INTRODUCTION

TOP ABSTRACT INTRODUCTION GENERAL METHODS SPECIFIC EXPERIMENTS DISCUSSION REFERENCES

CIRCADIAN RHYTHMS are a common feature of physiological systems and provide organisms with an internal temporal framework for coordinating physiological processes with periodic environmental events (23, 24). To be useful for predicting periodic events, however, circadian rhythms must be entrained or synchronized to environmental time. The daily light-dark cycle is the major environmental signal that entrains endogenous circadian rhythms to the environmental day. The experiments described here focus on several aspects of the sensitivity of the photic entrainment pathway in the golden hamster using a functional behavioral assay. Specifically, these experiments examine the changes in the sensitivity and responsiveness of this visual pathway after prior stimulation.

The golden hamster has been a model for studying the effects of light on mammalian circadian rhythms. The relative insensitivity of the hamster's photic entrainment pathway and it's ability to integrate light input over time both suggest that this visual pathway has evolved to transmit the light information for circadian entrainment without responding to photic "noise" in the environment that may disrupt circadian entrainment (20, 30). The ability to integrate light input over quite long durations of time suggests that light adaptation may not decrease the sensitivity of this pathway in a manner that is common in other visual pathways. Although numerous experiments have examined the sensitivity of the hamster photic entrainment pathway using single pulses, few studies have carefully examined the photic sensitivity to two or more light pulses or investigated how the photic sensitivity changes after an initial stimulus.

We have performed a series of three experiments to examine the visual sensitivity of the photic entrainment pathway to multiple pulses. First, we determined whether this visual system light adapts after prior stimulation. Second, we sought to determine whether response saturation completely blocks additional phase shifts of the pacemaker. Finally, we attempted to determine whether the number of light/dark transitions within a series of light stimuli affects the response of the photic entrainment pathway. Together these experiments support the hypothesis that the photic entrainment pathway in the hamster is a physiologically unique visual pathway and quite distinct from "image forming" visual pathways that mediate mammalian pattern vision. The hamster circadian pacemaker and the visual pathway that subserves it seem to integrate even multiple distinct light pulses delivered over extended durations of up to 1 h and respond to these pulses as a single stimulus.

	GENERAL METHODS

TOP ABSTRACT INTRODUCTION GENERAL METHODS SPECIFIC EXPERIMENTS DISCUSSION REFERENCES

Male golden hamsters [Mesocricetus auratus, Lak:LVG (SYR); 3-5 wk of age] were group housed in a 24-h light-dark cycle (14 h light, 10 h darkness). Illuminance inside the cages during the light phase was adjusted to ~250 lx (lumen/m²) using General Electric "cool white" fluorescent tubes (model F40CW). After at least 2 wk, hamsters were moved to individual cages equipped with running wheels and microswitches to monitor wheel running activity as described previously (20). After 7 days, the light-dark cycle was discontinued and the hamsters were kept in constant darkness. On the 7th day of darkness, a stimulus was delivered to each hamster at the appropriate phase of the rhythm. After stimulation, each hamster was returned to constant darkness for 2 wk, and the steady-state phase shift was measured.

Phase advances of the running wheel activity rhythm at circadian time 19 were used to measure the light sensitivity of the circadian system's phase-shifting mechanism after prior stimulation. Circadian time is determined by normalizing the endogenous period of an animal to 24 h using the onset of activity as a phase reference point (arbitrarily designated as circadian time 12 for the nocturnal hamster). The procedures for stimulating and measuring responses to light pulses have been detailed previously (20). The clock time for delivery of each light pulse was determined for each hamster on the day of stimulation. The mean, standard deviation (SD), and range of the actual circadian times of stimulus onset are reported for each experiment. The hamsters ranged from 6 to 12 wk of age at the time of photic stimulation and were used only once for these experiments.

Photic Stimulation

For stimulation, each hamster was transferred to a cylindrical (5.5 cm radius; 10.5 cm height) white-plastic stimulus chamber. All transfers were made without visible light using an infrared viewer (FJW Optical Systems, Palatine, IL). Irradiance inside the chamber was measured before and after each stimulus with a radiometer/photometer (model S350, probe 248, United Detector Technologies, Hawthorne, CA), with the probe inside the chamber centered 5 cm below the diffusing screen. The stimulus irradiance was the average of these two measurements, and the mean stimulus irradiance (±SD) is reported for each group of animals.

