Photoendocrine Transduction in Cultured Chick Pineal Cells: IV. What Do Vitamin A Depletion and Retinaldehyde Addition Do to the Effects of Light on the Melatonin Rhythm?

Abstract: Light has at least two distinguishable effects on the circadian rhythm of melatonin output displayed by dispersed chick pineal cells in static culture: acute suppression of melatonin output and entrainment (phase shifts) of the underlying pacemaker. Previous results indicated that these two effects of light are mediated by different mechanistic pathways. The pathways for the acute and phase-shifting effects of light either branch from the same, single photopigment or differ from the outset, starting from separate photopigments. If a single rhodopsin-like photopigment mediates both effects of light, then vitamin A depletion and retinoid addition should affect both responses in parallel, although not proportionately. We therefore compared the effects of vitamin A depletion and retinoid addition on the acute and phase-shifting effects of light under several experimental conditions. When chick pineal cells were depleted of vitamin A, acute responses to light were markedly reduced. Addition of 11-cis-retinaldehyde specifically restored (and enhanced) the acute response. When allowed to free run in constant red light, depleted cells displayed a rhythm of melatonin output with the same period as that of control cells. In contrast to the acute effects, phase shifts in response to 2- or 4-h light pulses did not differ between depleted and control cells. Addition of retinaldehyde to depleted cells did not, by itself, reduce melatonin output or induce phase shifts. Retinaldehyde did increase the acute response to 4-h light pulses but not the ensuing phase shifts. Responses increased with duration of the light pulse: Both the acute effect and the phase shifts induced by 4-h light pulses were considerably larger than those induced by 2-h (or 1-h) light pulses. Addition of retinaldehyde to depleted cells increased the acute effect of 2-h (or 1-h) light pulses to at least that seen with 4-h light pulses but did not Increase the size of the ensuing phase shifts. These results strongly confirm previous dissociations of the mechanistic pathways mediating the acute and phase-shifting effects of light on chick pineal cells. They also support a role for rhodopsin-like photopigment in the acute, but not phase-shifting, response. They favor, but do not prove, the conclusion that separate photopigments mediate the acute and entraining effects of light.     

Twenty-four hour oscillation of cAMP in chick pineal cells: role of cAMP in the acute and circadian regulation of melatonin production

Chick pineal cells contain circadian oscillators that regulate a rhythm of melatonin biosynthesis. We explored the role of cAMP in regulating this melatonin rhythm. Chick pineal cells expressed a 24 hr oscillation of cAMP efflux with a waveform similar to that of melatonin. Elevation of cAMP in chick pineal cells stimulated melatonin. These results suggest that an oscillation of cAMP regulates the rhythm of melatonin. We investigated whether cAMP was a component of the circadian oscillator by determining the effects of 8-Br cAMP pulses on the phase of the circadian melatonin rhythm. Six hour pulses of 8-Br cAMP did not cause steady-state phase shifts of the rhythm. The acute regulation of melatonin by cAMP, the 24 hr oscillation of cAMP, and the inability of cAMP to phase-shift the melatonin rhythm strongly suggest that cAMP acts as an output signal of the circadian oscillator.     

Does the circadian pacemaker act through cyclic AMP to drive the melatonin rhythm in chick pineal cells?

Cyclic AMP is a key regulator of melatonin production in the chick pineal gland. Agents that raise cyclic AMP levels (such as forskolin), or cyclic AMP analogues (such as 8-bromocyclic AMP), increase melatonin synthesis and release, whereas agents that lower cyclic AMP levels (including light) decrease melatonin synthesis and release. A circadian oscillator in these cells also raises and lowers melatonin output. We have been investigating the relationships between cyclic AMP and the circadian pacemaker in the regulation of melatonin production. In the chick pineal (unlike certain neuronal systems), the weight of the evidence indicates that cyclic AMP is not on an entrainment pathway to the circadian pacemaker. Instead, cyclic AMP appears to act downstream from the pacemaker. The pacemaker might itself act directly through cyclic AMP, regulating melatonin content by raising and lowering cyclic AMP levels. If this were the case, and if the effects of cyclic AMP levels on melatonin output are saturable (as they must be), then, in the face of such saturating levels of cyclic AMP, the pacemaker should no longer raise or lower melatonin output. To test this prediction, maximally effective concentrations of forskolin and 8-bromocyclic AMP were determined. Both agents markedly increased melatonin output. After 36 hr, cells were refractory to additional stimulation of melatonin output by addition of both agents together, or by higher concentrations of forskolin (although cyclic AMP levels could still be raised further). Nonetheless, the circadian pacemaker continued to raise and lower melatonin output: The rhythm persisted in the face of saturating levels of cyclic AMP. It is therefore suggested that the circadian pacemaker in chick pineal cells acts with, not through, cyclic AMP to regulate melatonin synthesis. Cyclic AMP and the pacemaker act synergistically to regulate serotonin N-acetyltransferase activity and the melatonin rhythm, with cyclic AMP mediating acute effects and amplitude regulation.     

