Activation of the central but not the medial and cortical amygdala during anticipation for daily nursing in the rabbit.

Rabbits have remarkable nursing behavior: after parturition, does visit daily their pups for nursing only once with circadian periodicity. Before the nursing events, they present increased activity and arousal, which shift according to the timing of scheduled nursing, either during the day or night. Brain areas related to maternal behavior and neuroendocrine cells for milk secretion are also entrained. The daily return of the doe for nursing at approximately the same hour suggests a motivational drive with circadian periodicity. Previously, we reported the activation of the mesolimbic system at the time of nursing, but not 12 h before that. Aiming at a better understanding of the mechanism of this anticipatory behavior, we explored the participation of the limbic regions of the amygdala and the bed nucleus of the stria terminalis, as well as the possible activation of the hypothalamic-pituitaryadrenal axis, specifically the corticotropin-releasing factor cells in the hypothalamic paraventricular nucleus of does at different times before and after nursing. The medial and cortical amygdala, the bed nucleus of the stria terminalis, and corticotropin cells showed activation only after nursing. However, the central amygdala was also activated before nursing. We conclude that the medial and the cortical amygdala form part of the afferent olfactory pathway for entrainment, and the central amygdala participates in the anticipatory motivational circuit of the control of periodic nursing. The lack of activation of corticotropin cells before nursing is consistent with the possible harmful effects of the doe's high glucocorticoid levels on the developing pups.

Rabbits can be conditioned in a food-induced place preference paradigm.

The conditioned place preference (CPP) paradigm has been employed in behavioral studies to investigate the responses to an environment where a reinforcing event occurs. It is applied to reveal incentive motivational responses to reward-related stimuli. It is standardized and widely applied in mice and rats, two of the most common species of laboratory animals. However, no studies using the CPP protocol have been performed in rabbits, even though this animal model is commonly used in pharmacological and behavioral research. There are important physiological and behavioral differences between rodents and rabbits. For example, rodents are spontaneous ovulators while rabbits are induced ovulators. In addition, lactation in the rabbit is circadian, which is unique among mammals. The present investigation aims to establish whether rabbits can be conditioned by using a food-induced CPP protocol in subjects with caloric restriction. Adult female rabbits were subjected to a three-compartment CPP protocol. The food produced place preference, demonstrating for the first time that rabbits can be conditioned using the CPP paradigm opening a new field of opportunities for behavioral studies of positive affective states in a species with important behavioral and physiological differences from rodents.

Oxytocinergic cells of the posterior hypothalamic paraventricular nucleus participate in the food entrained clock.

The mechanisms underlying food anticipatory activity are still poorly understood. Here we explored the role of oxytocin (OT) and the protein c-Fos in the supraoptic nucleus (SON), medial (PVNm) and posterior (PVNp) regions of the paraventricular hypothalamic nucleus. Adult rats were assigned to one of four groups: scheduled restricted feeding (RF), ad libitum (AL), fasting after restricted feeding (RF-F), to explore the possible persistence of oscillations, or ad libitum fasted (AL-F). In the SON and in the PVNm, OT cells were c-Fos positive after food intake; in contrast, OT cells in the PVNp showed c-Fos activation in anticipation to food access, which persisted in RF-F subjects. We conclude that OT and non-OT cells of the SON and PVNm may play a role as recipients of the entraining signal provided by food intake, whereas those of the PVNp which contain motor preautonomic cells that project to peripheral organs, may be involved in the hormonal and metabolic anticipatory changes in preparation for food presentation and thus, may be part of a link between central and peripheral oscillators. In addition, due to their persistent activation they may participate in the neuronal network for the clock mechanism that leads to food entrainment.

Oxytocinergic Cells of the Hypothalamic Paraventricular Nucleus Are Involved in Food Entrainment.

When food is presented at a specific time of day subjects develop intense locomotor behavior before food presentation, termed food anticipatory activity (FAA). Metabolic and hormonal parameters, as well as neural structures also shift their rhythm according to mealtime. Food-entrained activity rhythms are thought to be driven by a distributed system of central and peripheral oscillators sensitive to food cues, but it is not well understood how they are organized for the expression of FAA. The hormone Oxytocin plays an important role in food intake, satiety and homeostatic glucose metabolism and although it is recognized that food is the main cue for food entrainment this hormone has not been implicated in FAA. Here we investigated the activity of oxytocinergic (OTergic) cells of the hypothalamus in relation to the timing of feeding in rabbit pups, a natural model of food entrainment. We found that OTergic cells of the supraoptic nucleus and the main body of the paraventricular nucleus (PVN) are activated after feeding which suggests that OT may be an entraining signal for food synchronization. Moreover, a detailed analysis of the PVN revealed that OTergic cells of the caudal PVN and a subpopulation in the dorsal part of the main body of this nucleus shows activation before the time of food but not 12 h later. Moreover this pattern persists in fasted subjects at the time of the previous scheduled time of nursing. The fact that those OTergic cells of the dorsal and caudal part of the PVN contain preautonomic cells that project to the adrenal, pancreas and liver perhaps may be related to the physiological changes in preparation for food ingestion, and synchronization of peripheral oscillators, which remains to be determined; perhaps they play a main role in the central oscillatory mechanism of FAA as their activity persists in fasted subjects at the time of the next feeding time.

