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In Syrian and European hamsters, the duration of sensitive phase to light of the suprachiasmatic nuclei depends on the photoperiod
ELSEVIER
Nearoscienee Letters 208 (1996)37-40
NEURUSCIENCE LETTERS
In Syrian and European hamsters, the duration of sensitive phase to light of the suprachiasmatic nuclei depends on the photoperiod P a t r i c k Vuillez*, N a t h a l i e Jacob, R e b e c c a T e c l e m a r i a m - M e s b a h , Paul P6vet URA-CNRS 1332, 'Neurobiologie des fonctions rythmiques et saisonni~res', Universitd Louis Pasteur, 12 rue de l'Universitd, 67000 Strasbourg, France Received 8 January 1996; revised version received 7 March 1996; accepted 7 March 1996
Abstract
Light induction of the expression of Fos protein in the suprachiasmatic nuclei was used to investigate the photosensitive state of the clock in Syrian and European hamster kept under different photoperiods. We observed that the duration of the photosensitive phase is variable and tied to the length of the night. A maximal extension has been determined in both species studied. Finally, a 4 h lengthening of the phase of photosensitivity takes approximately 3 weeks, while 3 days only are needed for its shortening.
Keywords:Circadian rhythms; Light; c-fos; Photoperiod; Melatonin; Rodents
Mammalian suprachiasmatic nuclei (SCN) contain a pacemaker which generates oscillations with a period close to 24 h. Circadian rhythms persist under constant conditions such as permanent darkness (DD). External synchronizers, mainly the daily light/dark (LD) cycle, entrain circadian rhythms to precisely 24 h. Photic information is transmitted to the. SCN via two major pathways, the direct retinohypothal.'unic tract and the indirect geniculohypothalamic tract 116]. Light exposure during the night induces sharp expression of c-fos in the SCN, indicating activation of numerous cells. In DD, a light pulse during the subjective night, but not during the subjective day, causes induction of c-fos in the SCN and a permanent phase shift of the circadian locomotor activity [9]. Most studies on the sensitivity to light of the SCN have investigated only a few time points during the day or during the night. Data on the exact duration of the phase of photosensitivity of the SCN are very scarce [10,11], but changes in the sensitivity of different SCN cell populations have been shown to parallel that of the phaseshifting direction induced by light on locomotor activity [12]. Since the pattern of circadian locomotor activity can be dependent upon the photoperiod (relative duration of the light during the 24 h period [13]), we have hypothesized that the duration of the phase of photosensitivity * Corresponding author. Fax: +33 88 24 04 61.
would be also dependent upon the photoperiod. Using two photoperiodic species, the European (Cricetus cricetus) and Syrian (Mesocricetus auratus) hamster, we have studied, at different times of the nycthemer, under different photoperiodic regimes, the effect of light exposure on Fos expression in the SCN. Adult male European hamsters (250-450 g) caught in the fields near Strasbourg (France) in March were kept under natural environmental conditions until the beginning of the experiments. Adult male Syrian hamsters (90-100g), bred in LD 14:10 were obtained from Harlan (Netherlands). Animals were transferred, always at the L D transition, in different photoperiodic regimes detailed further on. For the experiment, animals were exposed to light for 15 min (identical to 'day' lighting). One hour after the onset of light stimulation, animals were anaesthetized in darkness with pentobarbital 6% (0.6 ml/100 g, i.p.) and perfused transcardially with NaCI 0.9% followed by 4% paraformaldehyde in phosphate buffer 0.1 M (pH 7.4). Dissected brains were postfixed for 4-8 h. Vibratome transversal sections (50/tm) throughout SCN were rinsed in phosphate-buffered saline (PBS), incubated overnight at 4°C with sheep anti-Fos antiserum (1:5000 in PBS containing 0.5% Triton X100; Cambridge Chemicals), then 1 h with a biotinylated secondary anti-sheep antibody (1:500, room temperature; Vector) followed by ABC reagent for 1 h (Vector). Peroxidase activity was
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All fights reserved PII: S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 1 252;5-9
P. Vuillez et al. / Neuroscience Letters 208 (1996) 37--40
38
