How can we know that zebrafish are asleep?

Sleep is a specific behavioral state that is defined as a period of inactivity, usually associated with a specific posture or resting location, that is accompanied by a change in arousal threshold. In other words,
a sleeping animal (or human) is less sensitive to environmental stimuli than when they are awake. In addition, sleep is under both circadian and homeostatic control. That is, the timing of sleep is governed by a 24-hour rhythm, but if you deprive an animal (or human) of sleep, they will also exhibit increased sleep pressure followed by longer or deeper sleep. Using these criteria, the pioneering work of Irene Tobler, Joan Hendricks, Paul Shaw, and others demonstrated that even insects like the fruit fly have a sleep-like state, thus paving the way for the experimental investigation of sleep in simple model systems.

Similarly, we and others have demonstrated that both adult and larval zebrafish have a sleep-like state. Using automated video-tracking software, we watch the sleep/wake behavior of hundreds of zebrafish larvae over several days and nights, up to two weeks.

This video shows zebrafish larvae automatically tracked in a 96-well plate. In this video, each larva's active (red) and inactive (blue) bouts are recorded. By recording the movements of each fish simultaneously, we can observe how experimental manipulations alter the larva's long-term behavioral dynamics.

To watch larvae continuously during the day or night, the plate is illumintated with infrared light, which they cannot see, and maintained at a constant temperature with a water bath.

The recorded activity of a single larva plotted over two days. The gray area is enlarged on the right (arrow indicates the timing of lights out). We take each larval dataset and extract parameters including sleep latency, number of sleep bouts, and sleep length. Combining this data into a behavioral fingerprint allows us to make statistical comparisons between datasets.