Presence of neuronal nitric oxide synthase in the suprachiasmatic nuclei of mouse and rat

Abstract

Nitric oxide has recently been identified as a major neural regulator. It is synthesized by the enzyme nitric oxide synthase. Whilst considerable functional evidence has pointed to an involvement of nitric oxide in circadian regulation, all previous morphological studies have failed to demonstrate the presence of nitric oxide synthase in the mammalian suprachiasmatic nucleus. By use of an antibody directed against whole recombinant rat neuronal nitric oxide synthase we have identified the presence of immunoreactivity for this enzyme in the suprachiasmatic nucleus of mouse and rat, to provide the first detailed report of this enzyme in the suprachiasmatic nucleus for any species of mammal. Immunoreactivity for neuronal nitric oxide synthase was found in neurons throughout the suprachiasmatic nucleus of mice and no difference could be detected between the ventrolateral and dorsomedial parts of the nucleus in terms of the optical density of the immunostaining. Very small, rounded neuronal cell bodies were immunopositive. Electron microscopy revealed that these neurons had relatively large nuclei and scant cytoplasm containing relatively few organelles, which sometimes included some 100 nm dense-cored peptidergic vesicles. Only about 5% of such neurons were not detectably immunoreactive. By contrast, in the rat suprachiasmatic nucleus, a much smaller number of neurons were immunopositive and these cells were aggregated in the ventrolateral part of the nucleus. The immunoreactive neurons were bipolar cells with scanty cytoplasm. Electron microscopy revealed diffuse immunoreactivity in the cytosol, but not within any organelles. In the surrounding neuropil immunoreactive dendrites and axons mingled with much larger numbers of immunonegative processes, but immunoreactive boutons were only identified just outside the dorsal margin of the nucleus. Astrocytes, oligodendrocytes and endothelial cells in the suprachiasmatic nucleus were immunonegative. NADPH-diaphorase activity was not detectable in the suprachiasmatic nucleus of either mouse or rat. This morphological evidence for nitric oxide synthase-immunoreactive cells in the suprachiasmatic nucleus supports the existing functional evidence for an involvement of nitric oxide in the transmission of light-induced signals to the suprachiasmatic nucleus in mammals.