这项工作是由威康信托基金赠款支持的LP

1。设备和材料<ol><li>这些实验中使用的一个刺激，刺激隔离病房，两个微电极放大器，数据采集系统和采集软件与计算机组成一个标准的电设置。附加设备包括一个法拉第笼，一个蓬勃发展的立场上的体视显微镜，振动隔离表，光源，和录音平台。 </li><li>五micromanipulators是使用。两个micromanipulators需要记录电极定位的精细控制，而其他三个micromanipulators只需要总值的控制地位的两个刺激电极和接地电极。 DLM的记录电极显微被放置在尾部的准备（实验者左）和显微为TTM记录电极之间的实验者和编制方（略实验者左侧）。两个micromanipulators将举行模拟电极放置在头（右）的实验者的准备。接地电极的显微操纵器是放置在远方的准备</li><li> 40-60MΩ的电阻上拉玻璃记录微电极，并存储在一盘由蜡的支持单位。刺激，两个电解（氢氧化钠）削尖的钨电极使用。一个钨丝，或第三电极电解编造用作地面。准备的刺激和接地电极和连接到micromanipulators实验会议开始前，不需要在会议期间所取代。 </li></ol> 2。准备了D.果蝇 <ol><li>一旦你的设备是成立的，它的时间来准备的苍蝇。冷却冰或使用的CO 2麻醉的苍蝇。如果使用的CO 2，然后让气体的影响有足够的时间（约20分钟），穿脱实验开始前。 </li><li>使用镊子，转移他们的腿苍蝇，轻轻一盘菜，含有约45 °角倾斜的软蜡平台。接下来的四个步骤完成在解剖显微镜下离（但接近）的录音设备。 </li><li>下一步是争取在蜡飞。东方飞腹面朝下，与前面临的斜坡上向上。使用一双细镊子，腿向外延伸，对他们推入蜡。 </li><li>熟悉自己的肌肉的位置，记录：背纵肌，或DLM的，和tergotrochanteral肌肉，或TTM。符合地区之间的胸廓中线和前背鬃（或刚毛）的DLMS subcuticular附着点。 TTM附着点是位于背部的后部和前超鼻翼刷毛。确保的翅膀会不会阻碍访问的DLM或TTM纤维，举行的翅膀向外，“胶水”蜡。 </li><li>使用产钳的罚款对拉长鼻向外仔细，并确保它浸入蜡。这是关键的一步，需要一定的练习，因为长鼻是软的，很容易从头部的其余部分分离。如果出现这种情况，弃飞，并重新开始。通过眼睛的刺激电极插入时，以这种方式来保护头部的失败导致问题。 </li></ol> 3。放置电极<ol><li>一旦飞是固定的蜡，转让的体视显微镜，这是位于内的法拉第笼底的附加飞盘。东方飞侧身实验者的权利飞头。 </li><li>接下来的步骤是插入电极。不通过显微镜，可以插入地面和刺激电极。良好的录音依靠精确的刺穿，因此，它是一个好主意实践处理micromanipulators。把电极接近插入部位micromanipulators的帮助，以促进其应有的配售及后续的录音。 </li><li>下进入调整车轮上使用的显微腹部的后部的接地电极。要削尖钨刺激电极放置在大脑中，使用的显微一个电极尖端的位置，所以它只是涉及苍蝇的眼睛。与其他同时使两个电极刚刚接触外面的每只眼睛。然后推电极，反过来，通过每只眼睛，使电极的技巧达到在头舱背（约2 - 3mm）位于大脑。 </li><li>正确放置电极，将激活巨大的光纤系统。为了测试，刺激电极放置正确，适用于短（0.03毫秒30-60跨刺激电极V）的刺激，看运动的翅膀和飞行/腿部肌肉抽搐“ </li><li>下一步是到后台填写与3M KCl的玻璃微电极，使用汉密尔顿或热拉的塑料注射器，并放入他们的优良控制micromanipulators。正确插入微电极，可用于几轮的实验。 </li><li>第一个记录电极将被插入到一个DLM的纤维。有两个双边对称DLMS;每一个六个人的肌纤维组成。录音，可从任何六个纤维做;然而，最常用的DLM纤维45A和45B，由于其良好的可通过胸角质层背侧，两种纤维是由同一motorneuron神经支配的事实。 </li><li>你从最远的一侧使用的显微操纵器，记录电极插入DLM纤维45A或B该平台的斜坡允许DLM的电极进入一个〜60-90 °角，其中艾滋病渗透背角质层。在示波器模式下使用的软件和电脑显示器的外观，同时记录电极插入胸部。当电极已进入肌肉的基线将下降到接近零或为负值。一个单一的刺激，看看您是否可以观察到肌肉反应测试。 </li><li>其他记录电极插入到离您最近的TTM。这种电极是横向插入，在你面前，由于肌肉附着部位的位置。同时做一个单一的刺激和测试一次跟踪指示电极是在肌肉，再次观察显示器。 </li></ol> 4。刺激和记录<ol><li>您现在准备开始从腿部和飞行的肌肉，刺激大脑和记录反应。适用于整个一个短期的刺激（0.03毫秒）在30 V开始增加至60 V，直到你看到一个响应（即肌肉抽搐，肌肉细胞的去极化，在计算机显示器上观察到）的刺激电极。