Cholinergic regulation of sleep in the upside-down jellyfish Cassiopea

Abstract

Perhaps nothing is stronger evidence of the importance of sleep than its conservation across animals [1], but the extent of its regulatory conservation is unknown. The upside-down jelly-fish Cassiopea xamachana sleeps [2], and this behavior is controlled by radially-spaced marginal ganglia. After defining a sleep-wake threshold, we compared gene expression profiles of ganglia from animals sleep-deprived for two nights and found differential expression in many sleep-related genes including GABAergic, melatonergic, and cholinergic receptors. We focused on a nicotinic acetylcholine receptor alpha subunit-like (Chrnal-E), based on its differential expression, and selected animals for a second round of RNAseq that included both light-based and mechanically-based sleep-deprivation. Combining datasets revealed a short list of differentially expressed genes, of which chrnal-E is the most recognizable and well-supported, so we investigated its potential role in sleep regulation. First, we found that chemical cholinergic neuromodulators positively regulate pacemaker activity. Then, we showed by in situ hybridization that chrnal-E is expressed primarily within the ganglia, and that the area of expression expands after sleep deprivation. Next, we developed RNAi for use in Cassiopea and determined that Chrnal-E promotes wakefulness. Finally, we sampled circadian timepoints in the field and found in control conditions, chrnal-E has lowest expression late at night, but in sleep deprived animals, chrnal-E peaks at this time, supporting a link to wakefulness. Our finding that Cassiopea sleep is regulated by the cholinergic system underscores that mechanisms of sleep conservation are deeply conserved in animal evolution.