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The orexin/hypocretin (ORX) system is involved in physiological processes such as feeding, energy metabolism, and the control of sleep and wakefulness. The ORX system may drive the aminergic and cholinergic activities that control sleep and wakefulness states because of the ORX fiber projections to the aminergic and cholinergic cell clusters. The biological mechanisms and relevance of the interactions between these neurotransmitter systems are poorly understood. We studied these systems in zebrafish, a model organism in which it is possible to simultaneously study these systems and their interactions. We cloned a zebrafish prepro-ORX gene that encodes for the two functional neuropeptides orexin-A (ORX-A) and orexin-B (ORX-B). The prepro-ORX gene of the zebrafish consisted of one exon in contrast to mammals. The sequence of the ORX-A peptide of the zebrafish was less conserved than the ORX-B peptide compared with other vertebrates. By using in situ hybridization and immunohistochemistry, we found that the organization of the ORX system of zebrafish was similar to the ORX system in mammals, including a hypothalamic cell cluster and widespread fiber projections. The ORX system of the zebrafish showed a unique characteristic with an additional putatively ORX-containing cell group. The ORX system innervated several aminergic nuclei, raphe, locus ceruleus, the mesopontine-like area, dopaminergic clusters, and histaminergic neurons. A reciprocal relationship was found between the ORX system and several aminergic systems. Our results suggest that the architecture of these neurotransmitter systems is conserved in vertebrates and that these neurotransmitter systems in zebrafish may be involved in regulation of states of wakefulness and energy homeostasis by similar mechanisms as those in mammals.

The Orexin/Hypocretin System in Zebrafish Is Connected to the Aminergic and Cholinergic Systems