in the field mouse mus booduga melatonin phase response curves (prcs) have a different time course...

J Pirirul Res 1999; ,76:153-157 Printed in Irrlund-uN rights resercrd.

In the field mouse Mus booduga melatonin phase response curves (PRCs) have a different time course and wave form relative to light PRC

Sharma VK, Chandrashekaran MK, Singaravel M, Subbaraj R. In the field mouse Mus boodugu melatonin phase response curves (PRCs) have a different time course and wave form relative to light PRC. J. Pineal Res. 1999; 26: 153- 157. Q Munksgaard, Copenhagen

Abstract: The phase shifting effects of the pineal hormone melatonin on the circadian locomotor activity rhythm of the field mouse Mus boodugu was examined at various phases of the circadian cycle using single melatonin injections of two concentrations ( 10 mg/kg, high dose; and 1 mg/kg, low dose) and two phase response curves (PRCs) were constructed. A single dose of melatonin administered during the early subjective day evoked maximum phase delays, and during the late subjective night evoked phase advances in the locomotor activity rhythm. Other phases of the circadian cycle also responded to melatonin. The interval between circadian time 19 (CT19) and CT2 of the high dose melatonin PRC is marked by significant phase advances, whereas the interval between CT2 and 19 is marked by significant phase delays. A single dose of melatonin of strength 10 mg/kg was found to evoke phase shifts that were of comparable magnitude to those of the phase shifts evoked by natural daylight pulses. Control animals, treated with 50%) dimethyl sulfoxide (DMSO), did not respond with phase shifts significantly greater than zero. Significant differences between the shapes of the two melatonin PRCs exist. Further melatonin PRCs appear to have a different time course and wave form relative to light-induced PRC.

Vijay Kumar Sharma', Maroli K. Chandrashekaran', Muniyandi Singarave12 and Ramanujam Subbaraj' 'Chronobiology Laboratory, Evolutionary and Organismal Biology Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Jakkur, Bangalore-560064, Kamataka; 'Department of Animal Behaviour and Physiology, School of Biological Sciences, Madurai Kamaraj University, Madurai-625 021, Tamilnadu. India

Key words: circadian rhythm - locomotor activity - melatonin - mice - phase response curve

Address reprint requests to V.K. Sharma, Chronobiology Laboratory, Evolutionary and Organismal Biology Unit. Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Jakkur, Bangalore-560064, Karnataka, India. E-mail: vsharmaQjncasr.ac.in

Received June 2, 1998: accepted August 31, 1998

Introduction

The influence of melatonin on circadian rhythms and the presence of melatonin receptors in the suprachiasmatic nucleus (SCN) suggest a direct action of the hormone on the circadian pacemaker [Cassone, 1990; Krause and Dubocovich, 1990; Reppert et al., 19941. Recurrent 24 hr administration of melatonin entrained the circadian activity rhythm in several species of mammals [Armstrong et al., 1986; Illnerova, 1991; Margraf and Lynch, 1993; Redman, 19971. A single dose of melatonin was also found to shift the phase of the activity rhythm in C3H/HeN mice [Benloucif and Dubo- covich, 19961, rats [Armstrong et al., 19891, and the sleep-wakefulness rhythm of humans [Lewy et al., 1992, 1,9981 according to a distinct phase response curve (PRC).

Although it is known that melatonin functions as a potential zeitgeber for the circadian timing system of mammals, sensitivity to melatonin has so far been systematically studied only in mice [Benloucif and Dubocovich, 19961, rats [Arm- strong et al., 19891, and humans [Lewy et al., 1992, 19981. In most of these previous studies on mammals, melatonin was observed to be effective during the late subjective day and late subjective night, with a diametrically opposite effect to that of light at the transitions of dusk and dawn [Arm- strong et al., 1989; Benloucif and Dubocovich, 1996; VanReeth et al., 19941. In rats melatonin was found to be effective only before the onset of activity [Armstrong et al., 19891. Hence, the circadian effects of melatonin appear to be diverse in mammals. Furthermore, the magnitude of phase shifts in all these studies was never greater than 1

1 53

Sharrna et al.

hr. In order to detect phase shifts of such small magnitude, the assay rhythm would have to be very precise. Moreover, the role of different doses of melatonin on the PRC shape remains an open question. We report effects of melatonin in two pharmacological doses (10 and 1 mg/kg) at various phases of a mammalian circadian system.

