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S22153, a melatonin antagonist, dissociates different aspects of photoperiodic responses in Syrian hamsters
Behavioural Brain Research 138 (2003) 145 /152 www.elsevier.com/locate/bbr
Research report
S22153, a melatonin antagonist, dissociates different aspects of photoperiodic responses in Syrian hamsters B. Pitrosky a, P. Delagrange b, M.C. Rettori b, P. Pe´vet a,* a
Neurobiologie des Rythmes, UMR-CNRS 7518, Universite´ Louis Pasteur, 12 rue de l’Universite´, 67000 Strasbourg, France b Institut de Recherches Internationales Servier (IRIS), Courbevoie, France Received 29 April 2002; received in revised form 5 August 2002; accepted 5 August 2002
Abstract In the Syrian hamster, short photoperiod (SP) induces changes in several physiological functions (body mass, reproduction, hibernation), and these responses involve the pineal hormone melatonin. The present study investigated the effects of a melatonin antagonist, S22153, on photoperiodic adaptation of male Syrian hamster. When constantly released from subcutaneous implants, S22153 had no effect on body or testes masses of animals kept in long photoperiod. S22153 decreased the total hibernation duration observed in animals exposed to SP and low temperature. The decrease in hibernation duration was due to a marked reduction in the number and duration of hypothermic bouts. Moreover, S22153 significantly inhibited the increase of interscapular brown adipose tissue (BAT) mass induced by SP. However, neither the gonadal atrophy nor the body mass increase induced by SP were affected by S22153. These results show that S22153 affects only part of the physiological changes controlled by SP and cold. Whether the decreases in BAT mass and hibernation duration are linked still remains an open question. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Photoperiod; Melatonin; Hibernation; Brown adipose tissue; Seasonal rhythms
1. Introduction In mammals living in the temperate zone, several physiological functions (such as breeding, moult, hibernation) exhibit seasonal changes important for survival during the cold period. In temperate regions, photoperiod is the most reliable environmental cue used to drive these seasonal adaptations. The photoperiodic message is transduced into a neuroendocrine message by the pineal gland through the pattern of melatonin secretion. Melatonin secretion is rhythmic with high levels during the night and low levels during the day. The duration of melatonin secretion is known to be proportional to the duration of the night in several mammalian species [31,34,39].
* Corresponding author. Tel.: /33-3-90-24-05-06; fax: /33-3-9024-05-28 E-mail address: [email protected] (P. Pe´vet).
Melatonin is known to control reproduction in photoperiodic seasonal breeders [1,25,43]. In Syrian hamsters, short photoperiod (SP, B/12.5 h of light per day) induces an inhibition of sexual activity [5,13], and this effect can be mimicked by daily infusion of melatonin [20,29]. Moreover, both SP and melatonin treatments are known to increase body mass and brown adipose tissue (BAT) mass in Syrian hamsters [2,12]. In combination with photoperiod, ambient temperature can modulate seasonal adaptations. In Syrian hamsters, cold exposure hastens gonadal atrophy induced by SP [28], which occurs in 4 weeks at a temperature of 5 8C while it requires 8 weeks at 20 8C. Moreover, low temperature is also able to increase BAT mass [12], and has an important role in the induction of hibernation [14,40]. Melatonin is also involved in the expression of the hibernating pattern by at least two ways. First, the photoperiodic inhibition of sexual activity is required to allow animals to hibernate
0166-4328/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 2 ) 0 0 2 3 5 - 8
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[27]. Second, melatonin is able to prolong hibernation bout when directly administered in the brain [42]. The exact mechanism of action of melatonin is still unclear. However, the understanding of the action of melatonin will benefit from the development of synthetic compounds, which will also open the field of therapeutic approaches. The effects of melatonin analogs have been largely tested on the circadian system [30,32,45,49]. In regard to photoperiodic responses, the antagonist S20928 has been shown to block the SP-induced body mass increase and to increase basal metabolism in garden dormouse [18]. Recently, S22153, a new melatonin ligand [24,36] has been characterized as a melatonin antagonist which blocks the phase-shifting effect of melatonin in mice [47], the behavioral changes in mice induced by short-day exposure [15,16] and the potentiation induced by melatonin of electrically evoked contraction of isolated rat tail arteries [6]. The aim of the present study was to investigate the physiological effects of subcutaneous implants of S22153 on the photoperiodic response and hibernation pattern in Syrian hamster.
