- Email: [email protected]
Resetting the rat circadian clock by ultra-short light flashes
Neuroscience Letters 261 (1999) 159–162
Resetting the rat circadian clock by ultra-short light flashes Andreas Arvanitogiannis, Shimon Amir* Center for Studies in Behavioral Neurobiology, Department of Psychology, Concordia University, 1455 de Maisonneuve Blvd West, Montreal, Quebec, Canada H3G 1M8 Received 9 November 1998; received in revised form 21 December 1998; accepted 21 December 1998
Abstract We examined the effects that ultra-brief, intense, light flashes have on the rat circadian clock, the suprachiasmatic nucleus of the hypothalamus (SCN). We found that as few as five intense flashes, each 10-ms in duration (1 per s), can produce both phase shifts in free-running activity rhythms and Fos expression in the SCN in rats kept in constant darkness. After pre-exposure to such flashes, phase shifts in response to a continuous light pulse delivered 2 h later were potentiated, but Fos expression in the SCN was decreased as following pre-exposure to continuous light. These results show that flashes induce behavioral and cellular effects indicative of clock resetting similar to those induced by light stimuli of longer duration. Extremely brief but intense, light stimuli may be much more important to clock resetting than had been previously known. 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Circadian rhythms; Phase shift; Suprachiasmatic nucleus; Fos
Stable entrainment of mammalian circadian rhythms requires daily photic resetting of the circadian clock located in the suprachiasmatic nucleus of the hypothalamus [9,12,16]. Although it is known that the effectiveness of light as a resetting stimulus is influenced by both duration and intensity, it has long been considered that the pathway mediating resetting by light is insensitive to very brief light stimuli, regardless of their intensity [4,7–10,12]. To further study this issue, we investigated the effect that ultrabrief, intense, light flashes have on the circadian clock in rats. Clock resetting was assessed using both a behavioral measure, phase shifts in free-running activity rhythms [3], and a cellular measure, Fos expression in the suprachiasmatic nucleus [5]. Male Wistar rats (300–350 g) were housed individually in plastic cages equipped with Nalgene running wheels and had free access to food and water. The cages were placed inside ventilated, light- and sound-tight enclosures. At the start of the experiment the rats were placed in constant darkness and their running-wheel activity was recorded
* Corresponding author. Tel.: +1-514-8482188; fax: +1-5148482817; e-mail: [email protected]
0304-3940/99/$ - see front matter PII: S03 04-3940(99)000 21-X
using Dataquest software (Mini Mitter, Sunriver, OR). To assess the effects of flashes on phase shifts in free-running activity rhythms, animals were assigned to one of three combinations of stimuli: (1) 45 × 10-ms flash/s delivered at a rate of 3 flash/s (total light duration = 450 ms); (2) 15 × 10-ms flash/s delivered at a rate of 1 flash/s (total light duration = 150 ms); (3) 5 × 10-ms flash/s delivered at a rate of 1 flash/s (total light duration = 50 ms). All animals were presented with these stimulus combinations at circadian time (CT) 13 (CT12 denotes time of activity onset). Light flash/s were generated by a Grass photic stimulator (PS22) placed on the side of the Plexiglas cages, at a distance of about 25 cm from their center (illuminance: ~2.4 × 106 lux; luminance: ~1.9 × 106 cd/m2; these numbers were derived from the nominal characteristics of the photic stimulator provided by the manufacturer; the perceived intensity of the flashes varied as a function of the position the subjects occupied in their cage as well as the differing degrees of approach or avoidance behavior they exhibited during the presentation of the flashes). The animals continued to free-run for at least 8 days and phase shifts were determined from graphic records of wheel-running behavior (actograms) by two independent observers. Phase shifts in the activity rhythms were calculated as the