对于实验的其余部分，设置的响应阈值以上的电压5-10 V。 </li><li>为了测量响应延迟，给予至少5单刺激5每个刺激之间的第二个休息时间。 </li><li>确定“以下的频率”通过提供不同的利率刺激列车。 10 10刺激的列车通常都在100Hz（10ms的每个刺激之间），200Hz的（每个刺激之间的毫秒）和300Hz的（3ms的每个刺激之间）。允许一个2秒的休息时间，彼此之间的刺激列车。 </li></ol> 5。结果：巨人纤维通路的响应延迟和频率<ol><li>响应延迟是刺激大脑和肌肉去极化之间的时间。这个数字比较DLM和TTM的响应延迟到一个单一的刺激。在0.7和1.2 GF - TTM途径和1.3和1.7毫秒之间的MS GF - DLM的途径之间的延迟显示一个健康的准备和正确的记录技术。可以随基因型，遗传背景，温度和年龄的延迟。 图1（A和B）。代表的痕迹，显示了从单一刺激大脑的TTMS和DLMS记录的反应。 </li><li>如上所示，录制的，TTM相比，那些从大的DLM纤维性突触后电位（PSP）的幅度和形状的多变性，这种增加的可变性是由于小尺寸的TTM肌纤维。然而，这种可变性，不影响巨人光纤到TTM途径的响应延迟值。 图1（C和D）。进一步的反应延迟“的痕迹，为TTM和DLM从​​4个人的苍蝇。请注意，TTM在PSP形状的痕迹表现出变异，但反应潜伏期不受影响。对于DLM的是PSP的形状变化较少。 <img src="/files/ftp_upload/2412/2412fig1.jpg" alt="图1 A - D" /> </li><li>比较在100赫兹，200赫兹和300赫兹以下的频率计算成功的反应（满分10分）的比例为DLM和刺激频率在每个TTM途径“”。在100赫兹，TTM和DLM都遵循1:1的刺激。刺激频率在100赫兹以上，DLM的反应开始出现的失败，因为两个interneurons之间的中介，化学突触没有足够的时间恢复之间的刺激。 TTM反应，但是，仍然1:1甚至超出了300Hz的刺激。 图2代表的痕迹，显示“以下的频率”录音。在100Hz，TTMS和DLMS应对所有10个刺激（左）。在200Hz，DLM的反应启动失败（星号）。 <img src="/files/ftp_upload/2412/2412fig2.jpg" alt="图2"> </li></ol> 6。代表性的成果野生型短潜伏期反应（刺激电极放置在眼睛上，绕过感受器，并直接引发了GF电路）在0.7和1.2 MS GF - TTM途径和之间，取决于基因型，遗传背景，温度和年龄，和范围1.3 and1.7毫秒为GF - DLM的途径（Tanouye和纬，1980年，托马斯和纬，1984;恩格尔和吴，1992年，艾伦和墨菲，2007年，费伦等，2008;。奥古斯丁等人 ，未发表。） 。这很短的TTM延迟是由于强劲的GF - TTMn电化学突触的突触途径和DLM的潜伏期更长的发生，因为disynaptic性质的途径，以及存在一个化学突触（PSI - DLMn）。中程和长潜伏期反应（&gt; 3毫秒）的GF传入激活和使用低强度的刺激，或提供了一个可视化的信号（“轻客”）取得的结果。在100Hz TTM和DLM都应该遵循的刺激1:1。 100Hz以上DLM的反应将开始出现故障，化学突触之间的PSI和DLMns没有足够的恢复时间小于10ms的除了之间的刺激。 TTM反应，但是，将保持甚至超越了300Hz的刺激（Tanouye和黄伟文，1980年恩格尔和吴，1992; Allen 等，2007; Martinez 等人 ，2007年）1:1。在shakB基因突变，编码一个果蝇间隙连接通道（“innexin”），显着增加GF - TTM的途径（1〜1.5毫秒）的响应延迟，而GF - DLM的分支响应（艾伦和墨菲，2007年，费伦等，2008）。突变体的反应，可以恢复直接刺激胸神经节，表明不因扰乱神经肌肉传递的延迟效应。苍蝇GF - DLM和GF - TTM途径通常是能够按照1:1的比例，分别为100赫兹和300赫兹10刺激的野生型相比，这些突变遵循高频刺激的能力也受损。重要的是要注意，这些频率大大高于正常刺激频率，持续飞行（3-10赫兹）（Hummon和Costello，1989年）期间收到由承包肌肉。 用来描述稳定的政府飞行服务队输出的另一个参数是“不应期”，或双刺激脉冲之间的最短的时间内，仍然从肌肉产生两种反应。耐火时间变化TTMS DLMS为7-15毫秒之间1-4毫秒。 DLMS比较长的不应期是由于相对不稳定的PSI - DLMn交界（Tanouye和纬，1980年化学突触; Gorczyca和霍尔，1984年恩格尔和吴，1992年，班纳吉等人 ，2004年，艾伦和Godenschwege 2010年）。

