Michael Verwey

Endogenous Dopamine Regulates the Rhythm of Expression of the Clock Protein PER2 in the Rat Dorsal Striatum via Daily Activation of D2 Dopamine Receptors

A role for dopamine (DA) in the regulation of clock genes in the mammalian brain is suggested by evidence that manipulations of DA receptors can alter the expression of some clock genes outside the suprachiasmatic nucleus (SCN), the master circadian clock. The role of endogenous DA in the regulation of clock gene expression is unknown. Here, we demonstrate a direct relationship between extracellular DA levels and the rhythm of expression of the clock protein PERIOD2 (PER2) in the dorsal striatum of the male Wistar rat. Specifically, we show that the peak of the daily rhythm of extracellular DA in the dorsal striatum precedes the peak of PER2 by ∼6 h and that depletion of striatal DA by 6-hydroxydopamine or α-methyl-para-tyrosine or blockade of D2 DA receptors by raclopride blunts the rhythm of striatal PER2. Furthermore, timed daily activation of D2 DA receptors, but not D1 DA receptors, restores and entrains the PER2 rhythm in the DA-depleted striatum. None of these manipulations had any effect on the PER2 rhythm in the SCN. Our findings are consistent with the idea that the rhythm of expression of PER2 in the dorsal striatum depends on daily dopaminergic activation of D2 DA receptors. These observations may have implications for circadian abnormalities seen in Parkinsons disease.

Differential regulation of the expression of period2 protein in the limbic forebrain and dorsomedial hypothalamus by daily limited access to highly palatable food in food-deprived and free-fed rats

Circadian clock genes are rhythmically expressed in many areas of the brain and body and are thought to underlie most endogenous circadian behaviors and physiological processes. Daily rhythms of clock gene expression throughout the brain and body are normally coordinated by the suprachiasmatic nucleus (SCN), but they are also strongly influenced by daily temporal restrictions of food availability. Here, we studied the effects of a daily restricted presentation of highly palatable complete meal replacement, chocolate Ensure Plus (Ensure) in food-deprived (restricted feeding, RF) and free-fed (restricted treat, RT) rats, on the expression of the clock protein, Period2 (PER2) in regions of the brain involved in motivational and emotional regulation; these include the oval nucleus of the bed nucleus of the stria terminalis (BNSTov), the central nucleus of the amygdala (CEA), the basolateral amygdala (BLA), the dentate gyrus (DG) and the dorsomedial hypothalamus (DMH). RF and RT rats consumed similar amounts of Ensure, but changes in the pattern of PER2 expression were seen only in the RF condition, suggesting that changes in PER2 expression in these regions are triggered by the daily alleviation of a negative metabolic state associated with RF and are independent of the positive incentive properties of the consumed substance, per se. In contrast, the expression of the immediate early gene, Fos, was increased in these regions by both RF and RT schedules, showing that signals concerning the incentive value of the consumed food reach these regions. No changes in either PER2 or Fos expression were observed in the SCN of RF or RT rats. These findings demonstrate that mechanisms leading to changes in the expression of PER2 and those affecting the induction of Fos under RF and RT are, at least in part, dissociable.

Food‐entrainable circadian oscillators in the brain

Circadian rhythms in mammalian behaviour and physiology rely on daily oscillations in the expression of canonical clock genes. Circadian rhythms in clock gene expression are observed in the master circadian clock, the suprachiasmatic nucleus but are also observed in many other brain regions that have diverse roles, including influences on motivational and emotional state, learning, hormone release and feeding. Increasingly, important links between circadian rhythms and metabolism are being uncovered. In particular, restricted feeding (RF) schedules which limit food availability to a single meal each day lead to the induction and entrainment of circadian rhythms in food‐anticipatory activities in rodents. Food‐anticipatory activities include increases in core body temperature, activity and hormone release in the hours leading up to the predictable mealtime. Crucially, RF schedules and the accompanying food‐anticipatory activities are also associated with shifts in the daily oscillation of clock gene expression in diverse brain areas involved in feeding, energy balance, learning and memory, and motivation. Moreover, lesions of specific brain nuclei can affect the way rats will respond to RF, but have generally failed to eliminate all food‐anticipatory activities. As a consequence, it is likely that a distributed neural system underlies the generation and regulation of food‐anticipatory activities under RF. Thus, in the future, we would suggest that a more comprehensive approach should be taken, one that investigates the interactions between multiple circadian oscillators in the brain and body, and starts to report on potential neural systems rather than individual and discrete brain areas.

