基础记录：一种分析果蝇味觉神经元反应的技术
Summary
一种很少使用的电生理记录方法，即基础记录，可以分析传统记录方法无法检查的味觉编码特征。基础记录还可以分析对疏水刺激的味觉反应，这是使用传统电生理学方法无法研究的。
Abstract
昆虫通过味觉毛发或感器来品尝外部世界，这些毛发或感器在其尖端有毛孔。当感器与潜在的食物来源接触时，食物来源的化合物会通过毛孔进入并激活其中的神经元。50 多年来，这些反应一直使用一种称为尖端记录的技术进行记录。然而，这种方法有很大的局限性，包括无法测量刺激接触之前或之后的神经活动，以及要求品尝剂可溶于水溶液。我们在这里描述了一种我们称之为基础录音的技术，它克服了这些限制。基础记录允许在刺激之前、期间和之后测量味觉神经元的活动。因此，它允许对味觉刺激后发生的OFF反应进行广泛分析。它可用于研究疏水性化合物，例如在水中溶解度非常低的长链信息素。总之，基础记录作为测量神经元活动的手段，具有单感子电生理学的优点 – 高空间和时间分辨率，无需遗传工具 – 并克服了传统尖端记录技术的关键限制。
Introduction
昆虫，包括果蝇，被赋予了复杂的味觉系统，使它们能够从周围环境中提取复杂的化学信息。该系统使他们能够辨别各种物质的化学成分，区分有营养的物质和有害的物质 1,2。
该系统的核心是被称为味觉毛发或感器的特殊结构，它们战略性地位于身体的各个部位。在果蝇中，这些感器位于标签上，标签是苍蝇头部的主要味觉器官1,2,3,4，以及腿和翅膀1,2,5,6。标签位于长鼻的尖端，包含两个裂片 4,7,8。每个叶上覆盖着 31 种味觉，分为短、长、中4、7、8。这些感觉器每个容纳 2-4 个味觉神经元 1,2,9,10。这些味觉神经元表达至少四个不同基因家族的成员，即味觉受体 （Gr）、离子受体 （Ir）、扒手 （Ppk） 和瞬时受体电位 （Trp） 基因 1,2,11,12,13.这种受体和通道的多样性使昆虫能够识别多种化合物，包括非挥发性和挥发性线索1,2,14。
50 多年来，科学家们使用一种称为尖端记录的技术量化了味觉神经元及其受体的反应：3,4,6,8,13,15,16,17,18,19,20,21,22,23,24 ，25,26,27,28，
29,30,31,32,33,34,35。然而，这种方法有很大的局限性。首先，神经活动只能在与刺激物接触期间测量，而不能在接触之前或之后测量。此限制排除了对自发尖峰活动的测量，并阻止了对 OFF 响应的测量。其次，只有可溶于水溶液的品尝剂才能进行测试。
这些限制可以通过一种很少使用的替代电生理技术来克服，该技术称为“碱基记录”。在这里，我们描述了这种技术，我们采用了 Marion-Poll 及其同事24 使用的方法，并展示了它现在可以方便地测量的关键味觉编码特征14。
Protocol
以下协议符合耶鲁大学的所有动物护理指南。 1. 苍蝇 将10-15只新出现的苍蝇在25°C和60%相对湿度的新鲜标准培养瓶中，在12：12小时的光暗循环中。 在3-7天大时使用苍蝇。 2. 化学感觉刺激 获得最高可用纯度的化学感觉刺激。按照供应商的建议存放它们直到使用。 溶解化学感觉刺激，并在水或其?…
Representative Results
图4A显示了由感器引起的自发性尖峰。根据振幅，它们分为两类，较大的尖峰来自对苦味化合物敏感的神经元，而较小的尖峰来自对糖有反应的神经元。基因实验证实了刺突振幅与功能特异性之间的关系 4,14,37,38,39 。 <strong class="xfig…
Discussion
在某些类型的感觉器的记录中，区分不同神经元的尖峰可能具有挑战性。例如，S 和 I sensilla 的糖神经元和机械感觉神经元产生相似振幅的尖峰，因此难以区分它们 4,14。我们发现，使用非常锋利的钨丝记录电极可以减少机械感觉神经元的放电，记录电极的明智放置也是如此。将记录电极插入感器插座的环中（即插入基部但不深）通常会导致机械?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 Zina Berman 的支持，感谢 Lisa Baik 对手稿的评论，以及 Carlson 实验室的其他成员进行讨论。这项工作得到了 NIH 资助 K01 DC020145 到 H.K.M.D 的支持;美国国立卫生研究院（NIH）向J.R.C.DC011697授予R01 DC02174、R01 DC04729和R01。
Materials
| Microscope | Olympus | BX51WI | equipped with a 50X objective (LMPLFLN 50X, Olympus) and 10X eyepieces. |
| Antivibration Table | TMC | 63-7590E | |
| motorized Micromanipulators | Harvard Apparatus and Märzhäuser Micromanipulators | Micromanipulator PM 10 Piezo Micromanipulator | |
| manual Micromanipulators | Märzhäuser Micromanipulators | MM33 Micromanipulator | |
| Magnetic stands | ENCO | Model #625-0930 | |
| Reference and recording Electrode Holder | Ockenfels Syntech GmbH | ||
| Stimulus glass capillary Holder | Ockenfels Syntech GmbH | ||
| Universal Single Ended Probe | Ockenfels Syntech GmbH | ||
| 4-CHANNEL USB ACQUISITION CONTROLLER , IDAC-4 | Ockenfels Syntech GmbH | ||
| Stimulus Controllers | Ockenfels Syntech GmbH | Stimulus Controller CS 55 | |
| Personal Computer | Dell | Vostro | Check for compatibility with digital acquisition system and software |
| Tungsten Rod | A-M Systems | Cat#716000 | |
| Aluminum Foil and/or Faraday Cage | Electromagnetic noise shielding | ||
| Borosilicate Glass Capillaries | World Precision Instruments | 1B100F-4 | |
| Pipette Puller | Sutter Instrument Company | Model P-97 Flaming/Brown Micropipette Puller | |
| Stereomicroscope | Olympus | VMZ 1x-4x | For fly preparation |
| p200 Pipette Tips | Generic | ||
| Microloader tips | Eppendorf | E5242956003 | |
| 1 ml Syringe | Generic | ||
| Crocodile clips | |||
| Power Transformers | STACO ENERGY PRODUCTS | STACO 3PN221B | Assembled from P1000 pipette tips, flexible plastic tubing, and mesh |
| Modeling Clay | Generic | ||
| Forceps | Generic | ||
| Plastic Tubing | Saint Gobain | Tygon S3™ E-3603 | |
| Standard culture vials | Archon Scientific | Narrow 1-oz polystyrene vails, each with 10 mL of glucose medium, preloaded with cellulose acetate plugs | |
| Berberine chloride (BER) | Sigma-Aldrich | Cat# Y0001149 | |
| Denatonium benzoate (DEN) | Sigma-Aldrich | Cat# D5765 | |
| N,N-Diethyl-m- toluamide (DEET) | Sigma-Aldrich | Cat# 36542 |
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Cite This Article
Dweck, H. K. M., Carlson, J. R. Base Recording: A Technique for Analyzing Responses of Taste Neurons in Drosophila. J. Vis. Exp. (205), e66665, doi:10.3791/66665 (2024).