We propose to develop and apply fluorescence-based polysomnography (fPSG) in zebrafish, a novel, non- invasive method allowing neurogenetic and pharmacological interrogations of nervous system function through whole brain and whole body imaging. fPSG combines custom light sheet microscopy with a new zebrafish line ?zPSG? carrying four transgenes expressing GCaMP7a [Tg(5xUAS:GCaMP7a)] in the brain [Tg(?-tubulin:nls-Kal4FF)] and trunk muscles [Et(gSAIzGFFD109A)], and GFP in the heart [Tg(cmlc2:GFP)] in order to capture brain wide Ca2+ activity (fEEG, fluorescent electroencephalogram), muscle Ca2+ activity (fEMG, fluorescent electromyogram), heart rate (fECG, fluorescent electrocardiogram) as well as eye movement (fEOG, fluorescent electrooculogram). Polysomnography (PSG) is a classic method used to characterize sleep and diagnose sleep disorders and sleep abnormalities in neurological and psychiatric disorders. Slow wave sleep (SWS, non-REM) and rapid eye movement sleep (REM, a.k.a. paradoxical sleep, PS) are defined by specific electrophysiological PSG signatures based on recordings from the surface of the neocortex (EEG), and voluntary or autonomous muscles (EMG+ECG+EOG). SWS-REM/PS have only been reported so far in the more evolutionary-recent amniotic vertebrates: mammals, birds and reptiles. It is unclear whether such neuronal and muscular dynamics are found in non-amniotic vertebrates such as fishes and amphibians. In a first study we have found slow synchronous neural activity and traveling waves of neural activity in the sleeping fish brain. We have coined these novel signatures: Slow Bursting Sleep (SBS) and Propagating Wave Sleep (PWS) which share remarkable commonalities with SWS and PS/REM states, respectively. We propose to develop and apply fPSG to fully characterize SBS (Aim 1) and PWS (Aim 2) at the whole brain, body scale levels. After this full characterization, we will next investigate the molecular and circuit underpinning of these dynamics by interrogating different neurogenetic contexts of melanin-concentrating hormone (MCH) signaling, a conserved neuropeptidergic system which is involved in mammalian sleep but whose role in fish sleep has been debated for over 30 years (Aim 3). Overall, this proposal will (i) develop a new PSG methodology with whole brain-single cell resolution imaging and body scale comprehension that could also be used with other fish models [e.g. cavefish, danionella, medaka], (ii) establish the first neural definition of sleep in fish, (iii) uncover the role of MCH in fish sleep, and finally (iv) shed light on whether common neural signatures of sleep emerged in the non-amniotic vertebrate brain over 450 million years ago. Importantly, fPSG tools and methodology can be extended to any neuroscience question in the awake or asleep animal requiring whole brain imaging with cardiovascular, ocular and voluntary muscles readouts (e.g. studies of the autonomic and non-autonomic systems).

Public Health Relevance

Here we develop and apply a novel method, fluorescent polysomnography, to characterize specific neuronal and muscular activity signatures in the sleeping zebrafish. This method allows whole brain imaging, including deep subcortical structures inaccessible in other animal models, combined with heart rate, eye movement, and voluntary muscle tone measures. This method can interrogate the impact of genes and drugs on nervous system functions at the entire body scale. This approach can be extended to other promising fish models for neurosciences such as cavefish Astyanax mexicanus or Danionella translucida. As fishes represent more than half of the vertebrate species (>30,000) and share many genetic and neural commonalities with humans, there is much to gain from understanding the integrated cellular dynamics of the fish brain and body with potentially exciting outcomes from medical neurogenetics to even ecology.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Emerging Imaging Technologies in Neuroscience Study Section (EITN)
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Laposky, Aaron D
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Stanford University
Schools of Medicine
United States
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