Windows in the clock: An in vitro study of melatonin action and signal transduction in the suprachiasmatic circadian pacemaker

A number of studies have suggested that the pineal hormone, melatonin, may act at the level of the suprachiasmatic nuclei (SCN) in mammals to regulate the functioning of this primary circadian pacemaker. By isolating the rat SCN in vitro in a hypothalamic brain slice preparation, we have demonstrated a direct resetting of the SCN pacemaker's rhythm of ensemble neuronal firing rate in response to exogenous melatonin treatment. This effect depends upon the phase of the pacemaker at the time of treatment: maximal responses to 1 hr treatment are seen at the times of transition in the animals' environmental lighting cycle. Bath application of 10$\sp{-9}$ M melatonin advanced the time-of-peak spontaneous electrical activity by 2-4 hr during a broad period surrounding lights-off at circadian time (CT) 10-14, while a more narrow window of sensitivity was found just prior to lights-on. Further, melatonin utilizes a pertussis toxin (PTX)-sensitive G protein pathway to reset the SCN pacemaker. A 6-hr pre-incubation with 1$\mu$g/ml PTX completely blocked melatonin's phase-advance at CT 10. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), directly resets the SCN firing rate rhythm with a congruent profile of temporal sensitivity, suggesting that melatonin may activate protein kinase C (PKC) to alter pacemaker function. The PKC inhibitor, staurosporine, completely blocks both melatonin and TPA phase-advances at each sensitive period, strengthening this hypothesis. However, melatonin did not alter forskolin-stimulated cAMP levels, nor SCN IP$\sb3$ levels, at CT 6, 10, or 23. These data indicate that melatonin directly resets the suprachiasmatic clock in vitro with two windows of sensitivity which correspond to the times of transition in the animals' environmental lighting cycle. Further, melatonin alters SCN cellular function via PTX- sensitive G protein pathway(s) that activate PKC and appears to be separate from influence of cyclic nucleotide and IP$\sb3$ pathways.U of I OnlyETDs are only available to UIUC Users without author permissio