revealed using 0.025% DAB (Sigma) in Tris 0.05 M (pH 7.6), containing 0.5% nickel ammonium sulphate and 0.1% H202. Fos-like immunoreactivity (Fos-ir) appears as nuclear black precipitate. According to the supplier's specification, the polyclonal anti-Fos antibody recognizes Fos and Fos-related proteins. For all experiments detailed later on, 2--4 animals per time point were studied, and at least one control hamster per point was sacrificed without light stimulation. Stimulated hamsters may show very large numbers of densely immunostained cells in the SCN. In contrast, the SCN of control animals contain relatively few labelled cells. These cells are mostly weakly stained but all of them were counted for quantitative studies. This explains the relatively high control values we show in histograms. In experiment Eh-LP, at the end of June (natural photoperiod is 16 h L/8 h D), European hamsters were transferred indoors under experimental long photoperiod (LP) conditions (LD 16:8, lights off at 2000 h, close to natural conditions) for 2 weeks. In the rooms (20 ___I°C), light is provided by fluorescent strip lights yielding approximately 300 lux at the bottom of cages. A constant dim red light (less than 1 lux) allows manipulation of animals in 'darkness'. D
D+4
D+8
D+12
D+16
The animals were then submitted to a light stimulus ($) during the 'normal' night (black bar) at either D + 1 (1 h after lights off), D + 4, D + 6, or D + 7. In this experiment as well as in others, in order to test the photosensibility of SCN after the normal end of the night, on the day of experiment hamsters were kept in darkness. At either D + 8, 9, 10, 11, 12, 13, 14, or 15, light stimulation was thus given in the prolonged night (grey bar). European hamsters submitted to a light stimulus during the normal night showed a great number of Fos-ir cells in the SCN. At D + 1, labelled cells extend rostrocaudally for 450/~m in the ventral part of the SCN. At D + 4, 6 or 7, cells are more numerous and are found distributed throughout 600/~m in the ventral and dorsal parts of the SCN. In animals stimulated at D + 8 (normal lights on), the same pattern of labelled cells in the SCN as in animals stimulated in the middle or at the end of the night was observed. However, animals stimulated from D + 9 to D + 15 showed only a few more labelled cells than controls sacrificed without prior light exposure. In experiment Eh-SP, at mid-December (natural photoperiod approximately 8 h L, 16 h D), European hamsters were transferred indoors to short photoperiod (SP, LD 8:16, lights off at 1700 h) for 2 weeks. They were then light-stimulated at either 1, 8, 9, 10, 11, 12, 13, 14 or 15 h after dark onset. At D + 1, animals showed Fos-ir cells throughout the SCN as in animals stimulated at the same
time from experiment Eh-LP. From D + 8 to D + 13 (Fig. 1), labelling was similar to that observed from D + 4 to D + 8 in Eh-LP. However, at D + 14 or D + 15 (Fig. 2), light-exposed animals showed only very few labelled cells, and the SCN of these animals were not distinguishable from the SCN of control hamsters. Here, the duration of the photosensitive period is increased from 8 to 14 h compared to animals kept in LP. In experiment Sh-LP, the SCN of Syrian hamsters kept in LP (LD 14:10) and stimulated by light at either D + 1 or D + 9 showed numerous Fos-ir cells. D+4
D
I"
D+8
D+12 I
? 'I" "I" $ 'I"
I
I
I
The distribution of labelled cells was similar to that previously reported [12]. At the beginning of the night, the main light-sensitive cell population was located in the ventral and lateral part of the caudal half of the SCN. At D + 9, light-induced Fos-ir also occurred dorsally to the first population and in the rostral SCN. At this time, numerous cells were also labelled laterally, dorsally and rostrally to the nuclei. Light stimuli were also applied at D + 10, 11, 12, or 13 in prolonged night on the day of experiment. At D + 10, a labelling similar to D + 9 was observed. From D + 11 on, only few scattered cells expressed Fos after light exposure. The total duration of the photosensitive period was thus 10 h. In experiment Sh-SP1, Syrian hamsters were maintained for 8 weeks in SP (LD 10:14). In SCN of animals stimulated at D + 1, 9 or 10 the labelling was identical to that obtained at the same times for Sh-LP. At D + 11, 12,
~i~i~~'~ i~i~!!!~i ~ ~ii~i~ ~!i~i~i!i~ i~i!:~?:~ %~iT~Ji!i~i~!i~i~i~J~!i Ji¸i~i~:i~ ii!ii!'
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Figs. 1-4. Representativecoronal sections through the SCN stained for Fos immunoreaetivity.Bar = 100/am. Figs. 1, 2, European hamstersin LD 8:16, light stimulated at D + 13 (Fig. !) or at D + 14 (Fig. 2). Figs. 3, 4. Syrian hamsters kept in LD 10:!4 and light stimulated at D+ 11 (Fig. 3) or sacrificedat the same time without prior light stimulus.
