Daytime Gating in the Syrian Hamster Pineal Gland

Journal of Neuroendocrinology From Molecular to Translational Neurobiology Journal of Neuroendocrinology 21, 760–769 ª 2009 The Authors. Journal Compilation ª 2009 Blackwell Publishing Ltd

ORIGINAL ARTICLE

Daytime Gating in the Syrian Hamster Pineal Gland A. Salingre, P. Klosen, P. Pe´vet and V. Simonneaux Institut des Neurosciences Cellulaires et Inte´gratives, De´partement de Neurobiologie des Rythmes, UMR CNRS 3212, Universite´ de Strasbourg, Strasbourg Cedex, France.

Journal of Neuroendocrinology

Correspondence to: Vale´rie Simonneaux, Institut des Neurosciences Cellulaires et Inte´gratives, De´partement de Neurobiologie des Rythmes, UMR CNRS 3212, Universite´ de Strasbourg, 5 rue Blaise Pascal, 67084 Strasbourg, Cedex, France (e-mail: [email protected]).

Melatonin synthesis in rodents is tightly regulated at the transcriptional level by stimulatory and inhibitory transcription factors. Among them, phosphorylated cAMP-related element binding protein (pCREB) and inducible cAMP early repressor (ICER), a strong inhibitor of cAMP-related element-driven genes, have an antagonistic action in activating ⁄ inhibiting the transcription of the Aa-nat gene, which is an important enzyme in melatonin synthesis. In the Syrian hamster, a rodent displaying a seasonal control of reproduction, melatonin synthesis is strongly gated to the second part of the night. Indeed, exogenous adrenergic stimulation is unable to stimulate Aa-nat gene transcription and melatonin synthesis during daytime. In the present study, we investigated whether ICER may be the cause of this daytime repression by comparing the dynamic of ICER and the adrenergic regulation of two genes whose expression is rapidly activated by cAMP-dependant mechanisms, c-fos and Icer. Adrenergic induction of c-fos and Icer expression was not possible during daytime, except at early day. ICER immunoreactivity was elevated throughout the daily cycle but reached the highest levels at early day, when gene expression can be induced by adrenergic agonists. Additionally, CREB phosphorylation was subjected to the same daily gating with an adrenergic induction occurring in the early but not in the late day. Taken together, our results indicate that the diurnal gating of pineal activity in the Syrian hamster is not caused by the repressor ICER and that it may occur at the level of noradrenergic receptor signalling. Key words: pineal gland, AA-NAT, ICER, c-fos, adrenergic signalling.

Regulation of gene transcription is the result of the antagonistic action of stimulatory and inhibitory transcription factors (TF). This is of prime importance for genes undergoing marked variations over a short period of time. The Arylalkylamine N-acetyltransferase (Aa-nat) coding gene in rodent pineal gland experiences such a dramatic day ⁄ night variation, with up to 150-fold more mRNA at night than during the day, which ultimately drives the daily and seasonal rhythm of melatonin production. This large nocturnal increase is mainly driven by the circadian clock of the hypothalamus inducing the release of norepinephrine (NE) at the beginning of the night, which in turn leads to a rapid and large increase in intracellular cAMP levels (1). Regulation of gene expression in response to the cAMP pathway activation is mostly mediated by TF of the cAMP responsive element binding protein (CREB) ⁄ CRE modulator (CREM) ⁄ activated TF (ATF) family (2). CREB is constitutively expressed and is activated to a stimulatory TF by phosphorylation at Ser133 (pCREB) by a cAMPactivated protein kinase (PKA). The CREM gene shows a complex

doi: 10.1111/j.1365-2826.2009.01897.x

organisation and its mRNA is subjected to various alternative splicing events leading to a variety of isoforms, especially inducible cAMP early repressor (ICER), which binds CRE sites to powerfully inhibit cAMP ⁄ CREB-induced transcription (3, 4). In the rat pineal gland, it was proposed that the daily variation in Aa-nat transcription primarily results from activation by pCREB at early night and inhibition by ICER at late night ⁄ early day, with this regulation being under the control of the b1 adrenergic ⁄ cAMP ⁄ PKA pathway (5). Recent studies, however, have pointed out that regulation of pineal Aa-nat gene transcription varies among species (6, 7). In the Syrian hamster, CREB activation is not sufficient for Aa-nat transcription at night because NE-induced de novo synthesis of inducible TFs, in particular c-Fos (8, 9), is also required. Moreover, adrenergic stimulation of Aa-nat transcription and melatonin synthesis in the Syrian hamster is not possible during the light phase of the daily cycle (10–14). Because it was hypothesised that ICER protein may be the cause of this restriction, we have examined the

Gating of NE-signalling in the hamster pineal gland

putative role this inhibitory TF in the daytime gating of melatonin synthesis in the Syrian hamster pineal gland. In previous studies, we reported a fast NE-driven induction of c-Fos protein (9) and Icer mRNA (15) at night-time in the Syrian hamster pineal gland. Moreover, both c-fos and Icer gene expression are subjected to the inhibitory action of ICER in other models: Icer (4, 16) and c-fos (17, 18). Therefore, the present study aimed to evaluate the putative role of ICER in the daily gating of pineal activity by matching cAMP-dependant genes induction (c-fos, Icer and Aa-nat) with ICER protein levels at different periods of the day.