The field mouse Milts hooduga was chosen for the present study because of its precise, robust, and easily measured circadian rhythm in locomotor activity. The response of activity rhythm to light stimuli has also been well studied in these mice [Sharma, 1996; Sharma and Chan- drashekaran, 1997; Sharma et al., 19971. The light- induced PRC is of type-1, with delays dominating during early subjective night, advances during late subjective night, and minimal but non-zero phase shifts during the subjective day [Sharma et al., 19971. Moreover, administration of melatonin to these mice at critically timed phases has been found to accelerate reentrainment to shifted light/ dark (LD) cycle [Singaravel et al., 19961.

Materials and methods

Male field mice (n = 216) 111. boodzrga, were cap- tured from cultivated fields near the Madurai Ka- inaraj University campus ( 9 9 8 ' N lat., 78"lO' E long.) and maintained in the laboratory for 2 15 days before being used in experiments. Adult male mice (age 2 90 days; weighing 8-13 g) were entrained to L D cycles of 12:12 hr (lights on at 06.00 hr and lights off at 18.00 hr) for 15 days and then released into continuous darkness ( D D ) for the duration of the experiments, wherein their locomotor activity was monitored using an activity running wheel (diameter 2 2 0 cm) attached to a transparent plexiglass cage of dimension 0.07 < 0.1 1 K 0.09 m, with a small opening of 0.02 m diameter. Fluorescent daylight (Philips, 240 V, 20 W, 6500 I() was used as the source of light during the LD cycle. Reed-relays attached to the wheels activated the writing stylets of an Esterline Angus A620X Event Recorder when the running mice caused revolutions of the wheel. The activity pat- terns of 18 mice in separate running wheels, placed on open shelves in the experimental room, could be monitored concurrently. The room was pro- tected from all types of external interference. The temperature inside these experimental rooms (3.05 x 2.43 x 4.01 m), which did not have win- dows but were gently ventilated, remained constant at 25 k 1°C and the relative humidity was 75 5 ' % ~ Food (millet and grain) and water were available ud libitum. The room was entered at irregular intervals on an average of once in two

1 54

days for purposes of cleaning cages, placing food and water, administering melatonin injections, etc. Care was taken that the animals were not dis- turbed except for inevitable entries that seldom lasted beyond 5 - 10 min. Red light of h > 640 nm obtained with a combination of red and orange filter fitted to a battery-operated torchlight was used inside the cubicle.

Circadian time 12 (CT12) denotes the onset of activity, and hence onset of subjective night. All phases and phase shifts given in the text are ex- pressed in hours of CT obtained by multiplying absolute (real time) phases by 24/2. After the at- tainment of a steady state free-run, z and phase shifts were calculated using linear regression lines through the onsets of activity. A minimum of 15 days of free-run was taken into account for such estimations. The average T of the animals used in the present experiment was 23.73 0.23 hr (meankS.D.) . One group of animals ( n = 7 2 ) kept in DD were then administered 2 pL of single subcutaneous (sc) injections of melatonin ( 10 mg/ kg, high dose). Another set of animals (n = 72) were also administered 2 pL of melatonin (sc) injections but of lower dose ( 1 mg/kg, low dose). Control animals (n = 72) also maintained under D D were administered the vehicle (50':<, dimethyl sulfoxide (DMSO)) at each tested CT i n order to estimate possible effects of DMSO, disturbances associated with handling, transfer, human interference, etc. Each mouse was given only one injection. At each of the twelve phases selected for injection, six animals were used. Melatonin was procured from Sigma, St Louis, MO. It was dis- solved in 500/0 DMSO. Golombik and Cardinali's protocol for preparation and the dosage of melatonin were followed in the present study [Goloni- bik and Cardinali, 19931.

