Effects of Histamine on Circadian Rhythms and Hibernation

Histamine appears to play a role in regulation of sleep and arousal as well as in synchronizing endogenous circadian rhythms with exogenous photic cues. Direct application of histamine to the suprachiasmatic nucleus (SCN), the site of the mammalian circadian pacemaker, phase shifts the circadian rhythm in neural activity. Intraventricular injections of histamine also phase shift circadian rhythms as do micro-injections directed towards the SCN. The magnitude and direction of the phase shifting effects of histamine depend on circadian phase in a manner similar to light. Depletion of brain histamine levels by inhibition of histamine synthesis reduces phase shifts to light. Histamine appears to influence phase shifts to light via a direct modulation of NMDA receptors in the SCN. Increased histamine levels and turnover observed in hibernating animals render it possible that histamine is a key regulator of hibernation. Thus histamine participates in an important link between sleep, circadian rhythms, and hibernation.     

Effects of Histamine on Circadian Rhythms and Hibernation

Histamine appears to play a role in regulation of sleep and arousal as well as in synchronizing endogenous circadian rhythms with exogenous photic cues. Direct application of histamine to the suprachiasmatic nucleus (SCN), the site of the mammalian circadian pacemaker, phase shifts the circadian rhythm in neural activity. Intraventricular injections of histamine also phase shift circadian rhythms as do micro-injections directed towards the SCN. The magnitude and direction of the phase shifting effects of histamine depend on circadian phase in a manner similar to light. Depletion of brain histamine levels by inhibition of histamine synthesis reduces phase shifts to light. Histamine appears to influence phase shifts to light via a direct modulation of NMDA receptors in the SCN. Increased histamine levels and turnover observed in hibernating animals render it possible that histamine is a key regulator of hibernation. Thus histamine participates in an important link between sleep, circadian rhythms, and hibernation.     

PPARalpha is a potential therapeutic target of drugs to treat circadian rhythm sleep disorders

Recent progress at the molecular level has revealed that nuclear receptors play an important role in the generation of mammalian circadian rhythms. To examine whether peroxisome proliferator-activated receptor alpha (PPARalpha) is involved in the regulation of circadian behavioral rhythms in mammals, we evaluated the locomotor activity of mice administered with the hypolipidemic PPARalpha ligand, bezafibrate. Circadian locomotor activity was phase-advanced about 3h in mice given bezafibrate under light-dark (LD) conditions. Transfer from LD to constant darkness did not change the onset of activity in these mice, suggesting that bezafibrate advanced the phase of the endogenous clock. Surprisingly, bezafibrate also advanced the phase in mice with lesions of the suprachiasmatic nucleus (SCN; the central clock in mammals). The circadian expression of clock genes such as period2, BMAL1, and Rev-erbalpha was also phase-advanced in various tissues (cortex, liver, and fat) without affecting the SCN. Bezafibrate also phase-advanced the activity phase that is delayed in model mice with delayed sleep phase syndrome (DSPS) due to a Clock gene mutation. Our results indicated that PPARalpha is involved in circadian clock control independently of the SCN and that PPARalpha could be a potent target of drugs to treat circadian rhythm sleep disorders including DSPS.     

Electrical and pharmacological properties of the suprachiasmatic nuclei

The suprachiasmatic nuclei (SCN) of the mammalian hypothalamus are in important circadian pacemaker. The electrical activity of these nuclei exhibits an intrinsic circadian rhythm. The rhythmicity of the SCN is also reflected in cyclic glucose consumption and serotonin metabolism. These rhythms are entrained to the light-dark cycle via the retinohypothalamic projection. This pathway, possibly together with a visual projection via the ventral lateral geniculate nuclei, innervates light-responsive SCN cells, which exhibit the functional properties of luminance detectors. The SCN contain various peptides, acetylcholine, and serotonin either intrinsically or in terminals of afferent projections. For acetylcholine it has been demonstrated that the SCN mediate the process of photic entrainment and light suppression of pineal synthetic activity. In the case of serotonin and vasopressin it seems certain that the SCN do not depend on their presence for generating circadian rhythms or for entrainment. Both substances may modulate the intrinsic pacemaker frequency through mechanisms that remain to be established.     

