由国防部（国防高级研究计划局，青年教师奖，项目编号N66001-09-1-2117）和NINDS（R15NS070734）的研究。

1。动物的外科下游 1。实验的主题使用小鼠的Tg（Thy1-COP4/eYFP）的的B6.Cg 18Gfng / J转线9; JAX应变＃7612）或其他小鼠表达的蓝色光敏感的阳离子通道，Channelrhodopsin-2，在大脑皮质的神经元。应用到大脑皮质的B6.Cg-TG（Thy1-COP4/eYFP）的18Gfng / J，转基因株系的蓝色光会导致锥体神经元去极化，并进行动作电位9,10表达Channelrhodopsin-2。作为一个结果，锥体细胞的活化，本地的interneurons被激活，和电势的变化传播到神经元刺激10以外的部位。执行手术遵守适用的监管指引：在无菌条件下的高压釜手术工具和softpacks（外科领域，纱布垫），热珠消毒所有热性植入的金属设备（套管，螺丝和电线）30秒，10秒浸在乙醇消毒热不耐受可植入设备（光纤电缆，塑料接头）应用的罐装空气干燥的打击乙醇浸项目。 2。麻醉，立体定位和颅骨的间隙使用IMPAC 6集成多病人系统（VET装备公司），麻醉鼠标。使用5％Isoflurane/95％氧诱导和3％Isoflurane/97％氧气维持在手术过程中。将动物设置为37℃，这是没有必要监测体温，如果在此温度下保持橡皮布上的循环水毯。 3。准备植入头骨准备切口部位剃所有毛皮从头骨的顶部。剃区域横向延伸，从耳朵到耳朵和前后从眼睛到后端的头骨。裸露的皮肤消毒有三个consecutivË拭子优碘，然后用乙醇。从两眼中间到后面的骷髅头骨的顶部，使内侧切口。与过氧化氢和无菌生理盐水（0.9％NaCl）中，清洁颅骨。控制出血烧灼工具。确定立体定向定位和标记前囟门和lambda。立体定向坐标的电极/ optogenetic刺激配置，我们已经使用的是在表1中给出，并在图1A中示意性地示出。 4。在头骨植入部位的制备使用高速牙钻0.5mm的球毛刺位来建立每个着床部位的头骨。每个使用了0.7毫米的球毛刺位，以方便插入螺丝孔倒角外部边缘。 5。插管插入和保护，肌电图（EMG）信息和EEG信息插入脑电图螺钉（0.8mm的长度，有2厘米的未涂覆30隔距镀锡铜总线线预焊接到旋拧头）插入孔中并驱动它们以一个开槽的手螺丝刀约4-5革命，以获得所需的深度。保持在一个立体的导管架，然后贴上他们的头骨和美女主播与丙烯酸水泥的引导插管。每个导管应包含一个虚拟的管/管芯（塑料件，部件编号：C312 DC \ 1）从实验的时间（10-14天），手术时保持通畅。 6。关闭手术部位一旦脑电图和引导插管的位置在头骨，把它们绑定一起牙骨水泥（郎牙科 - 邻水泥旋喷）。水泥台后，塑料连接器（品尼高技术部件编号：8402）以上的干水泥土墩的位置。焊锡从EEG引线的塑料连接器上的接触所产生的电线的端部。包住导线连接水泥。然后，通过颈部肌肉的肌电图线画s的滑入桶的21ga针穿透肌肉。领带的双重外科医生的结5-0尼龙缝线周围这些电线远端，他们退出肌肉。缝合到一起的皮肤，后退到访问的肌肉组织与单一中断的外科医生节，使用反向切割P-3针和5-0尼龙线。 7。手术后的镇痛和恢复 管理在手术后立即丁丙诺啡作为镇痛药（0.1毫克/千克，皮下）和氟胺烟酸葡胺（0.1毫克/千克，皮下）作为一种抗炎，并且每天在手术后1-2天。让动物恢复标准的殖民地的住房条件，在试验前至少7天。超越标准的殖民地房屋没有额外的照顾，插管留置钢丝固定到位，预计将保持至少两个星期的专利。 EEG和EMG传感器也可保留这个时间的功能。 2。代光脉冲定时 1。 MC刺激机组编程刺激MC-单元（多通道系统）​​连接，通过USB连接到一台台式电脑。使用类似电子表格的用户界面，刺激MC-MC刺激单元进行编程。输入所需的TTL（晶体管 - 晶体管逻辑）上的期间的电压，刺激的持续时间和关闭期间，每秒的周期需要的话，并重复循环的总数为刺激整个会话的数目。如需进一步详细信息，请参阅MC刺激技术手册： 3。制造/装配的光纤PR灵魂出窍光传输到大脑皮层<ol><li>用品和设备，被发现在： <a href="http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf" target="_blank">http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf</a> </li><li>切割和抛光的光纤电缆。 <a href="http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf" target="_blank">http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf</a> </li></ol> 4。大会的的啮齿动物Optogenetic刺激系统 1。 MC刺激组和激光接口一旦被编程的MC-刺激单元，它作为一个独立的一个5伏的ON / OFF信号的二进制信号发生器运行。将刺激MC-TTL功能的动力单元与BNC连接器的激光装置。 2。产生激光的输出，通过光纤电缆使用的数字显示和可变的激光功率单元的输出功率拨盘手册光年调整激光的输出功率。通过带状电缆连接的激光功率单元的激光。将阴（FC连接器）套圈的激光的男性的FC连接器电缆（3米长，直径为200μm的玻璃传导纤维包裹在光反射的丙烯酸酯聚合物包层）上的光纤活动光纤适配器。 3。输送的动物：激光旋转整流子连接始发的光纤电缆（3米长）和交付的光纤电缆（45厘米长）包住两个准直透镜（多利安镜片）的光纤旋转接头的相对的两端。光纤旋转接头作为换向器：作为动物有关它在笼的移动，旋转接头旋转，以防止破损的光纤电缆。使用塑料扎带固定换向器的金属，站在上面放置的圆柱笼中的动物被安置。 4。附加交货光纤电缆总目抑制鼠标用一只手到脚的鼠标下一个杯状的手掌。东方实验者的中间和食指之间的头。移除虚设插管从导向套管放置为光纤套管。清除指南套管碎片使用无菌25GA针的。用手工插入的光纤电缆，并把它固定在带有螺纹的螺丝帽从一个虚拟套管打捞光纤导向套管。控制由缝线结绑在纤维上的固定距离处的平面切割的端部的光缆的光纤电缆，在大脑中的插入深度。在图1B中示出的整个实验装置。 5。测量的的EEG定义的睡眠和乳酸浓度在Optogenetic刺激 1。预校准的乳酸传感器 在体外进行的乳酸传感器的预校准试剂盒（品尼高技术部件编号：7000-K1-T）。该传感器是平衡的磷酸盐缓冲盐水（pH 7.4）中，然后在每个制造商的协议的逐步的方式暴露于三种不同浓度的L-乳酸。 2。插入的的乳酸传感器和EEG电缆将预校准的传感器安装头骨乳酸引导套管的光纤电缆插入程序（第4.4节）相同的方式。乳酸传感器连接到品尼高8400双极型生物传感器插头连接器的前置放大器。将前置放大器的手术植入头戴8针连接器上。 3。建立Optogenetic刺激强度在收集数据之前，使用激光强度控制旋钮的调整力度的optogenetic刺激。的脑电反应的幅度各不相同的动物，由于还没有系统地研究了因素，<html> d必须通过目测验证，在发病的刺激。在实践中，一个强度80-120μWatts，将典型地产生的频率刺激脑电图振幅的约50％的增加。事后快速傅里叶变换分析（参见5.5节）是必要的，以量化的绝对星等的响应。 4。数据采集收集数据使用品尼高8400系统： <a href="http://pinnaclet.com/pal-8400.html" target="_blank">http://pinnaclet.com/pal-8400.html</a> 。 5。数据处理与Neuroscore 分类睡眠状态目视检查脑电图，肌电图数据的Neuroscore接口（DataSciences，国际<a href="http://www.datasci.com/products/software/NeuroScore_CNS_Software.asp" target="_blank">http://www.datasci.com/products/software/NeuroScore_CNS_Software.asp</a> ）的或海牛接口的（品尼高技术：F =“http://www.pinnaclet.com/sirenia.html”目标=“_blank”&gt; http://www.pinnaclet.com/sirenia.html）。在10秒的时代唤醒，非快速眼动睡眠，快速眼动睡眠脑电图和肌电图的基础上的过程数据。保存为Mic​​rosoft Excel电子表格的数据进一步分析和统计检验。