the implants. S22153 was obtained from IRIS, Courbevoie, France. 2.1. Experiment 1 To define the effects of S22153 on the photoperiodic response of male Syrian hamsters, animals were divided into four experimental groups and studied for 11 weeks: . LP group (n /5): animals in 14L:10D with empty implants . LP/S22153 group (n/7): animals in 14L:10D with S22153-filled implants . SP group (n/9): animals in 10L:14D with empty implants . SP/S22153 group (n /9): animals in 10L:14D with S22153-filled implants Animals were weighed on the first and the last days of experiment. At the end of the experiment, animals were killed by decapitation. Testes and interscapular BAT were dissected and weighed. 2.2. Experiment 2
2. Material and methods Adult male Syrian hamsters were purchased from a commercial supplier (Harlan, France) where they had been raised under long photoperiod (LP, 14L:10D). They were adapted to our laboratory conditions for 2 weeks (14L:10D; temperature: 209/1 8C). Food pellets and tap water were accessible ad libitum throughout the experiments. All experiments were performed in accordance with ‘Principles of laboratory animal care’ (NIH pub. No. 86/23, revised 1985) as well as in accordance with the French national laws. All the animals received a dorsal subcutaneous implant under halothane-gas anesthesia. The silastics implants were prepared according to the method of Turek et al. Two centimeter length silastic tubes (inner diameter 1.5 mm, medical grade, Dow-Corning, Medical Product Div., Midland, MI) were either filled with S22153 or left empty. Both ends of the implants were sealed with medical adhesive silicone. Implants filled with S22153 contained 20 mg each. For all animals, implants were changed every 6 weeks with the animals under halothane-gas anesthesia. Visual inspection of implants was performed during the implant’s replacement or at the end of the experiments in order to check the diffusion of S22153 during implantation. Previous tests showed that after 2, 4, 6 or 8 weeks of implantation the plasma concentrations (measured by GC /MS) of S22153 were still high, around 22, 24, 15 and 12 ng/ml, respectively (unpublished data, Servier internal report), demonstrating that S22153 is constantly released from
To determine the effects of S22153 on the hibernation pattern of Syrian hamsters, the animals received simultaneously an implant and a Mini-Mitter telemetry device (Mini-Mitter Co., Sunriver, OR), to record body temperature at 5-min intervals (Dataquest III acquisition system, Data Sciences Co., MN). After surgery, animals were transferred to individual cages in SP (10L:14D) at a room temperature of 59/1 8C, for 18 weeks (126 days). Animals were divided in two groups: one group consisted of animals bearing empty implants (Control group, n /7), and the second of animals bearing S22153-filled implants (S22153 group, n/5). Animals were weighed on the day of transfer and at the end of the experiment. At the end of the experiment, animals were killed by decapitation. Testes and interscapular BAT were dissected and weighed. A bout of hibernation (hypothermia) was considered to begin at the time when body temperature decrease reaches 32 8C (Fig. 3). Body temperature data are represented on a linear scale ranging from 32 /38 8C, (Fig. 4). Animals were followed for 126 days, but only 100 days were used to characterize hibernation. The first 25 days of experiment, which correspond to the time necessary for induction of the photoperiodic response, were not taken into account (no hibernation could be observed during this time). The last day was also ignored since animals were manipulated in order to be awaked on the day of sacrifice. Hypothermia duration corresponded to the time spent with a body temperature below 32 8C, which was determined by graphical analysis. Inversely, euthermia duration was defined as
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the time spent with a body temperature higher than 32 8C. This allowed determination of the time spent in hypothermia in regard to the total time studied (ratio hypothermia/total). The number of hypothermic bouts was also counted for each animal, as well as the mean duration of a hypothermic bout. 2.3. Statistical analysis In experiment 1, changes in body mass, BAT mass and testes mass were analysed by a two-way ANOVA (photoperiod and treatment), followed post-hoc by Duncan’s multiple range test. In experiment 2, changes in body mass were analysed by a two-way ANOVA (time and treatment), followed post-hoc by Duncan’s multiple range test. All other parameters were analysed by Student t -test. For determination of mean duration of hypothermic bout, one S22153-treated animals was not taken into account since this animals did not present any hypothermic episode.
3. Results 3.1. Experiment 1 After 11 weeks of treatment, all experimental groups showed an increase of body mass. For animals maintained in SP (SP and SP/S22153 groups), the body mass gain was greater than for the LP groups (P B/0.05) (Fig. 1A). However, the S22153 treatment did not modify the mass increase observed in both LP and SP, respectively. After 11 weeks, LP animals had fully developed testes and S22153 did not induce any changes (Fig. 1B). SPinduced a complete gonadal atrophy and this effect was not modified by S22153 treatment. S22153 had no effect on interscapular BAT mass in LP (Fig. 2). However, SP-induced an increase in BAT mass (P B/0.05) compared to LP conditions. This trophic effect was prevented by S22153 treatment, since treated animals presented a BAT mass similar to that observed in LP groups (Fig. 2).