Data Analyses

(1)

Statistical comparisons between the responses of individual groups were made using ANOVA or Student's t-test. When more than two groups were involved in a test, the responses of the groups in question were compared using the Tukey-Kramer method (ANOVA). A significance level of P < 0.05 was used to determine all significant differences between mean responses. SYSTAT (version 4.0) was used to perform both the ANOVA and the post hoc tests.

Terminology

Light adaptation changes the sensitivity in other visual pathways within milliseconds after the onset of a stimulus (1). We have tested for light adaptation over a much longer time course because of two unique characteristics of the hamster photic entrainment pathway (20). First, this pathway is very insensitive to brief light pulses of 30 s or less. Second, this pathway can fully or partially integrate the total photons in a single stimulus over durations of 300 s or more. For these reasons, the present study generally used stimuli of 300-s durations. A limitation of these experiments, therefore, is that only light adaptation with a long time course (300 s to 1 h) can be detected using these stimulus parameters. Light-adapting changes with a time course of milliseconds to seconds or hours to days would not be detected using the methods in this study. Light adaptation that is "functionally significant" to the hamster phase-shifting mechanism, however, would be detected using our methods.

"Initial stimulus" will be defined as the first of two stimuli when the second stimulus is used to measure the responsiveness after a prior stimulus. The "response" to this initial stimulus is the response to this stimulus presented alone (as a control). The "increment" or "test stimulus" will be defined as a subsequent light pulse used to measure the responsiveness or sensitivity of the pathway after the initial pulse. The "total response" will be the response to both the initial and the test stimulus. The "increment response" will be defined as the additional response induced by a test stimulus (the response to the initial stimulus subtracted from the total response).

	SPECIFIC EXPERIMENTS

TOP ABSTRACT INTRODUCTION GENERAL METHODS SPECIFIC EXPERIMENTS DISCUSSION REFERENCES

Part I: Assessing Light Adaptation and Response Saturation

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Phase-advance responses to 300-s light pulses delivered without prior stimulation () and immediately after a subsaturating stimulus of 4.5 × 1014 photons/cm2 (). A: increment responses (means ± SE) to light pulses are plotted as a function of photons in test pulse. For animals not receiving prior stimulation (3-8 per group), open circles represent mean responses (±SE) and dotted line is equation 1 fitted to data using ALLFIT (20). For this curve Rmin = 6 min (not significantly different from 0); N = 0.6;  (half-saturation constant) = 9.3 × 1013 photons/cm2; Rmax = 114 min. For animals receiving the test stimulus immediately after a subsaturating stimulus (; 4-10 per group), the initial stimulus was 4.5 × 1014 photons/cm2 and alone induced a phase advance of 80 min. This response was subtracted from total response to determine increment responses to test pulse. Solid line is a fit of equation 1 to data as described in SPECIFIC EXPERIMENTS. For this curve, Rmin = 3 min; N = 3.9;  = 6.3 × 1015 photons/cm2; and Rmax = 33 min. Maximum increment response and half-saturation constant were both significantly different from those measured without prior stimulation. B: increment responses (means ± SE) to light from A plotted as a function of total photons in both initial and test stimuli. , Mean responses for groups not receiving prior stimulation; , animals receiving an initial stimulus of 4.5 × 1014 photons/cm2, which induced a phase advance of 80 min in control animals. Dotted and solid lines are fits of equation 1 to data sets as described. Parameters for fit for animals receiving an initial stimulus are Rmin = 80 min; N = 0.5;  = 1.4 × 1014 photons/cm2; Rmax = 33 min. Value for  was not different from that measured for stimuli delivered without prior stimulus.

For animals receiving two light pulses, the total phase advance and the phase advance attributable to the test stimulus (the increment-phase advance) were determined to measure the system's light sensitivity after the initial stimulus. To determine the parameters of the function describing the stimulus-response relationship, ALLFIT was used to statistically fit equation 1 to the data. Together these experiments quantify the changes in sensitivity that occur after an initial stimulus.

