Histochemistry (1991) 95:397-402 030155649100025Q Histochemistrv © Springer-Verlag 1991 ~ The innervation of the gastrointestinal tract of a chelonian reptile, Pseudemys scripta elegans I. Structure and topography of the enteric nerve plexuses using neuron-specific enolase immunohistochemistry* J.-P. Timmermans 1, D.W. Scheuermann 1, R. Gabriel 2, D. Adriaensen 1, E. Fekete 2, and M.H.A. De Groodt-LasseeP 1 Institute of Histology and Microscopic Anatomy, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium 2 Department of Zoology, Jozsef Attila University, Sz6ged, Hungary Accepted November 12, 1990 Summary. The general morphological features of the in- tramural enteric nervous system of a chelonian species, i.e. the red-eared turtle, Pseudemys scripta elegans, has been studied in whole-mounts and cryosections by means of neuron-specific enolase immunohistochemis- try. A clear visualization of both neuronal cell bodies and nerve fibres allows the recognition of a myenteric plexus as well as a submucous plexus in several gut re- gions, namely the stomach, midgut and hindgut. The highest innervation density was found in the midgut por- tion. In contrast to other lower vertebrates, such as am- phibians and other reptilian groups, the present study clearly demonstrates the occurrence of neuronal cell bodies in the submucous plexus of all regions investigat- ed. The neurons stained for neuron-specific enolase har- boured smooth-contoured perikarya from which one or more processes emerge, as demonstrated for the mam- malian enteric nervous system. Introduction Heretofore, the knowledge of the general morphology of the enteric nervous system (ENS) is mainly deduced from studies on mammals. These are small laboratory animals (for review see Gabella 1979; Furness and Costa 1987), larger mammals (Gunn 1968; Stach 1977a, b, 1989; Mannl etal. 1986; Scheuermann etal. 1989a; Timmermans et al. 1989, 1990), and man (Hoyle and Burnstock 1989). In 1933 Kondratjew briefly com- mented on the presence of a submucosal nerve network in a turtle. Other, more recent, studies on the intramural innervation of the reptilian gastrointestinal tract dealt exclusively with such restricted topics as the demonstra- tion of adrenergic nerve fibres in the large intestine of a lizard (Read and Burnstock 1968), of bombesin immu- * Supported by grant no. 3.0005.89 (to D.W.S.) from the Fund for Medical Scientific Research (Belgium) Offprint requests to: D.W. Scheuermann noreactivity in the gut of a caiman (Holmgren et al. 1989), of VIP-immunoreactivity in the small intestine of a viper (Masini 1986) and of a number of regulatory peptides in the gastrointestinal tract of an alligator (Bu- chan et al. 1983). None of these investigations, however, discuss the general morphological features of the differ- ent enteric nerve networks in the reptiles studied. In the last decade, the study of the architecture and the structure of the ENS of lower vertebrates (for review see Nilsson and Holmgren 1989) and of some inverte- brates (Gabriel et al. 1988) has proved to be valid from an evolutionary and functional point of view because of the considerably lower number of intrinsic neurons involved. From a comparative morphological viewpoint, it is rather surprising that the reptiles, and in particular the chelonians, have been ignored so far. The present study offers the first extensive description of the archi- tecture and the topographical organization of the ENS of a chelonian species, i.e. the red-eared turtle, Pseu- demys scripta elegans, by means of immunocytochem- istry using an antiserum raised against neuron-specific enolase (NSE). Materials and methods Specimens of both sexes of the red-eared turtle, Pseudemys scripta elegans, weighing 10-400 g, were obtained from the Antwerp Zoo- logical Garden and from commercial dealers. The animals were fed ad libitum. Anesthesia was performed by an intraperitoneal injection of Nembutal (30 mg/kg body weight). Segments from several regions of the gastrointestinal tract, i.e. the stomach, midgut and hindgut, were prelevated, ligated and filled with phosphate buffered saline (PBS). The samples were then immersed in 4% paraformaldehyde containing 0.2% picric acid in phosphate buffer (pH = 7,2 ; 0.01 M) for 2 h at room temperature and subsequently processed as described earlier (Scheuermann et al. 1987). Accordingly, the whole-mount preparations contained either (1.) the tela submucosa or (2.) the tela submucosa with the circular muscle layer and the adhering myenteric plexus or (3.) the longitudinal muscle layer with the adhering myenteric plexus. The tissues were preincubated in 10% normal donkey serum (017- 000-121, Jackson Immunoresearch Laboratories) for 30 min, fot-