The tiny suprachiasmatic nucleus (SCN) of the hypothalamus plays a central role in the daily programming of organismic functions by regulating day-to-day oscillations of the internal milieu and synchronizing them to the changing cycles of day and night and of body state. This biological clock drives the daily expression of vital homeostatic functions as diverse as feeding, drinking, body temperature, and neurohormone secretion. It adaptively organizes these body functions into near-24-hour oscillations termed circadian rhythms. The SCN imposes temporal order 1) through generating output signals that relay time-of-day information, and 2) through gating its own sensitivity to incoming signals that adjust clock timing. Each of these properties, derived from the timebase of the SCN's endogenous near-24-hour pacemaker, persists when the SCN is maintained in a hypothalamic brain slice in vitro. Single-unit recording experiments demonstrate a spontaneous peak in the electrical activity of the ensemble of SCN neurons near midday. By utilizing this time of peak as a "pulse" of the clock, we have characterized a series of time domains, or windows of sensitivity, in which the SCN restricts its own sensitivity to stimuli that are capable of adjusting clock phase. Pituitary adenylyl cyclase-activating peptide (PACAP) and cAMP comprise agents that reset clock phase during the day time domain; both PACAP and membrane-permeable cAMP analogs cause phase advances only when applied during the day. In direct contrast to PACAP and cAMP, acetylcholine and cGMP analogs phase advance the clock only when applied during the night. Sensitivity to light and glutamate arises concomitant with sensitivity to acetylcholine and cGMP. Light and glutamate cause phase delays in the early night, by acting through elevation of intracellular Ca2+, mediated by activation of a neuronal ryanodine receptor. In late night, light and glutamate utilize a cGMP-mediated mechanism to induce phase advances. Finally, crepuscular domains, or dusk and dawn, are characterized by sensitivity to phase resetting by the pineal hormone, melatonin, acting through protein kinase C. Our findings indicate that the gates to both daytime and nighttime phase resetting lie beyond the level of membrane receptors; they point to critical gating within the cell, downstream from second messengers. The changing patterns of sensitivities in vitro demonstrate that the circadian clock controls multiple molecular gates at the intracellular level, to assure that they are selectively opened in a permissive fashion only at specific points in the circadian cycle. Discerning the molecular mechanisms that generate these changes is fundamental to understanding the integrative and regulatory role of the SCN in hypothalamic control of organismic rhythms.
