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

Abstract

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.

Organization of the Serotonergic System in the Central Nervous System of Two Basal Actinopterygian Fishes: the Cladistians <b><i>Polypterus senegalus</i></b> and <b><i>Erpetoichthys calabaricus</i></b>

“…No differences were observed between the immunolabeling of the sections treated with the antiserum alone or preadsorbed with BSA, thus indicating that the antibody does not cross-react with the carrier molecule alone. The antiserum has previously been used in several nonmammalian species, including different fishes and amphibians, and it has shown no cross-reaction with other monoamines [Stuesse and Cruce, 1992;Marín et al, 1998;Adrio et al, 1999;Antri et al, 2006;Abalo et al, 2007;Carrera et al, 2008;López et al, 2009;. The specificity of ChAT, CB, and CR antisera has been previously described in Polypterus [Graña et al, 2013;, and the distribution patterns for these markers are similar in the brain of Erpetoichthys (present results).…”

Spatiotemporal Development of the Orexinergic (Hypocretinergic) System in the Central Nervous System of <b><i>Xenopus laevis</i></b>

“…The antibodies against orexin-A and orexin-B used in the present study revealed patterns of immunoreactivity that were consistent among animals treated identically in each stage of development analyzed. No differences were observed in the pattern of immunoreactivity using anti-orexin-A or anti-orexin-B antisera, as has been reported in adult amphibians [López et al, 2009a] and other groups of vertebrates [López et al, 2009b[López et al, , 2014Domínguez et al, 2010b], and therefore we will refer in general to OX-ir structures. The ontogeny of Xenopus is divided into a relatively short embryonic phase and a long larval period, which starts with independent feeding and is generally subdivided into three sets of stages: (1) premetamorphic stages, (2) prometamorphic stages, and (3) metamorphic climax ( fig.…”

“…The close relationship between the orexinergic and catecholaminergic systems has been proposed in fishes [Kaslin et al, 2004;López et al, 2009b], adult amphibians [Suzuki et al, 2008;López et al, 2009a], reptiles [Domínguez et al, 2010b], and mammals, where reciprocal connections have been demonstrated between both systems. Thus, whereas the orexinergic neurons are directly inhibited by noradrenaline and dopamine [Yamanaka et al, 2003[Yamanaka et al, , 2006, orexins exert an excitatory effect via indirect presynaptic and direct postsynaptic mechanisms in noradrenergic and dopaminergic cells of the ventral tegmental area, the substantia nigra, the locus coeruleus, and the A1, A2, and A5 cell groups [Smith et al, 2002a;Baldo et al, 2003;Ciriello et al, 2003;Ferguson and Samson, 2003;Walling et al, 2004;Eriksson et al, 2010].…”

“…The present study provides the spatiotemporal sequence of the appearance of OX-ir cells and fibers through the brain development of X. laevis using antibodies against mammalian orexin-A and orexin B, which have been similarly used in previous studies in adult amphibian brains [López et al, 2009a], reptiles [Domínguez et al, 2010b], and some groups of fishes [López et al, 2009b;. The structure of orexins is highly preserved in all vertebrates, with only some variations in the amino acid sequence, with the C-terminal sequence being very conserved in the orexins of all of the vertebrate species analyzed [Sakurai et al, 1998;Shibahara et al, 1999;Kaslin et al, 2004;Panula, 2010;Wong et al, 2011;Yan et al, 2011].…”

“…In mammals, orexins are produced by a small population of neurons located in the lateral hypothalamus that profusely innervates all major subdivisions of the central nervous system Cutler et al, 1999;Nambu et al, 1999;McGranaghan and Piggins, 2001;Mintz et al, 2001;Novak and Albers, 2002;Sakurai, 2005;Nixon and Smale, 2007]. The highly phylogenetic conserved molecular structure of these neuropeptides [Shibahara et al, 1999;Wong et al, 2011] has allowed the immunohistochemical localization of orexinlike immunoreactive cells and fibers in the brain of nonmammalian vertebrates like birds [Ohkubo et al, 2002;Singletary et al, 2006;Miranda et al, 2013], reptiles [Domínguez et al, 2010b], amphibians [Shibahara et al, 1999;Galas et al, 2001;López et al, 2009a], and distinct fish groups like lungfishes [López et al, 2009b], teleosts [Kaslin et al, 2004;Huesa et al, 2005;Amiya et al, 2007;Panula, 2010;Matsuda et al, 2012;Pérez Sirkin et al, 2013], and cladistians [López et al, 2014]. All of these studies point to a conserved organization of the orexinergic system among vertebrates, with a general presence of orexin neurons in the hypothalamus (although the particular localization within this region can vary among vertebrate groups or even among species within the same group) and a pattern of extensive innervation on the main brain areas.…”