Sleep is a fundamental biological process conserved across the animal kingdom. The study of how sleep regulatory networks are conserved is needed to better understand sleep across evolution. We present a detailed description of a sleep state in adult zebrafish characterized by reversible periods of immobility, increased arousal threshold, and place preference. Rest deprivation using gentle electrical stimulation is followed by a sleep rebound, indicating homeostatic regulation. In contrast to mammals and similarly to birds, light suppresses sleep in zebrafish, with no evidence for a sleep rebound. We also identify a null mutation in the sole receptor for the wake-promoting neuropeptide hypocretin (orexin) in zebrafish. Fish lacking this receptor demonstrate short and fragmented sleep in the dark, in striking contrast to the excessive sleepiness and cataplexy of narcolepsy in mammals. Consistent with this observation, we find that the hypocretin receptor does not colocalize with known major wake-promoting monoaminergic and cholinergic cell groups in the zebrafish. Instead, it colocalizes with large populations of GABAergic neurons, including a subpopulation of Adra2a-positive GABAergic cells in the anterior hypothalamic area, neurons that could assume a sleep modulatory role. Our study validates the use of zebrafish for the study of sleep and indicates molecular diversity in sleep regulatory networks across vertebrates.

In mammals, hypocretin/orexin (HCRT) neuropeptides are important sleep-wake regulators and HCRT deficiency causes narcolepsy. In addition to fragmented wakefulness, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in the zebrafish HCRT receptor mutant (hcrtr؊/؊). We therefore used zebrafish to study the potential mediators of HCRT-mediated sleep consolidation. Similar to mammals, zebrafish HCRT neurons express vesicular glutamate transporters indicating conservation of the excitatory phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed parallels with established mammalian HCRT neuroanatomy, including projections to the pineal gland, where hcrtr mRNA is expressed. As pineal-produced melatonin is a major sleep-inducing hormone in zebrafish, we further studied how the HCRT and melatonin systems interact functionally. mRNA level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme of melatonin synthesis, is reduced in hcrtr؊/؊ pineal gland during the night. Moreover, HCRT perfusion of cultured zebrafish pineal glands induces melatonin release. Together these data indicate that HCRT can modulate melatonin production at night. Furthermore, hcrtr؊/؊ fish are hypersensitive to melatonin, but not other hypnotic compounds. Subthreshold doses of melatonin increased the amount of sleep and consolidated sleep in hcrtr؊/؊ fish, but not in the wild-type siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation. pineal gland ͉ sleep consolidation H ypocretin 1 and 2 (HCRT 1 and 2, also known as orexin A and B) are two neuropeptides originally isolated in rats, that are derived from a single gene precursor (Hcrt/Orx) (1, 2). HCRT preproprotein is exclusively expressed in neurons restricted to the lateral hypothalamus (LH) organized as a single compact cluster in each hemi-brain (1-3). HCRT neuron number may vary from a few thousand in a rodent LH to 50,000-80,000 in the human LH. This cluster organization is conserved in all mammals investigated (4). Despite its restricted expression, HCRT is a critical regulator of the sleep-wake cycle and is further implicated in food intake regulation, energy homeostasis, arousal, drug addiction, stress, and cardiovascular function. Interestingly, the complexity of HCRT physiological function is reflected in the diversity of HCRT anatomic projections and HCRT receptor expression sites in the central nervous system. From their discrete location in the LH, HCRT neurons send widespread projections throughout the brain and the spinal cord (3, 5). This broad fiber distribution is consistent with the diffuse expression patterns of the two HCRT G protein-coupled receptors (HCRTR1/OX1R and HCRTR2/OX2R) (2, 6).HCRT deficiencies produce narcolepsy, a disorder characterized in mammals by excessive sleepiness during the normal wake periods, direct transitions from wake to REM sleep, and sudden loss o...

