The retinohypothalamic tract (RHT) relays photic information from the eyes to the suprachiasmatic nucleus (SCN). Activation of this pathway by light plays a role in adjusting circadian timing via a glutamatergic pathway at night. Here we report a new signaling pathway by which the RHT may regulate circadian timing in the daytime as well. We used dual immunocytochemistry for pituitary adenylate cyclase-activating peptide (PACAP) and the in vivo tracer cholera toxin subunit B and observed intense PACAP-immunoreactivity (PACAP-IR) in retinal afferents in the rat SCN as well as in the intergeniculate leaflet (IGL) of the thalamus. This PACAP-IR in the SCN as well as in the IGL was nearly lost after bilateral eye enucleation. PACAP afferents originated from small ganglion cells distributed throughout the retina. The phase of circadian rhythm measured as SCN neuronal activity in vitro was significantly advanced (3.5 +/- 0.4 hr) by application of 1 x 10(-6) M PACAP-38 during the subjective day [circadian time (CT)-6] but not at night (CT14 and CT19). The phase-shifting effect is channeled to the clock via a PACAP-R1 receptor, because mRNA from this receptor was demonstrated in the ventral SCN by in situ hybridization. Furthermore, vasoactive intestinal peptide was nearly 1000-fold less potent in stimulating a phase advance at CT6. The signaling mechanism was through a cAMP-dependent pathway, which could be blocked by a specific cAMP antagonist, Rp-cAMPS. Thus, in addition to its role in nocturnal regulation by glutamatergic neurotransmission, the RHT may adjust the biological clock by a PACAP/cAMP-dependent mechanism during the daytime.

Synchronization between the environmental lighting cycle and the biological clock in the suprachiasmatic nucleus (SCN) is correlated with phosphorylation of the Ca 2ϩ /cAMP response element binding protein (CREB) at the transcriptional activating site Ser 133 . Mechanisms mediating the formation of phospho-CREB (P-CREB) and their relation to clock resetting are unknown. To address these issues, we probed the signaling pathway between light and P-CREB. Nocturnal light rapidly and transiently induced P-CREB-like immunoreactivity (P-CREB-lir) in the rat SCN. Glutamate (Glu) or nitric oxide (NO) donor administration in vitro also induced P-CREB-lir in SCN neurons only during subjective night. Clock-controlled sensitivity to phase resetting by light, Glu, and NO is similarly restricted to subjective night. The effects of NMDA and nitric oxide synthase (NOS) antagonists on Glu-mediated induction of P-CREB-lir paralleled their inhibition of phase shifting. Significantly, among neurons in which P-CREB-lir was induced by light were NADPH-diaphorase-positive neurons of the SCN's retinorecipient area. Glu treatment increased the intensity of a 43 kDa band recognized by anti-P-CREB antibodies in subjective night but not day, whereas anti-␣CREB-lir of this band remained constant between night and day. Inhibition of NOS during Glu stimulation diminished the anti-P-CREB-lir of this 43 kDa band. Together, these data couple nocturnal light, Glu, NMDA receptor activation and NO signaling to CREB phosphorylation in the transduction of brief environmental light stimulation of the retina into molecular changes in the SCN resulting in phase resetting of the biological clock.

Circadian clocks are complex biochemical systems that cycle with a period of approximately 24 hours. They integrate temporal information regarding phasing of the solar cycle, and adjust their phase so as to synchronize an organism's internal state to the local environmental day and night. Nocturnal light is the dominant regulator of this entrainment. In mammals, information about nocturnal light is transmitted by glutamate released from retinal projections to the circadian clock in the suprachiasmatic nucleus of the hypothalamus. Clock resetting requires the activation of ionotropic glutamate receptors, which mediate Ca2+ influx. The response induced by such activation depends on the clock's temporal state: during early night it delays the clock phase, whereas in late night the clock phase is advanced. To investigate this differential response, we sought signalling elements that contribute solely to phase delay. We analysed intracellular calcium-channel ryanodine receptors, which mediate coupled Ca2+ signalling. Depletion of intracellular Ca2+ stores during early night blocked the effects of glutamate. Activators of ryanodine receptors induced phase resetting only in early night; inhibitors selectively blocked delays induced by light and glutamate. These findings implicate the release of intracellular Ca2+ through ryanodine receptors in the light-induced phase delay of the circadian clock restricted to the early night.