A role for cyclic AMP in entrainment of the circadian oscillator in Xenopus retinal photoreceptors by dopamine but not by light

The circadian oscillator in Xenopus retinal photoreceptor layers can be reset in similar ways by light and agonists of D2-like dopamine receptors. Treatments that increase cyclic AMP levels act on this oscillator in an opposite fashion, mimicking darkness in the induction of phase shifts. Light and dopamine have each been reported to inhibit adenylate cyclase in photoreceptors. Together, these data suggest that the transduction pathways for entrainment by dopamine and/or light include suppression of cyclic AMP or a cyclic AMP-sensitive step. In these studies, we examined this hypothesis by measuring the effects of treatment with a cyclic AMP analogue on the phase shifts induced in photoreceptor melatonin rhythms by light or a D2 receptor agonist (quinpirole). When photoreceptor layers were treated simultaneously with 8-(4-chlorophenylthio)cyclic AMP (8-CPT-cAMP) and quinpirole at any of three different phases of the circadian cycle, the resulting phase shifts of the melatonin rhythm were always the same as those caused by 8-CPT-cAMP alone. This indicates that there is a cyclic AMP-sensitive step in the dopamine entrainment pathway. In contrast, light pulses did reset the oscillator in the presence of elevated cyclic AMP. This suggests a separate cyclic AMP-insensitive transduction pathway for entrainment by light. Quinpirole reduced basal levels of cyclic AMP in photoreceptors, but light did not. These data suggest that cyclic AMP plays a role in the entrainment pathway activated by dopamine but not in the entrainment pathway activated by light.     

Exogenous melatonin reduces the resynchronization time after phase shifts of a nonphotic zeitgeber in the house sparrow (Passer domesticus)

Continuous melatonin administration via silastic implants accelerates the resynchronization of the circadian locomotor activity rhythm in house sparrows (Passer domesticus) after exposure to phase shifts of a weak light-dark cycle. Constant melatonin might induce this effect either by increasing the sensitivity of the visual system to a light zeitgeber or by reducing the degree of self-sustainment of the circadian pacemaker. To distinguish between these two possible mechanisms, two groups of house sparrows, one carrying melatonin implants and the other empty implants, were kept in constant dim light and subjected to advance and delay shifts of a 12-h feeding phase. The resynchronization times of their circadian feeding rhythm following the phase shifts were significantly shorter when the birds carried melatonin implants than when they carried empty implants. In a second experiment, melatonin-implanted and control birds were released into food ad libitum conditions 2 days after either a delay or an advance phase shift. The number of hours by which the activity rhythms had been shifted on the second day in food ad libitum conditions was assessed. Melatonin-implanted house sparrows had significantly larger phase shifts in their circadian feeding rhythm than control birds. This is in accordance with the first experiment since a larger phase shift at a given time reflects accelerated resynchronization. Additionally, the second experiment also excludes any possible masking effects of the nonphotic zeitgeber. In conclusion, constant melatonin accelerates resynchronization even after phase shifts of a nonphotic zeitgeber, indicating that constant high levels of melatonin can reduce the degree of self-sustainment of the circadian pacemaker independent of any effects on the photoreceptive system.     

