这项工作是由聂和镍氢电池。

1。 VideoSwitcher 1.1。将VideoSwitcher连接到电脑 VideoSwitcher（图1）接收模拟视频（VGA）输入，用来驱动阴极射线管（CRT）的彩色监视器。使用的VideoSwitcher之前，确保计算机图形卡具有一个VGA接口（图2A）或数字可视接口（DVI-I），港口（图2B），传输数字和模拟视频信号。一个DVI至VGA转接器（图2C）是要连接的DVI-I端口的VideoSwitcher。 关闭计算机，监测和断开计算机图形卡的显示器。 连接VGA或计算机图形卡的DVI-I端口的VideoSwitcher输入端口使用提供的VGA线。用一个小螺丝刀，以确保连接。 显示器的VGA线连接到输出端口的VideoSwitcher 1ND确保连接。 连接的VideoSwitcher使用提供的电源适配器到电源。 打开电脑和显示器。 1.2。两个视频切换显示模式上VideoSwitcher开关按钮，允许用户来回切换两种显示模式。在彩色模式，VideoSwitcher不会改变从计算机图形卡的视频信号。在灰度模式，变成单色显示屏，其亮度主要取决于视频信号从显卡的蓝色通道，红色通道的信号轻微影响，并没有从绿色通道的贡献。这是因为内VideoSwitcher的电阻网络从红色通道由约128倍，从绿色通道的信号衰减的信号，不利于所有。蓝色的贡献率ð红色通道输出的VideoSwitcher被称为蓝到红比率（BRratio）的。 BRratio是视频切换器，显示设置独立的财产。它可确定实验为每个VideoSwitcher（见1.3）。 也可以通过使用Matlab中的函数调用的图形卡的绿色通道，通过发送一个特殊的信号切换显示模式: PsychVideoSwitcher（开关模式"，whichScreen，ToGrayMode）; 1或0表示ToGrayMode切换到灰度或彩色模式。 图3显示了一个VideoSwitcher原理。 1.3。衡量BRratio 可以衡量的BRratio每个VideoSwitcher使用方波光栅检测任务。方波两级分配的RGB值，如（0，0，40）和（X，0，39）。一个可以调整，直到x的值的方波光栅消失，此时x的值是BRratio ---在B通道的信号由一个单位生产的强度，导致在R-通道的信号幅度。虽然可以测量电子BRratio，例如衡量一个VideoSwitcher电压输出的RGB值（0，0，255）和（255，0，0）的比例，这里描述的是在心理的方法非常简单，准确。 1.4。显示校准对于大多数显示器，显示器的亮度不呈线性增加计算机图形卡指定像素的灰度水平。的关系可以描述为: <img src="/files/ftp_upload/3312/3312eq1.jpg" alt="式（1）" /> 其中L 最大 ，L 最小和γ伽玛校正程序确定，可以通过使用光度计或由心理过程和光度测量7,8组合。伽马校正是importanT到生产上显示的准确对比，它必须为每个显示设备进行。 光度计，可以简单地显示在不同像素graylevels的（例如，从0到255）大统一广场，广场中心的测量亮度，适合用公式1的结果，获得γ。 心理过程涉及创建，在观看表面的一个区域，作为统一的，尽可能的零和强度全像素和确定相邻的均匀区域在的像素graylevel价值的等量混合物（在空间和时间）（在所有的像素有相同的强度），产生心理的混合像素面积的亮度匹配。第一场比赛确定为最大亮度的1/2 graylevel价值。一个像素的亮度强度的1/2和1的混合物，用于确定的3/4的价值，并重复此过程，直到7值fROM的1/8至7/8已经确定。这些比赛是重复的，一致性的各种检查，如验证，3/4和1/4比赛1/2的混合物。在此过程中，无论是B和均质区域ŕ水平的调整，和U = B + R / BRratio。公式1的结果可以适应，获得γ。 后，我们获得最小的L，L 最大和γ值，我们可以设置背景亮度L 0 =（L 最大 + L 最小 ）/ 2，像素的对比度C（U）和像素的灰度水平U之间的关系是: <img src="/files/ftp_upload/3312/3312eq2.jpg" alt="公式（2）" />我们可以重写方程来解决需要一个给定的对比度灰度级: <img src="/files/ftp_upload/3312/3312eq3.jpg" alt="公式（3）" /> 其中，U通常是一个非整数。