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 D(2) DA receptors by raclopride blunts the rhythm of striatal PER2. Furthermore, timed daily activation of D(2) DA receptors, but not D(1) 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 D(2) DA receptors. These observations may have implications for circadian abnormalities seen in Parkinson's disease.

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.

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.

Carbachol injections into the intergeniculate leaflet induce nonphotic phase shifts.

Nonphotic phase shifts of the circadian clock in mammals are mediated by the intergeniculate leaflet (IGL) of the thalamus via a geniculohypothalamic projection to the suprachiasmatic nucleus. These shifts can be induced by arousing stimuli, such as wheel running, brain stimulation reward and foot shock. Because mesopontine cholinergic neurons are also activated by arousing stimuli, we tested the hypothesis that cholinergic input to the IGL mediates nonphotic phase shifts. Carbachol injected into the IGL of hamsters in their subjective day (CT8) induced phase advances similar to shifts that are induced by arousal at the same circadian time. Control injections of saline at CT8 did not advance phase similarly. Carbachol injections outside the IGL produced smaller shifts. Pre-injections of the muscarinic antagonist, atropine, reduced carbachol-induced phase advances relative to saline pre-injections. The results indicate that muscarinic input to the IGL can induce nonphotic phase shifts.

Individual differences in circadian locomotor parameters correlate with anxiety- and depression-like behavior.

Disrupted circadian rhythms are a core feature of mood and anxiety disorders. Circadian rhythms are coordinated by a light-entrainable master clock located in the suprachiasmatic nucleus. Animal models of mood and anxiety disorders often exhibit blunted rhythms in locomotor activity and clock gene expression. Interestingly, the changes in circadian rhythms correlate with mood-related behaviours. Although animal models of depression and anxiety exhibit aberrant circadian rhythms in physiology and behavior, it is possible that the methodology being used to induce the behavioral phenotype (e.g., brain lesions, chronic stress, global gene deletion) affect behavior independently of circadian system. This study investigates the relationship between individual differences in circadian locomotor parameters and mood-related behaviors in healthy rats. The circadian phenotype of male Lewis rats was characterized by analyzing wheel running behavior under standard 12h:12h LD conditions, constant dark, constant light, and rate of re-entrainment to a phase advance. Rats were then tested on a battery of behavioral tests: activity box, restricted feeding, elevated plus maze, forced swim test, and fear conditioning. Under 12h:12h LD conditions, percent of daily activity in the light phase and variability in activity onset were associated with longer latency to immobility in the forced swim test. Variability in onset also correlated positively with anxiety-like behavior in the elevated plus maze. Rate of re-entrainment correlated positively with measures of anxiety in the activity box and elevated plus maze. Lastly, we found that free running period under constant dark was associated with anxiety-like behaviors in the activity box and elevated plus maze. Our results provide a previously uncharacterized relationship between circadian locomotor parameters and mood-related behaviors in healthy rats and provide a basis for future examination into circadian clock functioning and mood.

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.

Mesocortical Dopamine Phenotypes in Mice Lacking the Sonic Hedgehog Receptor Cdon.

Motivated behaviors and many psychopathologies typically involve changes in dopamine release from the projections of the ventral tegmental area (VTA) and/or the substantia nigra pars compacta (SNc). The morphogen Sonic Hedgehog (Shh) specifies fates of midbrain dopamine neurons, but VTA-specific effects of Shh signaling are also being uncovered. In this study, we assessed the role of the Shh receptor Cdon in the development of VTA and SNc dopamine neurons. We find that Cdon is expressed in the proliferating progenitor zone of the embryonic ventral midbrain and that the number of proliferating cells in this region is increased in mouse Cdon(-/-) embryos. Consistent with a role of Shh in the regulation of neuronal proliferation in this region, we find that the number of tyrosine hydroxylase (TH)-positive neurons is increased in the VTA of Cdon(-/-) mice at birth and that this effect endures into adulthood. In contrast, the number of TH-positive neurons in the SNc is not altered in Cdon(-/-) mice at either age. Moreover, adult Cdon(-/-) mice have a greater number of medial prefrontal cortex (mPFC) dopamine presynaptic sites, and increased baseline concentrations of dopamine and dopamine metabolites selectively in this region. Finally, consistent with increased dopamine function in the mPFC, we find that adult Cdon(-/-) mice fail to exhibit behavioral plasticity upon repeated amphetamine treatment. Based on these data, we suggest that Cdon plays an important role encoding the diversity of dopamine neurons in the midbrain, influencing both the development of the mesocortical dopamine pathway and behavioral outputs that involve this neural circuitry.

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.

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.

Variable restricted feeding disrupts the daily oscillations of Period2 expression in the limbic forebrain and dorsal striatum in rats.

Predictable restricted feeding schedules limit food availability to a single meal at the same time each day, lead to the induction and entrainment of circadian rhythms in food-anticipatory activity, and shift daily rhythms of clock gene expression in areas of the brain that are important in the regulation of motivational and emotional state. In contrast, when food is delivered under a variable restricted feeding (VRF) schedule, at a new and unpredictable mealtime each day, circadian rhythms in food-anticipatory activity fail to develop. Here, we study the effects of VRF on the daily rhythm of plasma corticosterone and of clock gene expression in the limbic forebrain and dorsal striatum, of rats provided a 2-h access to a complete meal replacement (Ensure Plus) at an unpredictable time each day. VRF schedules varied the mealtimes within the 12 h of light (daytime VRF), the 12 h of dark (nighttime VRF), or across the 24 h light-dark cycle (anytime VRF). Our results show that contrary to the synchronizing effects of predictable restricted feeding, VRF blunts the daily corticosterone rhythm and disrupts daily rhythms of PER2 expression in a region-specific and mealtime-dependent manner.