Differential effects of acute and chronic treatment with the flavonoid chrysin on anxiety-like behavior and Fos immunoreactivity in the lateral septal nucleus in rats.

The aim of this study was to compare the effects of acute (a single injection) and chronic (21 consecutive days) treatments with chrysin 2, 4, and 8 µmol kg-1 on anxiety-like behavior and Fos immunoreactivity in the lateral septum nucleus (LSN), a structure that is involved in the regulation of anxiety, in male Wistar rats. These effects were compared with the clinically effective anxiolytic diazepam 7 µmol kg-1. The results showed that acute, but not chronic treatment, with 4 µmol kg-1 chrysin exerted anxiolytic- and anti- depressant-like effects with these effects being similar to that of diazepam. Also, none of the above-mentioned treatments did alter Fos immunoreactivity in the LSN, but a tendency towards the reduction of this variable was detected with chrysin 4 µmol kg-1 and diazepam 7 µmol kg-1. Altogether, results suggest that chrysin exerts anxiolytic-like effects, however, it can produce pharmacological tolerance after repeated use, similar to benzodiazepines.

Maternal care activates the ventral tegmental area but not dopaminergic cells in the rat.

The ventral tegmental area (VTA), together with the preoptic area, is part of a neural circuit necessary for the expression of maternal behaviour (MB); destruction of either area disrupts MB in postpartum rats. Central to the proposal of VTA activation are dopaminergic cells, for which the cell bodies lie in the VTA and project to forebrain structures. This mesolimbic system is a motivational circuit involved in rewarding behaviours such as sex and MB. Despite their recognised importance, surprisingly, unlike the preoptic area, there are no anatomical descriptions of the pattern of VTA activation or of the dopaminergic cell activation, specifically in relation to MB in the rat. In the present study, we explore the possible activation (as indicated by Fos protein via immunohistochemistry) of the anterior and medial portions of the VTA and in the dopaminergic cells in these regions, as well as in the medial preoptic area, in lactating rats, at postpartum day 7 (after a 12-hour mother/pups separation), and in dioestrous females. After 12 hours, mothers were perfused at that moment or after a 90 minutes of interaction, or not, with their pups. We found a strong significant Fos induction in both the preoptic area and in the anterior portion of VTA in dams that interacted with their pups. The number of dopaminergic cells that coexpressed Fos did not differ across groups. Additionally, we determined Fos and GABA colocalisation in the anterior part of the VTA and found dense GABAergic processes, possibly varicosities, in the area of increased Fos expression. The results of the present study support a proposed GABAergic pathway from medial preoptic area to VTA cells, critical for the expression of MB. Future experiments are warranted to explore the neurochemical identity of the Fos and no-Fos expressing cells that are recipients of GABAergic processes in the VTA, aiming to better understand the neural circuitry of the VTA in relation to MB.

Long-term ovariectomy increases anxiety- and despair-like behaviors associated with lower Fos immunoreactivity in the lateral septal nucleus in rats.

In woman, surgical menopause is associated with anxiety and depression symptoms. Ovariectomy in rats has been proposed as an experimental model of surgical menopause, but its long-term effects on anxiety- and depression-like behaviors and relationship with cellular changes in specific brain structures are unknown. The effects of ovariectomy on anxiety- and despair-like behavior 1, 3, 6, 9, 12, and 15-weeks postovariectomy were evaluated. Fos-immunoreactivity was evaluated in the lateral septal nucleus (LSN). The effects were compared with rats in the proestrus-estrus and metestrus-diestrus phases of the ovarian cycle and with ovariectomized rats that received 17ß-estradiol (OVXE). Three weeks postovariectomy, the rats exhibited an increase in anxiety-like behavior compared with PE and OVXE groups. Decreases in the locomotor activity and time spent grooming and rearing were detected in all the ovariectomized rats. In the forced swim test, the rats exhibited an increase in immobility time 6-weeks postovariectomy compared with control groups. The Fos-immunoreactivity in the LSN was significantly lower in all groups of ovariectomized rats compared with control groups. These findings indicate that rats develop anxiety-like behavior 3-weeks postovariectomy. Six weeks postovariectomy, the rats also developed despair-like behavior, which was associated with a reduction of Fos immunoreactivity in the LSN. Long-term ovariectomy may be considered a useful tool for understanding the development of neurobiological changes associated with surgical menopause. This model may also be useful for evaluating potential anxiolytic and antidepressant effects of diverse substances to ameliorate typical emotional and affective disorders during surgical menopause in women.