Materials and methods Animals Female Syrian hamsters were bred in our animal facilities under constant conditions of temperature and ambient humidity. They were raised under a 14 : 10 h light ⁄ dark cycle (L: 200 lux light intensity ⁄ D: 2 lux dim red light) (lights on 05.00 h) with food and water available ad lib. Each experimental group comprised three to six animals aged 2–3 months. All experiments were performed in accordance with the rules of the European Committee Council Directive of November 24, 1986 (86 ⁄ 609 ⁄ EEC) and the French Department of Agriculture (licence no. 67-250).

Experimental protocols Night-time regulation of c-fos and Icer expression To establish whether c-fos and Icer mRNA synthesis, similar to that of Aa-nat, depends on newly-synthesised proteins at early night, two groups of hamsters were injected i.p. with either cycloheximide (20 mg ⁄ kg; Sigma, St Louis, MO, USA) or vehicle (ethanol : saline, 25 : 100) at 19.00 h for c-fos or 21.00 h for Icer. Pineal glands were sampled either at 21.00, 22.00 or 23.00 h for c-fos or at 01.00 or 03.00 h for Icer in situ hybridisation. Time points were chosen according to the timing of the nocturnal peak of c-Fos protein (9) and Icer mRNA (15).

Daytime regulation of c-fos and Icer expression The objective of the first experiment was to determine whether c-fos and Icer transcription may be stimulated by an acute injection of an adrenergic agonist given at midday in different lighting conditions. Hamsters were kept under a 14 : 10 h light ⁄ dark cycle or placed for three consecutive days either in constant darkness (DD) or constant light (LL). For each experimental condition, three groups of animals were injected i.p. with isoproterenol (3 mg ⁄ kg; Sigma) at 12.00 h and then killed at the time of injection (12.00 h), 1 h (13.00 h) or 3 h (15.00 h) after injection. Pineal tissue was then processed for in situ hybridisation with the c-fos or Icer probes. The second experiment aimed at delineating whether c-fos and Icer mRNA induction is differentially regulated at early and late day. Groups of hamsters were injected i.p. with either isoproterenol and phenylephrine (3 mg ⁄ kg each, for c-fos analysis) or isoproterenol (3 mg ⁄ kg, for Icer analysis) or vehicle (Ringer) at 09.00 h (4 h after lights on) or at 14.00 h (9 h after lights on). Pineal expression of c-fos and Icer 1 h after injection was examined by in situ hybridisation. In this experiment, two adrenergic agonists were used for c-fos because both a- and b-adrenergic stimulation is required for its full induction (9); isoproterenol alone was used for Icer because a b-adrenergic stimulation is sufficient for its full induction (15).

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Daytime regulation of Aa-nat expression by repeated injections of adrenergic agonists Hamsters were injected i.p. with isoproterenol and phenylephrine (3 mg ⁄ kg each) or vehicle (Ringer) every 2 h, the first injection beginning at 09.00 h (4 h after lights on) or 14.00 h (9 h after lights on). Pineal glands were sampled 5 h after the first injection and Aa-nat expression was analysed by in situ hybridisation. Repeated injections were used because, in the Syrian hamster pineal, Aa-nat transcription is a process that requires at least 5 h of constant stimulation (8). One group of hamsters was killed at 00.00 h (5 h after lights off) as night-time control of expression.

Daytime induction of c-Fos and pCREB by adrenergic agonists Hamsters were injected i.p. with isoproterenol and phenylephrine (3 mg ⁄ kg each) or vehicle (Ringer) at 09.00 h (4 h after lights on) or 14.00 h (9 h after lights on). Two hours after injection, the brains were sampled and prepared for c-Fos and pCREB immunohistochemistry. In parallel, a group of hamsters was sacrificed at 22.00 h (i.e. a positive control with maximal contents of c-Fos and pCREB) (9). This experiment was reproduced once. A preliminary time-course experiment showed that both c-Fos and pCREB were at their highest values 2 h after the adrenergic agonist injection.

