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.

Immunohistochemical Localization of DARPP-32 in the Brain of Two Lungfishes: Further Assessment of Its Relationship with the Dopaminergic System.

The distribution of DARPP-32 (a phosphoprotein related to the dopamine D1 receptor) has been widely used as a means to clarify the brain regions with dopaminoceptive cells, primarily in representative species of tetrapods. The relationship between dopaminergic and dopaminoceptive elements is frequently analyzed using the catecholamine marker tyrosine hydroxylase (TH). In the present study, by means of combined immunohistochemistry, we have analyzed these relationships in lungfishes, the only group of sarcopterygian fishes represented by 6 extant species that are the phylogenetically closest living relatives of tetrapods. We used the Australian lungfish Neoceratodus forsteri and the African lungfish Protopterus dolloi. The DARPP-32 antibody yields a distinct and consistent pattern of neuronal staining in brain areas that, in general, coincide with areas that are densely innervated by TH-immunoreactive fibers. The striatum, thalamus, optic tectum, and torus semicircularis contain intensely DARPP-32-immunoreactive cell bodies and fibers. Cells are also located in the olfactory bulbs, amygdaloid complex, lateral septum, pallidum, preoptic area, suprachiasmatic nucleus, tuberal hypothalamic region, rostral rhombencephalic reticular formation, superior raphe nucleus, octavolateral area, solitary tract nucleus, and spinal cord. Remarkably, DARPP-32-immunoreactive fibers originating in the striatum reach the region of the dopaminergic cells in the mesencephalic tegmentum and represent a well-established striatonigral pathway in lungfishes. Double immunolabeling reveals that DARPP-32 is present in neurons that most likely receive TH input, but it is absent from the catecholaminergic neurons themselves, with the only exception of a few cells in the suprachiasmatic nucleus of Neoceratodus and the solitary tract nucleus of Protopterus. In addition, some species differences exist in the localization of DARPP-32 cells in the pallium, lateral amygdala, thalamus, prethalamus, and octavolateral area. In general, the present study demonstrates that the distribution pattern of DARPP-32, and its relationship with TH, is largely comparable to those reported for tetrapods, highlighting a shared situation among all sarcopterygians.

Immunohistochemical localization of DARPP-32 in the brain of the turtle, Pseudemys scripta elegans: further assessment of its relationship with dopaminergic systems in reptiles.

A previous study in the lizard Gekko gecko has revealed that the distribution of DARPP-32 (a phosphoprotein related to the dopamine D(1)-receptor) largely resembles the pattern observed in birds and mammals, at least as far as basal ganglia structures are concerned. On the other hand, several specific features of DARPP-32 distribution in the gekkonid brain were noted that deserved further attention, e.g. cellular co-localization of DARPP-32 and tyrosine hydroxylase (TH) immunoreactivity in hypothalamic and caudal rhombencephalic areas. To assess the primitive or derived character of these features, DARPP-32 and TH antibodies have been applied to the brain of the turtle, Pseudemys scripta elegans, which belongs to a different radiation of reptiles. Areas in Pseudemys that are densely innervated by TH-immunoreactive fibers, such as the striatum and amygdaloid complex, display strong immunoreactivity for DARPP-32 in somata and neuropil. Strongly immunoreactive fiber plexuses were found in the substantia nigra pars reticulata and in the ventromedial part of the rhombencephalon. Cellular co-localization of DARPP-32 and TH was observed in the ventral hypothalamus but, in contrast to Gekko, not at caudal rhombencephalic levels. Moreover, cellular DARPP-32 immunoreactivity was not seen in the raphe nuclei and spinal cord of Pseudemys. Other notable species differences in DARPP-32 distribution were found in the olfactory bulb, dorsal ventricular ridge and pretectum. In conclusion, the present account on the distribution of DARPP-32 in Pseudemys confirms and extends previous findings in a gekkonid lizard. At the same time, however, it demonstrates that substantial species differences exist, some of which may be related to differences previously observed in the dopaminergic systems.