39
P. Vuillez et aL /Neuroscience Letters 208 (1996) 37-40
13 or 14, a great number of cells were sensitive to light and a pronounced Fos-ir was observed throughout the SCN (compare Figs. 3 and 4). On the other hand, only a few cells, mostly weakly stained, were observed in the SCN in animals stimulated at D + 15. Like in the European hamster, an increase in duration of the photosensitive period is observed when animals are transferred from LP to SP. In experiment Sh-SP2, Syrian hamsters previously kept in LP were maintained for 8 weeks in SP (LD 8:16). Contrary to observations in animals kept under LD 10:14 or 14:10, animals stimulated at D + 1 showed only very few labelled cells. Those stimulated at D + 3, 12, 14, 15, or 16 showed an intense Fos labelling. The increased delay of the time of the switch from insensitivity to sensitivity to light in respect with the beginning of the night has already been demonstrated for other parameters such as circadian locomotor activity. It should be correlate with the so-called 'decompression' of the clock [2]. At D + 17 or D + 18 light has no effect on Fos expression. The duration of the light-sensitive period has thus increased and is approximately 15 h. In experiment Sh-SP3, Syrian hamsters previously kept under LP were maintained for 8 weeks in SP (LD 4:20) and light-stimulated at D + 1, 3, 15, 16, 17, 18, or 19. In this experiment, no labelled cells was detected in the SCN of animals stimulated at D + 1. Light stimulus from D + 3 to D + 17 produced an intense Fos-ir in the SCN. At D + 18, the same labelling was observed in one animal, when the second one, as well as hamsters stimulated at D + 19, showed only a few labelled cells in the SCN, similar to control animals. This demonstrates that, like in European hamster, the light-sensitive period has a limit in its extension. The system is no more sensitive 18 h after the beginning: of the night. Due to the 'decompression' of the clock, the exact beginning of the sensitive phase and thus its exact duration (between 15 and 18 h) cannot be precisely determined. Having observed that the duration of photosensitive phase was indeed longer in SP than in LP, the next two experiments were designed to determine the rate of extension/reduction of this photosensitive period when the animals were transferred from one photoperiod to another. In experiment Sh-LP to SP, Syrian hamsters raised and kept in LP (LD 14:10) were transferred in SP (LD 10:14). After 4, 11, 20 or 25 nycthemers in SP, animals were light-stimulated during the extended night either at D + 11 or at D + 13. Out of the 8-10 sections obtained from rostro-caudal SCN, four similar sections per animal were chosen and Fos-ir cells were counted on a monitoring video coupled to a microscope. Cells inside the SCN and in the hypothalamic area immediately adjacent to the dorso-lateral boundaries of the nuclei which have already been described to be sensitive to light [ 12] were counted. The number of cells expressing Fos in animals stimulated
at either D + 11 or D + 13 on the night of the photoperiod change was in the same range as that of control animals. The number of light-sensitive cells in the SCN and surrounding hypothalamus during the 4 added hours increased progressively until the 25th night after the photoperiod change. Then, the total values were similar to those after stimulus at equivalent times for animals maintained 8 weeks in SP (Fig. 5A). In experiment Sh-SP to LP, Syrian hamsters maintained for 8 weeks in SP (LD 10:14) were transferred to LP (LD 14:10) for 2, 3, 4 or 11 nycthemers. To test the photosensitivity of SCN cells after the normal end of the night (D + 10), animals were kept in darkness on the day of experiment. Light stimulus were applied at D + 11 or D + 13. After a change from SP to LP, the photosensitivity of SCN cells at the corresponding times D + 11 and D + 13 decreased rapidly. Four nights after the change, light stimulation was almost ineffective on the induction of Fos expression in the SCN (Fig. 5B). Our results show that the sensitivity of the SCN to light is dependent on the photoperiod. The duration of the photosensitive phase, as determined by Fos expression, increases with the lengthening of the night. Such increase
r-7
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--
Control
1600-
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400
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4
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20
25
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1200
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~
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in LID 10:14
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Fig. 5. Expression of Fos protein in the SCN of Syrian hamsters induced by a light pulse: (A) after transferfrom LP (LD 14:10) to SP (LD 10:14); (B) after transfer from SP (LD 10:14) to LP (LD 14:10). The dotted lines correspond to control hamsters of the time points D + 11 and D + 13, sacrificedwithout prior light stimulus.