Daily rhythm in pineal ICER protein expression under long or short photoperiod Hamsters were maintained either under long (14 : 10 h light ⁄ dark cycle, lights on 05.00 h, n = 40) or short (10 : 14 h light ⁄ dark cycle, lights on 09.00 h, n = 40) photoperiod for 10 weeks. Animals were then dispatched randomly into eight groups of five animals for each photoperiod and were prepared for immunohistochemical analysis of ICER and CREB expression at the time points: 07.00, 11.00, 14.00, 17.00, 20.00, 22.00, 01.00 and 04.00 h.

In situ hybridisation Brains with the pineal gland were removed, frozen on dry-ice, then stored at )80 C until sectioning for in situ hybridisation. Antisense and sense riboprobes were transcribed from a Syrian hamster c-fos cDNA (nucleotides 628–1025 of Genbank accession number AF061881) using T7 (antisense) and SP6 (sense) in presence of a[35S]-UTP (1250 Ci ⁄ mmol; NEN-Dupond, Zaventem, Belgium) according to the manufacturer’s instructions (MAXIscript; Ambion, St Austin, TX, USA). The synthesis of the Icer and Aa-nat riboprobes has been described previously [Icer (15); Aa-nat (8, 19)]. Coronal brain sections (18 lm) were cut at )18 C in a cryostat, thaw-mounted onto gelatin-coated slides, and stored at )80 C. For in situ hybridisation, tissue sections were fixed, acetylated, dehydrated and incubated overnight at 54 C in a hybridisation medium containing 400 pM of c-fos riboprobe, 80 pM of Aa-nat or Icer riboprobes. Post-hybridisation treatments consisted in stringency washes down to 0.2 · SSC, 62 C. Slides together with 14C standards were exposed to an autoradiographic film (Hyperfilm MP; Kodak, Orsay, France).

Immunohistochemistry Animals were deeply anaesthetised with isoflurane, rapidly injected with heparin (250 UI by animal) directly into the left ventricle, and immediately perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The head was removed, post-fixed for 12 h in the perfusion fixative. The brain was then carefully dissected out, further post-fixed for 12 h, rinsed with

ª 2009 The Authors. Journal Compilation ª 2009 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 21, 760–769

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phosphate-buffered saline (30 min then overnight) and finally dehydrated successively in 70% ethanol (1 h), 95% ethanol (1 h), 100% ethanol (2 · 1 h) and n-butanol (overnight). Polyethylene glycol (PEG; Acros, Fairlawn, NJ, USA) embedding consisted of incubations at 48–50 C in PEG 1000 overnight; PEG1000 ⁄ PEG1500 mix for 4–5 h, PEG1000 ⁄ PEG1500 mix infiltration overnight (20). Finally, the blocks were cast in fresh PEG1500 at room temperature. The PEG blocks were sectioned (8 lm) on a rotary microtome and mounted onto Super Frost Plus slides (Menzel-Glaser, Braunschweig, Germany) from a 2% sucrose solution. For immunohistochemistry, brain sections were blocked for 1 h by 3% of nonfat dry milk in Tris buffered saline ⁄ Tween 20 solution and incubated overnight with the specific primary antibodies for CREB and pCREB (dilution 1 : 2000; Upstate Biotechnology, Lake Placid, NY, USA), c-Fos (dilution 1 : 2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and ICER Ic (dilution 1 : 2000; a generous gift from Dr Franc¸oise Miot, University of Brussels, Brussels, Belgium). After washing for 30 min with Tris-buffered high-salt saline ⁄ Tween 20, the tissue sections were incubated for 1 h in donkey anti-rabbit biotinylated secondary antibody (Jackson) in 1 : 2000 dilution, then washed again for 30 min with Tris-buffered high-salt saline ⁄ Tween 20 and exposed for 1 h to streptavidinperoxidase (Roche Diagnostics, Basel, Switzerland) at a dilution of 1 : 2000. Immunolabelling was detected using diaminobenzidine (0.5 mg ⁄ ml; H2O2 0.003% in Tris buffered imidazole · 1). The rabbit polyclonal antibody for ICER used in the present study was made from bacterially expressed protein synthesised from expression vector encoding the mouse ICER Ic reverse transcript (21). Specificity of the ICER Ic antibody was previously verified by Uyttersprot et al. using enzyme-linked immunosorbent assays assays. This antibody was verified to recognise only ICER isoforms on western blot of rat pineal proteins (Fig. 1).

Data analysis Quantitative analysis of the autoradiographs Analysis was performed with NIH Image J software derived from the public domain NIH Image program (developed at the US National Institutes of Health; available at: http://rsb.info.nih.gov/nih-image). Specific labelling was determined as the difference between total (antisense) and nonspecific (sense) hybridisation, with both being run in parallel in each experiment.

