陈德良Huynh，安德鲁杰里林圣安东尼奥，我们感谢他们的技术援助。这项工作是由国家卫生赠款研究院DA023700和DA023700 - 04S1至第XX

1。脑片制备<ol><li>深深麻醉与戊巴比妥钠（&gt; 100毫克/千克，IP）的转基因小鼠，迅速断头，他们的大脑中提取到一个冷冻和含氧的裁剪解决方案。 </li><li>用于视觉画面，如果老鼠大脑确实表达绿色荧光蛋白GFP的护目镜。 </li><li> 400微米厚的初级躯体感觉皮层部分削减在vibratome蔗糖，含有人工脑脊液（ACSF）。片首先在培养蔗糖含学联在32℃，30分钟到1小时后最初的潜伏期，在室温下转移到录音学联。在整个孵化和录音，切片不断冒泡的95％O 2 -5％CO 2。 </li></ol> 2。设备启动整个系统实现如图1所示。 <ol><li>激光冷却系统和电源供应器打开，激光准备。 </li><li> photostimulation控制（包括扫描镜系统，光调制器，电子快门和一个光电二极管输入放大器）相关的所有硬件打开。 </li><li>电设备（包括一个Multiclamp 700B放大器和micromanipulators）开启。电生理记录，photostimulation，成像和切片准备做切片灌注室安装在显微镜的机动阶段。 </li><li>成像相机（Retiga 2000 Q -成像，美国德克萨斯州奥斯汀​​）打开。片红外微分干涉对比（DIC）和通过外延荧光光学成像系统的直立显微镜（BW51X奥林巴斯）的可视化。 </li><li>基于Matlab的EPHUS软件和Q - 捕捉摄像头软件启动。一个Ephus软件（Ephus，在提供定制修改后的版本<a href="https://openwiki.janelia.org/">https://openwiki.janelia.org/</a> ）是用来控制photostimulation协议，并获得photostimulation数据。 </li><li>系统后，应检查看看激光photostimulation系统的工作状态。由于4倍的显微镜下的目标是提供谷氨酸uncaging UV闪烁，一张白纸可以把下观察激光扫描模式，而系统的运行。 </li></ol> 3。设置切片灌注<ol><li>打开一个加压灌注系统送入切片室录音学联。护理是采取措施，确保恒定的2.0毫米的流体在会议厅内片以上的水平。 </li><li> MNI的笼谷氨酸原液等分添加到25毫升循环学联为0.2毫米笼谷氨酸浓度。请注意，将被刷新后5-6 h的试验，沐浴液和MNI的谷氨酸​​。 </li><li>脑片移入录音室。一个可以运行的成像系统检查切片质量和解剖学定位初级躯体感觉区。然后切片是挂靠一个特制的弦乐器铂金戒指。注意不要把打算录音的大脑区域的锚。 </li></ol> 4。全细胞膜片钳记录<ol><li>玻璃电极（4-6MΩ的电阻）被拉到和充满内部含有0.1％的生物胞素细胞标记和形态学鉴定的解决方案。 </li><li>要执行修补程序记录，细胞在60X目标可视化。首次发现抑制细胞类型，并选定一个DIC /奥林巴斯荧光显微镜下基于可视化表达GFP;随后通过红外DIC视频监控辅助视觉控制下进行录音。请注意，DIC和荧光模式之间切换几次要确认GFP细胞的位置，而接近电极的靶细胞。 </li><li>传统的膜片钳技术的应用。电极是充满积极的压力，移动到细胞表面附近建立后，有针对性的细胞膜明显的酒窝;然后负压迅速应用到形成一个gigaseal和突破，同时在视频监视器上的电流注入反应监测。 </li><li>打破后，在DIC和荧光模式下的60X的细胞图像上线验证。收集的photostimulation数据之前，超极化和去极化电流脉冲注入到每个细胞的基本电生理特性的研究。 </li><li>一旦稳定的全细胞记录，取得了良好的接入电阻（通常&lt;20MΩ），显微镜下的目标是从60X切换到激光扫描photostimulation 4倍。在4X的切片图像的收购，将用于指导和登记photostimulation网站。 </li></ol> 5。激光SCAnning photostimulation <ol><li> photostimulation和数据采集参数设置激活Ephus配置交换机模块。通常情况下，1毫秒的激光与刺激间隔为1秒（20兆瓦）是用于photostimulation映射。在10 kHz采样1秒的数据跟踪收购。 </li><li> Ephus映射器模块加载到4X切片图像。是指细胞胞体的位置，和一个16x16的模式（80微米× 80微米间距）photostimulation网站设立周围的细胞位置，涵盖所有的皮质层。 </li><li>研究应对photostimulation扣球的位置，在电流钳模式记录神经元的激发轮廓映射。我们的用户友好的软件上线的显示功能，方便的映射实验。 </li><li>本地兴奋电路连接映射检测记录细胞的兴奋性突触后电流（EPSC），同时在不同地点的激光扫描。