Neuronal nitric oxide synthase (nNOS) immunoreactivity in the olfactory system of a cartilaginous fish Sara Ferrando a, *, Lorenzo Gallus a , Chiara Gambardella a , Andrea Amaroli a , Alessia Cutolo a , Maria Angela Masini a , Mauro Vallarino a , Marino Vacchi b a DipTeRis, University of Genoa, Viale Benedetto XV 5, I-16132 Genoa, Italy b ISPRA, c/o Museo Nazionale dell’Antartide, University of Genoa, Viale Benedetto XV 5, I-16132 Genoa, Italy 1. Introduction Two systems are devoted to olfaction in vertebrates: the olfactory and the vomeronasal ones. In tetrapod, they are anatomically distinct while, in fish, they are morphologically lodged in the same organs, and they are commonly called ‘‘olfactory system’’ even if they bear both olfactory and vomer- onasal features (e.g. Hansen et al., 2003). Compared to the olfactory system of other fishes, the olfactory system of Chondrichtyans is characterized by some peculiar lacks: their olfactory epithelium (OE) lacks of ciliated sensory neurons (Holl, 1973; Theisen et al., 1986; Franceschini and Ciani, 1993; Takami et al., 1994); in their olfactory bulb (OB), the periglo- merular cells are absent or rare (Rodrı ´guez-Moldes et al., 1993; Dryer and Graziadei, 1996); the two main olfactory receptor families, olfactory receptors (ORs) and trace-amino associated receptors (TAARs), are almost missing in the genome of the elephant shark Callorhinchus milii (Grus and Zhang, 2009; Niimura, 2009); immunohistochemical detection of the olfactory-type G protein alpha subunit (a marker of the olfactory system) always failed to find any immunoreactivity (ir) in the olfactory system of investigated species (Ferrando et al., 2009, 2010a). Nitric oxide (NO) is a gas that in living organisms acts as a messenger and is likely to have important roles in olfaction, such as the regulation of neuronal precursor proliferation and synaptic maintenance (Chen et al., 2004), memory consolidation (Ju ¨ ch et al., 2009) and regulation of neurotransmitters release or uptake (Crespo et al., 2003). NO is synthesized from arginine by the enzymes NO synthases (NOS) and, in particular in the nervous system, by the neural NOS (nNOS) (Garthwaite, 2008). Although the fundamental role of NO in the olfactory system, the distribution of nNOS in the olfactory and vomeronasal systems of vertebrates is quite variable. In mammals, nNOS is present in the OB (in granule cells, cells of the plexiform layer, and weakly, in some periglomerular cells) and in the vomeronasal accessory OB (AOB) (in granule cells) (Kishimoto et al., 1993; Iwase et al., 1998; Matsuda et al., 1996; Kosaka and Kosaka, 2007b; Wenisch and Arnhold, 2010). Few data are reported in the literature about reptilians and birds: immunohistochemistry and histoenzymatic reaction for NOS, show the absence of such enzyme from the olfactory and vomeronasal systems (Bru ¨ ning et al., 1994a,b; Jiang and Tera- shima, 1996). In adult amphibian, nNOS ir has been observed in the OB (in granule cells) and in the anuran AOB (in granule cells) (Porteros et al., 1996). A peculiar distribution has been found in the neotenic adult Ambystoma mexicanum, where nNOS ir has been detected in sensory receptor neurons and some basal cells (BCs) of both olfactory and vomeronasal epithelium (Sa ´ nchez-Islas and Journal of Chemical Neuroanatomy 43 (2012) 133–140 A R T I C L E I N F O Article history: Received 16 November 2011 Received in revised form 14 February 2012 Accepted 5 March 2012 Available online 26 March 2012 Keywords: Elasmobranch Shark Olfaction Gamma-aminobutyric acid Nitric oxide A B S T R A C T Nitric oxide is a regulative molecule with important roles in the olfactory system of vertebrates. Chondrichtyans have a key position in vertebrate evolution and nothing is known about nitric oxide in their olfactory system. Aim of this work was to investigate the neuronal nitric oxide synthase (nNOS) immunoreactivity in the olfactory system of the shark Scyliorhinus canicula. Because nitric oxide is often related to GABA in the olfactory system, also the distribution of GABA and its synthesis enzyme GAD has been investigated. In the olfactory epithelium scattered cells in the basal and medial zone of the epithelium thickness presented nNOS-like immunoreactivity. In the olfactory bulb the nNOS-like immunoreactivity has been highlighted in nerve fibers around some blood vessels and in scattered GABAergic granule cells. The presence of nNOS in the olfactory system of S. canicula is overall lesser than that described in other vertebrates, even if nitric oxide probably keeps some essential functions. ß 2012 Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +39 010 35338256; fax: +39 010 3538047. E-mail address: sara131274@gmail.com (S. Ferrando). Contents lists available at SciVerse ScienceDirect Journal of Chemical Neuroanatomy jo ur n al ho mep ag e: www .elsevier .c om /lo cate/jc h emn eu 0891-0618/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2012.03.005