Semi-quantitative analysis of immunolabelled pineal tissue All the slides from an individual experiment were processed for immunohistochemistry at the same time with identical treatment (i.e. for the ICER, analysis of the slides of hamster pineal in long and short photoperiod were treated together). After slides-mounting, pictures of all immunolabelled pineal areas were recorded on a Leica DMRB microscope (Leica Microsystems, Rueil-Malmaison, France) equipped with an Olympus DP50 digital camera (Olympus France, Rungis, France). All lighting parameters on the microscope and the camera software (Viewfinder Lite; Olympus) were determined for the first pineal section from an individual experiment and kept constant for all subsequent samples. Images were then recorded into eight bit grey value (0– 255) resolution images with a 1392 · 1040 pixels spatial resolution. To avoid the problem of light homogeneity, a background image of the slide without section was recorded and substracted from all corresponding samples of the slide, leading to an inverted image with a homogeneous background. Selected immunolabelled areas were then analysed using NIH Image J software. Pineal section with the most intense immunoreactivity was used to adjust the colour balance that was applied to all the samples (grey levels: 0 for background and 255 for the most immunoreactive pixels) and then an area to be quantified was drawn. The inverted images were contrasted and segmented (threshold procedure) to analyse only the specific immunoreactive areas (i.e. the nuclei). The segmentation threshold was determined for measuring only the pixels with a value > 3 · background and held constant for each sample, ensuring that only pixels with values greater than threshold were analysed. The integrated density (sum of pixel values) of all the immunoreactive nuclei with an area superior or equal to 7–8 lm2 was measured. Finally, all of these values were summed to give the total integrated density (TID) of the specific labelling, with this value being divided by the quantification area in lm2 (TID ⁄ lm2). For ICER, c-Fos and pCREB density quantifications, the value obtained for each sample was divided by the value measured for CREB on an adjacent section aiming to minimise the variations among animals of the same group. In parallel, all immunoreactive (IR) nuclei were counted using NIH Image J software, and the obtained value was divided by the quantification area in mm2 (number of IR nuclei ⁄ 0.1 mm2). This method gives highly reproducible results because we obtained similar values and numbers of IR nuclei in the pineal of one animal quantified through three independent immunoreactions (with the same antibody) and in two different sets of animals (i.e. for ICER rhythm; data not shown).

Statistical analyses Statistical analyses were performed using one- or two-way ANOVA, depending on the experiment, followed by Tukey’s multicomparison test. P < 0.05 was considered statistically significant.

Results Night-time regulation of c-fos and Icer expression

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ICER Ig

Fig. 1. Western blot control of the inducible cAMP early repressor (ICER) I rabbit polyclonal antibody on protein extracted from night-time rat pineal. Two bands corresponding to the ICER I and ICER Ic isoforms are observed.

The nocturnal increase in c-Fos protein (9) and Icer mRNA (15) in the Syrian hamster pineal gland is known to depend on the b1 ⁄ cAMP pathway. The effect of a protein synthesis inhibitor was examined to find out whether this cAMP driven transcriptional activation depends solely upon pCREB or requires neosynthesised transcription factors, as reported for Aa-nat transcription (8). The protein synthesis inhibitor cycloheximide had no significant effect on the early night increase in c-fos and icer mRNA (Fig. 2) but enhanced the later contents of c-fos mRNA (P < 0.001 for treatment–time interaction at 22.00 and 23.00 h).



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Fig. 2. Effect of a protein synthesis inhibitor on nocturnal level of Icer and c-fos mRNA in Syrian hamster pineal gland. Hamsters were raised under a 14 : 10 h light ⁄ dark cycle (lights on 05.00 h). (A) At 19.00 h (lights off), two groups of hamsters received an i.p. injection of either cycloheximide (cyclo; 20 mg ⁄ kg) or vehicle (25% ethanol in saline) and were killed at 21.00, 22.00 or 23.00 h (2, 3 or 4 h later, maximal expression of c-fos). (B) At 21.00 h (2 h after lights off), two groups of hamsters received an i.p. injection of either cycloheximide (cyclo; 20 mg ⁄ kg) or vehicle (25% ethanol in saline) and were killed at 01.00 or 03.00 h (4 or 6 h later, maximal expression of Icer). Pineal tissue was processed for in situ hybridisation with the Icer or c-fos probes. Each point is the mean SEM of n = 5–6 animals. *P < 0.001 compared to vehicle-injected animals. The right panel shows representative images of in situ hybridisation for each condition at 21.00 and 22.00 h for c-fos and 01.00 and 03.00 h for Icer.

