我们要感谢瑞安女士对数据中显示的德索菲拉中枢神经系统的解剖和图像。

1. 设备和材料
<ol><li>准备电生理钻机所需的组件 (见材料表), 以对五血子核细胞进行吸电极记录。 注: 在实验之前, 有必要建造用于解剖五丁宁管的室, 并在录音过程中用于在盐水中沐浴神经节。下面提供了箱体结构的逐步轮廓。</li>
	<li>准备幼虫室。<ol>
<li>用热板融化黑蜡。</li>
		<li>将2毫升的熔融蜡倒进最大体积为 2-2.5 ml 的腔中。</li>
		<li>让蜡冷却和硬化约2小时。<...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxicology+of+bis(n)-tacrines+on+Blattella+germanica+and+Drosophila+melanogaster+acetylcholinesterase.">Neurotoxicology of bis(n)-tacrines on Blattella germanica and Drosophila melanogaster acetylcholinesterase.</a> Archives of Insect Biochemistry and Physiology. 83 (4), 180-194 (2013).</li><li>Chen, R., Swale, 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=Inwardly+rectifying+potassium+(Kir)+channels+represent+a+critical+ion+conductance+pathway+in+the+nervous+systems+of+insects.">Inwardly rectifying potassium (Kir) channels represent a critical ion conductance pathway in the nervous systems of insects.</a> Scientific Reports. 8 (1), 1617 (2018).</li><li>Francis, S. A., Taylor-Wells, J., Gross, A. D., Bloomquist, J. 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相关视频和文本中提供的详细信息提供了关键步骤, 以记录嗜酸性粒细胞的活性和穗放电频率. 解剖效果是该方法最关键的方面, 因为短或少量的降神经元将降低基线发射速率, 这将导致复制之间的巨大差异。然而, 一旦解剖技术已经掌握, 与此检测收集的数据是高度可重现性和可修正的各种学科。对所述方法的一个修改是纳入了一个自动浓缩系统, 该系统将防止需要将盐水和化学溶液?...

这种方法的总体目标是使研究人员能够快速测量模型昆虫果蝇中枢神经系统 (cns) 的电成因. 该方法可靠、快速、经济高效, 可进行生理和毒理学实验.中枢神经系统是生命所必需的, 因此, 在理解或改变神经功能的过程中, 对适当神经功能至关重要的生理途径已被广泛探索。节肢动物中枢神经系统内信号通路的特性使得发现了几种化学杀虫剂, 这些杀虫剂扰乱无脊椎动物的神经功能, 以诱发死亡率, 同时限制目标外的后果。因此, 测量昆虫神经活动的能力对昆虫毒理学和生理学领域非常感兴趣, 因为神经系统是大多数已部署杀虫剂1的目标组织。然而, 昆虫神经系统基础知识和应用知识的持续增长需要先进的神经生理技术, 这些技术的可行性有限, 因为目前的技术是劳动密集型的, 需要高昂的费用, 昆虫神经细胞系有限, 或对大多数节肢动物的中央突触的访问有限。目前, 大多数昆虫神经蛋白的特性要求对目标进行克隆, 并在随后的药物发现和电生理记录中异种表达, 如昆虫内整流钾通道2所述, 昆虫神经胺受体3, 蚊虫电压敏感 k+通道4, 等。为了满足异源表达的需求和低功能表达的可能性, bloomquist 和他的同事们的目的是诱导培养的 spopoptera (sf21) 细胞中的神经元表型, 作为一种新的方法。杀虫剂发现5,6。这些方法为新化学的发展提供了有效的方法, 但它们往往为药理剂的鉴定、杀虫剂抗药性的识别机制和特性的表征创造了不可逾越的瓶颈基本的生理原理。在这里, 我们描述了一种外体内方法, 它能够记录一种具有可塑性遗传学7、8、9 和已知神经表达模式的模型昆虫的电活动复合物 10,11, 12,以便能够表征神经水平的阻力机制, 新开发的药物的作用方式, 和其他毒理学研究。
果蝇 d . 黑色素瘤是定义昆虫神经系统或杀虫剂作用机理的常用模型生物, 已被确立为适合毒理学研究的模型生物13, 药理学 14 15、神经生理学16和病理生理学17,18, 19,20个过程的脊椎动物。d. 黑色素瘤是一种全代谢昆虫, 它进行完全的蜕变, 包括幼虫和木偶阶段, 才到达生殖成虫阶段。在整个发育过程中, 神经系统在不同的生命阶段经历了重大的重塑, 但幼虫中枢神经系统将是这一方法的重点。完全发育的幼虫中枢神经系统是解剖简单的胸腔和腹部段融合并形成腹侧神经节, 这代表了一系列重复的和几乎相同的神经元单位 21,22。下移运动神经来源于食管下神经节的尾端, 并下降到神经的身体壁肌肉和幼虫的内脏器官。图 1描述了幼虫嗜酸性粒细胞的大体解剖。
嗜酸性粒血脑屏障 (bbb) 在胚胎发生结束时发展, 由会阴间胶质细胞 (spg)21形成。spg 细胞形成了许多类似丝状的过程, 扩散到建立一个连续的, 非常平坦的, 内皮样的片, 涵盖整个嗜酸性粒细胞cns23。五合子bbb 与脊椎动物 bbb 有相似之处, 其中包括通过控制营养物质和异种生物进入 cns 21 来保持神经微环境的稳态.这是可靠的神经传递和功能的先决条件, 然而 bbb 对中枢神经系统的保护限制了合成药物、大多数肽和其他异生物的渗透 24,25, 这引入了潜在的潜力小分子调制器的优点时存在的问题。该方法使用一个简单的横截面来破坏这个屏障, 并提供随时的药理访问中央突触。 所述方法的最大优点是该系统固有的简单性、可重现性和相对较高的吞吐量。该协议相对容易掌握, 设置所需的空间很少, 只需要最初的财政投入, 这将减少到试剂和消耗品。此外, 所描述的方法是完全修正, 以记录家庭苍蝇的中央下降神经活动, 家蝇 26 .

