Functional whole-brain connectivity maps of sleep-regulating circuits
Final Report Abstract
Despite the importance of sleep, the neuronal regulation of sleep and wake remains elusive due to the complexity of mammalian brains and their poor amenability for live imaging. Zebrafish larvae, in contrast, are diurnal vertebrates that allow manipulation and visualization of entire neurons throughout the brain of intact animals. Sleep can be distinguished from inactivity using behavioral criteria, and zebrafish larvae fulfil these criteria when they show a sleep-like state. Brain regions and cell types implicated in regulating mammalian sleep are conserved in the much smaller and transparent zebrafish larva, providing a simpler and more accessible system to study neuronal sleep circuits. We use non-invasive optogenetic stimulation of neurons in freely behaving animals in combination with high-troughput locomotor activity tracking to characterize neuronal mechanisms that regulate vertebrate sleep/wake states. The Prober lab has identified several neuropeptides that regulate zebrafish sleep and wake, including the RFamide peptide Qrfp. Previous studies found that overexpression of Qrfp reduced locomotor activity, while mutation of qrfp or its receptors resulted in more locomotor activity and less sleep, although these effects were mild. Here we show that optogenetic stimulation of Qrfp neurons strongly reduces locomotor activity and increases sleep, whereas optogenetic inhibition of these neurons has the opposite effect. In contrast, neighboring cells that express the neuropeptide hypocretin (Hcrt) promote locomotor activity and reduce sleep. Surprisingly, sleep induced by stimulating Qrfp neurons persists in qrfp mutants, suggesting the presence of an unknown sleep-promoting factor in these cells. Recently, we found that a novel hormone is coexpressed in Qrfp cells. As soon as double-mutants for qrfp and this novel peptide are established, I will test whether mutating both peptides eliminates the optogenetic effect. The possible involvement of a new factor in sleep regulation suggested by the coexpression observed illustrates how much there is to learn about mechanisms of sleep regulation. I am also working on the additional question whether sleep/wake regulation is executed via common or different neuronal circuits using whole-brain neuronal activity imaging to identify the downstream targets of sleep/wake-regulating cells. Furthermore, I am reconstructing the individual morphology of Qrfp and Hcrt neurons using the Brainbow technique. The results of this project are broadening our understanding of the genes and neuronal pathways of sleep/wake regulation.
