我们感谢兰迪Chitwood博士，批判性阅读的手稿。这项工作是支持的白厅基金会和斯隆基金会的赠款HJK。

1。光学装置（图1） <ol><li>对于双光子激发的飞秒脉冲的红外脉冲激光系统被使用。高激光输出功率（在某些情况下，在890 nm波长大于2W）介绍了该系统的光学元件，以抵消巨大的损失。 </li><li>由两个棱镜组成的甲prechirper系统赋予负的群速度色散（GVD）到激光脉冲之前的声光偏转器（季节AOD）补偿时间色散引入由AODs患者1。 </li><li>两个季节AOD与大孔（10毫米的40倍水浸物镜NA 0.8）的激光束偏转，在两个维度。 </li><li> 100园/毫米的反射衍射光栅被置于13厘米背后的季节AOD补偿引入的季节AOD的空间色散，当使用短的激光脉冲。 </li><li>的激光束被引导与两个中继望远镜到一个直立microsco的相机端口PE。 </li><li>鸢尾花被放置在固定的时间间隔的光学元件的对准。 </li><li>的二色性分束器的前面的目标发送到试样的红外激发光，并反映到检测器上的试样的荧光。 </li><li>外延和transfluorescence的检测器（光电倍增管，光电倍增管）通过的目标和收集荧光信号 - 如果适用 - 通过冷凝器。 </li><li>有色玻璃滤光片（BG-39，3-5毫米）被放置在检测器的前面，以防止激发光到达检测器。 </li><li>数字 - 模拟转换器电路板（156.25 kHz的时钟速率），这反过来又驱动电压控制振荡器配备由计算机控制的AOD的偏转角。 </li><li>从光电倍增管的信号通过一个低通巴特沃斯滤波器（截止频率为100千赫）中继，由一个模拟 - 数字转换器（156.25 kHz的时钟速率）之前被存储在数字化进行分析的计算机。 </li><li>对准和电噪声通过记录荧光信号的分布的激光的光带和不带测试，在低和高的光电倍增管的增益，以及带和不带指示灯。扫描仪被正确设置和屏蔽时的荧光信号在高增益和带指示灯的分布的宽度比其他分布的荧光信号（图2）的宽度大得多。 </li></ol> 2。实验过程<ol><li>抖动随机存取扫描检测依赖于细胞内钙增加。甲大量的神经元，可以使用大剂量注射到神经组织5的酯形式的钙指示器（例如俄勒冈绿488 BAPTA-1 AM）被染色。 </li><li>从每一个神经元胞体的几个地点都记录下来，很短的时间（“抖动”4个位置，每个位置6.4μs的电子= 25.6微秒记录，时间在每个周期中，图的每个神经元。 3C）。选择感兴趣的神经元组成一个完整的帧的256×256像素收购（图3A）。手动选择每个胞体的中心，以被记录在此图像。控制软件会自动添加3个点，围绕这一中心的距离在2μm。 </li><li>荧光信号被记录在每个周期中，从各40个神经元（图3B）。这个过程被重复的整个持续时间的一个记录（5第二记录= 3255周期，1周期= 1.536毫秒）。 </li></ol> 3。在线软件工具，以最大限度地提高棘波检测效率<ol><li>穗从荧光体钙信号的检测依赖于一个高信号对噪声比（S / N）的体细胞荧光钙信号。通过增加激发光强度，可以实现高的S / N。然而，激发光强度，只能提高到一定的限度，因为光损伤。棘波检测是在一个很小的WINDO瓦特只激发强度的荧光信号具有高的S / N，但仅观察到非常小的光损伤3。为了确保所记录的信号是高的尖峰电压检测窗口内，我们在录音监控率（见方程式3.2）和下降的基线荧光光子使用在线分析。 </li><li>每个神经元计算光子率约，从基线噪音很短的时间窗口（100-200）。的光子数（Nλ）和光子率（λ=λ/Δt的）分布拟合（σ）的相对荧光变化，用下面的公式计算出的荧光值： </li></ol><img alt="式（1）" src="/files/ftp_upload/4052/4052eq1.jpg" /> 该方程表示光子散粒噪声的泊松分布与变量相对荧光变化的变化：ΔF/ F =（G * N 为λ（t）-G * N＆林北京经济技术开发区，0）/ G * Nλ，其中G代表的光电倍增管和所有其他电子元件的累计收益。请注意，这个方程不正确地确定体内录音检测的光子的数目，因为有其他的噪声源（运动工件），在除了光子散粒噪声。然而，这个方程是有用的在体内的记录来估计噪声。 <ol start="3"><li>计算基线荧光从相同的时间窗，并绘制为时间的函数或试验。调整激光功率的，因为秒杀检测迅速下降，当超过这个限额保持在低于0.0002 / s的平均降幅为基准。 </li><li>每10-20分钟再次获得一个完整的帧图像的神经元胞体的位置进行了验证。如果需要，记录位置被调节。可以调整一次，所有的神经元或单个神经元的位置。 </li></ol> 4。