- Email: [email protected]
A novel, associative process modulating photic resetting of the circadian clock
PII: S 0 3 0 6 - 4 5 2 2 ( 0 1 ) 0 0 1 6 2 - 2
Neuroscience Vol. 104, No. 3, pp. 615^618, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00
www.elsevier.com/locate/neuroscience
Letter to Neuroscience A NOVEL, ASSOCIATIVE PROCESS MODULATING PHOTIC RESETTING OF THE CIRCADIAN CLOCK A. ARVANITOGIANNIS2 and S. AMIR* Center for Studies in Behavioral Neurobiology, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, QC, Canada H3G 1M8 Key words : photic resetting, conditioning, circadian rhythms, Fos immunocytochemistry, rat.
Appropriate timing of physiological and behavioral processes requires that the circadian clock be reset daily by salient cues in the environment, particularly light. It is known that the ability of light to reset the clock depends both on its intensity and on the circadian time when it is applied (Daan and Pittendrigh, 1976; Moore-Ede et al., 1982). Here we show that the ability of a weak light stimulus to reset the clock is dramatically enhanced when it is presented daily at the same circadian time. Equivalent daily presentations of this light stimulus, but at di¡erent circadian times each day, do not lead to such enhancement. These ¢ndings suggest that the ability of light to reset the clock can be modi¢ed through a novel, and previously unrecognized, conditioning-like associative process in which circadian time serves as the conditioned stimulus and light as the unconditioned stimulus. The idea that circadian time can serve as a conditioned stimulus to modulate the e¡ectiveness of light provides a new perspective on the lasting impact that light schedules have on the circadian clock and, thus, may have implications for existing models of photic entrainment (Pittendrigh and Daan, 1976; Moore-Ede et al., 1982). ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.
onset of darkness, at zeitgeber time 15 (ZT15) every day, for 10 days (ZT12 denotes onset of the dark phase under entrained conditions). A second group (UNPAIRED) was given presentations of the same weak light stimulus, but at di¡erent times of the dark phase of the cycle each day. Thus, for this group the weak light stimulus was not associated with a speci¢c circadian time. A third group (CONTROL) was not exposed to the weak light stimulus. Following this phase of the experiment, animals were placed in constant darkness and the ability of the weak light stimulus to reset the clock was assessed 10 days later. For the test, all animals were given a short 3-min presentation of the weak light stimulus at circadian time 15 (CT15; CT12 denotes the onset of subjective night, the active phase, under constant darkness) and phase shifts in free-running activity rhythms were measured. Groups did not di¡er in the pattern of entrained wheel-running activity during the initial phase of the experiment. All animals exhibited stable 24-h activity rhythms regardless of presentations of the weak light stimulus, nor did they di¡er in pattern (Fig. 2a) or period (Fig. 2b) of free-running rhythms during the subsequent 10 days in constant darkness [analysis of variance (ANOVA), for period: F 2;33 = 0.29, P = 0.75]. This indicates that, under entrained conditions, repeated daily exposure to the weak light stimulus, whether at the same circadian time or at a di¡erent time each day, had no lasting e¡ect on the intrinsic period of the circadian clock. In contrast, phase shifts induced by the 3-min presentation of the weak light stimulus at CT15, after 10 days in constant darkness, varied signi¢cantly as a function of treatment group (Fig. 2a, c). Phase shifts induced in the PAIRED group were greatly enhanced compared to those in the CONTROL and UNPAIRED groups (ANOVA: F 2;33 = 5.86, P = 0.006). These results show that the response of the circadian clock to the initially weak light stimulus was enhanced after repeated exposure to that stimulus, but only when that stimulus had been presented at the same circadian time each day. In behavioral terms, this change in e¡ec-
The design of the experiment is outlined in Fig. 1. Brie£y, rats entrained to a 12-h:12-h light^dark cycle were assigned to one of three groups (n = 12/group). One group (PAIRED) was given a 30-min presentation of a distinct weak light stimulus (1 lux) 3 h after the
2 Present address: Department of Psychiatry, Harvard Medical School, McLean Hospital, Mailman Research Center, 115 Mill Street, Belmont, MA 02478, USA. *Corresponding author. Tel.: +1-514-848-2188; fax: +1-514-8482817. E-mail address: [email protected] (S. Amir). Abbreviations : ANOVA, analysis of variance; CT, circadian time ; SCN, suprachiasmatic nucleus; ZT, zeitgeber time.
