Phase Of Circadian Clock Research Articles

Metabotropic glutamate receptor modulation of glutamate responses in the suprachiasmatic nucleus.

Glutamate is the primary excitatory transmitter in the suprachiasmatic nucleus (SCN). Ionotropic glutamate receptors (iGluRs) mediate transduction of light information from the retina to the SCN, an important circadian clock phase shifting pathway. Metabotropic glutamate receptors (mGluRs) may play a significant modulatory role. mGluR modulation of SCN responses to glutamate was investigated with fura-2 calcium imaging in SCN explant cultures. SCN neurons showed reproducible calcium responses to glutamate, kainate, and N-methyl-D-aspartate (NMDA). Although the type I/II mGluR agonists L-CCG-I and t-ACPD did not evoke calcium responses, they did inhibit kainate- and NMDA-evoked calcium rises. This interaction was insensitive to pertussis toxin. Protein kinase A (PKA) activation by 8-bromo-cAMP significantly reduced iGluR inhibition by mGluR agonists. The inhibitory effect of mGluRs was enhanced by activating protein kinase C (PKC) and significantly reduced in the presence of the PKC inhibitor H7. Previous reports show that L-type calcium channels can be modulated by PKC and PKA. In SCN cells, about one-half of the calcium rise evoked by kainate or NMDA was blocked by the L-type calcium channel antagonist nimodipine. Calcium rises evoked by K+ were used to test whether mGluR inhibition of iGluR calcium rises involved calcium channel modulation. These calcium rises were primarily attributable to activation of voltage-activated calcium channels. PKC activation inhibited K+-evoked calcium rises, but PKC inhibition did not affect L-CCG-I inhibition of these rises. In contrast, 8Br-cAMP had no effect alone but blocked L-CCG-I inhibition. Taken together, these results suggest that activation of mGluRs, likely type II, modulates glutamate-evoked calcium responses in SCN neurons. mGluR inhibition of iGluR calcium rises can be differentially influenced by PKC or PKA activation. Regulation of glutamate-mediated calcium influx could occur at L-type calcium channels, K+ channels, or at GluRs. It is proposed that mGluRs may be important regulators of glutamate responsivity in the circadian system.

Conditioned stimulus control in the rat circadian system depends on clock resetting during conditioning.

The authors examined the ability of a conditioned stimulus (CS; mild air disturbance) previously paired with an entraining light pulse to reset the circadian pacemaker in rats. Rats were entrained to a single 30-min light stimulus delivered every 25 hr or 24 hr (T cycle). Each daily light presentation was paired with the CS. After at least 20 days of stable entrainment to each of the T cycles, the rats were allowed to free run and were then presented with the CS at circadian time 15. CS-induced phase shifts in wheel-running activity rhythms were taken as evidence for conditioning. For the most part, conditioning occurred after CS-light pairings on the 25-hr but not 24-hr T cycle. The results suggest that CS control of the circadian clock phase depends on the effect that the entraining light pulse has on the clock during conditioning.

Neuropeptide Y blocks serotonergic phase shifts of the suprachiasmatic circadian clock in vitro

The mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) can be reset in vitro by various neurochemical stimuli. This study investigated the phase-shifting properties of neuropeptide Y (NPY) and serotonin (5-HT) agonists when applied alone, as well as their combined effects on clock resetting. These neurotransmitters have both been shown to advance the SCN clock in vitro when applied during the daytime. By monitoring the SCN neuronal activity rhythm in vitro, I first confirm that the 5HT 1A/5HT 7 agonist (+)DPAT maximally advances the SCN clock when applied at zeitgeber time 6 (ZT6). Conversely, NPY only phase advances the neuronal activity rhythm when applied at ZT 10. This effect occurs through stimulation of Y 2 receptors. NPY, again acting through Y 2 receptors, blocks (+)DPAT-induced phase shifts at ZT 6, while neither (+)DPAT nor 5-HT affect NPY-induced phase shifts at ZT 10. NPY appears to block (+)DPAT-induced phase shifts by preventing increases in cyclic AMP. These data are the first to demonstrate in vitro interactions between daytime resetting stimuli in the rat, and provide critical insights into mechanisms controlling circadian clock phase.

