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     

Evidence that potassium channels mediate the effects of serotonin on the ocular circadian pacemaker of Aplysia

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. Serotonin is known to influence the phase of this ocular rhythm. The aim of the present study was to evaluate whether potassium channels are involved in effects on the ocular circadian rhythm. Our experimental approach was to study the effect of the potassium channel antagonist barium on serotonin-induced phase shifts of this rhythm. The application of barium was found to block serotonininduced phase shifts whereas barium alone did not cause significant phase shifts. The effects of barium were found to be dose dependent. In addition, barium blocked forskolin-induced phase advances but did not interfere with serotonin-induced increases in cAMP content. Finally, barium antagonized serotonin-induced suppression of compound action potential activity. These results are consistent with a model in which the application of serotonin phase shifts the ocular pacemaker by causing a membrane hyperpolarization which is mediated by a cAMP-dependent potassium conductance.Abbreviations ASW artificial seawater - Ba^{+ +} barium - CAP compound action potential - CT circadian time - 5-HT serotonin - TEA tetraethylammonium     

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.     

Calcium plays a central role in phase shifting the ocular circadian pacemaker of Aplysia

The eye of the marine mollusk Aplysia californica contains an oscillator that drives a 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 aim of the present study was to evaluate the role of extracellular calcium in both light and serotonin-mediated phase shifts. Low calcium treatments were found to cause phase shifts which resembled those produced by the transmitter serotonin. However, unlike serotonin, low calcium neither increased ocular cAMP levels nor could these phase shifts be prevented by increasing extracellular potassium concentration. Low calcium-induced phase shifts were prevented by the simultaneous application of the translational inhibitor anisomycin and low calcium treatment resulted in changes in [³⁵S]methionine incorporation into several proteins as measured by a two-dimensional electrophoresis gel analysis. Finally, light treatments failed to produce phase shifts in the presence of low calcium or the calcium channel antagonist nickel chloride. These results are consistent with a model in which serotonin phase shifts the ocular pacemaker by decreasing a transmembrane calcium flux through membrane hyperpolarization while light-induced phase shifts are mediated by an increase in calcium flux.Abbreviations ASW artificial seawater - EGTA ethylene glycol-bis(-amino-ethyl ester) N,N,N N-tetraacetic acid - CAP compound action potential - CT circadian time 5-HT serotonin - Ni⁺⁺ nickel     

The circadian rhythm and photosensitivity of small impulses of the Bulla eye

Summary The eye of the mollusk Bulla gouldiana contains a pacemaker that generates a circadian rhythm in compound action potentials (CAPs) in the optic nerve. In this paper, we present evidence of a second circadian rhythm in the optic nerve of the eye maintained in darkness at 15 °C. This is a rhythm in the frequency of small (10–40 V) neural impulses that occurs about 12 h out-of-phase with the rhythm in CAPs. Typically, the small-spike frequency is at a minimum within an hour of the peak in CAP frequency and is maximal during the subjective night. Like the CAP rhythm, the phase of the small-spike rhythm is determined by the prior light/dark cycle. A rebound in small-spike activity following the end of a light pulse and the presence of photoinhibited impulses in surgically reduced eyes suggests that the cells that generate the small-spikes may be photoreceptors that are inhibited by light. In addition, by using isolated nervous system preparations, we have found that smallspikes occur in the two optic nerves in a one-for-one relationship immediately following a light-to-dark transition. This inter-eye communication may be involved in the coupling of the ocular pacemakers.Abbreviations ASW artificial sea water - BRN basal retinal neuron - CAP compound action potential     

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.     

Phase-shifts of the Bulla ocular circadian pacemaker in the presence of calmodulin antagonists

Previous work has shown that light-induced phase shifts of the Bulla ocular circadian pacemaker require extracellular calcium, suggesting the possibility that the action of calcium as a second messenger via calmodulin is an element in the phase shifting mechanism. The calmodulin antagonists calmidazolium, trifluoperazine (TFP) and W7 were applied with phase shifting light pulses. Light phase shifts were not blocked by calmidazolium or TFP, suggesting that calmodulin does not mediate light-induced phase shifts. Period changes were observed with treatments of both TFP and W7, but not with calmidazolium and are probably not calmodulin-mediated.     

