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     

Increasing external K+ blocks phase shifts in a circadian rhythm produced by serotonin or 8-benzylthio-cAMP

Serotonin (5-HT) phase shifts the circadian rhythm from the isolated eye of Aplysia. The discovery of the mechanisms involved in phase shifting by 5-HT may help elucidate the nature of the circadian oscillator. We have found that 5-HT appears to phase shift by causing a change in membrane K+ conductance. Solutions containing zero K+(0-K+) phase shift the rhythm and the phase response curve (PRC) for 0-K+ is similar to one previously obtained for 5-HT. The similarity in PRCs for 0-K+ and 5-HT suggested that these treatments may be phase shifting the rhythm through a common mechanism. The nonadditivity of phase shifting by 0-K+ and 5-HT supports this suggestion. A common mechanism of action of 5-HT and 0-K+ might be effects on membrane potentials. The possible involvement of a membrane potential change in mediating the effect of 5-HT and the lack of an effect of large reductions in Na+, Cl-, and Ca2+ ions on phase shifting by 5-HT led us to examine the role of K+ ions in phase shifting by 5-HT. A change in K+ conductance may mediate the effects of 5-HT on the rhythm because HiK (30mM) solutions blocked the phase shift normally produced by 5-HT. The conductance change produced by 5-HT may be an increase in K+ conductance which would produce a hyperpolarization and not a decrease in K+ conductance which would produce a depolarization since depolarizing treatments, HiK (30-110mM), had no effect on the rhythm at the phase where 5-HT produces its largest phase shifts. Since we previously found that the effects of 5-HT appear to be mediated by cAMP, we examined whether HiK solutions could block the effects of 8-benzylthio-cAMP on the rhythm. HiK (40mM) blocked the phase shifts normally produced by 8-benylthio-cAMP. Our working hypothesis for the 5-HT phase-shifting pathway based on these results is 5-HT leads to increased cAMP leads to elevates K+ conductance leads to membrane hyperpolarization leads to phase shifts the rhythm.     

Light and serotonin interact in affecting the circadian system of Aplysia

Summary The eye of the marine mollusk Aplysia californica contains a photo-entrainable circadian pacemaker that drives an overt rhythm of spontaneous compound action potentials. The current study evaluated the influence of serotonin on light-induced phase shifts of this ocular rhythm. The application of serotonin in combination with light was found to have profound and interactive effects on the magnitude of the resulting phase shifts. Further, the phase shifts that resulted from the interaction between light and serotonin appeared to be phase dependent, i.e., the application of serotonin inhibited the phase shifting effects of light during one part of the circadian cycle but enhanced them during another. Finally, the results show that the interaction between light and serotonin is dependent upon the sequence in which these two treatments are paired. These data, coupled with previous findings, suggest that serotonin may act to modulate light's phase shifting effects on the ocular pacemaker in Aplysia.Abbreviations CAP compound action potential - ASW artificial sea water - CT circadian time - 5-HT serotonin     

Circadian and light-induced conductance changes in putative pacemaker cells of Bulla gouldiana

The ocular circadian rhythm of compound action potential frequency in Bulla gouldiana is driven by rhythmic changes in the membrane potential of putative circadian pacemaker cells. Changes in the membrane potential of these neurons is required for light-induced phase shifts of the rhythm. We have tested the proposition that these changes in membrane potential reflect underlying changes in ionic conductances. We have found that: 1. Membrane conductance in the dark is highest during the subjective night when the cells are hyperpolarized, decreases as the cells depolarize spontaneously near projected dawn and is lowest during the subjective day. The changes in membrane potential and conductance follow a similar time course. 2. Long pulses of light delivered to eyes during their subjective night produce a characteristic response: There is initially a large, phasic depolarization accompanied by a burst of CAPs; this is followed by a repolarizing phase during which CAP activity is reduced to zero; and finally a tonic depolarization develops that is accompanied by a resumption of CAP activity at a steady rate. 3. During the subjective night, the tonic depolarization is accompanied by a decrease in conductance compared to the previous dark value. However, light pulses of similar duration delivered to eyes during their subjective day causes tonic depolarizations and increased CAP activity, but no measurable change in conductance. 4. Membrane responses to light are sensitive to agents that reduce Ca2+ flux. Light pulses during the subjective night produce a phasic depolarization, but the repolarization phase is eliminated in low Ca2+/EGTA seawater and is reduced in 5 mM Ni2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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     

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     

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.     

