Oxytocinergic Cells of the Posterior Hypothalamic Paraventricular Nucleus Participate in the Food Entrained Clock

Oxytocinergic Cells of the Posterior Hypothalamic Paraventricular Nucleus Participate in the Food Entrained Clock | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Oxytocinergic Cells of the Posterior Hypothalamic Paraventricular Nucleus Participate in the Food Entrained Clock Mario Caba, Enrique Meza, Carolina Escobar, Angeles Jiménez, Mario Daniel Caba-Flores, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-622616/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract The mechanisms underlying food anticipatory activity is still not well understood. Here we explored the role of oxytocin (OT) and the protein c-Fos in the supraoptic nucleus (SON) and in the 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; contrasting, OT cells in the PVNp showed c-Fos activation in anticipation to food access, which persisted in RF-F subjects. We conclude that 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. Cellular & Molecular Neuroscience suprachiasmatic nucleus (SCN) oxytocin (OT) Ad libitum (AL) food entrainment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Organization of day and night activity is controlled by the suprachiasmatic nucleus (SCN), the master circadian clock located in the anterior hypothalamus. Light is the main environmental zeitgeber that entrains the SCN, which in turn controls behavioral and physiological rhythms in mammals. The SCN have a self-sustaining clockwork mechanism and through humoral and neural projections control local clocks through the body. Thenceforth, the feeding-fasting cycle is timely organized at the neuroendocrine, physiological and metabolic level at the appropriate time 1 leading that nocturnal rats eat at night during their active phase and rest during the day. Likewise, glucocorticoid hormones rise before the onset of activity to provide circulating glucose from the liver stores and to promote arousal. During the resting phase circulating glucocorticoid levels drop; glucose is stored as glycogen in the liver and the subjects rest 2 . This orderly sequence controlled by the SCN is challenged by feeding schedules, which are powerful zeitgebers, when the time of food access is restricted to a few hours per day. Therefore, when food is provided during a short period of time during the resting phase, subjects awake and exhibit arousal and forging during the resting phase in anticipation to the time of the scheduled meal in order to obtain food. 3 In addition to the intense locomotor behavior they show high glucocorticoid circulating levels in advance of meal timing, a phenomenon known as food anticipatory activity 4 . As mentioned above light is the main cue for the master clock. In the case of FAA, food is the main synchronizer, but in sharp contrast to the light entrainable system. To date, the neural network underlying a food entrainable oscillator is poorly understood. Moreover, the prevailing evidence better supports the proposal that several oscillators both at the central and the peripheral level are entrained by food and together may drive FAA 5 . The specific network is currently poorly understood as food affects central and peripheral organs, mainly those of the gastrointestinal system and glands that release hormones and metabolites in anticipation and as a response to the restricted meal. This complex of secretions is one of the reasons that have complicated the identification of specific signals and routes important for a possible locus critical for FAA. Among the several humoral responses elicited by food ingestion is the activation of hypothalamic cells that produce the hormone oxytocin (OT). It is well documented that food ingestion elicits an acute and sharp activation of OT cells in the hypothalamus and a concomitantly release of this hormone to the peripheral circulation 6,7 . Besides, the infusion of OT exerts anorexigenic effects and their projections from the paraventricular hypothalamic nucleus (PVN) to the brainstem are important for a satiety neural circuit 8,9 . The peripheral actions of OT in relation to food intake are not well explored because the attention to this hormone was centered on their well-known effects in reproductive behavior, particularly parturition and lactation. However, in the last years evidence is accumulating about an important role of OT in several metabolic processes (see below); furthermore, here we report that this hormone plays an important and differential process in FAA. In contrast to the previously well known activation of OT cells after food intake, recently we reported that a particular subpopulation of oxytocinergic cells in the posterior region of the paraventricular hypothalamus in the rabbit pup shows a clear activation in anticipation to their only daily episode of food ingestion 10 . Rabbit pups ingest food only once a day and had been recognized as a natural model of food entrainment 11,12 . The activation of these OT cells during FAA suggests the presence of a possible oscillatory mechanism in a particular region of the PVN that participates in the preparatory ingestion of the timely upcoming of meal. We suggested that the overall implication of this anticipatory activation of OT cells might be a link between a possible central oscillator and peripheral organs in preparation for metabolic and hormonal events that lead to FAA 10 . We believe that this observation in the rabbit pup is relevant for a better understanding of FAA; however, due to the developmental stage of the rabbit pups, it is not possible to contrast their condition with Ad libitum fed and fasted subjects. Likewise, it is not possible to determine the possible persistence of the food entrained patterns as in rodent models. Therefore, we considered it necessary to confirm whether this phenomenon occurs in the rat, which is a species commonly, used to study food entrainment. The present experiment explored OT producing cells in the hypothalamus of adult male rats following a regular restricted scheduled food access protocol to study food entrainment. Based on our previous observations in rabbit pups, our hypothesis was that OT producing cells in the paraventricular hypothalamic nucleus would have a differential activation, as determined by the c-Fos protein, before and after food ingestion in conditions of scheduled food restriction. Moreover, this experimental model allowed us to explore their activation in Ad libitum conditions and the persistence of the food entrained pattern after interruption of the scheduled food access. Materials And Methods Subjects and housing Adult male Wistar rats weighing 275±25 g from the bioterium of the Universidad Veracruzana, were maintained in a controlled light/dark (L/D) cycle (12/12-h, lights on at 07 h, defined as zeitgeber time 0 (ZT0), with regulated temperature (22 ± 1 ° C), and with free access to water, and standard rat chow (Rodent Laboratory Chow 5001; Purina, México). Rats were acclimated to environmental conditions for 1 week before starting the experimental procedures, which were approved by the Universidad Veracruzana and conducted according to the National Guide for the Production, Care and Use of laboratory animals (Official Mexican Norm: NOM-062-Z00-1999) and in accordance with the ARRIVE guidelines (https://arriveguidelines.org). Groups and food entrainment Rats were randomly assigned to one of four feeding conditions and were housed in groups of 4-6 rats of the same condition in transparent acrylic cages (40 x 50 x 20 cm). The Ad libitum (AL) group always had free access to food. The food restricted (RF) group had food available daily for 2 h, from ZT5-ZT7, during three weeks. A third group was exposed to the restricted food protocol followed by two days of fasting (RF-F), which was used to determine the possible persistence of c-Fos/OT immunoreactive (-ir) cells, after the termination of the scheduled feeding. Additionally, an Ad libitum fasted group (AL-F) was included as a control of the RF-F condition. In this group, after a continuous Ad libitum fed condition, food was removed at ZT7 and rats were fasted two days before perfusion. At the end of the manipulations rats were randomly perfused at one of 4 time points (n=5 per group/temporal time point): ZT1, ZT5, ZT7 and ZT13. ZT1 and ZT5 explored food anticipatory activation; ZT7 explored the response to food and ZT13 a late response to food intake. General activity was monitored automatically with tilt sensors placed under each individual cage, which detected continuously the movement of each subject. Data were recorded and stored in 15-s bins to generate double plotted actograms and then were grouped in 1 h bins for analysis at the time points indicated with the circadian recording system SPAD9 (Omnialva, México 11 ). Perfusion and immunohistochemistry Rats were anaesthetized with an overdose of sodium pentobarbital (Sedal-Vet, 65 mg/ml) and were perfused transcardially with ~250 ml of 0.9% saline followed by 250 ml of fixative 4% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.2). Brains were removed immediately after perfusion, postfixed over night and cryoprotected successively in 10, 20 and 30% sucrose in PB. Brains were frozen at -19 °C and cut coronally at 40 μm with a cryostat (Microm, Walldrof, Germany) from the diagonal band of Broca to the mammillary bodies and one set was processed for immunohistochemistry of c-Fos/OT following protocols previously established for double label immunohistochemistry 13,14 . For c-Fos immunohistochemistry tissue was washed three times for 5 min in PB in order to remove excess aldehydes and then exposed for 10 min in 0.5% hydrogen peroxide solution to eliminate endogenous peroxidase activity. Sections were incubated in the polyclonal c-Fos primary antibody raised in goat at a 1:2000 dilution (sc-52G, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in 3% normal horse serum with 0.3% Triton X-100 (Sigma, St. Louis, MO, USA) at 4°C. After 72 h tissue was incubated in biotinylated horse anti-goat serum (1:200, Vector Labs, Burlingame, CA) for 1 h and then incubated in the avidin-biotin-HRP complex (1: 250 ABC Vectastain elite, Vector Laboratories) for 2 hours. After incubations tissue was rinsed three times in PB/5 min each. Staining of c-Fos immunoreactivity (c-Fos-ir) was reacted with a solution of 0.05% diaminobenzidine in the presence of nickel sulfate (10 mg/ml, Fisher Scientific, Pittsburgh, PA), cobalt chloride (10 mg/ml, Fisher Scientific) and 0.01% hydrogen peroxide, which produced a black-purple precipitate. After 5 min the tissue was transferred to PB to stop the reaction. For OT immunohistochemistry sections were rinsed in PB and incubated for 24 h at 4°C in the monoclonal primary antibody made in mouse diluted at 1:5000 (MAB5296, MERCK) with 3% normal horse serum with 0.3% Triton x-100 (Sigma, St. Louis, MO). Sections were then incubated sequentially in biotinylated secondary anti-mouse serum IgG (1:200, Vector Laboratories) for 1 h, and incubated in the avidin-biotin-HRP complex (1: 250 ABC Vectastain elite, Vector Laboratories, Burlingame, CA) for 2 hours. After incubations tissue was rinsed three times in PB (5 min. each). OT antibody-peroxidase complex reaction was contrasted by using 0.05% diaminobenzidine, which produced a brown cytoplasmic precipitate. Sections were mounted on gelatin-coated slides, dehydrated and cover-slipped with Permount. In all cases, tissue sections from subjects of each ZT time were processed together. As control we used tissue sections processed as above but without primary antibody. Quantification of c-Fos, OT and c-Fos/OT cells Sections were coded, so that the observer was blind to the physiological condition of the subject, and were examined under bright-field illumination with an Olympus BX41 microscope at 10X and 20X magnification. c-Fos-ir was identified as a black-purple precipitate from the diaminobenzidine-nickel/cobalt reaction in the cell nucleus, while OT-ir was identified as a brown precipitate in the cytoplasm, whereas double-labeled cells had brown cytoplasm and black nuclei. Immunostaining was absent in tissue processed without primary antibodies. Sections for anatomical identification and quantification of OT-ir, c-Fos-ir and double-labeled c-Fos/OT-ir cells in the hypothalamus were analyzed in the main subdivision of the SON and in two subregions of the PVN, in the medial (PVNm) and the posterior region (PVNp). The SON contains magnocellular neurons that project to the posterior pituitary gland. The PVNm contains populations of both magnocellular and parvocellular neurons, and the area was selected on basis of two previous publication from our lab where a strong induction of c-Fos was found after feeding in the rabbit 14 and in the rat 15 . The PVNp contains preautonomic neurons, which project mainly to the brainstem and spinal cord 16 . The location of brain structures was determined according to the rat atlas of Paxinos and Watson 17 . Sections were analyzed at -1.40 mm (plate 24), -1.80 mm (plate 25) and -2.12 mm (plate 26) for the SON, PVNm and PVNp, respectively (Supplementary Fig. 1). Number of OT-ir, c-Fos-ir and c-Fos/OT-ir cells were counted bilaterally in one representative section at the levels indicated by two observers blind to the experimental condition of animals. Data Analysis Cell counts were grouped for each hypothalamic structure, experimental group and time (ZT) and are represented as mean  standard error of the mean (SEM). Cell data counts and locomotor behavior were compared with a two-way-analysis of