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Distribution of Orexin/Hypocretin Immunoreactivity in the Brain of the Lungfishes Protopterus dolloi and Neoceratodus forsteri

Lungfishes are currently considered the closest living relatives of tetrapods and represent an interesting group for the study of evolutionary traits in the transition from fishes to tetrapods. The brains of lungfishes have received little attention in comparative studies probably due to the difficulty of obtaining these unique animals. In the present study the distribution of orexin (hypocretin)-like immunoreactivity was studied in the brain of the African lungfish Protopterus dolloi and the Australian lungfish Neoceratodus forsteri by using antibodies directed against the mammalian orexin-A and orexin-B peptides. Simultaneous detection of orexins and tyrosine hydroxylase or serotonin was used to assess the precise location of the orexins in the brain and to evaluate the possible influence of the orexin system on the monoaminergic cell groups. Although some differences were noted, a common pattern for the distribution of orexins in the two lungfishes studied was observed. In both species, most immunoreactive neurons were observed in the suprachiasmatic nucleus and dorsal hypothalamus. Only in Neoceratodus, however, were important cell populations found in the preoptic area and infundibular hypothalamus, whereas small numbers of faintly reactive neurons were present in the lateral septum and ventral striatum. Fiber labeling was widely distributed in all main brain subdivisions, but was more abundant in regions such as the septum, preoptic area, suprachiasmatic nucleus, lateral hypothalamic area, thalamus, pretectum and tegmentum. Less conspicuous was the innervation of the pallial regions, habenula, optic tectum, rhombencephalic reticular formation and spinal cord. Orexinergic innervation was found in close contact with dopaminergic, noradrenergic and serotoninergic cell groups, homologous to the substantia nigra in the midbrain tegmentum, the locus coeruleus, the nucleus of the solitary tract and the raphe nuclei. Although unique features have been found for lungfishes, the location of orexin immunoreactive elements is largely consistent with that recently reported following a similar approach in amphibians and amniotes, suggesting that the general organization of this peptidergic system occurred in the common ancestor of lungfishes and tetrapods.