墨西哥河鱼睡眠和睡眠活动的自动测量
Summary
该协议详细介绍了在墨西哥的运动行为和睡眠的量化方法。以前的分析扩展到衡量这些行为在社会居住的鱼。该系统可广泛应用于其他鱼类的睡眠和活动研究。
Abstract
在整个植物区, 睡眠的特点是高度保守的行为特征, 包括提高唤醒阈值, 睡眠剥夺后反弹, 和巩固的行为不动期。墨西哥紫外鱼是研究环境扰动下性状演化的模型. mexicanus存在于眼睛表面居住的形态和多个盲孔居住种群中, 它们具有强烈的形态和行为差异。睡眠丧失发生在多个独立进化的空基鱼种群中。该方案描述了一种定量的方法, 在a. mexicanus洞穴和水面鱼类的睡眠和运动活动。一个经济高效的视频监控系统允许个人居住的幼虫或成年鱼的行为成像一周或更长时间。该系统可应用于成年后4天的鱼类。这种方法也可以用来测量社会交往对睡眠的影响, 在一个竞技场上记录多条鱼。在行为记录之后, 使用自动跟踪软件对数据进行分析, 并使用自定义脚本进行处理, 这些脚本量化多个睡眠变量, 包括持续时间、回合长度和回合数。该系统可用于测量睡眠, 生理行为, 和运动活动, 在几乎任何鱼类物种, 包括斑马鱼和粘滞。
Introduction
睡眠在生理、功能和行为水平1、2、3 上在整个动物王国高度保守。虽然哺乳动物实验室动物的睡眠通常是用脑电图来评估的, 但在小的基因间可利用的模型系统中, 电生理记录就不那么实用了, 因此睡眠通常是根据行为来衡量的,4. 与睡眠相关的行为特征在整个动物王国高度保守, 包括增加唤醒阈值、刺激可逆性和长时间的行为静止 5。这些措施可以用来描述动物的睡眠, 从线虫, 线虫, 通过人类 6 .
使用行为静止来描述睡眠需要自动跟踪软件。使用跟踪软件, 活动周期和不活动期在几天内确定, 长时间不活动被归类为睡眠 7,8。近年来, 开发了多个跟踪系统, 用于获取各种适合基因的小型模型系统中的活动数据;包括蠕虫、果蝇和鱼9,10,11。这些程序附有允许自动跟踪动物行为的软件, 包括开源免费软件和商业上可用的软件7、12、13、14.这些系统在灵活性上有所不同, 允许在许多基因修正模型中有效地筛选和表征睡眠表型。
斑马鱼睡眠的基因调查,达尼奥雷里奥, 已经导致识别了许多基因和神经回路, 调节睡眠15,16。虽然这提供了一个强大的系统, 用于调查在脊椎动物实验动物的睡眠的神经基础, 更不知道睡眠是如何演变, 以及自然变化如何有助于睡眠调节。墨西哥紫鱼,马西卡努斯(a。在睡眠、运动活动和生理节律17, 18 方面发生了巨大的差异。这些鱼作为眼睛表面鱼类存在于墨西哥和南部的得克萨斯州和墨西哥东北部的 sierra del abra 地区周围至少29个洞穴种群 19,20,21。值得注意的是, 许多行为差异, 包括睡眠不足, 似乎已经独立出现在多个空鱼群体 14,22。因此, 紫鱼为研究睡眠、生理和社会行为的收敛演变提供了一个模型。
该协议描述了一种测量a中睡眠和运动行为的系统。仙人掌幼虫和成虫。一个定制的基于红外的记录系统允许在光明和黑暗的条件下对动物进行录像。商业上可用的软件可用于测量活动, 自定义宏用于量化不活动的几个方面, 并确定睡眠时间。该协议还描述了用于跟踪水箱内多个动物活动的实验修改, 提供了检查睡眠和社会行为之间相互作用的能力。这些系统可用于测量睡眠, 生理行为, 和运动活动的其他种类, 包括斑马鱼和粘滞。
Protocol
注: 在幼虫和成虫中建立行为跟踪系统。 1. 构建幼虫睡眠系统 请注意:通过幼鱼在受精后4天通过 30 dpf a. mexicanus追踪幼虫的监测系统需要多种设备, 包括红外 (ir) 照明、丙烯酸红外光扩散器、自动照明控制 (定时器)、计算机、摄像机和二次材料, 如布线和电源控制器 (图 1a)。以下说明将说明如何建立一个系统, 以准确…
Representative Results
4-30 dpf 年龄的幼虫可以可靠地记录在图 1所述的自定义构建的闭合系统中。该系统包括红外和可见光照明, 以便在光线和黑暗条件下、在各种可见光条件下进行录制 (图 1a)。然后使用跟踪软件 (图 1b, c) 对视频进行分析, 并使用自定义睡眠宏进行后处理 (请参阅补充下载)。与表面鱼?…
Discussion
该协议描述了一个定制系统, 用于量化幼虫和成虫的睡眠和运动活动。紫鱼已成为研究睡眠演变的主要模型, 可用于研究睡眠调节1的遗传和神经基础。该协议中的关键步骤包括优化照明和视频质量, 以确保准确的跟踪, 这是量化睡眠所必需的。这里描述的采集和分析系统功能齐全, 许多其他商业和定制系统也是如此, 可以量化运动和行为28、29…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 NIGMS 奖 GM127872 ack 奖、NINSD 奖105072对 ERD 和 ACK 的支持, NSF 奖对 ack 的支持。
Materials
| 12V power adaptor | Environmental Lights | 24 Watt 12 VDC Power Supply | |