1999 Elsevier Science Ireland Ltd. All rights reserved.
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A. Arvanitogiannis, S. Amir / Neuroscience Letters 261 (1999) 159–162
difference between the eye-fitted lines connecting the time of onset of activity for a period of seven days before and after exposure to the flashes and/or continuous light [1,3]. To assess the effect of flashes on Fos expression in the SCN, the experiment was repeated after randomly reassigning the rats to the above three conditions. One hour after presentation of the stimuli (CT14), the rats were injected with a lethal dose of sodium pentobarbital (100 mg/kg) and perfused with 300 ml of 0.9% NaCl followed by 300 ml 0.1 M phosphate buffer containing 4% paraformaldehyde. Fos immunohistochemistry was carried out on free floating coronal brain sections (50 mm) using a mouse monoclonal antibody raised against residues 4–17 of the 55 kDa Fos protein N-terminal (1:8000; NCI/BCB Repository, Quality Biotech, Camden, NJ), as previously described [1]. Estimates of the number of nuclei expressing Fos were made from cell counts performed on serial sections taken from the SCN using a computerized image analysis system and the NIH Image software. Mean cell counts on one side of the SCN on the four sections exhibiting the highest number of Fos immunoreactive cells were taken from each animal. As shown in Fig. 1, exposure to each of the combinations of 10-ms flashes at CT13 resulted in both phase shifts in running wheel activity and Fos expression in the SCN. Although the phase shifts induced by each of the three combinations of flashes were of similar magnitude (Fig. 1a), progressively less Fos-IR was seen in the SCN as the total light exposure decreased (Fig. 1b). The graded Fos response argues against the possibility that the effects of the flashes were due to aberrant over-activation of the retina or the photic-entrainment pathway. Fig. 2a shows an example of a phase shift that resulted from exposure to five 10-ms flashes. Fig. 3 shows Fos-IR cells in the SCN in response to the three combinations of flashes. Notice that Fos was expressed predominantly in the ventrolateral portion of the SCN, the terminal region of retinal afferents. In control animals that did not receive light flashes, or that were exposed to the flashes at CT6, Fos expression in the SCN was negligible (,six Fos-IR cells). Light-induced phase shifts of behavioral rhythms in rodents are known to be positively correlated with the induction of Fos in the SCN when their respective magnitudes are below ceiling, and it has been suggested that Fos may mediate light induced phase shifts [2,5,6,11,13,17]. It is somewhat surprising, therefore, that the magnitude of the flash-induced phase shifts was similar across conditions, whereas Fos expression in the SCN decreased with decreasing light exposure. The apparent dissociation between the magnitude of flash-induced phase shifts and the magnitude of Fos expression could imply that such brief flashes exert their influence on the clock via mechanisms different from those involved in the effect of a continuous light pulse. As a first step toward evaluating this possibility we examined the effect that pre-exposure to flashes or continuous light had on phase shifts and Fos expression in response to a subsequently delivered continuous light pulse. For this experi-
ment, three groups of animals were maintained in constant darkness and were given, at CT13, either five 10-ms flashes (1 flash/s), a continuous light pulse delivered via an overhead cool white fluorescent lamp (illuminance: ~300 lux; duration: 75 s) or no light exposure. At CT15 all animals were exposed to the continuous light pulse. It can be seen from Fig. 1d that when animals were exposed to two continuous light pulses, one at CT13 and another at CT15, Fos expression in the SCN measured 1 h after the CT15 pulse was diminished compared to that seen in animals exposed only to a single continuous light pulse at CT15. Likewise, Fos expression in the SCN induced by the continuous light pulse at CT15 was decreased in animals pre-exposed to the flashes a CT13 (Fig. 1d). Thus, exposure to the flashes appears to modulate the Fos response in the SCN in a manner similar to that of the continuous light. The most parsimonious explanation for the decline in Fos staining in response to the second light pulse is that after the first exposure to light, whether flashed or continuous, the cells that expressed Fos in the SCN were, at least in part, unable
Fig. 1. (a,b) The effects of presenting, at CT13, one of three combinations of 10-ms flashes (45 flashes delivered at a rate of 3 flashes/s; 15 flashes delivered at a rate of 1 flash/s; five flashes delivered at a rate of 1 flash/s) on phase shifts in free-running activity rhythms (n = 4 per group) and Fos expression in the SCN (n = 4 per group). (c,d) The effects of pre-exposure at CT13 to either flashes or continuous light on phase shifts in free-running activity rhythms (n = 8 per group) and Fos expression in the SCN (n = 4 per group) in response to a continuous light pulse delivered at CT15. L, 75 s of continuous light delivered at CT15; FL, five flashes delivered at a rate of 1 flash/s at CT13 followed by 75 s of continuous light at CT15; LL, 75 s of continuous light delivered at both CT13 and CT15. All data are expressed as mean ± SEM. *P , 0.05, one-way analysis of variance/Scheffe post-hoc test.