The appearance of specific changes in the stimulus-response curve during light adaptation is dependent on the plotting convention used to plot the stimulus-response data (1, 21, 31). After an initial stimulus, the half-saturation constant for the stimulus response curves appears to shift to the right during light adaptation if the data are plotted as total stimulus vs. total response or increment response vs. increment stimulus. The apparent half-saturation constant may also shift without light-adaptive changes if the responses are plotted as a function of the increment stimuli. To circumvent this problem, we first plot the increment response as a function of the photons in the test stimulus to determine the minimum and maximum response of the curve using the ALLFIT curve-fitting program. Once these values are estimated, the total responses are plotted as a function of the total stimulus using the minimum and maximums for the curve determined in the initial fit. The resulting analysis compared the half-saturation constants and slopes of the curves to determine whether these parameters had changed significantly.

EXPERIMENT 1. To determine the light sensitivity immediately after an initial stimulus, test pulses were presented to five groups of hamsters (4-10 per group) that received a stimulus (300 s) immediately before the test stimulus. The initial stimulus was started at circadian time 19 [19:04 ± 16 min (mean ± SD); range = 18:28-19:32]. The irradiance of the initial stimulus was 1.5 (±0.06) × 10¹² photons · cm² · s¹ (4.5 × 10¹⁴ total photons/cm²) and, on the basis of the stimulus-response curve for single pulses, was estimated to induce a phase advance of ~75 min when delivered alone (see Fig. 1). As a control, an additional group of animals (n = 5) received only the first stimulus. For these experiments, test stimuli (300 s) were substituted for the first stimulus by removing or adding neutral density filters to the light path of the stimulus. This substitution changed the irradiance of the initial stimulus to that of the test pulse. No interruption of the stimulus occurred during this substitution.

EXPERIMENT 2. The sensitivity to light 1 h after a prior stimulus was determined by delivering test stimuli to five groups of hamsters (5-7 per group; total n = 22) that had received a 300-s stimulus 1 h earlier (64 ± 7 min). The initial stimulus was delivered at circadian time 19 (19:04 ± 10 min; range = 18:53-19:22) and was composed of 1.5 (±0.02) × 10¹² photons/cm². The initial stimulus was delivered alone to one group to determine its affect without the test stimulus. The total phase advance and the increment response were measured to quantify the sensitivity of the system to light 1 h after this initial stimulus.

EXPERIMENT 3. The sensitivity was also measured immediately after a "weak" stimulus (a pulse that induced <25% of the maximum response). Test stimuli (300 s) were presented to groups of hamsters that also received a stimulus immediately before the test stimulus (4-8 per group; total n = 32). The initial stimulus was 300 s in duration and started at circadian time 19 (18:59 ± 8 min; range = 18:40-19:29). The irradiance of this initial pulse was 3.5 (±0.18) × 10¹⁰ photons · cm² · s¹ and was chosen to induce a phase advance of ~20 min when delivered alone. The initial stimulus was also delivered to two groups of hamsters to determine the phase shift induced without the test stimulus (n = 4 and 7 per group). The total responses and the increment responses were estimated for each animal to measure the sensitivity of the system after this initial low-irradiance stimulus.

Results.EXPERIMENT 1: SENSITIVITY MEASUREMENT IMMEDIATELY AFTER SUBSATURATING PHOTIC STIMULI. The responsive range of the photic entrainment pathway was reduced immediately after the initial stimulation (Fig. 1). The initial stimulus induced a phase advance of 80 ± 5 min when delivered without a test stimulus. The additional test stimuli induced phase advances that increased the magnitude of the total phase shift in those animals receiving both pulses. The largest average response to both pulses was 113 ± 13 min.