Calcium channels mediate phase shifts of the Bulla circadian pacemaker

1. Light-induced phase advances of the activity rhythm of the Bulla ocular circadian pacemaker are blocked when the extracellular calcium concentration is reduced with EGTA to 0.13 microM. Phase advances are also blocked in low calcium solutions without EGTA [( Ca] less than 50 microM). 2. The dependence of light-induced phase delays on extracellular calcium concentration in EGTA-free seawater was determined. Phase delays are blocked at calcium concentrations below 400 microM, and reduced at concentrations of 1 mM and 3.5 mM (relative to shifts in normal ASW, [Ca] = 10 mM). Phase delays are also reduced and blocked at calcium concentrations higher than normal (60 mM and 110 mM, respectively). 3. Low calcium EGTA also blocked both phase delays and phase advances induced by pulses of depolarizing high K+ seawater. Low calcium EGTA pulses presented alone at the same times did not generate significant phase shifts. 4. The organic calcium channel antagonists verapamil, diltiazem and nitrendipine as well as the inorganic calcium channel antagonists La3+, Co2+, Cd2+, and Mn2+ were applied along with light pulses, however, the treated eyes were either phase shifted by these substances, or these substances were found to be toxic. 5. The inorganic calcium channel antagonist Ni2+ blocked both light-induced phase delays and advances at a concentration of 5 mM. Ni2+ applied alone did not generate significant phase shifts. Phase delays induced by high K+ seawater were blocked in the presence of 50 mM Ni2+ but not in 5 mM Ni2+. The light-induced CAP activity of the putative pacemaker cells was not inhibited by Ni2+, suggesting that its blocking action was probably via its known role as a calcium channel antagonist.     

S20098 AFFECTS THE FREE-RUNNING RHYTHMS OF BODY TEMPERATURE AND ACTIVITY AND DECREASES LIGHT-INDUCED PHASE DELAYS OF CIRCADIAN RHYTHMS OF THE RAT

Mammalian endogenous circadian rhythms are entrained to the environmental day-night cycle by light exposure. Melatonin is involved in this entrainment by signaling the day-night information to the endogenous circadian pacemaker. Furthermore, melatonin is known to affect the circadian rhythm of body temperature directly. A striking property of the endogenous melatonin signal is its synthesis pattern, characterized by long-term elevated melatonin levels throughout the night. In the present study, the influence of prolonged treatment with the melatonin agonist S20098 during the activity phase of free-running rats was examined. This was achieved by giving S20098 in the food. The free-running body temperature and activity rhythms were studied. The present study shows that enhancement of the melatonin signal, using S20098, affected the free-running rhythm by gradual phase advances of the start of the activity phase, consequently causing an increase in length of the activity phase. A well-known feature of circadian rhythms is its time-dependent sensitivity for light. Light pulse exposure of an animal housed under continuous dark conditions can cause a phase shift of the circadian pacemaker. Therefore, in a second experiment, the influence of melatonin receptor stimulation on the sensitivity of the pacemaker to light was examined by giving the melatonin agonist S20098 in the food during 1 day prior to exposure to a 60-min light pulse of 0, 1.5, 15, or 150 lux given at circadian time (CT) 14. S20098 pretreatment caused a diminished lightpulse- induced phase shift when a light pulse of low light intensity (1.5 lux) was given. S20098 treatment via the food was sufficient to exert chronobiotic activity, and S20098 treatment resulting in prolonged overstimulation of melatonin receptors is able to attenuate the effect of light on the circadian timing system. (Chronobiology International, 18(5), 781-799, 2001)     

