www.elsevier.com/locate/brainres

Brain Research 1048

Research report

Caloric restriction and melatonin substitution: Effects on murine

circadian parameters

David Resuehr1, James Olcese*

Institute for Hormone and Fertility Research, Centre for Innovative Medicine, University of Hamburg, Falkenried 88, 20251 Hamburg, Germany

Accepted 22 April 2005

Available online 23 May 2005

Abstract

Aging effects have been reported in endocrine, metabolic and behavioral circadian rhythms. The effects of age on the circadian system

have been investigated primarily in rats and hamsters and only seldom in mice. Our aim was to assess the effects of two common ‘‘anti-

aging’’ treatments, namely caloric restriction (CR) and melatonin substitution, on the circadian system of mice. Animals were subjected to

phase delays of the light–dark cycle and constant darkness (DD). The most pronounced change in the murine circadian system was the length

of the endogenous period, s, which increased with age regardless of treatment. CR had diverse effects e.g., enabling a more rapid phase shift

response while concomitantly leading to a fragmented circadian phenotype with considerable activity during the rest (light) phase. Melatonin

enforced the adaptation to the light/dark cycle, thus facilitating a rapid reentrainment to phase delayed lighting conditions. Interestingly, the

melatonin-substituted animals displayed an increase in locomotor activity under constant darkness and in 50% of all cases a biphasic (split)

activity pattern. These results contribute to the phenotypic evaluation of two very different approaches to intervene in the age-related

degeneration of the mammalian circadian system. As both CR and melatonin have negative and positive effects on the behavioral expression

of clock function (i.e., fragmentation of rhythms vs. faster reentrainment), their usefulness in managing age-related circadian disorders may

be limited.

D 2005 Elsevier B.V. All rights reserved.

Theme: Neuronal basis of behavior

Topic: Biological rhythms and sleep

Keywords: Melatonin; Caloric restriction; Circadian period s; SCN; Locomotor activity; DD; LD

1. Introduction

The synchronization of cellular, physiological and/or

behavioral rhythms to a cyclically changing environment is

adaptive, ubiquitous and probably evolved early in the

history of life. One of the most prominent rhythms among

animals is the circadian day/night activity rhythm [34].

0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2005.04.063

* Corresponding author. Current address: Department of Biomedical

Sciences, Florida State University College of Medicine, Tallahassee, FL

32306, USA. Fax: +1 850 644 5781.

E-mail address: james.olcese@med.fsu.edu (J. Olcese).

URL: http://www.med.fsu.edu (J. Olcese).1 The data represent a portion of the doctoral dissertation of D.R.

submitted to the University of Hamburg, Department of Biology.

In the aging animal, the circadian system undergoes

many changes at multiple levels. Adaptation to altered

daylengths takes longer, activity rhythms are more frag-

mented and the endogenous circadian period (s) changes ascompared to young animals [21,29]. Disruptions in the

circadian system lead to a reduced life expectancy in many

animal models [11,15,16].

Melatonin has diverse biological functions, one of which

is to serve as an output signal from the circadian clock [26].

In many animal species, it also controls more slowly

changing processes, such as fertility and fur growth (e.g.,

in hamsters, sheep, [12,37]). The synthesis and release of

pineal melatonin are greatly reduced in the aging individual

when compared to youthful secretory levels [25]. Melatonin

substitution in aging animals increases the maximum life

(2005) 146 – 152

D. Resuehr, J. Olcese / Brain Research 1048 (2005) 146–152 147

span [31] and can reduce the severity of other senescence-

accompanied changes [17,33].

Another approach toward reducing age-related physical

degeneration is caloric restriction (CR) which has been

shown in many model systems to delay the onset and

severity of age-related diseases [41,42]. However, the effect

of CR on the circadian system has not been investigated at

the behavioral level in terms of free-running periods or in

terms of reentrainment efficiencies after phase shifting nor

in terms of a comparison to melatonin effects on these

parameters [8,9]. In the present study, mice were maintained

for 10 months (beginning at 5 months of age) under one of

three treatment groups: CR, melatonin substitution or a

controlled normocaloric diet. During this time entrainment

to light:dark (LD) cycles, as well as free-running periods

under constant darkness (DD) and phase shifting efficiency

were assessed in all groups.

2. Materials and methods

2.1. Animals

Mice of the hybrid strain B6C3F1 were obtained at the

age of 6 weeks (N = 80) (Charles River, Wilmington, MA).

This strain has often been used in studies of aging [7,18]. In

contrast to many other laboratory mouse strains [13], the

B6C3F1 strain (derived from a C57BL/6 � C3H cross) is

melatonin-proficient (Olcese et al., unpublished). Young

control mice (N = 20) were kept for longitudinal assessment

of locomotor activity during the course of the study.

