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Entrainment of rat circadian rhythms by melatonin does not depend on the serotonergic afferents to the suprachiasmatic nuclei
Brain Research 876 (2000) 10–16 www.elsevier.com / locate / bres
Research report
Entrainment of rat circadian rhythms by melatonin does not depend on the serotonergic afferents to the suprachiasmatic nuclei ´ a ,* Helge A. Slotten a,b , Bruno Pitrosky a , Paul Pevet a
` , Universite´ Louis Pasteur, 12 rue de l’ Universite´ , F-67000 Strasbourg, UMR CNRS 7518, Neurobiologie des fonctions rythmiques et saisonnieres France b Department of Psychology, NTNU, N-7491 Trondheim, Norway Accepted 9 May 2000
Abstract Daily administration of melatonin (MEL) can entrain rat circadian rhythms free-running in constant darkness. The high MEL doses needed to obtain entrainment suggest the implication of other neural mechanisms than simply an effect on the hormone’s specific receptors detected in the SCN. Administration of serotonin receptor agonists can phase-shift the rodent circadian clock, and MEL is known to modulate release and reuptake of serotonin in nerve endings. This raises the question of a critical involvement of 5-HT-fibres in the entraining properties of MEL. The aim of the present study was to test this hypothesis. Bilateral neurotoxic (5,7-dihydroxytryptamine) lesions of the serotonergic fibres in the SCN were performed in animals kept in LD 12:12. Following the post-operative period, the animals were transferred to constant darkness to free-run. MEL was then administered by a 1 h daily infusion. Both well lesioned and intact animals entrained to MEL. No differences were observed between lesioned and control animals on parameters such as the phase-angles between MEL onset and activity onset, and core body temperature acrophase, respectively. Entrainment of rat circadian rhythms to exogenous MEL is thus not directly dependent on the 5-HT fibres in the SCN. 2000 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behaviour Topic: Biological rhythms and sleep Keywords: Melatonin; Serotonin; 5,7-dihydroxytryptamine; SCN; Entrainment; Rat
1. Introduction In mammals, circadian variations in biological functions such as core body temperature (CBT), pineal melatonin (MEL) secretion, and activity / quiescence are driven by the suprachiasmatic nuclei (SCN) of the hypothalamus [17]. Because the period of the SCN is slightly different from 24 h, the timekeeping system needs daily adjustment from zeitgebers (timegivers) to synchronize to the nychthemeral environment. The natural light / dark (LD) cycle is the strongest zeitgeber [23], but also non-photic stimuli such as daily MEL administration are known to entrain rat circadian rhythms free-running in DD [21,22,24]. It is a
*Corresponding author. Tel.: 133-3-8835-8509; fax: 133-3-88240461. ´ E-mail address: [email protected] (P. Pevet).
general belief that the hormone mediates this effect through high affinity MEL receptors which have been localized in the rat SCN [11], especially because in vitro administration of physiological doses of MEL cause phaseshifts of rat SCN cells [12,15,16]. Nevertheless, in vivo there is uncertainty as to whether these receptors are crucial for the hormone’s phase-shifting actions because supraphysiological doses are needed to induce entrainment [21,26]. Serotonergic (5-HT) fibres originate in the midbrain raphe nuclei and are present in a large amount in the SCN [2]. The 5-HT system in the SCN is involved in the mechanisms of entrainment to non-photic stimuli [7], and depletion of the SCN 5-HT fibres are known to modulate the entrainment effect of light [25]. Moreover, administration of 5-HT agonists induce phase-shifts in the activity rhythm [7,9], and when administered at high doses, MEL is known to act as a 5-HT reuptake inhibitor [4,18]. This
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02590-7
H. A. Slotten et al. / Brain Research 876 (2000) 10 – 16
suggest that mediation of MEL’s phase-shifting action may involve the 5-HT system. Thus, from what is presently known about MEL’s biological effects, there is reason to believe that the hormone, when administered in vivo, does not induce a chronobiotic effect exclusively via its own receptors located in the SCN. The aim of the present study was to determine if the 5-HT fibres in the SCN are necessary for the entrainment effect of exogenous MEL. Specifically, by depleting the 5-HT fibres in the SCN by the selective neurotoxin 5,7-dihydroxytryptamine (DHT) [7], we wanted to test if the MEL regulation of 5-HT secretion and reuptake in the SCN could be the critical mechanism underlying entrainment to the hormone.