variance (ANOVA) for factors group and time. Analysis was followed by a Tukey post hoc test with significant levels set at P<0.05. The data that did not passed the homogeneity of variance were rank-transformed before ANOVA analysis 18 . Statistical analyses were performed using Sigma Stat Software version 3.5, (Chicago, IL). Results Locomotor behavior Rats under AL and AL-F conditions showed an increase of locomotor behavior at the beginning of the night which is the onset of their normal activity phase. In general, it was low during the day with a sharp increase at ZT13 at the onset of the night and remained high along the dark phase. RF and RF-F groups had a completely different pattern. Rats under restricted feeding, RF and in RF-F groups developed FAA at the middle of the day at the time when they are normally sleeping. As shown in the actogram (Figure 1a) the locomotor behavior started increasing about 2 h before the mealtime (ZT3-4), reaching the maximal activity at the moment of food access. In both groups the highest value at ZT5 (at mealtime for RF or projected mealtime RF-F), was significantly higher than the time point before (ZT1; p< .05) and after feeding (ZT7; p< 0.05). The two-way ANOVA indicated that locomotor activity varied significantly among feeding conditions (F 3-48 =11.64, P< .001), time factor (F 3-48 = 28.72, p< .001) and the interaction between feeding condition and time (F 3-48 =11.52, P< .001; (Fig. 1b). The post hoc analysis between groups indicated that values at ZT5 for RF and RF-F groups were significantly higher than AL and AL-F conditions (p< .001). Supraoptic nucleus (SON) In the SON food intake induced an increase of c-Fos and also an activation of OT cells after feeding in food-entrained rats. In the AL group, OT values remained constant except at ZT5, which was significantly lower than value at ZT1 ( p =0.02). In the RF group, a significant increase at ZT7 was observed in comparison to values before feeding at ZT1 and at ZT5 (P<0.01). In the RF-F group the increase at ZT7 did not persist; values were similar in all time points except at ZT7, which was significantly lower than values at ZT1 and at ZT13 ( p <0.05). In the AL-F group, OT values were in general higher than in the other groups. The two-way ANOVA indicated that OT expression in the SON varied significantly due to the group condition (F 3-63 =37.98, P<0.001), and the interaction between feeding condition and time (F 9-63 = 9,011, P<0.001), but not by time factor (F 3-63 = 2,55 P<0.06; Fig. 2a). c-Fos values were low in all groups except in RF group where food intake induced a sharp increase of this protein at ZT7 (P<0.001). This activation by food ingestion did not persist in the RF-F group, which exhibited similar values as the AL group. In the AL-F group, the c-Fos expression was also low and similar to the AL in all time points. The two-way ANOVA indicated that c-Fos expression in the SON varied significantly with group condition (F 3-63 = 442.94; P<0.001), due to the time factor (F 3-63 = 232.79, P<0.001) and to the interaction between feeding condition and time (F 9-63 = 233.21, P<0.001; Figure 2b. Double-labelled c-Fos/OT cells were scarce in all groups except in the RF group where food intake induced at ZT7 ( p <0.001) a sharp increase of double-labelled cells (Figure 2c). In the RF-F group, the food entrained increase at ZT7 did not persist; the OT % activation was similar than in AL and AL-F groups. The two-way ANOVA indicated that the c-Fos/OT percentage of activation in the SON varied significantly among groups condition (F 3-63 = 483.75, P<0.001), time factor (F 3-63 = 221.37, p <0.001) and the interaction between groups and time (F 9-63 = 193.79, p <0.001; Figure 2c). In Figure 3a and b we show microphotographs of SON at the time of feeding (ZT5) and two hours later at ZT7. Median subregion of the Paraventricular hypothalamic nucleus (PVNm ) Similar as observed in the SON, in the PVNm food intake induced an increase of c-Fos and also an activation of OT cells after feeding in food-entrained rats. In the AL group, the number of OT positive cells was similar in all time points with only a significant increase at ZT13 in comparison to ZT1 and at ZT5 values ( p <0.001). In the RF group, there was an increase of OT expression at ZT7 that was significantly different from the remaining time points (P<0.001). In the RF-F group, values remained unchanged in most time points except at ZT13 which was significantly lower from remaining time points ( p <0.001). In the AL-F group, OT value at ZT7 was significantly higher than remaining time points ( p <0.001 in all cases). The two –way ANOVA indicated that OT expression in the PVNm varied significantly with group condition (F 3-63 = 235.289, p <0,001), time factor (F 3-63 = 56.755, p <0,001) and the interaction between feeding condition and time (F 9-63 = 36.760, p <0,001; Figure 4a). c-Fos values were low in all groups except in RF group where food intake induced a sharp increase of this protein at ZT7 ( p <0.001; Figure 4b). This increase did not persist in the RF-F group, which had low values at all time points similar to AL and AL-F groups. The two-way ANOVA indicated that in the AL, RF, RF-F and AL-F groups, c-Fos expression in the PVNm varied significantly with group condition (F 3-63 = 2847.694, p <0,001), time factor (F 3-63 = 3153.575, p <0,001) and the interaction between feeding condition and time (F 9-63 = 3263.682, p <0,001). In the PVNm the AL group showed high c-Fos/OT expression at ZT1 significantly different from the remaining time point values ( p <0.001; Figure 4c). In the RF group, food ingestion triggered expression of c-Fos in OT cells at ZT7 and ZT13, which were significantly higher than the values at ZT1 and ZT5 ( p <0.001) and also from all groups at ZT7 and ZT13 (P < 0.001). The increase in activation of OT cells did not persist in the RF-F group. In this group the highest OT % activation was observed at ZT1, which was significantly different from the remaining time point values (P<0.001 in all cases). In the AL-F group, the OT % activation was not significantly different at any time point ( p =0.250). The two-way ANOVA indicated that OT % activation in the PVNm varied significantly with group condition (F 3-63 = 335.60, P<0,001), time factor (F 3-63 = 74.92, p <0,001) and the interaction between feeding condition and time (F 9-63 = 116.54, p <0,001; Figure 4c). In Figure 3c and d we show microphotographs of the PVNm at the time of feeding at ZT5 and two hours after at ZT7. Posterior subregion of the paraventricular hypothalamic nucleus (PVNp) In contrast to the SON and PVNm, the PVNp exhibited a completely different pattern of c-Fos activation in OT cells. In the RF group, there was a sharp induction of c-Fos before food intake, at the time of FAA. Importantly, this increase persisted in the RF-F group in fasted conditions at the time of the previous scheduled mealtime. The number of OT immunoreactive cells was constant along time points for all experimental groups (Figure 5a). The two-way ANOVA indicated that OT expression in the PVNp did not vary with group condition (F 3-63 =1.48, p =0.23), time factor (F 3-63 = 1.39, p =0.25) nor with the interaction between feeding condition and time (F 9-63 =0.54, p =0.83). The c-Fos expression in the AL group was not significantly different at any time point ( p =0.146). In the RF group, a high c-Fos expression was observed at ZT7, significantly higher than the other time points ( p <0.001). In the RF-F and AL-F groups, c-Fos did not significantly vary among time points (P=0.203 and p=0 .753, respectively). The two-way ANOVA indicated that c-Fos expression in the PVNp varied significantly with group condition (F 3-63 = 9.83, P<0.001), time factor (F 3-63 = 4.60, p =0.007) and the interaction between feeding condition and time (F 9-63 = 9.94, P<0.001; Figure 5b). In contrast to the SON and PVNm, the PVNp showed an activation of c-Fos/OT cells before feeding at ZT5 coinciding with the time of FAA, and this activation persisted in the RF-F group (P<0.01; Figure 5c). In addition, food ingestion also showed an increase in activation of OT cells at ZT7, which was significantly higher than the values at ZT1 and ZT13 (P<0.01). This effect was not observed in AL and in AL-F groups (Figure 5c). Double-labelled c-Fos/OT cells in the PVNp at ZT5 are shown in Figure 6a and b and their persistence in RF-F in Figure 6c and 6d). No double-labelled c-fos/OT cells were observed at ZT5 in the AL-F group (Figs. 6e, f). The two-way ANOVA indicated that c-Fos/OT positive cells in the PVNp varied significantly with group condition (F 3-64 = 24.60, p <0,001), time factor (F 3-63 = 39,18, P<0.001) and the interaction between feeding condition and time (F 9-63 = 21.78, P<0.001). Discussion In agreement to our hypothesis, we found a differential activation of OT producing cells in the hypothalamus of adult rats in conditions of food restriction. In the RF group a high proportion of oxytocinergic cells in the SON and in the PVNm expressed c-Fos after food intake. More important, a specific population in the posterior part of the PVN showed c-Fos activation before food presentation at the time of FAA. Furthermore, this anticipatory activation persisted in entrained-fasted animals at the previous time of scheduled feeding. Present data indicate a differential role of the medial and posterior regions of the PVN in food entrainment. While the medial region appears to respond to the incoming entraining signal given by food intake, the posterior region was activated in anticipation to the feeding event. Overall our results add up to recent accumulating evidence about actions of OT on metabolism homeostasis and are relevant for a better understanding of the FAA phenomenon at both central and peripheral levels. The activation of OT cells after food intake is in agreement with previous reports showing a strong expression of c-Fos in both the SON 19 and PVN 15 in the rat hypothalamus. In the SON and PVN some c-Fos producing neurons were identified as oxytocinergic 19 similar to present results. In conditions of food entrainment a strong induction of c-Fos cells was detected in the main body of the PVN at a similar level as in the present study in the PVNm, but their phenotypical identity was not identified 15 . Here we report that many of those activated cells are oxytocinergic. Classical anatomical studies indicate that OT cells in the SON are magnocellular, project to the neurohypophysis and secrete large amounts of OT, and also vasopressin, to the peripheral circulation 7 . But, unlike the SON, the PVN showed to be more complex 16 . Here, we observed that OT cells in the PVNp, a region not explored before in food-restricted animals, exhibit a different response from the PVNm. SON and PVN have rhythms controlled by the SCN In AL rats significant high levels of c-Fos and c-Fos/OT double labelled cells were found at ZT1, during the morning in the SON and PVN, similar to a previous publication in Ad lib rats 15 . Both SON and PVN have a rhythmic activity controlled by the SCN 1,2 . Anatomical studies show that these two nuclei are targets of the master clock and show parallel c-Fos induction triggered by a light pulse in the SCN 20 . In vitro experiments demonstrate that both SON and PVN show rhythmic activity which rapidly dampens in absence of the SCN input, which confirms that their rhythmic activity is driven by the master clock 21 . But in conditions of food restriction both nuclei uncouple their oscillations from the SCN. Oxytocin, a key hormone as an entraining cue for food entrainment? There is a general agreement that food elicited metabolic responses may be the entraining cue in relation to food entrainment 5 . But unlike the light entrainable system, in the case of food the pathways and the entrained targets are broadly distributed at both central and peripheral level, which has complicated the understanding of this phenomenon. Also they have led to the proposal that instead of a single master clock as in the case of the light entrainable system, there are multiple possible food entrained oscillators responding to different humoral and neural pathways 5 . In this regard, recently it was recognized that very little is known about the synchronizing signals and pathways in food-restricted animals, once food, the main cue, is ingested 22 . On the basis of our present results we propose that the release of the hormone OT following food intake could be an important candidate to be considered as one of these synchronizing signals. Our study shows that in the condition of food restriction, oxytocinergic cells are activated by food intake and shift SON and PVN from their usual daily rhythms controlled by the SCN to a new phase entrained by periodic food intake. Although the SON shows a few c-Fos expressing cells in AL and RF groups at different time points, there is a strong induction of this protein after food intake in agreement to a previous report in the Ad libitum 7 and also in food entrained rats 15 . Moreover, previous work from our laboratory revealed that many of those induced cells are oxytocinergic, not only in the SON but also in the PVN in the rabbit pup 14 , as well as in the rat as observed in the present study, suggesting a massive release of OT to the bloodstream 7 . The attention to the peripheral OT release has been centered in the classical effects of oxytocin on the reproductive system as parturition and milk ejection 23 . Nonetheless, in the last years evidence is accumulating about metabolic effects of this hormone. Circulating OT has