40
P. Vuillez et al. / Neuroscience Letters 208 (1996) 37-40
has a limit which is 15-18 h in the Syrian hamster and 14 h in the European hamster. However, after the transfer from LP (LD 14:10) to SP (LD 10:14), a 4 h increase in the duration of the night, the time interval enabling light induction of Fos extends gradually, the full extension being achieved after more than 3 weeks. This indicates that the photoperiod has a slow and progressive effect on the SCN. It is thus not possible to exclude completely that the 15-18 h extension limit observed in Syrian hamster kept under LD 4:20 is because 8 weeks was not enough to integrate the 10 h night lengthening. This, however, does not apply to the experiment with European hamsters since they were in a natural SP close to the experimental one. Probably these extension limits would point to the already demonstrated role of the circadian system in the expression of photosensitive phases of which the duration only is dependent on the photoperiod. In a recent study done in the rat, Sumowi et al. [10,11] using a different paradigm, came to the same conclusion: a circadian property, namely, the photosensitivity state, of the SCN is influenced by the photoperiodic environment. The question is then how and by which pathway can the photoperiod influence SCN activity? We know that it is through changes in the duration of nocturnal melatonin secretion that the organism is able to read photoperiod [5]. Following transfer from LP (14:10) to SP (10:14), duration of melatonin secretion extends gradually and 3 weeks are also needed to obtain maximal duration. After transfer from SP to LP, the melatonin peak adjusts very rapidly to the shortened night [3]. With the same type of protocol we observed that the decrease in duration of the photosensitive period similarly takes only a few days. The SCN are also known to contain melatonin receptors [1,8]. It is thus possible that, throughout photoperiod-induced changes in the duration of melatonin peak, photoperiod acts on the SCN. The possible role of other photoperiod-dependent changes has also to be considered. The SCN receive photic information indirectly from the retina via NPYfibers originating in the intergeniculate leaflets [6]. The role of NPY in photic transduction is at present not well defined but in the jerboa ( J a c u l u s orientalis) the NPY innervation of the SCN shows photoperiod-dependent seasonal changes [4]. It is thus possible that this pathway through fluctuation of NPY content underlies the photosensitive phase duration of the SCN. As the rhythmic melatonin secretion is dependent on the SCN, the parallel observed between the time course of photoperiod-induced changes in the duration of melatonin production and in the duration of light-sensitive phase of the SCN might also reflect changes in the pacemaker itself. Following the theoretical model of Pittendrigh and
Daan [7] the clock would consist of a complex circadian pacemaker consisting of evening and morning components; the LD cycle would define a dawn and a dusk signal. In such a system, by measuring the changes in phase relationship between the morning and evening component, the SCN would be able to build itself a photoperiodic signal and would not need to perceive it. Our data, as well as those of Sumov~i et ai. [10,11], would correlate also with such a hypothesis. In conclusion, using two photoperiodic species and different experimental conditions, we have been able to demonstrate that the duration of the photosensitive phase of the SCN is not a set property of the circadian system but is dependent upon the photoperiod. The mechanisms involved are still to be elucidated.
[1] Gauer, F., Masson-P6vet, M. and P6vet, P., Melatonin receptor density is regulated in rat pars tuberalis and suprachiasmatic nuclei by melatonin itself, Brain Res., 602 (1993) 153-156. [2] lllnerovli, H., Circadian rhythms in the mammalian pineal gland, Rozpr. Cesk. Akad. Ved, 96(1) (1986) 3-105. [3] Illnerovri, H., Hoffmann, K. and Vanecek, J., Adjustment of the rat pineal N-acetyltransferaserhythm to change from long to short photoperiod depends on the direction of extension of the dark period, Brain Res., 362 (1986) 403-408. [4] Lakhdar-Ghazal, N., Oukouchoud, R. and Pgvet, P., Seasonal variation in NPY immunoreactivity in the suprachiasmatic nucleus of the jerboa (Jaculus orientalis), a desert hibernator, Neurosci. Lett., 193 (1995) 49-52. [5] Maywood, E.S., Hastings, M.H., Max, M., Ampleford, E., Menaker, M. and Loudon, A.S.I., Circadian and daily rhythms of melatonin in the blood and pineal gland of free-running and entrained Syrian hamsters, J. Endocrinol., 136 (1993) 65-73. [6] Morin, L.P., The circadian visual system, Brain Res. Rev., 67 (1994) 102-127. [7] Pittendrigh, C.S. and Daan, S., A functional analysis of circadian pacemakers in nocturnal rodents, J. Comp. Physiol. A,, 106 (1976) 333-355. [8] Reppert, S.M., Weaver, D.R. and Ebisawa, T., Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses, Neuron, 13 (1994) 11771185. [9] Rusak, B., Robertson, H.A., Wisden, W. and Hunt, S.P., Light pulses that shift rhythms induce gene expression in the suprachiasmatic nucleus, Science, 248 (1990) 1237-1240. [10] Sumovfi,A., Tr~ivnfckov~i,Z. and lllnerovfi, H., Memory on long but not on short days is stored in the rat suprachiasmatic nucleus, Neurosci. Lett., 200 (1995) 191-194. [11] Sumovi A., Tnivnfckov~i, Z., Peters, R., Schwartz, W.J. and lllnerov~i, H., The rat suprachiasmatic nucleus is a clock for all seasons, Proc. Natl. Acad. Sci. USA, 92 (1995) 7754-7758. [12] Teclemariam-Mesbah,R., Vuillez, P., Van Rossum, A. and P6vet, P., Time course of neuronal sensitivity to light in the circadian timing system of the golden hamster, Neurosci. Lett., 201 (1995) 5-8. [13] Wollnik, F., Breit, A. and Reinke D., Seasonal change in temporal organization of wheel-running activity of the European hamster, Cricetus cricetus, Naturwissenschaften, 78 ( 1991) 419-422.