Regulation of Circadian and Acute Activity Levels by the Murine Suprachiasmatic Nuclei

The suprachiasmatic nuclei (SCN) coordinate the daily sleep-wake cycle by generating a circadian rhythm in electrical impulse frequency. While period and phase of the SCN rhythm have been considered as major output parameters, we propose that the waveform of the rhythm of the SCN also has significance. Using implanted micro-electrodes, we recorded SCN impulse frequency in freely moving mice and manipulated its circadian waveform by exposing mice to light-dark (LD) cycle durations ranging from 22 hours (LD 11∶11) to 26 hours (LD 13∶13). Adaptation to long T-cycles (>24 h) resulted in a trough in electrical activity at the beginning of the night while in short T-cycles (<24 h), SCN activity reached a trough at the end of night. In all T-cycle durations, the intensity of behavioral activity was maximal during the trough of SCN electrical activity and correlated negatively with increasing levels of SCN activity. Interestingly, small changes in T-cycle duration could induce large changes in waveform and in the time of trough (about 3.5 h), and accordingly in the timing of behavioral activity. At a smaller timescale (minutes to hours), we observed a negative correlation between SCN activity and behavioral activity, and acute silencing of SCN neurons by tetrodotoxin (TTX) during the inactive phase of the animal triggered behavioral activity. Thus, the SCN electrical activity levels appear crucially involved in determining the temporal profile of behavioral activity and controls behavior beyond the circadian time domain.     

Cocaine modulates pathways for photic and nonphotic entrainment of the mammalian SCN circadian clock

Cocaine abuse is highly disruptive to circadian physiological and behavioral rhythms. The present study was undertaken to determine whether such effects are manifest through actions on critical photic and nonphotic regulatory pathways in the master circadian clock of the mouse suprachiasmatic nucleus (SCN). Impairment of SCN photic signaling by systemic (intraperitoneal) cocaine injection was evidenced by strong (60%) attenuation of light-induced phase-delay shifts of circadian locomotor activity during the early night. A nonphotic action of cocaine was apparent from its induction of 1-h circadian phase-advance shifts at midday. The serotonin receptor antagonist, metergoline, blocked shifting by 80%, implicating a serotonergic mechanism. Reverse microdialysis perfusion of the SCN with cocaine at midday induced 3.7 h phase-advance shifts. Control perfusions with lidocaine and artificial cerebrospinal fluid had little shifting effect. In complementary in vitro experiments, photic-like phase-delay shifts of the SCN circadian neuronal activity rhythm induced by glutamate application to the SCN were completely blocked by cocaine. Cocaine treatment of SCN slices alone at subjective midday, but not the subjective night, induced 3-h phase-advance shifts. Lidocaine had no shifting effect. Cocaine-induced phase shifts were completely blocked by metergoline, but not by the dopamine receptor antagonist, fluphenazine. Finally, pretreatment of SCN slices for 2 h with a low concentration of serotonin agonist (to block subsequent serotonergic phase resetting) abolished cocaine-induced phase shifts at subjective midday. These results reveal multiple effects of cocaine on adult circadian clock regulation that are registered within the SCN and involve enhanced serotonergic transmission.     

Daily changes of neuropeptide Y-like immunoreactivity in the suprachiasmatic nucleus of the rat

Most of the biochemical, physiological and behavioural events in living organisms show diurnal fluctuations, normally synchronized with 24-h environmental rhythms, such as the light-dark cycle. The suprachiasmatic nucleus (SCN) of the hypothalamus is considered to be a pacemaker of the circadian rhythms in several mammals. The light-dark cycle is the primary synchronizing agent for many of the circadian rhythms which are regulated by the SCN. The photic information reaches the SCN also through a neuropeptide Y(NPY)-like immunoreactive pathway from the ventro-lateral geniculate nucleus. We found that in 12-h-dark and 12-h-light housed rats the NPY-like immunoreactive innervation of the ventro-lateral part of the SCN shows a 24 h rhythm with values rising gradually during the light phase and falling during the dark phase. Besides this rhythm, we found two peaks corresponding to the switching on and switching off of the light. The average level of NPY-like immunoreactivity, as assessed by means of semiquantitative immunocytochemistry and expressed in 'arbitrary units', is reduced in rats housed in total darkness for 2 weeks. These results confirm the physiological role of NPY in the timing of the circadian activity of the SCN.     

Serotonin agonist quipazine induces photic-like phase shifts of the circadian activity rhythm and c-Fos expression in the rat suprachiasmatic nucleus

Nonphotic stimuli can reset and entrain circadian activity rhythms in hamsters and mice, and serotonin is thought to be involved in the phase-resetting effects of these stimuli. In the present study, the authors examined the effect of the serotonin agonist quipazine on circadian activity rhythms in three inbred strains of rats (ACI, BH, and LEW). Furthermore, they investigated the effect of quipazine on the expression of c-Fos in the mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN). Quipazine reduced the amount of running wheel activity for 3 h after treatment, however, no long-term changes in tau and in the activity level were observed. More important, quipazine induced significant phase advances of the activity rhythm and c-Fos production in the SCN at the end of the subjective night (Circadian Time [CT] 22), whereas neither phase shifts nor c-Fos induction were observed during the subjective day. Quipazine injections also resulted in moderate phase delays at the beginning of the subjective night (CT 14). A similar phase-response characteristic typically can be observed for photic stimuli. By contrast, nonphotic stimuli normally produce phase advances during the subjective day. The present results suggest species differences between the hamster and the rat with respect to the serotonergic action on circadian timekeeping and indicate that serotonergic pathways play a role in the transmission of photic information to the SCN of rats.     

