Ultraviolet-light-evoked phase shifts in the locomotor activity rhythm of the field mouse Mus booduga

Journal o f

Photochemistry and Photobiology B:Biology

E LS EV I E R

Journal of Photochemistry and Photobiology B: Biology 45 (1998) 83-86

Ultraviolet-light-evoked phase shifts in the locomotor activity rhythm of the field mouse Mus booduga V.K. Sharma a,,, M.K. Chandrashekaran a, M. Singaravel b, R. Subbaraj t, ~Chronobiology Laboratory, Animal Behaviour Unit, Jawaharlal Nehru Centrefor Advanced Scientific Research, Jakkur P.O., Jakkur, Bangalore 560 064, India bDepartment of Animal Behaviour and Physiology, School of Biological Sciences, Madurai Kamaraj UniversiO,,, Madurai 625 021, India Received 5 January 1998; accepted 8 July 1998

Abstract

The circadian rhythm of locomotor activity in the nocturnal field mouse Mus booduga has been monitored after an exposure to ultravioletA (UV-A; wavelength between 350 and 400 nm) radiation at various hours of the subject's cycle. The magnitude of the phase shift is used to determine the sensitivity of the circadian pacemaker underlying locomotor activity rhythm. It is observed that UV-A shifts the phase of the locomotor activity rhythm in a phase-dependent manner. A phase response curve (PRC) is constructed to depict the time course and waveform of the basic oscillator's sensitivity to UV-A radiation. The UV-A PRC qualitatively resembles the PRC constructed using diffused daylight as stimulus. Furthermore, the phase response characteristics of the circadian pacemaker for UV-A suggest a periodically varying sensitivity to UV-A similar to that to daylight. © 1998 Elsevier Science S.A. All rights reserved. Kevwords: Circadian rhythms; Phase response curves; Photoreceptors; Ultraviolet-A

I. Introduction

Extraretinal perception of light is a common phenomenon in vertebrates, with the exception of mammals [ 1 ]. In mammals, the retinal photoreceptors are the only known route for perception of light, and blinding results in free-running of their circadian rhythms [ 2,3 ]. It has also been experimentally demonstrated that there is no extraretinal photic entrainment in the diurnal squirrel Funambulus palmaram and the nocturnal mouse Mus booduga [4]. It is also known that in mammals a specialized retinal projection terminating in the suprachiasmatic nucleus (SCN) [ 5,6 ] conveys information about the circadian effects of light. In addition, at the level of photoreceptors, it is known that in some mammals, the action spectrum for entrainment resembles the spectral sensitivity curve for rhodopsin, with the sensitivity peaking in the bluegreen region [ 1,7,8]. These results suggest the involvement of a single class of retinal photoreceptor with rhodopsin-like pigments [9]. On the other hand, the presence of two classes ofphotoreceptors was speculated in the nocturnal cave-dwelling bat, Hipposideros speoris [ 10]. On the contrary, experiments with the nocturnal field mice M. booduga, the model * Corresponding author. Tel.: + 91-80-8462750 to 57 (8 lines); Fax: + 9180-8462766; E-mail: [email protected]

system for the present study, suggested the presence of a single class of photoreceptor [ 11 ]. The phase shifts evoked in M. booduga by three wavelengths of monochromatic light were similar in direction [11]. Furthermore, the phase response curves (PRCs) evoked in response to brief pulses from three different light sources (daylight, fluorescent light and incandescent light) were qualitatively similar. All the three light-pulse PRCs showed regions of delay phase shifts during the early subjective night, advance phase shifts during the late subjective night and minimal phase shifts during the subjective day [12]. However, there were significant differences in the shapes of the PRCs. Both D (area under the delay zone of the PRC) and A (area under the advance zone of the PRC) differed among all three light-pulse PRCs, with all pair-wise comparisons being significant [ 12]. The observed differences in the PRC shapes, when other factors (e.g., sample size, free-running period, duration and intensity of the light pulse, etc.) were comparable in the three PRC experiments, can only be attributed to differences in the spectral composition of the various components of the three light stimuli used. The spectral irradiances of the wavelengths in the visible region in the three light sources used were apparently similar. The only source of the observed differences in the PRC shapes could be wavelengths in either the