Circadian effects of light no brighter than moonlight

In mammals, light entrains endogenous circadian pacemakers by inducing daily phase shifts via a photoreceptor mechanism recently discovered in retinal ganglion cells. Light that is comparable in intensity to moonlight is generally ineffective at inducing phase shifts or suppressing melatonin secretion, which has prompted the view that circadian photic sensitivity has been titrated so that the central pacemaker is unaffected by natural nighttime illumination. However, the authors have shown in several different entrainment paradigms that completely dark nights are not functionally equivalent to dimly lit nights, even when nighttime illumination is below putative thresholds for the circadian visual system. The present studies extend these findings. Dim illumination is shown here to be neither a strong zeitgeber, consistent with published fluence response curves, nor a potentiator of other zeitgebers. Nevertheless, dim light markedly alters the behavior of the free-running circadian pacemaker. Syrian hamsters were released from entrained conditions into constant darkness or dim narrowband green illumination (~0.01 lx, 1.3 x 10(-9) W/cm(2), peak lambda = 560 nm). Relative to complete darkness, constant dim light lengthened the period by ~0.3 h and altered the waveform of circadian rhythmicity. Among animals transferred from long day lengths (14 L:10 D) into constant conditions, dim illumination increased the duration of the active phase (alpha) by ~3 h relative to complete darkness. Short day entrainment (8 L:16 D) produced initially long alpha that increased further under constant dim light but decreased under complete darkness. In contrast, dim light pulses 2 h or longer produced effects on circadian phase and melatonin secretion that were small in magnitude. Furthermore, the amplitude of phase resetting to bright light and nonphotic stimuli was similar against dimly lit and dark backgrounds, indicating that the former does not directly amplify circadian inputs. Dim illumination markedly alters circadian waveform through effects on alpha, suggesting that dim light influences the coupling between oscillators theorized to program the beginning and end of subjective night. Physiological mechanisms responsible for conveying dim light stimuli to the pacemaker and implications for chronotherapeutics warrant further study.     

TheBulla ocular circadian pacemaker

In an effort to understand the cellular basis of entrainment of circadian oscillators we have studied the role of membrane potential changes in the neurons which comprise the ocular circadian pacemaker of Bulla gouldiana in mediating phase shifts of the ocular circadian rhythm. We report that: 1. Intracellular recording was used to measure directly the effects of the phase shifting agents light, serotonin, and 8-bromo-cAMP on the membrane potential of the basal retinal neurons. We found that light pulses evoke a transient depolarization followed by a smaller sustained depolarization. Application of serotonin produced a biphasic response; a transient depolarization followed by a sustained hyperpolarization. Application of a membrane permeable analog of the intracellular second messenger cAMP, 8-bromo-cAMP, elicited sustained hyperpolarization, and occasionally a weak phasic depolarization. 2. Changing the membrane potential of the basal retinal neurons directly and selectively with intracellularly injected current phase shifts the ocular circadian rhythm. Both depolarizing and hyperpolarizing current can shift the phase of the circadian oscillator. Depolarizing current mimics the phase shifting action of light, while hyperpolarizing current produces phase shifts which are transposed approximately 180 degrees in circadian time to depolarization. 3. Altering BRN membrane potential with ionic treatments, depolarizing with elevated K+ seawater or hyperpolarizing with lowered Na+ seawater, produces phase shifts similar to current injection. 4. The light-induced depolarization of the basal retinal neurons is necessary for phase shifts by light. Suppressing the light-induced depolarization with injected current inhibits light-induced phase shifts. 5. The ability of membrane potential changes to shift oscillator phase is dependent on extracellular calcium. Reducing extracellular free Ca++ from 10 mM to 1.3 X 10(-7) M inhibits light-induced phase shifts without blocking the photic response of the BRNs. The results indicate that changes in the membrane potential of the pacemaker neurons play a critical role in phase shifting the circadian rhythm, and imply that a voltage-dependent and calcium-dependent process, possibly Ca++ influx, shifts oscillator phase in response to light.     

Melatonin rhythms: Trekking toward the heart of darkness in the chick pineal

Chick pineal cells make melatonin rhythmically, even in culture. Light pulses have two effects on these cells: acute suppression of melatonin synthesis and phase shifts (entrainment) of the underlying pacemaker. The two effects use different mechanistic pathways: the first goes through cAMP, and the second goes through the clock. Both converge on serotonin N-acetyltransferase, whose gene has recently been cloned, with cAMP acting on mRNA levels, but primarily on, enzyme activity, and the clock acting primarily on mRNA levels. Aspects of calcium regulation, not yet well-understood, may impinge on both pathways. These cells also have a novel calcium channel and a novel photopigment.     