Additionally, beginning at 5 months of age, another 60

mice were weighed and sorted into three groups of 20

animals each: controls (CT), melatonin-substituted and CR.

All groups of mice had the same average initial body

weight. Body weight was then measured weekly for the first

14 weeks and every 2 weeks thereafter to assess the impact

of CR.

Animals were housed individually in Plexiglas cages

with overhead infrared sensors to record locomotor activity

(see below for details). Ambient humidity was 68%, and

room temperature was 20 T 2 -C. At the beginning of the

experiment, the light cycle was set to 06:00 h lights on and

18:00 h lights off. For subsequent experimental purposes,

the light cycle was occasionally modified as described

below. Periods of constant darkness were imposed for a

minimum of 3 weeks to determine endogenous period s.

2.2. Feeding

Specially formulated mouse chow was purchased from

Teklad (Madison, Wisconsin). Control and melatonin-

substituted animals were fed standard chow (AIN-93M

adult maintenance precision pellets). CR animals were fed

an especially prepared, vitamin- and mineral-enhanced

restriction diet (based on AIN-93M) to assure proper

nutrition in the face of caloric restriction. This diet has

been employed frequently in studies on the effects of caloric

restriction [5,18,42]. At the beginning of the experiment,

feeding was ad libitum. The amount of food consumed on a

daily basis was assessed by offering each individual a pre-

weighed amount and measuring the uneaten amount

remaining on the following day. The average amount

consumed was 4.3 g/day which was defined as 100%.

Thereafter, food was provided three times a week (a

commonly used feeding strategy [6]). On Monday and

Wednesday, a 2-day portion was given, while on Friday the

mice were fed a 3-day amount of chow. In the case of the

CR mice, initial food amounts were reduced biweekly by

20% until a level of 60% was achieved which was then

maintained over the course of the study (65.8 kcal/week).

The melatonin mice received the same amount of food as

the control mice, whereas the former received melatonin in

their drinking water at a concentration of 5 Ag/ml.

Melatonin (Sigma, St. Louis, MO) stock solutions were

dissolved in 96% ethanol which were then diluted 1:1000 in

water. The CR and CT animals also received alcohol in their

drinking water (0.1%) as a vehicle control. The drinking

bottles were painted black to protect melatonin from

photodegradation.

2.3. Locomotor activity recordings

To record individual locomotor activity, passive infrared

detectors (Conrad-Elektronik, Art.-Nr.192236-3F) were

mounted on top of all cages (N = 20 per group). The

exact timing of the light–dark cycle in the facilities was

recorded with a photodiode (Conrad-Elektronik, Art.-Nr.

606863-3F). Locomotor activity and ambient lighting

signals were measured as TTL-Signals (electrical currents

between 0 V and 5 V) and relayed to a personal computer

(IBM PC 286, 500 MB HDD, 32 MB RAM) via a digital

I/O-Card (Conrad-Elektronik, Model Nr. PIO 24/48 II).

The absence of activity ‘‘0’’ was defined as 0–0.8 V,

whereas 2.4–5 V was defined as ‘‘1’’—or activity. A

refractory period of 7 s after each signal was established in

the computer program, during which time no further

signals could be recorded. Signals were recorded in 6

min bins per recording interval, i.e., 240 intervals per day.

The recording and analysis of locomotor activity and the

determination of endogenous period s (using a chi square

periodogram) were performed with a Quickbasic computer

program written by Dr. Haiko Dernbach (Tiermedizinische

Hochschule Hannover, Germany). Reentrainment analyses

after phase shifts were conducted by calculating the

amount of activity in the 40 6 min bins immediately

following the onset of darkness before and after shifting. A

threshold level was determined by forming the average

activity under normal LD conditions i.e., before the shift

(N = 20 animals per tested group). Attainment of this

amount of nocturnal activity after shifting was considered

as the threshold for reentrainment.

Fig. 1. Total average activity of animals from different treatment groups at

the age of 14 months (N = 10 per group) under LD (12 h light: 12 h dark) or

DD (constant dark) conditions. Night LD refers to nocturnal activity under

LD, whereas day LD refers to daytime activity. Note the higher total

activity of melatonin mice under DD (P < 0.05) and the elevated daytime

activity of CR mice under LD (P < 0.05). The data represented here are

derived from the electronic files displayed in actogram form in Fig. 2.

