Manuel Angeles-Castellanos

Food Intake during the Normal Activity Phase Prevents Obesity and Circadian Desynchrony in a Rat Model of Night Work

Shift work or night work is associated with hypertension, metabolic syndrome, cancer, and other diseases. The cause for these pathologies is proposed to be the dissociation between the temporal signals from the biological clock and the sleep/activity schedule of the night worker. We investigated the mechanisms promoting metabolic desynchrony in a model for night work in rats, based on daily 8-h activity schedules during the resting phase. We demonstrate that the major alterations leading to internal desynchrony induced by this working protocol, flattened glucose and locomotor rhythms and the development of abdominal obesity, were caused by food intake during the rest phase. Shifting food intake to the normal activity phase prevented body weight increase and reverted metabolic and rhythmic disturbances of the shift work animals to control ranges. These observations demonstrate that feeding habits may prevent or induce internal desynchrony and obesity.

Restricted feeding schedules phase shift daily rhythms of c-Fos and protein Per1 immunoreactivity in corticolimbic regions in rats.

Entrainment by daily restricted feeding schedules (RFS) produces food anticipatory activity (FAA) which involves motivational processes which may be regulated by corticolimbic structures and the nucleus accumbens. The present study aimed first to determine whether corticolimbic structures participate in the expression of FAA, therefore c-Fos immunoreactivity (Fos-IR) was employed as marker of neuronal activity. The second goal was to characterize diurnal rhythms of the clock protein protein Per1 (PER1) in corticolimbic structures and to determine the influence of RFS on the diurnal temporal pattern. Rats were maintained under RFS with food access for 2 h daily, a control group was fed ad libitum. Food entrainment produced a pattern of increased Fos-IR during FAA and after mealtime in the two sub-regions of the nucleus accumbens (ACC), in the basolateral and central amygdala, in the bed nucleus of the stria terminalis (BNST), in the lateral septum (LS), in the prefrontal cortex (PFC), and in the paraventricular thalamic nucleus (PVT). No increased Fos-IR was observed in the hippocampus. Under ad libitum conditions all structures studied showed daily oscillations of PER1, excluding both amygdalar nuclei and the PFC. RFS shifted and set the daily peaks at zeitgeber time (ZT) 12 for both sub-regions in the accumbens, the hippocampus, lateral septum and PFC. RFS enhanced the amplitude at ZT12 of the BNST and shifted the peak of the PVT to ZT6. No changes were observed in the amygdalar nuclei. Present data indicate that cellular activation of corticolimbic structures is associated with behavioral events related to food anticipatory activity and that mealtime is a relevant signal that shifts daily oscillations of PER1 in corticolimbic structures. Data suggest a relevant role of corticolimbic structures as oscillators for FAA.

Internal desynchronization in a model of night-work by forced activity in rats.

Individuals engaged in shift- or night-work show disturbed diurnal rhythms, out of phase with temporal signals associated to the light/dark (LD) cycle, resulting in internal desynchronization. The mechanisms underlying internal desynchrony have been mainly investigated in experimental animals with protocols that induce phase shifts of the LD cycle and thus modify the activity of the suprachiasmatic nucleus (SCN). In this study we developed an animal model of night-work in which the light-day cycle remained stable and rats were required to be active in a rotating wheel for 8 h daily during their sleeping phase (W-SP). This group was compared with rats that were working in the wheel during their activity phase (W-AP) and with undisturbed rats (C). We provide evidence that forced activity during the sleeping phase (W-SP group) alters not only activity, but also the temporal pattern of food intake. In consequence W-SP rats showed a loss of glucose rhythmicity and a reversed rhythm of triacylglycerols. In contrast W-AP rats did not show such changes and exhibited metabolic rhythms similar to those of the controls. The three groups exhibited the nocturnal corticosterone increase, in addition the W-SP and W-AP groups showed increase of plasma corticosterone associated with the start of the working session. Forced activity during the sleep phase did not modify SCN activity characterized by the temporal patterns of PER1 and PER2 proteins, which remained in phase with the LD cycle. These observations indicate that a working regimen during the sleeping period elicits internal desynchronization in which activity combined with feeding uncouples metabolic functions from the biological clock which remains fixed to the LD cycle. The present data suggest that in the night worker the combination of work and eating during working hours may be the cause of internal desynchronization.