Anterior paraventricular thalamus modulates light-induced phase shifts in circadian rhythmicity in rats

The reciprocal connections between the paraventricular thalamic nucleus (PVT) and the suprachiasmatic nuclei suggest that PVT may participate in the regulation of circadian rhythms. We studied in rats the effect of lesions of the anterior and midposterior regions of the PVT on phase shifts of drinking circadian rhythm induced by light pulses at circadian times 6, 12, and 23, as well as the phase shifts produced by electrical or glutamatergic stimulation of the anterior PVT at the same circadian times. Lesion of the anterior PVT abolishes the advances induced by light during late subjective night, whereas midposterior PVT lesions did not affect the phase shifts. Electrical stimulation or glutamate injections in the anterior PVT mimic the phase-shifting effects of light pulses. These results indicate the participation of the anterior PVT as a modulator of entrainment of circadian rhythms to light.     

High potassium treatment resets the circadian oscillator in Xenopus retinal photoreceptors

In vertebrate retina, light hyperpolarizes the photoreceptor membrane, and this is an essential cellular signal for vision. Cellular signals responsible for photic entrainment of some circadian oscillators appear to be distinct from those for vision, but it is not known whether changes in photoreceptor membrane potential play roles in photic entrainment of the photoreceptor circadian oscillator. The authors show that a depolarizing exposure to high potassium resets the circadian oscillator in cultured Xenopus retinal photoreceptor layers. A 4-h pulse of high [K(+)] (34 mM higher than in normal culture medium) caused phase shifts of the melatonin rhythm. This treatment caused phase delays during the early subjective day and phase advances during the late subjective day. In addition to the phase-shifting effect, high potassium pulses stimulated melatonin release acutely at all times. High [K(+)] therefore mimicked dark in its effects on oscillator phase and melatonin synthesis. These results suggest that membrane potential may play a role in photic entrainment of the photoreceptor circadian oscillator and in regulation of melatonin release.     

In search of the pathways for light-induced pacemaker resetting in the suprachiasmatic nucleus

Within the suprachiasmatic nucleus (SCN) of the mammalian hypothalamus is a circadian pacemaker that functions as a clock. Its endogenous period is adjusted to the external 24-h light-dark cycle, primarily by light-induced phase shifts that reset the pacemaker's oscillation. Evidence using a wide variety of neurobiological and molecular genetic tools has elucidated key elements that comprise the visual input pathway for SCN photoentrainment in rodents. Important questions remain regarding the intracellular signals that reset the autoregulatory molecular loop within photoresponsive cells in the SCN's retino-recipient subdivision, as well as the intercellular coupling mechanisms that enable SCN tissue to generate phase shifts of overt behavioral and physiological circadian rhythms such as locomotion and SCN neuronal firing rate. Multiple neurotransmitters, protein kinases, and photoinducible genes add to system complexity, and we still do not fully understand how dawn and dusk light pulses ultimately produce bidirectional, advancing and delaying phase shifts for pacemaker entrainment.     

Neurophysiological mechanisms involved in photo-entrainment of the circadian rhythm from the Aplysia eye

An attempt was made to identify the neurophysiological processes involved in entrainment of the circadian rhythm of spontaneous optic nerve potentials from the Aplysia eye by determining whether pharmacological agents or ion substitutions could block phase shifts produced by single light pulses. Knowing which physiological processes are involved in entrainment should help define the morphological pathway traveled by entrainment information. A secretory step does not appear to be involved in the flow of entrainment information from the environment to the circadian oscillator. A treatment (HiMg LoCa) capable of inhibiting secretion did not interfere with phase shifting by light. Furthermore, treating eyes with putative transmitters or extracts of eyes did not phase shift the free running rhythm. Also, the phase shifting information is not translated into action potentials before reaching the oscillator since TTX–HiMg LoCa solutions did not block the light-induced phase shift. The photoreceptor potential does seem to be important for light-induced phase shifts. A correlation was found between the effects of treatments on the ERG and their effects on the light-induced phase shift. Solutions which decreased the ERG by 90% or more blocked phase shifting whereas solutions which decreased the ERG by less than 74% had no effect on phase shifting by light. The results from these studies are consistent with two pathways for the flow of phase shifting information to the circadian oscillator. The circadian oscillator may be associated with receptor cells and the entrainment pathway would include a step involving the photoreceptor potential. Alternatively, the circadian oscillator may be associated with secondary cells and receive entrainment information via the photoreceptor potential and passive spread of current through a gap junction. Higher order cells than second-order ones are probably not involved in the entrainment pathway.     