615
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616
A. Arvanitogiannis and S. Amir
Fig. 1. A schematic outline of the experimental design (see Experimental procedures for details).
tiveness of the weak light stimulus could re£ect the outcome of an associative process whereby circadian time, serving as a conditioned stimulus, augmented the clock response to light, the unconditional stimulus. This interpretation ¢ts well with evidence that conditioned stimuli have modulating e¡ects on the magnitude of responses to unconditional stimuli (Hollis, 1982). For instance, conditioned contextual cues are known to enhance the magnitude of the behavioral activating e¡ects of stimulant drugs (Stewart, 1992). Interestingly, we showed recently that behavioral sensitization to amphetamine can likewise come to be controlled by time itself acting as the conditioned stimulus (Arvanitogiannis et al., 2000). To determine whether the enhanced e¡ectiveness of the weak light stimulus was truly time dependent, separate groups of similarly trained PAIRED, UNPAIRED and CONTROL rats (n = 4/group) were tested with the weak light stimulus at CT21, a time not previously associated with daily presentations of the weak light stimulus. Photic stimuli delivered at CT21 would be expected to result in phase advances, not phase delays. We found that a 3-min exposure to the weak light stimulus at CT21 induced equally strong phase advances in all three groups (PAIRED: 26.25 þ 7.18; UNPAIRED: 33.75 þ 3.75; CONTROL: 30 þ 10.61; F 2;9 = 0.237, P = 0.794). These results demonstrate that repeated presentations of a weak light stimulus at a speci¢c circadian time can subsequently enhance the ability of light to reset the clock only if it is presented at the same circadian time. Such a ¢nding lends support to the idea that circadian time can act as a conditioned stimulus that, through repeated pairing with a weak light, comes to facilitate the response to that stimulus. In separate groups of PAIRED, UNPAIRED and CONTROL animals (n = 8/group), we assessed the e¡ect of the weak light stimulus on the expression of the transcription factor Fos in the SCN, the master circadian clock in mammals (Klein et al., 1991; Hastings and Maywood, 2000). The ability of light to induce shifts in clock phase varies systematically with its ability to induce Fos expression in the SCN, and it has been suggested that Fos is part of the input mechanism mediating photically induced phase shifts (Hastings et al., 1995;
Kornhauser et al., 1996). In the present experiment, however, no signi¢cant di¡erences in Fos expression in the SCN in response to presentation of the weak light stimulus at CT15 were found between the three groups (Fig. 2d; ANOVA: F 2;21 = 1.06, P = 0.36). This indicates that the input from the retina to the SCN that mediates photic resetting was not enhanced, as would be expected, for example, if modi¢cations had occurred in the autonomous circadian oscillator within the eye (Tosini and Menaker, 1996) or in neural pathways mediating the transmission of photic information from the eye to the SCN. It can be pointed out as well, that in the absence of light, circadian time 15 did not act as a phase resetting stimulus; there were no di¡erences in the free-running periods of animals in the three groups. Importantly, environmental stimuli paired with light have been shown both to enhance the Fos response to light (Amir and Stewart, 1998) and to induce phase shifts and Fos expression in the SCN in the absence of light (Amir and Stewart, 1996; Arvanitogiannis and Amir, 1999; but see de Groot and Rusak, 2000). In the present experiment, however, there were no signi¢cant di¡erences in Fos expression to the weak light stimulus between groups. Hence, although circadian time appears to act as a conditioned stimulus to enhance clock resetting by light, it may do so by a mechanism di¡erent from that previously described for environmental cues that reliably precede the onset of light (Amir and Stewart, 1996; Arvanitogiannis and Amir, 1999). Our data demonstrate that the ability of light to reset the clock is signi¢cantly enhanced when it is presented repeatedly, over days, at the same circadian time. Circadian time is presumably represented by the state of the clock, itself, thus the modi¢cations that lead to this enhancement, that were brought about by the association of time and light, are likely to have occurred downstream from the input pathway by which light reaches the clock. The new knowledge that circadian time is encoded by oscillating levels of clock proteins (King and Takahashi, 2000; Shearman et al., 2000), and the recent identi¢cation of a discrete sub-nucleus in the SCN that is both light responsive and necessary for circadian timing (LeSauter and Silver, 1999) raise the enticing possibility that this association can be linked to an interaction between speci¢c molecules within identi¢ed clock neurons.