shown to improve insulin sensitivity and lipolysis, stimulates glucose uptake and lipid utilization in adipose tissue and skeletal muscle 24- 26 . Besides, the metabolic effects of OT on peripheral organs also can be achieved through indirect projections. The PVN is composed of at least 10 different subregions of magno and parvocellular neurons 27 . In addition to the neurohypophysis, they project to the brainstem and the spinal cord, specifically those in the posterior region of the PVN 16 , and transmit their signal through the autonomic nervous system. PVN have preautonomic motor neurons Electrophysiological studies identified some neurons in the PVNp as preautonomic motor neurons that project to autonomic cells in the brainstem or the spinal cord 28,29 . The attention to these pathways focused mainly on cardiovascular function 30,31 . Detailed studies by retrograde tracing, electrophysiological recordings and morphological reconstructions characterized the properties of these pre autonomic neurons, which were different from adjacent magnocellular neurosecretory neurons. Moreover, these pre autonomic neurons were identified in the posterior region of the PVN 32 in similar posterior levels as in the present contribution and in our previous report in the rabbit pup 10 . Further tract tracing studies with selective autonomic denervation identified separate populations of preganglionic sympathetic and parasympathetic motor neurons in the dorsal and posterior PVN, which through the intermediolateral column and the dorsal motor nucleus of the vagus innervate the liver, pancreas and adrenals. Importantly, they were identified as oxytocinergic cells 33,34 . In the rat a daily hypothalamic preautonomic control of plasma glucose concentrations and insulin (rev. in 35 ) has been reported. In agreement, denervation of the sympathetic input to the liver resulted in a disruption of the daily plasma glucose rhythmicity 36 , and it was demonstrated that this rhythmic activity of the PVN is controlled by the SCN 2,35 . PVN activation during FAA Recently 10 we reported in young rabbits a differential activation of the PVN in relation to restricted food access. The dorsal and the posterior part of the PVN showed an increase of c-Fos in OT neurons at the time of FAA, which persisted in fasted subjects. In contrast, OT cells in the ventral part of the main body of the PVN were activated only after food intake 10 . This differential action of PVN cells in relation to food entrainment had been explored in the rat by c-Fos mRNA 37 . These authors found activation of parvocellular cells in the dorsal and caudal portion of the PVN during FAA and in contrast magnocellular cells in the main body of the PVN, showed activation only after feeding at a similar level here reported. All together, these results reinforce the differential activation of subregions of the PVN at the time of FAA when subjects are hungry and undergoing a catabolic state 38 . Electrophysiological studies provide evidence about the specific properties of these subregions of the PVN. Patch-clamp recording of PVN neurons in the dorsal posterior portion identified glucosensing preautonomic neurons that are glucose-excited or glucose-inhibited 39 . These glucosensing cells are at a similar level where we reported activation of OT cells during FAA in the rabbit 10 . In sharp contrast, adjacent OT cells of the ventral portion of the PVN, below those activated OT cells, were not activated in the rabbit 10 , nor in the study of Melnick et al. 39 in rats, which were identified as neurosecretory, not preautonomic and not glucosensitive. Unfortunately, in the work of Melnick et al. 39 cells of the PVNp were not explored. Taken together, we propose that OT cells in the PVNp may play a main role during FAA as glucosensitive neurons that, perhaps, trigger gluconeogenesis through parasympathetic output to the liver. As mentioned during FAA animals are in a catabolic state and exhibit increase of plasma levels of corticosterone, free fatty acids and glucagon whereas there is a decrease in glycogen and insulin 38 which agree with a possible action of pre autonomic PVN cells on peripheral organs. Other neurochemicals besides OT In this study we have reported c-Fos activation in non-oxytocinergic cells in both, the PVNm and PVNp in RF animals. In a preliminary study we found that some of them colocalize with the peptide vasopressin (AVP), without any apparent tendency in the different restricted feeding conditions (data not shown). However, the PVN also contain cells that produce dopamine, corticotrophin releasing factor (CRF) and encephalin, in addition to other neurotransmitters 27 . Among them CRF cells are interesting as there is an increase of glucocorticoids during FAA in the rat 40 and the rabbit 41–43 and CRF cells are found in the PVN, but not in the SON, and those in the PVN express OT receptors 44 . Additionally OT, but not AVP cells, expresses the corticotrophin releasing factor receptor CRFR2 44 . These authors propose a regulation between OT and CRF release 44 that may be related to the drop of glucocorticoids in food restricted animals after feeding 38, 40, 41, 43 or during fasting after a restricted feeding protocol 40, 42 in both the rat 38, 40 and the rabbit 41-43 . Moreover, tract-tracing studies with pseudorabies virus in peripheral organs demonstrated CRF preautonomic cells in the PVN 45 which also support a possible influence of this nucleus in the increase of glucocorticoids during FAA. Interestingly, the same study 45 found that in the PVN there are no vasopressinergic preautonomic cells to the liver. Overall, we consider that this is important evidence that points out the importance of OT in the metabolic and hormonal changes during FAA and are also in agreement with the lack of activation of AVP cells in the PVN during FAA, as mentioned above. Interestingly, the injection of 6-Hydroxydopamine in the PVN suppressed the increase of CORT during FAA 46 ; this emphasize the importance of the PVN in FAA, their shift from the SCN influence in animals under food restricted protocols, and also the importance of the catecholaminergic system in the PVN for the hormonal changes during FAA. Future studies need to determine the precise interaction between food intake, OT and CRF cells and their receptors in the PVN. In addition , it is necessary to confirm whether these activated OT cells during FAA and at the time of the previous scheduled feeding time in fasted subjects are indeed pre-autonomic. Lastly, it is necessary to explore the phenotypical identity of c-Fos non-oxytocinergic cells, as dopaminergic cells, among other neurochemicals, and their possible participation in FAA. Concluding remarks The protocol of food entrainment evidenced that oxytocin cells in the PVN are a key element in the pathway elicited by food intake, while the PVNm responds to the input provided by meal on set, the PVNp anticipates to the scheduled feeding event. Both regions may constitute a possible link between brain and periphery in the coordination with FAA. In addition, cells of the posterior region of the PVN may be a part of the clockwork mechanism for FAA. Declarations Author contributions Conceived and designed the experiment: MC, EM, CE. Performed the experiments and analyzed the data: MC, CE, AJ, EM, MDCF, MLMC, AM. Wrote, edited and revised the manuscript: MC. EM, CE, MDCF, MLMC. Competing interests The authors declare no competing interests. References Van Drunen, R. & Eckel-Mahan, K. Circadian Rhythms of the Hypothalamus: From Function to Physiology. Clocks Sleep 3 , 189–226, 10.3390/clockssleep3010012 (2021). Yi, C., la Fleur, S. E., Fliers, E. & Kalsbeek, A. The role of the autonomic nervous liver innervation in the control of energy metabolism. Biochim. Biophys. Acta 1802 , 416–431, 10.1016/j.bbadis.2010.01.006 (2010). Silver, R., Balsam, P. D., Butler, M. P. & LeSauter, J. Food anticipation depends on oscillators and memories in both body and brain. Physiol. Behav. 104 , 562–571, 10.1016/j.physbeh.2011.05.034 (2011). Mistlberger, R. E. Circadian food-anticipatory activity: formal models and physiological mechanisms. Neurosci. Biobehav. Rev. 18 , 171–195, 10.1016/0149-7634(94)90023-x (1994). Mistlberger, R. E. Neurobiology of food anticipatory circadian rhythms. Physiol. Behav. 104 , 535–545, 10.1016/j.physbeh.2011.04.015 (2011). Renaud, L. P., Tang, M., McCann, M. J., Stricker, E. M. & Verbalis, J. G. Cholecystokinin and gastric distension activate oxytocinergic cells in rat hypothalamus. Am. J. Physiol. 253 , R661-665, 10.1152/ajpregu.1987.253.4.R661 (1987). Verbalis, J. G., McCann, M. J., McHale, C. M. & Stricker, E. M. Oxytocin secretion in response to cholecystokinin and food: differentiation of nausea from satiety. Science 232 , 1417–1419, 10.1126/science.3715453 (1986). Arletti, R., Benelli, A. & Bertolini, A. Influence of oxytocin on feeding behavior in the rat. Peptides 10 , 89–93, 10.1016/0196-9781(89)90082-x (1989). Rinaman, L. Oxytocinergic inputs to the nucleus of the solitary tract and dorsal motor nucleus of the vagus in neonatal rats. J. Comp. Neurol. 399 , 101–109, 10.1002/(sici)1096-9861(19980914)399:13.0.co;2-5 (1998). Caba, M., Huerta, C., Meza, E., Hernández, M. & Rovirosa-Hernández, M. J. Oxytocinergic Cells of the Hypothalamic Paraventricular Nucleus Are Involved in Food Entrainment. Front. Neurosci. 14 , 49, 10.3389/fnins.2020.00049 (2020). Caba, M. et al. Nature’s food anticipatory experiment: entrainment of locomotor behavior, suprachiasmatic and dorsomedial hypothalamic nuclei by suckling in rabbit pups. Eur. J. Neurosci. 27 , 432–443, 10.1111/j.1460-9568.2008.06017.x (2008). Morgado, E. et al. Artificial feeding synchronizes behavioral, hormonal, metabolic and neural parameters in mother-deprived neonatal rabbit pups. Eur. J. Neurosci. 34 , 1807–1816, 10.1111/j.1460-9568.2011.07898.x (2011). Jiménez, A., Caba, M. & Escobar, C. Food-entrained patterns in orexin cells reveal subregion differential activation. Brain. Res. 1513 , 41–50, 10.1016/j.brainres.2013.03.031 (2013). Caba, M., Rovirosa, M. J. & Silver, R. Suckling and genital stroking induces Fos expression in hypothalamic oxytocinergic neurons of rabbit pups. Dev. Brain. Res. 143 , 119–128, 10.1016/s0165-3806(03)00064-6 (2003). Angeles-Castellanos, M., Aguilar-Roblero, R. & Escobar, C. c-Fos expression in hypothalamic nuclei of food-entrained rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286 , R158-165, 10.1152/ajpregu.00216.2003 (2004). Swanson, L. W. & Kuypers, H. G. The paraventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. J. Comp. Neurol. 194 , 555–570, 10.1002/cne.901940306 (1980). Paxinos, G. & Watson, C. The rat Brain in stereotaxic coordinates . (Academic Press, 1986). Conover, W. J. & Iman, R. L. Rank Transformations as a Bridge Between Parametric and Nonparametric Statistics. Am. Stat. 35 , 124–129, 10.2307/2683975 (1981). Johnstone, L. E., Fong, T. M. & Leng, G. Neuronal activation in the hypothalamus and brainstem during feeding in rats. Cell Metab. 4 , 313–321, 10.1016/j.cmet.2006.08.003 (2006). Munch, I. C., Møller, M., Larsen, P. J. & Vrang, N. Light-induced c-Fos expression in suprachiasmatic nuclei neurons targeting the paraventricular nucleus of the hamster hypothalamus: phase dependence and immunochemical identification. J. Comp. Neurol. 442 , 48–62, 10.1002/cne.1421 (2002). Abe, M. et al. Circadian rhythms in isolated brain regions. J. Neurosci. 22 , 350–356, 10.1523/JNEUROSCI.22-01-00350.2002 (2002). Mistlberger, R. E. Food as circadian time cue for appetitive behavior. F1000Res. 9 , 10.12688/f1000research.20829.1 (2020). Burbach, J. P. H., Young, L. J. & Russell, J. A. Oxytocin: Synthesis, Secretion, and Reproductive Functions in Knobil and Neill’s Physiology of Reproduction (ed. Neill, J. D.) 3055–3128 (Academic Press, 2006). Spetter, M. S. & Hallschmid, M. Current findings on the role of oxytocin in the regulation of food intake. Physiol. Behav. 176 , 31–39, 10.1016/j.physbeh.2017.03.007 (2017). Ding, C., Leow, M. K. & Magkos, F. Oxytocin in metabolic homeostasis: implications for obesity and diabetes management. Obes. Rev. 20 , 22–40, 10.1111/obr.12757 (2019). Onaka, T. & Takayanagi, Y. Role of oxytocin in the control of stress and food intake. J. Neuroendocrinol. 31 , e12700, 10.1111/jne.12700 (2019). Koutcherov, Y., Mai, J. K., Ashwell, K. W. & Paxinos, G. Organization of the human paraventricular hypothalamic nucleus. J. Comp. Neurol. 423 , 299–318, 10.1002/1096-9861(20000724)423:23.0.CO;2-A (2000). Yamashita, H., Inenaga, K. & Koizumi, K. Possible projections from regions of paraventricular and supraoptic nuclei to the spinal cord: electrophysiological studies. Brain Res. 296 , 373–378, 10.1016/0006-8993(84)90077-5 (1984). Lawrence, D. & Pittman, Q. J. Interaction between descending paraventricular neurons and vagal motor neurons. Brain Res. 332 , 158–160, 10.1016/0006-8993(85)90399-3 (1985). Coote, J. H. Cardiovascular function of the paraventricular nucleus of the hypothalamus. Biol. Signals 4 , 142–149, 10.1159/000109434 (1995). Coote, J. H., Yang, Z., Pyner, S. & Deering, J. Control of sympathetic outflows by the hypothalamic paraventricular nucleus. Clin. Exp. Pharmacol. Physiol. 25 , 461–463, 10.1111/j.1440-1681.1998.tb02235.x (1998). Stern, J. E. Electrophysiological and morphological properties of pre-autonomic neurones in the rat hypothalamic paraventricular nucleus. J. Physiol. 537 , 161–177, 10.1111/j.1469-7793.2001.0161k.x (2001). Buijs, R. M., Chun, S. J., Niijima, A., Romijn, H. J. & Nagai, K. Parasympathetic and sympathetic control of the pancreas: a role for the suprachiasmatic nucleus and other hypothalamic centers that are involved in the regulation of food intake. J. Comp. Neurol. 