The average response to the initial pulse was subtracted from the total phase shift induced by both pulses to estimate increment responses. When increment responses are plotted as a function of the photons per square centimeter in the test stimuli (Fig. 1A), the minimum response (3 min) was not significantly different from the minimum response to pulses delivered without prior stimulation (ALLFIT). The slope of the curve (3.9) was also not different from the slope for the curve measured without prior stimuli. The half-saturation constant for the fit was 6.3 × 10¹⁵ photons/cm² and was significantly different from the one-half maximum for 300-s stimuli at circadian time 19 (ALLFIT; F = 24.65; df = 20). The maximum increment response after prior stimulation was only 33 min and was significantly smaller than that measured using single pulses at this phase (ALLFIT; F = 54.13; df = 20). Immediately after the first stimulus, therefore, there was an effective decrease in the response range available for the responses to the test stimuli. The decrease in the responsiveness range, however, can be almost entirely explained by the initial response to the test stimulus.

To compare the half-saturation constants for the stimulus-response curve measured immediately after prior stimulation and without prior stimulation, the increment responses were plotted as a function of the total number of photons in both the initial and the test stimuli (Fig. 1B). To analyze these data using ALLFIT, the minimum response for the Naka-Rushton curve fit to the data was held at 80 min (to correct for the response induced by the initial stimulus). The maximum was held constant at 33 min (the maximum increment response fit to the data as previously described). The half-saturation constant for the curve fit with these constraints was 1.4 × 10¹⁴ photons/cm², which was not different from that measured without prior stimulation. The slope (0.5) was also not significantly different. These data suggest that the half-saturation constant for the stimulus-response curve remained unchanged by the initial stimulation. There is no evidence for light adaptation (shifting of the stimulus-response curve on the stimulus irradiance axis).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Sensitivity to light 1 h after a subsaturating stimulus. Initial stimulus was 4.5 × 1014 photons/cm2 and induced a phase advance of 80 min when presented alone. Response to initial stimulus was subtracted from total response to determine increment responses. A: increment responses (means ± SE) to pulses delivered 1 h after initial stimulus are plotted as a function of photons in test stimuli (5-7 per group). Dashed line is stimulus-response curve measured without prior stimulation (from Fig. 1). Solid line is a fit of Naka-Rushton equation to the data (Rmin = 0 min; N = 0.4;  = 3 × 1016 photons/cm2; Rmax = 38 min). B: increment responses (means ± SE) to light pulses from Fig. 2A are plotted as a function of total photons on both initial and test stimuli. Dashed line is curve measured without prior stimulation (Fig. 1). Solid line is fit of equation 1 to data using ALLFIT (Rmin = 80 min; N = 0.2;  = 1.2 × 1013 photons/cm2; Rmax = 38 min). Half-saturation constant is not significantly different from that measured for stimuli delivered in constant darkness. Slope was significantly different slope of curve, describing responses to light without prior stimulation.

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View larger version (20K): [in this window] [in a new window]   Fig. 3.   Stimulus response curves for 300-s light pulses presented immediately after a "weak" initial stimulus. Initial stimulus was 1.1 × 1013 photons/cm2 and induced a average phase advance of 18 min. This response was subtracted from total response to determine increment responses. A: increment responses (means ± SE) to 300-s pulses delivered immediately after a prior stimulus are plotted as a function of total photons in test stimulus (4-8 animals/group). Dashed line is stimulus-response curve to pulses delivered without a prior stimulus (Fig. 1). Solid line is a fit of equation 1 to data points as described. Parameters for curve are Rmin = 4 min; N = 1.4;  = 2.1 × 1014 photons/cm2; Rmax = 78 min. Maximum increment response and one-half-saturation constant were significantly different from parameters measured without prior stimulation. B: increment responses (means ± SE) to light pulses measured immediately after 300-s initial stimulus (Fig. 3A) as a function of total photons on both initial and test stimuli. Dashed line is curve fit to responses without prior stimulation (Fig. 1). Solid line is equation 1 fit to data as described. Parameters for this curve are Rmin = 18 min; N = 0.6;  = 2.2 × 1014 photons/cm2; Rmax = 84 min. Half-saturation constant () was not significantly different from that measured without prior stimulation.

