Sleep–wake regulation and hypocretin–melatonin interaction in zebrafish

Abstract

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...

Sleep and Development in Genetically Tractable Model Organisms

“…Fish show brief periods during which they stop swimming and are immobile, with increased arousal threshold; these behaviors occur nearly exclusively during the night (zebrafish are diurnal) (Zhdanova et al 2001;Yokogawa et al 2007;Appelbaum et al 2009). Recent work leveraging the genetic tractability of this vertebrate model organism has uncovered a role for melatonin in conveying the circadian control of sleep timing (Zhdanova et al 2001;Gandhi et al 2015).…”

“…Moreover, zebrafish utilize the hypocretin/orexin signaling system (Zhdanova 2006;Appelbaum et al 2010;Panula et al 2010;Yelin-Bekerman et al 2015), abnormalities in which are known to cause the human sleep disorder narcolepsy. Similar to its mammalian analog, the zebrafish hypocretin system is located in the hypothalamus and regulates sleep-wake transitions (Prober et al 2006;Yokogawa et al 2007;Appelbaum et al 2009;Elbaz et al 2013). It comprises merely 20-60 hypocretin neurons, several orders of magnitude fewer than those found in mice (Kaslin et al 2004).…”

“…Capitalizing on this advantage, Appelbaum et al (2009Appelbaum et al ( , 2010 showed that synaptic outputs of the hypocretin system are under circadian control, perhaps offering a mechanism linking the sleep and circadian systems. Although the neural circuits that regulate sleep in fish are not well understood at this time, the innervation pattern of the zebrafish hypocretin system resembles mammalian circuits for regulating sleep and wakefulness.…”

Genetic Ablation of Hypocretin Neurons Alters Behavioral State Transitions in Zebrafish

“…In zebrafish, the neuronal circuit organization and the circadian and homeostatic (compensatory increased sleep following sleep deprivation) processes that regulate sleep are largely conserved with mammals (Zhdanova et al, 2001;Prober et al, 2006;Yokogawa et al, 2007;Rihel et al, 2010). The zebrafish HCRT neuronal network is simple and comprises only ϳ20 -60 hypothalamic neurons that project to several areas in the brain, including putative wake-and sleep-regulating neurons, such as the hindbrain and pineal gland, respectively (Kaslin et al, 2004;Faraco et al, 2006;Prober et al, 2006;Appelbaum et al, 2009Appelbaum et al, , 2010. Global overexpression of HCRT in larval zebrafish induces wakefulness (Prober et al, 2006) and the HCRT-receptor mutant (hcrtr Ϫ/Ϫ ) adult zebrafish exhibits sleep fragmentation during the night (Yokogawa et al, 2007).…”

Orexin-A influences mRNA expression of some glutamatergic receptor subtypes inCarassius auratus(Actinopterygii: Cyprinidae)

“…In this context, a recent study suggested a major role of iGluRs and ORX interactions since it has been shown that glutamate agonists initiate excitatory postsynaptic currents by increasing the number of c-Fos-positive hypothalamic ORX neurons (Eyigor et al 2012) as well as ORX-A-dependent anxiety-like behaviors in the bed nucleus of the stria terminalis (Lungwitz et al 2012). Although a growing amount of data regarding ORX and iGluRs cross-talking events has been reported in mammals, to date little is known on aquatic vertebrates, aside from the fact that glutamatergic phenotype of ORX neurons is well conserved across vertebrates, especially in fish (Appelbaum et al 2009). …”