Region-specific modulation of PER2 expression in the limbic forebrain and hypothalamus by nighttime restricted feeding in rats

Feeding schedules that restrict food access to a predictable daytime meal induce in rodents food-anticipatory behaviors, changes in physiological rhythms and shifts in the rhythm of clock gene expression in the brain and periphery. However, little is known about the effects of nighttime restricted feeding. Previously, we showed that daytime restricted access to a highly palatable complete meal replacement, Ensure Plus (Ensure), shifts the rhythm of expression of the clock protein PER2 in limbic forebrain areas including the oval nucleus of the bed nucleus of the stria terminalis (BNSTov), central nucleus of the amygdala (CEA), basolateral amygdala (BLA) and dentate gyrus (DG), and induces a rhythm in the dorsomedial hypothalamic nucleus (DMH) in food deprived (restricted feeding), but not free-fed rats (restricted treat). In the present study we investigated the effects of nighttime restricted feeding (Ensure only, 2 h/night) and nighttime restricted treats (Ensure 2 h/night+free access to chow) in order to determine whether these effects were dependent on the time of day the meal was provided. We found that nighttime restricted feeding, like daytime restricted feeding, shifted the rhythm of PER2 expression in the BNSTov and CEA and peak expression was observed approximately 12 h after the mealtime. Also consistent with previous work, nighttime restricted feeding induced a rhythm of PER2 expression in the DMH and these effects occurred without affecting the rhythm in the suprachiasmatic nucleus (SCN). In contrast to previous work with daytime restricted feeding, nighttime restricted feeding had no effect on PER2 rhythms in the BLA and DG. Finally, nighttime restricted treats, as was the case for daytime restricted treats, had no effect on PER2 expression in any of the brain areas studied. The present results together with our previous findings show that the effect of restricted feeding on PER2 rhythms in the limbic forebrain and hypothalamus depend on a negative energy balance and vary as a function of time of day in a brain region-specific manner.

Circadian rhythms of PERIOD1 expression in the dorsomedial hypothalamic nucleus in the absence of entrained food-anticipatory activity rhythms in rats

When food availability is restricted to a single time of day, circadian rhythms of behavior and physiology in rodents shift to anticipate the predictable time of food arrival. It has been hypothesized that certain food‐anticipatory rhythms are linked to the induction and entrainment of rhythms in clock gene expression in the dorsomedial hypothalamic nucleus (DMH), a putative food‐entrained circadian oscillator. To study this concept further, we made food availability unpredictable by presenting the meal at a random time each day (variable restricted feeding, VRF), either during the day, night or throughout the 24‐h cycle. Wheel running activity and the expression of the clock protein, Period1 (PER1), in the DMH and the suprachiasmatic nucleus (SCN) were assessed. Rats exhibited increased levels of activity during the portion of the day when food was randomly presented but, as expected, failed to entrain anticipatory wheel running activity to a single time of day. PER1 expression in the SCN was unchanged by VRF schedules. In the DMH, PER1 expression became rhythmic, peaking at opposite times of day in rats fed only during the day or during the night. In rats fed randomly throughout the entire 24‐h cycle, PER1 expression in the DMH remained arrhythmic, but was elevated. These results demonstrate that VRF schedules confined to the day or night can induce circadian rhythms of clock gene expression in the DMH. Such feeding schedules cannot entrain behavioral rhythms, thereby showing that food‐entrainment of behavior and circadian rhythms of clock gene expression in the DMH are dissociable.

Reward and aversive stimuli produce similar nonphotic phase shifts.

Circadian rhythms in rodents respond to arousing, nonphotic stimuli that contribute to daily patterns of entrainment. To examine whether the motivational significance of a stimulus is important for eliciting nonphotic circadian phase shirts in Syrian hamsters (Mesocricetus auratus), the authors compared responses to a highly rewarding stimulus (lateral hypothalamic brain stimulation reward [BSR]) and a highly aversive stimulus (footshock). Animals were housed on a 14:10-hr light-dark cycle until test day, when they were given a 1-hr BSR session (trained animals) or a 1-mA electric footshock at 1 of 8 circadian times, and were maintained in constant dark thereafter. Both BSR pulses and footshock produced nonphotic phase response curves. These results support the hypothesis that arousal resulting from the motivational significance of a stimulus is a major factor in nonphotic phase shifts.