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Obesity has more than doubled in children and tripled in adolescents in the past 30 yr. The association between metabolic disorders in offspring of obese mothers with diabetes has long been known; however, a growing body of research indicates that fathers play a significant role through presently unknown mechanisms. Recent observations have shown that changes in paternal diet may result in transgenerational inheritance of the insulin-resistant phenotype. Although diet-induced epigenetic reprogramming via paternal lineage has recently received much attention in the literature, the effect of paternal physical activity on offspring metabolism has not been adequately addressed. In the current study, we investigated the effects of long-term voluntary wheel-running in C57BL/6J male mice on their offspring's predisposition to insulin resistance. Our observations revealed that fathers subjected to wheel-running for 12 wk produced offspring that were more susceptible to the adverse effects of a high-fat diet, manifested in increased body weight and adiposity, impaired glucose tolerance, and elevated insulin levels. Long-term paternal exercise also altered expression of several metabolic genes, including Ogt, Oga, Pdk4, H19, Glut4, and Ptpn1, in offspring skeletal muscle. Finally, prolonged exercise affected gene methylation patterns and micro-RNA content in the sperm of fathers, providing a potential mechanism for the transgenerational inheritance. These findings suggest that paternal exercise produces offspring with a thrifty phenotype, potentially via miRNA-induced modification of sperm.

Biological clocks are intrinsic time-keeping systems that regulate behavior and physiological functions in most living organisms. Previous works suggested a possible link between the endogenous circadian clock and cell cycle regulation. The mammalian Period-2 gene (mPer2), an important component of the circadian clock mechanism, is recently demonstrated to play an important role in repressing tumor growth. In this study, we found that polyethylenimine-mediated intratumoral Per2 gene delivery had significant antitumor effects in C57BL/6 mice transplanted with Lewis lung carcinoma. Our data illustrated that the Per2 gene delivery inhibited PCNA expression and induced apoptosis. Our results support the emerging role of the circadian clock in critical aspects of tumorigenesis. These findings underscore the potential use of Per2 gene delivery as a novel therapeutic intervention for the treatment of malignant tumors.

Virag JA, Dries JL, Easton PR, Friesland AM, DeAntonio JH, Chintalgattu V, Cozzi E, Lehmann BD, Ding JM, Lust RM. Attenuation of myocardial injury in mice with functional deletion of the circadian rhythm gene mPer2.

Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells. Previous work raised the possibility that brain-derived neurotrophic factor (BDNF) and its high-affinity tropomyosin-related receptor kinase may be important as modulators of this excitatory input into the SCN. In order to test this possibility, we used whole-cell patch-clamp methods to measure spontaneous excitatory currents in mouse SCN neurons. We found that the amplitude and frequency of these currents were increased by BDNF and decreased by the neurotrophin receptor inhibitor K252a. The neurotrophin also increased the magnitude of currents evoked by application of N-methyl-d-aspartate and amino-methyl proprionic acid. Next, we measured the rhythms in action potential discharge from the SCN brain slice preparation. We found that application of K252a dramatically reduced the magnitude of phase shifts of the electrical activity rhythm generated by the application of glutamate. By itself, BDNF caused phase shifts that resembled those produced by glutamate and were blocked by K252a. The results demonstrate that BDNF and neurotrophin receptors can enhance glutamatergic synaptic transmission within a subset of SCN neurons and potentiate glutamate-induced phase shifts of the circadian rhythm of neural activity in the SCN.