Organization of the nitrergic neuronal system in the primitive bony fishesPolypterus senegalusandErpetoichthys calabaricus(Actinopterygii: Cladistia)

Abstract

Cladistians are a group of basal actinopterygian fishes that constitute a good model for studying primitive brain features, most likely present in the ancestral bony fishes. The analysis of the nitrergic neurons (with the enzyme nitric oxide synthase; NOS) has helped in understanding important aspects of brain organization in all vertebrates studied. We investigated the nitrergic system of two cladistian species by means of specific antibodies against NOS and NADPH-diaphorase (NADPH-d) histochemistry, which, with the exception of the primary olfactory and terminal nerve fibers, labeled only for NADPH-d, yielded identical results. Double immunohistochemistry was conducted for simultaneous detection of NOS with tyrosine hydroxylase, choline acetyltransferase, calbindin, calretinin, and serotonin, to establish accurately the localization of the nitrergic neurons and fibers and to assess possible interactions between these neuroactive substances. The pattern of distribution in both species showed only subtle differences in the density of labeled cells. Distinct groups of NOS-immunoreactive cells were observed in pallial and subpallial areas, paraventricular region, tuberal and retromammillary hypothalamic areas, posterior tubercle, prethalamic and thalamic areas, optic tectum, torus semicircularis, mesencephalic tegmentum, interpeduncular nucleus, superior and middle reticular nuclei, magnocellular vestibular nucleus, solitary tract nucleus, nucleus medianus magnocellularis, the spinal cord and amacrine cells in the retina. Large neurons in cranial nerve sensory ganglia were also labeled. The comparison of these results with those from other vertebrates, using a neuromeric analysis, reveals a conserved pattern of organization of the nitrergic system from this primitive fish group to amniotes, including mammals.

Pattern of Nitrergic Neuronal System Organization in the Brain of Two Holostean Fishes (Actinopterygii: Ginglymodi)

“…NOS-ir cells or fibers were not observed in the olfactory bulbs of gars, in line with results obtained in most actinopterygians [López et al, 2016]. However, nitrergic cells in the olfactory bulbs have been reported in a teleost fish [Singru et al, 2003], a cartilaginous fish [Ferrando et al, 2012], a lungfish [Northcutt, 2009a], and urodele and gymnophionan amphibians [González et al, , 2002b.…”

“…Antibody specificity against neuronal NOS was assessed by liquid-phase adsorption experiments . In addition, the specificity of the antiserum for the detection of NOS was warranted by the observation of a staining pattern that was identical to that in previous reports in which it matched the NADPH-d histochemical reaction Smeets et al, 1997;Muñoz et al, 2000;Brüning and Mayer, 2001;López and González, 2002;Moreno et al, 2002;Moreno and González, 2004, 2005a, 2005bLópez et al, 2016]. The immunoreactivity is abolished by absorption of the K205 antiserum with recombinant neuronal NOS protein (1 mM overnight at 4 ° C).…”

“…This is reinforced by the lack of NADPH-d staining observed in transgenic knockout mice for NOS [Huang et al, 1993]. The lack of correlation in the olfactory system, noted in several species, including cladistians, teleosts, amphibians, and mammals Porteros et al, 1996;Virgili et al, 2001;López and González, 2002;Moreno et al, 2002;Giráldez-Pérez et al, 2008López et al, 2016], was explained by the presence of cytochrome P-450 reductase, which has close homology with NOS [Bredt et al, 1991] and presents NADPH-d activity. It should be mentioned that although we have referred here to "terminal nerve" fibers, it might be the case that these fibers represent extrabulbar olfactory projections, demonstrated for other actinpterygians and amphibians [Pinelli et al, 2004;Gayoso et al, 2011].…”

“…In a previous review article, some information based on the NADPH-d method was provided for the telencephalon [Braford, 2009]. We used nNOS immunolocalization and NADPH-d histochemistry to confirm the consistency of both approaches in the detection of this gaseous neurotransmission system, as in previous studies conducted by our group in different vertebrates [González et al, , 2002bLópez and González, 2002;López et al, 2016]. This neuroanatomical information is a useful tool for distinguishing and defining classes of neurons and cell groups within the poorly investigated brain of gars.…”

“…Thus, the anatomy of the nitrergic system has been extensively studied by histochemical and immunohistochemical methods in the CNS of most groups of vertebrates [for references see Alonso et al, 2000;López et al, 2016]. In particular, several studies have described the organization of the nitrergic system in teleosts Arévalo et al, 1995;Brüning et al, 1995;Villani and Guarnieri, 1995;Anken and Rahmann, 1996;Bell et al, 1997;Virgili et al, 2001;Singru et al, 2003;Girál-dez-Pérez et al, 2008 but little information is available for other actinopterygian groups [López et al, 2016].…”