2. Materials and methods
2.1. Animals and surgery Twenty-three male Long–Evans rats obtained from Janvier (France) were used. Their age was approximately 10 weeks at the beginning of the experiment, and about 7 months at the end. The animals were maintained in a temperature-controlled (21628C) room with food (small rodent chow) and water available ad libitum. The experiment was performed in accordance with «Principles of Laboratory Animal Care» (NIH publication No. 86-23 revised 1985) as well as with the French national laws. Lesioning was performed in 16 animals when they weighed 280–310 g. About 30 min prior to surgery, the animals were pre-treated with 25 mg / kg desipramine hydrochloride (Sigma) dissolved in 0.9% NaCl to prevent uptake of neurotoxin into noradrenergic nerve terminals. The animals were then anaesthesized with equithesine (0.4 ml / 100 g) and placed in a Kopf small-animal stereotaxic instrument. Holes were drilled bilaterally 1.6 mm anterior to bregma, and a glass micropipette connected to a Hamilton syringe was lowered into the SCN (I55, DV52 8.3 mm from dura at 0.5 mm lateral to the midline with the injector positioned at a 28 angle). Each animal then received a bilateral injection of 25 mg DHT (Sigma) in 0.2 ml of 0.5% ascorbic acid, and each infusion was performed over 10 min. Under the same anaesthesia, a Mini Mitter (Sunriver, OR) telemetry device was implanted in the animals’ abdomen to allow continuous registration of CBT. The animals were then connected to an infusion system [14] by first fixing subcutaneously a silicone covered metal collar around the neck. From the neck the collar was connected to a flexible metal tube leading to a swivel which was connected to a bar at the top of the cage. A catheter was placed subcutaneously on the back of the animal and led outside of the cage through the hollow tube were the catheter was connected to a syringe placed on a timer-controlled pump. The chronically placed catheter permitted timed substance infusion, and the rats could
11
move freely in their cages. Unmanipulated control animals (n57) received telemetry device and were connected to the infusion system under halothane anaesthesia. This anaesthetic procedure is highly adequate for short-lasting surgical operations and, compared to the use of equithesine, it is somewhat better supported.
2.2. Experimental procedures, data recording and analysis Following surgery, the animals were placed in individual cages equipped with a running-wheel, and the experimental procedure started after a 2-week post-operative period in LD under vehicle infusion (NaCl / 1% alcohol). The animals were then transferred to DD and when free-running circadian rhythms were observed, MEL was infused for 1 h daily at a rate of 100 mg / h. Depending on each animal’s free-running period, the initial MEL infusion started 2–3 h following onset of the daily locomotor activity. Toward the end of the experiment, MEL was withdrawn and the vehicle re-administered. Fig. 2 illustrates this protocol. Two variables were used as markers of phase of the biological clock: Onset of locomotor activity, and CBT acrophase. Data from wheel revolutions (detected by a microswitch) and CBT (registered by the implanted thermistor) was recorded in bins of 5 min using the Dataquest III data aquisition system (Mini Mitter, Sunriver, OR). A non-linear regression equation described in Challet et al. [6] was used to determine CBT acrophase, and an eye-fitted straight line was drawn over at least ten successive days to mark onset of locomotor activity. We determined the phase-angle between MEL onset and activity onset, and CBT acrophase, respectively. The phase angle was then defined by expressing in hours the phase between the line indicating activity onset and markers of time on the actogram. On the basis of these data the group means (6S.E.M.) were calculated. For comparison between the two groups, two-tailed independent samples t-tests were used. Entrainment of circadian rhythms occurred when the animals expressed periods equal to the zeitgeber over at least 15 days consecutively.
2.3. Histology At the end of the experiment, the quality of the neurotoxic lesions were tested by an immunocytochemical control for the presence of 5-HT fibres within the SCN. The animals were killed with an overdose of pentobarbital and perfused transcardially with heparinized NaCl 0.9% to evacuate the blood before fixation of the brain. The first of three succeeding fixative solutions [27] was a phosphate buffer (PBS) of 4% paraformaldehyde (Merck), 0.2% picric acid, and 0.5% glutaraldehyde adjusted to pH 7.4. The second solution contained PBS with 4% paraformaldehyde and 0.2% picric acid (pH 6.3), while the last perfusate was a buffered 4% paraformaldehyde solution
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H. A. Slotten et al. / Brain Research 876 (2000) 10 – 16
(pH 7.4). Each animal received 100 ml of each perfusate before the brains were quickly removed from the skull. Following an overnight post fixation in the third fixative at 48C, transversal brain slices were sectioned (50 mm) on a vibratome. Sections were then incubated overnight at 48C with a 5-HT antiserum (rabbit anti-serotonin, INCSTAR, USA) diluted at 1:20,000 in a Tris–NaCl buffer (TBS) with 0.5% Triton X-100, followed by a 1-h incubation in biotinylated goat anti-rabbit IgG (Vectastain ABC kit, Vector Labs., CA). The last incubation implied a 1-h bath in the ABC kit reagents (avidin DH and biotinylated horseradish peroxidase H). Each incubation was interrupted by TBS rinsing, and tinting of the antigene–antibody complex was done with 0.05% 3-39 diaminobenzidine. The brain sections were then rinsed, mounted on slides, dried, and coverslipped by use of Eukitt (Kindler, Germany). The SCN were analysed for 5-HT immunoreactivity by use of optical densitometry (Biocom program RAG 200) against background of brain tissue outside SCN.
