Jorge E. Mendoza

Feeding Cues Alter Clock Gene Oscillations and Photic Responses in the Suprachiasmatic Nuclei of Mice Exposed to a Light/Dark Cycle

The suprachiasmatic nuclei (SCN) of the hypothalamus contain the master mammalian circadian clock, which is mainly reset by light. Temporal restricted feeding, a potent synchronizer of peripheral oscillators, has only weak influence on light-entrained rhythms via the SCN, unless restricted feeding is coupled with calorie restriction, thereby altering phase angle of photic synchronization. Effects of daytime restricted feeding were investigated on the mouse circadian system. Normocaloric feeding at midday led to a predominantly diurnal (60%) food intake and decreased blood glucose in the afternoon, but it did not affect the phase of locomotor activity rhythm or vasopressin expression in the SCN. In contrast, hypocaloric feeding at midday led to 2-4 h phase advances of three circadian outputs, locomotor activity rhythm, pineal melatonin, and vasopressin mRNA cycle in the SCN, and it decreased daily levels of blood glucose. Furthermore, Per1 and Cry2 oscillations in the SCN were phase advanced by 1 and 3 h, respectively, in hypocalorie- but not in normocalorie-fed mice. The phase of Per2 and Bmal1 expression remained unchanged regardless of feeding condition. Moreover, the shape of behavioral phase-response curve to light and light-induced expression of Per1 in the SCN were markedly modified in hypocalorie-fed mice compared with animals fed ad libitum. The present study shows that diurnal hypocaloric feeding affects not only the temporal organization of the SCN clockwork and circadian outputs in mice under light/dark cycle but also photic responses of the circadian system, thus indicating that energy metabolism modulates circadian rhythmicity and gating of photic inputs in mammals.

High‐fat feeding alters the clock synchronization to light

High‐fat feeding in rodents leads to metabolic abnormalities mimicking the human metabolic syndrome, including obesity and insulin resistance. These metabolic diseases are associated with altered temporal organization of many physiological functions. The master circadian clock located in the suprachiasmatic nuclei controls most physiological functions and metabolic processes. Furthermore, under certain conditions of feeding (hypocaloric diet), metabolic cues are capable of altering the suprachiasmatic clocks responses to light. To determine whether high‐fat feeding (hypercaloric diet) can also affect resetting properties of the suprachiasmatic clock, we investigated photic synchronization in mice fed a high‐fat or chow (low‐fat) diet for 3 months, using wheel‐running activity and body temperature rhythms as daily phase markers (i.e. suprachiasmatic clocks hands). Compared with the control diet, mice fed with the high‐fat diet exhibited increased body mass index, hyperleptinaemia, higher blood glucose, and increased insulinaemia. Concomitantly, high‐fat feeding led to impaired adjustment to local time by photic resetting. At the behavioural and physiological levels, these alterations include slower rate of re‐entrainment of behavioural and body temperature rhythms after ‘jet‐lag’ test (6 h advanced light–dark cycle) and reduced phase‐advancing responses to light. At a molecular level, light‐induced phase shifts have been correlated, within suprachiasmatic cells, with a high induction of c‐FOS, the protein product of immediate early gene c‐fos, and phosphorylation of the extracellular signal‐regulated kinases I/II (P‐ERK). In mice fed a high‐fat diet, photic induction of both c‐FOS and P‐ERK in the suprachiasmatic nuclei was markedly reduced. Taken together, the present data demonstrate that high‐fat feeding modifies circadian synchronization to light.

