TEMPORALLY RESTRICTED ROLE OF RETINAL PACAP: INTEGRATION OF THE PHASE-ADVANCING LIGHT SIGNAL TO THE SCN

Photic information embedded in the alternation between days and nights directly affects circadian systems, ensuring that organisms are in tune with the period imposed by the light:dark (LD) cycle (Pittendrigh and Daan, 1976). This entrainment is a fundamental property of circadian clocks and of rhythms they regulate. In mammals, the master circadian clock is localized within the cells of the hypothalamic suprachiasmatic nucleus (SCN) (Klein et al., 1991). The SCN receives photic information directly from the retina via a retinohypothalamic tract (RHT) projection that contains glutamate and pituitary adenylate cyclase-activating peptide (PACAP) (Hannibal et al., 2000; Moore and Lenn, 1972). The current model for light-induced clock resetting proposes that glutamate and PACAP are co-released in the SCN upon photic stimulation. Whereas the role of glutamate in clock resetting has received considerable attention (Ding et al., 1994; Ebling, 1996; Gillette and Mitchell, 2002), the contribution of PACAP to the photic response of the SCN is still unclear (Morin and Allen, 2006). Multiple animal models have been designed to clarify the contribution of PACAP to circadian function. Three independently developed strains of transgenic mice with null mutations for PACAP expression have been created, as well as a knockout of PAC1R, the primary PACAP receptor (reviewed in Hannibal, 2006) (Hannibal et al., 1997; Kopp et al., 1999; von Gall et al., 1998). Circadian behavioral responses to light of two of these mouse models differ, likely due to experimental idiosyncracies and subtleties of genetic backgrounds and lesions. Specifically, a PACAP peptide-null mouse, developed in the Institute of Cancer Research (ICR) mouse eliminating exon 5 of the PACAP gene-coding sequence (Kawaguchi et al., 2003), exhibits significant attenuations of light-induced phase advance at CT 21, but no significant alteration in behavioral responses to light during the early night (Kawaguchi et al., 2003). These PACAP-null mice also express lower light-induced c-Fos at CT15 compared to WT, but, interestingly, show identical light-induced c-Fos at CT 21, where the behavioral deficits are observed. The PACAP peptide-null mouse developed by Colwell and colleagues (Colwell et al., 2004) lacks exons 3, 4, and 5a of the PACAP gene, eliminating not only PACAP, but also the PACAP-related peptide (PRP) encoded by exons 3 and 4. Significant attenuations of light-induced phase resetting are seen in both phase delays and advances, in contrast to the reports of Kawaguchi et al. (2003). However, in spite of significant deficits in the response to the acute effects of light, these PACAP-null mice re-entrain to 8-h shifts in the LD cycle at normal rates (Colwell et al., 2004). The third PACAP-null mouse model has not been studied in a circadian context, and is the subject of the present report (Hamelink et al., 2002). To more thoroughly investigate the role of PACAP in circadian resetting, we evaluated the circadian responses to light of this third PACAP-null animal model. This animal was generated in the C57BL/6 strain with only the PACAP-coding sequence deleted, eliminating a potential source of confound in the results. Details and specificity of the genetic lesion, complete loss of PACAP expression and functional deficits in PACAP-mediated adrenal function have been described previously (Hamelink et al., 2002). We paid particular attention to the phase-advance portion of the circadian cycle, a time where models studied previously displayed common deficits in light-induced resetting. We report here that mice lacking PACAP show specific impairment in their ability to generate phase advances to a single brief light exposure when housed in darkness, whereas endogenous free-running period and phase-delaying capabilities are intact. PACAP-null mice are able to entrain to a 23-h T-cycle, although with longer phase-angle of entrainment, and with slower rates of entrainment following a 6-h step advance of the LD cycle. These data indicate that PACAP contributes to normal integration of the phase-advancing signal required for acute phase shifts and for entrainment.