431 , 405–423, 10.1002/1096-9861(20010319)431:43.0.co;2-d (2001). Buijs, R. M. et al. The suprachiasmatic nucleus balances sympathetic and parasympathetic output to peripheral organs through separate preautonomic neurons. J. Comp. Neurol. 464 , 36–48, 10.1002/cne.10765 (2003). Kalsbeek, A. et al. SCN outputs and the hypothalamic balance of life. J. Biol. Rhythms 21 , 458–469, 10.1177/0748730406293854 (2006). Cailotto, C. et al. The suprachiasmatic nucleus controls the daily variation of plasma glucose via the autonomic output to the liver: are the clock genes involved? Eur. J. Neurosci. 22 , 2531–2540, 10.1111/j.1460-9568.2005.04439.x (2005). Poulin, A. M. & Timofeeva, E. The dynamics of neuronal activation during food anticipation and feeding in the brain of food-entrained rats. Brain Res. 1227 , 128–141, 10.1016/j.brainres.2008.06.039 (2008). Díaz-Muñoz, M., Vázquez-Martínez, O., Aguilar-Roblero, R. & Escobar, C. Anticipatory changes in liver metabolism and entrainment of insulin, glucagon, and corticosterone in food-restricted rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 279 , R2048-2056, 10.1152/ajpregu.2000.279.6.R2048 (2000). Melnick, I. V., Price, C. J. & Colmers, W. F. Glucosensing in parvocellular neurons of the rat hypothalamic paraventricular nucleus. Eur. J. Neurosci. 34 , 272–282, 10.1111/j.1460-9568.2011.07742.x (2011). Kato, H., Saito, M. & Suda, M. Effect of starvation on the circadian adrenocortical rhythm in rats. Endocrinology 106 , 918–921, 10.1210/endo-106-3-918 (1980). Morgado, E. et al. Hormonal and metabolic rhythms associated with the daily scheduled nursing in rabbit pups. Am. J. Physiol. Regul. Integr. Comp. Physiol. 295 , R690-695, 10.1152/ajpregu.00162.2008 (2008). Morgado, E. et al. Persistence of hormonal and metabolic rhythms during fasting in 7- to 9-day-old rabbits entrained by nursing during the night. Horm. Behav. 58 , 465–472, 10.1016/j.yhbeh.2010.05.003 (2010). Rovirosa, M. J., Levine, S., Gordon, M. K. & Caba, M. Circadian rhythm of corticosterone secretion in the neonatal rabbit. Dev. Brain Res. 158 , 92–96, 10.1016/j.devbrainres.2005.06.007 (2005). Dabrowska, J. et al. Neuroanatomical evidence for reciprocal regulation of the corticotrophin-releasing factor and oxytocin systems in the hypothalamus and the bed nucleus of the stria terminalis of the rat: Implications for balancing stress and affect. Psychoneuroendocrinology 36 , 1312–1326, 10.1016/j.psyneuen.2011.03.003 (2011). Stanley, S. et al. Identification of neuronal subpopulations that project from hypothalamus to both liver and adipose tissue polysynaptically. Proc. Natl. Acad. Sci. U.S.A. 107 , 7024–7029, 10.1073/pnas.1002790107 (2010). Honma, K., Noe, Y., Honma, S., Katsuno, Y. & Hiroshige, T. Roles of paraventricular catecholamines in feeding-associated corticosterone rhythm in rats. Am. J. Physiol. 262 , E948-955, 10.1152/ajpendo.1992.262.6.E948 (1992). Mistlberger, R. E. & Rusak, B. Food-Anticipatory Circadian Rhythms in Rats with Paraventricular and Lateral Hypothalamic Ablations. J. Biol. Rhythms 3 , 277–291, 10.1177/074873048800300306 (1988). Kreier, F. et al. Tracing from fat tissue, liver, and pancreas: a neuroanatomical framework for the role of the brain in type 2 diabetes. Endocrinology 147 , 1140–1147, 10.1210/en.2005-0667 (2006). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1SR.pdf Additional information Supplementary Figure 1. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 06 Aug, 2021 Reviews received at journal 16 Jul, 2021 Reviewers agreed at journal 01 Jul, 2021 Reviewers invited by journal 28 Jun, 2021 Editor assigned by journal 17 Jun, 2021 Editor invited by journal 17 Jun, 2021 Submission checks completed at journal 17 Jun, 2021 First submitted to journal 14 Jun, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-622616","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":33799055,"identity":"eff728e2-89fd-48fa-a9d9-818fed6ea560","order_by":0,"name":"Mario Caba","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAiklEQVRIiWNgGAWjYHACNgaGCtK1nCFZC2MbKep12w8/e/Bz3uHEBrEzBsRpMTuTZm7Yuw2oRTotgUgtNxjMJHi3HTZmkE4+QKwW9m+Sf+eAtCQ2EKuFx0yat+GwHAm2nMkpN5Y5li7HRrxfjh/f9vBNjTUPv3QOkSEGB2wkqh8Fo2AUjIJRgA8AADIOJ1WKrWlOAAAAAElFTkSuQmCC","orcid":"","institution":"Universidad Veracruzana","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mario","middleName":"","lastName":"Caba","suffix":""},{"id":33799056,"identity":"84bafc36-d749-4bbc-9722-23f8eed16f0d","order_by":1,"name":"Enrique Meza","email":"","orcid":"","institution":"Universidad Veracruzana","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Enrique","middleName":"","lastName":"Meza","suffix":""},{"id":33799057,"identity":"1a3e429d-8e36-4d80-b3a9-daf8bc3fa171","order_by":2,"name":"Carolina Escobar","email":"","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Escobar","suffix":""},{"id":33799058,"identity":"6c39f203-7821-4693-9423-b3af7eff1c9a","order_by":3,"name":"Angeles Jiménez","email":"","orcid":"","institution":"CIRA, UAT-CINVESTAV","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Angeles","middleName":"","lastName":"Jiménez","suffix":""},{"id":33799059,"identity":"636f82a5-fc7e-4a60-ac73-e148a5651257","order_by":4,"name":"Mario Daniel Caba-Flores","email":"","orcid":"","institution":"Universidad Veracruzana","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mario","middleName":"Daniel","lastName":"Caba-Flores","suffix":""},{"id":33799060,"identity":"ab7f2934-19fb-4873-8883-73c577d5e3a0","order_by":5,"name":"María Luisa Moreno-Cortés","email":"","orcid":"","institution":"Universidad Veracruzana","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"María","middleName":"Luisa","lastName":"Moreno-Cortés","suffix":""},{"id":33799061,"identity":"4c1ab64a-a5b2-407f-a364-0a37ec29ba16","order_by":6,"name":"Angel I. Melo","email":"","orcid":"","institution":"CIRA, UAT-CINVESTAV","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Angel","middleName":"I.","lastName":"Melo","suffix":""}],"badges":[],"createdAt":"2021-06-14 22:44:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-622616/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-622616/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":10644427,"identity":"39efcb99-38d9-45fd-a4bd-6eb11beb5833","added_by":"auto","created_at":"2021-06-22 14:58:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":292034,"visible":true,"origin":"","legend":"See the Supplemental Files section for the complete figure caption.","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-622616/v1/da2a0c36ac1c534b7c43c904.png"},{"id":10645178,"identity":"3d09e018-d88b-4f13-a1ff-08d13f7bab12","added_by":"auto","created_at":"2021-06-22 15:01:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":50932,"visible":true,"origin":"","legend":"See the Supplemental Files section for the complete figure caption.","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-622616/v1/67c2fefc13a04bf9e9a2de93.png"},{"id":10644038,"identity":"36019872-2bae-4294-a5bc-b61d8123d4d9","added_by":"auto","created_at":"2021-06-22 14:55:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1412984,"visible":true,"origin":"","legend":"See the Supplemental Files section for the complete figure caption.","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-622616/v1/464e38f1ebfc16e7a4e6bacd.png"},{"id":10643739,"identity":"c25238ed-9164-4113-a8b8-cc4ac7098c08","added_by":"auto","created_at":"2021-06-22 14:52:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":47673,"visible":true,"origin":"","legend":"See the Supplemental Files section for the complete figure caption.","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-622616/v1/2447b8311d466343fb7f4d42.png"},{"id":10644039,"identity":"fa3d19b5-de43-4903-8589-64f84b410ad8","added_by":"auto","created_at":"2021-06-22 14:55:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74566,"visible":true,"origin":"","legend":"See the Supplemental Files section for the complete figure caption.","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-622616/v1/86efcb01781e5771fa58d42c.png"},{"id":10643736,"identity":"5ea18e0a-7f9e-4448-9b92-a36fe04ef434","added_by":"auto","created_at":"2021-06-22 14:52:06","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":558849,"visible":true,"origin":"","legend":"See the Supplemental Files section for the complete figure caption.","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-622616/v1/23a3aff0a075b9a3e58f985e.jpeg"},{"id":13699958,"identity":"f8a7c76f-e3bf-40bc-a0eb-844d11591978","added_by":"auto","created_at":"2021-09-17 13:22:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2797106,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-622616/v1/b7d6e9d2-b636-468c-9282-58f30a1dd313.pdf"},{"id":10644042,"identity":"0d4791c8-a525-422b-bcb3-be41cfe29f6a","added_by":"auto","created_at":"2021-06-22 14:55:06","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1177233,"visible":true,"origin":"","legend":"Additional information\nSupplementary Figure 1. \n","description":"","filename":"SupplementaryFigure1SR.pdf","url":"https://assets-eu.researchsquare.com/files/rs-622616/v1/61e76d214819a4baa4a92d92.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eOxytocinergic Cells of the Posterior Hypothalamic Paraventricular Nucleus Participate in the Food Entrained Clock\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOrganization of day and night activity is controlled by the suprachiasmatic nucleus (SCN), the master circadian clock located in the anterior hypothalamus. Light is the main environmental zeitgeber that entrains the SCN, which in turn controls behavioral and physiological rhythms in mammals. The SCN have a self-sustaining clockwork mechanism and through humoral and neural projections control local clocks through the body. Thenceforth, the feeding-fasting cycle is timely organized at the neuroendocrine, physiological and metabolic level at the appropriate time\u003csup\u003e1\u003c/sup\u003e\u0026nbsp; leading that nocturnal rats eat at night during their active phase and rest during the day. \u0026nbsp;Likewise, glucocorticoid hormones rise before the onset of activity to provide circulating glucose from the liver stores and to promote arousal. During the resting phase circulating glucocorticoid levels drop; glucose is stored as glycogen in the liver and the subjects rest\u003csup\u003e2\u003c/sup\u003e. This orderly sequence controlled by the SCN is challenged by feeding schedules, which are powerful zeitgebers, when the time of food access is restricted to a few hours per day. Therefore, when food is provided during a short period of time during the resting phase, subjects awake and exhibit arousal and forging during the resting phase in anticipation to the time of the scheduled meal in order to obtain food.\u003csup\u003e3\u003c/sup\u003e In addition to the intense locomotor behavior they show high glucocorticoid circulating levels in advance of meal timing, a phenomenon known as food anticipatory activity\u003csup\u003e4\u003c/sup\u003e. \u0026nbsp; As mentioned above light is the main cue for the master clock. In the case of FAA, food is the main synchronizer, but in sharp contrast to the light entrainable system. To date, the neural network underlying a food entrainable oscillator is poorly understood. Moreover, the prevailing evidence better supports the proposal that several oscillators both at the central and the peripheral level are entrained by food and together may drive FAA\u003csup\u003e5\u003c/sup\u003e. The specific network is currently poorly understood as food affects central and peripheral organs, mainly those of the gastrointestinal system and glands that release hormones and metabolites in anticipation and as a response to the restricted meal. This complex of secretions is one of the reasons that have complicated the identification of specific signals and routes important for a possible locus critical for FAA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the several humoral responses elicited by food ingestion is the activation of hypothalamic cells that produce the hormone oxytocin (OT). It is well documented that food ingestion elicits an acute and sharp activation of OT cells in the hypothalamus and a concomitantly release of this hormone to the peripheral circulation\u003csup\u003e6,7\u003c/sup\u003e. Besides, the infusion of OT exerts anorexigenic effects and their projections from the paraventricular hypothalamic nucleus (PVN) to the brainstem are important for a satiety neural circuit\u003csup\u003e8,9\u003c/sup\u003e. The peripheral actions of OT in relation to food intake are not well explored because the attention to this hormone was centered on their well-known effects in reproductive behavior, particularly parturition and lactation. However, in the last years evidence is accumulating about an important role of OT in several metabolic processes (see below); furthermore, here we report that this hormone plays an important and differential process in FAA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast to the previously well known activation of OT cells after food intake, recently we reported that a particular subpopulation of oxytocinergic cells in the posterior region of the paraventricular hypothalamus in the rabbit pup shows a clear activation\u003cem\u003e\u0026nbsp;in anticipation to\u0026nbsp;\u003c/em\u003etheir only daily episode of food ingestion\u003csup\u003e10\u003c/sup\u003e. Rabbit pups ingest food only once a day and had been recognized as a natural model of food entrainment\u003csup\u003e11,12\u003c/sup\u003e. The activation of these OT cells during FAA suggests the presence of a possible oscillatory mechanism in a particular region of the PVN that participates in the preparatory ingestion of the timely upcoming of meal. We suggested that the overall implication of this anticipatory activation of OT cells might be a link between a possible central oscillator and peripheral organs in preparation for metabolic and hormonal events that lead to FAA\u003csup\u003e10\u003c/sup\u003e. We believe that this observation in the rabbit pup is relevant for a better understanding of FAA; however, due to the developmental stage of the rabbit pups, it is not possible to contrast their condition with \u003cem\u003eAd libitum\u003c/em\u003e fed and fasted subjects. Likewise, it is not possible to determine the possible persistence of the food entrained patterns as in rodent models. Therefore, we considered it necessary to confirm whether this phenomenon occurs in the rat, which is a species commonly, used to study food entrainment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe present experiment explored OT producing cells in the hypothalamus of adult male rats following a regular restricted scheduled food access protocol to study food entrainment. Based on our previous observations in rabbit pups, our hypothesis was that OT producing cells in the paraventricular hypothalamic nucleus would have a differential activation, as determined by the c-Fos protein, before and after food ingestion in conditions of scheduled food restriction. Moreover, this experimental model allowed us to explore their activation in \u003cem\u003eAd libitum\u003c/em\u003e conditions and the persistence of the food entrained pattern after interruption of the scheduled food access.