Fig. 1. Effects of S22153 treatment on (A) body mass increase and (B) testes mass of male Syrian hamsters. Two groups were kept in LP with empty (LP group) or S22153-filled implants (LP/S22153 group), while two other groups were maintained in SP with the same treatments (SP and SP/S22153 groups). *P B/0.05 when compared to LP group (values are mean9/SEM).
3.2. Experiment 2 After 18 weeks under SP, the two groups showed a significant decrease in body mass (Table 1). Implants containing S22153 did not lead to a significant change in body mass. The decrease in body mass was rather linked to the time spent in SP at 5 8C (P B/0.05). In most animals, regardless of the implant content, testes mass was reduced, showing that experimental conditions were able to inhibit sexual activity (Table 1). No significant differences for testes mass were observed between the two groups. The mass of BAT was
Fig. 2. Effects of S22153 treatment on interscapular BAT mass of Syrian hamsters. *P B/0.05 when compared to LP group (For groups details, see legend Fig. 1) (values are mean9/SEM).
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Group
Control S22153
Body mass (g) Initial
Final
117.1493.63 126.494.63
104.2994.4.97* 113.695.97*
Testes mass (g)
BAT mass (mg)
Number of hypothermic bouts
Mean duration of hypothermic bouts (h)
Ratio hypothermia/total time
1.5190.42 2.3290.79
1573.7984.71 949.19106.5**
4.1491.12 3.8091.91**
88.7395.31 70.2191.70**
0.5490.07 0.1290.07**
* P B 0.05 when compared to the initial mass of the corresponding group. ** P B 0.05 when compared to the control group.
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Table 1 Effects of S22153 treatment on Syrian hamsters exposed to SP and cold for 18 weeks
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significantly lower in animals with S22153-filled implants (P B/0.05) (Table 1). The treatment of Syrian hamsters with S22153 implants did not modify the shape of the hypothermic bouts with body temperature decreasing up to 6/7 8C (Fig. 3). However, the number of hypothermic bouts was markedly decreased in S22153-treated animals (P B/ 0.05) (Table 1 and Fig. 4). Moreover, this phenomenon was accompanied by a decrease in the mean duration of hypothermic bouts (P B/0.05) (Table 1, Fig. 5). This led to a strong reduction in the time spent in hypothermia (P B/0.05) (Fig. 5). Moreover, in one animal, S22153 completely prevented hibernation (Fig. 4C). Conversely, the time spent in euthermia was significantly higher in S22153-treated animals (P B/0.05) (Fig. 5). Thus, the ratio of the time spent in hypothermia over the total recording time was significantly decreased in animals treated with S22153 implants (Table 1) (P B/0.05).
4. Discussion The present results show that, when constantly released from silastic implants, S22153 significantly alters the hibernation pattern of Syrian hamsters exposed to cold and SP. However, S22153, which has no effect on animals maintained in LP, did not fully block all the photoperiodic responses induced by SP. As previously reported [12], we observed a decrease in body mass of animals exposed to cold and SP. S22153 treatment did not influence this response. Moreover, gonadal activity, which is a highly photoperiodic regulated function (controlled by melatonin) was not significantly affected by S22153 after 11 or 18 weeks of treatment. Moreover, S22153 abolished the trophic
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effects of SP (and cold) on interscapular BAT mass. The photoperiod-induced trophic response of BAT is known to be also mediated by melatonin [46]. Thus, these observations suggest that S22153 might interfere only with a fraction of the physiological changes driven by the melatonin signal. In the Syrian hamster, sexual quiescence is a prerequisite for the occurrence of hibernation [14]. Furthermore, in hamsters, changes in androgenic pattern are known to facilitate or block hibernation [9,26,40]. However, in our experimental conditions, even though S22153 treatment did not significantly change the testes mass, the number of hypothermic bouts (as well as the mean duration of the bout) was strongly decreased which led to a decrease in the time spent in hibernation. These results suggest that the gonadal function is not the only factor controlling hibernation. Indeed, such a nongonadal-mediated effect of photoperiod has been previously suggested by other studies based on castration protocols [4,8,14,27]. Thus, in the present work the inhibition of hibernation cannot be linked to a blockade of the photoperiodinduced gonadal atrophy. These observations reinforce the idea of a dissociation of the different aspects of the photoperiodic response by S22153 treatment. Previous studies, based on physical lesions of brain structures, have shown that it was possible to dissociate the gonadotrophic response from the lactotrophic one, although both are controlled by the photoperiod and melatonin [19,21].The present study demonstrates that S22153 allows to dissociate pharmacologically different seasonal responses. In different rodent species, melatonin has been proposed to act directly on the hibernating pattern. Indeed, several studies reported a decrease of pineal
Fig. 3. Body temperature of Syrian hamsters exposed to SP and cold. Shape of an hypothermic bout of (A) a control animal (bearing empty implant), and (B) a S22153-treated animal.
