这项研究是由美国国立卫生研究院（R03DC009339-02，NIDCD的）和由国防高级研究计划局（DARPA）微系统技术办公室（MTO），杰克·W·朱迪博士的主持下，（支持jack.judy @ darpa.mil）作为可靠的神经科技计划的一部分，通过空间和海战系统司令部（SPAWAR）系统中心（SSC）太平洋批准号：N66001-11-1-4013。

1。设立电化学仪器<ol><li>电化学仪器，例如Methrohm的Autolab PGSTAT（乌得勒支，NL）对EIS，CV和复兴的需要。 FRA2添加使EIS的，附加的通道多路复用器（MUX）是用于测试多通道电极。 </li><li>建立一个探头适配器连接的探头通道MUX。 </li><li>进行连接。工作通道MUX和传感电极，参考和反电极连接到连接的电流返回路径，通常是植入骨钉的不锈钢或钛探头适配器连接。 </li></ol> 2。电阻抗图谱<ol><li>开始的频率响应分析仪（FRA）的软件，验证程序文件设置。应设置的程序来测试两个多正弦波波形每组15正弦同时从10赫兹到30不等千赫。施加电压应为25 mV或更低（见补充方法）。 </li><li>打开和编辑项目文件。该项目使用的程序文件，通过每个通道的循环，并保存结果（见补充方法）。 </li><li>连接一个无源探头（无放大器）动物主题。积极的探头将无法通过输入信号。 </li><li>执行该项目文件。每个通道需要几十秒钟的设定而异。 </li><li>显示和解释结果。解析输出的文本文件，用MATLAB（Natic​​k市，马萨诸塞州），使Nyquist图。一个半圆在更高的频率，表示组织的响应。 </li></ol> 3。循环伏安<ol><li>启动通用电化学系统（GPES）软件，并验证程序文件设置。程序应设置在50 mV / s的范围内水解的限制，这是典型的神经电极材料+0.8和-0.6之间至五扫电压（PT，IR，IROX）。应至少有三个扫描系统的运行，达到平衡。从最终的扫描结果保存（见补充方法）。扫描速度可提高到1 V / S，以减少测量时间，但是，IV曲线的形状可能会改变如果扫描速度比电极组织界面发生的电荷转移反应更快。 </li><li>打开和编辑项目文件。该项目使用的程序文件，通过每个通道的循环，并保存结果（见补充方法）。 </li><li>连接一个无源探头的动物主题。 </li><li>执行该项目文件。每个通道大约需要三分钟，根据设置。增加至1 V / s的扫描速率，减少了测量时间约10秒，每通道。 </li><li>显示和解释结果。解析输出的文本文件，用MATLAB绘制IV关系。通过整合区域承载能力的收费进行了量化在简历的阴极电流。 </li></ol> 4。复兴<ol><li>启动通用电化学系统（GPES）软件，并验证程序文件设置。使用步骤及彩票的方法，程序应加强持续4秒的电压为1.5 V（见补充方法）。 </li><li>打开和编辑项目文件。该项目使用的程序文件，通过每个通道的循环，并保存结果（见补充方法）。 </li><li>连接一个无源探头的动物主题。 </li><li>执行该项目文件。每个通道需要大约10秒。 4.5）EIS和CV的数据收集和解释结果。 </li></ol> 5。代表结果包括录音，EIS的，简历和复兴，是一个典型的工作流程，如图1所示。录音和EIS收集最频繁（每天或每周）通过各种渠道，而CV如果扣球活动没有再检测，可用于振兴。 EIS的变化，在天至数周后，电极植入。当EIS数据作为Nyquist图显示，一个半圆在更高的频率（原点附近），表明在电极站点（图2）组织反应。 CV产生的电流 - 电压（IV）曲线显示有些滞后。最相关的简历统计负责承载能力，正常化面积电极地盘面积内的IV曲线（图3）。充电容量大的微刺激电极的首选。 在复兴电压脉冲的应用，通常会导致充电容量增加和减少阻抗的大小（图3a及b）。扣球也可以恢复以前尖峰（图4a）的渠道。虽然复兴阻抗和信号没有只有短期效应ISE比（SNR），这种技术可以应用于日常生活。图4b＆C显示每天前和后的复兴1千赫阻抗幅度和豚鼠皮层植入了16个通道阵列的信噪比数据。复兴具有强大的效果上降低一个量级1千赫阻抗后，每个应用程序的大小。作为恢复信号和低阻抗，信噪比提高后，每一个复兴的会议。最终，所有的信号丢失后，植入后160天，复兴不再有效。 <img src="/files/ftp_upload/3566/3566fig1.jpg" alt="图1" /> 图1。EIS的测量后，每个录制会话。如果没有尖峰记录一个通道上，以前有尖峰，EIS显示了一个大的组织成分，增加随着时间的推移，简历和振兴试图在此通道上。 EIS和录音，然后使用，以确定是否治疗是成功的。 图2。EIS数据显示在一个电极植入后立即现场（蓝色），4个月后（绿色）的Nyquist图。 Nyquist图上的每个点代表在一个单一的频率阻抗的实部和虚。在更高的频率部分的半圆，由于在工地附近的组织是显而易见的。 <img src="/files/ftp_upload/3566/3566fig3.jpg" alt="图3" /> 图3。植入的铱氧化物电极前，后的复兴CV和EIS的变化。 （一）复兴增加面积相应增加的费用，承载能力的IV曲线。 （二）观察后复兴的重大转变一个阻抗specra的阻抗较低的水平一般。 <img src="/files/ftp_upload/3566/3566fig4.jpg" alt="图4" /> 图4。EFFECTS电压偏置在录音和阻抗。 （一）前，后复兴录音显示，此前一直保持沉默的渠道尖峰可以恢复。每天前和1千赫阻抗幅度和（C）在手术后约150天的信噪比增加一个强大的（乙）下降后复兴的结果。 errorbars代表豚鼠皮层植入了16个通道阵列收集的数据的标准误差。

