Photoperiod does not act on the suprachiasmatic ...

Neuroscience Letters 229 (1997) 117–120

Photoperiod does not act on the suprachiasmatic nucleus photosensitive phase through the endogenous melatonin, in the Syrian hamster Nathalie Jacob*, Patrick Vuillez, Paul Pe´vet URA-CNRS 1332, Neurobiologie des fonctions rythmiques et saisonnie`res, Universite´ Louis Pasteur, 12 rue de l’Universite´, 67000 Strasbourg, France Received 29 April 1997; accepted 9 June 1997

Abstract The duration of the sensitive phase to light of the suprachiasmatic nuclei, in terms of Fos protein expression, depends on the photoperiod. In Syrian hamsters, a 4 h lengthening of the photosensitive phase occurs within 3–4 weeks after a transfer from a long to a short photoperiod. The absence of endogenous melatonin following pinealectomy does not prevent the lengthening of the photosensitive phase. Thus, even if the pineal production is able to convey photoperiodic information, it does not feed back on the circadian clock to allow its integration.  1997 Elsevier Science Ireland Ltd. Keywords: Circadian rhythm; Suprachiasmatic nucleus; c-fos; Photoperiod; Pineal; Melatonin; Syrian hamster

In mammals, many daily rhythms like the locomotor activity, the body temperature and the pineal melatonin synthesis are endogenously generated by a circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Such rhythms show a precise 24 h period because the clock itself is entrained by daily variations of environmental parameters. The most effective synchronizer is the light/dark (L/D) cycle that resets the pacemaker every day [9]. Photic information is conveyed from the retina directly to the SCN by the retinohypothalamic tract and indirectly mainly via the neuropeptide Y (NPY) fibers originating from the intergeniculate leaflet [8]. Resetting light information induces the expression of immediate early genes, including c-fos, in several cell populations of the SCN [5,18]. In L/D conditions, these populations of SCN cells are activated by a light stimulation applied during the dark period of the nycthemer, whereas they do not seem to react to day light. Light appears to stimulate Fos expression only when administered at circadian times at which exposure causes a phase shift [10,13,18]. Using light-induced cfos mRNA or Fos protein expression as an in vivo marker of SCN intrinsic neural activity, recent studies have shown that the duration of the sensitive phase to light of the SCN

* Corresponding author. Fax: +33 3 88240461.

depends on the photoperiod. In European and Syrian hamsters [19] as well as in the rat [17], the duration of this photosensitive phase is tied to the length of the night. For instance, in Syrian hamsters kept in long photoperiod (LP; 14:10, 14 h of light and 10 h of night per 24 h) Fos expression in the SCN can be induced by a light stimulation throughout the 10 h of darkness. After a transfer from a long to a short photoperiod (SP; 10:14), a 4 h lengthening of the photosensitive phase occurs. The adjustment of the light sensitivity of the SCN to this new photoperiod takes 3– 4 weeks. Thus, the SCN whose rhythmic activity is well known to be regulated by daily information, also seem to integrate photoperiodic information. In the Syrian hamster, photoperiodic information which controls the seasonal rhythms is mediated through effects of light on the circadian clock system and the pineal gland. The daily rhythm of melatonin (Mel) production is characterized by a nocturnal secretion, whose duration is proportional to the length of the night. Moreover, Mel receptors have been identified within several brain regions including the SCN [3,11]. Neurophysiological recordings from SCN slices have identified neuronal firing rate rhythms which are responsive to Mel [7,15] and pinealectomy [12]. Furthermore, the time course of the lengthening of the photosensitive phase of the SCN, we observed in terms of lightinduced Fos expression, is similar to the one described

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N. Jacob et al. / Neuroscience Letters 229 (1997) 117–120