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Fig. 4. Double-plotted representation of body temperature of Syrian hamsters exposed to SP and cold. (A) Pattern of temperature of a control animal with an empty subcutaneous implant, showing regular hypothermic bouts. (B) Pattern of temperature of a S22153-treated animal, showing a reduced number of hypothermic bouts, and of (C) a S22153-treated animal showing no hypothermia. Body temperature is plotted on a linear scale ranging from 32 /38 8C; triangles indicate the time at which the subcutaneous implants were changed.
Fig. 5. Effects of S22153 on the duration of total hypothermia (filled bar) and total euthermia (open bar) of Syrian hamsters exposed to SP and cold. *P B/0.05 when compared to the control group (values are mean9/SEM).
melatonin content in hibernating animals [41,44], and intracerebroventricular infusion of melatonin prolonged the hibernation bout duration [42]. Melatonin is known to have an effect on thermoregulatory processes [37]. The thermoregulatory center controlling body temperature has been shown to be located in the preoptic area of the hypothalamus [38]. This structure lies close to the suprachiasmatic nucleus, which is involved in the
generation of the circadian rhythm of body temperature. However, both structures are known to have melatonin receptors [48]. These nuclei thus represent putative target sites for S22153 action on the hibernating pattern of Syrian hamsters. Nevertheless, numerous neuroendocrine changes have been observed in the brain of hibernating mammals [23], suggesting that other brain areas such as the lateral septum might be involved in the control of hibernation [10,26]. Moreover, hibernation has also been demonstrated to be influenced by peptidergic system, since a decrease in the duration of hibernation in squirrels has been observed after intraventricular injection of enkephalin [3]. The effects of S22153 on the expression of hibernation might then involve several brain structures and interact with other neuroendocrine systems. Mammalian hibernation is a regulated decline of body temperature, and hypothermic episodes are separated by periodic arousals. Since the shape of the hypothermic bouts was not altered by S22153, our results suggest that the mechanisms modulating the decrease in body temperature and rewarming were not changed. However, the BAT is known to be the major site for nonshivering thermogenesis and is a crucial component, in addition to shivering thermogenesis, for rewarming during arousal from hibernation [22]. Thus, the inhibition of the SP-induced increase of interscapular BAT
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mass might account for the decreased frequency of hypothermic bouts. The mechanisms of action of S22153 still remain an open question. Since there is a dissociation between the physiological functions studied, one can propose that S22153 acts specifically via one of the melatonin receptors subtypes, i.e. the MT1 or the MT2 [33]. However, as S22153 has been characterized as a putative antagonist of both melatonin receptor subtypes [6], the implication of another receptor subtype might not be excluded. Some melatonin binding sites have been characterized in the BAT of hamsters [17], but the exact nature of these receptors is still unknown. Nevertheless, two hypothesis can explain the inhibition of SP-induced increase of BAT mass. First, S22153 can block the trophic effect of SP by blocking the action of melatonin [2]. Second, S22153 can also increase the energy expenditure in the Syrian hamster, which can counteract the melatonin-induced increase in BAT mass [11,35]. Further studies providing more qualitative information on BAT (lipid composition, thermogenic capacity, GDP-binding, amount of uncoupling protein,. . .) are required to understand the effect of S22153 on BAT. Finally, a last possibility could be based on an interaction of S22153 with the endocrine function of BAT. Recently, it has been shown that the hormone leptin can inhibit the daily torpor of a nocturnal marsupial [7]. Thus, energy expenditure might be an important parameter to control in order to determine the action of S22153. In conclusion, our findings suggest that the melatonin antagonist S22153, when constantly released from subcutaneous implants, alters only a fraction of the physiological functions dependent of the photoperiodic information. Since S22153 strongly reduces hibernation and BAT mass increase induced by SP and cold, but does not alter other photoperiodic functions (body and testes mass), it is the first demonstration of a dissociation of photoperiodic-controlled seasonal functions. However, further studies are required to define the mechanism of action of both melatonin and S22153 on these functions.