TheBulla ocular circadian pacemaker

We have used intracellular recording to directly measure the effects of three experimental agents, light, elevated potassium seawater, and lowered sodium seawater on the membrane potential of the putative circadian pacemaker neurons of the Bulla eye. These agents were subsequently tested for effects on the free running period of the circadian pacemaker. We report that: 1. When applied to the eye, light and elevated potassium seawater depolarized the putative pacemaker neurons, while lowered sodium seawater hyperpolarized them. The membrane potential changes induced by these agents are sustained for at least one hour, suggesting that they produce persistent changes in the average membrane potential of the putative pacemaker neurons. 2. The amplitude of the membrane potential response to the depolarizing agents varies with the phase of the circadian cycle. Depolarizations induced by light and elevated potassium seawater are twice as large during the subjective night than they are during the subjective day. No significant difference was found in the response to lowered sodium seawater at different phases. 3. Continuous application of each of these agents caused a lengthening of the free running period of the Bulla eye. Constant light increased the period by 0.9 h, while the other depolarizing treatment (elevated potassium seawater) increased the free running period by 0.6 h. Both treatments increased the mean peak impulse frequency of treated eyes. The hyperpolarizing treatment also increased the period of the ocular pacemaker (+0.8 h), but had little effect on peak impulse frequency.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Photic resetting of the human circadian pacemaker in the absence of conscious vision

Ocular light exposure patterns are the primary stimuli for entraining the human circadian system to the local 24-h day. Many totally blind persons cannot use these stimuli and, therefore, have circadian rhythms that are not entrained. However, a few otherwise totally blind persons retain the ability to suppress plasma melatonin concentrations after ocular light exposure, probably using a neural pathway that includes the site of the human circadian pacemaker, suggesting that light information is reaching this site. To test definitively whether ocular light exposure could affect the circadian pacemaker of some blind persons and whether melatonin suppression in response to bright light correlates with light-induced phase shifts of thecircadian system, the authorsperformed experiments with 5 totally blind volunteers using a protocol known to induce phase shifts of the circadian pacemaker in sighted individuals. In the 2 blind individuals who maintained light-induced melatonin suppression, the circadian system was shifted by appropriately timed bright-light stimuli. These data demonstrate that light can affect the circadian pacemaker of some totally blind individuals--either by altering the phase of the circadian pacemaker or by affecting its amplitude. They are consistent with data from animal studies demonstrating that there are different neural pathways and retinal cells that relay photic information to the brain: one for conscious light perception and the other for non-image-forming functions.     

Distribution of chemoreceptors to quinine on the cell surface of Paramecium caudatum

The topological distribution of the chemoreceptors to quinine in the membrane of a ciliate Paramecium caudatum were examined by conventional electrophysiological techniques. A CNR-mutant specimen defective in voltage-gated Ca channels produced a transient depolarization followed by a transient hyperpolarization and a sustained depolarization when 1 mM quinine-containing solution was applied to its entirety. A Ni²⁺-paralyzed CNR-mutant specimen produced a simple membrane depolarization in response to a local application of 1 mM quinine-containing solution to its anterior end, whereas it produced a transient membrane hyperpolarization in response to an application to its posterior end. An anterior half fragment of a CNR specimen produced a membrane depolarization whereas a posterior half fragment of the specimen produced a transient hyperpolarization upon application of 1 mM quinine-containing solution. Both anterior depolarization and posterior hyperpolarization took place prior to the contraction of the cell body. It is concluded that Paramecium caudatum possesses two kinds of chemoreceptors or two kinds of coupling of the same receptor to different signal transduction pathways to quinine which are distributed in different locations on the cell surface. Activation of the anterior receptor produces a sustained depolarizing receptor potential while activation of the posterior receptor produces a transient hyperpolarizing receptor potential.Abbreviation CNR caudatum non reversal     

Ion mechanisms of hyperpolarizing afterpotentials accompanying epileptic discharges in neocortical neurons

Hyperpolarizing afterpotentials of penicillin-induced (local application) paroxysmal depolarizing shifts (PDS) in neurons of the sensorimotor cortex of the cat were studied. The pattern of membrane conductance changes within different segments of hyperpolarization and the data on the role of various ion currents in its generation allow us to conclude that hyperpolarizing afterpotentials accompanying PDS are of a composite nature and include the following components: (i) the initial component provided by an increased membrane permeability to chloride ions (presumably a synaptic GABA_A response); (ii) the second component resulting predominantly from a potassium current and representing presumably a GABA_B response; and (iii) the final component comprising mainly a calcium-activated potassium current. These components are present in all neurons, are not clearly demarcated as separate waves, and partially overlap with each other, thus forming a prolonged hyperpolarizing deflection of the potential.     

