755/3755fig8.jpg" /> 图8。 <img alt="图9" src="/files/ftp_upload/3755/3755fig9.jpg" /> 图9。 <img alt="图10" src="/files/ftp_upload/3755/3755fig10.jpg" /> 图10。

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我们什么都没有透露。

后头部约束系统的电生理及神经解剖实验小鼠的优势是可以进行麻醉的，清醒小鼠实验，消除潜在的响应由于麻醉药品的污染。此外，一套最多可以使用多天，以便更有效地使用动物。 头后的大部分组件是可用的现成的。只有头部后和安装设备的定制，这两者是相对简单的建设，是可以重复使用。如果一台机器的店是不是在一个机构，研究者可能使用<a href="http://www.emachineshop...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>牙科水泥前清洗头骨的蚀刻凝胶 </td><td>亨利施恩</td><td> 101-5396 </td><td> 12/pk用1.2毫升注射器</td></tr><tr><td>牙科水泥底漆 </td><td>克尔Optibond </td><td> 25881 </td><td> 8毫升瓶装</td></tr><tr><td>牙科水泥胶粘剂 </td><td>克尔Optibond </td><td> 25882 </td><td> 8毫升瓶装</td></tr><tr><td> 喷头的牙科水泥</td><td>克尔Optibond </td><td> 24680 </td><td> 200/pk </td></tr><tr><td> 牙科水泥 </td><td>魅力，贺利氏古莎</td><td> 66000085 </td><td> 4gm注射器笔芯</td></tr><tr><td>钨棒（ 接地引脚 ） </td><td>上午系统</TD&gt; <td> 717200 </td><td> 0.010"直径</td></tr><tr><td>经济紫外线光固化 </td><td>亨利施恩</td><td> CU-80 </td><td></td></tr><tr><td> 头后 </td><td>内置的内部</td><td></td><td></td></tr><tr><td> 安装酒吧 </td><td>内置的内部</td><td></td><td></td></tr><tr><td> 固定块 </td><td>内置的内部</td><td></td><td></td></tr><tr><td> 立体框架 </td><td>大卫Kopf仪器</td><td> 902 </td><td></td></tr><tr><td> 鼠标和新生大鼠适配器 </td><td> Stoelting </td><td> 51625 </td><td></td></tr><tr><td> Deltaphase恒温垫 </td><td>布伦特里科学公司</td><td> 39DP </td><td></td></tr></tbody></table>

preparation of an awake mouse for recording neural responses and injecting tracers

这是众所周知的麻醉，神经反应特性改变在大脑的不同区域。13。在听觉系统，脑干神经元，包括阈值，频率特异性，抑制边带根本性的响应特性，在麻醉下1-2显着的方式被改变。这些观察提示生理学家设法从单个神经元的记录，没有麻醉的污染影响。其结果之一是去大脑的准备，完全是在中脑4水平横断脑干。该制剂的弊端，是一项艰巨的手术，消除降的预测从脑，无法用感官刺激，检查以上的脑结构。不同的策略已植入电极阵列，长期记录单个神经元和多单元集群，而动物是清醒和/或行为5,6 </suP&gt;。然而，这些技术与示踪染料注射后，电生理特征的大脑结构不兼容。为了避免改变与麻醉药的神经反应性能，同时记录单个神经元的电生理反应特性，我们已经适应了长期用鼠标10-12蝙蝠7-9头部约束技术。使用这种方法，我们能够在麻醉的小鼠进行了几天的电生理记录。我们可以在录音结束，然后注入一种染料，重建电极位置和记录点，或注入示踪剂，所以可确定，并从记录位点的途径。这种方法无需使用麻醉剂以及隔离多天的单神经元记录。

Watch this Scientific Journal Video about Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers at JoVE.com

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

重要的是理解大脑功能和指导安置途径示踪染料神经反应的电特性。然而，许多研究都在麻醉动物。理解没有麻醉药的脑功能，我们开发了一种方法来记录神经元的反应特性，并在清醒小鼠注入染料。

记录神经反应和注射示踪剂一个清醒小鼠的制备

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

preparation-an-awake-mouse-for-recording-neural-responses-injecting

Research

JoVE Journal

Neuroscience

19.3K 次观看.  Johns Hopkins University. 重要的是理解大脑功能和指导安置途径示踪染料神经反应的电特性。然而，许多研究都在麻醉动物。理解没有麻醉药的脑功能，我们开发了一种方法来记录神经元的反应特性，并在清醒小鼠注入染料。

视频: Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

用于体内的无线双向接口记录和刺激自由行为大鼠的神经活动

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

科研

行为学

Polygraphic Recording Procedure for Measuring Sleep in Mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

印刷业记录程序在小鼠体内测量睡眠

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

神经科学

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

小鼠大脑皮层细胞组织的大尺度三维成像

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

免疫与感染

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 ...