Interaction between hypothalamic dorsomedial nucleus and the suprachiasmatic nucleus determines intensity of food anticipatory behavior

Food anticipatory behavior (FAA) is induced by limiting access to food for a few hours daily. Animals anticipate this scheduled meal event even without the suprachiasmatic nucleus (SCN), the biological clock. Consequently, a food-entrained oscillator has been proposed to be responsible for meal time estimation. Recent studies suggested the dorsomedial hypothalamus (DMH) as the site for this food-entrained oscillator, which has led to considerable controversy in the literature. Herein we demonstrate by means of c-Fos immunohistochemistry that the neuronal activity of the suprachiasmatic nucleus (SCN), which signals the rest phase in nocturnal animals, is reduced when animals anticipate the scheduled food and, simultaneously, neuronal activity within the DMH increases. Using retrograde tracing and confocal analysis, we show that inhibition of SCN neuronal activity is the consequence of activation of GABA-containing neurons in the DMH that project to the SCN. Next, we show that DMH lesions result in a loss or diminution of FAA, simultaneous with increased activity in the SCN. A subsequent lesion of the SCN restored FAA. We conclude that in intact animals, FAA may only occur when the DMH inhibits the activity of the SCN, thus permitting locomotor activity. As a result, FAA originates from a neuronal network comprising an interaction between the DMH and SCN. Moreover, this study shows that the DMH–SCN interaction may serve as an intrahypothalamic system to gate activity instead of rest overriding circadian predetermined temporal patterns.

Entrainment by a palatable meal induces food-anticipatory activity and c-Fos expression in reward-related areas of the brain.

Rats maintained under restricted feeding schedules (RFS) develop food-anticipatory activity and entrainment of physiological parameters. Food entrainment is independent of the suprachiasmatic nucleus and depends on food-entrainable oscillators (FEO). Restricted feeding schedules lead animals toward a catabolic state and to increase their food driven motivation, suggesting that in this process metabolic- and reward-related mechanisms are implicated. This study explored if motivation driven by a palatable meal is sufficient to produce food-entrainment. To address this question, we evaluated whether daily fixed access to a highly palatable meal entrained (PME) locomotor activity, serum glucose and free fatty acids concentrations in rats maintained without food deprivation. The entrained response of PME rats was compared with rats entrained to RFS. In a second experiment, we used c-Fos-IR to identify structures in the central nervous system involved with PME. Rats showed anticipatory activity to a daily palatable meal, with a lower intensity than rats entrained to RFS. Anticipatory activity persisted at least for four cycles after interrupting palatable meal, suggesting that this persistence depends on an endogenous oscillator. Glucose and free fatty acids were not entrained in PME rats. c-Fos expression in limbic system nuclei was in phase with PME time, but not in the hypothalamus. Results suggest 1) that food deprivation, i.e. a catabolic state is not necessary for the expression of anticipatory activity; 2) that an increase in the motivational state due to taste and/or nutritional contents of palatable meal is sufficient to entrain behavior; and 3) that structures in the limbic system are involved in this entrainment process. The present study indicates that metabolic and motivational mechanisms are involved in food entrainment, and suggests that the FEO may be a multi-oscillatory system distributed over different regulatory systems in the brain.

Peripheral oscillators: the driving force for food‐anticipatory activity

Food‐anticipatory activity (FAA) and especially the food‐entrained oscillator (FEO) have driven many scientists to seek their mechanisms and locations. Starting our research on FAA we, possibly like many other scientists, were convinced that clock genes held the key to the location and the underlying mechanisms for FAA. In this review, which is aimed especially at discussing the contribution of the peripheral oscillators, we have put together the accumulating evidence that the clock gene machinery as we know it today is not sufficient to explain food entrainment. We discuss the contribution of three types of oscillating processes: (i) within the suprachiasmatic nucleus (SCN), neurons capable of maintaining a 24‐h oscillation in electrical activity driven by a set of clock genes; (ii) oscillations in metabolic genes and clock genes in other parts of the brain and in peripheral organs driven by the SCN or by food, which damp out after a few cycles; (iii) an FEO which, we propose, is a system built up of different oscillatory processes and consisting of an as‐yet‐unidentified network of central and peripheral structures. In view of the evidence that clock genes and metabolic oscillations are not essential for the persistence of FAA we propose that food entrainment is initiated by a repeated metabolic state of scarcity that drives an oscillating network of brain nuclei in interaction with peripheral oscillators. This complex may constitute the proposed FEO and is distributed in our peripheral organs as well as within the central nervous system.

A daily palatable meal without food deprivation entrains the suprachiasmatic nucleus of rats.