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to do so again because of the negative feedback that Fos exerts on its own transcription [14]. In contrast to the effect on Fos expression, pre-exposure
Fig. 3. Representative photomicrographs showing Fos-labeled cells in one side of the SCN in response to: (a) 45 × 10-ms flashes delivered at a rate of 3 flashes/s; (b) 15 × 10-ms flashes delivered at a rate of 1 flash/s; (c) 5 × 10-ms flashes delivered at a rate of 1 flash/s. Scale bar, 100 mm.
Fig. 2. Representative actograms showing the free-running activity rhythms (wheel running) of animals that received: (a) 5 × 10-ms flashes delivered at a rate of 1 flash/s (CT13); (b) a 75 s continuous light pulse (CT15); (c) 5 × 10-ms flashes delivered at a rate of 1 flash/ s (CT13) and a 75 s continuous light pulse (CT15). The horizontal lines in each actogram represent a single 24-hour period; the vertical marks indicate periods of activity of at least 10 wheel revolutions/10 min. Successive days are plotted from top to bottom. The open triangles indicate the day on which the flashes and/or continuous light were delivered. Phase-shifts in the activity rhythm are demonstrated by the presence of a difference between the eye-fitted lines connecting the onset of activity for a period of 7 days before and after exposure to the light stimuli.
to the flashes at CT13 enhanced the phase shift produced in response to the continuous light pulse presented at CT15 (Figs. 1c and 2b,c). As can be seen in Fig. 1c, the magnitude of the phase shift in animals exposed to the flashes at CT13 and then to the continuous light at CT15 was greater than that seen in animals exposed only to the continuous light pulse given at CT15, and not different from that seen in animals exposed twice to the continuous light stimulus. There are at least two ways to account for this additive effect. First, the temporally distinct flashes and continuous light may have engaged the mechanisms involved in generating the phase shifts on both occasions. If so, the potentiated phase shift is an outcome of combining two discretely produced phase shifts. As mentioned before, Fos expression in response to the continuous light stimulus at CT15 was suppressed after pre-exposure to flashes. Similarly, the phase shift produced in response to the continuous light
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pulse may have also been suppressed. However, although suppressed, the phase shift induced by the continuous light combined with the one induced by the flash could well have resulted in the observed potentiated phase shift. Another possibility is that the clock integrates all photic information that reaches it over a relatively prolonged period (at least 2 h), and that only after that time is the phase shifting mechanism engaged. In this latter case the potentiated phase shift is an outcome of the total input to the clock integrated over time. Clearly, however, the integration is not expressed as increased Fos in the SCN. The diminished Fos response may suggest that the expression of Fos, though self-limiting, serves as a gateway for integration of additional photic input within some, yet to be specified, temporal interval. It remains to be determined whether the refractoriness in Fos expression is related to ongoing integration within the pacemaker. If so, the temporal interval between two light pulses at which Fos expression in response to the second pulse becomes as strong as if the second pulse were delivered alone, would define the temporal window of integration and provide a direct measure of the time it takes for a phase shift to be completed. To conclude, the present study demonstrates that as few as five intense, 10-ms light flashes delivered within 5 s produce phase shifts in free-running rhythms and induce Fos expression in the SCN. These results extend findings from a recent study demonstrating that repeated exposure (but not a single exposure) to 2-ms light pulses induced phase shifts in freerunning activity