Mammalian circadian timekeeping is a property of the suprachiasmatic nuclei (SCN) of the hypothalamus. This biological clock is responsible for the near-24 h endogenous rhythms of activity observable in all mammals. Because we live in an ever-changing environment that varies over the 24 h period of the day, internal synchronization requires daily adjustment of the SCN clock to keep its phase, and thus the animal's, in synchrony with the outside world. The effect of light, the most potent environmental timing signal, upon the clock is a function of the phase of stimulation within the 24 h cycle: Light will delay the clock during early night and advance it during late night, but is without effect during the day. How is this remarkable integration between external and internal time accomplished? Our broad objective is to understand the cellular mechanisms by which light acting through a retinohypothalamic tract (RHT) projection, regulates the SCN clock. Glutamate (GLU) is the putative transmitter of the RHT. From our preliminary studies, we have developed an hypothesis that the pathway through which light can entrain the circadian clock during late night includes: light -> RHT stimulation -> GLU release -> nitric oxide release -> transcription events -> phase shift. The thrusts of this proposal are to elucidate the initial events connecting RHT stimulation to phase shifting of the circadian clock, and to probe the differences in the RHT pathway between early and late night. Based upon our preliminary studies, the proposal has four major aims: 1. To determine whether RHT stimulation in vitro resets the clock in a light-like, GLU-dependent manner. 2. To determine whether nitric oxide synthase activation is part of this pathway. 3. To investigate the involvement of a transcriptional process. 4. To compare the role of these elements between early and late night. The SCN clock survives intact in the hypothalamic brain slice where it is accessible to experiments aimed at dissecting cellular mechanisms. the model species to be studied is the rat. The effect of treatments (afferent stimulation as well as reagents of the putative signal transduction elements applied focally at projection sites, sometimes with antagonists in the bath) upon the phase of the circadian rhythm SCN electrical activity will be determined over 2-3 days in vitro. Mechanisms of phase shifting will be probed using extracellular recording, immunocytochemistry, enzyme assays, western blots and nitric oxide measurements. Because the SCN integrate most circadian behavior, this study is basic to determining the mechanisms responsible for generation and regulation of biological timekeeping. It will have applied relevance to strategies for ameliorating internal desynchronization manifested as disordered sleep, depression, negative affective states and the physiological decline with aging, as well as chronobiological aspects of therapeutic treatment.