细胞是举行-70毫伏电压钳模式检测外来的兴奋性电流。 EPSC地图photostimulation模式旋转重复2-3次，或翻转。 </li><li>可选：本地抑制电路连接，也可以映射在接近0毫伏电压钳模式记录细胞检测离港抑制电流的抑制性突触后电流（IPSC）的检测。请注意，这是最好使用的IPSC映射铯取代钾电极内部的解决方案。 </li><li>毕竟生理检测完成后，电极轻轻地从录制的细胞。脑片取出，并在4％多聚甲醛固定过夜。对生物胞素染色细胞。焦或落射荧光显微镜，这也印证了每个记录的细胞的确是原本有针对性的表达GFP的中间神经细胞形态研究。 </li></ol> 6。 Photostimulation数据分析我们新开发的方法和软件实施 11适用于在原始数据的地图photostimulation诱发的突触事件的检测和测量。例如图2，彩色编码图的构造说明记录神经元的突触输入的模式。 7。代表性的成果： 电生理记录和photostimulation映射的示例结果如图2所示。具体抑制细胞类型的有针对性的录音成为可能使用在已知的抑制细胞类型表达绿色荧光蛋白的转基因小鼠。由于抑制interneurons的多样性，GFP的表达，内在的电生理和形态特征（图2A）分析相结合，得出最终的细胞类型分类。图2B - C的说明，激光扫描photostimulation允许局部皮层电路连接到单一的抑制性神经元的广泛映射。原始数据图显示在图2C。直接的uncaging反应，从数据分析（图2C）被排除在外。定量输入地图颜色编码，在图2D - F所示。例如快速扣球抑制interneurons收到强烈的兴奋性突触输入（EPSCs），从第4层和深层。有关细胞的突触输入组织定义的皮层通路，我们可以推断其可能发挥的作用（例如，反馈和前馈抑制）在皮层信息处理。 <img alt="图1" src="/files/ftp_upload/3109/3109fig1.jpg" /> 图1。激光扫描photostimulation一般系统工具 ，我们的整个系统的组成photostimulation控制，视频影像，电生理记录系统。我们采用的LSP系统的设计前面描述 7,17 。一个激光装置是用来产生355纳米紫外激光谷氨酸uncaging。激光束直接通过我们的系统的光路。持续时间短的激光闪烁（例如，1 - 3 MS）控制使用的电光调制器（即，普克尔斯盒）和机械快门。激光束的功率是由中立的密度梯度轮调制和挪用玻璃盖玻片一个光电二极管的激光束的一小部分的监测。激光扫描系统包括一个对扫描镜，扫描镜头，管镜头，物镜的XY。镜提供的激光束通过扫描镜头，然后光束通过分色镜进入显微镜，重点是由一个特制的紫外线发射管镜头。梁底部填充回的显微镜物镜孔径提供更柱状（而不是锥形）照明光束，二维尽可能减少轴向分辨率的映射。 4倍的目标下，激光束形成uncaging点，每个approximati吴高斯分布与横向焦平面（插入）153微米的宽度。各种激光刺激的位置，可以实现通过检流驱动的XY扫描镜，镜子和回光圈的客观共轭平面镜职位翻译成不同的扫描物镜焦平面的位置。 uncaging期间，一个图案的网站，涵盖了整场的数目可变顺序在一个nonraster刺激，非随机的顺序，以避免重新最近刺激的地点附近。更详细的信息，请参阅徐等人 （2010） 。 <img alt="图2" src="/files/ftp_upload/3109/3109fig2.jpg" /> 图2。本地兴奋电路连接映射的GFP表达在小鼠初级感觉皮层第4层的快速扣球中间神经。A1和A2显示下60X目标针对性的细胞活脑切片的DIC和GFP荧光图像，而A3显示揭示生物胞素染色后的记录，固定片的细胞形态。 A4纸显示记录的GFP细胞，快速扣球抑制性的特点，intrasomatic目前注射不同的优势，射击模式。 B显示的切片图像叠加16 X16 photostimulation的网站（*）。单元格的位置是由红色的小圆圈表示。 C显示了一个数组photostimulation诱发B所示的刺激位置的反应痕迹，而细胞在-70 mV的检测外来的兴奋性电流。 photostimulation反应的不同形式说明1和2的位置，这是扩大和单独显示在插入的痕迹。跟踪1是一个直接反应（红色箭头表示），以谷氨酸的胞体和promximal树突上uncaging的例子。 2其他的痕迹，是典型的兴奋性突触输入的响应（蓝色）例子。直接反应和突触输入，可以区分其振幅和响应延迟。 D，E和F的平均EPSC幅值的彩色编码图（16x16的网站），EPSC的号码，并首次发现每个站点EPSC延迟，分别为平均每输入幅度在C所示的原始数据映射刺激的部位是在分析窗口的平均EPSCs幅度，从同一站点photostimulation响应中减去基线自发响应。 EPSCs和到达时间或延迟首次发现每个站点EPSC的数量也测量和绘制。有关详细信息，请参阅（石等，2010）。