A role for serotonin in the circadian system revealed by the distribution of serotonin transporter and light-induced Fos immunoreactivity in the suprachiasmatic nucleus and intergeniculate leaflet

Components of the circadian system, the suprachiasmatic nucleus and the intergeniculate leaflet receive serotonin input from the raphe nuclei. Manipulations of serotonin neurotransmission disrupt cellular, electrophysiological, and behavioural responses of the circadian system to light, suggesting that serotonin plays a modulatory role in photic regulation of circadian rhythms. To study the relation between serotonin afferents and light-activated cells in the suprachiasmatic nucleus and intergeniculate leaflet, we used immunostaining for the serotonin transporter and for the transcription factor, Fos. Serotonin transporter, a plasma membrane protein located on serotonin neurons, regulates the amount of serotonin available for neurotransmission by re-accumulating released serotonin into presynaptic neurons; expression of Fos in the suprachiasmatic nucleus identifies light-activated cells involved in photic resetting of circadian clock phase. In the suprachiasmatic nucleus, immunostaining for serotonin transporter revealed a dense plexus of fibres concentrated primarily in the ventrolateral region. In the intergeniculate leaflet, serotonin transporter immunostaining identified vertically-oriented columns of fibres. Serotonin transporter immunostaining was abolished by pretreatment with the serotonin neurotoxin, 5,7-dihydroxytryptamine. Exposure to light for 30 min during the dark phase of the light cycle induced Fos expression in the ventrolateral suprachiasmatic nucleus and intergeniculate leaflet regions. In both structures the Fos-expressing cells were encircled by serotonin transporter-immunoreactive fibres often in close apposition to these cells. These results support the idea that serotonin activity plays a modulatory role in processing of photic information within the circadian system.

Conditioning in the Orcadian System

Light is the dominant environmental cue for entrainment of circadian rhythms. In mammals, light entrains rhythms by resetting the phase of a circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Until recently, the mechanism responsible for pacemaker resetting by light was thought to be exclusively sensitive to photic cues. New experiments indicate, however, that this mechanism is more plastic than once thought; is amenable to conditioned stimulus control; and is capable of acquiring, through conditioning, new response capabilities. These experiments showed that, in rats, a neutral stimulus paired with light in Pavlovian conditioning trials is capable of eliciting cellular and behavioral effects characteristic of circadian clock phase resetting by light, expression of Fos protein in the ventrolateral region of the SCN, and phase shifts of free-running rhythms. These novel results open up a previously unappreciated perspective on photic phase resetting and entrainment of circadian rhythms. Specifically, they suggest that the process normally initiated by light to reset the clock can be modified by learning and events in the environment that reliably precede the onset of light can assume the resetting function of light.

In vitro reconstruction of the corticospinal projection in organotypic slice coculture

The suprachiasmatic nucleus (SCN) prominently expresses polysialic acid (PSA), a carbohydrate polymer that is attached to neural cell adhesion molecule (NCAM) and promotes changes in cell interactions. Previous studies have shown that expression of PSA is important for circadian rhythm stability under constant darkness, and for photic entrainment of the SCN circadian clock. In the present study, immunoblot analyses of the Syrian hamster SCN revealed marked diurnal fluctuations in PSA under a 24-h light/dark cycle. PSA levels were reduced by >90% during the mid-to-late dark phase, and were elevated to maximal daytime levels ∼1 h after lights-on. A similar pattern of PSA fluctuation persisted under constant darkness. Exposure of animals under a 24-h light/dark cycle to a 30-min light pulse during the late dark phase dramatically increased SCN contents of PSA within 60 min, and these values returned to basal levels 1–2 h later. There was no effect of light-on expression of PSA in the hippocampus. Parallel studies revealed changes in the NCAM-180 isoform that carries PSA in the brain, suggesting that regulation of PSA may include protein as well as carbohydrate-associated mechanisms. Immunohistological analysis revealed light-induced enhancement of PSA in the SCN subregion containing calbindin D28K cells. PSA staining was also closely associated with the majority of SCN cells expressing light-inducible Fos protein. This rhythmic, light-inducible expression of PSA within the SCN suggests that dynamic cell interactions are important for the photic regulation of circadian clock phase.

Circadian food-anticipatory activity: Formal models and physiological mechanisms

Rats and other species exhibit food-anticipatory activity (FAA) to daily mealtime under circadian (24 h) food access schedules. A critical review of several explanatory models indicates that hourglass clocks and associative learning processes are inadequate to explain many properties of FAA in intact and suprachiasmatic nuclei ablated rodents. A computational learning model, involving circadian clock consultation and phase memory, accounts for some but not all of these properties. An entrainment model, invoking separate, compound food- and light-entrainable oscillators, provides a more complete account of FAA. However, FAA may be simulated best by a model that combines oscillator entrainment with clock consulation and memory for circadian phase. Species as diverse as bees, birds, and mammals appear to share many features of FAA in common; differences may be explained in terms of oscillator organization and the ability to represent multiple circadian phases memorially. Physiological mechanisms of FAA are largely unknown; strategies for localization of entrainment pathways and oscillators, and a modest data base, are reviewed.

