SUBCELLULAR-LOCALIZATION OF THE NEURONAL ISOFORM OF NITRIC-OXIDE SYNTHASE IN THE RAT-BRAIN - A CRITICAL-EVALUATION

In the aldehyde-fixed rat brain NADPH-diaphorase is suggested to be re lated to brain nitric oxide synthase but also to other isoforms of thi s enzyme as well as to several non-related types of NADPH-oxidoreducta ses. In this study NADPH-diaphorase histochemistry using the tetrazoli um salt BSPT olyl)-5-styryl-3-(4'-phthalhydrazidyl)-tetrazolium chlori de) (to yield an electron dense formazan) and immunocytochemistry were applied for the cellular and subcellular localization of brain nitric oxide synthase in the striatum and the pontine laterodorsal tegmental nucleus of the rat. Combining the two techniques, in both brain regio ns identical distribution patterns of heavily-stained neurons were obs erved at the light microscopic level. There are inconsistencies in the literature with regard to the subcellular localization of brain nitri c oxide synthase and NADPH-diaphorase in neurons. In our results brain nitric oxide synthase immunoreactivity in abundantly stained neurons was mainly cytosolically distributed, sometimes in a patch-like form a nd distant fi om membranes, whereas the NADPH-diaphorase reaction prod uct BSPT-formazan was closely attached to discrete portions of intrace llular membranes. Other neurons and glial cells including their proces ses showed also, but to a lesser extent, formazan-labelled membrane po rtions. In such cell populations brain nitric oxide synthase immunorea ctivity was not detectable. Possible reasons for these inconsistencies are discussed in detail. The strength but not the specificity of the NADPH-diaphorase related reaction was shown to be dependent on concent rations of Triton X-100 and tetrazolium salt. We suggest that, for ele ctron microscopical cytochemistry, the BSPT technique combined with ot her independent techniques, such as immunocytochemistry and in situ hy bridization, may be a viable means for the identification and subcellu lar localization of the different nitric oxide synthase isoforms, and to discriminate them from other types of NADPH-diaphorases. (C) 1997 I BRO. Published by Elsevier Science Ltd.