Circadian rhythms of house sparrows are phase-shifted by pharmacological manipulation of brain serotonin

Summary Fluoxetine, a specific serotonergic reuptake blocker and indirect agonist, and 5,6-dihydroxytryptamine, a serotonergic neurotoxin, affect the free-running locomotor rhythms of house sparrows,Passer domesticus. Both compounds caused phase-shifts in the circadian system that times locomotor activity of the birds. The magnitude and direction of the phase-shifts were dependent on the circadian phase of the drug administration, suggesting that serotonergic activity can modulate the circadian pacemaker of the house sparrow.Abbreviations 5HT serotonin, 5-hydroxytryptamine - 5,6-DHT 5,6-dihydroxytryptamine - DMI desmethylimipramine - SCN suprachiasmatic nuclei - CT circadian time - CAP compound action potential This paper is dedicated to Professor Colin S. Pittendrigh on the occasion of his 65th birthday     

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.     

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.     

Phase advances of circadian rhythms in somatostatin depleted rats: effects of cysteamine on rhythms of locomotor activity and electrical discharge of the suprachiasmatic nucleus

Somatostatin is synthesized in the suprachiasmatic nucleus (SCN), a circadian pacemaker in mammals. To explore the functional significance of somatostatin in the circadian system, we examined rhythms of rat locomotor activity and electrical firing rate of SCN neurons in the brain slice after temporal depletion of somatostatin levels in the SCN. Intraperitoneal administration of cysteamine (200 mg/kg), a somatostatin depletor, significantly reduced somatostatin level in the in vivo SCN 5 min after injection and kept low level as long as 3 to 4 days. This administration, on the other hand, induced significant phase advances of about 51 min in the subsequent free-running rhythm of locomotor activity of the rat. A marked phase advance in the circadian rhythm of firing rate in the SCN was also observed after administration of cysteamine in coronal hypothalamic slices. These persistent phase shifts after administration of a somatostatin depletor may suggest that the change of somatostatin level in the SCN have a feedback influence on the circadian pacemaker.Abbreviations SCN suprachiasmatic nucleus - AVP arginine-vasopressin - VIP vasoactive intestinal polypeptide - CT circadian time - ZT zeitgeber time - i.p. intraperitoneally - 12L:12D 12 h light and 12 h dark - ANOVA analysis of variance     

Cellular analysis of ocular circadian pacemaker coupling in Bulla: role of efferent impulses in phase shifting

The eyes of Bulla gouldiana, a marine snail, contain circadian oscillators that are coupled to each other. Obvious candidates for the coupling signals are the optic nerve compound action potentials (CAPs) that express the circadian rhythm and lead to efferent impulses in the contralateral optic nerve. In the present experiments, the role of the CAPs as coupling signals was evaluated. We found that, following desynchronization of the two ocular oscillators by phase-delaying one eye with manganese, subsequent phase shifts in the initially unshifted ocular rhythm only occurred during the time that efferent optic nerve signals were present. In addition, in the absence of ocular desynchrony, phase shifts of the ocular rhythm could still be effected by activation of the efferent pathway. The influence of efferent impulses on identified retinal cells was also evaluated. No effect of efferent signals on receptor layer cells was detected, while it was found that efferent impulses generated depolarizations in basal retinal neurons (BRNs), the putative circadian oscillator cells. Depolarization of the BRNs has been shown previously to be involved in the light entrainment pathway. Depolarization appears to be similarly involved in the coupling pathway, since membrane depolarizations that mimicked the efferent-induced postsynaptic potentials likewise generated phase shifts of the ocular rhythm.     

Phase Resetting of the Human Circadian Pacemaker with Use of a Single Pulse of Bright Light

Since the initial studies reporting that light can alter the phase position of the human circadian system, there has been increasing interest in the use of bright light as a tool for manipulating the phase position of the circadian pacemaker. Exposure protocols typically require subjects to receive 2-5 h of exposure over several circadian cycles. As a consequence, bright light treatment can involve a considerable time investment. However, recent studies indicate that a single pulse of bright light can produce significant phase shifts in the circadian pacemaker. If a single pulse of bright light can produce significant phase-shifting effects, multiple-pulse designs may be unnecessary. This study examined the phase-shifting effects of a single 4-h pulse of bright light (12,000 lux) in 14 male and one female subject aged between 19-45 years. With use of a “constant routine” to estimate circadian phase, a single 4-h pulse of light produced significant shifts in the phase of the core temperature rhythm. The timing of the exposure, relative to the core temperature rhythm, determined the degree and direction of the phase shift. Exposure immediately prior to habitual bedtime produced a mean phase delay in the core temperature of 2.39 h (SD = 1.37 h). In contrast, exposure immediately following habitual wake-up produced a mean phase advance of 1.49 h (SD = 2.06 h). In addition, the magnitude of the shift increased the closer the light pulse was to the individual's estimated endogenous core temperature minimum. There was, however, considerable interindividual variability in this relationship. Overall, these results confirm that a single pulse of bright light can produce significant phase shifts in the phase of the circadian pacemaker controlling core temperature. Key Words: Bright light—Circadian rhythm—Core body temperature—Sleep-wake disorders—Chronobiology.     