Serotonin and the mammalian circadian system: I. In vitro phase shifts by serotonergic agonists and antagonists.

The primary mammalian circadian clock, located in the suprachiasmatic nuclei (SCN), receives a major input from the raphe nuclei. The role of this input is largely unknown, and is the focus of this research. The SCN clock survives in vitro, where it produces a 24-hr rhythm in spontaneous neuronal activity that is sustained for at least three cycles. The sensitivity of the SCN clock to drugs can therefore be tested in vitro by determining whether various compounds alter the phase of this rhythm. We have previously shown that the nonspecific serotonin (5-HT) agonist quipazine resets the SCN clock in vitro, inducing phase advances in the daytime and phase delays at night. These results suggest that the 5-HT-ergic input from the raphe nuclei can modulate the phase of the SCN circadian clock. In this study we began by using autoradiography to determine that the SCN contain abundant 5-HT1A and 5-HT1B receptors, very few 5-HT1C and 5-HT2 receptors, and no 5-HT3 receptors. Next we investigated the ability of 5-HT-ergic agonists and antagonists to reset the clock in vitro, in order to determine what type or types of 5-HT receptor(s) are functionally linked to the SCN clock. We began by providing further evidence of 5-HT-ergic effects in the SCN. We found that 5-HT mimicked the effects of quipazine, whereas the nonspecific 5-HT antagonist metergoline blocked these effects, in both the day and night. Next we found that the 5-HT1A agonist 8-OH-DPAT, and to a lesser extent the 5-HT1A-1B agonist RU 24969, mimicked the effects of quipazine during the subjective daytime, whereas the 5-HT1A antagonist NAN-190 blocked quipazine's effects. None of the other specific agonists or antagonists we tried induced similar effects. This suggests that quipazine acts on 5-HT1A receptors in the daytime to advance the SCN clock. None of the specific agents we tried were able either to mimic or to block the actions of 5-HT or quipazine at circadian time 15. Thus, we were unable to determine the type of 5-HT receptor involved in nighttime phase delays by quipazine or 5-HT. However, since the dose-response curves for quipazine during the day and night are virtually identical, we hypothesize that the nighttime 5-HT receptor is a 5-HT1-like receptor.

Phase Determination of the Circadian Rhythm of Conidiation in Heterocaryons between two out-of-Phase Mycelia in Neurospora crassa

Neurospora grows vegetatively as a syncytium in which multiple nuclei exist within a connected cytoplasm. Because of the ability of separate and distinct mycelia to fuse, the possibility exists of generating heterocaryotic cultures in which the nuclei and cytoplasms of two different strains are comingled into the same syncytium. We have used such heterocaryons, in which the component parts differed with respect to their circadian clock phase, to examine whether or not clock-dominant phases exist in the circadian cycle. To this end, the phase subsequent to the formation of heterocaryons by pairs of mycelial discs that are initially at different circadian phases was examined in Neurospora crassa. The resulting phase was an average of the parent phases in many cases, but was sometimes observed to correspond more closely to just one of the original parental phases. In these cases, we did not observe any dominant phases in the circadian cycle; the phase of a particular parent disc was more dominant in the heterocaryon when the proportion of the nuclei from that parent was greater in the heterocaryon. In some instances, which occurred mostly when the difference in phase of the parental discs was large, the resultant phase could not be related in a simple way to the parental phases. An interpretation based on a limit cycle model of the circadian oscillation is possible.

Spatial Distribution of Circadian Clock Phase in Aging Cultures of Neurospora crassa

Neurospora crassa has been utilized extensively in the study of circadian clocks. Previously, the clock in this organism has been monitored by observing the morphological and biochemical changes occurring at the growing front of cultures grown on solid medium. A method has been developed for assaying the clock in regions of the culture behind the growing front, where no apparent morphological changes occur during the circadian cycle. Using this assay with Petri dish cultures that were 2 to 7 days old, the presence of a functional circadian clock not only at the growing front but in all other regions of the culture as well was demonstrated. Furthermore, the entire culture is not in the same phase, but shows a gradient of phases which is a function of the length of time the clock in a given part of the culture has been free-running. This gradient may be the result of a somewhat longer period of the oscillator behind the growing front compared to that at the growing front. The phase differences within a single culture of interconnected mycelium demonstrate the absence of total internal synchronization between adjacent regions of the hyphae under these conditions.