Fig. 2. Representative actograms of mouse locomotor activity at the ages of 7 (A–

DD (*) and subsequent reentrainment (triangle) (A–C). (A) Seven-month-old

melatonin-treated animal displaying biphasic activity. In panels (D–F), the light p

(arrow Y). The DD in all actograms began at the * and lasted until the reentrainm

(E) CR animal aged 11 months. (F) Melatonin animal aged 11 months.

D. Resuehr, J. Olcese / Brain Research 1048 (2005) 146–152148

Statistical data analysis was performed on a Dell

Dimension 2400\ PC using GraphPad Prism\ and MS

Excel\.

3. Results

The untreated CT group had an average body weight of

29.1 T 0.4 g, the melatonin-substituted animals had average

body weight of 29.4 T 0.4 g and the CR animals had an

average body weight of 28.6 T 0.3 g at the beginning of the

experiment. At the end of the experiment (40th week), the

control animals had an average body weight of 41.0 T 0.9 g

and the melatonin-substituted animals had an average body

weight of 35.0 T 0.9 g. The weight difference at the end of

the experiment between the CT and melatonin groups was

statistically significant after a two sided t test (P < 0.001).

The body weight of the CR animals decreased to 20.5 T 0.3

g in the 10th week and increased thereafter to 24.1 T 0.4 g inthe 40th week (P < 0.001).

The activity in all groups of mice in the dark phase was

higher than during the light phase (Fig. 1, P < 0.05).

However, the nocturnal activity of the CR animals was a

little lower (�11%, P < 0.05), while the daytime activity

was higher (+43%, P < 0.05) when compared to CT or

C) and 11 months (D–F). The light cycle was 06:00–18:00 h followed by

control animal. (B) Seven-month-old CR animal. (C) Seven-month-old

eriod was 10:00–22:00 h followed by a 4 h phase delay to 14:00–02:00 h

ent to the pre-DD conditions (triangle). (D) Control animal aged 11 months.

D. Resuehr, J. Olcese / Brain Research 1048 (2005) 146–152 149

melatonin animals. The total activity increased (+26.3%,

P < 0.05) during DD conditions in the melatonin-treated

animals (Fig. 1). There was no significant inter-group

difference in total activity between the examined groups

of the same age (Fig. 1, LD 24 h). Older control mice

(21 months) displayed significantly less activity under all

lighting conditions (data not shown).

CT animals displayed a robust circadian rhythmicity

under LD conditions and a free-running period <24 h in DD

(Figs. 2A and D). During LD conditions, but even more so

during DD conditions, there was a tendency for the main

bout of activity to split slightly into two separate bouts. The

major trait of the CR group was a strongly fragmented

activity rhythm with significant activity during the dark/

light transition (Figs. 2B and E upper portion of picture,

above * and Y). As was the case with control animals, CR

mice individuals often displayed two activity peaks, which

Fig. 3. Reentrainment after a 4 h phase delay in CT (A), CR (B) and

melatonin (C) mice. The letter B on the abscissa of each graph indicates the

average nocturnal activity in each group before the phase shift. The letters

A1 to A5 indicate the average nocturnal activity on the first five

consecutive days after applying the phase shift. Untreated CT animals

reached their average amount of nocturnal activity by the third night after

the phase shift (*P < 0.05). CR animals achieved resynchronization by the

second night after the phase delay (*P < 0.05), melatonin animals were also

resynchronized by the second night (*P < 0.05).

drifted apart under DD conditions (Fig. 2E). The main

characteristic of the melatonin-substituted animals was a

very robust entrainment to the LD cycle (Fig. 2F) and a

rapid adjustment to changes in LD phase. About 50% of the

animals treated with melatonin displayed a very strong

biphasic behavior (Fig. 2C) after onset of DD. A splitting of

the activity bouts with this severity was only observed in the

melatonin-treated group.

The effects of a 4 h phase delay (depicted in Figs. 2D, E,

F at the Y) were evaluated statistically to determine if CR

or melatonin treatment modulated the time necessary to

resynchronize to the novel lighting conditions (Fig. 3).

Untreated animals (Fig. 3A: CT) reached the pre phase shift

level of nocturnal activity (the criterion for reentrainment)

by the third night after the phase shift. CR animals (Fig. 3B)

achieved resynchronization by the second night after the

phase delay. The locomotor activity of CR continued to rise

from the second to fourth night before dropping to normal

levels (data not shown). Melatonin animals (Fig. 3C) also

resynchronized within two nights, comparable to the CR

animals.