EFFECT OF LIGHT ON THE DEVELOPMENT OF THE CIRCADIAN RHYTHM OF MOTOR ACTIVITY IN THE MOUSE

In previous experiments, we found that rats raised in constant light (LL) manifested a more robust circadian rhythm of motor activity in LL and showed longer phase shifts after a light pulse in constant darkness (DD) than those raised under constant darkness. In addition, we observed that the effects produced by constant light differed depending on the time of postnatal development in which it was given. These results suggest that both sensitivity to light and the functioning of the circadian pacemaker of the rat could be affected by the environmental conditions experienced during postembryonic development. Thus, the present experiment aimed to study whether postnatal exposure to light could also affect the circadian system of the mouse. Three groups of mice were formed: One group was raised under constant darkness during lactation (DD group), the second under constant light (LL group), and the third under light-dark cycles (LD group). After lactation, the three groups were submitted first to constant light of high intensity, then to LD cycles, and finally to constant darkness. In the DD stage, a light pulse was given. Finally, mice were submitted to constant light of low intensity. We observed that the circadian rhythm of the DD group was more disturbed under constant light than the rhythm of the LL group, and that, when light intensity increased, the period of the rhythm of the DD group lengthened more than that of the LL group. No significant differences among the groups were found in the phase shift induced by the light pulse. Therefore, it appears that DD mice are more sensitive to light than their LL counterparts. However, at present there is no evidence to affirm that the light environment experienced by the mouse during postnatal development affects the circadian pacemaker. (Chronobiology International, 18(4), 683–696, 2001)     

Photoentrainment, pharmacology, and phase shifts of the circadian rhythm in the rat pineal.

Photoentrainment of circadian rhythms in mammals is mediated by the retinohypothalamic projection to the suprachiasmatic nucleus of the hypothalamus. It should therefore be possible to mimic or block the effects of light on the circadian pacemaker with appropriate pharmacological agents. Such agents and their effects should be useful in identifying the neurotransmitters involved in photoentrainment and their mechanisms of action on the circadian pacemaker. The effects of light on the circadian rhythm in rat pineal serotonin N-acetyltransferase activity are described. Carbachol, a cholinergic agonist, was found to mimic the effects of light on this rhythm, including the acute reduction of nocturnal activity and phase-shifting of the free-running rhythm. These results raise the possibility that acetylcholine is involved in the photoentrainment of mammalian circadian rhythms.     

PROBING THE CIRCADIAN OSCILLATOR OF A MAMMAL BY TWO-PULSE PERTURBATIONS

When organisms are maintained under constant conditions of light and temperature, their endogenous circadian rhythms free run, manifesting their intrinsic period. The phases of these free-running rhythms can be shifted by stimuli of light, temperature, and drugs. The change from one free-running steady state to another following a perturbation often involves several transient cycles (cycles of free-running rhythm drifting slowly to catch up with the postperturbation steady state). Although the investigation of oscillator kinetics in circadian rhythms of both insects and mammals has revealed that the circadian pacemaker phase shifts instantaneously, the phenomenon of transient cycles has remained an enigma. We probed the phases of the transient cycles in the locomotor activity rhythm of the field mouse Mus booduga, evoked by a single light pulse (LP), using LPs at critically timed phases. The results of our experiments indicate that the transient cycles generated during transition from one steady state to another steady state do not represent the state of the circadian pacemaker (basic oscillator) controlling the locomotor activity rhythm in Mus booduga. (Chronobiology International, 17(2), 129–136, 2000)     

Melatonin rhythm observed throughout a three-cycle bright-light stimulus designed to reset the human circadian pacemaker.