Nearoscienee Letters 208 (1996)37-40
NEURUSCIENCE LETTERS
In Syrian and European hamsters, the duration of sensitive phase to light of the suprachiasmatic nuclei depends on the photoperiod P a t r i c k Vuillez*, N a t h a l i e Jacob, R e b e c c a T e c l e m a r i a m - M e s b a h , Paul P6vet URA-CNRS 1332, 'Neurobiologie des fonctions rythmiques et saisonni~res', Universitd Louis Pasteur, 12 rue de l'Universitd, 67000 Strasbourg, France Received 8 January 1996; revised version received 7 March 1996; accepted 7 March 1996
Abstract
Light induction of the expression of Fos protein in the suprachiasmatic nuclei was used to investigate the photosensitive state of the clock in Syrian and European hamster kept under different photoperiods. We observed that the duration of the photosensitive phase is variable and tied to the length of the night. A maximal extension has been determined in both species studied. Finally, a 4 h lengthening of the phase of photosensitivity takes approximately 3 weeks, while 3 days only are needed for its shortening.
Keywords:Circadian rhythms; Light; c-fos; Photoperiod; Melatonin; Rodents
Mammalian suprachiasmatic nuclei (SCN) contain a pacemaker which generates oscillations with a period close to 24 h. Circadian rhythms persist under constant conditions such as permanent darkness (DD). External synchronizers, mainly the daily light/dark (LD) cycle, entrain circadian rhythms to precisely 24 h. Photic information is transmitted to the. SCN via two major pathways, the direct retinohypothal.'unic tract and the indirect geniculohypothalamic tract 116]. Light exposure during the night induces sharp expression of c-fos in the SCN, indicating activation of numerous cells. In DD, a light pulse during the subjective night, but not during the subjective day, causes induction of c-fos in the SCN and a permanent phase shift of the circadian locomotor activity [9]. Most studies on the sensitivity to light of the SCN have investigated only a few time points during the day or during the night. Data on the exact duration of the phase of photosensitivity of the SCN are very scarce [10,11], but changes in the sensitivity of different SCN cell populations have been shown to parallel that of the phaseshifting direction induced by light on locomotor activity [12]. Since the pattern of circadian locomotor activity can be dependent upon the photoperiod (relative duration of the light during the 24 h period [13]), we have hypothesized that the duration of the phase of photosensitivity * Corresponding author. Fax: +33 88 24 04 61.
would be also dependent upon the photoperiod. Using two photoperiodic species, the European (Cricetus cricetus) and Syrian (Mesocricetus auratus) hamster, we have studied, at different times of the nycthemer, under different photoperiodic regimes, the effect of light exposure on Fos expression in the SCN. Adult male European hamsters (250-450 g) caught in the fields near Strasbourg (France) in March were kept under natural environmental conditions until the beginning of the experiments. Adult male Syrian hamsters (90-100g), bred in LD 14:10 were obtained from Harlan (Netherlands). Animals were transferred, always at the L D transition, in different photoperiodic regimes detailed further on. For the experiment, animals were exposed to light for 15 min (identical to 'day' lighting). One hour after the onset of light stimulation, animals were anaesthetized in darkness with pentobarbital 6% (0.6 ml/100 g, i.p.) and perfused transcardially with NaCI 0.9% followed by 4% paraformaldehyde in phosphate buffer 0.1 M (pH 7.4). Dissected brains were postfixed for 4-8 h. Vibratome transversal sections (50/tm) throughout SCN were rinsed in phosphate-buffered saline (PBS), incubated overnight at 4°C with sheep anti-Fos antiserum (1:5000 in PBS containing 0.5% Triton X100; Cambridge Chemicals), then 1 h with a biotinylated secondary anti-sheep antibody (1:500, room temperature; Vector) followed by ABC reagent for 1 h (Vector). Peroxidase activity was
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All fights reserved PII: S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 1 252;5-9
P. Vuillez et al. / Neuroscience Letters 208 (1996) 37--40
38
