Distribution and colocalization of choline acetyltransferase immunoreactivity and NADPH diaphorase reactivity in neurons within the medial septum and diagonal band of Broca in the rat basal forebrain

Abstract

NADPH diaphorase histochemistry and choline acetyltransferase immunocytochemistry were used to assess quantitatively the presence of nitric oxide synthase in the cholinergic neurons of the magnocellular basal forebrain complex. Virtually all (97%) NADPH diaphorase reactive magnocellular neurons in the medial septum and the vertical and horizontal limbs of the diagonal band of Broca were choline acetyltransferase immunoreactive, whereas only a proportion of the choline acetyltransferase immunoreactive neurons were NADPH diaphorase reactive. Thus NADPH diaphorase histochemistry identified a subpopulation of the magnocellular cholinergic neurons. Occasionally, NADPH diaphorase reactive neurons were observed within the medial septum and diagonal band of Broca that were not choline acetyltransferase immunoreactive, and in general were morphologically distinct from the magnocellular neurons; such neurons are probably representatives within the medial septum and diagonal band of more widely distributed phenotypically distinct populations of NADPH diaphorase reactive neurons. The proportions of the neurons in which choline acetyltransferase and NADPH diaphorase colocalized in the medial septum and in the diagonal bands of Broca were similar in any one coronal section, but there was a considerable difference in the proportions throughout the rostrocaudal extent of these nuclei. In the most rostral sections of the medial septum and diagonal band, approximately 70% of the choline acetyltransferase immunoreactive neurons were NADPH diaphorase reactive, whereas the proportion decreased progressively to about 30% at the level of the decussation of the anterior commissure. To examine further the extent of colocalization throughout the magnocellular basal forebrain complex, sections of the magnocellular preoptic nucleus, substantia innominata, and nucleus basalis magnocellularis were examined. While there was little total colocalization of choline acetyltransferase immunoreactivity and NADPH diaphorase reactivity in any particular section (approximately 18%), almost all of the double labelled neurons were in the substantia innominata, with very few in the other nuclei. Thus although there is a caudal to rostral gradient of the proportion of magnocellular cholinergic neurons that are NADPH diaphorase reactive throughout the entire basal forebrain magnocellular complex, subregions, such as the substantia innominata and magnocellular preoptic nucleus, may not follow this trend. The recent demonstration that the NADPH diaphorase histochemical reaction localizes a nitric oxide synthase suggests that attention should be given to the NADPH diaphorase subpopulation in pathological and experimentally induced alterations of the basal forebrain.