Daytime gating of c-fos and Icer expression To establish whether c-fos and Icer expression is submitted to a gating similar to that of Aa-nat (8), the mRNA content of both genes was examined in the pineal gland of hamsters kept under various lighting conditions and injected at midday with a b1-adrenergic agonist. Isoproterenol injection at 12.00 h did not increase the mRNA content of c-fos and Icer in the subsequent hours after injection when hamsters were kept under LD or DD conditions (Fig. 3). By contrast, in hamsters kept for 3 days in LL, isoproterenol induced a marked increase in c-fos and Icer gene expression (P < 0.001 compared to untreated animals kept under the same lighting condition) with a more sustained effect on Icer (Fig. 3). The next experiment was performed to determine whether the daytime blocking of gene expression occurs throughout the all day or only at some period of the day. Hamsters in LD were injected with adrenergic agonists either 3–4 or 9 h after lights on, and c-fos and Icer mRNA expression was measured 1 h after injection. There was a clear differential induction between both daytime periods (Fig. 4). The expression of both genes was markedly induced at early day (P < 0.001 between vehicle and adrenergic agonistsinjected animals), whereas it was not (c-fos) or slightly (Icer) increased at late day. The same results were obtained when analy-

sing adrenergic induction of c-Fos protein at early or late day. Interestingly, this differential induction was abolished when hamsters were kept under LL the night before because the adrenergic agonists induced a similar increase in c-fos and Icer mRNA at both periods (not shown).

Daytime gating of Aa-nat expression after repeated adrenergic stimulations Because induction of Aa-nat expression is known to require several hours of constant stimulation in the Syrian hamster pineal gland (8), the effect of repeated injections of adrenergic agonists given either at early or late day was investigated. Induction of Aa-nat expression displayed the same differential gating as observed for c-fos and Icer with a significant increase in mRNA content when injections started at 09.00 h and no effect at 14.00 h (Fig. 5). However, the levels obtained during the early day period did not reach the night control values.

Daily rhythm in pineal ICER protein expression under long or short photoperiod ICER being a strong repressor of cAMP-mediated gene expression, it was hypothesised that ICER protein could be present at a high


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Time after injection (h) Fig. 3. Effect of a daytime injection of a b-adrenergic agonist on c-fos (A) and Icer (B) mRNA levels in the pineal gland of Syrian hamsters kept under various lighting conditions. Hamsters were raised under a 14 : 10 h light ⁄ dark cycle (lights on 05.00 h) and were either kept under LD, or exposed to constant darkness (DD) or to constant light (LL) for 3 days. On the third day of exposure, Syrian hamsters were injected i.p. at 12.00 h with the b-adrenergic agonist isoproterenol (Iso, 3 mg ⁄ kg) and killed 1 or 3 h later. Data were compared with untreated animals killed at the time of injection (12.00 h). Pineal tissue was processed for in situ hybridisation with the Icer or c-fos probes. Each point is the mean SEM of n = 5–6 animals. *P < 0.01 compared to animals sacrificed at the time of injection. The right panel shows representative images of in situ hybridisation for each condition.

amount during the second part of the day to prevent induction of c-fos, Icer and Aa-nat expression in the Syrian hamster pineal. To test this hypothesis, the daily pattern of pineal ICER protein was determined by immunohistochemistry. ICER was expressed only within the nucleus of most pinealocytes (Fig. 6A). By contrast to Icer mRNA (15), ICER protein was expressed at all time points of the 24-h rhythm. CREB was constantly expressed over 24 h with no significant rhythm. Therefore, values obtained for ICER immunoreactivity were expressed as the ratio between ICER and CREB immunoreactivites to minimise the inter-individual variations. A significant rhythm of the mean nuclear density of ICER expression was detected (Fig. 6B): the protein content decreased slowly during the light period to reach minimal values at 22.00 h (P < 0.001 compared to 07.00 h), and then increased gradually during the second part of the night ⁄ early day to maximum values observed at 07.00–10.00 h. The number of ICER expressing cells did not vary over 24 h (Fig. 6C). An identical daily pattern was obtained in another independent experiment, demonstrating the robustness of the daily rhythm and the reproducibility of the analysis. Surprisingly, no significant difference was found in the daily rhythm of ICER protein expression between long and short photoperiod (two-way ANOVA, P = 0.247 for time–photoperiod interaction; Fig. 7).

Daytime gating of CREB phosphorylation after acute injection of adrenergic agonists This experiment was designed to determine whether CREB phosphorylation might be subjected to a differential daytime gating as observed here for c-fos, Icer and Aa-nat. pCREB content in the Syrian hamster pineal gland was examined 2 h after an acute injection of adrenergic agonists given either at early or late day. pCREB was strongly and significantly induced at early day (P < 0.01 compared to vehicle-injected animals; with a value similar to the night-time control value), but not at late day (Fig. 8B). The same results were found in parallel on c-Fos protein (Fig. 8A), confirming those obtained for c-fos mRNA (Fig. 4A). This experiment was reproduced once, with similar results being obtained.