<table>
	<tbody>
		<tr>
			<td>Drosophila melanogaster (strain OR)</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>2376</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration isolation table</td>
			<td>Kinetic Systems</td>
			<td>9200 series</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>Kinetic Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope on a Boom</td>
			<td>Nikon</td>
			<td>SMZ800N</td>
			<td>Multiple scopes can be used; boom stand is critical</td>
		</tr>
		<tr>
			<td>AC/DC differential amplifier</td>
			<td>ADInstruments</td>
			<td>AM3000H</td>
			<td>The model 1700 can be used instead of the model 3000</td>
		</tr>
		<tr>
			<td>audio monitor</td>
			<td>ADInstruments</td>
			<td>AM3300</td>
			<td></td>
		</tr>
		<tr>
			<td>Hum Bug Noise Eliminator</td>
			<td>A-M Systems</td>
			<td>726300</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition System (PowerLab)</td>
			<td>ADInstruments</td>
			<td>PL3504</td>
			<td>Multiple PowerLab models can be used.</td>
		</tr>
		<tr>
			<td>Lab Chart Pro Software</td>
			<td>ADInstruments</td>
			<td>N/A - Online Download</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber Optic Lights</td>
			<td>Edmund Optics</td>
			<td>89-740</td>
			<td>Different light sources can be used, but fiber optics are the most adaptable</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>M325</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Holder</td>
			<td>World Precision Instruments</td>
			<td>MEH715</td>
			<td>Different models are acceptable</td>
		</tr>
		<tr>
			<td>BNC cables</td>
			<td>World Precision Instruments</td>
			<td>multiple based on size</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>PG52151-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000</td>
			<td>Also can use Narashige PC-100</td>
		</tr>
		<tr>
			<td>Black Wax</td>
			<td>Carolina Biological Supply</td>
			<td>974228</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-coated insect pins, size #2</td>
			<td>Bioquip</td>
			<td>1208S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fince Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrophysiological recording of the central nervous system activity of third-instar drosophila melanogaster

目前可用的杀虫剂大多针对无脊椎动物神经蛋白的神经系统和基因突变, 往往会产生有害的后果, 然而目前记录个人神经系统活动的方法动物是昂贵和费力的。这种吸电极制剂制备的第三和第三世界星幼虫中枢神经系统,是一个可追踪的系统, 用于测试神经活性物质的生理效果, 确定各种神经的生理作用中枢神经系统活性的途径, 以及基因突变对神经功能的影响。这种体外制剂只需要适度的解剖技能和电生理专业知识, 就能产生昆虫神经元活动的重现性记录。然后, 包括多肽在内的多种化学调节剂可以直接应用于有生理盐水的溶液中的神经系统, 以测量对中枢神经系统活性的影响。此外, 基因技术, 如 GAL4/UAS 系统, 可以独立应用或与药理剂结合使用, 以确定特定离子通道、转运体或受体对节肢动物中枢神经系统功能的作用。在这种情况下, 本文所述的检测方法对杀虫剂毒理学家、昆虫生理学家和发育生物学家都有重要的意义, d . 黑色素瘤是其已建立的模型生物。该协议的目的是描述一种电生理方法, 以便能够测量模型昆虫果蝇的中枢神经系统的电发生, 这对于测试多种科学假设。

使用具有一致重现性的细胞外抽吸电极可以记录会角内神经下降引起的外周神经的自发活动。切除和横切的食心细胞壳体的自发活动产生了一种循环模式, 以1-2 秒的发射爆发, 约为1秒的近静止活动。例如, 中枢神经系统在 0.5-1 s 时接近静止 (1-2 hz), 然后在大约1秒内突然 (10-400 hz), 然后返回到近静止状态 (1-2 hz) 0.5-1 s (图 5a)。这种射击模式?...

