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Circadian rhythms in mammals are entrained to the environmental light cycle by daily adjustments in the phase of the circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Brief exposure of hamsters maintained under constant darkness to ambient light during subjective nighttime produces both phase shifts of the circadian activity rhythm and characteristic patterns of c-fos protein (Fos) immunoreactivity in the SCN. In this study, we demonstrate that light-induced phase shifts of the circadian activity rhythm are blocked by intracerebroventricular (i.c.v.) injection of the competitive nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME), but not by the inactive isomer, D-NAME. The effects of L-NAME are reversible and dose-related, and are countered by co-injection of arginine, the natural substrate for NOS. While effects on behavioral rhythms are pronounced, similar treatment does not alter the pattern of light-induced Fos immunoreactivity in the SCN. These results suggest that nitric oxide is a component of the signal transduction pathway that communicates photic information to the SCN circadian pacemaker, and that nitric oxide production is either independent of, or downstream from, pathways involved in induction of c-fos expression.

The mammalian suprachiasmatic nuclei (SCN) contain a circadian pacemaker that continues to keep 24-h time when isolated in vitro. We are investigating the role of cAMP in the cellular mechanisms underlying SCN function. We have previously shown that increasing intracellular cAMP during the subjective day resets the SCN pacemaker in the in vitro rat brain slice preparation. We now report that the level of cAMP fluctuates within the rat SCN under constant conditions in vitro. The level of endogenous cAMP is high during late day and late night, and low during early night. These changes in cAMP concentration are accompanied by opposite changes in phosphodiesterase activity; we detected no significant change in adenylate cyclase activity. These results provide further support for the hypothesis that cAMP is involved in circadian function in the SCN.

The environmental photoperiod regulates the synthesis of melatonin by the pineal gland, which in turn induces daily and seasonal adjustments in behavioral and physiological state. The mechanisms by which melatonin mediates these effects are not known, but accumulating data suggest that melatonin modulates a circadian biological clock, either directly or indirectly via neural inputs. The hypothesis that melatonin acts directly at the level of the suprachiasmatic nucleus (SCN), a central mammalian circadian pacemaker, was tested in a rat brain slice preparation maintained in vitro for 2-3 days. Exposure of the SCN to melatonin for 1 h late in the subjective day or early subjective night induced a significant advance in the SCN electrical activity rhythm; at other times melatonin was without apparent effect. These results demonstrate that melatonin can directly reset this circadian clock during the period surrounding the day-night transition.

Neurons of the suprachiasmatic nuclei (SCN) of the hypothalamus compose a primary oscillator which organizes circadian rhythms in mammals. In cultured hypothalamic slices from rat brain, the SCN diurnal oscillation in neuronal firing rate continued unperturbed when slices were prepared during the light phase of the donor's light/dark cycle. However, when slices were prepared during the donor's dark period, the rhythm was phase-shifted. The sign and shape of the phase-response relationship for resetting in the isolated oscillator is very similar to that for intact animals, except that in isolation the SCN oscillator undergoes large shifts during the first cycle. The finding that a phase-shifting stimulus at the time of brain slice preparation causes normal phase readjustment in vitro demonstrates that the underlying mechanism is endogenous to the SCN and can be probed in the brain slice.

Cellular mechanisms underlying the primary circadian pacemaker in mammals were investigated by isolating rat suprachiasmatic nuclei in brain slices and maintaining them in vitro for up to 3 days. The circadian rhythm of neuronal firing rate was used to assess the phase of the pacemaker. This rhythm was rapidly reset by bath application of cAMP analogs. Moreover, the pacemaker demonstrated circadian sensitivity to analog treatment: the rhythm was advanced by application during the donor's day, but not during the donor's night. These results suggest that cAMP-mediated events may stimulate pacemaker afferents within the SCN or may directly influence the pacemaker mechanism.