The lower entrainable limit of rat circadian rhythm to sinusoidal light intensity cycles: A preliminary study
Abstract
The lower entrainable limit of the circadian behavioral rhythm was examined in rats exposed to sinusoidal light intensity cycles with maximum illuminance of 20 lux and the minimum of 0.01 lux. The period (T) of the light intensity cycle was initially kept at 23.5 h for 20 cycles, and then shortened to 23 h for 33 cycles. Thereafter the rats were released into constant darkness. Five out of 10 rats entrained their circadian rhythms to T = 23.5-h cycle, and they also entrained to the T = 23-h cycle. The phase angle of entrainment was almost unchanged when T was shortened from 23.5 h to 23 h. These results suggest that the T = 23-h cycle was close to the lower limit of entrainment.
INTRODUCTION
Environmental light–dark alternation is a primary zeitgeber for circadian rhythms in most species. Abrupt alternation of the light period and the dark period by means of lights-on and -off has been commonly used in laboratories for circadian rhythm research. Under this condition, it is generally accepted that entrainment is achieved by resetting of the circadian pacemaker at the lights-on and/or -off time.1,4 In natural conditions, environmental light intensity changes gradually throughout the day. The circadian system has evolved to use daily changes in light intensity as a zeitgeber to adjust circadian phases. However, there have been few studies investigating the role of illuminance changes in the entrainment of circadian rhythms. We previously reported that rat circadian rhythms entrained to sinusoidal light intensity cycles with a period of 24 h.2 In the present study, the period of the sinusoidal light intensity cycles was shortened from 23.5 h to 23 h to assess the lower entrainable limit of rat circadian rhythms.
METHOD
Ten male Sprague-Dawley rats were used. Each animal was housed individually in a stainless steel grid cage with a tilting floor for detecting ambulatory activity, and a drinking spout connected with a drinkometer. The cage was placed in a sound-attenuated chamber with constant temperature (25 ± 1°C) and humidity (40 ± 10%). Laboratory food and water were given ad lib throughout the experiment. Drinking and ambulatory activities were continuously monitored. Light was provided by an incandescent bulb fixed 36 cm above the top of the cage. The light intensity measured at the top of the cage was automatically controlled in 1-min steps by a programmable illuminance-controlling system.2,3
The rats were put under the 12-h light and 12-h dark condition (LD 12 : 12) for 13 days, and then they were exposed to the sinusoidal light intensity cycle with a period (T) of 23.5 h for 20 cycles. Maximum illuminance was 20 lux, and the minimum was 0.01 lux. Thereafter T was shortened to 23 h, and the T-cycle continued for 33 cycles. Then the rats were released into constant darkness (DD) for 50 days. The free-running period in DD was calculated from the data obtained during the last 20 days in DD.
RESULT
Five out of 10 rats entrained their circadian rhythms to T = 23.5-h cycle, and the five rats also entrained to T = 23-h cycle (Fig. 1). Circadian behavioral rhythms in the remaining five rats showed relative coordination or free-running patterns during exposure to the sinusoidal light intensity cycle. The mean τDD± SD was 24.04 ± 0.05 h in the entrained rats (n = 5) and was 24.27 ± 0.12 h in the unentrained rats (n = 5). The mean τDD was significantly longer in the unentrained rats than in the entrained rats (P < 0.05, t-test). The phase angle difference between the light intensity cycle and the entrained activity period was almost unchanged when T was shortened from 23.5 h to 23 h (Fig. 2). In the rats entrained to T = 23 h, their free-running rhythms in DD showed after-effects of entrainment to T = 23 h; circadian periods lengthened gradually after release into DD (Fig. 1).
A double plotted record of drinking activity of a rat. For LD 12 : 12 cycles, rectangles superimposed on the record indicate the light period. During sinusoidal light intensity cycles, superimposed thin lines indicate timings of maximum and minimum illuminance. DD: constant darkness.
Records of (a) ambulatory and (b) drinking activities of a rat exposed to sinusoidal light intensity cycles of T = 23.5 h and T = 23 h, and constant darkness (DD). To visualize easily the phase of circadian rhythm entrained to the T-cycles, both activities were plotted per T-cycle on a time scale of the zeitgeber, where the time of minimum illuminance was defined as 12 h of zeitgeber time.
DISCUSSION
Since 50% of rats entrained to the T = 23-h cycle of sinusoidal light fluctuation, the T = 23-h cycle seems to be close to the lower limits of entrainment. Recently, we examined the lower entrainable limit of rat circadian rhythm to rectangular light–dark (LD) cycles. The LD cycles had maximum illuminance, minimum illuminance, and accumulated illuminance per cycle identical to those of sinusoidal light intensity cycles used in the present study. We found that none of the 10 rats examined could entrain their rhythms to T = 23-h cycle of LD alternation. These results suggest that the sinusoidal light intensity cycle may be a strong zeitgeber compared with LD alternation.
The rats entrained to the T = 23-h cycle had the short mean τDD compared to the unentrained rats. It is expected that a rat with a shorter τDD can entrain to shorter T-cycles. However, the shorter mean τDD observed in the rats entrained to the T = 23-h cycle may reflect the after-effect of entrainment to the light cycle. Thus, it would seem better to measure a τDD before T-experiments.
Several studies suggested that rhythm-entraining properties of sinusoidal light intensity cycles might be different from the LD alternation cycles.2,5 The present results also suggest the difference in rhythm-entraining power between the light intensity cycle and LD alternation. However, it remains to be clarified whether the circadian clock is set only once or twice per day at a certain threshold level of light intensity or if it is continuously modulated by light throughout the light intensity cycle. Further studies are necessary for understanding entrainment by light intensity cycles.