\u003c/p\u003e"},{"header":"Materials And Methods","content":" \u003cp\u003e\u003cstrong\u003eSubjects and housing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdult male Wistar rats weighing 275\u0026plusmn;25 g from the bioterium of the Universidad Veracruzana, were maintained in a controlled light/dark (L/D) cycle (12/12-h, lights on at 07 h, defined as zeitgeber time 0 (ZT0), with regulated temperature (22 \u0026plusmn; 1 \u0026deg; C), and with free access to water, and standard rat chow (Rodent Laboratory Chow 5001; Purina, M\u0026eacute;xico). Rats were acclimated to environmental conditions for 1 week before starting the experimental procedures, which were approved by the Universidad Veracruzana and conducted according to the National Guide for the Production, Care and Use of laboratory animals (Official Mexican Norm: NOM-062-Z00-1999) and in accordance with the ARRIVE guidelines (https://arriveguidelines.org).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroups and food entrainment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats were randomly assigned to one of four feeding conditions and were housed in groups of 4-6 rats of the same condition in transparent acrylic cages (40 x 50 x 20 cm). The \u003cem\u003eAd libitum\u003c/em\u003e (AL) group always had free access to food. The food restricted (RF) group had food available daily for 2 h, from ZT5-ZT7, during three weeks. A third group was exposed to the restricted food protocol followed by two days of fasting (RF-F), which was used to determine the possible persistence of c-Fos/OT immunoreactive (-ir) cells, after the termination of the scheduled feeding. Additionally, an \u003cem\u003eAd libitum\u003c/em\u003e fasted group (AL-F) was included as a control of the RF-F condition. In this group, after a continuous \u003cem\u003eAd libitum\u003c/em\u003e fed condition, food was removed at ZT7 and rats were fasted two days before perfusion. At the end of the manipulations rats were randomly perfused at one of 4 time points (n=5 per group/temporal time point): ZT1, ZT5, ZT7 and ZT13. ZT1 and ZT5 explored food anticipatory activation; ZT7 explored the response to food and ZT13 a late response to food intake. General activity was monitored automatically with tilt sensors placed under each individual cage, which detected continuously the movement of each subject. Data were recorded and stored in 15-s bins to generate double plotted actograms and then were grouped in 1 h bins for analysis at the time points indicated with the circadian recording system SPAD9 (Omnialva, M\u0026eacute;xico\u003csup\u003e11\u003c/sup\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePerfusion and immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats were anaesthetized with an overdose of sodium pentobarbital (Sedal-Vet, 65 mg/ml) and were perfused transcardially with ~250 ml of 0.9% saline followed by 250 ml of fixative 4% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.2). Brains were removed immediately after perfusion, postfixed over night and cryoprotected successively in 10, 20 and 30% sucrose in PB. Brains were frozen at -19 \u0026deg;C and cut coronally at 40 \u0026mu;m with a cryostat (Microm, Walldrof, Germany) from the diagonal band of Broca to the mammillary bodies and one set was processed for immunohistochemistry of c-Fos/OT following protocols previously established for double label immunohistochemistry\u003csup\u003e13,14\u003c/sup\u003e. For c-Fos immunohistochemistry tissue was washed three times for 5 min in PB in order to remove excess aldehydes and then exposed for 10 min in 0.5% hydrogen peroxide solution to eliminate endogenous peroxidase activity. Sections were incubated in the polyclonal c-Fos primary antibody raised in goat at a 1:2000 dilution (sc-52G, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in 3% normal horse serum with 0.3% Triton X-100 (Sigma, St. Louis, MO, USA) at 4\u0026deg;C. After 72 h tissue was incubated in biotinylated horse anti-goat serum (1:200, Vector Labs, Burlingame, CA) for 1 h and then incubated in the avidin-biotin-HRP complex (1: 250 ABC Vectastain elite, Vector Laboratories) for 2 hours. After incubations tissue was rinsed three times in PB/5 min each. Staining of c-Fos immunoreactivity (c-Fos-ir) was reacted with a solution of 0.05% diaminobenzidine in the presence of nickel sulfate (10 mg/ml, Fisher Scientific, Pittsburgh, PA), cobalt chloride (10 mg/ml, Fisher Scientific) and 0.01% hydrogen peroxide, which produced a black-purple precipitate. After 5 min the tissue was transferred to PB to stop the reaction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor OT immunohistochemistry sections were rinsed in PB and incubated for 24 h at 4\u0026deg;C in the monoclonal primary antibody made in mouse diluted at 1:5000 (MAB5296, MERCK) with 3% normal horse serum with 0.3% Triton x-100 (Sigma, St. Louis, MO). Sections were then incubated sequentially in biotinylated secondary anti-mouse serum IgG (1:200, Vector Laboratories) for 1 h, and incubated in the avidin-biotin-HRP complex (1: 250 ABC Vectastain elite, Vector Laboratories, Burlingame, CA) for 2 hours. After incubations tissue was rinsed three times in PB (5 min. each). OT antibody-peroxidase complex reaction was contrasted by using 0.05% diaminobenzidine, which produced a brown cytoplasmic precipitate. Sections were mounted on gelatin-coated slides, dehydrated and cover-slipped with Permount. In all cases, tissue sections from subjects of each ZT time were processed together. As control we used tissue sections processed as above but without primary antibody.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of c-Fos, OT and c-Fos/OT cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSections were coded, so that the observer was blind to the physiological condition of the subject, and were examined under bright-field illumination with an Olympus BX41 microscope at 10X and 20X magnification. c-Fos-ir was identified as a black-purple precipitate from the diaminobenzidine-nickel/cobalt reaction in the cell nucleus, while OT-ir was identified as a brown precipitate in the cytoplasm, whereas double-labeled cells had brown cytoplasm and black nuclei. Immunostaining was absent in tissue processed without primary antibodies. Sections for anatomical identification and quantification of OT-ir, c-Fos-ir and double-labeled c-Fos/OT-ir cells in the hypothalamus were analyzed in the main subdivision of the SON and in two subregions of the PVN, in the medial (PVNm) and the posterior region (PVNp). The SON contains magnocellular neurons that project to the posterior pituitary gland. The PVNm contains populations of both magnocellular and parvocellular neurons, and the area was selected on basis of two previous publication from our lab where a strong induction of c-Fos was found after feeding in the rabbit\u003csup\u003e14\u003c/sup\u003e and in the rat\u003csup\u003e15\u003c/sup\u003e. \u0026nbsp;The PVNp contains preautonomic neurons, which project mainly to the brainstem and spinal cord\u003csup\u003e16\u003c/sup\u003e. The location of brain structures was determined according to the rat atlas of Paxinos and Watson\u003csup\u003e17\u003c/sup\u003e. Sections were analyzed at -1.40 mm (plate 24), -1.80 mm (plate 25) and -2.12 mm (plate 26) \u0026nbsp;for the SON, PVNm and PVNp, respectively (Supplementary Fig. 1). Number of OT-ir, c-Fos-ir and c-Fos/OT-ir cells were counted bilaterally in one representative section at the levels indicated by two observers blind to the experimental condition of animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell counts were grouped for each hypothalamic structure, experimental group and time (ZT) and are represented as mean\u0026nbsp;\u0026nbsp;standard error of the mean (SEM). Cell data counts and locomotor behavior were compared with a two-way-analysis of variance (ANOVA) for factors group and time. Analysis was followed by a Tukey \u003cem\u003epost hoc\u003c/em\u003e test with significant levels set at P\u0026lt;0.05.\u0026nbsp;The data that did not passed the homogeneity of variance were rank-transformed before ANOVA analysis\u003csup\u003e18\u003c/sup\u003e. Statistical analyses were performed using Sigma Stat Software version 3.5, (Chicago, IL).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eLocomotor behavior\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats under AL and AL-F conditions showed an increase of locomotor behavior at the beginning of the night which is the onset of their normal activity phase. In general, it was low during the day with a sharp increase at ZT13 at the onset of the night and remained high along the dark phase. RF and RF-F groups had a completely different pattern. Rats under restricted feeding, RF and \u0026nbsp;in RF-F groups developed FAA at the middle of the day at the time when they are normally sleeping. As shown in the actogram (Figure 1a) the locomotor behavior started increasing about 2 h before the mealtime (ZT3-4), reaching the maximal activity at the moment of food access. In both groups the highest value at ZT5 (at mealtime for RF or projected mealtime RF-F), was significantly higher than the time point before (ZT1; p\u0026lt; .05) and \u0026nbsp;after feeding (ZT7; p\u0026lt; 0.05). The two-way ANOVA \u0026nbsp;indicated that locomotor activity varied significantly among feeding conditions (F\u003csub\u003e3-48\u003c/sub\u003e=11.64,\u0026nbsp;P\u0026lt; .001), time factor\u0026nbsp;(F\u003csub\u003e3-48\u003c/sub\u003e= 28.72, p\u0026lt; .001)\u0026nbsp;and\u0026nbsp;the interaction between feeding condition and time\u0026nbsp;(F\u003csub\u003e3-48\u003c/sub\u003e=11.52, P\u0026lt; .001; (Fig. 1b). \u0026nbsp;The post hoc analysis between groups indicated that values at ZT5 for RF and RF-F groups were significantly higher than AL and AL-F conditions (p\u0026lt; .001). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupraoptic nucleus (SON)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the SON food intake induced an increase of c-Fos and also an activation of OT cells after feeding in food-entrained rats.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the AL group, OT values remained constant except at ZT5, which was significantly lower than value at ZT1 (\u003cem\u003ep\u003c/em\u003e=0.02). In the RF group, a significant increase at ZT7 was observed in comparison to values before feeding at ZT1 and at ZT5 (P\u0026lt;0.01). In the RF-F group the increase at ZT7 did not persist; values were similar in all time points except at ZT7, which was significantly lower than values at ZT1 and at ZT13 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). In the AL-F group, OT values were in general higher than in the other groups.\u0026nbsp;The\u0026nbsp;two-way ANOVA indicated that OT expression in the SON varied significantly due to the group condition (F\u003csub\u003e3-63\u003c/sub\u003e=37.98, P\u0026lt;0.001), and the interaction between feeding condition and time (F\u003csub\u003e9-63\u003c/sub\u003e = 9,011, P\u0026lt;0.001), but not by time factor (F\u003csub\u003e3-63\u003c/sub\u003e = 2,55 P\u0026lt;0.06; Fig. 2a).\u003c/p\u003e\n\u003cp\u003ec-Fos values were low in all groups except in RF group where food intake induced a sharp increase of this protein at ZT7\u0026nbsp;(P\u0026lt;0.001).\u0026nbsp;This activation by food ingestion did not persist in the RF-F group, which exhibited similar values as the AL group. In the AL-F group, the c-Fos expression was also low and similar to the AL in all time points.\u0026nbsp;The two-way ANOVA indicated that c-Fos expression in the SON varied significantly with group condition (F\u003csub\u003e3-63\u0026nbsp;\u003c/sub\u003e= 442.94; P\u0026lt;0.001), due to the time factor (F\u003csub\u003e3-63\u003c/sub\u003e = 232.79, P\u0026lt;0.001) and to the interaction between feeding condition and time (F\u003csub\u003e9-63\u003c/sub\u003e = 233.21, P\u0026lt;0.001; Figure 2b.