These results indicate that the amplitude increase in KO explants is caused by VIP-dependent enhancement of coupling in SCN cells. Next, to test whether the VPAC2 antagonist can reverse the faster entrainment behavioral phenotype in 4E-BP1 null mice, Eif4ebp1 KO mice were infused with PG99-465 (100 μM, 4 μl) or vehicle (physiological saline, 4 μl) into the lateral ventricle at ZT15, before the light cycle was advanced for 6 hr (light on at ZT18). Both groups of mice re-entrained to the new LD cycle. Notably, however, the mice infused with PG99-465 re-entrained more slowly than those Epigenetic Reader Domain inhibitor infused with saline ( Figure 6G).

From day 2 to day 4 following the LD cycle shift, PG99-465-infused mice exhibited a smaller phase advance than control

(PG99-465 versus vehicle, p < 0.05, ANOVA, Figure 6H). Together, these results demonstrate that VIP overexpression in the SCN underlies the phenotypes of Eif4ebp1 KO mice. mTOR phosphorylates 4E-BP1 and decreases its translational inhibitory activity in the SCN (Cao et al., 2008). To corroborate Baf-A1 molecular weight the regulation of VIP and the clock function by 4E-BP1, we utilized an Mtor+/− mouse strain. In the Mtor+/− SCN, VIP was decreased by ∼50% (Mtor+/− versus Mtor+/+, p < 0.05, Student’s t test; Figure 7A). In the Mtor+/− brain, mTOR activity was decreased, as indicated by decreased phosphorylation of 4E-BP1 (normalized band intensities: Mtor+/− versus Mtor+/+, at Thr70: 0.69 ± 0.04 versus 1 ± 0.07; at Ser65: 0.67 ± 0.07 versus

1 ± 0.19, p < 0.05, Student’s t test), and prepro-VIP was reduced (normalized band intensities: Mtor+/− Oxyphenisatin versus Mtor+/+, 0.43 ± 0.08 versus 1 ± 0.08, p < 0.05, Student’s t test; Figure 7B). To investigate the effects of lower VIP level on the circadian clock function, we monitored circadian behavior of the Mtor+/− mice in LL. Mice were housed in regular cages in LL (200 lx) for 14 days and then transferred to cages equipped with running wheels in LL (55 lx) to record their circadian behavior for 14 days. LL induced three types of behavior (R, AR, and WR) in both Mtor+/− and Mtor+/+animals. A larger percentage of Mtor+/− mice (47.4%, 9/19) exhibited arrhythmic behavior than did Mtor+/+ mice (16.7%, 3/18) (p < 0.05, χ2 test; Figures 7C and 7D), indicating increased susceptibility to LL-induced clock desynchrony in Mtor+/− mice. Taken together, the results demonstrate that mTOR/4E-BP1 signaling bidirectionally regulates VIP level and susceptibility of the SCN clock to desynchronizing effect of LL. In the present study, we found that mTOR signaling promotes Vip mRNA translation by repressing 4E-BP1. Consequently, in Eif4ebp1 KO mice, VIP is increased in the SCN, which is associated with a larger amplitude of PER2 rhythms, accelerated circadian clock entrainment, and enhanced synchrony.