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.

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.

Rhythmic feeding in rabbit pups is a natural model to study food entrainment because, similar to rodents under a schedule of food restriction, these animals show food-anticipatory activity (FAA) prior to daily nursing. In rodents, several brain systems, including the orexinergic system, shift their activity to the restricted feeding schedule, and remain active when subjects are hungry. As the lamina terminalis and regions of the preoptic area participate in the control of behavioral arousal, it was hypothesized that these brain regions are also activated during FAA. Thus, the effects of daily milk ingestion on FOS protein expression in the organum vasculosum of lamina terminalis (OVLT), median preoptic nucleus (MnPO), and medial preoptic area (MPOA) were examined using immunohistochemistry before and after scheduled time of nursing in nursed and fasted subjects. Additionally, FOS expression was explored in orexin (ORX) cells in the lateral hypothalamic area and in the supraoptic nucleus (SON) because of their involvement in arousal and fluid ingestion, respectively. Pups were entrained by daily nursing, as indicated by a significant increase in locomotor behavior before scheduled time of nursing in both nursed and fasted subjects. FOS was significantly higher in the OVLT, MnPO, and MPOA at the time of nursing, and decreased 8 h later in nursed pups. In fasted subjects, this effect persisted in the OVLT, whereas in the MnPO and MPOA, values did not drop at 8 h later, but remained at the same level or higher than those at the time of scheduled nursing. In addition, FOS was significantly higher in ORX cells during FAA in nursed pups in comparison with 8 h later, but in fasted subjects it remained high during most fasting time points. Additionally, OVLT, SON, and ORX cells were activated 1.5 h after nursing. We conclude that the OVLT, MnPO, and MPOA, but not SON, may participate in FAA, as they show activation before suckling of periodic milk ingestion, and that sustained activation of the OVLT, MnPO, and MPOA by fasting may contribute to the high arousal state associated with food deprivation. In agreement with this, ORX cells also remain active after expected nursing, which is consistent with reports in other species.