Distribution of NADPH-diaphorase staining and light-induced Fos expression in the rat suprachiasmatic nucleus region supports a role for nitric oxide in the circadian system

Abstract

Nitric oxide serves as a messenger molecule in some neuronal systems that use glutamate as a transmitter and it has been shown that glutamate mediates the transmission of photic signals by retinal ganglion cell axons terminating in the hypothalamic suprachiasmatic nucleus, site of the circadian pacemaker in rodents. Recent experiments have demonstrated that pharmacological treatments which block nitric oxide synthesis by nitric oxide synthase prevent glutamate-induced phase shifts of the cell firing rhythm in suprachiasmatic nucleus slice preparation in vitro; similar treatments were found to inhibit light transmission to the suprachiasmatic nucleus as well as light-induced phase shifts in activity rhythms in vivo, implicating nitric oxide in circadian light signalling in vivo. There is limited information, however, about the presence and function of nitric oxide synthase-containing neurons within retinorecipient regions of the rodent suprachiasmatic nucleus. In the present study we used NADPH-diaphorase histochemistry and immunostaining for the nuclear phosphoprotein Fos to assess the co-distribution of nitric oxide synthase-containing neurons and light-responsive cells in the rat suprachiasmatic nucleus region. A strong convergence between NADPH-diaphorase-stained cell bodies and fibres and cells that expressed Fos in response to photic stimulation was noted in the anterior periventricular nucleus, suprachiasmatic preoptic nucleus, retrochiasmatic area, the inter-suprachiasmatic nucleus region, and the dorsal aspect of the optic chiasm, below the suprachiasmatic nucleus. A similar convergence between NADPH-diaphorase-stained fibres and Fos-immunoreactive cells was noted inside the suprachiasmatic nucleus, but the number of NADPH-diaphorase-stained elements found in this region was substantially low compared with that found in retinorecipient regions bordering the nucleus. In many cases both inside and outside the suprachiasmatic nucleus, the Fos-immunoreactive cells appeared to make direct contact with NADPH-diaphorase-stained cells or fibres, but no co-localization of Fos immunoreactivity and NADPH-diaphorase histochemical activity within individual cells was detected. Extensive co-distribution of NADPH-diaphorase-stained cells and fibres and cells that express Fos in response to photic stimulation in the suprachiasmatic nucleus region is in line with the hypothesis that nitric oxide participates in the mechanism mediating circadian light signalling in the suprachiasmatic nucleus. However, lack of co-localization of the two markers to individual cells rules out the possibility that retinorecipient cells in the suprachiasmatic region synthesize and release nitric oxide when photically-activated. Instead, the results support the possibility that photic stimulation triggers nitric oxide synthesis in nitric oxide synthase-containing neurons located near the photically-activated cells.