rhythms in mice [15]. Contrary to previous suggestions [10], extremely brief light stimuli in the environment that could arise, for example, from lightning, may influence the circadian clock and cannot be considered mere ‘noise’. It is worth noting that the flashes in the present study had a significantly lower luminance value than lightning [10] (1.9 × 106 cd/m2, flashes VS. 8 × 1010 cd/m2, lightning). The double light-pulse experiments provide strong support for the idea that flashes can exert strong influence on clock phase resetting. Phase shifts produced by the flashes summed with those produced by a continuous light pulse delivered 2 h later. Furthermore, preexposure to flashes or continuous light suppressed the induction of Fos in the SCN in response to the subsequently administered continuous light pulse. The fact pre-exposure to either flashes or continuous light similarly modulate the effects of a subsequent continuous light pulse suggests that both stimulus events affect clock resetting via a common mechanism. We thank Jane Stewart for helpful comments on the manuscript. This work was supported by grants from the
Medical Research Council of Canada, the Natural Sciences and Engineering Research Council of Canada, and the Fonds pour la Formation de Chercheurs et l’Aide a la Recherche (Quebec). [1] Amir, S. and Stewart, J., Resetting of the circadian clock by a conditioned stimulus, Nature, 379 (1996) 542–545. [2] Aronin, N., Sagar, S.M., Sharp, F.R. and Schwartz, W.J., Light regulates expression of a Fos-related protein in rat suprachiasmatic nuclei, Proc. Natl. Acad. Sci. USA, 87 (1990) 5959–5962. [3] Daan, S. and Pittendrigh, C.S., A functional analysis of circadian pacemakers in nocturnal rodents. II. The variability of phase response curves, J. Comp. Physiol. A, 106 (1976) 253–266. [4] Groos, G.A. and Meijer, J.H., Effects of illumination on suprachiasmatic nucleus electrical discharge, Ann. N.Y. Acad. Sci., 453 (1985) 134–146. [5] Kornhauser, J.M., Mayo, K.E. and Takahashi, J.S., Light, immediate-early genes, and circadian rhythms, Behav. Genet., 26 (1996) 221–240. [6] Kornhauser, J.M., Nelson, D.E., Mayo, K.E. and Takahashi, J.S., Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus, Neuron, 5 (1990) 127–134. [7] Meijer, J.H., Watanabe, K., Schaap, J., Albus, H. and De´ta´ri, L., Light responsiveness of the suprachiasmatic nucleus: longterm multiunit and single-unit recordings in freely moving rats, J. Neurosci., 18 (1998) 9078–9087. [8] Meijer, J.H. and Rietveld, W.J., Neurophysiology of the suprachiasmatic circadian pacemaker in rodents, Physiol. Rev., 69 (1989) 671–707. [9] Morin, L.P., The circadian visual system, Brain Res. Rev., 67 (1994) 102–127. [10] Nelson, D.E. and Takahashi, J.S., Sensitivity and integration in a visual pathway for circadian entrainment in the hamster (Mesocricetus auratus), J. Physiol., 439 (1991) 115–145. [11] Rea, M.A., Light increases Fos-related protein immunoreactivity in the rat suprachiasmatic nuclei, Brain Res. Bull., 23 (1989) 577–581. [12] Roenneberg, T. and Foster, R.G., Twilight times: light and the circadian system, Photochem. Photobiol., 66 (1997) 549–561. [13] Rusak, B., Robertson, H.A., Wisden, W. and Hunt, S.P., Light pulses that shift rhythms induce gene expression in the suprachiasmatic nucleus, Science, 248 (1990) 1237–1240. [14] Sassone-Corsi, P., Sisson, J.S. and Verma, I.M., Transcriptional autoregulation of the proto-oncogene fos, Nature, 334 (1988) 314–319. [15] Van den pol, A.N., Cao, V. and Heller, H.C., Circadian system of mice integrates brief light stimuli, Am. J. Physiol., 275 (1998) R654–R657. [16] Weaver, D.R., The suprachiasmatic nucleus: a 25-year retrospective, J. Biol. Rhythms, 13 (1998) 100–112. [17] Wolluik, F., Brysch, W., Uhlmann, E., Gillardon, F., Bravo, R. and Zimmermann, M., Schlingensiepen, K.H. and Herdegen, T., Block of c-Fos and JunB expression by antisense oligonucleotides inhibits light-induced phase shifts of the mammalian circadian clock, Eur. J. Neurosci., 7 (1995) 388–393.