Discussion In most vertebrate species, the nocturnal peak of melatonin displays seasonal variations that convey the annual changes in night duration. This endocrine message is extremely important for the synchronisation of annual physiology, especially reproduction, with the seasons (22–25). This implies a tight regulation of melatonin synthesis in seasonal species whose survival depends on an appropri-



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Fig. 4. Effect of a single injection of adrenergic agonists given at early or late day on c-fos (A) and Icer (B) mRNA levels in Syrian hamster pineal gland. Hamsters were raised under a 14 : 10 h light ⁄ dark cycle (lights on 05.00 h). (A) For c-fos analysis, hamsters were injected i.p. with a solution of isoproterenol and phenylephrine (Iso ⁄ Phe; 3 mg ⁄ kg) at 09.00 or 14.00 h. (B) For Icer analysis, hamsters were injected i.p. with a solution of Iso (3 mg ⁄ kg) at 08.00 or 14.00 h. Vehicle (Ringer) treated animals were also employed at each time point. Pineal tissue was processed for in situ hybridisation with the Icer or c-fos probes. Data are presented as the mean SEM of values in n = 4–5 animals. *P< 0.01 compared to the vehicle-injected animals at each time point. §P < 0.01 between both conditions. The right panel shows representative images of in situ hybridisation for each condition.

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Fig. 5. Effect of repeated injections of adrenergic agonists given at early or late day on Aa-nat mRNA levels in Syrian hamster pineal gland. Hamsters, raised under a 14 : 10 h light ⁄ dark cycle (lights on 05 h), were injected i.p. with isoproterenol and phenylephrine (Iso ⁄ Phe; 3 mg ⁄ kg) or vehicle (Ringer) every 2 h, with the first injection beginning either at 09.00 h or at 14.00 h. Pineal tissue was sampled 5 h after the first injection and processed for in situ hybridisation with Aa-nat riboprobe. White and black boxes represent day and night periods, respectively. Arrows represent the time of injection. Data are presented as the mean SEM of values in n = 4–5 animals. *P < 0.01 compared to the vehicle-injected animals at each time point. §P < 0.001 between both conditions. The right panel shows representative images of in situ hybridisation for each condition.

ateness of offspring birth and weaning during optimal environmental conditions. In the highly seasonal Syrian hamster, we have reported that the regulation of the rhythm-generating AA-NAT enzyme is different from that described in rat or mouse (8). In the rat, the large nocturnal increase in Aa-nat gene transcription mostly depends on the rapid phosphorylation of CREB into pCREB,

which in turns binds the CRE site on the Aa-nat promoter to trigger gene transcription (5, 26–28). Furthermore, rat pineal Aa-nat transcription is highly responsive to adrenergic stimulation at any time of the daily cycle (27, 29, 30). By contrast, different mechanisms appear to govern Aa-nat gene transcription in the Syrian hamster pineal gland (8, 9). The nocturnal increase in Aa-nat mRNA


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Fig. 6. Daily rhythm in inducible cAMP early repressor (ICER) protein in Syrian hamster pineal gland. Hamsters were raised under a 14 : 10 h light ⁄ dark cycle (lights on 05.00 h). At various time points of the daily cycle (07.00, 11.00, 14.00, 17.00, 20.00, 22.00, 01.00 and 04.00 h), brains were processed for immunohistochemistry analysis of ICER and CREB proteins in the pineal gland. (A) Representative pictures of nuclear ICER and CREB immunoreactivity (IR) in pineal gland of hamsters at 07.00, 14.00, 20.00 and 01.00 h. Scale bar = 25 lm. (B) Quantification of pineal ICER protein throughout a daily cycle; the level of ICER was quantified on each slide as the density of the labelling in the nuclei and is presented as the ratio of ICER-IR ⁄ CREB-IR values obtained on adjacent slices. (C) Quantification of the number of pineal ICER expressing nuclei throughout a daily cycle (given as number of IR nuclei per 0.1 mm2 of pineal surface). White and black boxes represent day and night periods, respectively. Data are expressed as the mean SEM of values obtained in the pineal of n = 4–5 animals at each condition. *P < 0.01 compared to 07.00 h.

Fig. 7. Daily rhythm in inducible cAMP early repressor (ICER) protein in the pineal gland of Syrian hamster raised under long or short photoperiod. Hamsters were raised either under long (14 : 10 h light ⁄ dark cycle, lights on 05.00 h; black circles) or short (10 : 14 h light ⁄ dark cycle, lights on 09.00 h; white circles) for 10 weeks. Animals were sacrificed at various time points of the 24-cycle (07.00, 11.00, 14.00, 17.00, 20.00, 22.00, 01.00 and 04.00 h) and brains were processed for analysis of ICER and cAMP-related element binding protein (CREB) immunoreactivity (IR) in the pineal gland. Data are presented as ICER-IR ⁄ CREBIR ratio values obtained on adjacent slices (A) and as the number of IR nuclei (B) throughout the daily cycle. Data are expressed as the mean SEM of values measured in the pineal of n = 4–5 animals at each time point. White boxes represent daytime and grey ones represent night (light grey for LD and dark grey for SD). No significant difference was found between both profiles (two-way ANOVA, Time-photoperiod interaction).

requires de novo transcription and synthesis of stimulatory TF, resulting in a long delay between dark onset and melatonin synthesis. Additionally, exogenous adrenergic stimulation of Aa-nat transcription is blocked during daytime.

