Regional study of the co-localization of neuronal nitric oxide synthase with muscarinic receptors in the rat cerebral cortex

Abstract

There is increasing evidence that nitric oxide is an important molecular messenger involved in a wide variety of biological processes including the regulation of the cerebral circulation. For instance, it has been implicated in the vascular response to nucleus basalis magnocellularis stimulation, a structure which is widely recognized as the predominant source of cholinergic fibres projecting to the neocortex. The present investigation was carried out to determine if muscarinic receptors are present on cortical neurons expressing neuronal nitric oxide synthase (nitric oxide-producing enzyme). To this aim, double labelling of both neuronal nitric oxide synthase/vessels and neuronal nitric oxide synthase/muscarinic receptors was performed on free-floating cryosections obtained from rat brain. The observations were made by confocal laser scanning microscopy. The double labelling of neuronal nitric oxide synthase with the arterioles demonstrated the presence of nitroxidergic fibres in the wall of intraparenchymal vessels. A rich network of nitroxidergic fibres independent of the vessels was also seen in the parenchyma. Since the maximal surface of a square of tissue without any nitroxidergic fibres corresponded to 1400 +/- 105 microns2, the distance separating any cortical point from its closest neuronal nitric oxide synthase-positive fibre was never higher than 25 microns (half diagonal of square). According to models of the diffusional spread of nitric oxide, it is likely that nitric oxide can reach the whole cortical volume. Our results on the regional study of neuronal nitric oxide synthase/muscarinic receptors showed a high density of neuronal nitric oxide synthase-positive neurons principally in the frontal and perirhinal cortices and a low density in the occipital cortex. These data fit well with the known pattern of cortical projections from the nucleus basalis magnocellularis as revealed by anterogradely transported markers. The double labelling showed that about 10% of neuronal nitric oxide synthase-positive neurons were co-localized with muscarinic receptors in the frontoparietal cortex. In agreement with previous papers, the vascular innervation by nitroxidergic neuronal processes was often found to lie near the branching points of arterioles. Such localization allows neuronal nitric oxide synthase-positive neurons an extensive control of the vascular tree without requiring a large number of neuronal commands. Therefore, despite the low level of neuronal nitric oxide synthase/muscarinic receptor co-localization, this neuronal subpopulation could represent a possible relay implicated in the vascular effects of the nucleus basalis magnocellularis.