LL induced all three types of behavior in both WT and Eif4ebp1 KO animals. Most WT mice were either arrhythmic (AR) or weakly rhythmic (WR), while most KO mice were rhythmic (R) in LL ( Figure 4B). Distribution of the three types of behavior (AR, WR, and R) in both genotypes is quantified in Figure 4C. Strikingly, a smaller percentage of KO mice (6.3%, 1/16) exhibited arrhythmic behavior than did WT mice (38.5%, 5/13) (KO versus WT, p < 0.05, χ2 test). The pooled periodograms from all the mice used in the experiment are shown in Figure 4D. The main peak of the periodogram is higher in the KO mice than in the

WT mice, demonstrating stronger rhythmicity in the KO mice in LL. To verify that the rhythms of clock protein expression are disrupted in behaviorally arrhythmic mice, PER2 was immunostained in the SCN at CT0 and CT12 Rapamycin ic50 for the rhythmic mice and at two random time points 12 hr apart for the arrhythmic mice. CT12 was defined as the onset time of the active phase, and CT0 was defined as the time point 12 hr apart from CT12. As expected, PER2 was not rhythmic in the SCN of behaviorally arrhythmic mice (KO or WT), as compared to the rhythmic mice ( Figure 4E). Thus, these data show that Eif4ebp1

KO mice are more resistant to LL-induced disruption of circadian behavioral and PER2 rhythms, consistent with Ipatasertib in vivo enhanced synchrony in the SCN cells. VIP plays a critical role in mediating synchrony in SCN cells. To investigate the

mechanisms of enhanced re-entrainment and synchrony of the SCN clock in Eif4ebp1 KO mice, we first studied VIP expression in these animals. Using double immunofluorescent labeling, we first examined the expression pattern of VIP and arginine vasopressin Phosphoprotein phosphatase (AVP) in the SCN. AVP is generally used as a neuropeptide marker for the dorsolateral SCN ( Abrahamson and Moore, 2001). Confocal microscopic imaging revealed that VIP was expressed in a subset of ventromedial (core) SCN neurons, while AVP was expressed in some cells in the dorsal and lateral (shell) SCN ( Figure 5A). The spatial distribution of VIP and AVP was similar in the SCN of KO and WT animals. Immunohistochemical staining also revealed robust VIP expression in the SCN ( Figure 5B and Figure S4A). In both the WT and the KO mice, expression of VIP at ZT12 was decreased compared to ZT0 (ZT12 versus ZT0, p < 0.05, ANOVA), which is consistent with a previous report ( Takahashi et al., 1989). Interestingly, VIP level was increased by ∼1-fold in the Eif4ebp1 KO mice at both ZT0 and ZT12 (KO versus WT, p < 0.05, ANOVA) ( Figure 5B), suggesting constitutive repression of VIP expression by 4E-BP1. To investigate the mechanisms underlying VIP increase in Eif4ebp1 KO mice, we examined the expression of the VIP precursor protein, prepro-VIP, in the brain.