The comparisons among the melatonin PRCs and also with the vehicle PRC were done using the area under delay ( D ) and advance (A) zones of the PRCs. In order to get an estimate of the variation in D and A within each experiment, the following scheme of resampling was followed [Sharma et al., 19971. From the six data points at each phase available in each experiment (melatonin: 10 mg/ kg, 1 mg/kg, and 50'% DMSO), one was chosen at random and a PRC constructed using the 12 data points chosen. This sampling process was re- peated, with replacement, 12 times, thus generat- ing 12 PRCs per experiment, which, in turn, yielded 12 values of D and A for each experiment. These 12 values of D and A per experiment can be used to construct confidence intervals about the estimated mean of D and A for each experiment. These 36 values of D and A were used as data for

Melatonin PRCs

one-way analysis of variance (ANOVA), treating experiment (dose type) as a fixed factor. Differ- ences among the shapes of the two PRCs were tested for significance by Scheffe's test (with family type I error rate fixed at 0.05).

Results

The magnitude and direction of phase shifts induced by single melatonin injections of two concentrations (10 and 1 mg/kg) were computed in the field mice M. booduga, and PRCs constructed (Fig. 1). The effects of the vehicle injection (50% DMSO) were also estimated in separate experiments at various phases of the circadian cycle (Fig. 1). Melatonin was observed to shift the phase of the circadian rhythm of locomotor activity in a phase-dependent manner. High dose of melatonin (10 mg/kg) injected at phases between CT2 and 19 evoked phase delays, whereas melatonin injected at phases between CT19 and 2 evoked phase advances. However, maximum delay responses were obtained by administering melatonin at CT4. PRC constructed using low dose of melatonin (1 mg/kg) did not show significant responses other than at

2 t

I I I 1 I I

0 4 0 12 16 20 24

CIRCADIAN TIME

Fig. 1. Phase response curve (PRC) evoked by a single dose of melatonin (10 mg/kg, a), ( I mg/kg, 0 ) and 50% DMSO ( 0 )

for the circadian rhythm in the locomotor activity of M. baoduga. 0-12 CT (circadian time), subjective day; 12-24 CT, subjective night. Error bars about the mean phase shifts a t each phase of: the circadian cycle represent 95% confidence intervais and can be used for statistical convention of visual hypothesis testing.

TIME OF DAY(ht1

b

Fig. 2. Data for wheel-running activity rhythms of two adult male mice administered 10 mg/kg of melatonin at CT14 (A) and CT4 (B).

three phases of the circadian cycle (CT4, 14, and 22). The delay responses of one animal each for the two doses (10 and 1 mg/kg) at CT14 and 4 are illustrated in Figs. 2 and 3, respectively.

A single dose of melatonin administered at CT22 elicited maximum advances. Such phase advances of one animal each for the two doses (10 and 1 mg/kg) at CT20 and 22 are illustrated in Figs. 4 and 5, respectively. The control animals treated with 50% DMSO also responded with small phase shifts (Fig. 1).

The areas under D and A zones of the high dose melatonin PRC (1 0 mg/kg) were 13.1 1 k 3.29 and 4.43 f 0.78 hr2 as compared to the same for the

TIME OF DAY (hr.) 0000 1200 0000 1200 0000

I I

a I I I I 1


155

Sharma et al.

