Organization of physiology and behavior with recurring daily environmental conditions is an adaptation that occurs in essentially all living organisms. Circadian rhythms are regulated by three components: the circadian pacemaker, an input mechanism and an output mechanism. In mammals, the master pacemaker driving circadian rhythms resides in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. Environmental light-dark cycles entrain the SCN clock to the 24-hr day. Synchronization of SCN neurons leads to coordinated circadian outputs that regulate expressed rhythms. A clear view of molecular clock mechanisms within the SCN has emerged. The molecular clockwork of the SCN circadian clock consists of auto-regulatory transcriptional and translational feedback loops that have both positive and negative elements. Similarly, the signal pathway of how light input resets SCN clock to synchronize with the environmental light/dark cycle is also emerging. In contrast to molecular mechanisms of SCN pacemaker clockwork and input pathway, relatively little is known about the output mechanism by which the SCN circadian pacemaker sends timing information to control physiological and behavioral rhythms. Prokineticin 2 (PK2), a cysteine-rich protein, has recently been shown as a SCN output molecule that transmits the circadian locomotor rhythm. PK2 fulfill the criteria expected for a bona fide output molecule from SCN circadian clock: 1) PK2 is a secreted molecule; 2) The transcription of PK2 is regulated by core clock genes, and PK2 mRNA oscillates in the SCN with high magnitude; 3) The production of PK2 is responsive to light entrainment; 4) Receptor for PK2 is expressed in primary SCN output target areas; and 5) Intracerebroventricular (ICV) administration of PK2 at subjective night, when endogenous PK2 levels are low, suppressed high nocturnal wheel-running activity. We propose to further investigate the role of PK2 signaling in the output mechanism of the SCN circadian clock. Specifically, the rhythms of PK2 protein in the cell bodies, terminal areas of SCN neurons and cerebral spinal fluid will be investigated by quantitative immunocytochemistry and/or radioimmunoassay. Whether PK2 is the common signal that mediates the output of SCN circadian clock and light masking will be investigated. How the PK2 rhythmic output from the SCN responds to abrupt shifts of light/dark cycle will also be investigated. Moreover, the effects of PK2 on SCN circadian clock-controlled locomotor and sleep/wake rhythms will be investigated by acute and chronic infusion of PK2 and PK2 antagonist in rats. Furthermore, the PK2 gene will be disrupted in mice and its effect on SCN-controlled circadian behavioral rhythms as well as core SCN circadian loops will be examined. Finally, the SCN PK2 output pathway will be investigated by a genetic approach. These proposed studies should help us gain further insight into the mechanism of PK2 signaling in mediating the output of timing information from the SCN circadian crock, and could have a major impact on the future treatment of a number of circadian disorders such as jet-lag, shift work syndrome, and chronic insomnia ? ?
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