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没有利益冲突的声明。

这里介绍的方法允许一个以前是不可能实现的时间尺度上测量大脑中的浓度之间的关系的睡眠和变化的糖酵解中间乳酸。动物自发唤醒，的NREMS和REMS之间的转换。此外，我们当动物接受这些转变可以申请optogenetic刺激。最新收集到的数据表明，自发和诱发波影响的乳酸氧化酶为基础的生物传感器的读出。 人们可以构建实验系统，类似于此处所述的设备和软件从其他来源。多导...

<table border="1"><tbody><tr><td> 元件 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>巴斯鼠标导管</td><td>品尼高技术/奥林巴斯公司</td><td> 7032 </td><td></td></tr><tr><td>乳酸生物传感器</td><td>品尼高技术</td><td> 7004 </td><td></td></tr><tr><td>头山</td><td>品尼高技术</td><td> 8402 </td><td></td></tr><tr><td>睡眠/生物传感器记录系统</td><td>品尼高技术</td><td> 8400-K1-SL </td><td> 2 EEG通道，1个EMG通道及1生物传感器</td></tr><tr><td>栓鼠标在体外校准套件</td><td>品尼高技术</td><td> 7000-K1-T </td><td></td></tr><tr><td>光纤导管</td><td>塑料件</td><td> C312G </td><td> 21号导管</td></tr><tr><td>虚拟套管</td><td>塑料件</td><td> C312DC </td><td> 21号假</td></tr><tr><td>纤维钻石抄写员</td><td> Thorlabs公司</td><td> S90W </td><td></td></tr><tr><td>光纤连接器压接工具</td><td> Thorlabs公司</td><td> CT042 </td><td></td></tr><tr><td>分叉管</td><td> Thorlabs公司</td><td> FT030 </td><td> 03.0毫米</td></tr><tr><td></td><td> Thorlabs公司</td><td> T10S13 </td><td>最大直径。 0.012 </td></tr><tr><td>分叉管汽提塔</td><td> Thorlabs公司</td><td> FTS3 </td><td></td></tr><tr><td>裸硬包层多模光纤</td><td> Thorlabs公司</td><td> BFL37-200 </td><td> 200微米的核心，0.37 NA </td></tr><tr><td>电线剪刀/芳纶剪</td><td> Thorlabs公司</td><td> T865 </td><td></td></tr><tr><td>光纤环氧树脂</td><td> Thorlabs公司</td><td> F112 </td><td></td></tr&gt; <tr><td>光纤剥线工具</td><td> Thorlabs公司</td><td></td><td></td></tr><tr><td>玻璃抛光板</td><td> Thorlabs公司</td><td> CTG913 </td><td></td></tr><tr><td>橡胶软磨片</td><td> Thorlabs公司</td><td> NRS913 </td><td></td></tr><tr><td>眼睛放大镜</td><td> Thorlabs公司</td><td> JEL10 </td><td></td></tr><tr><td>金湿巾</td><td> Thorlabs公司</td><td> KW32 </td><td></td></tr><tr><td>压缩空气</td><td> Thorlabs公司</td><td> CA3 </td><td></td></tr><tr><td>抛光普克</td><td> Thorlabs公司</td><td> D50-XX </td><td></td></tr><tr><td>纤维检验范围</td><td> Thorlabs公司</td><td> CL-200 </td><td></td></tr><tr><td>抛光电影</td><td> Thorlabs公司</td><td> LFG5P，LFG3P，LFG1P，LFG03P </td><td></td></tr><tr><td> FC / PC连接器端</td><td> Thorlabs公司</tD&gt; <td> 30126G2-240 </td><td> 240微米的孔，SS卡套</td></tr><tr><td> MC刺激单位</td><td>多通道系统</td><td> STG-4002 </td><td></td></tr><tr><td> MC刺激软件</td><td>多通道系统</td><td> MC刺激V 2.1.5 </td><td></td></tr><tr><td>蓝色激光</td><td> CrystaLaser </td><td> CL473-050-0 </td><td></td></tr><tr><td>激光电源</td><td> CrystaLaser </td><td> CL2005 </td><td></td></tr><tr><td>光纤旋转接头</td><td>多立克镜头</td><td> FRJ-V4 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表2。用品和设备。 </td></tr></tbody></table>

simultaneous electroencephalography, real-time measurement of lactate concentration and optogenetic manipulation of neuronal activity in the rodent cerebral cortex