Hypocretins/orexins are neuropeptides involved in the regulation of sleep and energy balance in mammals. Conservation of gene sequence, hypothalamic localization of cell bodies, and projection patterns in adult zebrafish suggest that the architecture and function of the hypocretin system are conserved in fish. We report on the complete genomic structure of the zebrafish and Tetraodon hypocretin genes and the complete predicted hypocretin protein sequences from five teleosts. Using whole mount in situ hybridization, we have traced the development of hypocretin cells in zebrafish from onset of expression at 22 h post-fertilization through the first week of development. Promoter elements of similar size from zebrafish and Tetraodon were capable of driving efficient and specific expression of enhanced green fluorescent protein in developing zebrafish embryos, thus defining a minimal promoter region able to accurately mimic the native hypocretin pattern. This enhanced green fluorescent protein expression also revealed a complex pattern of projections within the hypothalamus, to the midbrain, and to the spinal cord. To further analyze the promoter, a series of deletion and substitution constructs were injected into embryos, and resulting promoter activity was monitored in the first week of development. A critical region of 250 base pairs was identified containing a core 13-base pair element essential for hypocretin expression.The hypocretin/orexin peptides (HCRT-1 and HCRT-2) 3 are neurotransmitters involved in the regulation of sleep and energy balance (1). HCRT-1 and HCRT-2 have significant homology and result from the cleavage of a common precursor encoded by the hcrt locus. In mammals, the hypocretin expressing cell clusters are located in the lateral hypothalamus and send widespread projections that innervate multiple brain areas and the spinal cord. The population is relatively small and involves ϳ70,000 neurons in the human brain (2). Projections are especially dense on several aminergic and cholinergic nuclei, most notably the adrenergic locus coeruleus and histaminergic tuberomammilary neurons (3).Loss of HCRT transmission causes the sleep disorder narcolepsy in humans, canines, and rodents (4 -6). The symptoms of narcolepsy in humans typically present in the second decade of life, usually in association with undetectable levels of HCRT-1 in the cerebrospinal fluid (7,8). Post-mortem studies indicate a 90 -95% loss of hypocretin-producing cells in human narcolepsy (2, 6, 9, 10). The process leading to cell death is unknown, but because of a strong association with human leukocyte antigen DQB1*0602 (11, 12), it is generally presumed that hypocretin cells are the target of an autoimmune process (13). This cell destruction in human narcolepsy is likely triggered by environmental factors interacting with a specific genetic background.An understanding of the processes underlying hcrt cell specification, differentiation, and maintenance as well as why they are susceptible to loss are key questions of both basic and...

In juvenile dermatomyositis (JDM), the most common pediatric inflammatory myopathy, weakness is accompanied by a characteristic rash that often becomes chronic and is associated with vascular damage. We hoped to understand the molecular underpinnings of JDM, particularly when untreated, which would facilitate the identification of novel mechanisms and clinical targets that might disrupt disease progression. We studied the RNA-Seq data from untreated JDM peripheral blood mononuclear cells (PBMCs; n = 11), PBMCs from a subset of the same patients when clinically inactive (n = 8/11), and separate samples of untreated JDM skin and muscle (n = 4 each). All JDM samples were compared to non-inflammatory control tissues. The untreated JDM PBMCs showed a strong signature for type1 interferon response, along with IL-1, IL-10, and NF-κB. Surprisingly, PBMCs from clinically inactive JDM individuals had persistent immune activation that was enriched for IL-1 signaling. JDM skin and muscle both showed evidence for type 1 interferon activation and genes related to antigen presentation and decreased expression of cellular respiration genes. Additionally, we found that PBMC gene expression correlates with disease activity scores (DAS; skin, muscle, and total domains) and with nailfold capillary end row loop number (an indicator of microvascular damage). This included otoferlin, which was significantly increased in untreated JDM PBMCs and correlated with all 3 DAS domains. Overall, these data demonstrate that PBMC transcriptomes are informative of molecular disruptions in JDM and provide transcriptional evidence of chronic inflammation despite clinical quiescence.