EXPERIMENTAL PROCEDURES
The experimental procedures followed the guidelines of the Canadian Council on Animal Care and were approved by the Animal Care Committee, Concordia University. Three groups of male Wistar rats (n = 16/group, 300^325 g, Charles River, St. Constant, QC, Canada) were housed individually in cages equipped with running wheels, under a 12-h light:12-h dark cycle (light: 300 lux at cage level) and had continuous access to food and water. The cages were placed inside individual ventilated light- and sound-tight enclosures. The PAIRED group was presented with a distinctive weak light stimulus (duration: 30 min; green, Omni-Glow Neon pilot light, Electro Sonic Inc., Ville St. Laurent, QC, Canada; illuminance at the center of the cage, 15 cm from the light source: 1 lux) 3 h after the onset of
NSC 5007 26-6-01
Novel associative process for clock resetting
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Fig. 2. (a) Actograms showing the free-running activity rhythms in constant darkness of two animals from each of the PAIRED, UNPAIRED and CONTROL groups before and after exposure to the 3-min weak light stimulus at CT15. The horizontal lines in each actogram represent 24-h periods; the vertical marks indicate bouts of activity of at least 10 wheel revolutions/10 min. Successive days are plotted from top to bottom. T indicates day of test; the white circles indicate the times of exposure to the weak light stimulus during the test (CT15). Phase shifts in the activity rhythm are demonstrated by the horizontal de£ection of the line indicating the onset of activity after exposure to the weak light stimulus. (b) Periods (in hours, mean þ S.E.M.) of free-running activity rhythms in constant darkness of animals from the PAIRED, UNPAIRED and CONTROL groups (n = 12/group). Periods were calculated from activity data for the ¢rst 10 days in constant darkness using Periodgram analysis (Circadian). (c) Phase delays (mean þ S.E.M.) in free-running activity rhythms of animals from the PAIRED, UNPAIRED and CONTROL groups induced by exposure to a 3-min weak light stimulus at CT15. The magnitude of the phase delay was signi¢cantly enhanced in the PAIRED group (*P 6 0.01, Tukey post hoc test). (d) Mean þ S.E.M. number of Fos-immunoreactive cells in the suprachiasmatic nucleus (SCN) in animals from the PAIRED, UNPAIRED and CONTROL groups that were housed in constant darkness for 10 days and killed 60 min after exposure to the 3-min weak light stimulus at CT15 (n = 8/group).
dark (ZT15) for 10 days. The UNPAIRED group was also presented with the weak light stimulus for 10 days but at di¡erent times of the dark phase each day. The CONTROL group was never exposed to the weak light stimulus during this phase of the experiment. Following this phase, all animals were placed in constant darkness for 10 days and were then presented with the weak light stimulus, once, at CT15 (n = 12/group) or CT21 (n = 4/group). The duration of the weak light stimulus used to test clock resetting was 3 min, a duration short enough to produce sub-maximal phase shifts. The animals continued to freerun in constant darkness for 10 additional days. The ability of the weak light stimulus to reset the clock was assessed by measuring phase shifts in free-running activity rhythms using graphic records (actograms) of wheel-running behavior. Phase shifts were calculated as the di¡erence between the onset of activity before and after exposure to the weak light stimulus (Amir and Stewart, 1996). Statistical analysis was carried out using a one-way ANOVA followed by post hoc comparisons using the Tukey post hoc test (signi¢cance set at P 6 0.05). The e¡ect of the weak light stimulus on Fos expression in the SCN was studied in separate animals from the PAIRED, UNPAIRED and CONTROL groups (n = 8/group). The rats
were housed in constant darkness for 10 days and killed 60 min after they were exposed to the 3-min weak light stimulus at CT15. Immunostaining for Fos was carried out as previously described (Amir and Stewart, 1996) using a mouse monoclonal antibody raised against the N-terminal sequence of Fos (corresponding to N-terminal residues 4^17 of human Fos protein; NCI/BCB Repository, Quality Biotech, Camden, NJ, USA; 1:8000). Estimates of the number of nuclei expressing Fos were made from cell counts performed on serial sections using a computerized image analysis system and the NIH Image software. Mean cell counts on one side of the brain on the ¢ve sections exhibiting the highest number of Fos-immunoreactive cells were taken from each animal. Statistical analysis was carried out using a one-way ANOVA. Signi¢cance was set at P 6 0.05.
