In nature and under laboratory conditions, dams nurse rabbit pups once daily for a duration of fewer than 5 min. The present study explored neural mechanisms mediating the timing of nursing in this natural model of food anticipatory activity, focussing on the suprachiasmatic nucleus (SCN), the locus of the master circadian clock and on the dorsomedial hypothalamic nucleus (DMH), a region implicated in timing of food-entrained behavior. Rabbit pups are born in the dark, with eyelids closed. Nursing visits to the litters also occurs during the dark phase. To explore the effect of the timing of feeding, pups were maintained in constant darkness, while females housed in a light-dark cycle were permitted to nurse their pups either during the night (night-fed group) or day (day-fed group). All pups exhibited anticipatory locomotor activity before daily nursing. In the SCN, PER1 and FOS peaked during the night in both groups, with a longer duration of elevated protein expression in the night-fed group. In contrast, DMH peak PER1 expression occurred 8 h after pups were fed, corresponding to the shift in timing of nursing. Comparison of nursed and 48 h fasted pups indicates that the timing of PER1 expression was similar in the SCN and DMH, with fewer PER1-positive cells in the latter group. The results indicate that rabbit pups show food anticipatory activity, and that timing of nursing differentially affects PER1 expression in the SCN and DMH.

Young rabbits are nursed every 24 h for a period of 3-5 min. As a consequence, pups are synchronized to this nursing event; this synchronization is characterized by increased locomotor activity and a peaking of core temperature and plasma corticosterone in anticipation of the daily meal. Ghrelin is a hormone suggested to play a role in meal initiation and to promote food intake. The present study explored the role of ghrelin in food-entrained conditions. Newborn rabbits were maintained in constant darkness and nursed once daily at 1000 by the lactating dam. On postnatal day 7, rabbits were killed at six different time points to complete a 24-h cycle. All pups developed locomotor rhythms entrained by mealtime and exhibited anticipatory activity. Food-entrained rhythms in plasma corticosterone and free fatty acids were observed even if two meals were omitted. In contrast, daily food-driven rhythms in stomach weight, plasma glucose, liver glycogen, and ghrelin did not persist when two meals were omitted. Peak ghrelin levels were observed at the moment in the cycle when the stomach weight was lowest, i.e., before initiation of anticipation. The present data are in agreement with previous data from rabbit pups maintained in light-dark conditions and provide evidence that 7- to 9-day-old rabbits in constant darkness can exhibit metabolic and hormonal rhythms mainly driven by the restricted daily nursing.

Rabbit does nurse their litter once every 24 h during the night. We hypothesized that corticosterone, ghrelin, leptin and metabolites like glucose, liver glycogen and free fatty acids could be affected in the pups by the time at which does nurse them. Therefore, we measured these parameters in pups nursed at 02:00 h (nighttime for the doe) to compare them with results from a previous study where does nursed at 10:00 h, during daytime. From postnatal day 7, pups were sacrificed either just before their scheduled time of nursing or at 4, 8, 12, 16 or 20 h after nursing (n = 6 at each time point); additional pups were sacrificed at 4 h intervals between 48 and 72 h after nursing to study the persistence of oscillations during fasting. All pups developed locomotor anticipatory activity to nursing. Corticosterone, ghrelin and free fatty acids exhibited a rhythm that persisted in fasted pups. Glucose concentrations were lower in fasted than in nursed pups, and glycogen was only detected in nursed subjects. Leptin values were stable and low in nursed subjects but increased significantly in fasted subjects up to 72 h after the expected nursing time. The rhythm of ghrelin persisted during fasting, contrary to our previous findings in pups nursed during daytime (i.e. outside the natural time of nursing for this species). Therefore, in 7-day-old rabbit pups, night nursing is a strong zeitgeber for corticosterone, ghrelin, free fatty acids and energy metabolites but not for leptin.

Nursing in the rabbit is a circadian event during which mother and pups interact for a period of < 5 min every day. Here we explored behavioral and neuronal changes in the mother by analyzing the suprachiasmatic nucleus (SCN), and oxytocinergic (OT) neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). We maintained lactating does in a light-dark cycle (lights on at 07 : 00 hours; ZT0); they were scheduled to nurse during either the day (ZT03) or the night (ZT19). Groups of intact and nursing females was perfused, one at each 4-h point through a 24-h cycle. We explored, by immunohistochemistry, the PER1 expression and double-labeling, with OT antibody, of neurons in the PVN and SON at lactation on day 7. In the SCN, intact and lactating groups had peak PER1 expression at ZT11; however, there was a reduction in PER1 at peak time in the nursing groups. There was a locomotor activity rhythm with increased activity around the time of lights-on in intact subjects and around the time of suckling in lactating does. There was an induction of PER1 in OT cells in the PVN and SON that shifted in phase with timing of nursing. We further explored the maintenance of the PER1 expression in OT cells in nursing-deprived does and found a significant decrease at 24 and 48 h after the last nursing. We conclude that suckling induced PER1 in the PVN and SON, but not in the SCN, in nursing does, and also shifted their locomotor behavior.

Nursing in the rabbit is under circadian control, and pups have a daily anticipatory behavioral arousal synchronized to this unique event, but it is not known which signal is the main entraining cue. In the present study we hypothesized that food is the main entraining signal. Using mother-deprived pups we tested the effects of artificial feeding on the synchronization of locomotor behavior, plasma glucose, corticosterone, FOS and PER1 protein rhythms in suprachiasmatic, supraoptic, paraventricular and tuberomammillary nuclei. At postnatal day 1 an intragastric tube was placed by gastrostomy. The next day and for the rest of the experiment pups were fed with a milk formula through the cannula at either 02:00 or 10:00 h (feeding time = zeitgeber time (ZT) 0). At postnatal days 5–7 pups exhibited behavioral arousal with a significant increase in locomotor behavior 60 min before feeding. Glucose levels increased after feeding, peaking at ZT4–ZT12 and then declining. Corticosterone was highest around the time of feeding then decreased to trough concentrations at ZT12–ZT16, increasing again in anticipation of next feeding bout. In the brain, the suprachiasmatic nucleus had a rhythm of FOS and PER1 that was not significantly affected by the feeding schedule. Conversely, the supraoptic, paraventricular and tuberomammillary nuclei had rhythms of both FOS and PER1 induced by the time of scheduled feeding. We conclude that the nursing rabbit pup is a natural model of food entrainment, since food, in this case milk formula, is a strong synchronizing signal for behavioral, hormonal, metabolic and neural parameters.