重构n的荧光信号（反褶积穗定时） <ol><li>荧光信号的钙瞬变是因为衰变产生的神经活动往往summate时间长（几百毫秒为单位）。一个反褶积方法重建穗和穗计时的荧光信号。 </li><li>要确定最有可能的穗列车相关记录的荧光信号，不同的车型进行了比较。在这里，我们用遗传算法来确定模型 - 秒杀火车和尖峰时序 - 最大似然。 </li><li>在不均匀的神经元群体的，秒杀诱发的钙信号可以改变神经元之间。对于无监督数据集的分析中，我们设计了一个算法，考虑到穗诱发的钙信号从神经元到神经元的变化。 </li><li>为了避免大量的假阳性检测的收缩允许的振幅和衰减时间常数的m，它是非常有用的ODEL穗诱发的钙信号。被记录在一组单独的神经元在相同的实验条件下，使用同步电和光学记录相同类型的实验，从单穗诱发钙瞬变的幅度和衰减时间常数的联合分布。 </li><li>到账户基线变化缓慢，并减少计算成本deconvolving，较长的录音分为几个较短的1-5秒的痕迹。 </li><li>对于每个神经元和每个记录，去卷积算法可能测试了大量的模型（高达1,000,000个不同的模型或更多）。一个实验要加快反褶积，反褶积了10个不同的并行计算机。 </li><li>解卷积后，尖峰数据进行分析，并进行检查。一个刺激周围的直方图，穗概率，发射率（平均穗数神经元）以自动的方式计算。 </li></ol> 5。代表性的成果成功的棘波检测铰链上所记录的荧光体细胞钙信号的高信号噪声比。只需使用高的激发率（激光功率），可能会导致在的不利影响photoeffects，生物材料（光损伤）。在抖动随机存取扫描光损伤表现为基线荧光减少，并降低穗诱发的钙离子荧光信号。减少在秒杀诱发的信号，可以迅速在故障检测峰值。只有一个非常小的窗口，激发棘波检测荧光信号强度高。在此窗口中的较高端是有限的光损伤，下端上的荧光信号，有一个低的信号 - 噪声比。对于急性片的皮层神经元光子率约400,000-1,500,000光子/ s的记录片表面约100微米以下时，我们使用的激光功率。当使用高亲和力指示器 - 这里俄勒冈绿488 BAPTA - 1 - 这足以检测个别尖峰信号。图。 3E示出了一个例子，在非常低的激发速率，检测窗口内的记录的一个例子，和在非常高的激发速率之一记录的荧光信号。 与其他技术相比，与单细胞和单穗分辨率记录神经活动，抖动随机存取扫描可以记录从一个较大数目的神经元从相同的，本地的人口，并例如是微创四极/多极录音相比。因此抖动随机存取扫描可用于记录许多神经元的神经活动信号的阈上活动（图4A），在人群中的神经元（大脑皮质可塑性）的神经活动的变化来衡量互信息，并传播的阈上活动通过种群的神经元14（图4B） <img<html> ALT =“图1”SRC =“/ files/ftp_upload/4052/4052fig1.jpg”/&gt; 图1的抖动的随机访问扫描设置的光学设计。 <img alt="图2" src="/files/ftp_upload/4052/4052fig2.jpg" /> 图2。对齐和测试：记录在不同的条件下荧光信号的分布。 A）没有激光和光电倍增管增益低，B）在较高的PMT增益，但没有激光光，由于光电倍增管的暗电流的分布更宽。 C）上的激光记录高PMT的增益。在B中所示的分布，该分布之间的差别将表示该激发光到达光电倍增管探测器。 D）的分布记录的荧光信号在神经元的胞体的高收益。如果没有其他噪声源的贡献，这种分布产生光子散粒噪声。 文件，“/&gt; 图3：A）全画幅荧光图像检测并选择神经元胞体的位置，B）为一个周期的扫描路径，C）插图的抖动原则，在每个SOMA（圈）的几个地点之前记录的光束移动到下一个躯体，D）插图的两个D / A通道的输出。对于每一个神经元的胞体，荧光信号被记录在每个SOMA（S1-S4）从4个不同的点。各点的位置，由下式给出的x和y位置。的x和y可用于所有斑点的位置和所有的神经元被发送到数字 - 模拟转换器，在一个连续的方式。而梁之间移动两个神经元胞体，无信号获取（空白）。 E）的荧光信号的例子。注意每个例子一穗（与电生理细胞贴附记录）。 <img alt="图4" src="/files/ftp_upload/4052/4052fig4.jpg" /> 图4。研究CORTI校准功能，使用随机存取扫描抖动。 A）测量信号通过神经元群体的共同信息。上的图像显示的一种急性脑切片的显微照片和放置在相同的皮质层4（L4）的列中的两个刺激移液器。中心的图表显示了每次重复刺激的神经反应。下图显示了香农人口的神经元信号的互信息。 B）测量繁殖种群之间的皮层神经元的阈上扣球活动（信号传输）。上面的图示出的实验设计，中心的图像显示的荧光图像，虚线表示桶边界，低级的图表显示探测到的峰值响应于丘脑纤维（三角形）的电刺激。