Watch this Scientific Journal Video about Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster at JoVE.com

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

该协议描述了一种方法来记录果蝇中枢神经系统的电活动下降, 以实现对药理剂、神经蛋白基因突变的经济高效和方便的检测,和尚未探索的生理途径的作用。

第三星果蝇中枢神经系统活动的电生理记录

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

该方法通过测量嗜血杆菌黑色素中枢神经系统的电发生，有助于回答昆虫毒理学和昆虫生理学领域关键问题。这允许人们测试广泛的科学假设，并协助发现新的杀虫剂行动模式。该技术的主要优点是，它提供了一个简单、可重复和相对较高的通量系统，以最少的财政投入来研究果蝇的神经系统。要启动，请打开采集/分析软件。单击主工具栏中的"设置"，然后选择"频道设置"，这将打开一个对话框。将总通道数减至三个。通道一将记录来自放大器的原始信号，其范围为 100 毫伏。通道 2 将用于计算通道 1 中高于给定阈值的事件数。为此，单击"计算"选项卡可打开通道 2 的下拉菜单。选择循环测量，这将打开第二个对话框。选择通道 1 作为源。然后，在下拉菜单测量中，选择"事件时的单位峰值"。最后，在检测设置区域中，从下拉菜单中选择"简单阈值"。阈值级别在检测调整窗口上输入，该窗口要设置在背景噪声之上。要将电活动转换为以赫兹表示的速率图，请选择第三通道上的算术。在新窗口中，输入 1000 倍平滑秒，括号通道两个逗号 2。然后，在单位下拉菜单中选择频率单位赫兹。关闭对话框以返回到主屏幕。通道三的 y 轴应以赫兹表示，x 轴应及时表示。通过首先识别和提取一种游荡的三星嗜血杆菌黑色素，将其放入200微升的盐水中，对幼虫果蝇CNS进行解剖。然后，用一对细钳抓住嘴钩，用第二对钳子抓住虫子的腹部，用轻压，避免撕裂薄薄的切层。轻轻地将嘴钩和腹部向不同方向拉，将虫子的角端与头部区域分开，并露出虫子的内脏。CNS 将与气管和消化道交织在一起。用钳子将 CNS 从消化道和气管中调出。从虫子中解剖果蝇中枢神经系统是成功的关键一步。解剖的神经道必须完好无损，对下降的马达神经没有任何重大损伤。连接到神经的电机神经数量增加基线发射频率。如有必要，用Vannas弹簧剪刀手动将CNS后部转到脑叶，从而破坏血脑屏障。转节应基于使用化学品的物理化学性质进行。红线是建议的转流点和横穿的CNS与完整的下降周围神经干干显示这里。首先，首先将玻璃移液器电极从硅酸盐玻璃毛细管拉到 5 到 15 兆欧姆的电阻。将转接的 CNS 插入包含 200 微升盐水的蜡室。用鳄鱼夹将未涂层的昆虫针夹夹在接地线上，并将针插入盐水以完成电路。使用微操纵器，将电极定向到横切的 CNS 的极点。在连接外周神经干干之前，通过调整采集/分析软件中的阈值水平来消除背景噪声。对注射器施加轻微的负压，将外围神经吸入吸盘。在收集基准触发速率数据之前，开始对数据采集软件进行记录，并平衡基准触发速率五分钟。五分钟后，添加200微升盐水和车辆，使腔室的总量达到400微升，开始记录控制发射速率。如果控制处理的触发模式与此处所示的示例不相似，请放弃准备和记录。当在记录三到五分钟后确定基线时，提取200微升盐水，并加入200微升在盐水中溶解的实验剂。在获取/分析软件中标记此时间点的药物应用，包括包含药物和最终浓度的注释。此处显示的是暴露于 DMSO 之前和之后具有代表性的神经放电痕迹。箭头指示应用时间。看到对 DMSO 的响应有限。增加泛氧剂的剂量以浓度依赖的方式放大了转眼果蝇CNS的峰值放电频率，而神经抑制剂GABA则以浓度依赖的方式降低了尖峰放电频率。在尝试此程序时，重要的是要记住，这是一个外体实验，因此盐水的条件，如pH值和温度，是中枢神经系统准备的长期活动的关键。同样，缺乏无关的电活动、有效的法拉第笼和减少 60 赫兹的噪音是这次测定成功的关键。通过这种测定收集的数据有助于确定杀虫剂的具体作用模式。通过电压夹电生理学、生化分析和其他药理学分析等方法对这些数据进行后续验证。

Research

JoVE Journal

Neuroscience

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

科研

神经科学

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

调查中枢神经系统修复中受伤范式<em&gt;果蝇</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

电生理记录从<em&gt;果蝇</em&gt; Labellar味感器

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

在自发活动的测定<em&gt;果蝇</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

鼠标中枢神经系统的光透明使用被动CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...