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ultraviolet (UV) or infrared region. Light of wavelength beyond 650 nm was found to be safe when used for brief durations [ 12]. We also know that the circadian systems of mice [ 13] and of the golden hamster [14] are sensitive to both green light ( -- 500 nm) and near-UV irradiation. These are the only two previous studies illustrating the sensitivity of the circadian pacemaker to near-UV irradiation. However, it is not known whether the circadian pacemaker is sensitive to these irradiations at all times of the animal's day. Consequently, we undertook the study reported here, in order to investigate the phase-resetting effect of low doses of UV-A (A between 350 and 400 nm) in the locomotor activity rhythm of the nocturnal field mouse M. booduga.

2. Materials and methods Male field mice, M. booduga, were captured from cultivated fields near the Madurai Kamaraj University campus (lat. 9o58 ' N, long. 78010 ' E) and maintained in the laboratory for about 15 days before being used in experiments. Adult male mice (age--90 days; weight 8-13 g) were entrained to light--dark (LD) cycles of 12:12 h (fights on at 06.00 h and lights off at 18.00 h) for 15 days and then released into continuous darkness (DD) for the duration of the experiments wherein their locomotor activity was monitored using an activity running wheel (diameter = 20 cm) attached to a transparent plexiglass cage of dimensions 0.07 × 0.11 × 0.09 m, with a small opening of 0.02 m diameter. Reed-relays attached to the wheels activated the writing stylus of an Esterline Angus A620X Event Recorder when the running mice caused revolutions of the wheel. The activity patterns of as many as 18 mice in separate running wheels, placed on open shelves in the experimental room, could be assayed concurrently. The temperature inside these rooms (3.05 X 2.44 × 4.01 m), which did not have windows but were gently ventilated, remained more or less constant at 25 + I°C and the relative humidity was 75 + 5%. Food (millet and grain) and water were available ad libitum. The room was entered at irregular intervals on an average of once in two days for the purposes of cleaning cages, placing food and water, administering light pulses, etc. Care was taken that the animals were not disturbed during such entries, which seldom lasted beyond 5-10 min. Red light of A > 640 nm, obtained with a combination of red and orange filters fitted to a battery-operated torch light, was used inside the cubicle. Actograms were obtained by pasting 24 h activity/rest strips chronologically one below the other in the standard manner and were double plotted. The onset of activity was used for computation of z during free runs. The individual values of ~"were derived using linear regression on the onsets of locomotor activity, which were clear enough for unambiguous determination. These calculations were based on consecutive samples of data. Phase shifts were computed using two steady states, one prior to light-pulse administration and another following it.

CT12 denotes the onset of activity and hence the onset of subjective night. All phases and phase shifts given in the figures and text are expressed in hours of circadian time (CT) obtained by multiplying absolute (real-time) phases by 24/ ~'. A UV-A lamp (TL-33/12; Philips, Netherlands) was used as the source of light. The light intensity at eye level was measured using a spectroradiometer (IL-700, USA) equipped with a broadband light sensor (Type 400). UV light pulses of 30 min duration and 2.5 W / m 2 intensity were administered to experimental animals at various phases of the circadian cycle. After exposure to UV light pulses, the animals were returned to their running wheels and the recording resumed in constant darkness. Control animals at each tested CT were also transported in light-tight containers of the same size as the plexiglass cages (wrapped additionally in black cloth) in order to establish that UV light pulses per se, and not the disturbances associated with handling, transfer and human interference caused the phase shifts. In order to get an estimate of the variation in D and A within each experiment, the following scheme of resampling was followed. From the six data points at each of the phases available in each experiment, one was chosen at random and a PRC constructed using the 12 data points thus chosen. This sampling process was repeated, with replacement, 12 times, thus generating 12 PRCs per experiment, which, in ram, yielded 12 values of D and A for each experiment. These 12 values of D and A per experiment were used to construct confidence intervals about the estimated mean o l D andA for each experiment. These 24 values of D and A were used as data for the test of significance. Differences among the shapes of the two PRCs (UV-A and diffused-daylight PRCs) were tested for significance by Student's t-test.