Exposure to light and darkness can rapidly induce phase shifts of the human circadian pacemaker. A type 0 phase response curve (PRC) to light that has been reported for humans was based on circadian phase data collected from constant routines performed before and after a three-cycle light stimulus, but resetting data observed throughout the entire resetting protocol have not been previously reported. Pineal melatonin secretion is governed by the hypothalamic circadian pacemaker via a well-defined neural pathway and is reportedly less subject to the masking effects of sleep and activity than body temperature. The authors reasoned that observation of the melatonin rhythm throughout the three-cycle light resetting trials could provide daily phase-resetting information, allowing a dynamic view of the resetting response of the circadian pacemaker to light. Subjects (n = 12) living in otherwise dim light (approximately 10-15 lux) were exposed to a noncritical stimulus of three cycles of bright light (approximately 9500 lux for 5 h per day) timed to phase advance or phase delay the human circadian pacemaker; control subjects (n = 11) were scheduled to the same protocols but exposed to three 5-h darkness cycles instead of light. Subjects underwent initial and final constant routine phase assessments; hourly melatonin samples and body temperature data were collected throughout the protocol. Average daily phase shifts of 1 to 3 h were observed in 11 of 12 subjects receiving the bright light, supporting predictions obtained using Kronauer's phase-amplitude model of the resetting response of the human circadian pacemaker. The melatonin rhythm in the 12th subject progressively attenuated in amplitude throughout the resetting trial, becoming undetectable for >32 hours preceding an abrupt reappearance of the rhythm at a shifted phase with a recovered amplitude. The data from control subjects who remained in dim lighting and darkness delayed on average -0.2 h per day, consistent with the daily delay expected due to the longer than 24-h intrinsic period of the human circadian pacemaker. Both temperature and melatonin rhythms shifted by equivalent amounts in both bright light-treated and control subjects (R = 0.968; p<0.0001; n = 23). Observation of the melatonin rhythm throughout a three-cycle resetting trial has provided a dynamic view of the daily phase-resetting response of the human circadian pacemaker. Taken together with the observation of strong type 0 resetting in humans in response to the same three-cycle stimulus applied at a critical phase, these data confirm the importance of considering both phase and amplitude when describing the resetting of the human circadian pacemaker by light.     

Light-induced c-Fos expression in the SCN and behavioural phase shifts of Djungarian hamsters with a delayed activity onset

C-Fos expression in the suprachiasmatic nucleus (SCN) and phase shifts of the activity rhythm following photic stimulation were investigated in Djungarian hamsters (Phodopus sungorus) of two different circadian phenotypes. Wild-type (WT) hamsters display robust daily patterns of locomotor activity according to the light/dark conditions. Hamsters of the DAO (delayed activity onset) phenotype, however, progressively delay the activity onset, whereas activity offset remains coupled to “light-on”. Although the exact reason for the delayed activity onset is not yet clarified, it is connected with a disturbed interaction between the light/dark cycle and the circadian clock. The aim was to test the link between photoreception and the behavioral output of the circadian system in hamsters of both phenotypes, to get further insight in the underlying mechanism of the DAO phenomenon. Animals were exposed to short light pulses at different times during the dark period to analyze phase shifts of the activity rhythm and expression of Fos protein in the SCN. The results indicate that the photosensitive phase in DAO hamsters is shifted like the activity onset. Also, phase shifts were significantly smaller in DAO hamsters. At the same time, levels of Fos expression did not differ between phenotypes regarding the circadian phase. The results provide evidence that the shifted photosensitivity of the circadian system in DAO hamsters does not differ from that of WT animals, and lead us to conclude that processes within the SCN that enable light information to reset the circadian pacemaker might offer an explanation for the DAO phenomenon.     

Light and serotonin phase-shift the circadian clock in the cricket optic lobe in vitro

Light and serotonin were found to cause phase shifts of the circadian neural activity rhythm in the optic lobe of the cricket Gryllus bimaculatus cultured in vitro. The two phase-shifting agents yielded phase-response curves different in shape. Light induced phase delay and advance in the early and late subjective night, respectively, and almost no shifts in the subjective day, whereas serotonin phase-advances the clock during the subjective day and induced delay shifts during the subjective night. The largest phase advance and delay occurred at circadian time 21 and 12, respectively, for light, and circadian time 3 and 18, respectively, for serotonin. Quipazine, a nonspecific serotonin agonist, induced phase advance and phase delay at circadian time 3 and 18, respectively, like serotonin. (±)8-OH-DPAT, a specific 5-HT_1A agonist, phase delayed by 2 h at the subjective night, but produced no significant phase shifts at the subjective day. When NAN-190, a specific 5-HT_1A antagonist, was applied together with quipazine, it completely blocked the phase delay at circadian time 18, whereas it had no effect on the advance shifts induced by quipazine. The results suggest that the phase dependency of serotonin-induced phase shifts of the clock may be partly attributable to the daily change in receptor type. Accepted: 4 July 1999