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没有利益冲突的声明。

间接地抖动随机存取扫描检测阈上扣球活动的增加与每穗在一个神经元的胞体在体细胞内的钙。胞内钙的增加，检测到由荧光钙染料。主要是从有限的信号 - 噪声比的钙荧光信号产生抖动的随机存取扫描的局限性。信号对噪声比是依次由光损伤，不允许使用高激发率限制。因为有限的信号 - 噪声比，穗的检测失败时，在某些神经元中，并也可能失败为持续和高频活动。例如，记录在体内时?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td></td><td></td><td></td><td>光学元件是按顺序排列，从激光器</td></tr><tr><td>土卫六：蓝宝石激光</td><td>相干公司</td><td>变色龙超2 </td><td>推荐输出功率（&gt; 2W在900 nm） </td></tr><tr><td>消色差镜头f = 30毫米</td><td>雷神实验室</td><td> AC254-030-B </td><td>为650-1050纳米涂层防反射（AR） </td></tr><tr><td>消色差镜头f = 100毫米</td><td>雷神实验室</td><td> AC254-100-B的</td><td> AR 650-1050纳米</td></tr><tr><td>镜头f = 75毫米</td><td>雷神实验室</td><td> LA1608-B </td><td> AR 650-1050纳米</td></tr><tr><td>镜头f = 175毫米</td><td>雷神实验室</td><td> LA1229-B </td><td> AR 650-1050纳米</td></tr><tr><td>消色差镜头f = 300毫米</td><td>雷神实验室</td><td> AC254-300-B的</td><td> AR 650-1050纳米</td></tr><tr><td>消色差镜头f = 100毫米</td><td>雷神实验室</td><td> AC254-100-B的</td><td> AR 650-1050纳米</td></tr><tr><td>消色差镜头f = 100毫米</td><td>雷神实验室</td><td> AC254-100-B的</td><td> AR 650-1050纳米</td></tr><tr><td>声光偏转器</td><td> Intraaction公司</td><td> ATD 6510CD2 </td><td></td></tr><tr><td>反射型衍射光栅</td><td>新港</td><td> 53-011R </td><td> 100线/ mm与65 MHz的带宽和扫描角度为45毫弧度的季节AOD </td></tr><tr><td> 21.6毫米布鲁斯特棱镜</td><td>拉姆达研究光学公司</td><td> IBP21.6SF10 </td><td></td></tr><tr><td>有色玻璃</td><td>肖特</td&gt; <td> BG-39 </td><td></td></tr><tr><td>分色镜</td><td>色度科技股份有限公司</td><td> Z532RDC </td><td></td></tr><tr><td>光电倍增管模块</td><td>滨松</td><td> H9305-03 </td><td></td></tr><tr><td> DAC，ADC板</td><td>美国国家仪器公司</td><td> PCI-6115 </td><td></td></tr><tr><td>俄勒冈绿488 BAPTA-AM </td><td> Invitrogen公司</td><td> O-6807 </td><td></td></tr></tbody></table>

optical recording of suprathreshold neural activity with single-cell and single-spike resolution