Forebrain oscillators ticking with different clock hands

Clock proteins like PER1 and PER2 are expressed in the brain, but little is known about their functionality outside the main suprachiasmatic clock. Here we show that PER1 and PER2 were neither uniformly present nor identically phased in forebrain structures of mice fed ad libitum. Altered expression of the clock gene Cry1 was observed in respective Per1 or Per2 mutants. In response to hypocaloric feeding, PERs timing was not markedly affected in few forebrain structures (hippocampus). In most other forebrain oscillators, including those expressing only PER1 (e.g., dorsomedial hypothalamus), PER2 (e.g., paraventricular hypothalamus) or both (e.g., paraventricular thalamus), PER1 was up-regulated and PER2 largely phase-advanced. Cry1 expression was selectively modified in the forebrain of Per mutants challenged with hypocaloric feeding. Our results suggest that there is not one single cerebral clock, but a system of multiple brain oscillators ticking with different clock hands and differentially sensitive to nutritional cues.

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.

The Cerebellum Harbors a Circadian Oscillator Involved in Food Anticipation

The cerebellum participates in motor coordination as well as in numerous cerebral processes, including temporal discrimination. Animals can predict daily timing of food availability, as manifested by food-anticipatory activity under restricted feeding. By studying ex vivo clock gene expression by in situ hybridization and recording in vitro Per1-luciferase bioluminescence, we report that the cerebellum contains a circadian oscillator sensitive to feeding cues (i.e., whose clock gene oscillations are shifted in response to restricted feeding). Food-anticipatory activity was markedly reduced in mice injected intracerebroventricularly with an immunotoxin that depletes Purkinje cells (i.e., OX7-saporin). Mice bearing the hotfoot mutation (i.e., Grid2ho/ho) have impaired cerebellar circuitry and mild ataxic phenotype. Grid2ho/ho mice fed ad libitum showed regular behavioral rhythms and day–night variations of clock gene expression in the hypothalamus and cerebellum. When challenged with restricted feeding, however, Grid2ho/ho mice did not show any food-anticipatory rhythms, nor timed feeding-induced changes in cerebellar clock gene expression. In hypothalamic arcuate and dorsomedial nuclei, however, shifts in Per1 expression in response to restricted feeding were similar in cerebellar mutant and wild-type mice. Furthermore, plasma corticosterone and metabolites before mealtime did not differ between cerebellar mutant and wild-type mice. Together, these data define a role for the cerebellum in the circadian timing network and indicate that the cerebellar oscillator is required for anticipation of mealtime.

Brain Clocks: From the Suprachiasmatic Nuclei to a Cerebral Network

Circadian timing affects almost all life’s processes. It not only dictates when we sleep, but also keeps every cell and tissue working under a tight temporal regimen. The daily variations of physiology and behavior are controlled by a highly complex system comprising of a master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus, extra-SCN cerebral clocks, and peripheral oscillators. Here are presented similarities and differences in the molecular mechanisms of the clock machinery between the primary SCN clock and extra-SCN brain clocks. Diversity of secondary clocks in the brain, their specific sensitivities to time-giving cues, as their differential coupling to the master SCN clock, may allow more plasticity in the ability of the circadian timing system to integrate a wide range of temporal information. Furthermore, it raises the possibility that pathophysiological alterations of internal timing that are deleterious for health may result from internal desynchronization within the network of cerebral clocks.

Serotonergic activation potentiates light resetting of the main circadian clock and alters clock gene expression in a diurnal rodent

The main circadian clock, localized in the suprachiasmatic nuclei (SCN) in mammals, can be synchronized by light and non-photic factors such as serotonergic cues. In nocturnal rodents, injections during the subjective day of the 5-HT1A/7 receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) or its positive enantiomer, induce behavioral phase-advances in correlation with decreased expression of two clock genes, Per1/2. In addition, 8-OH-DPAT and the selective serotonin reuptake inhibitor fluoxetine reduce light-induced phase-shifts during the subjective night. Beside the chronobiotic effects of serotonin, changes of serotonergic activity in humans have been involved in mood disorders, that are often associated with alterations in circadian rhythmicity. To get insights into the circadian role of serotonin in diurnal species, we investigated its modulation of the SCN in Arvicanthis ansorgei housed in constant darkness. In striking contrast to nocturnal rodents, daily serotonin content in Arvicanthis SCN peaked during daytime while the sensitivity window of its SCN to (+)8-OH-DPAT occurred essentially during the subjective night. Moreover, fluoxetine produced behavioral phase-advances at circadian time (CT) 0 and CT12. Expression of Per1/2, Rev-erbalpha/beta and Roralpha/beta in the SCN was not modified after fluoxetine or (+)8-OH-DPAT injection. Furthermore, both treatments enhanced light-induced phase-advances and delays. Light responses of Per1 and Rorbeta expression at CT0 and those of Per2 and Rev-erbalpha at CT12 were markedly altered by serotonergic activation. The present findings demonstrate that the serotonergic modulation of the SCN clock appears to differ between nocturnal species and the diurnal Arvicanthis. The potentiating effects of fluoxetine on light resetting in a diurnal rodent may be clinically relevant.