AcknowledgementsöThis work was supported by grants from the Canadian Institutes for Health Research, and the Fonds pour la Formation de Chercheurs et l`Aide a© la Recherche (Que¨bec).
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A. Arvanitogiannis and S. Amir REFERENCES
Amir, S., Stewart, J., 1996. Resetting of the circadian clock by a conditioned stimulus. Nature 379, 542^545. Amir, S., Stewart, J., 1998. Induction of Fos expression in the circadian system by unsignaled light is attenuated as a result of previous experience with signaled light : a role for Pavlovian conditioning. Neuroscience 83, 657^661. Arvanitogiannis, A., Amir, S., 1999. Conditioned stimulus control in the rat circadian system depends on clock resetting during conditioning. Behav. Neurosci. 113, 1297^1300. Arvanitogiannis, A., Sullivan, J., Amir, S., 2000. Time acts as a conditioned stimulus to control behavioral sensitization to amphetamine in rats. Neuroscience 101, 1^3. Daan, S., Pittendrigh, C.S., 1976. Functional analysis of circadian pacemakers in nocturnal rodents. II. The variability of phase response curves. J. Comp. Physiol. 106, 253^266. de Groot, M.H., Rusak, B., 2000. Responses of the circadian system of rats to conditioned and unconditioned stimuli. J. Biol. Rhythms 15, 277^ 291. Hastings, M.H., Maywood, E.S., 2000. Circadian clocks in the mammalian brain. Bioessays 22, 23^31. Hastings, M.H., Ebling, F.J., Grosse, J., Herbert, J., Maywood, E.S., Mikkelsen, J.D., Sumova, A., 1995. Immediate-early genes and the neural bases of photic and non-photic entrainment. Ciba Found. Symp. 183, 175^189. Hollis, K.L., 1982. Pavlovian conditioning of signal-centered action patterns and autonomic behavior: a biological analysis of function. In: Rosenblatt, J.S., Hinde, R.A., Beer, C., Busnel, M.-C. (Eds.), Advances in the Study of Behavior. Academic Press, New York, pp. 1^64. King, D.P., Takahashi, J.S., 2000. Molecular genetics of circadian rhythms in mammals. Annu. Rev. Neurosci. 23, 713^742. Klein, D., Moore, R.Y., Reppert, S.M., 1991. Suprachiasmatic Nucleus: The Mind's Clock. Oxford University Press, Oxford. Kornhauser, J.M., Mayo, K.E., Takahashi, J.S., 1996. Light, immediate-early genes, and circadian rhythms. Behav. Genet. 26, 221^240. LeSauter, J., Silver, R., 1999. Localization of a suprachiasmatic nucleus subregion regulating locomotor rhythmicity. J. Neurosci. 19, 5574^5585. Moore-Ede, M.C., Sulzman, F.M., Fuller, C.A., 1982. The Clocks That Time Us. Harvard University Press, Cambridge. Pittendrigh, C.S., Daan, S., 1976. A functional analysis of circadian pacemakers in nocturnal rodents. IV. Entrainment Pacemaker as clock. J. Comp. Physiol. 106, 291^331. Shearman, L.P., Sriram, S., Weaver, D.R., Maywood, E.S., Chaves, I., Zheng, B., Kume, K., Lee, C.C., van der Horst, G.T., Hastings, M.H., Reppert, S.M., 2000. Interacting molecular loops in the mammalian circadian clock. Science 288, 1013^1019. Stewart, J., 1992. In: Gormezano, I., Wasserman, E.A. (Eds.), Learning and Memory: Behavioral and Biological Substrates. Erlbaum, Hillsdale, pp. 129^151. Tosini, G., Menaker, M., 1996. Circadian rhythms in cultured mammalian retina. Science 272, 419^421. (Accepted 5 April 2001)
NSC 5007 26-6-01
Neuroscience Vol. 104, No. 3, pp. 615^618, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00
www.elsevier.com/locate/neuroscience
Letter to Neuroscience A NOVEL, ASSOCIATIVE PROCESS MODULATING PHOTIC RESETTING OF THE CIRCADIAN CLOCK A. ARVANITOGIANNIS2 and S. AMIR* Center for Studies in Behavioral Neurobiology, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, QC, Canada H3G 1M8 Key words : photic resetting, conditioning, circadian rhythms, Fos immunocytochemistry, rat.