Circadian dynamics of cytosolic and nuclear Ca2+ in single suprachiasmatic nucleus neurons

Intracellular free Ca(2+) regulates diverse cellular processes, including membrane potential, neurotransmitter release, and gene expression. To examine the cellular mechanisms underlying the generation of circadian rhythms, nucleus-targeted and untargeted cDNAs encoding a Ca(2+)-sensitive fluorescent protein (cameleon) were transfected into organotypic cultures of mouse suprachiasmatic nucleus (SCN), the primary circadian pacemaker. Circadian rhythms in cytosolic but not nuclear Ca(2+) concentration were observed in SCN neurons. The cytosolic Ca(2+) rhythm period matched the circadian multiple-unit-activity (MUA)-rhythm period monitored using a multiple-electrode array, with a mean advance in phase of 4 hr. Tetrodotoxin blocked MUA, but not Ca(2+) rhythms, while ryanodine damped both Ca(2+) and MUA rhythms. These results demonstrate cytosolic Ca(2+) rhythms regulated by the release of Ca(2+) from ryanodine-sensitive stores in SCN neurons.     

Behavioral responses of Vipr2-/- mice to light

Vasoactive intestinal polypeptide and its receptor, VPAC2, play important roles in the functioning of the dominant circadian pacemaker, located in the hypothalamic suprachiasmatic nuclei (SCN). Mice lacking VPAC2 receptors (Vipr2-/-) show altered circadian rhythms and impaired synchronization to environmental lighting cues. However, light can increase phosphoprotein and immediate early gene expression in the Vipr2-/- SCN demonstrating that the circadian clock is readily responsive to light in these mice. It is not clear whether these neurochemical responses to light can be transduced to behavioral changes as seen in wild-type (WT) animals. In this study we investigated the diurnal and circadian wheel-running profile of WT (C57BL/6J) and Vipr2-/- mice under a 12-h light:12-h complete darkness (LD) lighting schedule and in constant darkness (DD) and used 1-h light pulses to shift the activity of mice in DD. Unlike WT mice, Vipr2-/- mice show grossly altered locomotor patterns making the analysis of behavioral responses to light problematic. However, analyses of both the onset and the offset of locomotor activity reveal that in a subset of these mice, light can reset the offset of behavioral rhythms during the subjective night. This suggests that the SCN clock of Vipr2-/- mice and the rhythms it generates are responsive to photic stimulation and that these responses can be integrated to whole animal behavioral changes.     

Damped oscillation of the lateral hypothalamic multineuronal activity synchronized to daily feeding schedules in rats with suprachiasmatic nucleus lesions.

In male Wistar rats with chronically implanted electrodes, multiple-unit activity (MUA) was recorded from the lateral hypothalamus (LH) and ventromedial hypothalamus (VMH). Blinded rats with bilateral suprachiasmatic nucleus (SCN) lesions showed no circadian rhythm in MUA or motor activity when food was available ad libitum. However, under a restricted-feeding schedule (food was available from 1400 to 1600 hr; water was always available) lasting for 10 days, a gradual increase of MUA of the LH developed, starting 3-4 hr prior to the feeding time. The elevated MUA lasted up to 6-7 hr after feeding and subsequently returned to the baseline level. This circadian rhythm of MUA of the LH persisted up to 4 days under total food deprivation, with quickly decreasing amplitude after termination of the schedule. MUA rhythm in VMH was less obvious than that in LH. Also, general motor activity showed a rhythm comparable to that of MUA, but it was less prominent. The elevated MUA in the LH prior to the feeding time may have been neural substrate of anticipatory activity appearing under the restricted-feeding schedule. These findings may suggest the existence of a quickly damping oscillator mechanism in the brain, presumably in the LH, which can be induced by daily feeding cues in the absence of the SCN.     