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+outputs+of+giant+nerve+fiber+in+Drosophila.">Motor outputs of giant nerve fiber in Drosophila.</a> J Neurophysiol. 44, 405-421 (1980).</li><li>Thomas, J. B., Wyman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+altering+synaptic+connectivity+between+identified+neurons+in+Drosophila.">Mutations altering synaptic connectivity between identified neurons in Drosophila.</a> J Neurosci. 4, 530-538 (1984).</li></ol>

一是要注意当试图获得高品质的录音最重要的事情之一，是正确的方向和健康的准备。在理想的情况下，飞仍然活着录音会议结束，电刺激作出反应。对于记录电极，以最有效地穿透胸腔外骨骼，飞应粘在这样一种方式表面与电极形成一个直角，如有必要，插入电极，可通过删除部分的便利钨手术刀背与胸角质层暴露DLM的飞行肌肉（这一步提供一个额外的优势，使其更难打破玻璃电极的提示）。此外，必须小心，以避免通过subcuticularly位于DLMS和TTMS推电极。飞的头应该得到很好的保护，允许要正确插入到大脑的刺激电极，以防止他们在录制过程中会被拉。 由于其规模和良好描述的形态，政府飞行服务队代表在果蝇中最容易获得的神经途径之一。电突触小分子量的示踪染料的渗透性，允许电耦合的神经元的可视化，和几个可用GAL4线，使人们有可能操纵基因的表达水平在细胞或细胞群 （Jacobs 等人，2000年的一个子集;。艾伦等人，2006年除了上述优点，电路显示属性，如习惯性的，自发的复苏和dishabituation传入和胸椎的组成部分，使果蝇的飞行服务队方便的模型系统，为研究神经可塑性（恩格尔和吴） 1996年）。

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electrophysiological recordings from the giant fiber pathway of d. melanogaster