Beside Aa-nat, NE stimulates the transcription of a large array of other genes (1, 31, 32). Among these transcripts is the pineal specific Crem-Icer mRNA (whose synthesis is driven by the same cAMP ⁄ PKA ⁄ pCREB pathway), which is translated into ICER, a small



(A) c-FOS IR (relative to CREB)

0.30 Vehicle Iso/phe

0.25

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0.20 0.15 0.10 0.05 0.00 6

8

10 12 14 16 18 20 22 24 Time (h)

pCREB IR (relative to CREB)

(B) Vehicle Iso/phe

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§

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10 12 14 16 18 20 22 24 Time (h)

Fig. 8. Effect of a single injection of adrenergic agonists given at early or late day on c-Fos (A) and phosphorylated cAMP-related element binding protein (pCREB) (B) proteins in Syrian hamster pineal gland. Hamsters, raised under a 14 : 10 h light ⁄ dark cycle (lights on 05.00 h), were injected i.p. with a solution of isoproterenol and phenylephrine (iso ⁄ phe 3 mg ⁄ kg) or vehicle (Ringer) either at 09.00 h or at 14.00 h. Two hours after injection, brains were processed for analysis of inducible cAMP early repressor (ICER), pCREB and CREB protein immunoreactivity (IR) in the pineal gland. White and black boxes represent day and night periods, respectively. Arrows represent the time of injection. Data are presented as c-Fos-IR ⁄ CREB-IR (A) and pCREBIR ⁄ CREB-IR (B) ratio values obtained on adjacent slices and are expressed as the mean SEM of measured values of n = 3–4 animals. *P < 0.01 compared to the vehicle-injected animals in each condition. §P < 0.01 between both conditions.

protein with a potent inhibitory action on Aa-nat gene transcription (4, 5, 33). We previously reported that Icer mRNA is also expressed in the Syrian hamster pineal with a NE-driven nocturnal increase (15). In the present study, in agreement with the recent report of Maronde et al. (2007), we confirm the presence of high levels of ICER protein in the hamster pineal. This suggests that ICER may be involved in the daytime gating of gene expression in the Syrian hamster pineal gland. To test this hypothesis, we searched for markers of norepinephrine-driven gene expression with an induction faster than that of Aa-nat. We recently reported a rapid and transient night time synthesis of c-Fos protein in the Syrian hamster pineal (9), indicating that c-fos gene may be a good candidate. Indeed, c-fos mRNA levels are markedly increased as soon as 2 h after night onset (data not shown). This early night increase was not prevented by cycloheximide administration (Fig. 2), although c-Fos protein induction was abolished (9), indicating that, in contrast to Aa-nat, new protein synthesis is not required for the induction of c-fos transcription.

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Additionally, we also observed that the early night induction of Icer mRNA is not inhibited by cycloheximide. Therefore, c-fos and Icer were used as markers of NE-induced pineal gene activation in the subsequent studies. As already reported for Aa-nat, transcription of c-fos and Icer is resistant to daytime adrenergic stimulation even in constant darkness, indicating that this mechanism is not dependent on light inhibition, but rather controlled by the central circadian clock or endogenous to the pineal. The observation that the daytime gating of gene expression is abolished in animals kept for 3 days in constant light could be explained by different hypotheses. First, the observed effect could have derived from an injection made at a different internal circadian phase after the LL; however, the same effect was observed in animals placed only one day in LL (data not shown), excluding this hypothesis. Second, in the absence of endogenous release of NE, the adrenergic signalling may have become supersensitive to NE, leading to a visible response. Third, our privileged hypothesis is that the LL condition may have prevented the initiation of the gating, suggesting that this mechanism is controlled by the central circadian clock via NE. Overall, these observations demonstrate that the gating impinges on general NE-induced gene expression in the Syrian hamster pineal gland, and that c-fos and Icer appear to comprise good markers for investigating the mechanisms involved in the gating of pineal activation. One hypothesis explaining the daily gating would be the presence of an inhibitory TF synthesised during the night and active during daytime to prevent NE-induced gene transcription. ICER is a powerful inhibitor of CRE-driven gene transcription, including its own gene (3, 4, 16) and c-fos transcription (17, 18). Therefore, we reasoned that the blockade of daytime NE ⁄ cAMP induction of gene transcription would be the consequence of a high amount of the ICER protein. Examination of the daily pattern of ICER protein in the Syrian hamster pineal shows that, in contrast to the mRNA (15), protein content is elevated throughout the 24-h cycle, but nevertheless display significant daily variations. Semi-quantitative analysis demonstrates that ICER protein levels are highest at the end of the night ⁄ early day and lowest at the beginning of the night. This daily pattern is similar to that described in the rat pineal (5), although with higher values during daytime. According to this daily pattern of ICER, the gating should occur all along the photophase. However, in the present study, we report that c-fos and Icer expression is subjected to a clear differential daytime induction, with mRNA levels being increased when the adrenergic stimulation is given 4 h and not 7–9 h after lights on. Additionally, Aa-nat gene expression can also be induced during the early day (at a time when endogenous levels are at a minimum) after repeated administration of adrenergic agonists but not later in the day. These results demonstrate that the blockade of pineal gene expression is restricted to the late part of the day. Notably, the highest daytime levels of ICER protein are observed during the early part of the day, when c-fos, Icer and Aa-nat gene transcription can be induced. This strongly indicates that ICER is not the cause of the daytime blockade of transcriptional activity in the Syrian hamster pineal gland. In agreement with this conclusion, a recent study (34) reported that specific inhibition of Icer expression did not alter the NE-inducted Aa-nat expression in cultured rat pinealocytes.