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy (pCLE) of Hippocampal Capillaries in Awake Mice

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application to elucidate capillary blood flow effects during seizures, driven by mural cells. In vitro and in vivo cortical imaging have shown that capillary constrictions driven by pericytes can result from functional local neural activity, as well as from drug application, in healthy animals. Here, a protocol is presented on how to use pCLE to determine the role of microvascular dynamics in neural degeneration in epilepsy, at any tissue depth (specifically in the hippocampus). We describe a head restraint technique that has been adapted to record pCLE in awake animals, to address potential side-effects of anesthetics on neural activity. Using these methods, electrophysiological and imaging recordings can be conducted over several hours in deep neural structures of the brain.

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy pCLE of Hippocampal Capillaries in Awake Mice

Whereas multiphoton imaging is only effective at limited depths from the tissue&#8217;s surface, it is possible to achieve 3 &#181;m resolution imaging at any depth via pCLE. Here, we present a protocol to conduct pCLE imaging to measure microvascular dynamics in the hippocampus of ictal and wild-type mice.

清醒小鼠海马毛细血管的体内纤维耦合临床前共聚焦激光扫描内窥镜 （pCLE）

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application ...

<meta charset="utf8"></head><body>小鼠深部脑干结构的倾斜硅多电极记录

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many brain regions. However, recording neuronal activity from deep caudal structures like the brainstem using multi-channel probes remains challenging. A significant concern is finding a trajectory for probe insertion that avoids large blood vessels, such as the superior sagittal venous sinus and the transverse venous sinus. Injuring these large veins can cause extensive bleeding, damage to the underlying brain tissue, and potentially death. This approach describes targeting brainstem structures by coupling anterior coordinates with an angled approach, allowing the recording probe to penetrate the brain below high-risk vascular structures. Compared to a strictly vertical approach, the angled approach maximizes the number of brain regions that can be targeted. Using this strategy, the ventrolateral periaqueductal gray (vlPAG), a brainstem region associated with REM sleep, can be reproducibly and reliably accessed to obtain single-unit, multi-electrode recordings in head-fixed mice before and during sevoflurane anesthesia. The ability to record neuronal activity in the vlPAG and surrounding nuclei with high temporal resolution is a step forward in advancing the understanding of the relationship between REM sleep and anesthesia.

<meta charset="utf8"></head><body>作者聚焦：研究小鼠麻醉诱导的睡眠通路和神经元兴奋性

This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

来自头部固定小鼠脑干的急性单单位多电极记录

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many ...

记录神经反应和注射示踪剂一个清醒小鼠的制备

摘要

探索更多视频

此视频中的章节

用于体内的无线双向接口记录和刺激自由行为大鼠的神经活动

印刷业记录程序在小鼠体内测量睡眠

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

小鼠大脑皮层细胞组织的大尺度三维成像

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

清醒小鼠海马毛细血管的体内纤维耦合临床前共聚焦激光扫描内窥镜（pCLE）

来自头部固定小鼠脑干的急性单单位多电极记录

来自头部固定小鼠脑干的急性单单位多电极记录

来自头部固定小鼠脑干的急性单单位多电极记录

记录神经反应和注射示踪剂一个清醒小鼠的制备

摘要

探索更多视频

此视频中的章节

用于体内的无线双向接口记录和刺激自由行为大鼠的神经活动

印刷业记录程序在小鼠体内测量睡眠

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

小鼠大脑皮层细胞组织的大尺度三维成像

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

清醒小鼠海马毛细血管的体内纤维耦合临床前共聚焦激光扫描内窥镜 （pCLE）

来自头部固定小鼠脑干的急性单单位多电极记录

来自头部固定小鼠脑干的急性单单位多电极记录

来自头部固定小鼠脑干的急性单单位多电极记录

清醒小鼠海马毛细血管的体内纤维耦合临床前共聚焦激光扫描内窥镜（pCLE）