Nucleus-specific effects of meal duration on daily profiles of Period1 and Period2 protein expression in rats housed under restricted feeding

Restricted feeding (RF) schedules provide a cycle of fasting and feeding each day and induce circadian rhythms in food-anticipatory activity. In addition, daily rhythms in the expression of circadian clock genes, such as rhythms in Period1 (PER1) or Period2 (PER2), are also shifted in many brain areas that are important for the regulation of motivation and emotion. In order to differentiate brain areas that respond to the time of food presentation from areas that are sensitive to the degree of restriction, the present study compared RF schedules that provided rats with either a 2 h-meal (2hRF) or a 6 h-meal (6hRF) each day. As expected, 2hRF was associated with less food-consumption, more weight-loss, and more food-anticipatory running-wheel activity than 6hRF. In association with these metabolic and behavioral differences, the daily pattern of PER1 and PER2 expression in the dorsomedial hypothalamic nucleus (DMH), which has been proposed to be integral to the generation and/or maintenance of food-anticipatory activities, peaked earlier in the 2hRF group and later in the 6hRF group. Because both RF groups exhibited approximately synchronous food-anticipatory activity, but phase shifted rhythms of PER1 and PER2 expression in the DMH, it suggests that the phase of food-anticipatory activity is not directly regulated by this brain area. Next, daily rhythms of PER2 expression in the limbic forebrain responded to each RF schedule in a nucleus-specific manner. In some brain areas, the amplitude of the PER2 rhythm was differentially adjusted in response to 2hRF and 6hRF, while other areas, responded similarly to both RF schedules. These findings demonstrate that daily rhythms of clock gene expression can be modulated by the motivational state of the animal, as influenced by meal duration, weight loss and food-consumption.

Timed restricted feeding restores the rhythms of expression of the clock protein, Period2, in the oval nucleus of the bed nucleus of the stria terminalis and central nucleus of the amygdala in adrenalectomized rats

Feeding schedules that limit food availability to a set time of day are powerful synchronizers of the rhythms of expression of the circadian clock protein Period 2 (PER2) in the limbic forebrain in rats. Little is known, however, about the mechanisms that mediate the effect of such timed restricted feeding (TRF) schedules on the expression of PER2. Adrenal glucocorticoids have been implicated in the circadian regulation of clock genes expression in peripheral tissues as well as in the control of the rhythms of expression of PER2 in certain limbic forebrain regions, such as the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) and central nucleus of the amygdala (CEA) in rats. To study the possible involvement of glucocorticoids in the regulation of PER2 expression by TRF, we assessed the effect of adrenalectomy on TRF-entrained PER2 rhythms in the limbic forebrain in rats. Adrenalectomy selectively abolished the rhythms of PER2 in the BNSTov and CEA in normally fed rats, as previously shown, but had no effect on TRF-entrained PER2 rhythms in the same structures. These findings show that the effect of TRF on PER2 rhythms in the limbic forebrain is independent of adrenal glucocorticoids and demonstrate that the involvement of glucocorticoids in the regulation PER2 rhythms in the limbic forebrain is not only region specific, as previously shown, but also state dependent.

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT). Although circadian rhythms in running-wheel activity are typically linked to the SCN clock, circadian oscillators in many other regions of the brain and body could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN and instead, are correlated with changes in the activity of extra-SCN oscillators. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian influences on dopamine circuits of the brain: regulation of striatal rhythms of clock gene expression and implications for psychopathology and disease

Circadian clock proteins form an autoregulatory feedback loop that is central to the endogenous generation and transmission of daily rhythms in behavior and physiology. Increasingly, circadian rhythms in clock gene expression are being reported in diverse tissues and brain regions that lie outside of the suprachiasmatic nucleus (SCN), the master circadian clock in mammals. For many of these extra-SCN rhythms, however, the region-specific implications are still emerging. In order to gain important insights into the potential behavioral, physiological, and psychological relevance of these daily oscillations, researchers have begun to focus on describing the neurochemical, hormonal, metabolic, and epigenetic contributions to the regulation of these rhythms. This review will highlight important sites and sources of circadian control within dopaminergic and striatal circuitries of the brain and will discuss potential implications for psychopathology and disease . For example, rhythms in clock gene expression in the dorsal striatum are sensitive to changes in dopamine release, which has potential implications for Parkinson’s disease and drug addiction. Rhythms in the ventral striatum and limbic forebrain are sensitive to psychological and physical stressors, which may have implications for major depressive disorder. Collectively, a rich circadian tapestry has emerged that forces us to expand traditional views and to reconsider the psychopathological, behavioral, and physiological importance of these region-specific rhythms in brain areas that are not immediately linked with the regulation of circadian rhythms.