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDouble-labelled c-Fos/OT cells were scarce in all groups except in the RF group where food intake induced at ZT7 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001) a sharp increase of double-labelled cells (Figure 2c). In the RF-F group, the food entrained increase at ZT7 did not persist; the OT % activation was similar than in AL and AL-F groups.\u0026nbsp;The two-way ANOVA indicated that the c-Fos/OT percentage of activation in the SON varied significantly among groups condition (F\u003csub\u003e3-63\u003c/sub\u003e = 483.75, P\u0026lt;0.001), time factor (F\u003csub\u003e3-63\u003c/sub\u003e = 221.37, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001) and the interaction between groups and time (F\u003csub\u003e9-63\u003c/sub\u003e = 193.79, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001; Figure 2c). In Figure 3a and b we show microphotographs of SON at the time of feeding (ZT5) and two hours later at ZT7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMedian subregion of the Paraventricular hypothalamic nucleus (PVNm\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eSimilar as observed in the SON, in the PVNm food intake induced an increase of c-Fos and also an activation of OT cells after feeding in food-entrained rats.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the AL group, the number of OT positive cells was similar in all time points with only a significant increase at ZT13 in comparison to ZT1 and at ZT5 values (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001). In the RF group, there was an increase of OT expression at ZT7 that was significantly different from the remaining time points (P\u0026lt;0.001). In the RF-F group, values remained unchanged in most time points except at ZT13 which was significantly lower from remaining time points (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001). In the AL-F group, OT value at ZT7 was significantly higher than remaining time points \u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 in all cases). The two \u0026ndash;way ANOVA indicated that OT expression in the PVNm varied significantly with group condition (F\u003csub\u003e3-63\u003c/sub\u003e = 235.289, \u003cem\u003ep\u003c/em\u003e\u0026lt;0,001), time factor (F\u003csub\u003e3-63\u003c/sub\u003e = 56.755, \u003cem\u003ep\u003c/em\u003e\u0026lt;0,001) and the interaction between feeding condition and time (F\u003csub\u003e9-63\u003c/sub\u003e = 36.760, \u003cem\u003ep\u003c/em\u003e\u0026lt;0,001; Figure 4a).\u003c/p\u003e\n\u003cp\u003ec-Fos values were low in all groups except in RF group where food intake induced a sharp increase of this protein at ZT7\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001; Figure 4b).\u0026nbsp;This increase did not persist in the RF-F group, which had low values at all time points similar to AL and AL-F groups.\u0026nbsp;The two-way ANOVA indicated that in the AL, RF, RF-F and AL-F groups, c-Fos expression in the PVNm varied significantly with group condition (F\u003csub\u003e3-63\u003c/sub\u003e = 2847.694, \u003cem\u003ep\u003c/em\u003e\u0026lt;0,001), time factor (F\u003csub\u003e3-63\u0026nbsp;\u003c/sub\u003e= 3153.575, \u003cem\u003ep\u003c/em\u003e\u0026lt;0,001) and the interaction between feeding condition and time (F\u003csub\u003e9-63\u003c/sub\u003e = 3263.682, \u003cem\u003ep\u003c/em\u003e\u0026lt;0,001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the PVNm the AL group showed high c-Fos/OT expression at ZT1 significantly different from the remaining time point values (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001; Figure 4c). In the RF group, food ingestion triggered expression of c-Fos in OT cells at ZT7 and ZT13, which were significantly higher than the values at ZT1 and ZT5 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001) and also from all groups at ZT7 and ZT13 \u0026nbsp;(P\u003cem\u003e\u0026lt;\u003c/em\u003e0.001). The increase in activation of OT cells did not persist in the RF-F group. In this group the highest OT % activation was observed at ZT1, which was significantly different from the remaining time point values (P\u0026lt;0.001 in all cases). In the AL-F group, the OT % activation was not significantly different at any time point (\u003cem\u003ep\u003c/em\u003e=0.250). The two-way ANOVA indicated that OT % activation in the PVNm varied significantly with group condition (F\u003csub\u003e3-63\u003c/sub\u003e = 335.60, P\u0026lt;0,001), time factor (F\u003csub\u003e3-63\u003c/sub\u003e = 74.92, \u003cem\u003ep\u003c/em\u003e\u0026lt;0,001) and the interaction between feeding condition and time (F\u003csub\u003e9-63\u003c/sub\u003e = 116.54, \u003cem\u003ep\u003c/em\u003e\u0026lt;0,001; Figure 4c). In Figure 3c and d we show microphotographs of the PVNm at the time of feeding at ZT5 and two hours after at ZT7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePosterior subregion of the paraventricular hypothalamic nucleus (PVNp)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn contrast to the SON and PVNm, the PVNp exhibited a completely different pattern of c-Fos activation in OT cells. In the RF group, there was a sharp induction of c-Fos before food intake, at the time of FAA. Importantly, this increase persisted in the RF-F group in fasted conditions at the time of the previous scheduled mealtime.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe number of OT immunoreactive cells was constant along time points for all experimental groups (Figure 5a). The two-way ANOVA indicated that OT expression in the PVNp did not vary with group condition (F\u003csub\u003e3-63\u003c/sub\u003e =1.48, \u003cem\u003ep\u003c/em\u003e=0.23), time factor (F\u003csub\u003e3-63\u003c/sub\u003e = 1.39, \u003cem\u003ep\u003c/em\u003e=0.25) nor with the interaction between feeding condition and time (F\u003csub\u003e9-63\u003c/sub\u003e =0.54, \u003cem\u003ep\u003c/em\u003e=0.83).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe c-Fos expression in the AL group was not significantly different at any time point (\u003cem\u003ep\u003c/em\u003e=0.146). In the RF group, a high c-Fos expression was observed at ZT7, significantly higher than the other time points (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001). In the RF-F and AL-F groups, c-Fos did not significantly vary among time points (P=0.203 and \u003cem\u003ep=0\u003c/em\u003e.753, respectively).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe two-way ANOVA indicated that c-Fos expression in the PVNp varied significantly with group condition (F\u003csub\u003e3-63\u003c/sub\u003e = 9.83, P\u0026lt;0.001), time factor (F\u003csub\u003e3-63\u003c/sub\u003e = 4.60, \u003cem\u003ep\u003c/em\u003e=0.007) and the interaction between feeding condition and time (F\u003csub\u003e9-63\u003c/sub\u003e = 9.94, P\u0026lt;0.001; Figure 5b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast to the SON and PVNm, the PVNp showed an activation of c-Fos/OT cells \u003cem\u003ebefore\u003c/em\u003e feeding at ZT5 coinciding with the time of FAA, and this activation persisted in the RF-F group (P\u0026lt;0.01; Figure 5c). In addition, food ingestion also showed an increase in activation of OT cells at ZT7, which was significantly higher than the values at ZT1 and ZT13 (P\u0026lt;0.01). This effect was not observed in AL and in AL-F groups (Figure 5c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDouble-labelled c-Fos/OT cells in the PVNp at ZT5 are shown in Figure 6a and b and their persistence in RF-F in Figure 6c and 6d). No double-labelled c-fos/OT cells were observed at ZT5 in the AL-F group (Figs. 6e, f).\u0026nbsp;The two-way ANOVA indicated that c-Fos/OT positive cells in the PVNp varied significantly with group condition (F\u003csub\u003e3-64\u003c/sub\u003e = 24.60, \u003cem\u003ep\u003c/em\u003e\u0026lt;0,001), time factor (F\u003csub\u003e3-63\u003c/sub\u003e = 39,18, P\u0026lt;0.001) and the interaction between feeding condition and time (F\u003csub\u003e9-63\u003c/sub\u003e = 21.78, P\u0026lt;0.001). \u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn agreement to our hypothesis, we found a differential activation of OT producing cells in the hypothalamus of adult rats in conditions of food restriction. In the RF group a high proportion of oxytocinergic cells in the SON and in the PVNm expressed c-Fos after food intake. More important, a specific population in the posterior part of the PVN showed c-Fos activation \u003cem\u003ebefore\u003c/em\u003e food presentation at the time of FAA. \u0026nbsp;Furthermore, this anticipatory activation persisted in entrained-fasted animals at the previous time of scheduled feeding. Present data indicate a differential role of the medial and posterior regions of the PVN in food entrainment. While the medial region appears to respond to the incoming entraining signal given by food intake, the posterior region was activated in anticipation to the feeding event. Overall our results add up to recent accumulating evidence about actions of OT on metabolism homeostasis and are relevant for a better understanding of the FAA phenomenon at both central and peripheral levels.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe activation of OT cells after food intake is in agreement with previous reports showing a strong expression of c-Fos in both the SON\u003csup\u003e19\u003c/sup\u003e and PVN\u003csup\u003e15\u003c/sup\u003e in the rat hypothalamus. In the SON and PVN some c-Fos producing neurons were identified as oxytocinergic\u003csup\u003e19\u003c/sup\u003e similar to present results. In conditions of food entrainment a strong induction of c-Fos cells was detected in the main body of the PVN at a similar level as in the present study in the PVNm, but their phenotypical identity was not identified\u003csup\u003e15\u003c/sup\u003e. Here we report that many of those activated cells are oxytocinergic. Classical anatomical studies indicate that OT cells in the SON are magnocellular, project to the neurohypophysis and secrete large amounts of OT, and also vasopressin, to the peripheral circulation\u003csup\u003e7\u003c/sup\u003e. But, unlike the SON, the PVN showed to be more complex\u003csup\u003e16\u003c/sup\u003e. Here, we observed that OT cells in the PVNp, a region not explored before in food-restricted animals, exhibit a different response from the PVNm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSON and PVN have rhythms controlled by the SCN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn AL rats significant high levels of c-Fos and c-Fos/OT double labelled cells were found at ZT1, during the morning in the SON and PVN, similar to a previous publication in \u003cem\u003eAd lib\u003c/em\u003e rats\u003csup\u003e15\u003c/sup\u003e. \u0026nbsp; Both SON and PVN have a rhythmic activity controlled by the SCN\u003csup\u003e1,2\u003c/sup\u003e. \u0026nbsp;Anatomical studies show that these two nuclei are targets of the master clock and show parallel c-Fos induction triggered by a light pulse in the SCN\u003csup\u003e20\u003c/sup\u003e. \u003cem\u003eIn vitro\u003c/em\u003e experiments demonstrate that both SON and PVN show rhythmic activity which rapidly dampens in absence of the SCN input, which confirms that their rhythmic activity is driven by the master clock\u003csup\u003e21\u003c/sup\u003e. \u0026nbsp; But in conditions of food restriction both nuclei uncouple their oscillations from the SCN. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxytocin, a key hormone as an entraining cue for food entrainment?\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is a general agreement that food elicited metabolic responses may be the entraining cue in relation to food entrainment\u003csup\u003e5\u003c/sup\u003e. \u0026nbsp; But unlike the light entrainable system, in the case of food the pathways and the entrained targets are broadly distributed at both central and peripheral level, which has complicated the understanding of this phenomenon. Also they have led to the proposal that instead of a single master clock as in the case of the light entrainable system, there are multiple possible food entrained oscillators responding to different humoral and neural pathways\u003csup\u003e5\u003c/sup\u003e. In this regard, recently it was recognized that very little is known about the synchronizing signals and pathways in food-restricted animals, once food, the main cue, is ingested\u003csup\u003e22\u003c/sup\u003e. On the basis of our present results we propose that the release of the hormone OT following food intake could be an important candidate to be considered as one of these synchronizing signals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur study shows that in the condition of food restriction, oxytocinergic cells are activated by food intake and shift SON and PVN from their usual daily rhythms controlled by the SCN to a new phase entrained by periodic food intake. Although the SON shows a few c-Fos expressing cells in AL and RF groups at different time points, there is a strong induction of this protein after food intake in agreement to a previous report in the \u003cem\u003eAd libitum\u003c/em\u003e\u003csup\u003e7\u003c/sup\u003e and also in food entrained rats\u003csup\u003e15\u003c/sup\u003e. \u0026nbsp; Moreover, previous work from our laboratory revealed that many of those induced cells are oxytocinergic, not only in the SON but also in the PVN in the rabbit pup\u003csup\u003e14\u003c/sup\u003e, as well as in the rat as observed in the present study, suggesting a massive release of OT to the bloodstream\u003csup\u003e7\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe attention to the peripheral OT release has been centered in the classical effects of oxytocin on the reproductive system as parturition and milk ejection\u003csup\u003e23\u003c/sup\u003e. \u0026nbsp;Nonetheless, in the last years evidence is accumulating about metabolic effects of this hormone. Circulating OT has shown to improve insulin sensitivity and lipolysis, stimulates glucose uptake and lipid utilization in adipose tissue and skeletal muscle \u003csup\u003e24-\u003c/sup\u003e\u003csup\u003e26\u003c/sup\u003e. Besides, the metabolic effects of OT on peripheral organs also can be achieved through indirect projections. The PVN is composed of at least 10 different subregions of magno and parvocellular neurons\u003csup\u003e27\u003c/sup\u003e. In addition to the neurohypophysis, they project to the brainstem and the spinal cord, specifically those in the posterior region of the PVN\u003csup\u003e16\u003c/sup\u003e, and transmit their signal through the autonomic nervous system.