revealed using 0.025% DAB (Sigma) in Tris 0.05 M (pH 7.6), containing 0.5% nickel ammonium sulphate and 0.1% H202. Fos-like immunoreactivity (Fos-ir) appears as nuclear black precipitate. According to the supplier's specification, the polyclonal anti-Fos antibody recognizes Fos and Fos-related proteins. For all experiments detailed later on, 2--4 animals per time point were studied, and at least one control hamster per point was sacrificed without light stimulation. Stimulated hamsters may show very large numbers of densely immunostained cells in the SCN. In contrast, the SCN of control animals contain relatively few labelled cells. These cells are mostly weakly stained but all of them were counted for quantitative studies. This explains the relatively high control values we show in histograms. In experiment Eh-LP, at the end of June (natural photoperiod is 16 h L/8 h D), European hamsters were transferred indoors under experimental long photoperiod (LP) conditions (LD 16:8, lights off at 2000 h, close to natural conditions) for 2 weeks. In the rooms (20 ___I°C), light is provided by fluorescent strip lights yielding approximately 300 lux at the bottom of cages. A constant dim red light (less than 1 lux) allows manipulation of animals in 'darkness'. D
D+4
D+8
D+12
D+16
The animals were then submitted to a light stimulus ($) during the 'normal' night (black bar) at either D + 1 (1 h after lights off), D + 4, D + 6, or D + 7. In this experiment as well as in others, in order to test the photosensibility of SCN after the normal end of the night, on the day of experiment hamsters were kept in darkness. At either D + 8, 9, 10, 11, 12, 13, 14, or 15, light stimulation was thus given in the prolonged night (grey bar). European hamsters submitted to a light stimulus during the normal night showed a great number of Fos-ir cells in the SCN. At D + 1, labelled cells extend rostrocaudally for 450/~m in the ventral part of the SCN. At D + 4, 6 or 7, cells are more numerous and are found distributed throughout 600/~m in the ventral and dorsal parts of the SCN. In animals stimulated at D + 8 (normal lights on), the same pattern of labelled cells in the SCN as in animals stimulated in the middle or at the end of the night was observed. However, animals stimulated from D + 9 to D + 15 showed only a few more labelled cells than controls sacrificed without prior light exposure. In experiment Eh-SP, at mid-December (natural photoperiod approximately 8 h L, 16 h D), European hamsters were transferred indoors to short photoperiod (SP, LD 8:16, lights off at 1700 h) for 2 weeks. They were then light-stimulated at either 1, 8, 9, 10, 11, 12, 13, 14 or 15 h after dark onset. At D + 1, animals showed Fos-ir cells throughout the SCN as in animals stimulated at the same
time from experiment Eh-LP. From D + 8 to D + 13 (Fig. 1), labelling was similar to that observed from D + 4 to D + 8 in Eh-LP. However, at D + 14 or D + 15 (Fig. 2), light-exposed animals showed only very few labelled cells, and the SCN of these animals were not distinguishable from the SCN of control hamsters. Here, the duration of the photosensitive period is increased from 8 to 14 h compared to animals kept in LP. In experiment Sh-LP, the SCN of Syrian hamsters kept in LP (LD 14:10) and stimulated by light at either D + 1 or D + 9 showed numerous Fos-ir cells. D+4
D
I"
D+8
D+12 I
? 'I" "I" $ 'I"
I
I
I
The distribution of labelled cells was similar to that previously reported [12]. At the beginning of the night, the main light-sensitive cell population was located in the ventral and lateral part of the caudal half of the SCN. At D + 9, light-induced Fos-ir also occurred dorsally to the first population and in the rostral SCN. At this time, numerous cells were also labelled laterally, dorsally and rostrally to the nuclei. Light stimuli were also applied at D + 10, 11, 12, or 13 in prolonged night on the day of experiment. At D + 10, a labelling similar to D + 9 was observed. From D + 11 on, only few scattered cells expressed Fos after light exposure. The total duration of the photosensitive period was thus 10 h. In experiment Sh-SP1, Syrian hamsters were maintained for 8 weeks in SP (LD 10:14). In SCN of animals stimulated at D + 1, 9 or 10 the labelling was identical to that obtained at the same times for Sh-LP. At D + 11, 12,
~i~i~~'~ i~i~!!!~i ~ ~ii~i~ ~!i~i~i!i~ i~i!:~?:~ %~iT~Ji!i~i~!i~i~i~J~!i Ji¸i~i~:i~ ii!ii!'
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Figs. 1-4. Representativecoronal sections through the SCN stained for Fos immunoreaetivity.Bar = 100/am. Figs. 1, 2, European hamstersin LD 8:16, light stimulated at D + 13 (Fig. !) or at D + 14 (Fig. 2). Figs. 3, 4. Syrian hamsters kept in LD 10:!4 and light stimulated at D+ 11 (Fig. 3) or sacrificedat the same time without prior light stimulus.