Neurogenetics of food anticipation

Circadian clocks enable the organisms to anticipate predictable cycling events in the environment. The mechanisms of the main circadian clock, localized in the suprachiasmatic nuclei of the hypothalamus, involve intracellular autoregulatory transcriptional loops of specific genes, called clock genes. In the suprachiasmatic clock, circadian oscillations of clock genes are primarily reset by light, thus allowing the organisms to be in phase with the light–dark cycle. Another circadian timing system is dedicated to preparing the organisms for the ongoing meal or food availability: the so‐called food‐entrainable system, characterized by food‐anticipatory processes depending on a circadian clock whose location in the brain is not yet identified with certainty. Here we review the current knowledge on food anticipation in mice lacking clock genes or feeding‐related genes. The food‐entrainable clockwork in the brain is currently thought to be made of transcriptional loops partly divergent from those described in the light‐entrainable suprachiasmatic nuclei. Possible confounding effects associated with behavioral screening of meal anticipation in mutant mice are also discussed.

An evolutionary-based decision support system for vehicle routing: The case of a public utility

Customer-related processes in a public utility, such as meter replacement programs, demand a large number of auditing visits to customer sites. The proposed decision support system (DSS) helps the operating manager to plan these visits by integrating commercial systems such as SAP/R3 and ArcGIS with a custom-made distance-constrained routing module. This module includes a modified Clarke and Wright savings heuristic and two memetic algorithms, along with two integer-programming clustering models whose function is to balance the workload. The system was tested on ten real-world distance-constrained vehicle routing instances ranging from 323 to 601 nodes.

Circadian and photic regulation of clock and clock-controlled proteins in the suprachiasmatic nuclei of calorie-restricted mice

In mammals, behavioural and physiological rhythms as well as clock gene expression in the central suprachiasmatic clock (SCN) are phase‐shifted by a timed calorie restriction (T‐CR; animals receiving at midday 66% of their daily food intake). The molecular mechanism of SCN depends on feedback loops involving clock genes and their protein products. To understand how T‐CR mediates its synchronizing effects, we examined the rhythmic expression of three clock proteins, PERIOD (PER) 1, 2 and CLOCK, and one clock‐controlled protein (i.e. vasopressin; AVP) in the SCN of mice either fed ad libitum (AL) or with T‐CR. Moreover, we evaluated expression of these proteins in the SCN of AL and T‐CR mice following a 1‐h light pulse. The results indicate that, while PER1 and AVP rhythms were phase‐advanced in T‐CR mice, the PER2 rhythm showed an increased amplitude. CLOCK was expressed constitutively in AL mice while in T‐CR it was significantly reduced, especially after feeding time. A light pulse produced a delayed increase in PER1 and a larger increase in PER2 expression in the SCN of T‐CR mice than in AL animals. In addition, light exposure triggered an increase in AVP‐ir cells in both AL and T‐CR mice, and also of CLOCK expression but in T‐CR mice only. The circadian changes in clock and clock‐controlled proteins and their acute responses to light in the SCN of T‐CR mice demonstrate that metabolic cues induced by a calorie restriction modulate the translational regulation of the SCN clock.