Mechanisms underlying burst generation of the pyloric muscle in the mantis,shrimp, Squilla oratoria

The pyloric constrictor muscles of the stomach in Squilla can generate spikes by synaptic activation via the motor nerve from the stomatogastric ganglion. Spikes are followed by slow depolarizing afterpotentials (DAPs) which lead to sustained depolarization during a burst of spikes. 1. The frequency of rhythmic bursts induced by continuous depolarization is membrane voltage-dependent. A brief depolarizing or hyperpolarizing pulse can trigger or terminate bursts, respectively, in a threshold-dependent manner. 2. The conductance increases during the DAP response. The amplitude of DAP decreases by imposed depolarization, whereas it increases by hyperpolarization. DAPs from successive spikes sum to produce a sustained depolarizing potential capable of firing a burst. 3. The spike and DAP are reduced in amplitude by decreasing [Ca]o, enhanced by Sr2+ or Ba2+ substituted for Ca2+, and blocked by Co2+ or Mn2+. DAPs are selectively blocked by Ni2+, and the spike is followed by a hyperpolarizing afterpotential. 4. The spike and DAP are prolonged by intracellular injection of the Ca2+ chelator EGTA. A hyperpolarizing afterpotential is abolished by EGTA and enhanced by increasing [Ca]o. The DAP is diminished in Na(+)-free saline and reduced by tetrodotoxin. 5. It is concluded that the muscle fiber is endowed with endogenous oscillatory properties and that the oscillatory membrane events result from changes of a voltage- and time-dependent conductance to Ca2+ and Na+ and a Ca2+ activated conductance to K+.     

The visual input stage of the mammalian circadian pacemaking system: I. Is there a clock in the mammalian eye?

Threads of evidence from recent experimentation in retinal morphology, neurochemistry, electrophysiology, and visual perception point toward rhythmic ocular processes that may be integral components of circadian entrainment in mammals. Components of retinal cell biology (rod outer-segment disk shedding, inner-segment degradation, melatonin and dopamine synthesis, electrophysiological responses) show self-sustaining circadian oscillations whose phase can be controlled by light-dark cycles. A complete phase response curve in visual sensitivity can be generated from light-pulse-induced phase shifting. Following lesions of the suprachiasmatic nuclei, circadian rhythms of visual detectability and rod outer-segment disk shedding persist, even though behavioral activity becomes arrhythmic. We discuss the converging evidence for an ocular circadian timing system in terms of interactions between rhythmic retinal processes and the central suprachiasmatic pacemaker, and propose that retinal phase shifts to light provide a critical input signal.     

Spinal ganglion neurons of rats--a model for the study of the central serotonin receptors

Application of 5-hydroxytryptamine (5-HT) (3 x 10(-5) M) on the rat lumbar dorsal ganglia (RDG) induced membrane depolarization with increased input resistance in 30% of neurons, hyperpolarization with decreased input resistance in 30% of neurons and mixed responses in 40% of neurons. Methysergide and amitriptyline (10(-6) M) blocked depolarizing but not hyperpolarizing effects of 5-HT. Propranolol (3 x 10(-6) M) was inactive in respect to both 5-HT responses. 5-HT depolarizing responses of RDG neurons were mediated by 5-HT2 receptors activation and decreased membrane potassium conductivity; 5-HT hyperpolarizing responses were mediated by 5-HT1A receptor activation and increased potassium conductivity. RDG neurons seem to be an interesting model for the investigation of central 5-HT receptor mechanism.     

A study of the crustacean axon repetitive response. 3. A comparison of the effect of veratrine sulfate solution and potassium-rich solutions

