NADPH diaphorase histochemistry

The gaseous free radical nitric oxide (NO), a non-conventional neural messenger, is synthesized in neurons by the enzyme nitric oxide synthase (NOS), which can be revealed in histological sections by NADPH-diaphorase histochemistry or NOS immunohisto-chemistry. The role of NO in neural signaling has raised considerable interest (see, for example, Schmidt and Walter, 1994), stemming also from the finding that NOS has a discrete distribution in subsets of brain neurons, including intense expression in neuronal subsets of the striatum (Vincent, 2000). 

The existence of the semilunar nucleus was established on the basis of NADPH-diaphorase histochemistry (Paxinos etal., in press [a]). We acknowledge assistance of R. Harlan and P.-Y. Wang in the identification of this structure (Ahima and Harlan, 1990 Wang and Zhang, 1995). 

Carrive, R, and Paxinos, G. (1994). The supraoculomotor cap A region revealed by NADPH diaphorase histochemistry. NeuroReport 5, 2257-2260. 

Wu, W. (1993). Expression of nitric oxide synthase in injured CNS neurons as shown by NADPH diaphorase histochemistry. Exp. Neurol. 120, 153-159. 

Localization of Nitric Oxide Synthase Using NADPH Diaphorase Histochemistry... 

NADPH diaphorase is an enzyme that catalyzes NADPH-dependent reduction of a tetrazoUum salt, such as nitro blue tetrazohum (NBT), into an insoluble colored formazan. In theory, any NADPH-requinng enzyme could exhibit NADPH diaphorase activity. However, when NADPH diaphorase histochemistry was applied to aldehyde-fixed mammalian brain tissue, only a specific population of neurons stained (6). These NADPH-diaphorase-positive neurons did not correspond to any of the known classical neurotransmitter systems and they remained a mystery until the discovery of NO-producing neurons in the brain. 

NADPH diaphorase histochemistry is now the most widely used method for revealing the distribution of NOS in the nervous system. Using this approach, the anatomy of putative NO-producing neurons has been described in the nervous systems of several mammalian species, a variety of other vertebrate species, and invertebrates (7—11). A major advantage of the NADPH diaphorase method over such alternatives as NOS immunocytochemistry and in situ hybridization is that the only specific reagents required are NBT and NADPH, both of which can be easily purchased Moreover, to date anti-NOS antibodies have been raised only to forms of the enzyme obtained from mammals and... 

Scherer-Singler, U, Vincent, S. R., Kimura, H., and McGeer, E. G. (1983) Demonstration of a unique population of neurons with NADPH-diaphorase histochemistry. J. Neurosci. Methods 9,229-234. 

Grozdanovic, Z., Baumgarten, H. G., and Brttning, G. (1992) Histochemistry of NADPH diaphorase, a marker for neuronal nitric oxide synthase, in the penpheral autonomic nervous system of the mouse. Neuroscience 48,225-235. 

Histochemistry Several NADPH consuming enzymes including NOS show an activity similar to NADPH diaphorase. The latter uses NADPH to convert soluble tetrazolium salts into insoluble visible formazan. Nitro blue tetrazolium is a widely used blue stain for NADPH diaphorase activity, and it has been observed that staining correlates well with NOS activity. The NOS contribution to the staining cannot be quantified, as several enzymes show diaphorase activity. 

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