<ol>
	<li>Szarowski, D. H., Andersen, M. D., Retterer, S., Spence, A. J., Isaacson, M., Craighead, H. G., Turner, J. N., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+responses+to+micro&#45;machined+silicon+devices.">Brain responses to micro&#45;machined silicon devices.</a> Brain Res. 983, 23-35 (2003).</li><li>Vetter, R. J., Williams, J. C., Hetke, J. F., Nunamaker, E. A., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+neural+recording+using+silicon&#45;substrate+microelectrode+arrays+implanted+in+cerebral+cortex.">Chronic neural recording using silicon&#45;substrate microelectrode arrays implanted in cerebral cortex.</a> IEEE Trans. Biomed. Eng. 51, 896-904 (2004).</li><li>Williams, J. C., Hippensteel, J. A., Dilgen, J., Shain, W., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+impedance+spectroscopy+for+monitoring+tissue+responses+to+inserted+neural+implants.">Complex impedance spectroscopy for monitoring tissue responses to inserted neural implants.</a> J. Neural Eng. 4, 410-423 (2007).</li><li>Johnson, M. D., Otto, K. J., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+voltage+biasing+improves+unit+recordings+by+reducing+resistive+tissue+impedances.">Repeated voltage biasing improves unit recordings by reducing resistive tissue impedances.</a> IEEE Trans. Neural Syst. Rehabil. Eng. 13, 160-165 (2005).</li><li>Otto, K. J., Johnson, M. D., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+pulses+change+neural+interface+properties+and+improve+unit+recordings+with+chronically+implanted+microelectrodes.">Voltage pulses change neural interface properties and improve unit recordings with chronically implanted microelectrodes.</a> IEEE Trans. Biomed. Eng. 53, 333-340 (2006).</li><li>Pickup, P. G., Birss, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+anodic+hydrous+oxide&#45;growth+at+iridium.">A model for anodic hydrous oxide&#45;growth at iridium.</a> J. Electroanal. Chem. 220, 83-100 (1987).</li></ol>

没有利益冲突的声明。

神经假肢录音系统显示功能寿命有限，录音功能，随着时间的推移植入后减小。减少性能的可能因素是无组织反应的植入装置作为一个紧凑的胶质鞘功能隔离异物从健康组织1。随着神经录音，电化学测试（EIS和CV）通常用于纵向监测电极组织界面2,3。 EIS实际上是在评估接口的录音功能非常有用。植入后提示无组织反应，随着时间的推移改变接口3，电气性能，阻抗迅速增加。此外，EIS数据，可以用来模拟植入电极3-5相邻的细胞组成。循环伏安法可用于录音和EIS的变化，以进一步探讨。在ELECTR的颂歌材料和粗糙度以及电化学反应，与周围组织影响的IV曲线的形状。大电荷的承载能力，决定从IV曲线的面积，通常是首选，特别是微电机刺激。往往伴有低充电容量增加EIS。在CV应用的潜力，本身可能改变充电容量和EIS，尤其是如果电压范围足够大的驱动氧化还原反应。 偏置电压，或复兴的应用，可以用增加负责承载能力的目的，减少阻抗，并增加5记录尖峰渠道。氧化在电极界面可能发生在复兴过程中，与铱的材料，含水氧化物单层形成阳极电位为1.2 V的6。它已建议，这个单层的形成可能会删除蜂窝和ACELlular材料附着到电极，低阻抗接口5。而复兴，可以恢复丢失的神经信号，它是最有效的，如果在几天之内，以前尖峰前通道使用。录音，EIS的，简历，振兴最能作为辅助工具，在监测的神经接口和改善长期植入设备的功能。