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没有利益冲突的声明。

Photostimulation基础测绘技术已有效地应用于分析皮质电路。激光扫描photostimulation全细胞记录相结合，使高分辨率的映射到单个神经元突触前输入源层分布的，因为从突触后神经元的突触前神经元在许多不同的地点的集群photostimulation同步录音提供兴奋性的空间分布的定量措施或抑制性输入。使用在已知的抑制细胞类型表达绿色荧光蛋白的转基因小鼠资助这种技术大大促进了我们目前的工作，对阐明...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>转基因小鼠线</td><td>杰克逊实验室或其他来源</td><td></td><td>请参阅徐和Callaway（2009） </td></tr><tr><td> GFP的护目镜</td><td> BLS的有限公司，匈牙利</td><td></td><td></td></tr><tr><td> vibratome </td><td>徕卡系统</td><td> VT1200S </td><td></td></tr><tr><td> MNI的笼中的谷氨酸（4 - 甲氧基- 7 - nitroindolinyl笼L -谷氨酸） </td><td> Tocris生物科学，Ellisville，莫</td><td>猫号1490 </td><td></td></tr><tr><td>生物胞素</td><td></td><td> B4261 </td><td></td></tr><tr><td>电极拉马</td><td>萨特仪器，诺瓦托，CA </td><td> P - 97 </td><td></td></tr><tr><td>使电极的玻璃试管</td><td></td><td> BF150 - 86 - 10 </td><td></td></tr><tr><td> Multiclamp 700B放大器</td><td>分子器件，加利福尼亚州桑尼维尔， </td><td> Multiclamp 700B </td><td></td></tr><tr><td>数字CCD相机</td><td> Q -成像，美国德克萨斯州奥斯汀</td><td> Retiga 2000 </td><td></td></tr><tr><td>研究显微镜</td><td>奥林巴斯，东京，日本</td><td> BW51X </td><td></td></tr><tr><td>紫外激光装置</td><td> DPSS激光器，加利福尼亚州圣克拉拉</td><td>模型3501 </td><td></td></tr><tr><td>其他设备激光扫描phostimulation </td><td></td><td></td><td>请参考Xu 等 （2010） </td></tr></tbody></table>解决方案： <ul><li>含蔗糖片切割的人工脑脊液（ACSF）（单位：mm：85氯化钠，75蔗糖，2.5氯化钾，25葡萄糖，1.25的NaH 2 PO 4，4，0.5 MgCl 2的氯化钙2，和24 碳酸氢钠 3） 。 </li><li>拍摄学联（单位：mm：126氯化钠，2.5氯化钾，26碳酸氢钠3，2 2 2氯化钙，氯化镁，1.25的NaH 2 PO 4和10葡萄糖） </li><li>电极内部解决方案（单位：mm：126的K -葡萄糖，4氯化钾，10 HEPES，4个ATP镁，0.3 GTP娜和10磷酸，pH值7.2，300 mOsm）。 </li></ul>

