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.
This protocol can be used to quantify sleep and activity in any fish population and has the potential to expand the number of model systems used to study sleep regulation. The advantage of this technique is its flexibility. For example, fish can be tracked in a variety of arena sizes or in singly-housed or group-housed experiments.
A strength of measuring sleep in this system is the amenability of the method for high throughput drug or genetic screening. We have applied this method to the study of the evolution of sleep in two different populations of cavefish that demonstrate dramatically different sleep phenotypes. Although it can be difficult to establish a system for behavioral imaging, our system provides a cost-effective method for studying larval and adult fish.
To build the system to accurately track locomotor behavior for studying sleep and circadian rhythms in larval A.mexicanus, use 0.33-centimeter-thick white sign acrylic to construct a platform for the larval tracking system. Place the recording platform on top of the lighted square heat sink upon which fish-containing arenas will reside during the behavioral tracking experiments, and place a second acrylic inside the box between the lights in the animal housing to diffuse the infrared for optimal lighting and contrast. Place the entire larval tracking stage and lighting set up within an enclosed plastic tube.
Use a rotary tool to remove the lens from an experimentally-suitable webcam. Remove the small screws on the back of the camera to remove the inner housing, and remove the small screws inside the body of the camera to loosen the remainder of the lens. Use a small screwdriver to remove any portions of the lens housing that are left after cutting the lens.
And remove the blue light-emitting diode on the top part of the charge-coupled device or CCD housing. Next, return the inner housing to its original orientation before screwing the two outer screws back into their original positions. Use a small saw to route the inside of the camera to allow the camera to be fitted with a rounded plastic routing bit smoothing down the extra plastic until it can accompany a lens adapter.
Install an infrared pass filter inside the camera as close to the CCD as possible without making direct contact with the camera, and screw the adapter of a 35-millimeter fixed lens in the front of the camera into the back of the lens. Then place the camera and lens into the hole drilled into the lid at the top of the tube that houses the stage and lights, and attach the USB to the computer on which the animals will be recorded. To construct a sleep system for adult fish, first cut infrared strips at approximately 46-centimeter intervals.
And place a 46 by five by 0.32-centimeter 9%light pass white sign acrylic sheet directly in front of each infrared light strip to diffuse the infrared light. Then place all of the tanks on a rack that supports rear-mounted infrared lighting, and use opaque plastic dividers and 10-liter glass tanks to create individual arenas. To record the locomotor activity of the Mexican tetra fish place day four to six post-fertilization fish into individual wells of a 24-well tissue plate, and days 20 to 30 post-fertilization in individual wells of a six-well tissue plate.
To record adult tetra sleep and activity in a social setting house up to five adults per 10-liter tank with or without the dividers. In this representative experiment larval fish from three independent cave fish populations displayed a significant reduction in sleep compared to surface fish. This sleep production is consistent across all of the ages tested as sleep was also significantly reduced in adult Pachon, Molino, and Tinaja cave fish compared to surface fish.
Notably, social housing robustly reduced sleep in surface fish without affecting sleep in Pacho cave fish likely due to a basement effect in the cave fish populations. When recording the fish care should be taken to ensure that the camera lighting is optimal to enhance the signal of the fish relative to the background. Some basic command line coding is required for this protocol that is fully described in the text and should be amenable to any lab setting.
This protocol has led to numerous questions about sleep evolution and is currently being used for drug screens, track sleep behavior, and understand how social behavior affects sleep.