3. Results
3.1. Verification of DHT lesions Immunohistochemical staining of 5-HT fibres in the SCN of control animals revealed a high concentration of fibres in the nuclei (Fig. 1A). Approximately 100 days after lesioning the amount of 5-HT fibres was heavily reduced in the 16 DHT treated animals. In nine of these animals optical densitometry revealed no immunoreactivity for 5-HT fibres (Fig. 1B). These animals, with a complete lesion, were therefore chosen as the lesioned group from which the circadian rhythm data were analyzed. The seven partially lesioned animals were excluded from further analysis.
3.2. Effects of MEL administration on circadian rhythms The lesioned animals rapidly recovered from the surgery, and despite a complete absence of 5-HT fibres in the SCN we were unable to detect any effect of the lesion either on locomotor activity or on CBT. Following a post-operative period where the animals were well entrained to the LD cycle, they were transferred to DD. In free-running conditions, all animals in both groups expressed t s.24 h, and they all entrained when activity onset coincided with MEL onset (Fig. 2A–C). During entrainment, the phase-angle between activity onset and MEL onset was slightly negative and equal for both groups (controls: 20.5060.10 h; lesioned: 20.3760.06 h, t(14)5 21.21; P50.25). The CBT rose in parallel with an increase in activity (Fig. 2B–D), but the phase-angle between MEL onset and CBT acrophase was the same for the two groups (controls: 25.7260.22 h; lesioned:
25.3460.28 h, t(14)521.03; P50.32). When MEL treatment ceased, all animals free-ran with t s.24 h.
4. Discussion At concentrations as high as those used in studies of MEL’s effect on circadian rhythms, the hormone is known to inhibit 5-HT reuptake in nerve fibres [4,18]. To test the possible implication of the 5-HT fibres in the MEL effect, however, a complete lesion of these fibres within the SCN is needed. In a previous study [5] it was reported that a daily injection of MEL was able to entrain the free-running period of 5-HT depleted rats kept under DD. The approach used, bilateral injection of DHT into the lateral ventricle, is known as insufficient to totally deplete SCN 5-HT fibres. Cutrera et al. [7] demonstrated that in the Syrian hamster an almost total destruction of 5-HT fibres was obtained by use of bilateral application of DHT into the SCN, and that such complete lesions were necessary to block the phaseshifting effect of certain non-photic stimuli. This technique is also suitable to deplete totally the 5-HT fibres within the rat SCN ([6] the present work). In these conditions we observe that MEL is still able to entrain to a 24-h period the animals’ free-running rhythms. This clearly establish that, under the present experimental condition, the 5-HT fibres are not necessary for the circadian response to MEL. Following administration of DHT into the lateral ventricles of Syrian hamsters [25], it was observed differences in phase angles between L offset (LD 12:12) and activity onset for lesioned versus intact control animals. Although the present experiment was not designed to study the formal properties of entrainment to light, a qualitative analysis of the actograms of wheel-running revealed no differences on any parameter under entrainment either to LD or to MEL. Again, this contradiction might be explained by the quality and extent of the lesions. Our data clearly demonstrate that the well-established inhibiting effect of MEL on the 5-HT reuptake by nerve endings is not crucial in the hormone’s effect on circadian rhythms. The present negative results support the concept that such an effect is mediated through the MEL receptors present in the SCN. This concept is also sustained by the high correlation in mammals between the density of MEL receptors within the SCN and the ability to obtain entrainment to daily MEL administration. Contrary to the rat, where a high density of MEL binding is observed within the SCN, the mink (Mustela vison) does not seem to have specific MEL receptors located in these nuclei and this animal does not entrain to MEL [3]. Newborn Syrian hamsters express MEL receptors in the SCN, but shortly after birth the amount of receptors decrease. Young hamsters are entrainable by MEL, while in the adult hamster MEL is known as unable to entrain [13] or to entrain under particular experimental conditions ([14], Schuhler et al., unpublished).
H. A. Slotten et al. / Brain Research 876 (2000) 10 – 16
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Fig. 1. Sagittal sections of rat brains stained for 5-HT immunoreactivity. A dense plexus of terminals and fine fibres are present in the SCN of control animals (A), while DHT treated animals have a complete abscence of 5-HT fibres in the nuclei (B). SCN5suprachiasmatic nucleus; III5third ventricle, OC5optic chiasm, scale bar5100 mm.