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P. Vuillez et aL /Neuroscience Letters 208 (1996) 37-40
13 or 14, a great number of cells were sensitive to light and a pronounced Fos-ir was observed throughout the SCN (compare Figs. 3 and 4). On the other hand, only a few cells, mostly weakly stained, were observed in the SCN in animals stimulated at D + 15. Like in the European hamster, an increase in duration of the photosensitive period is observed when animals are transferred from LP to SP. In experiment Sh-SP2, Syrian hamsters previously kept in LP were maintained for 8 weeks in SP (LD 8:16). Contrary to observations in animals kept under LD 10:14 or 14:10, animals stimulated at D + 1 showed only very few labelled cells. Those stimulated at D + 3, 12, 14, 15, or 16 showed an intense Fos labelling. The increased delay of the time of the switch from insensitivity to sensitivity to light in respect with the beginning of the night has already been demonstrated for other parameters such as circadian locomotor activity. It should be correlate with the so-called 'decompression' of the clock [2]. At D + 17 or D + 18 light has no effect on Fos expression. The duration of the light-sensitive period has thus increased and is approximately 15 h. In experiment Sh-SP3, Syrian hamsters previously kept under LP were maintained for 8 weeks in SP (LD 4:20) and light-stimulated at D + 1, 3, 15, 16, 17, 18, or 19. In this experiment, no labelled cells was detected in the SCN of animals stimulated at D + 1. Light stimulus from D + 3 to D + 17 produced an intense Fos-ir in the SCN. At D + 18, the same labelling was observed in one animal, when the second one, as well as hamsters stimulated at D + 19, showed only a few labelled cells in the SCN, similar to control animals. This demonstrates that, like in European hamster, the light-sensitive period has a limit in its extension. The system is no more sensitive 18 h after the beginning: of the night. Due to the 'decompression' of the clock, the exact beginning of the sensitive phase and thus its exact duration (between 15 and 18 h) cannot be precisely determined. Having observed that the duration of photosensitive phase was indeed longer in SP than in LP, the next two experiments were designed to determine the rate of extension/reduction of this photosensitive period when the animals were transferred from one photoperiod to another. In experiment Sh-LP to SP, Syrian hamsters raised and kept in LP (LD 14:10) were transferred in SP (LD 10:14). After 4, 11, 20 or 25 nycthemers in SP, animals were light-stimulated during the extended night either at D + 11 or at D + 13. Out of the 8-10 sections obtained from rostro-caudal SCN, four similar sections per animal were chosen and Fos-ir cells were counted on a monitoring video coupled to a microscope. Cells inside the SCN and in the hypothalamic area immediately adjacent to the dorso-lateral boundaries of the nuclei which have already been described to be sensitive to light [ 12] were counted. The number of cells expressing Fos in animals stimulated
at either D + 11 or D + 13 on the night of the photoperiod change was in the same range as that of control animals. The number of light-sensitive cells in the SCN and surrounding hypothalamus during the 4 added hours increased progressively until the 25th night after the photoperiod change. Then, the total values were similar to those after stimulus at equivalent times for animals maintained 8 weeks in SP (Fig. 5A). In experiment Sh-SP to LP, Syrian hamsters maintained for 8 weeks in SP (LD 10:14) were transferred to LP (LD 14:10) for 2, 3, 4 or 11 nycthemers. To test the photosensitivity of SCN cells after the normal end of the night (D + 10), animals were kept in darkness on the day of experiment. Light stimulus were applied at D + 11 or D + 13. After a change from SP to LP, the photosensitivity of SCN cells at the corresponding times D + 11 and D + 13 decreased rapidly. Four nights after the change, light stimulation was almost ineffective on the induction of Fos expression in the SCN (Fig. 5B). Our results show that the sensitivity of the SCN to light is dependent on the photoperiod. The duration of the photosensitive phase, as determined by Fos expression, increases with the lengthening of the night. Such increase
r-7
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Fig. 5. Expression of Fos protein in the SCN of Syrian hamsters induced by a light pulse: (A) after transferfrom LP (LD 14:10) to SP (LD 10:14); (B) after transfer from SP (LD 10:14) to LP (LD 14:10). The dotted lines correspond to control hamsters of the time points D + 11 and D + 13, sacrificedwithout prior light stimulus.