Circadian Rhythms and Clock Genes in Reproduction: Insights From Behavior and the Female Rabbit's Brain.

Clock gene oscillations are necessary for a successful pregnancy and parturition, but little is known about their function during lactation, a period demanding from the mother multiple physiological and behavioral adaptations to fulfill the requirements of the offspring. First, we will focus on circadian rhythms and clock genes in reproductive tissues mainly in rodents. Disruption of circadian rhythms or proper rhythmic oscillations of clock genes provoke reproductive problems, as found in clock gene knockout mice. Then, we will focus mainly on the rabbit doe as this mammal nurses the young just once a day with circadian periodicity. This daily event synchronizes the behavior and the activity of specific brain regions critical for reproductive neuroendocrinology and maternal behavior, like the preoptic area. This region shows strong rhythms of the PER1 protein (product of the Per1 clock gene) associated with circadian nursing. Additionally, neuroendocrine cells related to milk production and ejections are also synchronized to daily nursing. A threshold of suckling is necessary to entrain once a day nursing; this process is independent of milk output as even virgin does (behaving maternally following anosmia) can display circadian nursing behavior. A timing motivational mechanism may regulate such behavior as mesolimbic dopaminergic cells are entrained by daily nursing. Finally, we will explore about the clinical importance of circadian rhythms. Indeed, women in chronic shift-work schedules show problems in their menstrual cycles and pregnancies and also have a high risk of preterm delivery, making this an important field of translational research.

Induced cytotoxic damage by exposure to gasoline vapors: a study in Sinaloa, Mexico.

Gasoline is a blend of organic compounds used in internal combustion engines. Gasoline-station attendants are exposed to gasoline vapors, which pose a potentially mutagenic risk. According to the International Agency for Research on Cancer, exposure to gasoline and engine exhaust is possibly carcinogenic to humans. We determined the frequency of micronucleus and other nuclear abnormalities, such as pyknotic nuclei, chromatin condensation, cells with nuclear buds, karyolytic cells, karyorrhexis, and binucleated cells in buccal mucosal smears of 60 gasoline-station attendants and 60 unexposed controls. In addition, we explored if factors such as smoking habits, alcohol consumption, and worked years exert an additional synergistic cytotoxic effect. There were statistically significant higher frequencies (p < 0.05) of nuclear abnormalities among exposed attendants compared to the controls. No statistical significant (p > 0.05) additional effect of lifestyle habits such as smoking and alcohol consumption or worked years on the cytotoxicity was observed. The results showed that from the beginning exposure to gasoline vapors increased the frequency of nuclear abnormalities in buccal epithelial cells. Our results provide valuable information on cytotoxic damage for an early pre-symptomatic diagnosis.

Cytogenetic biomonitoring of occupationally exposed workers to ashes from burning of sugar cane in Ahome, Sinaloa, México.

Burning the sugar cane field before harvesting has a negative impact on both air and human health, however this issue had not been explored in Mexico. The objective of this work was to determine the chromosomal damage in workers from sugar cane burning fields in Sinaloa, México. To this purpose, we analyzed 1000 cells of buccal exfoliated epithelia from 60 exposed workers and 60 non-exposed controls to determine micronucleus frequencies and other nuclear abnormalities. The results indicated significant higher values of micronucleus and nuclear abnormalities such as binucleate cells, pyknosis, karyolysis, chromatin condensation and nuclear buds frequencies in the exposed subjects compared to those that were not exposed. Our data indicates that sugar cane burning, that generates polycyclic aromatic hydrocarbons, represents a genotoxic risk for workers in this important sugar cane producing area in Mexico.

Inverse correlation among organochlorine pesticide levels to total lipid serum contents: a preliminary study in Veracruz, México.