Despite these strong arguments we have to face the problem that to entrain, in vivo, the circadian activity of the rat kept under DD, high doses of MEL have to be used. It has been suggested that endogenous MEL might make receptors less responsive to exogenous MEL [26]. However, the necessity for a high dose is probably not due to such a downregulation, neither is it a consequence of the hormone’s rapid metabolism because a photoperiodic
response is obtained when MEL is administered via a subcutaneous infusion system which mimics the hormone’s natural secretion profile [20]. It is thus difficult to consider that in these conditions all the effect of MEL would be mediated through an action on MEL receptors within the SCN. Very probably the hormone acts at the circadian system also through another mechanism. The present data only exclude an entraining action through induced changes
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H. A. Slotten et al. / Brain Research 876 (2000) 10 – 16
Fig. 2. Representative double plotted actograms of running-wheel (Fig. 2A–C) and core body temperature (CBT; range 36.5–39.58C) rhythms (Fig. 2B–D) of an intact control animal and an animal which in the beginning of the study underwent neurotoxic lesioning of the SCN 5-HT fibres. Following entrainment to the initial LD cycle (not shown), the animals were transferred to DD. The daily 1-h MEL administration (indicated by the shaded bars) started when free-run was observed, and led to entrainment of the circadian rhythms in all animals in both groups. When MEL treatment was discontinued, free-run re-occurred. Note how the onset in activity is parallelled with an increase in CBT.
H. A. Slotten et al. / Brain Research 876 (2000) 10 – 16
in 5-HT release from the nerve endings. They do not exclude a possible interaction between MEL and the 5-HT system. For example, MEL might act at the level of the post synaptic 5-HT receptors. This idea is supported by the work of Dugovic et al. [8] who have shown that, in vivo, exogenous MEL counteracts the sleep induced by 5-HT 2A agonists (DOM [1-2,5-dimethoxy-4-methylphenyl)-2aminopropane] and antagonists (ritanserin). Also, Eison et al. [10] demonstrated a modulation of 5-HT 2A receptormediated behavioral responses by exogenous MEL (high doses), and Ying et al. [28] found that high doses of the hormone exerted inhibitory effects on firing rates in IGL cells by mimicking the effects of 5-HT agonists. An indirect effect of MEL must also be considered, since increased levels of 5-HT within the brain have been reported after MEL administration [1,4,18,19]. In conclusion, we have demonstrated that the presence of 5-HT fibres in the SCN are unnecessary for the entraining effect of exogenous MEL. Thus, entrainment to MEL is not caused by a reduction in reuptake of 5-HT in SCN nerve endings, and the question of why high MEL doses are needed to entrain remains unanswered.
[8]
[9]
[10]
[11]
[12] [13]
[14]
[15]
Acknowledgements The authors thank Sylviane Gourmelen for technical assistance. This work was partially funded by IRIS, Courbevoie.
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serotonergic afferents to the suprachiasmatic nuclei prevents triazolam induced phase-advances of hamster activity rhythms, Behav. Brain Res. 62 (1994) 21–28. C. Dugovic, J.E. Leysen, A. Wauquier, Melatonin modulates the sensitivity of 5-hydroxytryptamine-2-receptor-mediated sleep–wakefulness regulation in the rat, Neurosci. Lett. 104 (1989) 320–325. D.M. Edgar, J.D. Miller, R.A. Prosser, R.R. Dean, W.C. Dement, Serotonin and the mammalian circadian system: II. Phase-shifting rat behavioral rhythms with serotonergic agonists, J. Biol. Rhythms 8 (1993) 17–31. A.S. Eison, R.P. Freeman, V.B. Guss, U.L. Mullins, R.N. Wright, Melatonin agonists modulate 5HT 2A receptor-mediated neurotransmission: behavioral and biochemical studies in the rat, J. Pharmacol. Exp. Therap. 273 (1995) 304–308. ´ ´ F. Gauer, M. Masson-Pevet, D. Skene, B. Vivien-Roels, P. Pevet, Daily rhythms of melatonin binding sites in the rat pars tuberalis and suprachiasmatic nuclei; evidence for a regulation of melatonin receptors by melatonin itself, Neuroendocrinology 57 (1993) 120– 126. M.U. Gillette, A.J. McArthur, Circadian actions of melatonin at the suprachiasmatic nucleus, Behav. Brain Res. 73 (1996) 135–139. M.H. Hastings, S.M. Mead, R.R. Vindlacheruvu, F.J.P. Ebling, E.S. Maywood, J. Grosse, Non-photic phase shifting of the circadian activity rhythm of Syrian hamsters: the relative potency of arousal and melatonin, Brain Res. 591 (1992) 20–26. B. Rusak, The mammalian circadian system: Models and physiology, in: S. Daan, E. Gwinner (Eds.), Biological Clocks and Environmental Time, The Guilford Press, New York, 1989, pp. 9–22. A.J. McArthur, M.U. Gillette, R.A. Prosser, Melatonin directly resets the rat suprachiasmatic circadian clock in vitro, Brain Res. 565 (1991) 158–161. A.J. McArthur, A.E. Hunt, M.U. Gillette, Melatonin action and signal transduction in the rat suprachiasmatic circadian clock: activation of protein kinase C at dusk and dawn, Endocrinology 138 (1997) 627–634. J.H. Meijer, W.J. Rietveld, Neurophysiology of the suprachiasmatic circadian pacemaker in rodents, Physiol. Rev. 69 (1989) 671–707. ´ J.M. Miguez, V. Simonneaux, P. Pevet, Evidence for a regulatory role of melatonin on serotonin release and uptake in the pineal gland, J. Neurochem. 