mapping inhibitory neuronal circuits by laser scanning photostimulation

抑制性神经元皮质功能的关键。他们皮层神经元的整个人口约占20％，还可以进一步细分到他们的免疫组化，形态和生理特性1-4为基础的多元化亚型。虽然以前的研究发现，个别类型的抑制性神经元的内在属性，对他们的局部电路连接知识还比较有限3,5,6。由于每一个人的神经元的功能是通过其兴奋性和抑制性突触内皮层电路输入形，我们已经使用激光扫描photostimulation者（LSP）地图的本地电路连接到特定的抑制细胞类型。 LSP的与传统的电刺激或谷氨酸粉扑刺激相比，具有独特的优势，允许广泛的测绘和定量分析的功能单独记录的神经元3,7-9当地投入。激光通过谷氨酸uncaging photostimulation选择性激活的神经元perisomatically，不激活的情况下通过轴突或远端树突，以确保子层的映射决议。非常适合皮质电路分析的灵敏度和效率，从在一个大区域的许多刺激站点的映射输入的LSP。 在这里，我们引进的技术，并结合本地抑制电路映射，全细胞膜片夹紧的LSP。有针对性的具体的抑制细胞类型的录音方便使用在有限的抑制神经元群表达绿色荧光蛋白（GFP）的转基因小鼠皮层3,10，使记录的细胞类型一致的抽样针对性的细胞类型，并明确识别。由于LSP的映射，我们概述系统仪器，描述实验过程和数据采集，和鼠标初级躯体感觉皮层的电路映射的例子。在我们的实验显示，笼谷氨酸在脑片的紫外激光光解空间限制的区域被激活;同时电压钳记录允许photostimulation诱发的突触反应的检测。无论是兴奋性或抑制性突触输入有针对性的神经元的生成地图，通过扫描激光束激发数百个潜在的突触前网站。因此，选择LSP可以经过反复实验撞击到特定类型的抑制性神经元的突触输入的详细地图的建设。两者合计，photostimulation为基础的技术提供神经学家确定的局部皮层电路功能组织一个强大的工具。

Watch this Scientific Journal Video about Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation at JoVE.com

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

本文介绍了激光扫描photostimulation在表达绿色荧光蛋白在有限的抑制性神经元的人口的转基因小鼠的全细胞记录相结合的方法。该技术可以广泛的测绘和定量分析当地具体的抑制皮层神经元的突触电路。

测绘激光扫描Photostimulation抑制神经回路

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into ...

mapping-inhibitory-neuronal-circuits-laser-scanning

Research

JoVE Journal

Neuroscience

17.2K 次观看.  University of California, Irvine. 本文介绍了激光扫描photostimulation在表达绿色荧光蛋白在有限的抑制性神经元的人口的转基因小鼠的全细胞记录相结合的方法。该技术可以广泛的测绘和定量分析当地具体的抑制皮层神经元的突触电路。

视频: Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

Optogenetically有针对性的前脑电路的激光扫描光刺激

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

科研

神经科学

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 ...

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

设计，表面处理，电镀细胞和神经元的模块化网络功能组成跨接电路的培养

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

激光制导神经跟踪在脑外植体

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

重建神经元电路：快速神经延伸和功能性神经元连接的新方法

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

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 ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

体外楔形切片准备模拟在Vivvo神经元电路连接

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.