Acknowledgements The authors are specially gratefull to Ms S. Gourmelen and Mr R. Brenkle´ for their excellent technical assistance in the care to the animals and telemetry devices.
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Research report
S22153, a melatonin antagonist, dissociates different aspects of photoperiodic responses in Syrian hamsters B. Pitrosky a, P. Delagrange b, M.C. Rettori b, P. Pe´vet a,* a
Neurobiologie des Rythmes, UMR-CNRS 7518, Universite´ Louis Pasteur, 12 rue de l’Universite´, 67000 Strasbourg, France b Institut de Recherches Internationales Servier (IRIS), Courbevoie, France Received 29 April 2002; received in revised form 5 August 2002; accepted 5 August 2002
Abstract In the Syrian hamster, short photoperiod (SP) induces changes in several physiological functions (body mass, reproduction, hibernation), and these responses involve the pineal hormone melatonin. The present study investigated the effects of a melatonin antagonist, S22153, on photoperiodic adaptation of male Syrian hamster. When constantly released from subcutaneous implants, S22153 had no effect on body or testes masses of animals kept in long photoperiod. S22153 decreased the total hibernation duration observed in animals exposed to SP and low temperature. The decrease in hibernation duration was due to a marked reduction in the number and duration of hypothermic bouts. Moreover, S22153 significantly inhibited the increase of interscapular brown adipose tissue (BAT) mass induced by SP. However, neither the gonadal atrophy nor the body mass increase induced by SP were affected by S22153. These results show that S22153 affects only part of the physiological changes controlled by SP and cold. Whether the decreases in BAT mass and hibernation duration are linked still remains an open question. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Photoperiod; Melatonin; Hibernation; Brown adipose tissue; Seasonal rhythms
1. Introduction In mammals living in the temperate zone, several physiological functions (such as breeding, moult, hibernation) exhibit seasonal changes important for survival during the cold period. In temperate regions, photoperiod is the most reliable environmental cue used to drive these seasonal adaptations. The photoperiodic message is transduced into a neuroendocrine message by the pineal gland through the pattern of melatonin secretion. Melatonin secretion is rhythmic with high levels during the night and low levels during the day. The duration of melatonin secretion is known to be proportional to the duration of the night in several mammalian species [31,34,39].
* Corresponding author. Tel.: /33-3-90-24-05-06; fax: /33-3-9024-05-28 E-mail address: [email protected] (P. Pe´vet).
Melatonin is known to control reproduction in photoperiodic seasonal breeders [1,25,43]. In Syrian hamsters, short photoperiod (SP, B/12.5 h of light per day) induces an inhibition of sexual activity [5,13], and this effect can be mimicked by daily infusion of melatonin [20,29]. Moreover, both SP and melatonin treatments are known to increase body mass and brown adipose tissue (BAT) mass in Syrian hamsters [2,12]. In combination with photoperiod, ambient temperature can modulate seasonal adaptations. In Syrian hamsters, cold exposure hastens gonadal atrophy induced by SP [28], which occurs in 4 weeks at a temperature of 5 8C while it requires 8 weeks at 20 8C. Moreover, low temperature is also able to increase BAT mass [12], and has an important role in the induction of hibernation [14,40]. Melatonin is also involved in the expression of the hibernating pattern by at least two ways. First, the photoperiodic inhibition of sexual activity is required to allow animals to hibernate
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[27]. Second, melatonin is able to prolong hibernation bout when directly administered in the brain [42]. The exact mechanism of action of melatonin is still unclear. However, the understanding of the action of melatonin will benefit from the development of synthetic compounds, which will also open the field of therapeutic approaches. The effects of melatonin analogs have been largely tested on the circadian system [30,32,45,49]. In regard to photoperiodic responses, the antagonist S20928 has been shown to block the SP-induced body mass increase and to increase basal metabolism in garden dormouse [18]. Recently, S22153, a new melatonin ligand [24,36] has been characterized as a melatonin antagonist which blocks the phase-shifting effect of melatonin in mice [47], the behavioral changes in mice induced by short-day exposure [15,16] and the potentiation induced by melatonin of electrically evoked contraction of isolated rat tail arteries [6]. The aim of the present study was to investigate the physiological effects of subcutaneous implants of S22153 on the photoperiodic response and hibernation pattern in Syrian hamster.