7 (1995) 949–956. J.M. Miguez, F.J. Martin, M. Lema, M. Aldegunde, Changes in serotonin level and turnover in discrete hypothalamic nuclei after pinealectomy and melatonin administration to rats, Neurochem. Int. 29 (1996) 651–658. ´ B. Pitrosky, M. Masson-Pevet, R. Kirsch, B. Vivien-Roels, B. ´ Canguilhem, P. Pevet, Effects of different doses and durations of melatonin infusions on plasma melatonin concentrations in the pinealectomized Syrian hamsters: Concequences at the level of sexual activity, J. Pineal Res. 11 (1991) 149–155. ´ B. Pitrosky, R. Kirsch, A. Malan, E. Mocaer, P. Pevet, Organization of rat circadian rhythms after daily infusion of melatonin or S20098, a melatonin agonist, Am. J. Physiol. 277 (1999) R812–R828. J. Redman, S. Armstrong, K.T. Ng, Free-running activity rhythms in the rat: Entrainment by melatonin, Science 219 (1983) 1089–1091. B. Rusak, The mammalian circadian system: Models and physiology, in: S. Daan, E. Gwinner (Eds.), Biological Clocks and Environmental Time, The Guilford Press, New York, 1989, pp. 9–22. ´ H.A. Slotten, B. Pitrosky, P. Pevet, Influence of the Mode of Daily Melatonin Administration on Entrainment of Rat Circadian Rhythms, J. Biol. Rhythms 14 (1999) 347–353. L. Smale, K.M. Michels, R.Y. Moore, L.P. Morin, Destruction of the hamster serotonergic system by 5,7-DHT: Effects on circadian rhythm phase, entrainment and response to triazolam, Brain Res. 515 (1990) 9–19.
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[26] W.S. Warren, D.B. Hodges, V.M. Cassone, Pinealectomized rats entrain and phase-shift to melatonin injections in a dose-dependent manner, J. Biol. Rhythms 8 (1993) 233–245. [27] H. Yamada, S. Mori, S. Ueda, M. Kawata, Y. Sano, Improvement of technique of immunohistochemical demonstration of bioactive sub-
stances in the central nervous system, Acta Histochem. Cytochem. 20 (1987) 629–637. [28] S.-W. Ying, D.-X. Zhang, B. Rusak, Effects of serotonin agonists and melatonin on photic responses of hamster IGL neurons, Brain Res. 628 (1993) 8–16.
Research report
Entrainment of rat circadian rhythms by melatonin does not depend on the serotonergic afferents to the suprachiasmatic nuclei ´ a ,* Helge A. Slotten a,b , Bruno Pitrosky a , Paul Pevet a
` , Universite´ Louis Pasteur, 12 rue de l’ Universite´ , F-67000 Strasbourg, UMR CNRS 7518, Neurobiologie des fonctions rythmiques et saisonnieres France b Department of Psychology, NTNU, N-7491 Trondheim, Norway Accepted 9 May 2000
Abstract Daily administration of melatonin (MEL) can entrain rat circadian rhythms free-running in constant darkness. The high MEL doses needed to obtain entrainment suggest the implication of other neural mechanisms than simply an effect on the hormone’s specific receptors detected in the SCN. Administration of serotonin receptor agonists can phase-shift the rodent circadian clock, and MEL is known to modulate release and reuptake of serotonin in nerve endings. This raises the question of a critical involvement of 5-HT-fibres in the entraining properties of MEL. The aim of the present study was to test this hypothesis. Bilateral neurotoxic (5,7-dihydroxytryptamine) lesions of the serotonergic fibres in the SCN were performed in animals kept in LD 12:12. Following the post-operative period, the animals were transferred to constant darkness to free-run. MEL was then administered by a 1 h daily infusion. Both well lesioned and intact animals entrained to MEL. No differences were observed between lesioned and control animals on parameters such as the phase-angles between MEL onset and activity onset, and core body temperature acrophase, respectively. Entrainment of rat circadian rhythms to exogenous MEL is thus not directly dependent on the 5-HT fibres in the SCN. 2000 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behaviour Topic: Biological rhythms and sleep Keywords: Melatonin; Serotonin; 5,7-dihydroxytryptamine; SCN; Entrainment; Rat
1. Introduction In mammals, circadian variations in biological functions such as core body temperature (CBT), pineal melatonin (MEL) secretion, and activity / quiescence are driven by the suprachiasmatic nuclei (SCN) of the hypothalamus [17]. Because the period of the SCN is slightly different from 24 h, the timekeeping system needs daily adjustment from zeitgebers (timegivers) to synchronize to the nychthemeral environment. The natural light / dark (LD) cycle is the strongest zeitgeber [23], but also non-photic stimuli such as daily MEL administration are known to entrain rat circadian rhythms free-running in DD [21,22,24]. It is a
*Corresponding author. Tel.: 133-3-8835-8509; fax: 133-3-88240461. ´ E-mail address: [email protected] (P. Pevet).