Organochlorine pesticides, due to their hydrophobic nature and persistence, accumulate in tissues rich in lipids, which had been used as a biomarker for environmental pollution. In humans, organochlorine pesticides are continuously circulating and equilibrating among body compartments. The objective of the study was to evaluate the concentrations of organochlorine pesticides in blood serum and compare their levels to the total lipid contents in Veracruz, México inhabitants. Our hypothesis is that concentrations of organochlorine pesticides will increase just as lipid concentrations. Levels of organochlorine pesticides were divided in ascending tertils according to their total lipid content. The linear trend model applied surprisingly reveals that the average level of all organochlorine pesticides decreases as the lipid concentration increases. From one tertil to the next ß-HCH, it shows a decrease of -3.19 mg kg(-1) on lipid basis, pp.'DDE levels decrease by -3.70 mg kg(-1) on lipid basis and pp.'DDT levels decrease -1.13 mg kg(-1) on lipid basis. We conclude that the levels and the orderly sequence of organochlorine pesticide distributions in the blood serum maintain an inverse relationship to total lipid blood serum concentrations.

Suckling induces a daily rhythm in the preoptic area and lateral septum but not in the bed nucleus of the stria terminalis in lactating rabbit does.

Maternal behavior in the rabbit is restricted to a brief nursing period every day. Previously, we demonstrated that this event induces daily rhythms of Period1 (PER1) protein, the product of the clock gene Per1, in oxytocinergic and dopaminergic populations in the hypothalamus of lactating rabbit does. This is significant for the periodic production and ejection of milk, but the activation of other areas of the brain has not been explored. Here, we hypothesised that daily suckling would induce a rhythm in the preoptic area, lateral septum, and bed nucleus of the stria terminalis, which are important areas for the expression of maternal behavior in mammals, including the rabbit. To this end, we analysed PER1 expression in those areas through a complete 24-h cycle at lactation day 7. Does were scheduled to nurse during either the day at 10:00 h [zeitgeber time (ZT)03] or the night at 02:00 h (ZT19). Non-pregnant, non-lactating females were used as controls. In contrast to control females, lactating does showed a clear, significant rhythm of PER1 that shifted in parallel with the timing of nursing in the preoptic area and lateral septum. We determined that the maximal expression of PER1 at 8 h after scheduled nursing decreased significantly at 24 and 48 h after the absence of suckling. This effect was more pronounced in the lateral septum than in the preoptic area. We conclude that daily suckling is a powerful stimulus inducing rhythmic activity in brain structures in the rabbit that appear to form part of a maternal entrainable circuit.

Main and accessory olfactory bulbs and their projections in the brain anticipate feeding in food-entrained rats.

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.

The median preoptic nucleus exhibits circadian regulation and is involved in food anticipatory activity in rabbit pups.

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.

Activation of organum vasculosum of lamina terminalis, median preoptic nucleus, and medial preoptic area in anticipation of nursing in rabbit pups.

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.

GLAST/EAAT1-induced glutamine release via SNAT3 in Bergmann glial cells: evidence of a functional and physical coupling.

Glutamate, the major excitatory transmitter in the vertebrate brain, is removed from the synaptic cleft by a family of sodium-dependent glutamate transporters profusely expressed in glial cells. Once internalized, it is metabolized by glutamine synthetase to glutamine and released to the synaptic space through sodium-dependent neutral amino acid carriers of the N System (SNAT3/slc38a3/SN1, SNAT5/slc38a5/SN2). Glutamine is then taken up by neurons completing the so-called glutamate/glutamine shuttle. Despite of the fact that this coupling was described decades ago, it is only recently that the biochemical framework of this shuttle has begun to be elucidated. Using the established model of cultured cerebellar Bergmann glia cells, we sought to characterize the functional and physical coupling of glutamate uptake and glutamine release. A time-dependent Na⁺-dependent glutamate/aspartate transporter/EAAT1-induced System N-mediated glutamine release could be demonstrated. Furthermore, D-aspartate, a specific glutamate transporter ligand, was capable of enhancing the co-immunoprecipitation of Na⁺-dependent glutamate/aspartate transporter and Na⁺-dependent neutral amino acid transporter 3, whereas glutamine tended to reduce this association. Our results suggest that glial cells surrounding glutamatergic synapses may act as sensors of neuron-derived glutamate through their contribution to the neurotransmitter turnover.

Development of retinal projections and response to photic input in the suprachiasmatic nucleus of New Zealand White Rabbits.