Appropriate timing of physiological and behavioral processes requires that the circadian clock be reset daily by salient cues in the environment, particularly light. It is known that the ability of light to reset the clock depends both on its intensity and on the circadian time when it is applied (Daan and Pittendrigh, 1976; Moore-Ede et al., 1982). Here we show that the ability of a weak light stimulus to reset the clock is dramatically enhanced when it is presented daily at the same circadian time. Equivalent daily presentations of this light stimulus, but at di¡erent circadian times each day, do not lead to such enhancement. These ¢ndings suggest that the ability of light to reset the clock can be modi¢ed through a novel, and previously unrecognized, conditioning-like associative process in which circadian time serves as the conditioned stimulus and light as the unconditioned stimulus. The idea that circadian time can serve as a conditioned stimulus to modulate the e¡ectiveness of light provides a new perspective on the lasting impact that light schedules have on the circadian clock and, thus, may have implications for existing models of photic entrainment (Pittendrigh and Daan, 1976; Moore-Ede et al., 1982). ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.
onset of darkness, at zeitgeber time 15 (ZT15) every day, for 10 days (ZT12 denotes onset of the dark phase under entrained conditions). A second group (UNPAIRED) was given presentations of the same weak light stimulus, but at di¡erent times of the dark phase of the cycle each day. Thus, for this group the weak light stimulus was not associated with a speci¢c circadian time. A third group (CONTROL) was not exposed to the weak light stimulus. Following this phase of the experiment, animals were placed in constant darkness and the ability of the weak light stimulus to reset the clock was assessed 10 days later. For the test, all animals were given a short 3-min presentation of the weak light stimulus at circadian time 15 (CT15; CT12 denotes the onset of subjective night, the active phase, under constant darkness) and phase shifts in free-running activity rhythms were measured. Groups did not di¡er in the pattern of entrained wheel-running activity during the initial phase of the experiment. All animals exhibited stable 24-h activity rhythms regardless of presentations of the weak light stimulus, nor did they di¡er in pattern (Fig. 2a) or period (Fig. 2b) of free-running rhythms during the subsequent 10 days in constant darkness [analysis of variance (ANOVA), for period: F 2;33 = 0.29, P = 0.75]. This indicates that, under entrained conditions, repeated daily exposure to the weak light stimulus, whether at the same circadian time or at a di¡erent time each day, had no lasting e¡ect on the intrinsic period of the circadian clock. In contrast, phase shifts induced by the 3-min presentation of the weak light stimulus at CT15, after 10 days in constant darkness, varied signi¢cantly as a function of treatment group (Fig. 2a, c). Phase shifts induced in the PAIRED group were greatly enhanced compared to those in the CONTROL and UNPAIRED groups (ANOVA: F 2;33 = 5.86, P = 0.006). These results show that the response of the circadian clock to the initially weak light stimulus was enhanced after repeated exposure to that stimulus, but only when that stimulus had been presented at the same circadian time each day. In behavioral terms, this change in e¡ec-
The design of the experiment is outlined in Fig. 1. Brie£y, rats entrained to a 12-h:12-h light^dark cycle were assigned to one of three groups (n = 12/group). One group (PAIRED) was given a 30-min presentation of a distinct weak light stimulus (1 lux) 3 h after the
2 Present address: Department of Psychiatry, Harvard Medical School, McLean Hospital, Mailman Research Center, 115 Mill Street, Belmont, MA 02478, USA. *Corresponding author. Tel.: +1-514-848-2188; fax: +1-514-8482817. E-mail address: [email protected] (S. Amir). Abbreviations : ANOVA, analysis of variance; CT, circadian time ; SCN, suprachiasmatic nucleus; ZT, zeitgeber time.