The crustacean single nerve fiber gives rise to trains of impulses during a prolonged depolarizing stimulus. It is well known that the alkaloid veratrine itself causes a prolonged depolarization; and consequently it was of interest to investigate the effect of this chemically produced depolarization on repetitive firing in the single axon and compare it with the effect of depolarization by an applied stimulating current or by a potassium-rich solution. It was found that veratrine depolarization, though similar in some respects to a potassium-rich depolarization of depolarizing current effect, was in many respects quite different. (1) At low veratrine concentration, less than 1 Mg%, the negative after potential following a spike action potential was prolonged and augmented. At higher concentrations or after a long period of time, veratrine caused a prolonged steady state depolarization of the membrane, the “veratrine response”. The prolonged plateau depolarization response could be elicited with or without an action potential spike by a short or long duration stimulating pulse, but only if the veratrine depolarization was prevented or offset by an applied conditioning hyperpolarizing inward current. (2) The “veratrine response” resembled the potassium-rich solution response in the plateau-like contour of the depolarization and the very low membrane resistance during this plateau phase. Like the potassium response, it was possible to obtain a typical hyperpolarizing response with an inwardly directed current pulse if applied during the plateau phase. During the negative after potential augmented with veratrine, however, this hyperpolarizing response was not observed. (3) In contrast to the potassium response, however, the “veratrine response” is intimately associated with the sodium concentration in the external medium. The depolarization in millivolts is linearly related to the log of the concentration of external sodium. Moreover, during veratrine action there is a continuous and progressive inactivation of the sodium mechanism which ultimately terminates repetitive firing and abolishes the spike action potential. Then even with conditioning hyperpolarization only the slow response may be elicited in veratrine, occasionally with a spike superimposed if sodium is present, but without repetitive firing. (4) It is concluded that veratrine action is the result of a chemical or metabolic reaction by the alkaloid in the membrane. It is suggested that veratrine may inhibit the sodium extrusion mechanism, or may itself compete for sites in the membrane with calcium and/or sodium. This explains the inhibiting effect of high calcium, the abolition of the “veratrine response” with low temperature and high calcium combined and the progressive inactivation of the sodium system.     

Electrophysiological Evidence for Intrinsic Pacemaker Currents in Crayfish Parasol Cells

I used sharp intracellular electrodes to record from parasol cells in the semi-isolated crayfish brain to investigate pacemaker currents. Evidence for the presence of the hyperpolarization-activated inward rectifier potassium current was obtained in about half of the parasol cells examined, where strong, prolonged hyperpolarizing currents generated a slowly-rising voltage sag, and a post-hyperpolarization rebound. The amplitudes of both the sag voltage and the depolarizing rebound were dependent upon the strength of the hyperpolarizing current. The voltage sag showed a definite threshold and was non-inactivating. The voltage sag and rebound depolarization evoked by hyperpolarization were blocked by the presence of 5–10 mM Cs²⁺ ions, 10 mM tetraethyl ammonium chloride, and 10 mM cobalt chloride in the bathing medium, but not by the drug ZD 7288. Cs⁺ ions in normal saline in some cells caused a slight increase in mean resting potential and a reduction in spontaneous burst frequency. Many of the neurons expressing the hyperpolarization-activated inward potassium current also provided evidence for the presence of the transient potassium current I_A, which was inferred from experimental observations of an increased latency of post-hyperpolarization response to a depolarizing step, compared to the response latency to the depolarization alone. The latency increase was reduced in the presence of 4-aminopyridine (4-AP), a specific blocker of I_A. The presence of 4-AP in normal saline also induced spontaneous bursting in parasol cells. It is conjectured that, under normal physiological conditions, these two potassium currents help to regulate burst generation in parasol cells, respectively, by helping to maintain the resting membrane potential near a threshold level for burst generation, and by regulating the rate of rise of membrane depolarizing events leading to burst generation. The presence of post-burst hyperpolarization may depend upon I_A channels in parasol cells.     

Acute and phase-shifting effects of ocular and extraocular light in human circadian physiology

Light can influence physiology and performance of humans in two distinct ways. It can acutely change the level of physiological and behavioral parameters, and it can induce a phase shift in the circadian oscillators underlying variations in these levels. Until recently, both effects were thought to require retinal light perception. This view was challenged by Campbell and Murphy, who showed significant phase shifts in core body temperature and melatonin using an extraocular stimulus. Their study employed popliteal skin illumination and exclusively considered phase-shifting effects. In this paper, the authors explore both acute effects and phase-shifting effects of ocular as well as extraocular light. Twelve healthy males participated in a within-subject design and received all of three light conditions--(1) dim ocular light/no light to the knee, (2) dim ocular light/bright extraocular light to the knee, and (3) bright ocular light/no light to the knee--on separate nights in random order. The protocol consisted of an adaptation night followed by a 26-h period of sustained wakefulness, during which a 4-h light pulse was presented at a time when maximal phase delays were expected. The authors found neither immediate nor phase-shifting effects of extraocular light exposure on melatonin, core body temperature (CBT), or sleepiness. Ocular bright-light exposure reduced the nocturnal circadian drop in CBT, suppressed melatonin, and reduced sleepiness significantly. In addition, the 4-h ocular light pulse delayed the CBT rhythm by -55 min compared to the drift of the CBT rhythm in dim light. The melatonin rhythm shifted by -113 min, which differed significantly from the drift in the melatonin rhythm in the dim-light condition (-26 min). The failure to find immediate or phase-shifting effects in response to extraocular light in a within-subjects design in which effects of ocular bright light are confirmed strengthens the doubts raised by other labs of the impact of extraocular light on the human circadian system.