TIME OF DAY(hr.1

ul

r I I I


low dose melatonin PRC ( 1 mg/kg) ( D = 4 . 6 0 + 1.63 hr2; A = 3.39 f 0.84 hr') and vehicle PRC (50% DMSO) (D = 0.13 & 0.20 hr'; A = 0.28 + 0.27 hr2). The area under D zone of the PRCs differed among the two melatonin and thc vehicle PRCs, with all pair-wise comparisons being significant (Scheffe's test, minimum significant difference = 2.22, P < 0.0001). The area under A zone of the PRCs also differed among the two melatonin and the vehicle PRCs, with all pair-wise comparisons being significant (Scheffe's test, minimum significant difference = 0.7 1, P < 0.0001). Al- though the maximum phase delay and advance

TIME OF DAY (hr.) oom 12m oom 124 00"

I I 1

a a

20

I I I I I


occur in the early subjective day and late subjective night, respectively, the circadian pacemaker of M . hooduga remains sensitive to melatonin throughout the subjective day, thus exhibiting a lack of a distinctive refractory zone. A higher dose of melatonin (10 mg/kg) evokes phase shifts of magnitude comparable to the magnitude of light- induced phase shifts (Sharma et al., 1997).

Discussion

Previous reports [Benloucif and Dubocovich, 19961 suggest species differences in sensitivity to melatonin. In rats, melatonin administrated at CTlO is known to evoke phase advance, but a single dose of melatonin at dawn (CT22-2) remains ineffective [Armstrong et al., 1989; Warren et al., 19931. On the contrary. sensitivity to melatonin at dawn in the rat SCN is supported by melatonin-induced alteration in metabolic activity in vitro [Cassone et al., 1988; Gillette and McArthur. 1996; McArthur et al., 19971. In humans, melatonin PRC was found to be nearly a mirror image of light pulse PRC [Lewy et al., 19921. Recently, Lewy et al. [1998] have reported that in humans melatonin PRC is z 12 hr out of phase with the light PRC. On the contrary, the circadian system of the nocturnal field mouse M . hoodugu responds with phase delays during the early subjective day, and phase advances during the late subjective night. The considerably large variability in the melatonin PRCs may be a conse- quence of the variation among individuals in the responses to melatonin and may also be due to human introduced variation in the process of administering injections. When compared with the light-induced PRC in these mice, the melatonin PRC appears to have a different time course and wave form relative to the light-induced PRC. Other factors such as average free-running period and experimental conditions remaining comparable in the melatonin and light-induced PRCs - such differences can only be attributed to the stimuli used. Taken together with our observa- tions on M . booduga reported here, it appears that circadian effects of melatonin in mammals are diverse. The cause for such differences may be due to existing differences in melatonin binding sites within the SCN, and/or profiles of endogenous melatonin rhythm [Margraf and Lynch, 19931.

In the present study, the circadian pacemaker of the field mouse M . booduga (Fig. 1) responded to a single dose of melatonin with a PRC exhibiting both advance and delay phase shifts. The locomotor activity rhythm of these mice free-running in constant darkness exhibited an average t of

156

Melatonin PRCs

23.73 f 0.23 hr (mean f S.D.). Therefore, in order to facilitate entrainment of the locomotor activity rhythm in these mice, using a single dose of melatonin, a recurrent phase delay of ca. 0.23 hr would be needed. As can be seen in the melatonin-induced PRC, there exist phases at which a single dose of melatonin can phase delay up to ca. 2.96 hr. This supports the fact that melatonin may also function as a potential zdtgeber for the circadian timing system of the field mice M. booduga.

Acknowledgments

Financial support from Jawaharlal Nehru Centre for Ad- vanced Scientific Research to VKS and M K C is acknowledged.

Literature cited

ARMSTRONG, S.M., V.M. CASSONE, M.J. CHESWORTH, J. REDMAN. R.V. SHORT (1986) Synchronization of mammalian circadian rhythms by melatonin. J. Neural Transm.

ARMSTRONG, S.M., E.M.V. THOMAS, M.J. CHESWORTH (1989) Melatonin induced phase-shifts of rat circadian rhythms. In: Advances in Pineal Research, D. Evered, S. Clark, eds. John Libbey, London, pp. 263-274.

BENLOUCIF, S., M.L. DUBOCOVICH (1996) Melatonin and light induce phase shifts of circadian activity rhythms in the C3H/HeN mouse. J. Biol. Rhythms 11:113-125.