在脊椎动物的中枢神经系统的信息的信号往往是人口，而不是单个神经元的神经元。传播的阈上扣球活动涉及人群的神经元。直接寻址皮质功能的实证研究，因此需要高分辨率的神经元群体的录音。在这里，我们描述的光学方法和去卷积算法从多达100个神经元，单细胞和单穗分辨率记录神经活动。这种方法依赖于检测的瞬态与大脑皮质神经元的阈上电尖峰（动作电位）在体细胞内的钙离子浓度增加。高时间分辨率的光学记录的是，实现快速的随机访问扫描技术使用声光偏转器（AODs患者）1。双光子激发的钙敏感染料的查询结果在较高的空间分辨率在不透明的脑那朵起诉2。尖峰的荧光钙录音重构实现由最大似然方法。同时电和光学记录表明，我们的方法可以可靠地检测峰值（&gt; 97％峰值检测效率），低利率的假阳性棘波检测（&lt;0.003尖峰/秒），时间精度和高（约3 ms）3。可以用于这种秒杀检测的光学方法， 在体外和在体内的麻醉动物3,4的记录神经活动。

Watch this Scientific Journal Video about Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution at JoVE.com

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

了解脊椎动物的中枢神经系统的功能，需要从许多神经元的录音，因为皮层的功能所产生的，在神经元中的人口水平。在这里，我们描述了一个光学的方法来记录阈上的神经活动与单细胞和单穗分辨率，抖动随机存取扫描。这种方法记录体荧光钙信号与高时间分辨率从多达100个神经元。最大似然的算法deconvolves的基本阈上的神经活动，自体荧光钙信号。这种方法可以可靠地检测具有较高的检测效率和低误报率的尖峰，可以用来研究神经元群<em在体外</em&gt;和<em在体内</em&gt;。

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

optical-recording-suprathreshold-neural-activity-with-single-cell

Research

JoVE Journal

Neuroscience

11.8K 次观看.  The University of Texas at Austin. 了解脊椎动物的中枢神经系统的功能，需要从许多神经元的录音，因为皮层的功能所产生的，在神经元中的人口水平。在这里，我们描述了一个光学的方法来记录阈上的神经活动与单细胞和单穗分辨率，抖动随机存取扫描。这种方法记录体荧光钙信号与高时间分辨率从多达100个神经元。最大似然的算法deconvolves的基本阈上的神经活动，自体荧光钙信号。这种方...

视频: Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

用于记录单机活动和Electrocorticographic信号的慢性神经电极植入手术

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

科研

神经科学

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

光遗传学的扰动激光照射半完好的神经活动<em&gt;果蝇</em&gt;幼虫运动

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

神经活动的传播的展开海马准备具有穿透微电极阵列

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

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 ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

高接触密度的制作，平面接口神经电极用于记录和刺激的应用

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

单细胞分辨荧光活成像在幼虫脑培养中的果蝇昼夜节律时钟

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Analyzing Neural Activity and Connectivity Using Intracranial EEG Data with SPM Software

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive neuroscience. Intracranial electroencephalography (EEG) can directly record electrical neural activity and has the unique potential to accomplish this goal. Traditionally, averaging analysis has been applied to analyze intracranial EEG data; however, several new techniques are available for depicting neural activity and intra- and inter-regional connectivity. Here, we introduce two analytical protocols we recently applied to analyze intracranial EEG data using the Statistical Parametric Mapping (SPM) software: time-frequency SPM analysis for neural activity and dynamic causal modeling of induced responses for intra- and inter-regional connectivity. We report our analysis of intracranial EEG data during the observation of faces as representative results. The results revealed that the inferior occipital gyrus (IOG) showed gamma-band activity at very early stages (110 ms) in response to faces, and both the IOG and amygdala showed rapid intra- and inter-regional connectivity using various types of oscillations. These analytical protocols have the potential to identify the neural mechanisms underlying cognitive functions with high spatial and temporal profiles.

We present two analytical protocols that can be used to analyze intracranial electroencephalography data using the Statistical Parametric Mapping (SPM) software: time-frequency statistical parametric mapping analysis for neural activity, and dynamic causal modeling of induced responses for intra- and inter-regional connectivity.

利用 SPM 软件分析颅内脑电图数据的神经活动和连接性

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

使用电压敏感染料在脑切片中进行宽场单光子光记录

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...