general belief that the hormone mediates this effect through high affinity MEL receptors which have been localized in the rat SCN [11], especially because in vitro administration of physiological doses of MEL cause phaseshifts of rat SCN cells [12,15,16]. Nevertheless, in vivo there is uncertainty as to whether these receptors are crucial for the hormone’s phase-shifting actions because supraphysiological doses are needed to induce entrainment [21,26]. Serotonergic (5-HT) fibres originate in the midbrain raphe nuclei and are present in a large amount in the SCN [2]. The 5-HT system in the SCN is involved in the mechanisms of entrainment to non-photic stimuli [7], and depletion of the SCN 5-HT fibres are known to modulate the entrainment effect of light [25]. Moreover, administration of 5-HT agonists induce phase-shifts in the activity rhythm [7,9], and when administered at high doses, MEL is known to act as a 5-HT reuptake inhibitor [4,18]. This
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02590-7
H. A. Slotten et al. / Brain Research 876 (2000) 10 – 16
suggest that mediation of MEL’s phase-shifting action may involve the 5-HT system. Thus, from what is presently known about MEL’s biological effects, there is reason to believe that the hormone, when administered in vivo, does not induce a chronobiotic effect exclusively via its own receptors located in the SCN. The aim of the present study was to determine if the 5-HT fibres in the SCN are necessary for the entrainment effect of exogenous MEL. Specifically, by depleting the 5-HT fibres in the SCN by the selective neurotoxin 5,7-dihydroxytryptamine (DHT) [7], we wanted to test if the MEL regulation of 5-HT secretion and reuptake in the SCN could be the critical mechanism underlying entrainment to the hormone.
2. Materials and methods
2.1. Animals and surgery Twenty-three male Long–Evans rats obtained from Janvier (France) were used. Their age was approximately 10 weeks at the beginning of the experiment, and about 7 months at the end. The animals were maintained in a temperature-controlled (21628C) room with food (small rodent chow) and water available ad libitum. The experiment was performed in accordance with «Principles of Laboratory Animal Care» (NIH publication No. 86-23 revised 1985) as well as with the French national laws. Lesioning was performed in 16 animals when they weighed 280–310 g. About 30 min prior to surgery, the animals were pre-treated with 25 mg / kg desipramine hydrochloride (Sigma) dissolved in 0.9% NaCl to prevent uptake of neurotoxin into noradrenergic nerve terminals. The animals were then anaesthesized with equithesine (0.4 ml / 100 g) and placed in a Kopf small-animal stereotaxic instrument. Holes were drilled bilaterally 1.6 mm anterior to bregma, and a glass micropipette connected to a Hamilton syringe was lowered into the SCN (I55, DV52 8.3 mm from dura at 0.5 mm lateral to the midline with the injector positioned at a 28 angle). Each animal then received a bilateral injection of 25 mg DHT (Sigma) in 0.2 ml of 0.5% ascorbic acid, and each infusion was performed over 10 min. Under the same anaesthesia, a Mini Mitter (Sunriver, OR) telemetry device was implanted in the animals’ abdomen to allow continuous registration of CBT. The animals were then connected to an infusion system [14] by first fixing subcutaneously a silicone covered metal collar around the neck. From the neck the collar was connected to a flexible metal tube leading to a swivel which was connected to a bar at the top of the cage. A catheter was placed subcutaneously on the back of the animal and led outside of the cage through the hollow tube were the catheter was connected to a syringe placed on a timer-controlled pump. The chronically placed catheter permitted timed substance infusion, and the rats could
11
move freely in their cages. Unmanipulated control animals (n57) received telemetry device and were connected to the infusion system under halothane anaesthesia. This anaesthetic procedure is highly adequate for short-lasting surgical operations and, compared to the use of equithesine, it is somewhat better supported.