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePVN have preautonomic motor neurons\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElectrophysiological studies identified some neurons in the PVNp as preautonomic motor neurons that project to autonomic cells in the brainstem or the spinal cord\u003csup\u003e28,29\u003c/sup\u003e. The attention to these pathways focused mainly on cardiovascular function\u003csup\u003e30,31\u003c/sup\u003e. Detailed studies by retrograde tracing, electrophysiological recordings and morphological reconstructions characterized the properties of these pre autonomic neurons, which were different from adjacent magnocellular neurosecretory neurons. Moreover, these pre autonomic neurons were identified in the posterior region of the PVN\u003csup\u003e32\u003c/sup\u003e in similar posterior levels as in the present contribution and in our previous report in the rabbit pup\u003csup\u003e10\u003c/sup\u003e. Further tract tracing studies with selective autonomic denervation identified separate populations of preganglionic sympathetic and parasympathetic motor neurons in the dorsal and posterior PVN, which through the intermediolateral column and the dorsal motor nucleus of the vagus innervate the liver, pancreas and adrenals. Importantly, they were identified as oxytocinergic cells\u003csup\u003e33,34\u003c/sup\u003e. In the rat a daily hypothalamic preautonomic control of plasma glucose concentrations and insulin (rev. in \u003csup\u003e35\u003c/sup\u003e) has been reported. In agreement, denervation of the sympathetic input to the liver resulted in a disruption of the daily plasma glucose rhythmicity\u003csup\u003e36\u003c/sup\u003e, and \u0026nbsp;it was demonstrated that this rhythmic activity of the PVN is controlled by the SCN\u003csup\u003e2,35\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePVN activation during FAA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecently\u003csup\u003e10\u003c/sup\u003e we reported in young rabbits a differential activation of the PVN in relation to restricted food access. The dorsal and the posterior part of the PVN showed an increase of c-Fos in OT neurons at the time of FAA, which persisted in fasted subjects. In contrast, OT cells in the ventral part of the main body of the PVN were activated only after food intake\u003csup\u003e10\u003c/sup\u003e. This differential action of PVN cells in relation to food entrainment had been explored in the rat by \u003cem\u003ec-Fos\u003c/em\u003e mRNA\u003csup\u003e37\u003c/sup\u003e. These authors found activation of parvocellular cells in the dorsal and caudal portion of the PVN during FAA and in contrast magnocellular cells in the main body of the PVN, showed activation only after feeding at a similar level here reported. All together, these results reinforce the differential activation of subregions of the PVN at the time of FAA when subjects are hungry and undergoing a catabolic state\u003csup\u003e38\u003c/sup\u003e. Electrophysiological studies provide evidence about the specific properties of these subregions of the PVN. Patch-clamp recording of PVN neurons in the dorsal posterior portion identified glucosensing preautonomic neurons that are glucose-excited or glucose-inhibited \u003csup\u003e39\u003c/sup\u003e. These glucosensing cells are at a similar level where we reported activation of OT cells during FAA in the rabbit\u003csup\u003e10\u003c/sup\u003e. In sharp contrast, adjacent OT cells of the ventral portion of the PVN, below those activated OT cells, were not activated in the rabbit \u003csup\u003e10\u003c/sup\u003e, nor in the study of Melnick et al.\u003csup\u003e39\u003c/sup\u003e in rats, which were identified as neurosecretory, not preautonomic and not glucosensitive. Unfortunately, in the work of Melnick et al.\u003csup\u003e39\u003c/sup\u003e cells of the PVNp were not explored.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaken together, we propose that OT cells in the PVNp may play a main role during FAA as glucosensitive neurons that, perhaps, trigger gluconeogenesis through parasympathetic output to the liver. As mentioned during FAA animals are in a catabolic state and exhibit increase of plasma levels of corticosterone, free fatty acids and glucagon whereas there is a decrease in glycogen and insulin\u003csup\u003e38\u003c/sup\u003e which agree with a possible action of pre autonomic PVN cells on peripheral organs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOther neurochemicals besides OT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study we have reported c-Fos activation in non-oxytocinergic cells in both, the PVNm and PVNp in RF animals. In a preliminary study we found that some of them colocalize with the peptide vasopressin (AVP), without any apparent tendency in the different restricted feeding conditions (data not shown). However, the PVN also contain cells that produce dopamine, corticotrophin releasing factor (CRF) and encephalin, in addition to other neurotransmitters\u003csup\u003e27\u003c/sup\u003e. Among them CRF cells are interesting as there is an increase of glucocorticoids during FAA in the rat\u003csup\u003e40\u003c/sup\u003e and the rabbit\u003csup\u003e41\u0026ndash;43\u003c/sup\u003e and CRF cells are found in the PVN, but not in the SON, and those in the PVN express OT receptors\u003csup\u003e44\u003c/sup\u003e. Additionally OT, but not AVP cells, expresses the corticotrophin releasing factor receptor CRFR2\u003csup\u003e44\u003c/sup\u003e. These authors propose a regulation between OT and CRF release\u003csup\u003e44\u003c/sup\u003e that may be related to the drop of glucocorticoids in food restricted animals after feeding\u003csup\u003e38, 40, 41, 43\u0026nbsp;\u003c/sup\u003eor during fasting after a restricted feeding protocol\u003csup\u003e40, 42\u003c/sup\u003e in both the rat\u003csup\u003e38, 40\u0026nbsp;\u003c/sup\u003eand the rabbit \u003csup\u003e41-43\u003c/sup\u003e. Moreover, tract-tracing studies with pseudorabies virus in peripheral organs demonstrated CRF preautonomic cells in the PVN\u003csup\u003e45\u003c/sup\u003e which also support a possible influence of this nucleus in the increase of glucocorticoids during FAA. Interestingly, the same study\u003csup\u003e45\u003c/sup\u003e found that in the PVN there are no vasopressinergic preautonomic cells to the liver. Overall, we consider that this is\u003cs\u003e\u0026nbsp;\u003c/s\u003eimportant evidence that points out the importance of OT in the metabolic and hormonal changes during FAA and are also in agreement with the lack of activation of AVP cells in the PVN during FAA, as mentioned above. Interestingly, the injection of 6-Hydroxydopamine in the PVN suppressed the increase of CORT during FAA\u003csup\u003e46\u003c/sup\u003e; this emphasize the importance of the PVN in FAA, their shift from the SCN influence in animals under food restricted protocols, and also the importance of the catecholaminergic system in the PVN for the hormonal changes during FAA. Future studies need to determine the precise interaction between food intake, OT and CRF cells and their receptors in the PVN. In addition\u003cs\u003e,\u003c/s\u003e it is necessary to confirm whether these activated OT cells during FAA and at the time of the previous scheduled feeding time in fasted subjects are indeed pre-autonomic. Lastly, it is necessary to explore the phenotypical identity of c-Fos non-oxytocinergic cells, as dopaminergic cells, among other neurochemicals, and their possible participation in FAA.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConcluding remarks\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocol of food entrainment evidenced that oxytocin cells in the PVN are a key element in the pathway elicited by food intake, while the PVNm responds to the input provided by meal on set, the PVNp anticipates to the scheduled feeding event. Both regions may constitute a possible link between brain and periphery in the coordination with FAA. In addition, cells of the posterior region of the PVN may be a part of the clockwork mechanism for FAA.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived and designed the experiment: MC, EM, CE. Performed the experiments and analyzed the data: MC, CE, AJ, EM, MDCF, MLMC, AM. Wrote, edited and revised the manuscript: MC. EM, CE, MDCF, MLMC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eVan Drunen, R. \u0026amp; Eckel-Mahan, K. Circadian Rhythms of the Hypothalamus: From Function to Physiology. \u003cem\u003eClocks Sleep\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 189\u0026ndash;226, 10.3390/clockssleep3010012 (2021).\u003c/li\u003e\n \u003cli\u003eYi, C., la Fleur, S. E., Fliers, E. \u0026amp; Kalsbeek, A. The role of the autonomic nervous liver innervation in the control of energy metabolism. \u003cem\u003eBiochim. Biophys. Acta\u003c/em\u003e \u003cstrong\u003e1802\u003c/strong\u003e, 416\u0026ndash;431, 10.1016/j.bbadis.2010.01.006 (2010).\u003c/li\u003e\n \u003cli\u003eSilver, R., Balsam, P. D., Butler, M. P. \u0026amp; LeSauter, J. Food anticipation depends on oscillators and memories in both body and brain. \u003cem\u003ePhysiol. Behav.\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 562\u0026ndash;571, 10.1016/j.physbeh.2011.05.034 (2011).\u003c/li\u003e\n \u003cli\u003eMistlberger, R. E. Circadian food-anticipatory activity: formal models and physiological mechanisms. \u003cem\u003eNeurosci. Biobehav. Rev.\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 171\u0026ndash;195, 10.1016/0149-7634(94)90023-x (1994).\u003c/li\u003e\n \u003cli\u003eMistlberger, R. E. Neurobiology of food anticipatory circadian rhythms. \u003cem\u003ePhysiol. Behav.\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 535\u0026ndash;545, 10.1016/j.physbeh.2011.04.015 (2011).\u003c/li\u003e\n \u003cli\u003eRenaud, L. P., Tang, M., McCann, M. J., Stricker, E. M. \u0026amp; Verbalis, J. G. Cholecystokinin and gastric distension activate oxytocinergic cells in rat hypothalamus. \u003cem\u003eAm. J. Physiol.\u003c/em\u003e \u003cstrong\u003e253\u003c/strong\u003e, R661-665, 10.1152/ajpregu.1987.253.4.R661 (1987).\u003c/li\u003e\n \u003cli\u003eVerbalis, J. G., McCann, M. J., McHale, C. M. \u0026amp; Stricker, E. M. Oxytocin secretion in response to cholecystokinin and food: differentiation of nausea from satiety. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e232\u003c/strong\u003e, 1417\u0026ndash;1419, 10.1126/science.3715453 (1986).\u003c/li\u003e\n \u003cli\u003eArletti, R., Benelli, A. \u0026amp; Bertolini, A. Influence of oxytocin on feeding behavior in the rat. \u003cem\u003ePeptides\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 89\u0026ndash;93, 10.1016/0196-9781(89)90082-x (1989).\u003c/li\u003e\n \u003cli\u003eRinaman, L. Oxytocinergic inputs to the nucleus of the solitary tract and dorsal motor nucleus of the vagus in neonatal rats. \u003cem\u003eJ. Comp. Neurol.\u003c/em\u003e \u003cstrong\u003e399\u003c/strong\u003e, 101\u0026ndash;109, 10.1002/(sici)1096-9861(19980914)399:1\u0026lt;101::aid-cne8\u0026gt;3.0.co;2-5 (1998).\u003c/li\u003e\n \u003cli\u003eCaba, M., Huerta, C., Meza, E., Hern\u0026aacute;ndez, M. \u0026amp; Rovirosa-Hern\u0026aacute;ndez, M. J. Oxytocinergic Cells of the Hypothalamic Paraventricular Nucleus Are Involved in Food Entrainment. \u003cem\u003eFront. Neurosci.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 49, 10.3389/fnins.2020.00049 (2020).\u003c/li\u003e\n \u003cli\u003eCaba, M. \u003cem\u003eet al.\u003c/em\u003e Nature\u0026rsquo;s food anticipatory experiment: entrainment of locomotor behavior, suprachiasmatic and dorsomedial hypothalamic nuclei by suckling in rabbit pups. \u003cem\u003eEur. J. Neurosci.\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 432\u0026ndash;443, 10.1111/j.1460-9568.2008.06017.x (2008).\u003c/li\u003e\n \u003cli\u003eMorgado, E. \u003cem\u003eet al.\u003c/em\u003e Artificial feeding synchronizes behavioral, hormonal, metabolic and neural parameters in mother-deprived neonatal rabbit pups. \u003cem\u003eEur. J. Neurosci.\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 1807\u0026ndash;1816, 10.1111/j.1460-9568.2011.07898.x (2011).\u003c/li\u003e\n \u003cli\u003eJim\u0026eacute;nez, A., Caba, M. \u0026amp; Escobar, C. Food-entrained patterns in orexin cells reveal subregion differential activation. \u003cem\u003eBrain. Res.