相应的像素的RGB值是:r = BRratio *的[U-INT（U）的地下= 0，乙=INT（美），INT（）将其输入的整数值。 1.5。演示正弦波光栅增加灰度分辨率的影响，可以通过比较两个正弦波光栅。左侧的光栅，使用适当的R和B的值指定每个像素强度的VideoSwitcher能力。在右边的光栅除左边的一个，只有圆润的U是作为每个像素的B值，模仿一个8位的显示设备的输出是相同的。 2。 RTbox 2.1。连接的RTbox计算机连接的RTbox的（图4）提供的电缆与计算机的USB端口。如果需要的话，请从设备的驱动程序<a href="http://www.ftdichip.com/Drivers/VCP.htm">http://www.ftdichip.com/Drivers/VCP.htm</a> 。安装的驱动程序后，在网站上的指示。 2.2。使用的RT框来衡量响应时间 RTbox可以使用两种不同的方式。 如果用户的程序可以返回可靠的刺激如通过屏幕返回的时间，起效时间（"翻转"）和PsychPortAudio Psychtoolbox [8]，在R​​Tbox就像一个键盘可以使用。因为是基于刺激起效时间和RTbox按钮倍的主机和RTbox的时钟，分别，我们必须同步两个时钟。这是每个刺激发病，以清除缓冲区中的有害事件之前函数调用RTBox的（"清除"）。可以简单地测量响应时间ţRESP = T - ţ 发病 ，其中tonset是从用户程序返回的刺激，t是按钮时，由函数调用[T，按钮返回= RTBox（超时）。这是，因为RTBox驱动自动转换从按钮的RTbox时间时间举办电脑的时间。 如果用户的程序不能返回可靠的刺激起效时间，必须提供触发信号，表明刺激声母的RTbox。一种解决方法是使用光传感器来检测发病的刺激或轻的补丁同步刺激。光传感器的输出连接到光端口的RTbox的。第二个方案是使用的VideoSwitcher的触发输出。触发可以直接连接到的RTbox的脉冲端口。第三个解决方案是连接的主机要健全的RTbox的端口的音频信号。 ，到产生的VideoSwitcher的刺激发病触发，我们"打开"绿色通道的中央部分的像素，在视觉刺激的第一帧。因为它不利于电阻网络，在绿色通道的信号是无形的主题。一个函数调用[T，按钮] = RTBox（"脉冲"，超时）返回的响应时间，&#39;脉冲&#39;指示的RTbox，计算t<html>他响应时间相对于触发。用于声音触发相同的函数调用。光传感器的触发器，函数调用，按钮[T] = RTBox（"轻"，超时）。 3。整合VideoSwitcher和RTbox 我们编写一个演示程序，使用的VideoSwitcher控制显示，RTbox收集准确的响应时间，事件代码和响应发送到ERP系统的RTbox。该方案可以从<a href="http://lobes.usc.edu/videoswitcher/VideoSwitcherRTBoxERP_demo.m">http://lobes.usc.edu/videoswitcher/VideoSwitcherRTBoxERP_demo.m</a>下载。 4。代表结果<img src="/files/ftp_upload/3312/3312fig1.jpg" alt="图1" /> 图1。一个视频切换图片。它具有VGA输入和输出端口（均为女性），一个按钮之间切换两种显示模式，并触发输出端口。 内容】"&gt; <img src="/files/ftp_upload/3312/3312fig2.jpg" alt="图2" /> 图2。图片的VGA（A）和（乙）的DVI-I端口和一个DVI-I至VGA转接器（三）。 <img src="/files/ftp_upload/3312/3312fig3.jpg" alt="图3" /> 图3。一个视频切换器的原理。多路复用器有两种输入模式，通过控制电压水平。当它是在原理图中所示的模式中，显示器的RGB通道接收相同的信号，所以显示是在灰度模式。由CPLD芯片，在G-通道和开关按钮信号控制开关的电压产生。触发也被控制在G-通道的输入。 <img src="/files/ftp_upload/3312/3312fig4.jpg" alt="图4" /> 图4一个RTbox的图片。它有一个USB-B的端口，光输入端口，脉冲/声音输入端口，外部按钮输入端口，以及一个TTL输出端口。