the implants. S22153 was obtained from IRIS, Courbevoie, France. 2.1. Experiment 1 To define the effects of S22153 on the photoperiodic response of male Syrian hamsters, animals were divided into four experimental groups and studied for 11 weeks: . LP group (n /5): animals in 14L:10D with empty implants . LP/S22153 group (n/7): animals in 14L:10D with S22153-filled implants . SP group (n/9): animals in 10L:14D with empty implants . SP/S22153 group (n /9): animals in 10L:14D with S22153-filled implants Animals were weighed on the first and the last days of experiment. At the end of the experiment, animals were killed by decapitation. Testes and interscapular BAT were dissected and weighed. 2.2. Experiment 2
2. Material and methods Adult male Syrian hamsters were purchased from a commercial supplier (Harlan, France) where they had been raised under long photoperiod (LP, 14L:10D). They were adapted to our laboratory conditions for 2 weeks (14L:10D; temperature: 209/1 8C). Food pellets and tap water were accessible ad libitum throughout the experiments. All experiments were performed in accordance with ‘Principles of laboratory animal care’ (NIH pub. No. 86/23, revised 1985) as well as in accordance with the French national laws. All the animals received a dorsal subcutaneous implant under halothane-gas anesthesia. The silastics implants were prepared according to the method of Turek et al. Two centimeter length silastic tubes (inner diameter 1.5 mm, medical grade, Dow-Corning, Medical Product Div., Midland, MI) were either filled with S22153 or left empty. Both ends of the implants were sealed with medical adhesive silicone. Implants filled with S22153 contained 20 mg each. For all animals, implants were changed every 6 weeks with the animals under halothane-gas anesthesia. Visual inspection of implants was performed during the implant’s replacement or at the end of the experiments in order to check the diffusion of S22153 during implantation. Previous tests showed that after 2, 4, 6 or 8 weeks of implantation the plasma concentrations (measured by GC /MS) of S22153 were still high, around 22, 24, 15 and 12 ng/ml, respectively (unpublished data, Servier internal report), demonstrating that S22153 is constantly released from
To determine the effects of S22153 on the hibernation pattern of Syrian hamsters, the animals received simultaneously an implant and a Mini-Mitter telemetry device (Mini-Mitter Co., Sunriver, OR), to record body temperature at 5-min intervals (Dataquest III acquisition system, Data Sciences Co., MN). After surgery, animals were transferred to individual cages in SP (10L:14D) at a room temperature of 59/1 8C, for 18 weeks (126 days). Animals were divided in two groups: one group consisted of animals bearing empty implants (Control group, n /7), and the second of animals bearing S22153-filled implants (S22153 group, n/5). Animals were weighed on the day of transfer and at the end of the experiment. At the end of the experiment, animals were killed by decapitation. Testes and interscapular BAT were dissected and weighed. A bout of hibernation (hypothermia) was considered to begin at the time when body temperature decrease reaches 32 8C (Fig. 3). Body temperature data are represented on a linear scale ranging from 32 /38 8C, (Fig. 4). Animals were followed for 126 days, but only 100 days were used to characterize hibernation. The first 25 days of experiment, which correspond to the time necessary for induction of the photoperiodic response, were not taken into account (no hibernation could be observed during this time). The last day was also ignored since animals were manipulated in order to be awaked on the day of sacrifice. Hypothermia duration corresponded to the time spent with a body temperature below 32 8C, which was determined by graphical analysis. Inversely, euthermia duration was defined as
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the time spent with a body temperature higher than 32 8C. This allowed determination of the time spent in hypothermia in regard to the total time studied (ratio hypothermia/total). The number of hypothermic bouts was also counted for each animal, as well as the mean duration of a hypothermic bout. 2.3. Statistical analysis In experiment 1, changes in body mass, BAT mass and testes mass were analysed by a two-way ANOVA (photoperiod and treatment), followed post-hoc by Duncan’s multiple range test. In experiment 2, changes in body mass were analysed by a two-way ANOVA (time and treatment), followed post-hoc by Duncan’s multiple range test. All other parameters were analysed by Student t -test. For determination of mean duration of hypothermic bout, one S22153-treated animals was not taken into account since this animals did not present any hypothermic episode.
3. Results 3.1. Experiment 1 After 11 weeks of treatment, all experimental groups showed an increase of body mass. For animals maintained in SP (SP and SP/S22153 groups), the body mass gain was greater than for the LP groups (P B/0.05) (Fig. 1A). However, the S22153 treatment did not modify the mass increase observed in both LP and SP, respectively. After 11 weeks, LP animals had fully developed testes and S22153 did not induce any changes (Fig. 1B). SPinduced a complete gonadal atrophy and this effect was not modified by S22153 treatment. S22153 had no effect on interscapular BAT mass in LP (Fig. 2). However, SP-induced an increase in BAT mass (P B/0.05) compared to LP conditions. This trophic effect was prevented by S22153 treatment, since treated animals presented a BAT mass similar to that observed in LP groups (Fig. 2).