40
P. Vuillez et al. / Neuroscience Letters 208 (1996) 37-40
has a limit which is 15-18 h in the Syrian hamster and 14 h in the European hamster. However, after the transfer from LP (LD 14:10) to SP (LD 10:14), a 4 h increase in the duration of the night, the time interval enabling light induction of Fos extends gradually, the full extension being achieved after more than 3 weeks. This indicates that the photoperiod has a slow and progressive effect on the SCN. It is thus not possible to exclude completely that the 15-18 h extension limit observed in Syrian hamster kept under LD 4:20 is because 8 weeks was not enough to integrate the 10 h night lengthening. This, however, does not apply to the experiment with European hamsters since they were in a natural SP close to the experimental one. Probably these extension limits would point to the already demonstrated role of the circadian system in the expression of photosensitive phases of which the duration only is dependent on the photoperiod. In a recent study done in the rat, Sumowi et al. [10,11] using a different paradigm, came to the same conclusion: a circadian property, namely, the photosensitivity state, of the SCN is influenced by the photoperiodic environment. The question is then how and by which pathway can the photoperiod influence SCN activity? We know that it is through changes in the duration of nocturnal melatonin secretion that the organism is able to read photoperiod [5]. Following transfer from LP (14:10) to SP (10:14), duration of melatonin secretion extends gradually and 3 weeks are also needed to obtain maximal duration. After transfer from SP to LP, the melatonin peak adjusts very rapidly to the shortened night [3]. With the same type of protocol we observed that the decrease in duration of the photosensitive period similarly takes only a few days. The SCN are also known to contain melatonin receptors [1,8]. It is thus possible that, throughout photoperiod-induced changes in the duration of melatonin peak, photoperiod acts on the SCN. The possible role of other photoperiod-dependent changes has also to be considered. The SCN receive photic information indirectly from the retina via NPYfibers originating in the intergeniculate leaflets [6]. The role of NPY in photic transduction is at present not well defined but in the jerboa ( J a c u l u s orientalis) the NPY innervation of the SCN shows photoperiod-dependent seasonal changes [4]. It is thus possible that this pathway through fluctuation of NPY content underlies the photosensitive phase duration of the SCN. As the rhythmic melatonin secretion is dependent on the SCN, the parallel observed between the time course of photoperiod-induced changes in the duration of melatonin production and in the duration of light-sensitive phase of the SCN might also reflect changes in the pacemaker itself. Following the theoretical model of Pittendrigh and
Daan [7] the clock would consist of a complex circadian pacemaker consisting of evening and morning components; the LD cycle would define a dawn and a dusk signal. In such a system, by measuring the changes in phase relationship between the morning and evening component, the SCN would be able to build itself a photoperiodic signal and would not need to perceive it. Our data, as well as those of Sumov~i et ai. [10,11], would correlate also with such a hypothesis. In conclusion, using two photoperiodic species and different experimental conditions, we have been able to demonstrate that the duration of the photosensitive phase of the SCN is not a set property of the circadian system but is dependent upon the photoperiod. The mechanisms involved are still to be elucidated.
[1] Gauer, F., Masson-P6vet, M. and P6vet, P., Melatonin receptor density is regulated in rat pars tuberalis and suprachiasmatic nuclei by melatonin itself, Brain Res., 602 (1993) 153-156. [2] lllnerovli, H., Circadian rhythms in the mammalian pineal gland, Rozpr. Cesk. Akad. Ved, 96(1) (1986) 3-105. [3] Illnerovri, H., Hoffmann, K. and Vanecek, J., Adjustment of the rat pineal N-acetyltransferaserhythm to change from long to short photoperiod depends on the direction of extension of the dark period, Brain Res., 362 (1986) 403-408. [4] Lakhdar-Ghazal, N., Oukouchoud, R. and Pgvet, P., Seasonal variation in NPY immunoreactivity in the suprachiasmatic nucleus of the jerboa (Jaculus orientalis), a desert hibernator, Neurosci. Lett., 193 (1995) 49-52. [5] Maywood, E.S., Hastings, M.H., Max, M., Ampleford, E., Menaker, M. and Loudon, A.S.I., Circadian and daily rhythms of melatonin in the blood and pineal gland of free-running and entrained Syrian hamsters, J. Endocrinol., 136 (1993) 65-73. [6] Morin, L.P., The circadian visual system, Brain Res. Rev., 67 (1994) 102-127. [7] Pittendrigh, C.S. and Daan, S., A functional analysis of circadian pacemakers in nocturnal rodents, J. Comp. Physiol. A,, 106 (1976) 333-355. [8] Reppert, S.M., Weaver, D.R. and Ebisawa, T., Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses, Neuron, 13 (1994) 11771185. [9] Rusak, B., Robertson, H.A., Wisden, W. and Hunt, S.P., Light pulses that shift rhythms induce gene expression in the suprachiasmatic nucleus, Science, 248 (1990) 1237-1240. [10] Sumovfi,A., Tr~ivnfckov~i,Z. and lllnerovfi, H., Memory on long but not on short days is stored in the rat suprachiasmatic nucleus, Neurosci. Lett., 200 (1995) 191-194. [11] Sumovi A., Tnivnfckov~i, Z., Peters, R., Schwartz, W.J. and lllnerov~i, H., The rat suprachiasmatic nucleus is a clock for all seasons, Proc. Natl. Acad. Sci. USA, 92 (1995) 7754-7758. [12] Teclemariam-Mesbah,R., Vuillez, P., Van Rossum, A. and P6vet, P., Time course of neuronal sensitivity to light in the circadian timing system of the golden hamster, Neurosci. Lett., 201 (1995) 5-8. [13] Wollnik, F., Breit, A. and Reinke D., Seasonal change in temporal organization of wheel-running activity of the European hamster, Cricetus cricetus, Naturwissenschaften, 78 ( 1991) 419-422.