2.2. Experimental procedures, data recording and analysis Following surgery, the animals were placed in individual cages equipped with a running-wheel, and the experimental procedure started after a 2-week post-operative period in LD under vehicle infusion (NaCl / 1% alcohol). The animals were then transferred to DD and when free-running circadian rhythms were observed, MEL was infused for 1 h daily at a rate of 100 mg / h. Depending on each animal’s free-running period, the initial MEL infusion started 2–3 h following onset of the daily locomotor activity. Toward the end of the experiment, MEL was withdrawn and the vehicle re-administered. Fig. 2 illustrates this protocol. Two variables were used as markers of phase of the biological clock: Onset of locomotor activity, and CBT acrophase. Data from wheel revolutions (detected by a microswitch) and CBT (registered by the implanted thermistor) was recorded in bins of 5 min using the Dataquest III data aquisition system (Mini Mitter, Sunriver, OR). A non-linear regression equation described in Challet et al. [6] was used to determine CBT acrophase, and an eye-fitted straight line was drawn over at least ten successive days to mark onset of locomotor activity. We determined the phase-angle between MEL onset and activity onset, and CBT acrophase, respectively. The phase angle was then defined by expressing in hours the phase between the line indicating activity onset and markers of time on the actogram. On the basis of these data the group means (6S.E.M.) were calculated. For comparison between the two groups, two-tailed independent samples t-tests were used. Entrainment of circadian rhythms occurred when the animals expressed periods equal to the zeitgeber over at least 15 days consecutively.
2.3. Histology At the end of the experiment, the quality of the neurotoxic lesions were tested by an immunocytochemical control for the presence of 5-HT fibres within the SCN. The animals were killed with an overdose of pentobarbital and perfused transcardially with heparinized NaCl 0.9% to evacuate the blood before fixation of the brain. The first of three succeeding fixative solutions [27] was a phosphate buffer (PBS) of 4% paraformaldehyde (Merck), 0.2% picric acid, and 0.5% glutaraldehyde adjusted to pH 7.4. The second solution contained PBS with 4% paraformaldehyde and 0.2% picric acid (pH 6.3), while the last perfusate was a buffered 4% paraformaldehyde solution
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(pH 7.4). Each animal received 100 ml of each perfusate before the brains were quickly removed from the skull. Following an overnight post fixation in the third fixative at 48C, transversal brain slices were sectioned (50 mm) on a vibratome. Sections were then incubated overnight at 48C with a 5-HT antiserum (rabbit anti-serotonin, INCSTAR, USA) diluted at 1:20,000 in a Tris–NaCl buffer (TBS) with 0.5% Triton X-100, followed by a 1-h incubation in biotinylated goat anti-rabbit IgG (Vectastain ABC kit, Vector Labs., CA). The last incubation implied a 1-h bath in the ABC kit reagents (avidin DH and biotinylated horseradish peroxidase H). Each incubation was interrupted by TBS rinsing, and tinting of the antigene–antibody complex was done with 0.05% 3-39 diaminobenzidine. The brain sections were then rinsed, mounted on slides, dried, and coverslipped by use of Eukitt (Kindler, Germany). The SCN were analysed for 5-HT immunoreactivity by use of optical densitometry (Biocom program RAG 200) against background of brain tissue outside SCN.
3. Results
3.1. Verification of DHT lesions Immunohistochemical staining of 5-HT fibres in the SCN of control animals revealed a high concentration of fibres in the nuclei (Fig. 1A). Approximately 100 days after lesioning the amount of 5-HT fibres was heavily reduced in the 16 DHT treated animals. In nine of these animals optical densitometry revealed no immunoreactivity for 5-HT fibres (Fig. 1B). These animals, with a complete lesion, were therefore chosen as the lesioned group from which the circadian rhythm data were analyzed. The seven partially lesioned animals were excluded from further analysis.
3.2. Effects of MEL administration on circadian rhythms The lesioned animals rapidly recovered from the surgery, and despite a complete absence of 5-HT fibres in the SCN we were unable to detect any effect of the lesion either on locomotor activity or on CBT. Following a post-operative period where the animals were well entrained to the LD cycle, they were transferred to DD. In free-running conditions, all animals in both groups expressed t s.24 h, and they all entrained when activity onset coincided with MEL onset (Fig. 2A–C). During entrainment, the phase-angle between activity onset and MEL onset was slightly negative and equal for both groups (controls: 20.5060.10 h; lesioned: 20.3760.06 h, t(14)5 21.21; P50.25). The CBT rose in parallel with an increase in activity (Fig. 2B–D), but the phase-angle between MEL onset and CBT acrophase was the same for the two groups (controls: 25.7260.22 h; lesioned:
25.3460.28 h, t(14)521.03; P50.32). When MEL treatment ceased, all animals free-ran with t s.24 h.
