NADPH-diaphorase reactive pyramidal neurons in Ammon's horn and the subiculum of the rat hippocampal formation

NADPH-diaphorase histochemistry has been shown to stain cells which contain nitric oxide synthase, an enzyme responsible for the biosynthesis of the freely diffusable gas nitric oxide. A number of studies have mapped the distribution of NADPH-diaphorase-reactive neurons in the hippocampal formation but they have failed to yield consistent data. The major point of controversy concerns the presence of NADPH-diaphorase-reactive pyramidal cells in the CA1 subfield of the rat hippocampal formation. The present results show that CA1 pyramidal neurons do contain nitric oxide synthase (NOS) which can be reliably demonstrated with the appropriate histochemical procedure. One of the critical determinants of CA1 pyramidal cell NADPH-diaphorase activity is shown to be incubation of brain's in sucrose solution prior to histochemical processing. Subicular pyramidal cells were also found to contain NOS and to possess NADPH-diaphorase activity. These results explain a number of contradictory reports in the literature relating to the presence of NADPH-diaphorase activity in hippocampal principal cells. Additionally, densitometric analysis carried out on 20 μm thick sections, from brains incubated in sucrose solution, indicated that there were characteristic gradients. The intensity of NADPH-diaphorase activity in pyramidal cells located in the ventral subiculum was found to be greater than those in the dorsal subiculum. A similar, yet marginal, trend was apparent for pyramidal cells in CA1 and CA3, as well as nonpyramidal cells in CA1. At both dorsal and ventral levels, NADPH-diaphorase-positive subicular pyramidal cells and CA1 nonpyramidal cells also show a greater intensity than CA1 or CA3 reactive pyramidal neurons. This study also showed that tissue incubation in sucrose solution prior to immunocytochemistry, enhanced immunoreactivity of the endothelial isoform of NOS whilst having little effect on neuronal NOS reactivity.