Part II: Responsiveness Measurement After a Saturating Stimulus

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An important control for this experiment was also performed. If the initial stimulus had induced a phase advance of 2 h, then the phase of the oscillator may be approximately circadian time 21 (circadian time 19 + 2 h = circadian time 21) at the time of the second pulse. To test the responsiveness of the hamster circadian system at this later phase without prior stimulation, a third group of hamsters (n = 6) was given a saturating pulse at circadian time 21 (21:06 ± 3 min). The irradiance of this 300-s light pulse was also 2.6 × 10¹⁴ photons · cm² · s¹.

RESULTS. The single light pulse delivered at circadian time 19 induced a steady-state phase advance of 113 ± 7 min (Fig. 4). The average phase shift induced in those hamsters that received a light pulse at circadian time 21 was 70 ± 4 min. Each of these saturating pulses alone induced the maximum possible shift at their respective phases of delivery (20). Animals that received a light pulse at circadian time 19 and again after 10 min of darkness displayed phase advances averaging 109 ± 5 min. This shift was not significantly different from the phase advances induced by single pulses delivered at circadian time 19. There was no significant additional response induced by the second light stimulus. It is important to note that the system was not less responsive to light simply because the phase-response curve had already shifted to a later phase (circadian time 21) of reduced responsiveness due to the initial pulse. The phase shift expected at circadian time 21 is ~70 min, and our data demonstrate that there was no additional response due to the second stimulus. This result, coupled with the results of Part 1, suggests that the circadian photic entrainment pathway of the golden hamster may only be capable of phase advancing ~2 h within a single circadian cycle. If an initial stimulus induces a maximum phase advance of ~2 h, then subsequent stimuli will probably not induce a phase advance. Similarly, an initial light pulse that induces a half-saturating phase shift probably reduces the maximum response to subsequent stimuli by this amount (see experiments 1-3).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Phase advances (means ± SE) of hamster circadian pacemaker induced by individual light pulses at circadian times (CT) 19 and 21 as well as by a pair of stimuli at CT 19. Average response to 2 pulses at CT 19 was not significantly different from response to a single saturating stimulus at this phase. ISI, interstimulus interval.

Part III: Responsiveness to Multiple Stimuli Containing Multiple Light/Dark Transitions

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Although the total duration of the light stimulation was equal for each group, the duration of the entire stimulus train (from the onset of the first stimulus to the offset of the last) was slightly different. For example, for the 3-s stimuli, (3 s/stimulus × 100 stimuli) + (3 s between stimuli × 99 interstimulus intervals) = 597 s of duration for the stimulus train. For the 30-s pulses, the total duration of the stimulus train was 570 s. None of the durations of stimulation exceeded 600 s of stimulation. As a control for these different stimulus durations, one group of hamsters (n = 8) received a stimulus of the same irradiance but with a duration of 600 s.

We also measured the responsiveness to multiple stimuli using a saturating total number photons in the stimulus train. For this test, a stimulus irradiance of 3.8 ± 0.01 × 10¹⁴ photons · cm² · s¹ was presented as either a single 300-s stimulus (n = 5) or as 100 stimuli (n = 6) of 3 s in duration. The total photons delivered in these stimulus trains was 1.1 × 10¹⁷ photons/cm².

RESULTS. The phase shifts induced by 8.1 × 10¹³ photons/cm² divided into different numbers of individual pulses were not significantly different from one another (Fig. 5). The average phase advance induced by the single 300-s pulse was 56 ± 3 min and was approximately one-half of the maximum response that can be induced by a 5-min pulse at this phase. The mean response to the 600-s stimulus of the same irradiance was 68 ± 6 min and was not significantly different from the response to the 300-s pulse. The responses to this total number of photons divided into 10, 100, or 1,000 pulses were 40 ± 4, 45 ± 6, and 49 ± 8 min, respectively.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Responsiveness to single pulses and trains of light pulses. Phase-shift responses are plotted for stimulus trains composed of different numbers of individual light pulses at circadian time 19. Each stimulus train was equivalent in irradiance and contained an equal number of photons. , Means ± SE of responses to a "half-saturating" stimulus (irradiance = 2.7 × 1011 photons · cm2 · s1; total photons = 8.1 × 1013 photons/ cm2) and are not significantly different from one another. , Means ± SE of responses to a "saturating" stimulus of 1.1 × 1017 photons/cm2 presented as 1 and 100 stimuli, and there is no significant difference between these points. There appears to be no effect of number of dark-light transitions or number of separate stimuli making up each train of pulses. Lines represent average overall phase advance induced by stimulus trains.