Resetting the rat circadian clock by ultra-short light flashes Andreas Arvanitogiannis, Shimon Amir* Center for Studies in Behavioral Neurobiology, Department of Psychology, Concordia University, 1455 de Maisonneuve Blvd West, Montreal, Quebec, Canada H3G 1M8 Received 9 November 1998; received in revised form 21 December 1998; accepted 21 December 1998
Abstract We examined the effects that ultra-brief, intense, light flashes have on the rat circadian clock, the suprachiasmatic nucleus of the hypothalamus (SCN). We found that as few as five intense flashes, each 10-ms in duration (1 per s), can produce both phase shifts in free-running activity rhythms and Fos expression in the SCN in rats kept in constant darkness. After pre-exposure to such flashes, phase shifts in response to a continuous light pulse delivered 2 h later were potentiated, but Fos expression in the SCN was decreased as following pre-exposure to continuous light. These results show that flashes induce behavioral and cellular effects indicative of clock resetting similar to those induced by light stimuli of longer duration. Extremely brief but intense, light stimuli may be much more important to clock resetting than had been previously known. 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Circadian rhythms; Phase shift; Suprachiasmatic nucleus; Fos
Stable entrainment of mammalian circadian rhythms requires daily photic resetting of the circadian clock located in the suprachiasmatic nucleus of the hypothalamus [9,12,16]. Although it is known that the effectiveness of light as a resetting stimulus is influenced by both duration and intensity, it has long been considered that the pathway mediating resetting by light is insensitive to very brief light stimuli, regardless of their intensity [4,7–10,12]. To further study this issue, we investigated the effect that ultrabrief, intense, light flashes have on the circadian clock in rats. Clock resetting was assessed using both a behavioral measure, phase shifts in free-running activity rhythms [3], and a cellular measure, Fos expression in the suprachiasmatic nucleus [5]. Male Wistar rats (300–350 g) were housed individually in plastic cages equipped with Nalgene running wheels and had free access to food and water. The cages were placed inside ventilated, light- and sound-tight enclosures. At the start of the experiment the rats were placed in constant darkness and their running-wheel activity was recorded
* Corresponding author. Tel.: +1-514-8482188; fax: +1-5148482817; e-mail: [email protected]
0304-3940/99/$ - see front matter PII: S03 04-3940(99)000 21-X
using Dataquest software (Mini Mitter, Sunriver, OR). To assess the effects of flashes on phase shifts in free-running activity rhythms, animals were assigned to one of three combinations of stimuli: (1) 45 × 10-ms flash/s delivered at a rate of 3 flash/s (total light duration = 450 ms); (2) 15 × 10-ms flash/s delivered at a rate of 1 flash/s (total light duration = 150 ms); (3) 5 × 10-ms flash/s delivered at a rate of 1 flash/s (total light duration = 50 ms). All animals were presented with these stimulus combinations at circadian time (CT) 13 (CT12 denotes time of activity onset). Light flash/s were generated by a Grass photic stimulator (PS22) placed on the side of the Plexiglas cages, at a distance of about 25 cm from their center (illuminance: ~2.4 × 106 lux; luminance: ~1.9 × 106 cd/m2; these numbers were derived from the nominal characteristics of the photic stimulator provided by the manufacturer; the perceived intensity of the flashes varied as a function of the position the subjects occupied in their cage as well as the differing degrees of approach or avoidance behavior they exhibited during the presentation of the flashes). The animals continued to free-run for at least 8 days and phase shifts were determined from graphic records of wheel-running behavior (actograms) by two independent observers. Phase shifts in the activity rhythms were calculated as the
