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

Abstract

The study of the nitrergic system, formed by the networks of neurons containing the enzyme nitric oxide synthase (NOS), has been extremely useful in unraveling neuroanatomical features of the organization of the central nervous system of vertebrates. Thus, data are available for representatives of most vertebrate classes and, in particular, several studies have detailed the organization of this system in teleosts. In contrast, no information is available regarding this neurotransmission system in the brains of holosteans, an early diverged and poorly understood group of actinopterygian fishes, currently considered a sister group of teleosts that contains only 8 species. The present study provides the first detailed information on the distribution of nitrergic cell bodies and fibers in 2 holostean species of the genus Lepisosteus, the spotted gar L. oculatus and the Florida gar L. platyrhincus. NOS immunohistochemistry and the NADPH diaphorase (NADPH-d) histochemical reaction were used, and both techniques yielded identical results, with the exception of the primary olfactory and terminal nerve fibers, which only labeled for NADPH-d exclusively in L. oculatus. Double immunohistochemistry was conducted for the simultaneous detection of NOS with tyrosine hydroxylase, choline acetyltransferase, calbindin, calretinin, and serotonin to accurately establish the localization of the nitrergic neurons and fibers in the brain of holosteans, the neuroanatomy of which has been mostly neglected, and to assess possible interactions between these neuroactive substances. Distinct groups of nitrergic cells were located in subpallial areas, the basal hypothalamus, posterior tubercle, optic tectum and mesencephalic tegmentum, reticular formation, solitary tract nucleus, spinal cord, and amacrine cells in the retina. In addition, low numbers of nitrergic cells were observed in the pallium, suprachiasmatic nucleus, prethalamic and thalamic areas, torus lateralis and torus semicircularis, cerebellar and laterodorsal tegmental nuclei, and the ventral octavolateral area. Comparison of these results with those from other classes of vertebrates, and including a segmental analysis to correlate cell populations, reveals that the pattern of the nitrergic system in holosteans is very close to that in ancestral actinopterygian fishes and highlights conserved and derived traits.

Pattern of nitrergic cells and fibers organization in the central nervous system of the Australian lungfish,Neoceratodus forsteri(Sarcopterygii: Dipnoi)

“…Antibody specificity against neuronal NOS was assessed by liquid‐phase adsorption experiments (Simonian & Herbison, ). In addition, the specificity of the antiserum for 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 diaphorase histochemical reaction (Brüning & Mayer, ; González et al, ; López et al, ; López & González, ; López, Lozano, Morales, & González, ; Moreno & González, , ; Moreno, López, Sánchez‐Camacho, & González, ; Muñoz, Marín, & González, ; Smeets et al, ). The immunoreactivity is abolished by absorption of the K205 antiserum with recombinant neuronal NOS protein (1 mM overnight at 4°C).…”

“…Alternatively, immunohistochemical detection of NOS directly reveals the nitrergic structures, although some mismatches between the two methodological approaches have been reported (Kharazia, Schmidt, & Weinberg, ; Sobreviela & Mufson, ; Vizzard, Erdman, Roppolo, Forstermann, & de Groat, ). Thus, the anatomy of the nitrergic system has been extensively studied by histochemical and immunohistochemical methods in the brain of most groups of vertebrates (for references see Alonso et al, ; López, Lozano, Morona, & González, ; López, Lozano, Morales, & González, ). In particular, several studies have described the organization of the nitrergic system in ray‐finned fishes, primarily teleosts (Anken & Rahmann, ; Arévalo et al, ; Bell, Pereda, & Faber, ; Brüning, Katzbach, & Mayer, ; Giráldez‐Pérez, Gaytan, & Pasaro, ; Giráldez‐Pérez, Gaytan, Ruano, Torres, & Pasaro, ; Holmqvist et al, ; Holmqvist, Ostholm, Alm, & Ekstrom, ; Singru, Sakharkar, & Subhedar, ; Villani & Guarnieri, ; Virgili, Poli, Beraudi, Giuliani, & Villani, ).…”

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

“…After rinsing 3 times in PB, the sections were mounted on glass slides and coverslipped with UltraCruz TM mounting medium containing DAPI for fluorescence labeling of DNA (Santa Cruz Biotechnology, sc-24941). The specificity of the TH and 5-HT antisera has been previously described [Morona et al, 2013;González et al, 2014;López et al, 2017], and the pattern of staining observed for NPY was consistent with that reported for L. ocu latus [Chiba, 2005].…”

“…The nomenclature is the same used in our previous studies on holosteans [Morona et al, 2013;López et al, 2017], and essentially follows previously used terms in these species [Northcutt, 1982;Parent and Northcutt, 1982;Northcutt, 1991a, 1991b;Butler and Northcutt, 1992;Northcutt and Butler, 1993;Heijdra and Nieuwenhuys, 1994;Collin and Northcutt, 1995;Meek and Nieuwenhuys, 1998;Chiba and Oka, 1999;Baker and Bird, 2002;Chiba, 2005], but incorporates the terminology of the prosomeric model for the forebrain Rubenstein, 2003, 2015].…”

“…We have described the cholinergic [Morona et al, 2013] and nitrergic systems [López et al, 2017] and some transcription factors in the subpallium of holosteans . 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).…”