The phase advance to the total number of photons in the saturating stimulus of 1.1 × 10¹⁷ photons/cm² was also unrelated to the number of individual stimuli (or dark-to-light and light-to-dark transitions) in the stimulus train. The phase advance induced by the single 300-s pulse was 113 ± 8 min. The response to the train of 100 stimuli with the identical total number of photons was also 113 ± 8 min and not significantly different from the response to the single pulse (Student's t-test). Neither the number of light pulses in a stimulus train nor the number of dark/light transitions associated with the stimulus (at least within a duration 600 s) appear to be significant parameters of the stimulus for influencing the magnitude of phase advances.

	DISCUSSION

TOP ABSTRACT INTRODUCTION GENERAL METHODS SPECIFIC EXPERIMENTS DISCUSSION REFERENCES

In prior experiments, we defined the ability of the hamster circadian entrainment pathway to integrate the total photons in single light pulses (20). This pathway appears to be capable of integrating the total photons in single pulses for extended durations as large as 300 s or more (with a maximum sensitivity to 300-s pulses). Experiments outlined here extend our prior results to reveal how this photic entrainment pathway responds to multiple light pulses. These results demonstrate that the hamster photic entrainment pathway does not respond independently to multiple light pulses delivered within 1-h durations. Instead, this system appears to integrate the photons within even multiple pulses over durations of up to 1 h. This temporal integration of inputs appears to be accompanied by a saturation of the photic entrainment pathway (or specifically the phase-shifting mechanism) without evidence for adaptation-like changes in light sensitivity. These results define several intriguing and fundamental characteristics of the hamster circadian pacemaker and the visual pathway that carries light information to it. Knowledge of these characteristics is important for designing and interpreting results from future studies to dissect the cellular and molecular nature of the mammalian circadian clock and how visual information affects this cellular clock during entrainment.

Temporal Integration and Saturation Without Light Adaptation

View larger version (23K): [in this window] [in a new window]   Fig. 6.   A: total responses (means ± SE) and their associated best-fit curves (experiments 1 and 3) have been plotted as total phase shift (response to both first stimulus and test stimulus) as a function of number of photons in test stimulus. , Total responses from experiment 3 (weak initial stimulus: 1.1 × 1013 photons/cm2). , Total responses from experiment 1 (strong initial stimulus: 4.5 × 1014 photons/cm2). , Responses obtained without prior stimulation. Dashed and thin solid lines are best-fit curves (from experiments 3 and 1, respectively) adjusted to represent total response to both pulses. Thick solid line is equation 1 fit to responses measured without prior stimulation (from Fig. 1). B: curves represent a model for saturation of hamster photic entrainment pathway by light. Thick solid line represents a stimulus-response curve from animals receiving a single light pulse. Dotted line represents a curve expected after an initial stimulus of 1.1 × 1013 photons/cm2 that induced an initial 18-min response (Rmin = 18 min; N = 0.6;  = 9.3 × 1013 photons/cm2; Rmax = 114 min). Thin solid line represents a curve expected after an initial stimulus of 4.5 × 1014 photons/cm2 that induced an initial response of 80 min (Rmin = 80 min; N = 0.6;  = 9.5 × 1013 photons/cm2; Rmax = 114 min). Initial light pulse induces a phase shift that effectively reduces response available to second light stimulus. There is an obvious increase in Rmin with increasing levels of initial stimulation, whereas there is no increase in Rmax.

Light adaptation has been discussed in the context of circadian photic entrainment in several previous studies. It has been suggested that the circadian systems of animals may light adapt when maintained in constant light for long periods of time (7, 22, 33). Without some type of light adaptation, it is reasoned a circadian pacemaker would effectively "stop" during constant light because it would experience continuous phase delays in the delay region of the phase response curve. Our results suggest that response saturation may be responsible for the reduced responsiveness of the pacemaker in constant light. The pacemaker may be responsive to light until the response saturates (the maximum shift has been achieved). After saturation, the pacemaker may be insensitive to additional light and continue its forward motion out of the phase delay region of the phase response curve. Additional light would be effectively "ignored" by the saturated pacemaker.