3. Results The phase shifts evoked due to a single exposure to UV-A at various phases of the circadian cycle were computed in the field mice M. booduga and a PRC was constructed (Fig. 1). UV-A was observed to shift the phase of the circadian rhythm of locomotor activity in a phase-dependent manner. The PRC constructed for UV-A qualitatively resembled diffused-daylight pulse PRC for the same species. However, the delay amplitude in the diffused-daylight PRC was of much larger magnitude compared to the UV-A PRC (Fig. 1 ). The area under delay differed between the two PRCs, with the pairwise comparisons being significant (p <0.05). The UV-A PRC shows regions of phase delay during the subjective day and early subjective night and regions of phase advance during the late subjective night. In the UV-A PRC, maximum delay responses were obtained by exposing the animals to UV-A at CT14 (Fig. 2, lower panel). A single exposure to UV-A at CT20 evokes maximum advances (Fig. 4, upper panel). The circadian system of the field mice M. booduga responded to UV-A radiation with non-zero phase shift at all tested phases of the circadian cycle

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V.K. Sharma et al. / Journal of Photochemistry and Photobiology B: Biology 45 (1998) 83-86

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Fig. 3. Data for wheel-running activity rhythm of two adult male mice administereda UV-Apulse at CT16 (upper panel) and CT18 (lower panel). The phase shift for the upper panel is -0.88 h, and for the lower panel is + 0.24 h.

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CIRCADIAN TIME Fig. 1. PRCs (phase response curves) constructed for the circadian rhythm in the locomotor activity of the field mouse M. booduga evoked by diffuseddaylight stimuli of 15 min duration and 5.06 W/m2 intensity (O) and UVA stimuli of 2.5 W/m 2 intensity and 30 rain duration (1). Error bars represent 95% confidence interval about the mean phase shift for 6-7 animals.

.,.

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Fig. 4. Data for wheel-running activity rhythm of one adult male mouse administereda UV-A pulse at CT20 ( upper panel) and CT22 (lower panel). The phase shift for the upper panel is +0.80 h, and for the lower panel is +0.64 h.

4. Discussion

¢B >.

Fig. 2. Data for wheel-running activity rhythms of two adult male mice administereda UV-A pulse at CTI2 (upper panel) and CT14 (lower panel). The phase shift for the upper panel is -0.48 h, and for the lower panel -0.92 h. ( Figs. 2 - 4 ) . The controls, wrapped in black light-tight boxes and taken along with the experimental animals to the site of the experiment, did not undergo any phase shift, thus establishing that disturbance stress did not cause any measurable phase shift in the locomotor activity rhythm.

The daylight PRC in the field mouse M. booduga, constructed by subjecting these animals to diffuse-daylight pulses o f 15 min duration and 5.06 W / m 2 intensity, showed amplified delay and advance peaks and shift in the zero transition when compared to the PRCs constructed for fluorescent and incandescent light pulses of comparable intensity and duration [ 12]. Although the shapes o f the three PRCs looked fairly similar, there existed significant differences in the areas under advancing and delaying zones o f the PRCs. Both D and A differed among all the three light-pulse PRCs, with all pair-wise comparisons being significant [ 12]. Such differences in the PRC shapes seem to be of ecological as well as physiological significance and can only be attributed to the differences in the spectral composition o f the various components o f the three light stimuli used. Also, the differences in the amplitudes o f the PRCs might have arisen due to lowdose UV radiation in the daylight treatment. W e investigated the phase-shifting effects o f U V - A on the circadian rhythm in locomotor activity of the field mouse M.

