Ultrastructural localization of neuronal nitric oxide synthase in the laterodorsal tegmental nucleus of wild-type and knockout mice.

摘要

The cellular and subcellular distribution of neuronal nitric oxide synthase and its related reduced beta-nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was compared in wild-type and homozygous knockout mice, in which the gene for neuronal nitric oxide synthase has been disrupted, resulting in a lack of the predominant splice isoform alpha. In the laterodorsal tegmental nucleus, used as a model structure, the cholinergic principal neurons also exhibited an intensive neuronal nitric oxide synthase immunoreactivity. Using the tetrazolium salt 2-(2-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl)-tetrazo++ +-lium chloride (BSPT), these neurons were filled with NADPH-diaphorase reaction product, whereas the equivalent neurons of knockout mice showed, if at all, only traces of neuronal nitric oxide synthase immunoreactivity in parallel to a diminished NADPH-diaphorase labelling. Subcellularly, the neuronal nitric oxide synthase-related diaminobenzidine product was, apparently owing to diffusion artifact, more or less evenly distributed in the cytosol of the neuronal perikarya and dendrites of wild-type mice. In contrast, the BSPT reaction product formazan was closely and discretely attached to endocellular membranes. In the intensely NADPH-diaphorase stained neurons of wild-type mice, 85% of the mitochondria were, at least partly, labelled for BSPT-formazan, whilst in the equivalent neurons of mutant mice, only 13% of mitochondria were NADPH-diaphorase positive. Related to the NADPH-diaphorase activity in the principal neurons of wild-type mice, only 10% of membranes of the endoplasmic reticulum, 27% of mitochondrial membranes and 26% of the nuclear envelope exhibited NADPH-diaphorase activity in the mutant mice. Our findings with the BSPT histochemistry suggest that residues of NADPH-diaphorase positivity in mutant mice are attributed to the alternative splice isoforms beta and/or gamma of neuronal nitric oxide synthase. The splice isoform a is located predominantly at the membranes of the endoplasmic reticulum.