<ol>
	<li>Li, X., Lu, Z. -. L., Xu, P., Jin, J., Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+high+gray-level+resolution+monochrome+displays+with+conventional+computer+graphics+cards+and+color+monitors.">Generating high gray-level resolution monochrome displays with conventional computer graphics cards and color monitors.</a> Journal of Neuroscience Methods. 130 (1), 9-18 (2003).</li><li>Pelli, D. G., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+control+of+contrast+on+microcomputer+displays.">Accurate control of contrast on microcomputer displays.</a> Vision Research. 31, 1337-1350 (1991).</li><li>Li, X., Liang, Z., Kleiner, M., Lu, Z. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RTbox:+A+device+for+highly+accurate+response+time+measurements.">RTbox: A device for highly accurate response time measurements.</a> Behavior Research Methods. 42 (1), 212-225 (2010).</li><li>Lu, Z. L., Sperling, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-order+reversed+phi.">Second-order reversed phi.</a> Perception Psychophysics. 61, 1075-1088 (1999).</li><li>Colombo, E., Derrington, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+calibration+of+CRT+monitors.">Visual calibration of CRT monitors.</a> Displays. 22, 87-95 (2001).</li></ol>

没有利益冲突的声明。

视频切换器提供了一个紧凑，价格低廉，易于使用的方法来产生高亮度高分辨率显示器使用传统的计算机图形卡和模拟彩色显示器。它是模拟输出的显卡和显示器之间的连接。它也可以单色和彩色显示模式之间切换，提供额外的便利。它的触发通道，使研究人员能够同步其他设备，以视觉显示器。 RTbox是结构紧凑，价格低廉，易于使用的硬件/软件解决方案，准确的响应时间?...

enabling high grayscale resolution displays and accurate response time measurements on conventional computers

基于传统的计算机图形卡的显示系统能够产生8位灰度级分辨率的图像。然而，在视觉研究中的大多数实验需要超过12位亮度分辨率的显示器。几种解决方案。位+ + 1 2 DataPixx的使用显卡和高分辨率的数字视频接口（DVI）输出（14或16位）的数字-模拟转换器驱动模拟显示设备。 VideoSwitcher 3这里描述相结合的模拟视频信号从显卡的红色和蓝色通道使用不同重量的被动电阻网络和有源电路提供相同的视频信号的彩色显示器的三个通道。该方法提供了一种廉价的方式，使高分辨率单色显示器，使用传统的图形卡和模拟监视器。它也可以提供触发信号，可以用来标记刺激声母，因此很容易与生理记录或响应时间测量同步可视化显示。 虽然经常用于测量响应时间（RT）的计算机键盘和鼠标，这些测量的精确度是相当低的。 RTbox是一个专门的硬件和软件解决方案，准确的实时测量。通过USB连接到电脑主机连接，驱动程序的RTbox是与所有传统的操作系统兼容。它采用了微处理器和高清晰度时钟记录按钮事件，这是缓冲，直到主机计算机检索的身份和时机。记录的按钮事件不会影响在主机上的数据传输和处理相关的潜在的时序不确定性或偏见。异步存储，大大简化了用户程序的设计。有几种方法可用，到同步的RTbox的钟表和主机COMPUT呃。还可以接收的RTbox外部触发器，可用于测量外部事件RT。 VideoSwitcher和RTbox供用户购买。有关的资料和许多演示程序，可以发现在<a href="http://lobes.usc.edu/">http://lobes.usc.edu/</a> 。

Watch this Scientific Journal Video about Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers at JoVE.com

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

传统的计算机硬件，不能产生足够高的灰度具有足够精度的分辨率和测量响应时间的视觉刺激。我们描述了如何使用的VideoSwitcher产生高分辨率单色显示器，响应时间测量精度高，与传统的计算机硬件和RTbox。

启用高灰度分辨率的显示器和传统计算机准确的响应时间测量

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments ...

enabling-high-grayscale-resolution-displays-accurate-response-time

Universidad de Cartagena UDEC

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Neuroscience

Unit 1

Transient and Steady-state Response Analysis

SCIENTISTS IN ACTION

9.3K 次观看.  The Ohio State University. 传统的计算机硬件，不能产生足够高的灰度具有足够精度的分辨率和测量响应时间的视觉刺激。我们描述了如何使用的VideoSwitcher产生高分辨率单色显示器，响应时间测量精度高，与传统的计算机硬件和RTbox。

视频: Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

科研

神经科学

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

高分辨率人类中脑功能磁共振成像方法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

在对小鼠坐骨神经体内的电测量

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

高分辨率结构磁共振人类皮质下的成像<I&gt;在体内</I&gt;与死亡

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

高分辨率定量免疫膜受体在视网膜带状突触分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

触发细胞应激和死亡使用传统的紫外线激光共聚焦显微镜

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

启用高灰度分辨率的显示器和传统计算机准确的响应时间测量

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