当一震成人D 。 果蝇的反应，跳跃到空中飞走。在许多无脊椎动物，包括D。果蝇 ，“越狱”（或“不惊人死不休”）在成年阶段的反应是介导的多组分的神经电路，称为巨型纤维系统（ GFS）。比较大尺寸的神经元，其独特的形态，简单地连接，使政府飞行服务队有吸引力的模型系统，为研究神经回路。政府飞行服务队的途径是由两个双边对称的巨型纤维（GF）interneurons，其轴突从大脑通过宫颈结缔组织胸神经节沿中线下降。腹侧神经节政府飞行服务队的形式电化学突触1）内侧的大枝晶的同侧motorneuron（TTMn）驱动tergotrochanteral肌肉（TTM），主要mesothoracic股骨/腿伸在mesothoracic neuromere（T2） 2）对侧外周突触中间神经（PSI），反过来形式背纵肌（DLMS）motorneurons（DLMns），机翼depressors突触化学（胆碱）。神经通路（S）（DVMs）dorsovental肌肉，翼电梯，尚未制定出（DLMS和DVMs共同被称为间接飞行肌 - 他们不直接连接的翅膀，而是移动翅膀间接地扭曲了附近的胸角质层）（国王和黄伟文，1980年，艾伦等人，2006年） 。的DLMS DI -突触激活（通过PSI），导致在这些肌肉的收缩，相对的TTM突触激活（0.5毫秒），让TTMS首先向股骨和推动时间的小而重要的延迟飞了地面。该TTMS同时舒展激活DLMS这反过来又相互牵拉激活飞行时间DVMs。的GF途径可以被激活或者间接地运用感官（如“空调卖自夸”或“灯关闭”）的刺激，或直接由一个超阈值电刺激大脑（详见这里）。在这两种情况下，动作电位达到仅通过政府飞行服务队，PSIS，TTM / DLM的运动神经元的TTMS和DLMS，TTMns和DLMns虽然也有其他的，身份不明的，感觉输入。测量“延迟反应”（之间的刺激和肌肉去极化时）和“下面的高频刺激”（成功应对一定数量的高频刺激）提供了一种方法，重复性和定量评估的功能状态政府飞行服务队的组成部分，包括中央突触（GF - TTMn，GF - PSI，PSI - DLMn）和化学（谷氨酸）神经肌肉接头（TTMn - TTM DLMn - DLM的）。它已被用于确定中央突触的形成有关的基因，并评估中枢神经系统功能。

Watch this Scientific Journal Video about Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster at JoVE.com

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

巨人的光纤系统是一个简单的成人神经电路<em&gt;果蝇</em&gt;包含在飞最大的神经元。我们描述了通过这一途径，通过记录背侧纵器（DLM）和tergotrochanteral（TTM）肌肉巨人光纤interneurons直接刺激后突触电位监测突触传递的协议。

从巨纤维通路的电生理记录 D。果蝇</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

electrophysiological-recordings-from-giant-fiber-pathway-d

Research

JoVE Journal

Neuroscience

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

14.4K 次观看.  University College London - UCL. 巨人的光纤系统是一个简单的成人神经电路果蝇包含在飞最大的神经元。我们描述了通过这一途径，通过记录背侧纵器（DLM）和tergotrochanteral（TTM）肌肉巨人光纤interneurons直接刺激后突触电位监测突触传递的协议。

视频: Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

在昆虫单感器记录果蝇按蚊</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

科研

神经科学

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

成人整装准备果蝇巨纤维染料注射腹侧神经索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

电检测屏幕的配对Nanoinjection和生物活性化合物的使用果蝇巨人纤维系统

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

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

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

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

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

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

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

设计和超轻重量的制作，可调节的多电极电生理记录小鼠探头

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly.

光感受器细胞光谱灵敏度在从昆虫模型决定<em&gt;在体内</em&gt;细胞内记录

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular ...

A Visual Guide to Sorting Electrophysiological Recordings Using 'SpikeSorter'

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be user friendly with a graphical user interface, able to read data files in a variety of formats, and provide users with a flexible set of tools giving them the ability to detect and sort extracellular voltage waveforms from different units with some degree of reliability. Previously published spike sorting methods are now available in a software program, SpikeSorter, intended to provide electrophysiologists with a complete set of tools for sorting, starting from raw recorded data file and ending with the export of sorted spikes times. Procedures are automated to the extent this is currently possible. The article explains and illustrates the use of the program. A representative data file is opened, extracellular traces are filtered, events are detected and then clustered. A number of problems that commonly occur during sorting are illustrated, including the artefactual over-splitting of units due to the tendency of some units to fire spikes in pairs where the second spike is significantly smaller than the first, and over-splitting caused by slow variation in spike height over time encountered in some units. The accuracy of SpikeSorter's performance has been tested with surrogate ground truth data and found to be comparable to that of other algorithms in current development.

A Visual Guide to Sorting Electrophysiological Recordings Using &#039;SpikeSorter&#039;

The article shows how to use the program SpikeSorter to detect and sort spikes in extracellular recordings made with multi-electrode arrays.

可视化指南排序的电生理记录使用“SpikeSorter”

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

全细胞贴片记录用于电泳生理测定通道视紫质中的离子选择性

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...