1999 Elsevier Science Ireland Ltd. All rights reserved.
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A. Arvanitogiannis, S. Amir / Neuroscience Letters 261 (1999) 159–162
difference between the eye-fitted lines connecting the time of onset of activity for a period of seven days before and after exposure to the flashes and/or continuous light [1,3]. To assess the effect of flashes on Fos expression in the SCN, the experiment was repeated after randomly reassigning the rats to the above three conditions. One hour after presentation of the stimuli (CT14), the rats were injected with a lethal dose of sodium pentobarbital (100 mg/kg) and perfused with 300 ml of 0.9% NaCl followed by 300 ml 0.1 M phosphate buffer containing 4% paraformaldehyde. Fos immunohistochemistry was carried out on free floating coronal brain sections (50 mm) using a mouse monoclonal antibody raised against residues 4–17 of the 55 kDa Fos protein N-terminal (1:8000; NCI/BCB Repository, Quality Biotech, Camden, NJ), as previously described [1]. Estimates of the number of nuclei expressing Fos were made from cell counts performed on serial sections taken from the SCN using a computerized image analysis system and the NIH Image software. Mean cell counts on one side of the SCN on the four sections exhibiting the highest number of Fos immunoreactive cells were taken from each animal. As shown in Fig. 1, exposure to each of the combinations of 10-ms flashes at CT13 resulted in both phase shifts in running wheel activity and Fos expression in the SCN. Although the phase shifts induced by each of the three combinations of flashes were of similar magnitude (Fig. 1a), progressively less Fos-IR was seen in the SCN as the total light exposure decreased (Fig. 1b). The graded Fos response argues against the possibility that the effects of the flashes were due to aberrant over-activation of the retina or the photic-entrainment pathway. Fig. 2a shows an example of a phase shift that resulted from exposure to five 10-ms flashes. Fig. 3 shows Fos-IR cells in the SCN in response to the three combinations of flashes. Notice that Fos was expressed predominantly in the ventrolateral portion of the SCN, the terminal region of retinal afferents. In control animals that did not receive light flashes, or that were exposed to the flashes at CT6, Fos expression in the SCN was negligible (,six Fos-IR cells). Light-induced phase shifts of behavioral rhythms in rodents are known to be positively correlated with the induction of Fos in the SCN when their respective magnitudes are below ceiling, and it has been suggested that Fos may mediate light induced phase shifts [2,5,6,11,13,17]. It is somewhat surprising, therefore, that the magnitude of the flash-induced phase shifts was similar across conditions, whereas Fos expression in the SCN decreased with decreasing light exposure. The apparent dissociation between the magnitude of flash-induced phase shifts and the magnitude of Fos expression could imply that such brief flashes exert their influence on the clock via mechanisms different from those involved in the effect of a continuous light pulse. As a first step toward evaluating this possibility we examined the effect that pre-exposure to flashes or continuous light had on phase shifts and Fos expression in response to a subsequently delivered continuous light pulse. For this experi-
ment, three groups of animals were maintained in constant darkness and were given, at CT13, either five 10-ms flashes (1 flash/s), a continuous light pulse delivered via an overhead cool white fluorescent lamp (illuminance: ~300 lux; duration: 75 s) or no light exposure. At CT15 all animals were exposed to the continuous light pulse. It can be seen from Fig. 1d that when animals were exposed to two continuous light pulses, one at CT13 and another at CT15, Fos expression in the SCN measured 1 h after the CT15 pulse was diminished compared to that seen in animals exposed only to a single continuous light pulse at CT15. Likewise, Fos expression in the SCN induced by the continuous light pulse at CT15 was decreased in animals pre-exposed to the flashes a CT13 (Fig. 1d). Thus, exposure to the flashes appears to modulate the Fos response in the SCN in a manner similar to that of the continuous light. The most parsimonious explanation for the decline in Fos staining in response to the second light pulse is that after the first exposure to light, whether flashed or continuous, the cells that expressed Fos in the SCN were, at least in part, unable
Fig. 1. (a,b) The effects of presenting, at CT13, one of three combinations of 10-ms flashes (45 flashes delivered at a rate of 3 flashes/s; 15 flashes delivered at a rate of 1 flash/s; five flashes delivered at a rate of 1 flash/s) on phase shifts in free-running activity rhythms (n = 4 per group) and Fos expression in the SCN (n = 4 per group). (c,d) The effects of pre-exposure at CT13 to either flashes or continuous light on phase shifts in free-running activity rhythms (n = 8 per group) and Fos expression in the SCN (n = 4 per group) in response to a continuous light pulse delivered at CT15. L, 75 s of continuous light delivered at CT15; FL, five flashes delivered at a rate of 1 flash/s at CT13 followed by 75 s of continuous light at CT15; LL, 75 s of continuous light delivered at both CT13 and CT15. All data are expressed as mean ± SEM. *P , 0.05, one-way analysis of variance/Scheffe post-hoc test.