If light adaptation cannot be detected in this functional output of the hamster circadian pacemaker, it is unlikely that light adaptation changes the sensitivity at any point along the pathway between the photoreceptor to the pacemaker's phase-shifting mechanism over the durations (300 s to 1 h) tested here. On the other hand, light adaption may be occurring with a time course that is much slower or more rapid than our experiments can detect. For example, if light adaptation occurred in <1 s, a time course for light adaptation measured for the rat electroretinogram (12), then the resolution of our test for light adaptation may be inadequate to identify true light adaptation. If light adaptation occurred rapidly, then the 300-s "dark-adapted" stimulus-response curve measured without prior stimulation (Fig. 1) may actually represent already light-adapted responses. However, because this photic entrainment pathway is relativity insensitive to brief stimuli (20) it is unlikely that adaptation occurring with time course of <1 s would be functionally important for changing the sensitivity of this circadian oscillation to light. Light adaptation, therefore, does not appear to be functionally important for the induction of phase advances in hamsters.

The absence of functional light adaptation in this pathway may provide an additional means of physiologically identifying the elements (photoreceptors and ganglion cells) of the neural pathway that delivers photic information from the eye to the pacemaker. For example, few retinal ganglion cells possess the physiological properties that are required of this photic pathway. "Luminance units" (4) and "tonic W-cells" (29) appear to possess physiological properties that would enable these cells to mediate the affects of light to the mammalian circadian pacemaker. Luminance units respond to maintained illumination with a sustained change in firing rate and make up a very small proportion of the retinal ganglion cells in the cat (<1%). Interestingly, these cells also have a relatively high threshold for light-induced changes in mean firing rate compared with that measured for other retinal ganglion cells. This high threshold for stimulation of luminance cells is similar to the high thresholds measured for electrophysiological responses and phase shifts in the photic entrainment pathway (14, 17-20). Interestingly, there is little evidence for either light adaptation or temporal integration in light-induced electrophysiological responses recorded from the hypothalamic suprachiasmatic nucleus (SCN), which is the site of the mammalian circadian pacemaker. Many cells in this nucleus demonstrate a transient response component at the onset of a photic stimulus, but a large sustained component is also present (14, 18). In addition, the photic sensitivity of SCN neurons is not reduced by prior illumination in a manner consistent with light adaptation (13, 18, 19).

Implications of Temporal Integration for Circadian Systems

Single light pulses have been found to induce a maximum phase advance of 2-3 h in the golden hamster in a number of studies (8, 9, 30), and our current results suggest that this maximum may also exist for multiple light pulses delivered within durations up to 1 h. DeCoursey (10) has also suggested that a response ceiling may also limit phase delay responses to multiple pulses in the flying squirrel. Larger light-induced shifts are observed in hamsters after pretreatments such as administration of 5-hydroxytryptamine 1A receptor antagonists (25) or dim constant light (Nelson and Takahashi, unpublished). In tau-mutant hamsters, pretreatments of constant darkness for long durations (49 days) causes the light responsiveness to increase from ~2 to 12 h or more (28). Because two or more pulses falling outside the limit for temporal integration may induce larger total shifts (5), it is possible that pretreatments that cause the photic entrainment pathway to be more responsive may act by shortening the duration of temporal integration within this pathway in addition to increasing the sensitivity of the pathway to irradiance.