615
NSC 5007 26-6-01
616
A. Arvanitogiannis and S. Amir
Fig. 1. A schematic outline of the experimental design (see Experimental procedures for details).
tiveness of the weak light stimulus could re£ect the outcome of an associative process whereby circadian time, serving as a conditioned stimulus, augmented the clock response to light, the unconditional stimulus. This interpretation ¢ts well with evidence that conditioned stimuli have modulating e¡ects on the magnitude of responses to unconditional stimuli (Hollis, 1982). For instance, conditioned contextual cues are known to enhance the magnitude of the behavioral activating e¡ects of stimulant drugs (Stewart, 1992). Interestingly, we showed recently that behavioral sensitization to amphetamine can likewise come to be controlled by time itself acting as the conditioned stimulus (Arvanitogiannis et al., 2000). To determine whether the enhanced e¡ectiveness of the weak light stimulus was truly time dependent, separate groups of similarly trained PAIRED, UNPAIRED and CONTROL rats (n = 4/group) were tested with the weak light stimulus at CT21, a time not previously associated with daily presentations of the weak light stimulus. Photic stimuli delivered at CT21 would be expected to result in phase advances, not phase delays. We found that a 3-min exposure to the weak light stimulus at CT21 induced equally strong phase advances in all three groups (PAIRED: 26.25 þ 7.18; UNPAIRED: 33.75 þ 3.75; CONTROL: 30 þ 10.61; F 2;9 = 0.237, P = 0.794). These results demonstrate that repeated presentations of a weak light stimulus at a speci¢c circadian time can subsequently enhance the ability of light to reset the clock only if it is presented at the same circadian time. Such a ¢nding lends support to the idea that circadian time can act as a conditioned stimulus that, through repeated pairing with a weak light, comes to facilitate the response to that stimulus. In separate groups of PAIRED, UNPAIRED and CONTROL animals (n = 8/group), we assessed the e¡ect of the weak light stimulus on the expression of the transcription factor Fos in the SCN, the master circadian clock in mammals (Klein et al., 1991; Hastings and Maywood, 2000). The ability of light to induce shifts in clock phase varies systematically with its ability to induce Fos expression in the SCN, and it has been suggested that Fos is part of the input mechanism mediating photically induced phase shifts (Hastings et al., 1995;
Kornhauser et al., 1996). In the present experiment, however, no signi¢cant di¡erences in Fos expression in the SCN in response to presentation of the weak light stimulus at CT15 were found between the three groups (Fig. 2d; ANOVA: F 2;21 = 1.06, P = 0.36). This indicates that the input from the retina to the SCN that mediates photic resetting was not enhanced, as would be expected, for example, if modi¢cations had occurred in the autonomous circadian oscillator within the eye (Tosini and Menaker, 1996) or in neural pathways mediating the transmission of photic information from the eye to the SCN. It can be pointed out as well, that in the absence of light, circadian time 15 did not act as a phase resetting stimulus; there were no di¡erences in the free-running periods of animals in the three groups. Importantly, environmental stimuli paired with light have been shown both to enhance the Fos response to light (Amir and Stewart, 1998) and to induce phase shifts and Fos expression in the SCN in the absence of light (Amir and Stewart, 1996; Arvanitogiannis and Amir, 1999; but see de Groot and Rusak, 2000). In the present experiment, however, there were no signi¢cant di¡erences in Fos expression to the weak light stimulus between groups. Hence, although circadian time appears to act as a conditioned stimulus to enhance clock resetting by light, it may do so by a mechanism di¡erent from that previously described for environmental cues that reliably precede the onset of light (Amir and Stewart, 1996; Arvanitogiannis and Amir, 1999). Our data demonstrate that the ability of light to reset the clock is signi¢cantly enhanced when it is presented repeatedly, over days, at the same circadian time. Circadian time is presumably represented by the state of the clock, itself, thus the modi¢cations that lead to this enhancement, that were brought about by the association of time and light, are likely to have occurred downstream from the input pathway by which light reaches the clock. The new knowledge that circadian time is encoded by oscillating levels of clock proteins (King and Takahashi, 2000; Shearman et al., 2000), and the recent identi¢cation of a discrete sub-nucleus in the SCN that is both light responsive and necessary for circadian timing (LeSauter and Silver, 1999) raise the enticing possibility that this association can be linked to an interaction between speci¢c molecules within identi¢ed clock neurons.