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Strikingly, we observed that the adrenergic induction of CREB phosphorylation, which does not require gene transcription, is also subjected to the same differential restriction in the daytime. This observation is in agreement with earlier studies showing that adrenergic agonists are unable to stimulate cAMP production (10, 11) or pinealocyte electrical activity (35) in the Syrian hamster pineal gland in vitro. Taken together, these observations suggest that the daytime gating of the Syrian hamster pineal activity is located upstream of transcriptional regulation. Whether this blockade occurs at the membrane receptor or along the signalling pathway remains unknown. Notably, a similar gating mechanism was shown to occur with respect to adenylyl cyclase activity in the rat retina (36). Moreover, AC1 and AC6 mRNA and all adenylyl cyclase proteins were found to be regulated by the light ⁄ dark cycle and to oscillate in the rat pineal gland (37, 38). Understanding the molecular mechanisms underpinning the Syrian hamster pineal gating will be important because such gating is a general phenomenon that also occurs in the mouse (39, 40) and human (41) pineal gland, as well as at the level of the circadian clock (42). Because gating mechanisms are often explained by a restriction of gene transcription, the results obtained in the present study with respect to the blockade of CREB activation suggest that these mechanisms should be investigated at the level of the receptor signalling pathway. The daily pattern of ICER reported in the present study suggests that it has a minimal impact on the daily regulation of melatonin synthesis in the Syrian hamster pineal gland, as already suggested for the rat (35, 43). The elevated level of ICER throughout the day ⁄ night cycle suggests that it may exert a constant and general dampening of cAMP-driven gene expression. Such a function would imply that long-term modification of ICER protein synthesis might regulate the amplitude of the nocturnal elevation of Aa-nat mRNA and eventually melatonin synthesis. Photoperiodic variation in AA-NAT and ⁄ or melatonin peak amplitude has been observed in the Syrian hamster and other seasonal rodent species (44–47). We hypothesised that ICER might be involved in the photoperiodic variation of Aa-nat gene expression and therefore we compared the daily pattern of ICER protein in long and short photoperiods. ICER levels, however, were very similar in both photoperiod (48, present study), suggesting that it is not responsible for the photoperiodic variation in the amplitude of the nocturnal peak of Aa-nat expression. As proposed earlier, the main role of ICER would be to participate in the decline of Aa-nat transcription towards the end of the night ⁄ early day because the ICER ⁄ pCREB ratio is increasing at late night in the rodent pineal gland (5, 35, present study). In the present study, we have shown that the noradrenergic-mediated transcription of several genes is gated in the Syrian hamster pineal gland with a blockade during the second part of the daytime. Notably, the inducibility of the adrenergic signalling pathway, as represented by CREB phosphorylation, displays a similar daytime gating. By contrast, ICER protein, a potent inhibitor of cAMP gene transcription, appears to play a minimal role in this mechanism. Taken together, these data indicate that mechanisms involved in the diurnal gating of melatonin synthesis in the Syrian hamster pineal occur at some stage of the adrenergic receptor signalling.

Acknowledgements The authors are indebted to Dr Franc¸oise Miot (University of Brussels, Brussels, Belgium) for the generous supply of ICER antibody, to Jerome Mutterer for his help for immunolabelling quantification and to Dr Jerome Menet for the c-fos probe. The authors are also grateful to Daniel Bonn for providing animal care and to Dr David Hicks for correction of the language.

Received: 24 March 2009, revised 12 June 2009, accepted 16 June 2009

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