[4,14] to get a maximal extension of the nocturnal Mel peak after a change from a long to a short photoperiod. These different points suggest that photoperiod-induced change of the photosensitive phase of the SCN could be mediated through Mel. The present study aimed at testing this hypothesis. Using pinealectomized hamsters kept in LP, we have studied the effect of a light stimulation on Fos expression in the SCN after transfer from LP to SP. Adult male Syrian hamsters Mesocricetus auratus (Charles River, Canada) were maintained in LP 14:10 (lights on from 0700 h to 2100 h, 200 lx) for at least 8 weeks prior to surgery. A constant dim red light was on throughout the experiment. Water and food were available ad libitum. Hamsters (approximately 100–140 g) were anesthetized during the light phase with i.p. Equithesine (0.4 ml/100 g). After being placed into a stereotaxic instrument, a cranial incision was made to expose the pineal gland. The pineal was removed with fine forceps, the skull cap replaced and the incision closed. After the surgery, the hamsters were transferred to SP 10:14 (lights on from 1100 h to 2100 h), at the L/D transition. After 4, 20, 25 and 60 nycthemers in SP, animals were light stimulated 13 h after the beginning of the night (D + 13). The lengthening of the photosensitive phase was investigated using a light stimulation of 15 min (identical to ‘day’ lighting). Then, Fos expression was followed by immunohistochemistry. At D + 14, 1 h after the onset of the light stimulation animals were anesthetized in darkness with 6% pentobarbital (0.6 ml/100 g, i.p.; Sanofi) and perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde in phosphate buffer 0.1 M (pH 7.4). Dissected brains were postfixed for 4–6 h, and microwaved 20 min at 100 W in a bucket of ice according to Buijs et al. [1]. Coronal 50 mm thick sections were cut throughout the SCN with a vibratome (Leica), rinsed in phosphate-buffered saline (PBS), incubated overnight at 4°C with sheep antiFos antiserum (1:5000 in PBS containing 0.5% Triton X100; Cambridge research Biochemicals, Wilmington, DE, USA), then for 1 h with a biotinylated secondary anti-sheep antibody (1:500, at room temperature; Vector Labs., Biosys SA, Compie`gne, France) followed by ABC reagent for 1 h (Vector). Peroxidase activity was visualized using 0.025% DAB (Sigma) in 0.05 M Tris (pH 7.6), containing 0.5% ammonium nickel sulfate and 0.1% H2O2. Fos-like immunoreactivity (Fos-ir) appears as a black nuclear precipitate. According to the supplier’s specification, the polyclonal anti-Fos antibody recognizes Fos and Fos-related proteins. The stained sections were then gathered on gelatinized slides, dehydrated and embedded in Eukitt (Kindler). Control intact and pinealectomized hamsters were sacrificed in darkness at D + 14, without prior light stimulation. For intact hamsters, each night, four stimulated and two to four controls have been studied. For pinealectomized hamsters, each night, six stimulated and four control hamsters

have been studied (except for the 4th night where n = 3 in both groups). 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 (Leica) [19]. Cells inside the SCN and in the hypothalamic area immediately adjacent to the dorsolateral boundaries of the nuclei, which have already been described to be sensitive to light [18], were counted. Stimulated hamsters may show very large numbers of densely immunostained cells in the SCN. In contrast, the SCN of control animals contain relatively few labeled cells. These cells are mostly weakly stained but all of them were counted for quantitative studies, which explains the quite high control values obtained. Results were expressed as the mean ± SEM. The analysis of data was performed using the best-fit sigmoidal curve (with a Hill’s coefficient k = 0.2) to represent the effect of pinealectomy on Fos expression in the SCN. Pinealectomy does not have any effect on SCN Fos expression in non-light stimulated animals. The mean of labeled cells of all control animals is relatively weak (370 ± 52) and similar to the one of intact animals (391 ± 67). After a photoperiod change of pinealectomized hamsters from LP 14:10 to SP 10:14, the number of lightinduced Fos-ir cells in the SCN and surrounding hypothalamus increases progressively until the 25th night. By that time, the total number of Fos-ir cells was similar to that observed after a light stimulus at the same time of the night in animals maintained in SP for 8 weeks (1883 ± 64; see Fig. 1). Likewise, these Fos-ir cells were observed in the ventral and dorsal subdivision throughout the rostro-caudal SCN. In point of fact, the same pattern of increase of Fos-ir cells is observed for the pinealectomized and non-pinealectomized animals. In both groups the number of labeled cells as well as their localization are comparable whatever the night of the stimulation after the photoperiod change (see Fig. 2).