Circadian Locomotor Rhythms in Mice with Targeted Disruption of the Gene for the Carbon Monoxide Synthesizing Enzyme, Heme Oxygenase-2

Carbon monoxide (CO), generated in neurons by the enzyme heme oxygenase-2 (HO2), is postulated to be a gaseous signaling molecule in the mammalian brain. Because of the recent evidence suggesting an important role of another endogenously produced gas, nitric oxide (NO), in entrainment of circadian rhythms in mammals, we hypothesized that CO may also be involved in regulating these rhythms. Consistent with this idea, others have found a circadian rhythm of heme turnover and CO synthesis can be induced by bright light. Furthermore, HO2 is co-localized with guanylyl cyclase, the putative target of CO, throughout the brain, with high amounts of staining in the suprachiasmatic nucleus (SCN) of the hypothalamus. The goal of the present study was to evaluate the role of CO in photic entrainment in wild-type and HO2 deficient mice. HO2-/- mice did not display any abnormalities in circadian rhythmicity. Entrainment to a light-dark cycle, the ability to phase delay locomotor activity after a four hour phase shift in photoperiod, and the period of the free running rhythm (t) were similar between HO2-/- and wild-type mice. Taken together, these data suggest that CO does not play a major role in regulating circadian activity rhythms in mice.     

Circadian Locomotor Rhythms in Mice with Targeted Disruption of the Gene for the Carbon Monoxide Synthesizing Enzyme,Heme Oxygenase-2

Carbon monoxide (CO), generated in neurons by the enzyme heme oxygenase-2 (HO2), is postulated to be a gaseous signaling molecule in the mammalian brain. Because of the recent evidence suggesting an important role of another endogenously produced gas, nitric oxide (NO), in entrainment of circadian rhythms in mammals, we hypothesized that CO may also be involved in regulating these rhythms. Consistent with this idea, others have found a circadian rhythm of heme turnover and CO synthesis can be induced by bright light. Furthermore, HO2 is co-localized with guanylyl cyclase, the putative target of CO, throughout the brain, with high amounts of staining in the suprachiasmatic nucleus (SCN) of the hypothalamus. The goal of the present study was to evaluate the role of CO in photic entrainment in wild-type and HO2 deficient mice. HO2–/– mice did not display any abnormalities in circadian rhythmicity. Entrainment to a light–dark cycle, the ability to phase delay locomotor activity after a four hour phase shift in photoperiod, and the period of the free running rhythm (t) were similar between HO2–/– and wild-type mice. Taken together, these data suggest that CO does not play a major role in regulating circadian activity rhythms in mice.     

Aging does not compromise in vitro oscillation of the suprachiasmatic nuclei but makes it more vulnerable to constant light

Circadian regulation of behavior worsens with age, however, the mechanism behind this phenomenon is still poorly understood. Specifically, it is not clear to what extend the ability of the circadian clock in the suprachiasmatic nuclei (SCN) to generate the rhythm is affected by aging. This study aimed to ascertain the effect of aging on the functioning of the SCN of mPer2^Luciferase mice under unnatural lighting conditions, such as constant light (LL). Under LL, which worsened the age-induced effect on behavioral rhythms, a marginal age-dependent effect on in vitro rhythmicity in explants containing the middle, but not the rostral/caudal, regions of the SCN was apparent; the proportion of mice in which middle-region SCN explants were completely arrhythmic or had an extremely long period (>30 h) was 47% in aged mice and 27% in adults. The results suggest that in some of the aged animals, LL may weaken the coupling among oscillators in specific sub-regions of the SCN, leaving other sub-regions better synchronized. In the standard light/dark cycle and in constant darkness, the SCN ability to produce bioluminescence rhythms in vitro was not compromised in aged mice although aging significantly affected their SCN-driven locomotor activity rhythms. Therefore, our results demonstrate that although age worsened the SCN output rhythm, the SCN molecular core clock mechanism itself was relatively resilient to aging in these same animals. The results suggest the involvement of pathways downstream of the core clock mechanism which are responsible for this phenomenon.     

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^1-5. 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^6-8, circadian oscillators in many other regions of the brain and body^9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN^15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators^17-20. 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.     

Dissociation between circadian Per1 and neuronal and behavioral rhythms following a shifted environmental cycle

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus contains a major circadian pacemaker that imposes or entrains rhythmicity on other structures by generating a circadian pattern in electrical activity. The identification of "clock genes" within the SCN and the ability to dynamically measure their rhythmicity by using transgenic animals open up new opportunities to study the relationship between molecular rhythmicity and other well-documented rhythms within the SCN. We investigated SCN circadian rhythms in Per1-luc bioluminescence, electrical activity in vitro and in vivo, as well as the behavioral activity of rats exposed to a 6-hr advance in the light-dark cycle followed by constant darkness. The data indicate large and persisting phase advances in Per1-luc bioluminescence rhythmicity, transient phase advances in SCN electrical activity in vitro, and an absence of phase advances in SCN behavioral or electrical activity measured in vivo. Surprisingly, the in vitro phase-advanced electrical rhythm returns to the phase measured in vivo when the SCN remains in situ. Our study indicates that hierarchical levels of organization within the circadian timing system influence SCN output and suggests a strong and unforeseen role of extra-SCN areas in regulating pacemaker function.