The endogenous free-running period (s) under DD was

shorter than 24 h in all mice of the strain B6C3F1. In all

treatment groups, we observed a significant increase of swith age (Fig. 4). Neither melatonin nor CR reversed or

attenuated the lengthening of s, although the circadian clock

tended to run a little slower in CR mice compared to CT and

melatonin (two sided t test, N = 10, P < 0.05).

4. Discussion

The starting weight of the animals in all groups was

similar (ca. 29 g). During the experimental period, however,

the CR animals significantly lost weight until the 10th week,

at which time body weight stabilized or even increased

slightly by the end of the experiment. This weight loss was

anticipated and is comparable to that which has been

reported previously [41,42]. It should perhaps be noted that

the CR regime established in the present study was

somewhat less severe than that employed by others [7,18].

Interestingly, there was a significant decrease in body

weight between the melatonin-treated and CT animals. In

female SHR mice [1], rats [31] and hamsters [4], a reduction

in body mass due to melatonin has been reported, but not in

male mice of our strain. One logical explanation for this

melatonin effect might be the increase of locomotor activity

(Figs. 1, 2), leading to increased calorie consumption.

However, as increased activity was only observed under DD

conditions, this is unlikely to be the cause. In earlier studies

in which young female BALB/c (melatonin deficient) mice

were substituted with melatonin at a concentration of 10 Ag/ml drinking water, no significant difference in body weight

between control- and melatonin-substituted animals was

seen [27,28]. However, a life-extending effect of melatonin

was noted [28]. The amount of food consumed by both

Fig. 4. The endogenous free-running period (s) of mice in DD. In all groups, s showed a significant increase with age. Neither melatonin nor CR treatment

reversed or attenuated this change. By the age of 14 months, the period is significantly longer in CR and melatonin animals as compared to controls (two sided t

test, N = 10, P < 0.05). At 8 and 14 months of age, the CR animals display the longest s as compared to both CT and melatonin animals.

D. Resuehr, J. Olcese / Brain Research 1048 (2005) 146–152150

melatonin-treated and CT animals did not appear to differ;

therefore, food intake does not appear to be a likely

explanation for the weight difference.

Recent studies reveal that the B6C3F1 strain is rather

long lived with a median life span of 30.7 months, which

can be increased up to 35.4 months by a 40% caloric

restriction [7], such as used here. In the present study, we

only assessed animals up to an age of 14 months. Never-

theless, it has been shown that even short term CR or CR

beginning in the middle of the murine life span leads to a

significant change in aging-related physiological parame-

ters, toward a more juvenile character [5,18]. Furthermore,

oral administration of melatonin beginning later in life leads

to prominent changes in mouse physiology, even at levels

less than half the concentration of those used in our study

[23].

Our results indicate a significant effect of melatonin

substitution, and even more dramatically, CR on the

locomotor activity of B6C3F1 mice. The endogenous free-

running period s (under DD conditions) and the pattern of

activity under LD conditions clearly lengthened over the

course of 14 months in control animals. However, neither

CR nor melatonin treatments prevented this trend (Fig. 4).

Valentinuzzi et al. [38] observed a lengthening of s in aging

mice (C57BL/6, 6–22 months), an effect comparable to our

results. However, the reported effects of aging on the

endogenous free-running circadian period are not always

consistent, even in the same strain of mice. For example,

Possidente and coworkers [30] reported a lengthening of swith age in C57BL/6 mice, while Teena and Wax [40]

observed no changes in this strain. This heterogeneity of

results may be explained by the use of different illumination

protocols before or after the measurement of s or may be

due to varying starting ages of the test animals. The age-

related increase in circadian period of our B6C3F1 mice is

comparable to previously described changes in s as a

function of age in rats [43]. Thus, a change in s seems to be

a common trait of the aging circadian system, consistent

with reports that the endogenous clock in the supra-

chiasmatic nuclei (SCN) itself undergoes changes during

senescence [42].

Under LD conditions CR mice exhibited an increased

daytime and a decreased nighttime locomotor activity

compared to the melatonin and CT mice (Figs. 1 and 2B).

This appears to reflect a fragmented circadian rhythmicity

and the animals’ tendency to search for food even during the

daytime hours [9]. Restrictive feeding can be expected to

result in a certain amount of mild stress and an increased

excitability as is seen in semi-starved rats and anorexia

nervosa patients [14]. In fact, we observed that even small

disturbances in the animal housing area were sufficient to

cause the entire CR group to become very active. Reduced

caloric availability is known to increase spontaneous day-

time wheel-running activity in rodents, which has been

suspected to be foraging activity [24]. It is likely that

hypoleptinemia could be the cause for this phenomenon

because the exogenous application of leptin abolishes the

anorexia-induced increase of activity in semi-starved rats

[10,14]. In order to minimize this masking effect of hunger,

feeding times in our experiments were randomly varied and

limited to the dark phase. The decreased nighttime activity

in CR mice is probably a compensatory increased nocturnal

D. Resuehr, J. Olcese / Brain Research 1048 (2005) 146–152 151

rest phase due to excessive daytime activity. The total

locomotor activity of all three treatment groups under LD

conditions is however not significantly different. In contrast,

under DD conditions, many of the melatonin-treated

animals displayed an increase in total activity (Figs. 1,

2C), in many cases due to rhythm splitting (see discussion

below).

