Development of NADPH-diaphorase/nitric oxide synthase in the brain of the urodele amphibian Pleurodeles waltl.

In the present study, the ontogenesis of nitrergic neurons has been studied in the urodele amphibian Pleurodeles waltl by means of NADPH-diaphorase (NADPHd) histochemistry and neuronal nitric oxide synthase (NOS) immunohistochemistry. Embryonic and larval stages were studied. Except for the olfactory fibers and glomeruli, both methods were equally suitable to reveal nitrergic structures in the brain. The earliest positive neurons were observed in the inferior reticular nucleus (Ri) in the caudal rhombencephalon at embryonic stage 30. At stage 33b, weakly reactive cells appeared in the tegmentum of the mesencephalon and isthmus, in the ventral hypothalamus (VH), and in the proximity of the solitary tract (sol). At initial larval stages (stages 34-38), two new groups appeared in the caudal telencephalon (future amygdaloid complex (Am)) and in the middle reticular nucleus (Rm) of the rhombencephalon. During the active larval life (stages 39-55c) the nitrergic system developed progressively both in number of cells and fiber tracts. At stages 39-42 reactive cells were found in the inner granular layer (igl) of the olfactory bulb, the telencephalic pallium, the pretectal region, the optic tectum (OT) and retina. New populations of nitrergic cells appear during the second half of the larval period (stages 52-55). Rostrally, reactive cells were found in the telencephalic diagonal band (DB) nucleus, medial septum and in the thalamic eminence (TE), whereas caudally cells appeared in the raphe (Ra) and the descending trigeminal nucleus (Vd). The last changes occurred during the juvenile period (metamorphic climax), when cells of the spinal cord (sc) and the preoptic area became positive. The sequence of appearance of nitrergic cells revealed a first involvement of this system in reticulospinal control, likely influencing locomotor behavior. As development proceeds, cells in different sensory systems expressed progressively nitric oxide synthase in a pattern that shows many similarities with amniotes.