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to do so again because of the negative feedback that Fos exerts on its own transcription [14]. In contrast to the effect on Fos expression, pre-exposure
Fig. 3. Representative photomicrographs showing Fos-labeled cells in one side of the SCN in response to: (a) 45 × 10-ms flashes delivered at a rate of 3 flashes/s; (b) 15 × 10-ms flashes delivered at a rate of 1 flash/s; (c) 5 × 10-ms flashes delivered at a rate of 1 flash/s. Scale bar, 100 mm.
Fig. 2. Representative actograms showing the free-running activity rhythms (wheel running) of animals that received: (a) 5 × 10-ms flashes delivered at a rate of 1 flash/s (CT13); (b) a 75 s continuous light pulse (CT15); (c) 5 × 10-ms flashes delivered at a rate of 1 flash/ s (CT13) and a 75 s continuous light pulse (CT15). The horizontal lines in each actogram represent a single 24-hour period; the vertical marks indicate periods of activity of at least 10 wheel revolutions/10 min. Successive days are plotted from top to bottom. The open triangles indicate the day on which the flashes and/or continuous light were delivered. Phase-shifts in the activity rhythm are demonstrated by the presence of a difference between the eye-fitted lines connecting the onset of activity for a period of 7 days before and after exposure to the light stimuli.
to the flashes at CT13 enhanced the phase shift produced in response to the continuous light pulse presented at CT15 (Figs. 1c and 2b,c). As can be seen in Fig. 1c, the magnitude of the phase shift in animals exposed to the flashes at CT13 and then to the continuous light at CT15 was greater than that seen in animals exposed only to the continuous light pulse given at CT15, and not different from that seen in animals exposed twice to the continuous light stimulus. There are at least two ways to account for this additive effect. First, the temporally distinct flashes and continuous light may have engaged the mechanisms involved in generating the phase shifts on both occasions. If so, the potentiated phase shift is an outcome of combining two discretely produced phase shifts. As mentioned before, Fos expression in response to the continuous light stimulus at CT15 was suppressed after pre-exposure to flashes. Similarly, the phase shift produced in response to the continuous light
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pulse may have also been suppressed. However, although suppressed, the phase shift induced by the continuous light combined with the one induced by the flash could well have resulted in the observed potentiated phase shift. Another possibility is that the clock integrates all photic information that reaches it over a relatively prolonged period (at least 2 h), and that only after that time is the phase shifting mechanism engaged. In this latter case the potentiated phase shift is an outcome of the total input to the clock integrated over time. Clearly, however, the integration is not expressed as increased Fos in the SCN. The diminished Fos response may suggest that the expression of Fos, though self-limiting, serves as a gateway for integration of additional photic input within some, yet to be specified, temporal interval. It remains to be determined whether the refractoriness in Fos expression is related to ongoing integration within the pacemaker. If so, the temporal interval between two light pulses at which Fos expression in response to the second pulse becomes as strong as if the second pulse were delivered alone, would define the temporal window of integration and provide a direct measure of the time it takes for a phase shift to be completed. To conclude, the present study demonstrates that as few as five intense, 10-ms light flashes delivered within 5 s produce phase shifts in free-running rhythms and induce Fos expression in the SCN. These results extend findings from a recent study demonstrating that repeated exposure (but not a single exposure) to 2-ms light pulses induced phase shifts in freerunning activity rhythms in mice [15]. Contrary to previous suggestions [10], extremely brief light stimuli in the environment that could arise, for example, from lightning, may influence the circadian clock and cannot be considered mere ‘noise’. It is worth noting that the flashes in the present study had a significantly lower luminance value than lightning [10] (1.9 × 106 cd/m2, flashes VS. 8 × 1010 cd/m2, lightning). The double light-pulse experiments provide strong support for the idea that flashes can exert strong influence on clock phase resetting. Phase shifts produced by the flashes summed with those produced by a continuous light pulse delivered 2 h later. Furthermore, preexposure to flashes or continuous light suppressed the induction of Fos in the SCN in response to the subsequently administered continuous light pulse. The fact pre-exposure to either flashes or continuous light similarly modulate the effects of a subsequent continuous light pulse suggests that both stimulus events affect clock resetting via a common mechanism. We thank Jane Stewart for helpful comments on the manuscript. This work was supported by grants from the
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