第三星果蝇中枢神经系统活动的电生理记录
Summary
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。
Abstract
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。
Introduction
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 .
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Drosophila melanogaster (strain OR) | Bloomington Drosophila Stock Center | 2376 | |
| Vibration isolation table | Kinetic Systems | 9200 series | |
| Faraday Cage | Kinetic Systems | N/A | |
| Dissecting Microscope on a Boom | Nikon | SMZ800N | Multiple scopes can be used; boom stand is critical |
| AC/DC differential amplifier | ADInstruments | AM3000H | The model 1700 can be used instead of the model 3000 |
| audio monitor | ADInstruments | AM3300 | |
| Hum Bug Noise Eliminator | A-M Systems | 726300 | |
| Data Acquisition System (PowerLab) | ADInstruments | PL3504 | Multiple PowerLab models can be used. |
| Lab Chart Pro Software | ADInstruments | N/A – Online Download | |
| Fiber Optic Lights | Edmund Optics | 89-740 | Different light sources can be used, but fiber optics are the most adaptable |
| Micromanipulator | World Precision Instruments | M325 | |
| Microelectrode Holder | World Precision Instruments | MEH715 | Different models are acceptable |
| BNC cables | World Precision Instruments | multiple based on size | |
| Glass Capillaries | World Precision Instruments | PG52151-4 | |
| Microelectrode Puller | Sutter Instruments | P-1000 | Also can use Narashige PC-100 |
| Black Wax | Carolina Biological Supply | 974228 | |
| Non-coated insect pins, size #2 | Bioquip | 1208S2 | |
| Fince Forceps | Fine Science Tools | 11254-20 | |
| Vannas Spring Scissors | Fine Science Tools | 15000-03 |
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