In rabbit pups, nursing by the mother is the prevailing entraining signal for their circadian rhythms during at least the first two weeks of life. Therefore, they are considered a natural model of food anticipatory activity. However, the photic entrainment of the circadian system in rabbit pups during this developmental stage is not well understood. The present study examined the retinal projections to the suprachiasmatic nucleus (SCN) and the functional responses of the SCN to light exposure. Using the anterograde tracer cholera toxin-B, we examined the retinal projections to the SCN at postnatal days (PD) 1, 9, 19 and in adult animals. The results revealed that the retinal projections were present at PD1 with a bilateral symmetry, and with a contralateral tendency at PD19 and adults. We also explored the response of the SCN to a light pulse by assessing the induction of FOS protein, a marker of neuronal activation, at PD1, 12, 19 and in adults. Light-induced FOS was observed during day and night at PD1, but mainly during night at PD12, 19 and adults. We conclude that in the SCN there is a "gating" mechanism to FOS induction by light that develops several days after birth, as in other mammals, and in the rabbit is already present at PD12. Moreover, in contrast to other altricial mammals, the circadian visual system, although not essential for entraining the rhythm during first two weeks of life, is present and functional in rabbit pups from birth.

A circadian clock in the olfactory bulb anticipates feeding during food anticipatory activity.

Rabbit pups ingest food, in this case milk, once a day with circadian periodicity and are a natural model of food anticipatory activity. During nursing, several sensory systems receive information about properties of the food, one of them being the olfactory system, which has received little attention in relation to synchronization by food. In addition, the olfactory bulb has a circadian pacemaker that exhibits rhythms independently of the suprachiasmatic nucleus, but the biological functions of these rhythms are largely unknown. In the present contribution, we hypothesized that circadian suckling of milk synchronizes rhythms in the olfactory bulb. To this aim we explored by immunohistochemistry, rhythms of FOS and PER1 proteins, as indicators of activation and reporter of oscillations, respectively, through a complete 24-h cycle in periglomerular, mitral and granular cell layers of both the main and the accessory olfactory bulb. Subjects were 7-day-old rabbit pups scheduled to nurse during the night (02:00 h) or day (10:00 h), and also fasted subjects, to explore the possible persistence of oscillations. In the three layers of the main olfactory bulb, FOS was high at time of nursing, then further increased 1.5 h afterward, and then decreased to increase again in advance of the next nursing bout. This pattern persisted, without the postprandial increase, in fasted subjects with a shift in subjects nursed at 02:00. PER1 was increased 2-8 h after nursing and this increase persisted in most cell layers, with a shift, in fasted subjects. In the accessory olfactory bulb we only observed a consistent pattern of FOS expression in the mitral cell layer of nursed subjects, similar to that of the main olfactory bulb. We conclude that the main olfactory bulb is synchronized during milk ingestion, but during fasting its oscillations perhaps are modulated by the suprachiasmatic nucleus, as proposed for rodents.

Synchronization of PER1 protein in parabrachial nucleus in a natural model of food anticipatory activity.

Rabbit pups represent a natural model of food anticipatory activity (FAA). FAA is the behavioral output of a putative food entrainable oscillator (FEO). It had been suggested that the FEO is comprised of a distributed system of clocks that work in concert in response to gastrointestinal input by food. Scheduled food intake synchronizes several nuclei in the brain, and the hypothalamus has received particular attention. On the contrary, brainstem nuclei, despite being among the brain structures to first receive food cues, have been scarcely studied. Here we analysed by immunohistochemistry possible oscillation of FOS and PER1 proteins through a complete 24-h cycle in the dorsal vagal complex (DVC) and parabrachial nucleus (PBN) of 7-8-day-old rabbit pups scheduled to nurse during the night (02:00 h) or day (10:00 h), and also in fasted subjects to explore the possible persistence of oscillations. We found a clear induction of FOS that peaks 1.5 h after nursing in all nuclei studied. PER1 was only synchronized in the PBN, reaching highest values 12 h after nursing. Only PER1 oscillations persisted, with a shift, in fasted subjects. We conclude that the DVC nuclei are probably more related to the transmission of food cues to other brain regions, but that the PBN participates in the integration of information essential for FAA. Our results support previous findings suggesting that the DVC nuclei, but not PBN, are not essential for FAA. We suggest that PBN is a key component of the proposed distributed system of clocks involved in FAA.

Artificial feeding synchronizes behavioral, hormonal, metabolic and neural parameters in mother-deprived neonatal rabbit pups.

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, c-Fos (FOS) and PERIOD1 (PER1) 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 h 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 levels were highest around the time of feeding, and then decreased to trough concentrations at ZT12-ZT16, increasing again in anticipation of the 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, as food, in this case milk formula, is a strong synchronizing signal for behavioral, hormonal, metabolic and neural parameters.