CASSONE, V.M., M.H. ROBERTS, R.Y. MOORE (1988) Effects of melatonin on 2-deoxy-[l-I4C] glucose uptake within rat suprachiasmatic nucleus. Am. J. Physiol. 255:331-337.

CASSONE, V.M. (1990) Effects of melatonin on vertebrate circadian systems. TINS 13:457-464.

GILLETTE, M.U., A.J. MCARTHUR (1996) Circadian actions of melatonin at the suprachiasmatic nucleus. Behav. Brain. Res. 73:135-139.

GOLOMBIK, D.A., D.P. CARDINALI (1993) Melatonin acceler- ates re-entrainment after phase advance of the light/dark cycle in Syrian hamsters: Antagonism by flumazenil. Chronobiol. Internat. 10:435-441.

ILLNEROVA, H. (1991) The suprachiasmatic nucleus and rhyth- mic pineal melatonin production. In: Suprachiasmatic Nu- cleus: The Mind Clock, D.C. Klein, R.Y. Moore, S.M. Re- ppert, eds. Oxford University Press, New York, pp. 197-216.

2 1 : 375 - 394.

KRAUSE, D., M.L. DLJBOCOVICH (1990) Regulating sites on the melatonin system of mammals. TINS 1332-95.

LEWY, A.J., S. AHMED, J.M.L. JACKSON, R.L. SACK (1992) Melatonin shifts human circadian rhythms according to a phase response curve. Chronobiol. Internat. 9:380-392.

LEWY, A,J., V.K BAUER, S. AHMED, K.H. THOMAS, N.L. CUTLER, C.M. SINGER, M.T. MOFFIT, R.L. SACK (1998) The human phase response curve (PRC) is about I2 hours out of phase with the -PRC to light. Chronobiol. Internat.

MARGRAF, R.R., G.R. LYNCH (1993) 'Melatonin injections affect circadian behaviour and SCN neurophysiology in Djungarian hamsters. Am. J. Physiol. 264:615-621.

MCARTHUR, A.J., A.K. HUNT, M.U. GILLETTE (1997) Mela- tonin action and signal transduction in the rat suprachiasmatic circadian clock: Activation of protein kinase C at dusk and dawn. Endocrinology 138:627-634.

REDMAN, J.R. (1997) Circadian entrainment and phase shifting in mammals with melatonin. J. Biol. Rhythms 12:581- 587.

REPPERT, S.M., D.R. WEAVER, T. EBISAWA (1994) Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses. Neuron 13:1177-1185.

SHARMA, V.K. (1996) Light-induced phase response curves of the circadian activity rhythm in individual field mice Mus boodugu. Chronobiol. Internat. 13:401-409.

SHARMA, V.K, M.K CHANDRASHEKARAN, P. NONGKYNRIH (1997) Daylight and artificial light phase response curves for the circadian rhythm in locomotor activity of the field mouse Mu3 boodugu. Biol. Rhythm Res. 28:39-49.

SHARMA, V.K., M.K. CHANDRASHEKARAN (1 997) Rapid phase resetting of a mammalian circadian rhythm by brief light pulses. Chronobiol. Internat. 14537-548.

SINGARAVEL, M., V.K SHARMA, R. SUBBARAJ, N. GOPUKU- MAR NAIR (1996) Chronobiotic effect of melatonin following phase shift of light/dark cycles on the locomotor activity rhythm of the field mouse Mus boodugu. J. Biosci. 21:789- 795.

VANREETH, O., Y . OLIVARES, F.W. ZHANG, F.W. TUREK, R. DEFRANCE, E. MOCAER (1994) S-20098, a melatonin-ago- nist, phase shifts the circadian clock of mice and Syrian hamsters according to a phase response curve. SOC. Neurosci. Abstr. 20:1440.

WARREN, W.S., D.B. HODGES, V.M. CASSONE (1993) Pinealectomized rats entrain and phase shift to melatonin injections in a dose-dependent manner. J. Biol. Rhythms 8233-245.

15:7 1 -84.

157

in the field mouse mus booduga melatonin phase response curves (prcs) have a different time course...

Documents