An opsin-based photopigment mediates phase shifts of the Bulla circadian pacemaker

1. The spectral response of the circadian pacemaker of the eye of the mollusk Bulla gouldiana was examined in two ways: by using the latency of the first light-evoked compound action potential (CAP) as an acute photoresponse of the putative pacemaker cells of the eye, the basal retinal neurons (BRNs), and by measuring the effectiveness of monochromatic light pulses at resetting the pacemaker. 2. Through measurements of the spectral sensitivity of the acute response of the BRNs, a photopigment absorbing maximally near 490 nm (lambda max) was described. Action spectra of the acute response following isolation of the BRNs, by surgical removal of the distal photoreceptor layer or the use of low Ca2+ media to block chemical synapses on the BRNs, further suggested that a 490 nm lambda max photopigment is used in generating the acute light response. The spectral sensitivity of eyes adapted to a dim background illumination also agreed with the expected absorption of a 490 lambda max rhodopsin. 3. The effectiveness of monochromatic light pulses at shifting the phase of the circadian rhythm in CAP frequency suggested that the photopigment used in the entrainment of the pacemaker is the opsin based molecule identified through acute response measurements.     

Cyclic AMP Resets the Circadian Clock in Cultured Xenopus Retinal Photoreceptor Layers

Abstract: The Xenopus retinal photoreceptor layer contains a circadian oscillator that regulates melatonin synthesis in vitro. The phase of this oscillator can be reset by light or dopamine. The phase-response curves for light and dopamine are similar, with transitions from phase delays to phase advances in the mid-subjective night. Light and dopamine each can inhibit adenylate cyclase in retinal photoreceptors, suggesting cyclic AMP as a candidate second messenger for entrainment of the circadian oscillator. We report here that treatments that increase intracellular cyclic AMP reset the phase of the photoreceptor circadian oscillator, and that the phase-response curves for these treatments are 180° out of phase with the phase-response curves for light and dopamine. Activation of adenylate cyclase by forskolin during the late subjective day or early subjective night caused phase advances. The same treatment during the late subjective night or early subjective day caused phase delays. Similar phase shifts were induced by 3-isobutyl-1-methyl-xanthine (a phosphodiesterase inhibitor) or 8-(4-chlorophenylthio)cyclic AMP. All of these treatments also acutely increased melatonin release. Forskolin and 3-isobutyl-1-methylxanthine increased the accumulation of intracellular cyclic AMP, but not cyclic GMP, in photoreceptor layers. The results indicate that cyclic AMP-dependent pathways regulate the photoreceptor circadian oscillator and suggest that a decrease in cyclic AMP may be involved in circadian entrainment by light and/or dopamine.     

Circadian phase shifting induced by clonidine injections in Syrian hamsters

Abstract

The mammalian circadian pacemaker can be phase shifted by photic, pharmacological, and behaviorally‐derived stimuli. The phase‐response curves (PRCs) characterizing these diverse stimuli may comprise two distinct families; a photic PRC typified by the response to brief light pulses, and a non‐photic PRC, typified by the response to dark pulses and to behavioral activation. The present study examined the phase shifting effects of acute systemic treatment with the alpha2‐adrenoceptor agonist, clonidine, in Syrian hamsters. Clonidine injections (0.25 mg/kg, ip) delivered during subjective night mimicked the phase shifting effects of light pulses in animals housed in both constant darkness (DD) and constant red light (RR), but similar effects were not seen in saline‐treated controls. Both clonidine and saline injections resulted in phase advances during subjective day, but only in RR‐housed animals. Clonidine‐induced phase shifting was dose‐dependent, but rather high doses were required to induce phase shifts. Pretreatment with the selective noradrenergic neurotoxin, DSP‐4, blocked clonidine‐induced phase shifting. These results suggest that clonidine acts at presynaptic alpha2‐adrenergic autoreceptors to disinhibit spontaneous and/or evoked activity in the photic entrainment pathway.     

