Organization of the Orexin/Hypocretin System in the Brain of Holostean Fishes: Assessment of Possible Relationships with Monoamines and Neuropeptide Y.

Holosteans form a small group of actinopterygian fishes considered the sister group of teleosts. Despite this proximity to the biggest group of vertebrates, relatively few studies have been conducted to investigate the organization of the central nervous system of this group of fishes. In this study, the neuroanatomical distribution of orexin/hypocretin-like immunoreactive (OX-ir) cell bodies and fibers was analyzed in the brain of 3 representative species of the 2 orders of extant holosteans, the spotted gar Lepisosteus oculatus, the Florida gar Lepisosteus platyrhincus, and the bowfin Amia calva. Antibodies against orexin-A (OXA) and orexin-B (OXB) were used, which labeled the same cells and fibers throughout the brain. In addition, double immunohistofluorescence was performed for the simultaneous detection of OXA and OXB with tyrosine hydroxylase, serotonin, and neuropeptide Y (NPY), in an attempt to localize the orexinergic structures precisely and study the possible interactions between these neuroactive substances. The pattern of distribution of OX-ir cells in the 3 species was largely similar, showing labeled cells in the preoptic area (POA), and the tuberal and retrotuberal hypothalamic regions, with only subtle differences between species in the density of labeled cells. OX-ir fibers were found in all main brain subdivisions of the 3 species, mostly in the ventral subpallial areas, POA, hypothalamus, posterior tubercle, thalamus, and mesencephalic tectum. Different densities of orexinergic fibers were observed in relation to catecholaminergic and serotoninergic cell groups, as well as an absence of colocalization between orexins and NPY in the same hypothalamic neurons. The comparison of these results with those obtained in other vertebrates highlights a constant pattern of distribution of this system of neurotransmission among different groups of actinopterygian fishes, especially in teleosts. Conserved features shared by all vertebrates studied were also observed, such as the presence of OX-ir cells in the basal hypothalamus, reflecting the preserved functions of these neuropeptides over the course of evolution.