In the future, it will be interesting to use the responsiveness to multiple light pulses to dissect the mechanism and function of the mammalian photic entrainment pathway. For example, the apparent insensitivity to a second pulse after a saturating stimulus probably represents response saturation at the level of the phase-shifting mechanism and not saturation of the retinal visual system that subserves it. Behavioral phase shifts have been compared with electrophysiological SCN responses using trains of light pulses (18). Stimulus trains of 2, 4, and 8 light pulses (1-min pulses of light, each separated by 1 min of darkness) induced increasing magnitudes of phase advances at circadian time 18. The total shifts observed, however, were smaller than predicted by simple linear addition of the responses to individual pulses. This result is consistent with the evidence for stimulus integration and response saturation of the phase-shifting mechanism demonstrated in our study. Interestingly, electrophysiological results suggested that the responsiveness of SCN neurons to retinal illumination did not change over the course of a stimulus train. SCN neurons responded similarly to each pulse of the stimulus train. These findings suggest that the decreased responsiveness after an initial stimulus probably originates "downstream" from the electrophysiological responses of SCN neurons to light.

One mechanism that may account for the integrating ability of this system and the maximum response ceiling may be the "motion" of the circadian pacemaker itself. The pacemaker presumably moves through the phase advance region in the late subjective night with a time course that is determined by the normal forward motion of the clock. The time course of this movement may also change due to the phase advance of the pacemaker by light. The motion of the pacemaker through the subjective night could act as a sort of "memory" for light stimulation of the pacemaker and allow for the temporal integration of single and multiple stimuli presented to the animal. In addition, this movement through the phase advance region of the phase-response curve may also limit the maximum response to light stimuli delivered during this time because the pacemaker could be shifted (by light) out of the region of responsiveness and into an unresponsive region during the subjective day. On the other hand, our results in experiment 4 show that even though the pacemaker should be sensitive to light after the initial light pulse (whether the second stimulus is delivered at circadian time 19 or 21), the second light pulse is ineffective at inducing an additional phase shift. There is clearly more to the response saturation than the motion of the pacemaker through the phase advance region. The pacemaker's motion could allow temporal integration of stimuli, but the maximum response ceiling of 2 h is not due to the simple movement of the pacemaker out of the phase advance region of the phase-response curve. Another mechanism must be reducing the responsiveness of the photic entrainment pathway to subsequent simulation.

Finally, the behavioral results presented here also reveal a great deal about the nature of molecular and cellular events that must mediate the effects of light to the clock. At the cellular level, it is interesting to speculate that photic stimulation causes some cellular process (such as the induction of an intracellular signal) to be less responsive to stimulation after an initial photic stimulus. Recent studies have demonstrated that visual stimulation induces the expression of immediate early genes (3, 15, 16, 26) and putative clock genes (2, 27) within SCN neurons. The most recent of these results have demonstrated that mPer1 message is induced in mouse SCN neurons by light stimulation that also causes phase shifts in the mouse circadian pacemaker. The photic threshold for inducing mPer1 message is very similar to the threshold for inducing phase delays in the mouse behavioral rhythm (27). There is also a rhythm of mPer1 production in constant darkness, which can be shifted by a light pulse (2, 27). Taken together, these results suggest that mPer1 may be a central component of the circadian oscillation mechanism and also mediate the effects of light to the circadian oscillator in the mouse. It is possible that a gene such as mPer1 may play a role in the temporal integration of photic information observed for single and multiple light pulses. The kinetics for light induction of mPer1 or a similar gene could explain the extended time course for integration of single and multiple photic pulses observed in the hamster entrainment pathway. In mouse, mPer1 message is rapidly induced by light at circadian time 16, and the peak of mPer1 is reached ~60 min after stimulus initiation. Levels return to near baseline by 180 min after stimulus onset. This time course for mPer1 induction may explain the time course of 300 s to 1 h for temporal integration of single light pulses observed for the hamster photic entrainment pathway (20). The kinetics of mPer1 induction by light may also explain the apparent integration of multiple light pulses observed in the present study. Additional studies are needed to determine whether the characteristics of light sensitivity for mPer1 induction may explain the unique characteristics of response saturation and temporal integration of multiple stimuli observed in behavioral studies of the mammalian circadian system. Behavioral studies continue to show that the photic entrainment pathway, which mediates the effects of light to the mammalian circadian pacemaker, is functionally very different from pathways that mediate pattern vision. These unique functional characteristics, in turn, provide useful tools to further dissect the cellular and molecular mechanism of the mammalian photic entrainment pathway.