A phase dynamics model of human circadian rhythms

Nonphotic entrainment of an overt sleep-wake rhythm and a circadian pacemaker-driving temperature/melatonin rhythm suggests existence of feedback mechanisms in the human circadian system. In this study, the authors constructed a phase dynamics model that consisted of two oscillators driving temperature/melatonin and sleep-wake rhythms, and an additional oscillator generating an overt sleep-wake rhythm. The feedback mechanism was implemented by modifying couplings between the constituent oscillators according to the history of correlations between them. The model successfully simulated the behavior of human circadian rhythms in response to forced rest-activity schedules under free-run situations: the sleep-wake rhythm is reentrained with the circadian pacemaker after release from the schedule, there is a critical period for the schedule to fully entrain the sleep-wake rhythm, and the forced rest-activity schedule can entrain the circadian pacemaker with the aid of exercise. The behavior of human circadian rhythms was reproduced with variations in only a few model parameters. Because conventional models are unable to reproduce the experimental results concerned here, it was suggested that the feedback mechanisms included in this model underlie nonphotic entrainment of human circadian rhythms.     

Light pulses suppress responsiveness within the mouse photic entrainment pathway

We have characterized a decrease in photic responsiveness of the mammalian circadian entrainment pathway caused by light stimulation. Phase delays of the running-wheel activity rhythm were used to quantify the photic responsiveness of the circadian system in mice (C57BL/6J). In an initial experiment, the authors measured the responsiveness to single "saturating" light pulses ("white" fluorescent light; approximately 1876 [microW; 15 min). In two additional experiments, the authors measured responses to this stimulus at several time points following a saturating pulse at CT 14 or CT 16. Data from these experiments were analyzed in two manners. Experiment 2 was analyzed assuming that the phase of the circadian pacemaker was unchanged by an initial pulse, and Experiment 3 was analyzed assuming that the initial pulse induced an instantaneous phase delay. Results reveal a significant reduction in responsivity to light that persists for at least 2 h and possibly up to 4 h after the initial stimulus. Immediately after the stimulus, the responsiveness of the photic entrainment pathway was reduced to levels < or = 12% of normal. After 2 h, the responsiveness was < or = 42% of normal, and by 4 h, responsiveness had recovered to levels that were < or = 60% of normal (levels not statistically different from controls). By the following circadian cycle, responsiveness was more completely recovered, although the magnitude of some phase delays remained < or = 85% of normal. These major reductions in the magnitude of phase delays (and phase response curve amplitude) caused by saturating light pulses confound interpretations of two-pulse experiments designed to measure the rate of circadian phase delays. In addition, the time course for this reduced responsiveness may reflect the time course of cellular and molecular events that underlie light-induced resetting of the mammalian circadian pacemaker.     

FMRFamide modulates the action of phase shifting agents on the ocular circadian pacemakers of Aplysia and Bulla

Summary The eye of the marine mollusk Aplysia californica contains a photo-entrainable circadian pacemaker that drives an overt circadian rhythm of spontaneous compound action potentials in the optic nerve. Both light and serotonin are known to influence the phase of this ocular rhythm. The current study evaluated the effect of FMRFamide on both light and serotonin induced phase shifts of this rhythm. The application of FMRFamide was found to block serotonin induced phase shifts but, by itself, FMRFamide did not cause significant phase shifts. Furthermore, the effects of FMRFamide on light-induced phase shifts appeared to be phase dependent (i.e., the application of FMRFamide inhibited light-induced phase delays but actually enhanced the magnitude of phase advances). As in Aplysia, the eye of Bulla gouldiana also contains a circadian pacemaker. In Bulla, FMRFamide prevented light-induced phase advances and delays. Although FMRFamide alone generated phase dependent phase shifts, it did not cause phase shifts at the phases where it blocked the effects of light. These data demonstrate that FMRFamide can have pronounced modulatory effects on phase shifting inputs to the ocular pacemakers of both Aplysia and Bulla.Abbreviations ASW artificial seawater - CAP compound action potential - CT circadian time - 5-HT serotonin