4. Discussion At concentrations as high as those used in studies of MEL’s effect on circadian rhythms, the hormone is known to inhibit 5-HT reuptake in nerve fibres [4,18]. To test the possible implication of the 5-HT fibres in the MEL effect, however, a complete lesion of these fibres within the SCN is needed. In a previous study [5] it was reported that a daily injection of MEL was able to entrain the free-running period of 5-HT depleted rats kept under DD. The approach used, bilateral injection of DHT into the lateral ventricle, is known as insufficient to totally deplete SCN 5-HT fibres. Cutrera et al. [7] demonstrated that in the Syrian hamster an almost total destruction of 5-HT fibres was obtained by use of bilateral application of DHT into the SCN, and that such complete lesions were necessary to block the phaseshifting effect of certain non-photic stimuli. This technique is also suitable to deplete totally the 5-HT fibres within the rat SCN ([6] the present work). In these conditions we observe that MEL is still able to entrain to a 24-h period the animals’ free-running rhythms. This clearly establish that, under the present experimental condition, the 5-HT fibres are not necessary for the circadian response to MEL. Following administration of DHT into the lateral ventricles of Syrian hamsters [25], it was observed differences in phase angles between L offset (LD 12:12) and activity onset for lesioned versus intact control animals. Although the present experiment was not designed to study the formal properties of entrainment to light, a qualitative analysis of the actograms of wheel-running revealed no differences on any parameter under entrainment either to LD or to MEL. Again, this contradiction might be explained by the quality and extent of the lesions. Our data clearly demonstrate that the well-established inhibiting effect of MEL on the 5-HT reuptake by nerve endings is not crucial in the hormone’s effect on circadian rhythms. The present negative results support the concept that such an effect is mediated through the MEL receptors present in the SCN. This concept is also sustained by the high correlation in mammals between the density of MEL receptors within the SCN and the ability to obtain entrainment to daily MEL administration. Contrary to the rat, where a high density of MEL binding is observed within the SCN, the mink (Mustela vison) does not seem to have specific MEL receptors located in these nuclei and this animal does not entrain to MEL [3]. Newborn Syrian hamsters express MEL receptors in the SCN, but shortly after birth the amount of receptors decrease. Young hamsters are entrainable by MEL, while in the adult hamster MEL is known as unable to entrain [13] or to entrain under particular experimental conditions ([14], Schuhler et al., unpublished).
H. A. Slotten et al. / Brain Research 876 (2000) 10 – 16
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Fig. 1. Sagittal sections of rat brains stained for 5-HT immunoreactivity. A dense plexus of terminals and fine fibres are present in the SCN of control animals (A), while DHT treated animals have a complete abscence of 5-HT fibres in the nuclei (B). SCN5suprachiasmatic nucleus; III5third ventricle, OC5optic chiasm, scale bar5100 mm.
Despite these strong arguments we have to face the problem that to entrain, in vivo, the circadian activity of the rat kept under DD, high doses of MEL have to be used. It has been suggested that endogenous MEL might make receptors less responsive to exogenous MEL [26]. However, the necessity for a high dose is probably not due to such a downregulation, neither is it a consequence of the hormone’s rapid metabolism because a photoperiodic
response is obtained when MEL is administered via a subcutaneous infusion system which mimics the hormone’s natural secretion profile [20]. It is thus difficult to consider that in these conditions all the effect of MEL would be mediated through an action on MEL receptors within the SCN. Very probably the hormone acts at the circadian system also through another mechanism. The present data only exclude an entraining action through induced changes
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Fig. 2. Representative double plotted actograms of running-wheel (Fig. 2A–C) and core body temperature (CBT; range 36.5–39.58C) rhythms (Fig. 2B–D) of an intact control animal and an animal which in the beginning of the study underwent neurotoxic lesioning of the SCN 5-HT fibres. Following entrainment to the initial LD cycle (not shown), the animals were transferred to DD. The daily 1-h MEL administration (indicated by the shaded bars) started when free-run was observed, and led to entrainment of the circadian rhythms in all animals in both groups. When MEL treatment was discontinued, free-run re-occurred. Note how the onset in activity is parallelled with an increase in CBT.
H. A. Slotten et al. / Brain Research 876 (2000) 10 – 16
in 5-HT release from the nerve endings. They do not exclude a possible interaction between MEL and the 5-HT system. For example, MEL might act at the level of the post synaptic 5-HT receptors. This idea is supported by the work of Dugovic et al. [8] who have shown that, in vivo, exogenous MEL counteracts the sleep induced by 5-HT 2A agonists (DOM [1-2,5-dimethoxy-4-methylphenyl)-2aminopropane] and antagonists (ritanserin). Also, Eison et al. [10] demonstrated a modulation of 5-HT 2A receptormediated behavioral responses by exogenous MEL (high doses), and Ying et al. [28] found that high doses of the hormone exerted inhibitory effects on firing rates in IGL cells by mimicking the effects of 5-HT agonists. An indirect effect of MEL must also be considered, since increased levels of 5-HT within the brain have been reported after MEL administration [1,4,18,19]. In conclusion, we have demonstrated that the presence of 5-HT fibres in the SCN are unnecessary for the entraining effect of exogenous MEL. Thus, entrainment to MEL is not caused by a reduction in reuptake of 5-HT in SCN nerve endings, and the question of why high MEL doses are needed to entrain remains unanswered.
[8]
[9]
[10]
[11]
[12] [13]
[14]
[15]
Acknowledgements The authors thank Sylviane Gourmelen for technical assistance. This work was partially funded by IRIS, Courbevoie.
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