The time required to reentrain to a shifted LD after DD

has been reported to increase with age in mice [38]. In the

present study, we observed that the melatonin-treated and

CR animals adjusted to a 4 h phase delay in the lighting

conditions faster than control mice (Figs. 2A–C and 3A–

C). However, the CR animals were unusual in that 3–5 days

after the phase delay they showed a remarkable excess of

activity (Fig. 3B). This overcompensation is likely to be

artificial and may be related to the increased excitability of

the CR mice subsequent to refeeding after a 3-day (week-

end) interval.

Melatonin’s effects on the circadian system are well

known. In several species, melatonin induces phase shifts

and entrainment of the circadian clock [32]. Melatonin

treatment can also entrain the circadian system of humans

[36]. In the present study, melatonin facilitated adjustment

to a 4 h phase delay, such that mice were resynchronized

within 48 h as opposed to 72 h for the control animals.

Similar positive effects of melatonin on synchronization to

new lighting conditions have been reported previously in

hamsters [39].

Quite unexpectedly, this mouse strain frequently devel-

oped a ‘‘split’’ free-running rhythm under DD. Splitting is

commonly seen in nocturnal rodents when they are

maintained under LL conditions [21]. It is defined as the

spontaneous separation of the main activity bout into two

smaller bouts with different free-running periods. Pro-

nounced splitting of the free-running locomotor activity

rhythm was seldom seen in either CT or CR animals (Figs.

2A, B, D, E); however, it was common in the melatonin-

treated animals, especially under DD conditions. Only

unsplit animals were used for the determination and

graphical illustration of s (Fig. 4). As depicted in Fig. 2C,

the rhythm splitting in melatonin-treated animals was often

substantial, resulting in two free-running rhythms with

different periods (23 vs. 23.6 h, calculated by v2 periodo-

gram analysis). Only 20% of the CT animals displayed

biphasic free-running activity under DD, while the inci-

dence of splitting in the melatonin animals was 50%. In

view of its known effects on the SCN clock [19, 20], it

seems conceivable that pharmacological levels of melatonin,

self-administered in a regular nocturnal manner to B6C3F1

mice, may paradoxically facilitate the uncoupling of dual

SCN oscillators under Zeitgeber-free conditions (i.e., DD),

which would manifest itself as rhythm splitting. Further

studies at the molecular level are needed to better under-

stand this unusual behavior. It would be informative to

determine whether the SCN expression of the MT1 and/or

MT2 melatonin receptor is vital for the splitting phenom-

enon that we observed in the B6C3F1 mice. This could, for

example, be assessed by backcrossing this strain with

melatonin receptor knockout mice [35].

In conclusion, the present study has compared two

anti-aging treatments, which are likely to influence the

endogenous circadian system. Melatonin’s use as a

chronobiotic has been well explored in rodents and

humans [2,3]. Timed restricted feeding under the con-

ditions of caloric restriction [22] is a potent synchronizer

of peripheral and central oscillators. In the present report,

we deliberately alternated the feeding times and fed mice

in the dark phase to avoid offering a light coupled

‘‘Zeitgeber’’ i.e., feeding in the light phase which could

lead to animals associating light with food. Nevertheless,

on a behavioral level, we could detect a direct effect of

caloric restriction on the circadian clock (in terms of

locomotor activity), rendering it more sensitive to

changed light cycles. A comparable effect was seen in

animals administered melatonin. The mechanisms through

which melatonin and caloric restriction exert their action

on the circadian clock however are probably completely

different, that is, melatonin is likely to work through G-

protein-coupled receptors [35], while caloric restriction is

thought to operate via other pathways, such as the

orexigenic system and leptin [10,14].

Acknowledgments

We would like to thank Dr. Haiko Dernbach for

assistance in setting up the locomotor activity electronics

and for his generosity in donating the software for the

activity recordings. We are also grateful to Jenny Behrens

and Helena Fischer for assistance in maintaining the

animals. This project was financed by the Leidenberger-

Muller Foundation and the Graduiertenkolleg 336 (DFG).

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