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.

The olfactory bulb (OB) has a circadian clock independent of the suprachiasmatic nucleus, but very little is known about the functional significance of its oscillations. The OB plays a major role in food intake as it contributes to the evaluation of the hedonic properties of food, it is necessary for a normal pattern of locomotor behavior and their ablation disrupts feeding patterns. Previously we demonstrated that OB of rabbit pups can be entrained by periodic nursing but it was not clear whether food was the entraining signal. Here we hypothesized that OB can be entrained by a food pulse during the day in adult rats under a restricted feeding schedule. Then we expect that OB will have a high activation before food presentation when animals show food anticipatory activity (FAA). To this aim we determined by immunohistochemistry the expression of FOS protein, as an indicator of neural activation, in the mitral and granular cell layers of the main and accessory OB. Additionally we also explored two of the OB brain targets, the piriform cortex (PC) and bed nuclei of the accessory olfactory tract (BAOT), in three groups: ad libitum (ALF), restricted feeding (RF), and fasted rats after restricted feeding (RF-F). In ALF group FOS levels in both main and accessory OB were low during the day and high during the night at the normal onset of the increase of activity, in agreement with previous reports. On the contrary in RF and RF-F groups FOS was high at the time of FAA, just before food presentation, when animals are in a state of high arousal and during food consumption but was low during the night. In their brain targets, we observed a similar pattern as OB in all groups with the only difference being that FOS levels remained high during the night in RF-F group. We conclude that the OB is entrained by food restriction by showing high activation at the time of food presentation, which persists during fasting and impose a similar FOS pattern to the two brain targets explored only in fed animals.

Rabbit pups are a natural model to study food anticipatory activity (FAA). Recently, we reported that three areas in the forebrain - the organum vasculosum of lamina terminalis, median preoptic nucleus (MnPO) and medial preoptic area - exhibit activation during FAA. Here, we examined the PER1 protein profile of these three forebrain regions in both nursed and fasted subjects. We found robust PER1 oscillations in the MnPO in nursed subjects, with high PER1 levels during FAA that persisted in fasted subjects. In conclusion, our data indicate that periodic nursing is a strong signal for PER1 oscillations in MnPO and future experiments are warranted to explore the specific role of this area in FAA.