Background

Sleep is a conserved behavior despite introducing some disadvantages to the organism, such as limiting responsiveness to the environment and preventing food foraging, which are critical activities for animals [1]. We spend nearly one-third of our lifetime sleeping. Although negative consequences of chronic sleep deprivation have been well-documented [1], the function of sleep is not fully known [2]. Major topics in sleep research include the investigation of why we sleep, what biological processes occur during sleep, and how sleep is regulated. Model organisms including zebrafish have been instrumental in addressing these fundamental questions in sleep research. Zebrafish meet all the behavioral criteria for defining sleep [3]. Because electrophysiological recordings of brain activity are not accessible in zebrafish, sleep is defined based on activity; one minute of inactivity in larvae [3,4] and six seconds of inactivity in adults [5] are defined as sleep. Sleep depth and arousability are possible to measure in larvae as well [6,7]. We and others have successfully identified sleep phenotypes in larval zebrafish when drug compounds were administered to their environment [8,9] and when particular genes were targeted via CRISPR/Cas9 mutation [6,10–12]. Nevertheless, there is a need to measure activity patterns and sleep in adult stages to seek answers to the following questions [13]:

1) Do sleep disturbances persist in juvenile mutant fish and adult mutant fish?

2) Do the genes of interest or the administered drug have a developmental (only at larval stage) role or a regulatory role (also at adult stages) in sleep?

For example, overexpression of the gene of interest via heat shock induction in a knockout model can be tested to see if sleep phenotypes at different stages of life are rescued. If the phenotype is rescued in larvae but not in adult fish, it indicates that the mutation irreversibly affects developmental circuits controlling sleep. Heat shock induction to mediate conditional gene expression has been applicable in juvenile [14] and adult zebrafish [15]. Sleep/wake patterns in adult zebrafish have been reported in previous studies that employed homemade solutions [5,7,16]. The methodology we propose here includes the use of a commercially available system, which ensures standardized and reproducible measurements. Sleep and activity phenotyping in larval zebrafish is high throughput; it is possible to include 96 animals in one assay [4]. Such assays in older age groups require the use of much larger tanks to allow normal swimming and exploration; therefore, one can phenotype behaviors of only 6–8 animals at once. We employed this automated video tracking in adult zebrafish (5 months old) and validated our protocol by administering melatonin, a known sleep-promoting compound. We demonstrated a sharp decrease in activity and wakefulness in adult zebrafish compared to the DMSO controls. Further studies are needed to carry out more detailed analyses of sleep parameters such as sleep bout length, sleep latency, and sleep bout numbers in age groups older than larval stages.