Fig. 1. Expression of light-induced Fos protein in the SCN of intact and pinealectomized hamsters, after transfer from LP 14:10 to SP 10:14. Each point represents the mean ± SEM for 3–6 animals.


Whatever the night of the stimulation is like there are no significant variations of Fos expression in pinealectomized hamsters in comparison with intact animals. The same pattern of increase is observed in the two groups. This indicates that like in intact animals after transfer from LP to SP, the duration of the photosensitive phase extends gradually in the SCN of pinealectomized animals. The full extension of the photosensitive phase is reached after 3–4 weeks. Thus, this photoperiod-dependent extension is independent of the Mel

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signal. Similar results have recently been obtained by Sumova´ and Illnerova´ [16] in the rat. Our results clearly show that, in the Syrian hamster which presents a Mel dependent photoperiodic control of some functions such as reproduction, the photoperiod does not act on the SCN through variations of Mel secretion. The SCN appear to be the only, or at least the first described, mammalian structure able to integrate photoperiodic information without a transduced hormonal signal produced by the pineal gland.

Fig. 2. Representative coronal sections through the SCN stained for Fos-ir of intact hamsters (a,c,e,g) and of pinealectomized hamsters (b,d,f,h). Animals were light stimulated 13 h after lights off (D + 13), the 4th (a,b), 20th (c,d), 25th (e,f) or 60th (g,h) night after transfer from LP 14:10 to SP 10:14.

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The adjustment of the duration of the light sensitive phase of the SCN to one photoperiod can be explained by the theoretical model of functioning of the circadian clock defined by Pittendrigh and Daan [9]. In this model, the SCN may contain two components, an evening one and a morning one being synchronized to L/D and D/L transition, respectively. By measuring the phase relationship between these two components, the whole SCN would be able to build itself a photoperiodic signal and then to distribute it to the organism, especially through the rhythmic secretion of Mel. As far as we know, no data have ever permitted to refute or to confirm such an hypothesis which is used by Sumova´ et al. to explain their observation in the rat. We cannot exclude that such a concept might also explain our present data but before it is concluded definitively, the possible role of other photoperiod-dependent cues has to be considered. In the Syrian hamster, the observed SP-induced increase in duration of the light sensitive period of the SCN is associated with an inhibition of gonadal activity. Although no steroid receptors have been described within the SCN, an indirect action via neurotransmitters themselves dependent on annual changes in circulating levels of sex steroids could explain the data. Presently however, we cannot favor such an hypothesis. Indeed, when hamsters were transferred from LP to SP the maximal extension of the light sensitive period is obtained within 3–4 weeks. After 3–4 weeks in SP, the sexual axis in Syrian hamsters is far from being completely regressed. The SCN is known to receive photic information from the retina not only by the retinohypothalamic tract but also indirectly via NPY-fibers originating from the intergeniculate leaflets [8]. The role of NPY in photic transduction is at present not well defined. But a clear seasonal variation of NPY-ir in the SCN has been observed in the Jerboa (Jaculus orientalis) [6]. So, this suggests the role of NPY innervation to transduce photoperiodic information to the SCN. Experiments are presently in progress to test this hypothesis. Our present results of light-induced Fos expression in pinealectomized hamsters also show that the presence of Mel is not a prerequisite for the clock cells to be sensitive to light. This raises the question of the exact role of Mel on the functioning of the clock [2]. In conclusion, the present study demonstrates that the lengthening of the photosensitive phase of the SCN after the shortening of the photoperiod occurs in pinealectomized Syrian hamsters in the same manner as in intact animals. The circadian clock is thus able to integrate photoperiodic information independently on endogenous pineal hormonal signals. The way by which photoperiodic changes are integrated has still to be determined. [1] Buijs, R.M., Nunes-Cardozo, B., Hou, Y.-X. and Shinn, S., Postembedding GABA and glutamate immunogold electron microscopy: a combination with anterograde Pha-L tracing, Neurosci. Prot., 10 (1993) 1–11.

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