&p.1:Abstract The distribution of neurons containing the en- zymes NADPH-diaphorase (NADPH-d) and nitric oxide synthase (NOS) has been studied in the gastrointestinal tract of lizard (Podarcis s. sicula) and snake (Thamnop- his sirtalis). The techniques employed were the NADPH- d/nitroblue tetrazolium histochemical method, and the indirect immunofluorescence applied to cryostat sections and to whole-mount preparations. The colocalization of NADPH-d with NOS, with vasoactive intestinal polypep- tide (VIP) and with galanin (Gal) was also studied, and a Western blot analysis using an antibody directed against mammalian Gal was performed on lizard stomach ex- tracts. NADPH-d positive nerve cell bodies and fibres were found in the myenteric and submucous plexuses throughout the gastrointestinal tract of both reptiles. These nerve structures were also present in the other in- tramural nerve plexuses, although in smaller quantities. Both in lizard and snake, the stomach revealed a positive nerve population that was more dense than elsewhere in the gut. The population of the NADPH-d-positive neu- rons observed in the lizard was larger than that observed in the snake. The distribution of both populations was similar to those that have been described in the gut of several mammalian and non-mammalian vertebrates. Both in lizard and snake, a one-to-one correspondence was noted between NOS- and NADPH-d-containing nerve cell bodies, and the nitrergic neurons containing Gal appeared to be more numerous than those containing VIP. Western blot analysis recognised a single band with a molecular weight (3.4 kDa) very similar to that of por- cine Gal. It is hypothesised that at least some of the ni- trergic neurons of the lizard and snake gut are inhibitory motor neurons innervating the circular smooth muscula- ture. In addition, the colocalization of NOS and VIP in neurons enhances their inhibitory action. The role of the neurons containing both NOS and Gal remains unknown. &kwd:Key words Reptiles · Gastrointestinal tract · Nitric oxide · VIP · Galanin&bdy: Introduction Nicotinamide adenine dinucleotide phosphate reduced diaphorase (NADPH-d) and nitric oxide synthase (NOS) are enzymes contained in the nitrergic neurons of the central and peripheral nervous system, i.e. those that pro- duce nitric oxide (NO). The discovery that these two en- zymes are colocalized in the nitrergic neurons was the first step in making clear that they form parts of a single molecule (Dawson et al. 1991; Hope et al. 1991). Conse- quently, the histochemical method for detecting NADPH-d enzymatic activity is used for detecting NOS in neuronal populations. The myenteric nitrergic neurons of the mammalian gastrointestinal tract are non-adrenergic, non-cholinergic cells (Belai et al. 1992; Ward et al. 1992; Young et al. 1992); they project towards the circular muscle of the splanchnic organs, and their axons are craniocaudally orientated. Functionally, they are considered to be inhibi- tory motor neurons (Bult et al. 1990; Li and Rand 1990; Costa et al. 1992; McConalogue and Furness 1993). As is well known, the impulse of inhibitory neurons is medi- ated not only by NO, but also by neuropeptides such as vasoactive intestinal polypeptide (VIP), one of the most studied (Li and Rand 1990; Fahrenkrug 1991). VIP and NOS have often been found colocalized in nerve cell bodies and fibres intrinsic to the gastrointestinal tract (Costa et al. 1992; Aimi et al. 1993). Recently, galanin (Gal), a 29 amino acid peptide abundantly present in gut neurons has been found to play an inhibitory role in gas- C. Lamanna · A. Costagliola · A. Vittoria · A. Cecio ( ✉ ) Department of Biological Structures, Functions and Technology, University of Naples “Federico II”, Via Veterinaria 1, I-80137 Naples, Italy e-mail: cecio@unina.it B. Mayer Institute of Pharmacology and Toxicology, Karl-Franzens University, Graz, Austria L. Assisi · V. Botte Department of Zoology, University of Naples “Federico II”, Naples, Italy&/fn-block: Anat Embryol (1999) 199:397–405 © Springer-Verlag 1999 ORIGINAL ARTICLE &roles:C. Lamanna · A. Costagliola · A. Vittoria · B. Mayer L. Assisi · V. Botte · A. Cecio NADPH-diaphorase and NOS enzymatic activities in some neurons of reptilian gut and their relationships with two neuropeptides &misc:Accepted: 18 September 1998