Topographical relationship between neuronal nitric oxide synthase immunoreactivity and cyclic 3',5'-guanosine monophosphate accumulation in the brain of the adult Xenopus laevis.

Previous immunohistochemical staining procedures of the brain and pituitary in Xenopus laevis, using an antiserum against neuronal nitric oxide (NO) synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry, have revealed NOS activity in neurons and fibers in a number of brain areas, as well as in fibers in the pituitary. In the present study we have localized the target structures of the NOergic system in the Xenopus brain by visualizing the sites of NO-sensitive cyclic 3',5'-guanosine monophosphate (cGMP) accumulation, according to a method for cGMP visualization in rat brain slices. Brain slices of unfixed Xenopus are incubated in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine and the NO donor sodium nitroprusside, followed by fixation and cryosectioning. Sections were then processed for immunohistochemistry using rabbit and sheep antisera against cGMP and a sheep antiserum against nNOS. Visualization of single and double labeling of cGMP immunoreactive and/or nNOS immunoreactive structures was performed with combined CY3/fluorescein isothiocyanate fluorescence microscopy. Following this procedure, we provide immunohistochemical evidence for the distribution of cGMP-accumulating neurons in the brain of adult Xenopus. In most brain areas, the distribution of nNOS and cGMP immunoreactive structures (neuron somata and fibers) is distinct and separate, for instance in the dorsal pallium, the lateral thalamic nuclei, the optic tectum, the locus coeruleus and the reticular formation. However, nNOS and cGMP immunoreactive structures are often found in the vicinity of each other, and in the optic tectum even in adjacent neuron fibers and somata. The present observations are in line with the presence of an NO-dependent soluble guanylate cyclase in distinct brain areas of Xenopus laevis, corroborating similar data in the mammalian brain. Further, our observations may add to the understanding of the anatomical connectivity pattern and functional relevance of the NOergic system in the amphibian brain.