Diurnal pattern of clock gene expression in the hypothalamus of the newborn rabbit

In the European rabbit (Oryctolagus cuniculus) nursing acts as a strong non-photic synchronizer of circadian rhythmicity in the newborn young. Rabbits only nurse for a few minutes once every 24 h and previous studies have shown that the pups, blind at birth, display endogenous circadian rhythms in behavior and physiology entrained by this regular daily event. As a further step toward understanding the neural organization of the rabbit's early circadian system, we investigated the expression of clock genes in the suprachiasmatic nucleus of the hypothalamus (SCN; the principal circadian pacemaker in adult mammals) across the pups' 24-h day. We used 43 pups from seven litters maintained in constant darkness and entrained non-photically by nursing at the same time each day until P7. After nursing on day 7, pups were killed in the dark at 3-h intervals so as to obtain eight groups (n=5-6 pups/group) distributed evenly across the 24 h before the next scheduled nursing. Profiles in the expression of the clock genes Per1, Per2, Cry1 and Bmal1 were determined using in situ hybridization in brain sections through the hypothalamus at the level of the SCN. We report for the first time: 1) that Per1, Per2, Cry1 and Bmal1 are all expressed in the SCN of the newborn rabbit, 2) that the expression of Per1, Per2 and Bmal1 but not Cry1 shows diurnal rhythmicity similar to that in adult mammals, and 3) that the expression of Per1, Per2 and Bmal1 is consistent with the strong entraining effect of nursing found in previous studies. Unexpectedly, and contrasting somewhat to the pattern in the SCN, we also found diurnal rhythmicity in the expression of Cry1 and Bmal1 but not of Per1 in the anterior ventromedial hypothalamic nucleus. Overall, our findings suggest that the SCN is a functional part of the newborn rabbit's circadian system and that it can be entrained by non-photic cues associated with the mother's daily nursing visit.