尽管大脑表示小于5质量％的身体时，它利用身体所利用的葡萄糖在休息1的约四分之一。非快速眼动睡眠（NREMS），睡眠时间的最大部分的功能​​是不确定的。然而，一个显着的特点NREMS是一个显着的减少的速率相对于觉醒2-4脑葡萄糖利用率。这与其他研究结果已广泛持有的信念，睡眠提供相关的脑代谢的功能。然而，仍有待澄清NREMS期间脑葡萄糖代谢的降低机制。 与的NREMS可能影响脑代谢率相关联的一种现象是慢波，在小于4赫兹的频率的振荡的发生，在脑波5,6。这些慢波在颅骨或大脑皮质表面的水平反映了检测相关的神经元去极化/状态和超极化/下的状态7之间振荡。在向下状态，细胞不发生动作电位到几百毫秒的间隔。后续动 ​​作电位的离子浓度梯度的恢复是一个重要的代谢负荷对细胞8，动作电位与NREMS在掉电状态的情况下可能会有助于减少代谢相关唤醒的。 为了要测试这个假设的关系，必须解决两个技术挑战。首先，它是必要的测量脑糖酵解代谢与脑EEG的动态反射的时间分辨率（即，超过秒，而不是分钟）。要做到这一点，我们测得的乳酸浓度，有氧糖酵解的产物，因此，在老鼠的大脑中的葡萄糖代谢率的读数。乳酸是采用基于乳酸氧化酶的实时传感器嵌入的额叶皮层。感测机构包括由乳酸氧化酶分子的层包围的铂 - 铱电极。由乳酸氧化酶的乳酸代谢产生的过氧化氢，其产生的电流中的铂 - 铱电极。因此，一个加速脑糖酵解提供乳酸氧化酶，然后被反射在增加的电流在检测电极中的底物浓度的增加。这是另外需要这些变量，而操纵大脑皮质的兴奋性，以找出这个变量的NREMS从其他方面来衡量。 我们设计了一个实验系统的神经元活动的同时测量通过大脑皮质神经元的活动的elecetroencephalogram，通过乳酸生物传感器的糖酵解流量测量，操作通过optogenetic激活的金字塔midal神经元。我们已经利用本系统之间的睡眠相关的脑电图波形和在大脑皮质的乳酸浓度的时刻到时刻动态文件的关系。该协议可能是有用的任何个人感兴趣，学习，在自由活动的啮齿动物，在脑电图能量大脑内的水平和细胞之间的关系测定的神经元活性。

<html>正如在图2中所示， 经历了自发的睡眠/唤醒状态转换，同时鼠标配备optogenetic刺激和乳酸/ EEG / EMG数据收集的脑电图，肌电图和脑乳酸浓度连续监测。目前在乳酸传感器增加了在低振幅脑电图的时期，期间，高振幅EEG下降。正如在图3中所示，两个通道的脑电图响应于optogenetic刺激交付的额叶皮层。 <img alt="...

Watch this Scientific Journal Video about Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral C

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

一个过程的描述，操纵大脑皮质锥体神经元的活动optogenetically而脑电图，肌电图，脑乳酸浓度监测。进行实验记录，电缆绳系小鼠，而他们自发的睡眠/觉醒周期。 optogenetic设备被组装在我们的实验室;记录设备是市售的。

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

simultaneous-electroencephalography-real-time-measurement-lactate

Research

JoVE Journal

Neuroscience

20.1K 次观看.  Washington State University. 一个过程的描述，操纵大脑皮质锥体神经元的活动optogenetically而脑电图，肌电图，脑乳酸浓度监测。进行实验记录，电缆绳系小鼠，而他们自发的睡眠/觉醒周期。 optogenetic设备被组装在我们的实验室;记录设备是市售的。

视频: Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

同时在啮齿动物脑功能磁共振成像和电

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

科研

神经科学

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

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 ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic field pulses. The initiation of cortical activation or its modulation depends on the background activation of the neurons of the cortical region activated, the characteristics of the coil, its position and its orientation with respect to the head. TMS combined with simultaneous electrocephalography (EEG) and neuronavigation (nTMS-EEG) allows for the assessment of cortico-cortical excitability and connectivity in almost all cortical areas in a reproducible manner. This advance makes nTMS-EEG a powerful tool that can accurately assess brain dynamics and neurophysiology in test-retest paradigms that are required for clinical trials. Limitations of this method include artifacts that cover the initial brain reactivity to stimulation. Thus, the process of removing artifacts may also extract valuable information. Moreover, the optimal parameters for dorsolateral prefrontal (DLPFC) stimulation are not fully known and current protocols utilize variations from the motor cortex (M1) stimulation paradigms. However, evolving nTMS-EEG designs hope to address these issues. The protocol presented here introduces some standard practices for assessing neurophysiological functioning from stimulation to the DLPFC that can be applied in patients with treatment resistant psychiatric disorders that receive treatment such as transcranial direct current stimulation (tDCS), repetitive transcranial magnetic stimulation (rTMS), magnetic seizure therapy (MST) or electroconvulsive therapy (ECT).

The protocol presented here is for TMS-EEG studies utilizing intracortical excitability test-retest design paradigms. The intent of the protocol is to produce reliable and reproducible cortical excitability measures for assessing neurophysiological functioning related to therapeutic interventions in the treatment of neuropsychiatric diseases such as major depression.

侧前额叶皮质联合经颅磁刺激与脑电图的研究

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

多鼠脑的同时 Cryosectioning

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...