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V.K. Sharma et al. / Journal of Photochemistry and Photobiology B: Biology 45 (1998) 83-86

booduga and

found that the phases of their circadian pacemakers were influenced by UV-A in a phase-dependent manner (Fig. 1 ). McGuire et al. [ 8 ] reported that the circadian rhythm in body temperature of rats entrains to near-UV irradiations of appropriate intensity under an LD schedule of 12:12 h. Some recent studies have also shown that the circadian systems of mice [13] and the golden hamster [ 14] are sensitive to both green light ( = 500 nrn) and near-UV irradiation. Taken together with the results of our experiments, this strongly suggests that the circadian systems of mammals are sensitive to UV-A irradiation.

Acknowledgements Financial support from the Jawaharlal Nehru Centre for Advanced Scientific Research to V.K.S., the Centre for Scientific and Industrial Research to M.S., the Indian Council of Medical Research to R.S., and the Department of Science and Technology, Government of India, to M.K.C. is acknowledged. We are also grateful to two anonymous referees and Dr Amitabh Joshi for critically reading and suggesting improvements in the manuscript.

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[3] R.J. Nelson, I. Zucker, Absence of extraocular photoreception indiurnal and nocturnal rodent exposed to direct sunlight, Comp. Biochem. Physiol. 69A ( 1981 ) 145-148. [4] K. Navaneethakannan, P. Kumarasamy, Absence of extraretinal photic entrainment in a diurnal squirrel Funambulus pabnarum and a nocturnal mouse Mus booduga, Indian J. Exp. Biol. 24 (1986) 730731. [5] R.Y. Moore, Central neural control of circadian rhythms, in: W.F. Ganong, L. Martini (Eds.), Frontiers in Neuroendocrinology, vol. 5, Raven Press, New York, 1978, pp. 185-206. [6] B. Rusak, I. Zucker, Neural regulation of circadian rhythms, PhysioL Rev. 59 (1979) 449-526. [7] S.A. Gordon, G.A. Brown, Observations on spectral sensitivities for the phasing of circadian temperature rhythm in Perognathus penicillatus, in: M. Menaker (Ed.), Biochronometry, Natl. Acad. Sci., USA, Washington, DC, 197 l, pp. 363-371. [8] R.A. McGuire, W.M. Rand, R.J. Wurtman, Entrainment of the body temperature rhythm in rats: effect of color and intensity of environmental light, Science 181 (1973) 956-957. [9] J.W. Hastings, B. Rusak, Z. Boulos, Circadian rhythms: the physiology of biological timing, in: Neural and Integrative Animal Physiology, Wiley-Liss, Amsterdam, 1991, pp. 535-546. [ 10l D. Joshi, M.K. Chandrashekaran, Spectral sensitivity of the photoreceptors responsible for phase shifting the circadian rhythm of activity of the bat Hipposideros speoris, J. Comp. Physiol. 156 (1985) 189198. [ l l I L. Geetha, R. Subbaraj, Green light evokes maximum phase shifts in the locomotor activity of the field mouse Mus booduga, J. Photochem. Photobiol. B: Biology 33 (1995) 79-82. [ 12] V.K. Sharma, M.K. Chandrashekaran, P. Nongkynrih, Daylight and artificial light phase response curves for the circadian rhythm in locomotor activity of the field mouse Mus booduga, Biol. Rhythm. Res. 28 (1997) 39-49. [ 13] I. Provencio, R.G. Foster, Circadian rhythms in mice can be regulated by photoreceptors with cone-like characteristics, Brain Res. 694 (1995) 183-190. [ 14] M. yon Schantz, S. Argamaso-Hernan, A. Szel, R.G. Foster, Photopigments and photoentrainment in the Syrian hamster, Brain Res., in press.