Food is considered a potent Zeitgeber for peripheral oscillators but not for the suprachiasmatic nucleus (SCN), which is entrained principally by the light–dark cycle. However, when food attains relevant properties in quantity and quality, it can be a potent Zeitgeber even for the SCN. Here we evaluated the entrainment influence of a daily palatable meal, without regular food deprivation, on the circadian rhythm of locomotor activity and the c‐Fos and PER‐1 protein expression in the SCN. Rats fed ad libitum, in constant darkness, received a palatable meal for 6 weeks starting in the middle of the subjective day. Locomotor activity showed entrainment when the offset of activity coincided with the palatable meal‐time. In the SCN, the peak expression of c‐Fos was observed at palatable meal‐time and PER‐1 showed a peak during the onset of subjective night, as predicted according to the behavioural entrained pattern. In addition, c‐Fos and PER‐1 expression in the paraventricular thalamic nucleus (PVT) showed increased expression at palatable meal‐time, while the intergeniculate leaflet did not, suggesting that the PVT may be involved as an input pathway of palatable food‐entrainment to the SCN. These results demonstrate that daily access to a palatable meal can entrain the SCN; several stimuli can be implicated in this process, including motivation and arousal.

Differential role of the accumbens Shell and Core subterritories in food-entrained rhythms of rats

Restricted feeding schedules (RFS) entrain behavioral and physiological rhythms even in suprachiasmatic nucleus ablated animals, suggesting the existence of a food-entrained oscillator. The nucleus accumbens is an important structure for the expression of motivational behaviors and because its anatomical subterritories, Shell (Acc-Sh) and Core (Acc-Co) establish connections with different functional systems, they may participate in a differential way in food-entrainment. A first experiment, explored the role of Acc-Sh and Acc-Co in food-entrainment using the immunohistochemical detection of the protein c-Fos as a transcriptional activation marker. Experiment 2 tested the differential effect of Acc-Sh and Acc-Co, NMDA excitotoxic lesions. Lesioned rats were entrained to RFS and locomotor activity and free fatty acids (FFA) concentrations were evaluated. Results data show that in the Acc-Sh there is an increase of c-Fos immunoreactivity in food-entrained rats principally during feeding, whereas c-Fos expression in the Acc-Co region was increased during feeding and also anticipating mealtime. FFA were entrained in both lesioned groups, but the basal level was lower in Core-lesion rats. All rats exhibited food anticipatory activity (FAA). However, FAA was increased in Shell-lesioned animals and was almost abolished in the Core-lesion rats. These data indicate that the accumbens nucleus is involved with behavioral and metabolic food-entrainment, and that there is a differential role between both subregions.

Expectancy for food or expectancy for chocolate reveals timing systems for metabolism and reward

The clock gene protein Per 1 (PER1) is expressed in several brain structures and oscillates associated with the suprachiasmatic nucleus (SCN). Restricted feeding schedules (RFS) induce anticipatory activity and impose daily oscillations of c-Fos and clock proteins in brain structures. Daily access to a palatable treat (chocolate) also elicits anticipatory activity and induces c-Fos expression mainly in corticolimbic structures. Here the influence of daily access to food or chocolate was explored by the analysis of the oscillatory patterns of PER1 in hypothalamic and corticolimbic structures. Wistar rats were exposed to RFS or to daily access to chocolate for 3 weeks. Persistence of food or chocolate entrained rhythms was determined 8 days after cessation of the feeding protocols. RFS and chocolate induced a phase shift in PER1 rhythmicity in corticolimbic structures with peak values at zeitgeber time 12 and a higher amplitude in the chocolate group. Both RFS and chocolate groups showed an upregulation of PER1 in the SCN. Food and chocolate entrained rhythms persisted for 8 days in behavior and in PER1 expression in the dorsomedial hypothalamic nucleus, accumbens, prefrontal cortex and central amygdala. The present data demonstrate the existence of different oscillatory systems in the brain that can be activated by entrainment to metabolic stimuli or to reward and suggest the participation of PER1 in both entraining pathways. Persistence and amplification of PER1 oscillations in structures associated with reward suggest that this oscillatory process is fundamental to food addictive behavior.

Disruption of circadian rhythms due to chronic constant light leads to depressive and anxiety-like behaviors in the rat.

Depression is strongly associated with the circadian system, disruption of the circadian system leads to increased propensity to disease and to mood disorders including depression. The present study explored in rats the effects of circadian disruption by constant light on behavioral and hormonal indicators of a depressive-like condition and on the biological clock, the suprachiasmatic nucleus (SCN). Exposure to constant light for 8 weeks resulted in loss of circadian patterns of spontaneous general activity, melatonin and corticosterone. Moreover these rats exhibited anhedonia in a sucrose consumption test, and increased grooming in the open-field test, which reflects an anxiety-like condition. In the SCN decreased cellular activation was observed by c-Fos immunohistochemistry. In rats exposed to constant darkness, circadian behavioral and hormonal patterns remained conserved, however mild depressive-like indicators were observed in the anhedonia test and mild anxiety-like behaviors were observed in the open field test. Data indicate that chronic conditions of LL or DD are both disruptive for the activity of the SCN leading to depression- and anxiety-like behavior. Present results point out the main role played by the biological clock and the risk of altered photoperiods on affective behavior.