| Acrylic dividers (adults) | TAP Plastic | Order sheets in sizes as needed | |
| Adult infrared light power source | Environmnental Lights 24 Watt 12 VDC Power Supply | ||
| Battery pack | CyberPower | CP850PFCLCD | |
| Camera lens (adult) | Navitar Zoom 7000 | Zoom 7000 | |
| Camera lens (larval) | Fujian 35mm f/1.7 | B01CHX7668 | Purchase on Amazon |
| Camera lens adapter | d | 1524219 | |
| Camera mount | CowboyStudio Super Clamp | B002LV7X1K | Purchase on Amazon |
| Fish tank | Deep Blue Professional | ADB11006 | |
| Heat sink (adult) | M-D Building products | SKU: 61085 | Cut to fit |
| Heat sink (larval) | M-D Building products | SKU: 57000 | Cut to fit |
| Infrared lights (adults) | Environmental Lights Infrared 850 nm 5050 LED strip | irrf850-5050-60-reel | Cut to fit |
| Infrared lights (larval) | LED World | B00MO9H7H4 | Purchase on Amazon |
| IR-diffusing acrylic | TAP Plastic | Order sheets in sizes as needed | |
| Laptop/computer | N/A | N/A | Any laptop will work. |
| LED light | Chanzon 10 High Power Led Chip 3W White (6000K-6500K/600mA-700mA/DC 3V-3.4V/3 Watt) | B06XKTRSP7 | Use with Chanzon 25pcs 1W 3W 5W LED Heat Sink (2 pin Black) Aluminum Base Plate Panel |
| light timer | Century 24 Hour Plug-in Mechanical Timer Grounded | ||
| Plastic wall mount for IR | Everbilt Plastic pegboard | Model # 17961 | |
| Power cable | BNTECHGO 22 Gauge Silicone Wire | B01K4RPE0Y | |
| Power source | Rapid LED | MOONLIGHT DRIVER (350MA) | |
| Tissue culture plates | Fisherbrand | 12-well (FB012928) 24-well (FB012929) | |
| Tripod Ball head | Demon DB-44 | B00TQ54CZO | Purchase on Amazon |
| USB Hardrive | Seagate 3TB backup | STDT3000100 | |
| USB Webcam | Microsoft LifeCam | Q2F-00014 | Purchase on Amazon |
| Wall mount for camera | LDR Industries 1/2" Steel pipe | 307 12X36 | Mounted on wall with Flange and 90 degree pipe elbow. Could also use a tripod to hold camera. |
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Cite This Article
Jaggard, J. B., Lloyd, E., Lopatto, A., Duboue, E. R., Keene, A. C. Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish. J. Vis. Exp. (145), e59198, doi:10.3791/59198 (2019).