\u003c/em\u003e \u003cstrong\u003e1513\u003c/strong\u003e, 41\u0026ndash;50, 10.1016/j.brainres.2013.03.031 (2013).\u003c/li\u003e\n \u003cli\u003eCaba, M., Rovirosa, M. J. \u0026amp; Silver, R. Suckling and genital stroking induces Fos expression in hypothalamic oxytocinergic neurons of rabbit pups. \u003cem\u003eDev. Brain. Res.\u003c/em\u003e \u003cstrong\u003e143\u003c/strong\u003e, 119\u0026ndash;128, 10.1016/s0165-3806(03)00064-6 (2003).\u003c/li\u003e\n \u003cli\u003eAngeles-Castellanos, M., Aguilar-Roblero, R. \u0026amp; Escobar, C. c-Fos expression in hypothalamic nuclei of food-entrained rats. \u003cem\u003eAm. J. Physiol. Regul. Integr. Comp. Physiol.\u003c/em\u003e \u003cstrong\u003e286\u003c/strong\u003e, R158-165, 10.1152/ajpregu.00216.2003 (2004).\u003c/li\u003e\n \u003cli\u003eSwanson, L. W. \u0026amp; Kuypers, H. G. The paraventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. \u003cem\u003eJ. Comp. Neurol.\u003c/em\u003e \u003cstrong\u003e194\u003c/strong\u003e, 555\u0026ndash;570, 10.1002/cne.901940306 (1980).\u003c/li\u003e\n \u003cli\u003ePaxinos, G. \u0026amp; Watson, C. \u003cem\u003eThe rat Brain in stereotaxic coordinates\u003c/em\u003e. (Academic Press, 1986).\u003c/li\u003e\n \u003cli\u003eConover, W. J. \u0026amp; Iman, R. L. Rank Transformations as a Bridge Between Parametric and Nonparametric Statistics. \u003cem\u003eAm. Stat.\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 124\u0026ndash;129, 10.2307/2683975 (1981).\u003c/li\u003e\n \u003cli\u003eJohnstone, L. E., Fong, T. M. \u0026amp; Leng, G. Neuronal activation in the hypothalamus and brainstem during feeding in rats. \u003cem\u003eCell Metab.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 313\u0026ndash;321, 10.1016/j.cmet.2006.08.003 (2006).\u003c/li\u003e\n \u003cli\u003eMunch, I. C., M\u0026oslash;ller, M., Larsen, P. J. \u0026amp; Vrang, N. Light-induced c-Fos expression in suprachiasmatic nuclei neurons targeting the paraventricular nucleus of the hamster hypothalamus: phase dependence and immunochemical identification. \u003cem\u003eJ. Comp. Neurol.\u003c/em\u003e \u003cstrong\u003e442\u003c/strong\u003e, 48\u0026ndash;62, 10.1002/cne.1421 (2002).\u003c/li\u003e\n \u003cli\u003eAbe, M. \u003cem\u003eet al.\u003c/em\u003e Circadian rhythms in isolated brain regions. \u003cem\u003eJ. Neurosci.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 350\u0026ndash;356, 10.1523/JNEUROSCI.22-01-00350.2002 (2002).\u003c/li\u003e\n \u003cli\u003eMistlberger, R. E. Food as circadian time cue for appetitive behavior. \u003cem\u003eF1000Res.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 10.12688/f1000research.20829.1 (2020).\u003c/li\u003e\n \u003cli\u003eBurbach, J. P. H., Young, L. J. \u0026amp; Russell, J. A. Oxytocin: Synthesis, Secretion, and Reproductive Functions in \u003cem\u003eKnobil and Neill\u0026rsquo;s Physiology of Reproduction\u003c/em\u003e (ed. Neill, J. D.) 3055\u0026ndash;3128 (Academic Press, 2006).\u003c/li\u003e\n \u003cli\u003eSpetter, M. S. \u0026amp; Hallschmid, M. Current findings on the role of oxytocin in the regulation of food intake. \u003cem\u003ePhysiol. Behav.\u003c/em\u003e \u003cstrong\u003e176\u003c/strong\u003e, 31\u0026ndash;39, 10.1016/j.physbeh.2017.03.007 (2017).\u003c/li\u003e\n \u003cli\u003eDing, C., Leow, M. K. \u0026amp; Magkos, F. Oxytocin in metabolic homeostasis: implications for obesity and diabetes management. \u003cem\u003eObes. Rev.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 22\u0026ndash;40, 10.1111/obr.12757 (2019).\u003c/li\u003e\n \u003cli\u003eOnaka, T. \u0026amp; Takayanagi, Y. Role of oxytocin in the control of stress and food intake. \u003cem\u003eJ. Neuroendocrinol.\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, e12700, 10.1111/jne.12700 (2019).\u003c/li\u003e\n \u003cli\u003eKoutcherov, Y., Mai, J. K., Ashwell, K. W. \u0026amp; Paxinos, G. Organization of the human paraventricular hypothalamic nucleus. \u003cem\u003eJ. Comp. Neurol.\u003c/em\u003e \u003cstrong\u003e423\u003c/strong\u003e, 299\u0026ndash;318, 10.1002/1096-9861(20000724)423:2\u0026lt;299::AID-CNE8\u0026gt;3.0.CO;2-A (2000).\u003c/li\u003e\n \u003cli\u003eYamashita, H., Inenaga, K. \u0026amp; Koizumi, K. Possible projections from regions of paraventricular and supraoptic nuclei to the spinal cord: electrophysiological studies. \u003cem\u003eBrain Res.\u003c/em\u003e \u003cstrong\u003e296\u003c/strong\u003e, 373\u0026ndash;378, 10.1016/0006-8993(84)90077-5 (1984).\u003c/li\u003e\n \u003cli\u003eLawrence, D. \u0026amp; Pittman, Q. J. Interaction between descending paraventricular neurons and vagal motor neurons. \u003cem\u003eBrain Res.\u003c/em\u003e \u003cstrong\u003e332\u003c/strong\u003e, 158\u0026ndash;160, 10.1016/0006-8993(85)90399-3 (1985).\u003c/li\u003e\n \u003cli\u003eCoote, J. H. Cardiovascular function of the paraventricular nucleus of the hypothalamus. \u003cem\u003eBiol. Signals\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 142\u0026ndash;149, 10.1159/000109434 (1995).\u003c/li\u003e\n \u003cli\u003eCoote, J. H., Yang, Z., Pyner, S. \u0026amp; Deering, J. Control of sympathetic outflows by the hypothalamic paraventricular nucleus. \u003cem\u003eClin. Exp. Pharmacol. Physiol.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 461\u0026ndash;463, 10.1111/j.1440-1681.1998.tb02235.x (1998).\u003c/li\u003e\n \u003cli\u003eStern, J. E. Electrophysiological and morphological properties of pre-autonomic neurones in the rat hypothalamic paraventricular nucleus. \u003cem\u003eJ. Physiol.\u003c/em\u003e \u003cstrong\u003e537\u003c/strong\u003e, 161\u0026ndash;177, 10.1111/j.1469-7793.2001.0161k.x (2001).\u003c/li\u003e\n \u003cli\u003eBuijs, R. M., Chun, S. J., Niijima, A., Romijn, H. J. \u0026amp; Nagai, K. Parasympathetic and sympathetic control of the pancreas: a role for the suprachiasmatic nucleus and other hypothalamic centers that are involved in the regulation of food intake. \u003cem\u003eJ. Comp. Neurol.\u003c/em\u003e \u003cstrong\u003e431\u003c/strong\u003e, 405\u0026ndash;423, 10.1002/1096-9861(20010319)431:4\u0026lt;405::aid-cne1079\u0026gt;3.0.co;2-d (2001).\u003c/li\u003e\n \u003cli\u003eBuijs, R. M. \u003cem\u003eet al.\u003c/em\u003e The suprachiasmatic nucleus balances sympathetic and parasympathetic output to peripheral organs through separate preautonomic neurons. \u003cem\u003eJ. Comp. Neurol.\u003c/em\u003e \u003cstrong\u003e464\u003c/strong\u003e, 36\u0026ndash;48, 10.1002/cne.10765 (2003).\u003c/li\u003e\n \u003cli\u003eKalsbeek, A. \u003cem\u003eet al.\u003c/em\u003e SCN outputs and the hypothalamic balance of life. \u003cem\u003eJ. Biol. Rhythms\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 458\u0026ndash;469, 10.1177/0748730406293854 (2006).\u003c/li\u003e\n \u003cli\u003eCailotto, C. \u003cem\u003eet al.\u003c/em\u003e The suprachiasmatic nucleus controls the daily variation of plasma glucose via the autonomic output to the liver: are the clock genes involved? \u003cem\u003eEur. J. Neurosci.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 2531\u0026ndash;2540, 10.1111/j.1460-9568.2005.04439.x (2005).\u003c/li\u003e\n \u003cli\u003ePoulin, A. M. \u0026amp; Timofeeva, E. The dynamics of neuronal activation during food anticipation and feeding in the brain of food-entrained rats. \u003cem\u003eBrain Res.\u003c/em\u003e \u003cstrong\u003e1227\u003c/strong\u003e, 128\u0026ndash;141, 10.1016/j.brainres.2008.06.039 (2008).\u003c/li\u003e\n \u003cli\u003eD\u0026iacute;az-Mu\u0026ntilde;oz, M., V\u0026aacute;zquez-Mart\u0026iacute;nez, O., Aguilar-Roblero, R. \u0026amp; Escobar, C. Anticipatory changes in liver metabolism and entrainment of insulin, glucagon, and corticosterone in food-restricted rats. \u003cem\u003eAm. J. Physiol. Regul. Integr. Comp. Physiol.\u003c/em\u003e \u003cstrong\u003e279\u003c/strong\u003e, R2048-2056, 10.1152/ajpregu.2000.279.6.R2048 (2000).\u003c/li\u003e\n \u003cli\u003eMelnick, I. V., Price, C. J. \u0026amp; Colmers, W. F. Glucosensing in parvocellular neurons of the rat hypothalamic paraventricular nucleus. \u003cem\u003eEur. J. Neurosci.\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 272\u0026ndash;282, 10.1111/j.1460-9568.2011.07742.x (2011).\u003c/li\u003e\n \u003cli\u003eKato, H., Saito, M. \u0026amp; Suda, M. Effect of starvation on the circadian adrenocortical rhythm in rats. \u003cem\u003eEndocrinology\u003c/em\u003e \u003cstrong\u003e106\u003c/strong\u003e, 918\u0026ndash;921, 10.1210/endo-106-3-918 (1980).\u003c/li\u003e\n \u003cli\u003eMorgado, E. \u003cem\u003eet al.\u003c/em\u003e Hormonal and metabolic rhythms associated with the daily scheduled nursing in rabbit pups. \u003cem\u003eAm. J. Physiol. Regul. Integr. Comp. Physiol.\u003c/em\u003e \u003cstrong\u003e295\u003c/strong\u003e, R690-695, 10.1152/ajpregu.00162.2008 (2008).\u003c/li\u003e\n \u003cli\u003eMorgado, E. \u003cem\u003eet al.\u003c/em\u003e Persistence of hormonal and metabolic rhythms during fasting in 7- to 9-day-old rabbits entrained by nursing during the night. \u003cem\u003eHorm. Behav.\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 465\u0026ndash;472, 10.1016/j.yhbeh.2010.05.003 (2010).\u003c/li\u003e\n \u003cli\u003eRovirosa, M. J., Levine, S., Gordon, M. K. \u0026amp; Caba, M. Circadian rhythm of corticosterone secretion in the neonatal rabbit. \u003cem\u003eDev. Brain Res.\u003c/em\u003e \u003cstrong\u003e158\u003c/strong\u003e, 92\u0026ndash;96, 10.1016/j.devbrainres.2005.06.007 (2005).\u003c/li\u003e\n \u003cli\u003eDabrowska, J. \u003cem\u003eet al.\u003c/em\u003e Neuroanatomical evidence for reciprocal regulation of the corticotrophin-releasing factor and oxytocin systems in the hypothalamus and the bed nucleus of the stria terminalis of the rat: Implications for balancing stress and affect. \u003cem\u003ePsychoneuroendocrinology\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 1312\u0026ndash;1326, 10.1016/j.psyneuen.2011.03.003 (2011).\u003c/li\u003e\n \u003cli\u003eStanley, S. \u003cem\u003eet al.\u003c/em\u003e Identification of neuronal subpopulations that project from hypothalamus to both liver and adipose tissue polysynaptically. \u003cem\u003eProc. Natl. Acad. Sci. U.S.A.\u003c/em\u003e \u003cstrong\u003e107\u003c/strong\u003e, 7024\u0026ndash;7029, 10.1073/pnas.1002790107 (2010).\u003c/li\u003e\n \u003cli\u003eHonma, K., Noe, Y., Honma, S., Katsuno, Y. \u0026amp; Hiroshige, T. Roles of paraventricular catecholamines in feeding-associated corticosterone rhythm in rats. \u003cem\u003eAm. J. Physiol.\u003c/em\u003e \u003cstrong\u003e262\u003c/strong\u003e, E948-955, 10.1152/ajpendo.1992.262.6.E948 (1992).\u003c/li\u003e\n \u003cli\u003eMistlberger, R. E. \u0026amp; Rusak, B. Food-Anticipatory Circadian Rhythms in Rats with Paraventricular and Lateral Hypothalamic Ablations. \u003cem\u003eJ. Biol. Rhythms\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 277\u0026ndash;291, 10.1177/074873048800300306 (1988).\u003c/li\u003e\n \u003cli\u003eKreier, F. \u003cem\u003eet al.\u003c/em\u003e Tracing from fat tissue, liver, and pancreas: a neuroanatomical framework for the role of the brain in type 2 diabetes. \u003cem\u003eEndocrinology\u003c/em\u003e \u003cstrong\u003e147\u003c/strong\u003e, 1140\u0026ndash;1147, 10.1210/en.2005-0667 (2006).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"suprachiasmatic nucleus (SCN), oxytocin (OT), Ad libitum (AL), food entrainment","lastPublishedDoi":"10.21203/rs.3.rs-622616/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-622616/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe mechanisms underlying food anticipatory activity is still not well understood. Here we explored the role of oxytocin (OT) and the protein c-Fos in the supraoptic nucleus (SON) and in the medial (PVNm) and posterior (PVNp) regions of the paraventricular hypothalamic nucleus. Adult rats were assigned to one of four groups: scheduled restricted feeding (RF), \u003cem\u003eAd libitum\u003c/em\u003e (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; contrasting, OT cells in the PVNp showed c-Fos activation \u003cem\u003ein anticipation to\u003c/em\u003e food access, which persisted in RF-F subjects. We conclude that 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.\u003c/p\u003e","manuscriptTitle":"Oxytocinergic Cells of the Posterior Hypothalamic Paraventricular Nucleus Participate in the Food Entrained Clock","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-06-22 14:52:04","doi":"10.21203/rs.3.rs-622616/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-08-06T16:51:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-07-16T10:01:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"625ff189-5646-45f8-9f65-d7f79139c96c","date":"2021-07-01T06:46:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-06-28T06:43:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-06-17T18:57:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-06-17T18:01:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-06-17T07:28:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2021-06-14T22:36:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c98d6d43-bc12-4934-8214-024d64bff964","owner":[],"postedDate":"June 22nd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":5175916,"name":"Cellular \u0026 Molecular Neuroscience"}],"tags":[{"value":"featured","date":"2021-06-25 16:48:48"}],"updatedAt":"2021-09-22T10:44:08+00:00","versionOfRecord":[],"versionCreatedAt":"2021-06-22 14:52:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-622616","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-622616","identity":"rs-622616","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}