The development of nitric oxide synthase (NOS) expression in the brain of Xenopus laevis tadpoles was studied by means of immunohistochemistry using specific antibodies against NOS and enzyme histochemistry for nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase. Both techniques yielded identical results and were equally suitable for demonstrating the nitrergic system in the brain. The only mismatches were observed in the olfactory nerve and glomeruli and in the terminal nerve; they were intensely labeled with the NADPH-diaphorase technique but failed to stain with NOS immunohistochemistry. As early as stage 33, nitrergic cells were observed in the caudal rhombencephalon within the developing inferior reticular nucleus. At later embryonic stages, different sets of reticular and tegmental neurons were labeled in the middle reticular nucleus and, more conspicuously, in the laterodorsal and pedunculopontine tegmental nuclei. As development proceeded, new nitrergic cell groups gradually appeared in the mesencephalon, diencephalon, and telencephalon. A general caudorostral temporal sequence was observed, both in the whole brain and within each main brain subdivision. The premetamorphic period was mainly characterized by the maturation of the cell populations developed in the embryonic period. During prometamorphosis, the nitrergic system reached an enormous development, and many new cell groups were observed for the first time, in particular in the telencephalon. By the climax of metamorphosis, the pattern of organization of nitrergic cells and fibers observed in the brain was similar to that present in the adult brain. Transient expression of NOS was not detected in any brain region. Our data suggest that nitric oxide plays an important role during brain development of Xenopus. Comparison with the developmental pattern of nitrergic systems in other vertebrates shows that amphibians possess more common features with amniotes than with anamniotes.

As a further step in unraveling the organization of the basal ganglia of amphibians, the efferent connections of the striatum and the nucleus accumbens have been studied in the brains of the anurans, Rana perezi and Xenopus laevis, and the urodele, Pleurodeles waltl, by using biotinylated or fluorescent dextran amines as anterograde tracers. A common pattern of efferent connections was observed in both groups of amphibians, but those in the anurans were more elaborate. Striatal efferent fibers were found to reach the lateral and medial amygdala, the anterior and posterior entopeduncular nuclei, several thalamic nuclei, the dorsomedial posterior tubercle, the pretectum, the optic tectum, the torus semicircularis, the pontomesencephalic reticular formation, and the caudal brainstem. Efferent fibers of the nucleus accumbens project to the medial amygdala, the preoptic area, the ventral hypothalamic nucleus, the dorsomedial posterior tubercle, the medial tegmental area, the pontomesencephalic reticular formation, and the raphe. In addition, the study has revealed the existence of intrinsic connections within the ventral telencephalic wall, suggesting a possible further compartmentalization of the amphibian basal forebrain. In conclusion, the results of the present study corroborate the notion that the basal ganglia of amphibians share many features with their presumed homologues in amniotes.