Modulation of Melatonin secretion and Sleep consolidation by the Hypocretin-Pineal Gland circuit

Lior Appelbaum¹, Gordon Wang¹, Géraldine Maro¹, Rotem Mori², Adi Tovin², Skariah Gemini¹, Tohei Yokogawa¹, Koichi Kawakami³, Stephen Smith¹, Yoav Gothilf²,Emmanuel Mignot¹,^○Philippe Mourrain⁴

1)Stanford University, CA, USA, 2)Tel Aviv University, Israel, 3)National Institute of Genetics, Mishima, Japan, 4)Stanford University, USA & INSERM, France

In mammals, hypocretin (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 wildtype siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation. This circuit is conserved in mammals as HCRT fibers and HCRT receptors were also found in the pineal gland. The use of melatonin for the treatment of insomnia in human narcolepsy has only been reported in a single case and a more systematic evaluation is needed.