Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish

Summary

This protocol details the methodology for quantifying locomotor behavior and sleep in the Mexican cavefish. Previous analyses are extended to measure these behaviors in socially-housed fish. This system can be widely applied to study sleep and activity in other fish species.

Abstract

Across phyla, sleep is characterized by highly conserved behavioral characteristics that include elevated arousal threshold, rebound following sleep deprivation, and consolidated periods of behavioral immobility. The Mexican cavefish, Astyanax mexicanus (A. mexicanus), is a model for studying trait evolution in response to environmental perturbation. A. mexicanus exist as in eyed surface-dwelling forms and multiple blind cave-dwelling populations that have robust morphological and behavioral differences. Sleep loss has occurred in multiple, independently-evolved cavefish populations. This protocol describes a methodology for quantifying sleep and locomotor activity in A. mexicanus cave and surface fish. A cost-effective video monitoring system allows for behavioral imaging of individually-housed larval or adult fish for periods of a week or longer. The system can be applied to fish aged 4 days post fertilization through adulthood. The approach can also be adapted for measuring the effects of social interactions on sleep by recording multiple fish in a single arena. Following behavioral recordings, data is analyzed using automated tracking software and sleep analysis is processed using customized scripts that quantify multiple sleep variables including duration, bout length, and bout number. This system can be applied to measure sleep, circadian behavior, and locomotor activity in almost any fish species including zebrafish and sticklebacks.

Introduction

Sleep is highly conserved throughout the animal kingdom at the physiological, functional, and behavioral levels^1,2,3. While sleep in mammalian laboratory animals is typically assessed using electroencephalograms, electrophysiological recordings are less practical in small genetically amenable model systems and thus sleep is typically measured based on behavior^3,4. Behavioral characteristics associated with sleep are highly conserved throughout the animal kingdom and include increased arousal threshold, reversibility w....

Protocol

NOTE: Set up systems for behavioral tracking in larvae and adults.

1. Constructing a Sleep System for Larvae

NOTE: The monitoring system for tracking larval through juvenile fish aged 4 days post fertilization (dpf) through 30 dpf A. mexicanus requires multiple pieces of equipment including infrared (IR) lighting, acrylic IR light diffusers, automated light controls (timers), computers, cameras, and secondary materials such as wiring and power controllers (Figure 1A). The following instructions will inform how to build a system to accurately track locomotor be....

Results

Larvae ages 4-30 dpf can be reliably recorded in the custom-build closed system described in Figure 1. The system includes both IR and visible lighting to allow for recordings under light and dark conditions, under various visible light conditions (Figure 1A). The videos are then analyzed using tracking software (Figure 1B,C) and post-processed using a custom sleep macro (See

Discussion

This protocol describes a custom system for quantifying sleep and locomotor activity in larval and adult cavefish. Cavefish have emerged as a leading model for studying the evolution of sleep that can be used to investigate the genetic and neural basis of sleep regulation¹. The critical steps in this protocol include optimization of lighting and video quality in order to assure accurate tracking that is necessary to quantify sleep. The system for acquisition and analysis described here are fully f.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12 V 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 25 pcs 1 W, 3 W, 5 W 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° pipe elbow. Could also use a tripod to hold camera.