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

Abstract

The present immunohistochemical study represents a detailed spatiotemporal analysis of the localization of orexin-immunoreactive (OX-ir) cells and fibers throughout development in the brain of the anuran amphibian Xenopus laevis, a model frequently used in developmental studies. Anurans undergo remarkable physiological changes during the early life stages, and very little is known about the ontogeny and the localization of the centers that control functions such as appetite and feed ingestion in the developing brain. We examined the onset of the orexinergic system, demonstrated to be involved in appetite regulation, using antibodies against mammalian orexin-A and orexin-B peptides. Simultaneous detection of orexins with other territorial markers was used to assess the precise location of the orexinergic cells in the hypothalamus, analyzed within a segmental paradigm. Double staining of orexins and tyrosine hydroxylase served to evaluate possible interactions with the catecholaminergic systems. At early embryonic stages, the first OX-ir cells were detected in the hypothalamus and, soon after, long descending projections were observed through the brainstem to the spinal cord. As brain development proceeded, the double-staining techniques demonstrated that this OX-ir cell group was located in the suprachiasmatic nucleus within the alar hypothalamus. Throughout larval development, the number of OX-ir cells increased notably and a widespread fiber network that innervated the main areas of the forebrain and brainstem was progressively formed, including innervation in the posterior tubercle and mesencephalon, the locus coeruleus, and the nucleus of the solitary tract where catecholaminergic cells are present. In addition, orexinergic cells were detected in the preoptic area and the tuberal hypothalamus only at late prometamorphic stages. The final distribution pattern, largely similar to that of the adult, was achieved through metamorphic climax. The early expression of orexins in Xenopus suggests important roles in brain development in the embryonic period before feeding, and the progression of the temporal and spatial complexity of the orexinergic system might be correlated to the maturation of appetite control regulation, among other functions.

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

“…These results follow previous studies conducted by our group in cladistian fishes, lungfishes, amphibians, and reptiles [López et al, 2009a[López et al, , 2009b[López et al, , 2016Domínguez et al, 2010]. The specificity of the antibodies was confirmed with control series, suggesting that the substances recognized are only orexins, although the presence of orexins in holostean fishes remains unknown.…”

“…In addition, it has been demonstrated that the synthetic human OXA has physiological activity in teleosts [Volkoff et al, 1999[Volkoff et al, , 2003Volkoff and Peter, 2001;Nakamachi et al, 2006;Yokogawa et al, 2007], which indicates that, despite the changes in the structure of orexins in vertebrates, there is still a high similarity between orexins in teleosts and humans. The anti-OXA and anti-OXB antisera used in this study were raised against a peptide mapping at the C-terminus of human OXA or OXA, whose invariability, together with the fact that orexins do not have structural homology with other biologically active peptides [Shibahara et al, 1999], accounts for the consistency in the immunolabeling pattern obtained with both antibodies in this and previous studies [Singletary et al, 2005[Singletary et al, , 2006López et al, 2009aLópez et al, , 2009bLópez et al, , 2014López et al, , 2016Domínguez et al, 2010;Miranda et al, 2013].…”

“…Following this research, we have conducted this study to provide detailed information about the location of OX-ir cells and fibers in the brain of 3 representative species of holosteans: the spotted gar (Lepisosteus oculatus), the Florida gar (L. platyrhin cus), and the bowfin (A. calva). We carried out immunohistochemical studies with the same methodology previously used in the brain of several tetrapods [López et al, 2009a[López et al, , 2016Domínguez et al, 2010], lungfishes [López et al, 2009b], and cladistians ; this reinforces the reliability of results and facilitates their comparison across vertebrates.…”

“…The distribution of orexins in the brain has been reported in birds [Ohkubo et al, 2002;Singletary et al, 2006;Miranda et al, 2013], reptiles [Domínguez et al, 2010], amphibians [Shibahara et al, 1999;Galas et al, 2001;López et al, 2009bLópez et al, , 2016, and lungfishes [López et al, 2009a]. In the case of teleosts, there are many studies regarding the orexinergic system [Kaslin et al, 2004;Huesa et al, 2005;Amiya et al, 2007;Panula, 2010;Matsuda et al, 2012;Pérez Sirkin et al, 2013] and its functions [Volkoff et al, 2005;Matsuda et al, 2012;Volkoff, 2012;Chiu and Prober, 2013;Nisembaum et al, 2014;Shahjahan et al, 2014], which were demonstrated to be basically comparable to those in mammals.…”

“…These experiments were prompted by previous studies on tetrapods and fishes, which reported interactions with some catecholaminergic groups in the brainstem, serotoninergic cells in the raphe nuclei Baldo et al, 2003;Wang et al, 2003;Kaslin et al, 2004;Zhang et al, 2004;Balcita-Pedicino and Sesack, 2007;López et al, 2009aLópez et al, , 2009bLópez et al, , 2014López et al, , 2016Domínguez et al, 2010;Panula, 2010], and NPY elements in the hypothalamus [Volkoff, 2006;López et al, 2014].…”