Fig. 1. Effects of S22153 treatment on (A) body mass increase and (B) testes mass of male Syrian hamsters. Two groups were kept in LP with empty (LP group) or S22153-filled implants (LP/S22153 group), while two other groups were maintained in SP with the same treatments (SP and SP/S22153 groups). *P B/0.05 when compared to LP group (values are mean9/SEM).
3.2. Experiment 2 After 18 weeks under SP, the two groups showed a significant decrease in body mass (Table 1). Implants containing S22153 did not lead to a significant change in body mass. The decrease in body mass was rather linked to the time spent in SP at 5 8C (P B/0.05). In most animals, regardless of the implant content, testes mass was reduced, showing that experimental conditions were able to inhibit sexual activity (Table 1). No significant differences for testes mass were observed between the two groups. The mass of BAT was
Fig. 2. Effects of S22153 treatment on interscapular BAT mass of Syrian hamsters. *P B/0.05 when compared to LP group (For groups details, see legend Fig. 1) (values are mean9/SEM).
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Group
Control S22153
Body mass (g) Initial
Final
117.1493.63 126.494.63
104.2994.4.97* 113.695.97*
Testes mass (g)
BAT mass (mg)
Number of hypothermic bouts
Mean duration of hypothermic bouts (h)
Ratio hypothermia/total time
1.5190.42 2.3290.79
1573.7984.71 949.19106.5**
4.1491.12 3.8091.91**
88.7395.31 70.2191.70**
0.5490.07 0.1290.07**
* P B 0.05 when compared to the initial mass of the corresponding group. ** P B 0.05 when compared to the control group.
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Table 1 Effects of S22153 treatment on Syrian hamsters exposed to SP and cold for 18 weeks
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significantly lower in animals with S22153-filled implants (P B/0.05) (Table 1). The treatment of Syrian hamsters with S22153 implants did not modify the shape of the hypothermic bouts with body temperature decreasing up to 6/7 8C (Fig. 3). However, the number of hypothermic bouts was markedly decreased in S22153-treated animals (P B/ 0.05) (Table 1 and Fig. 4). Moreover, this phenomenon was accompanied by a decrease in the mean duration of hypothermic bouts (P B/0.05) (Table 1, Fig. 5). This led to a strong reduction in the time spent in hypothermia (P B/0.05) (Fig. 5). Moreover, in one animal, S22153 completely prevented hibernation (Fig. 4C). Conversely, the time spent in euthermia was significantly higher in S22153-treated animals (P B/0.05) (Fig. 5). Thus, the ratio of the time spent in hypothermia over the total recording time was significantly decreased in animals treated with S22153 implants (Table 1) (P B/0.05).
4. Discussion The present results show that, when constantly released from silastic implants, S22153 significantly alters the hibernation pattern of Syrian hamsters exposed to cold and SP. However, S22153, which has no effect on animals maintained in LP, did not fully block all the photoperiodic responses induced by SP. As previously reported [12], we observed a decrease in body mass of animals exposed to cold and SP. S22153 treatment did not influence this response. Moreover, gonadal activity, which is a highly photoperiodic regulated function (controlled by melatonin) was not significantly affected by S22153 after 11 or 18 weeks of treatment. Moreover, S22153 abolished the trophic
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effects of SP (and cold) on interscapular BAT mass. The photoperiod-induced trophic response of BAT is known to be also mediated by melatonin [46]. Thus, these observations suggest that S22153 might interfere only with a fraction of the physiological changes driven by the melatonin signal. In the Syrian hamster, sexual quiescence is a prerequisite for the occurrence of hibernation [14]. Furthermore, in hamsters, changes in androgenic pattern are known to facilitate or block hibernation [9,26,40]. However, in our experimental conditions, even though S22153 treatment did not significantly change the testes mass, the number of hypothermic bouts (as well as the mean duration of the bout) was strongly decreased which led to a decrease in the time spent in hibernation. These results suggest that the gonadal function is not the only factor controlling hibernation. Indeed, such a nongonadal-mediated effect of photoperiod has been previously suggested by other studies based on castration protocols [4,8,14,27]. Thus, in the present work the inhibition of hibernation cannot be linked to a blockade of the photoperiodinduced gonadal atrophy. These observations reinforce the idea of a dissociation of the different aspects of the photoperiodic response by S22153 treatment. Previous studies, based on physical lesions of brain structures, have shown that it was possible to dissociate the gonadotrophic response from the lactotrophic one, although both are controlled by the photoperiod and melatonin [19,21].The present study demonstrates that S22153 allows to dissociate pharmacologically different seasonal responses. In different rodent species, melatonin has been proposed to act directly on the hibernating pattern. Indeed, several studies reported a decrease of pineal
Fig. 3. Body temperature of Syrian hamsters exposed to SP and cold. Shape of an hypothermic bout of (A) a control animal (bearing empty implant), and (B) a S22153-treated animal.