EXPERIMENTAL PROCEDURES
The experimental procedures followed the guidelines of the Canadian Council on Animal Care and were approved by the Animal Care Committee, Concordia University. Three groups of male Wistar rats (n = 16/group, 300^325 g, Charles River, St. Constant, QC, Canada) were housed individually in cages equipped with running wheels, under a 12-h light:12-h dark cycle (light: 300 lux at cage level) and had continuous access to food and water. The cages were placed inside individual ventilated light- and sound-tight enclosures. The PAIRED group was presented with a distinctive weak light stimulus (duration: 30 min; green, Omni-Glow Neon pilot light, Electro Sonic Inc., Ville St. Laurent, QC, Canada; illuminance at the center of the cage, 15 cm from the light source: 1 lux) 3 h after the onset of
NSC 5007 26-6-01
Novel associative process for clock resetting
617
Fig. 2. (a) Actograms showing the free-running activity rhythms in constant darkness of two animals from each of the PAIRED, UNPAIRED and CONTROL groups before and after exposure to the 3-min weak light stimulus at CT15. The horizontal lines in each actogram represent 24-h periods; the vertical marks indicate bouts of activity of at least 10 wheel revolutions/10 min. Successive days are plotted from top to bottom. T indicates day of test; the white circles indicate the times of exposure to the weak light stimulus during the test (CT15). Phase shifts in the activity rhythm are demonstrated by the horizontal de£ection of the line indicating the onset of activity after exposure to the weak light stimulus. (b) Periods (in hours, mean þ S.E.M.) of free-running activity rhythms in constant darkness of animals from the PAIRED, UNPAIRED and CONTROL groups (n = 12/group). Periods were calculated from activity data for the ¢rst 10 days in constant darkness using Periodgram analysis (Circadian). (c) Phase delays (mean þ S.E.M.) in free-running activity rhythms of animals from the PAIRED, UNPAIRED and CONTROL groups induced by exposure to a 3-min weak light stimulus at CT15. The magnitude of the phase delay was signi¢cantly enhanced in the PAIRED group (*P 6 0.01, Tukey post hoc test). (d) Mean þ S.E.M. number of Fos-immunoreactive cells in the suprachiasmatic nucleus (SCN) in animals from the PAIRED, UNPAIRED and CONTROL groups that were housed in constant darkness for 10 days and killed 60 min after exposure to the 3-min weak light stimulus at CT15 (n = 8/group).
dark (ZT15) for 10 days. The UNPAIRED group was also presented with the weak light stimulus for 10 days but at di¡erent times of the dark phase each day. The CONTROL group was never exposed to the weak light stimulus during this phase of the experiment. Following this phase, all animals were placed in constant darkness for 10 days and were then presented with the weak light stimulus, once, at CT15 (n = 12/group) or CT21 (n = 4/group). The duration of the weak light stimulus used to test clock resetting was 3 min, a duration short enough to produce sub-maximal phase shifts. The animals continued to freerun in constant darkness for 10 additional days. The ability of the weak light stimulus to reset the clock was assessed by measuring phase shifts in free-running activity rhythms using graphic records (actograms) of wheel-running behavior. Phase shifts were calculated as the di¡erence between the onset of activity before and after exposure to the weak light stimulus (Amir and Stewart, 1996). Statistical analysis was carried out using a one-way ANOVA followed by post hoc comparisons using the Tukey post hoc test (signi¢cance set at P 6 0.05). The e¡ect of the weak light stimulus on Fos expression in the SCN was studied in separate animals from the PAIRED, UNPAIRED and CONTROL groups (n = 8/group). The rats
were housed in constant darkness for 10 days and killed 60 min after they were exposed to the 3-min weak light stimulus at CT15. Immunostaining for Fos was carried out as previously described (Amir and Stewart, 1996) using a mouse monoclonal antibody raised against the N-terminal sequence of Fos (corresponding to N-terminal residues 4^17 of human Fos protein; NCI/BCB Repository, Quality Biotech, Camden, NJ, USA; 1:8000). Estimates of the number of nuclei expressing Fos were made from cell counts performed on serial sections using a computerized image analysis system and the NIH Image software. Mean cell counts on one side of the brain on the ¢ve sections exhibiting the highest number of Fos-immunoreactive cells were taken from each animal. Statistical analysis was carried out using a one-way ANOVA. Signi¢cance was set at P 6 0.05.
AcknowledgementsöThis work was supported by grants from the Canadian Institutes for Health Research, and the Fonds pour la Formation de Chercheurs et l`Aide a© la Recherche (Que¨bec).
NSC 5007 26-6-01
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A. Arvanitogiannis and S. Amir REFERENCES
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