<table border="1"><tbody><tr><td> 设备 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>电化学仪器</td><td>万通的Autolab </td><td> PGSTAT128N </td><td>添加组件：FRA2，通道MUX </td></tr><tr><td>无源探头</td><td>塔克 - 戴维斯技术</td><td></td><td>模型依赖于连接器和通道数</td></tr><tr><td> 26-pin母接头</td><td>安普</td><td> 5749069-2 </td><td>探头适配器或替代适当的连接器的探头</td></tr><tr><td>香蕉插孔</td><td> digikey </td><td> J151-ND </td><td>探头适配器的Autolab通道MUX香蕉插头</td></tr></tbody></table>

voltage biasing, cyclic voltammetry, &amp; electrical impedance spectroscopy for neural interfaces

电阻抗谱（EIS）和循环伏安法（CV）的电极组织界面，无需额外的侵入性程序的措施，属性，可以用于长期监视电极性能。 EIS的措施，在多频率电阻抗，阻抗的增加表明增加设备周围的胶质瘢痕形成，而循环伏安测量充电电极的承载能力，并指出如何在不同的电压等级转移费。作为电极植入年龄，EIS和CV数据的变化，和以前录制的扣球神经元的电极网站往往表现出神经录音疗效显着降低。植入电极阵列，作为振兴，一个简短的电压脉冲的应用可以带回扣球否则一段时间的沉默电极网站上的活动。复兴改变了EIS和简历，并可以通过这些辅助方法监测。通常情况下，EIS是每天测量作为电极部位的组织反应的迹象。如果尖峰通道，以前有尖峰缺席，则CV被用来确定充电电极站点的承载能力，复兴可应用于改善界面疗效。 CV和EIS，然后反复检查电极组织界面的变化，并收集神经录音。复兴的总体目标是延长植入阵列功能寿命。

Watch this Scientific Journal Video about Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces at JoVE.com

Voltage Biasing, Cyclic Voltammetry, &amp; Electrical Impedance Spectroscopy for Neural Interfaces

组织神经记录电极的电极接口，可与电阻抗谱（EIS）和循环伏安法（CV）的特点。电压偏置变化的电极组织界面的电化学性能，并能提高录音功能的应用。电压偏置，EIS的CV，和神经录音是相辅相成的。

偏置电压，循环伏安，神经接口电阻抗光谱

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive ...

voltage-biasing-cyclic-voltammetry-electrical-impedance-spectroscopy

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24.4K 次观看.  Purdue University. 组织神经记录电极的电极接口，可与电阻抗谱（EIS）和循环伏安法（CV）的特点。电压偏置变化的电极组织界面的电化学性能，并能提高录音功能的应用。电压偏置，EIS的CV，和神经录音是相辅相成的。

视频: Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

突触前多巴胺纹状体脑片的快速扫描循环伏安动态

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

科研

神经科学

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

神经肌肉功能的应用经皮神经电刺激评估

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is implicated in diseases and disorders including attention deficit hyperactivity disorder, Parkinson's disease, and traumatic brain injury. Thus, DA neurotransmission and the methods to study it are of intense scientific interest. In vivo fast-scan cyclic voltammetry (FSCV) is a method that allows for selectively monitoring DA concentration changes with fine temporal and spatial resolution. This technique is commonly used in conjunction with electrical stimulations of ascending DAergic pathways to control the impulse flow of dopamine neurotransmission. Although the stimulated DA neurotransmission paradigm can produce robust DA responses with clear morphologies, making them amenable for kinetic analysis, there is still much debate on how to interpret the responses in terms of their DA release and clearance components. To address this concern, a quantitative neurobiological (QN) framework of stimulated DA neurotransmission was recently developed to realistically model the dynamics of DA release and reuptake over the course of a stimulated DA response. The foundations of this model are based on experimental data from stimulated DA neurotransmission and on principles of neurotransmission adopted from various lines of research. The QN model implements 12 parameters related to stimulated DA release and reuptake dynamics to model DA responses. This work describes how to simulate DA responses using QNsim1.0 and also details principles that have been implemented to systematically discern alterations in the stimulated dopamine release and reuptake dynamics.

Fast-scan cyclic voltammetry can monitor in vivo dopamine neurotransmission in the context of drugs, disease, and other experimental manipulations. This work describes the implementation of QNsim1.0, a software to model electrically stimulated dopamine responses according to the quantitative neurobiological model to quantify estimates of dopamine release and reuptake dynamics.

使用QNsim1.0从电刺激多巴胺神经传递数据建模快速扫描循环伏安数据

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

全细胞电压钳记录的电生理方法<em&gt;果蝇</em&gt;光感受器

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

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 ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

自由运动大鼠皮质局部电位、心电图、肌电图和呼吸节律的同步记录

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

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 ...