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Fig. 4. Double-plotted representation of body temperature of Syrian hamsters exposed to SP and cold. (A) Pattern of temperature of a control animal with an empty subcutaneous implant, showing regular hypothermic bouts. (B) Pattern of temperature of a S22153-treated animal, showing a reduced number of hypothermic bouts, and of (C) a S22153-treated animal showing no hypothermia. Body temperature is plotted on a linear scale ranging from 32 /38 8C; triangles indicate the time at which the subcutaneous implants were changed.
Fig. 5. Effects of S22153 on the duration of total hypothermia (filled bar) and total euthermia (open bar) of Syrian hamsters exposed to SP and cold. *P B/0.05 when compared to the control group (values are mean9/SEM).
melatonin content in hibernating animals [41,44], and intracerebroventricular infusion of melatonin prolonged the hibernation bout duration [42]. Melatonin is known to have an effect on thermoregulatory processes [37]. The thermoregulatory center controlling body temperature has been shown to be located in the preoptic area of the hypothalamus [38]. This structure lies close to the suprachiasmatic nucleus, which is involved in the
generation of the circadian rhythm of body temperature. However, both structures are known to have melatonin receptors [48]. These nuclei thus represent putative target sites for S22153 action on the hibernating pattern of Syrian hamsters. Nevertheless, numerous neuroendocrine changes have been observed in the brain of hibernating mammals [23], suggesting that other brain areas such as the lateral septum might be involved in the control of hibernation [10,26]. Moreover, hibernation has also been demonstrated to be influenced by peptidergic system, since a decrease in the duration of hibernation in squirrels has been observed after intraventricular injection of enkephalin [3]. The effects of S22153 on the expression of hibernation might then involve several brain structures and interact with other neuroendocrine systems. Mammalian hibernation is a regulated decline of body temperature, and hypothermic episodes are separated by periodic arousals. Since the shape of the hypothermic bouts was not altered by S22153, our results suggest that the mechanisms modulating the decrease in body temperature and rewarming were not changed. However, the BAT is known to be the major site for nonshivering thermogenesis and is a crucial component, in addition to shivering thermogenesis, for rewarming during arousal from hibernation [22]. Thus, the inhibition of the SP-induced increase of interscapular BAT
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mass might account for the decreased frequency of hypothermic bouts. The mechanisms of action of S22153 still remain an open question. Since there is a dissociation between the physiological functions studied, one can propose that S22153 acts specifically via one of the melatonin receptors subtypes, i.e. the MT1 or the MT2 [33]. However, as S22153 has been characterized as a putative antagonist of both melatonin receptor subtypes [6], the implication of another receptor subtype might not be excluded. Some melatonin binding sites have been characterized in the BAT of hamsters [17], but the exact nature of these receptors is still unknown. Nevertheless, two hypothesis can explain the inhibition of SP-induced increase of BAT mass. First, S22153 can block the trophic effect of SP by blocking the action of melatonin [2]. Second, S22153 can also increase the energy expenditure in the Syrian hamster, which can counteract the melatonin-induced increase in BAT mass [11,35]. Further studies providing more qualitative information on BAT (lipid composition, thermogenic capacity, GDP-binding, amount of uncoupling protein,. . .) are required to understand the effect of S22153 on BAT. Finally, a last possibility could be based on an interaction of S22153 with the endocrine function of BAT. Recently, it has been shown that the hormone leptin can inhibit the daily torpor of a nocturnal marsupial [7]. Thus, energy expenditure might be an important parameter to control in order to determine the action of S22153. In conclusion, our findings suggest that the melatonin antagonist S22153, when constantly released from subcutaneous implants, alters only a fraction of the physiological functions dependent of the photoperiodic information. Since S22153 strongly reduces hibernation and BAT mass increase induced by SP and cold, but does not alter other photoperiodic functions (body and testes mass), it is the first demonstration of a dissociation of photoperiodic-controlled seasonal functions. However, further studies are required to define the mechanism of action of both melatonin and S22153 on these functions.
Acknowledgements The authors are specially gratefull to Ms S. Gourmelen and Mr R. Brenkle´ for their excellent technical assistance in the care to the animals and telemetry devices.
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