Melatonin influences Fos expression in the rat suprachiasmatic

47

Molecular Brain Research, 16 (1992) 47-56 Elsevier Science Publishers B.V.

BRESM ~ 8

Melatonin influences Fos expression in the rat suprachiasmatic T h o m a s S. Kilduff, H o l l i s t e r B. L a n d e l , G. S o n i a N a g y , E l l e n L. Sutin, W . C . D e m e n t , and H.C. Heller Sleep Research Center, Departments of Psychiatry and Biological Sciences, Stanford University, Stanford, CA 94305 (USA) (Accepted 16 June 1992)

Key words: Immediate early gene; Melatonin; Circadian rhythm; Suprachiasmatic nucleus

Administration of the pineal hormone melatonin to rats induces expression of Fos, the protein product of the c-fos proto-oncogene, in the suprachiasmatic nucleus (SCN), the putative biological clock of mammals. Expression of the Fos protein is dependent on circadian phase: injections in the late subjective night (circadian time (CI') 22) induce Fos expression in cells within the ventral SCN whereas injections during the subjective day are ineffective. Since melatonin injections in the late subjective day have previously been shown to phase advance circadian rhythms, these results indicate that phase-advances of the circadian system can occur without increased expression of Fos protein in the SCN, at least at levels detectable by immunohistochemistry. In support of in situ hybridization histochemical evidence obtained previously, immunocytochemical data from vehicle-injected control rats suggest that the Fos protein undergoes an endogenous fluctuation with peak levels in the SCN occurring during the subjective night. These observations indicate that melatonin can affect immediate early gene expression within the SCN.

INTRODUCTION

The suprachiasmatic nuclei (SCN) of the mammalian hypothalamus contain a circadian pacemaker that regulates behavioral and physiological rhythmicity t8'34. The SCN receive a direct retinal projection through the retinohypothalamic tract 12'19 and an indirect projection through the geniculo-hypothalamic tractS'22; photic stimulation is an effective agent for resetting the phase of the pacemaker within the SCN and for the entrainment of circadian rhythms. Several laboratories have recently reported that photic stimulation also induces expression of c-los mRNA or Fos-like immunoreactivity (Fos-LI) in the SCN of hamsters 14'28 and rats 4'11'23'28'a3 and in the retina 11. Fos is an inducible DNA-binding protein known to be a transcriptional activator and is rapidly and transiently expressed in neural tissue consequent to a variety of physiological and pharmacological stimuli. Fos and other immediateearly genes (IEGs) have been proposed to act as 'third messengers', coupling short-term membrane events to long-term changes in gene expression in the central nervous system 2°'a°. Although the experimental proee-

dures varied among laboratories, c-los mRNA or FosLI was consistently observed in the SCN when photic stimulation occurred during the animal's subjective night, the time which corresponds to the 'active' portion of the circadian pacemaker's phase-response curve. These observations are of interest because they may lead to insights into the molecular events which underlie the phase-shifting response due to light and the mechanism of entrainment of circadian rhythms. However, the neurochemical processes coupling photic stimulation to IEG expression in the SCN remain obscure. The pineal hormone melatonin has a circadian rhythm of synthesis and release that peaks during the subjective night and is under control of the SCN 34. Melatonin has at least three actions related to the circadian system. In photoperiodic species such as hamsters and sheep, the phase or duration of the nocturnal rise in melatonin plasma concentration can provide an internal temporal signal of the photoperiod length and thereby influence the reproductive systemaS. Secondly, melatonin acts as a hormone to communicate daylength information between mother and

Correspondence: T.S. Kilduff, Sleep Research Center, Department of Psychiatry TD-I14, Stanford University School of Medicine, Stanford, CA 94305, USA. Fax: (1) (415) 723-5882.

48 fetus 25'~'. Lastly, daily injections of melatonin can entrain activity rhythms in rats 2'24 and cause phase advances when administered in the subjective day:~; these effects are dependent on the integrity of the biological clock in the SCN 6. Melatonin has also been shown to have direct effects on the SCN itself, presumably mediated through a high-affinity melatonin receptor that has been characterized and is present in the SCN, the median emin e n c e / p a r s tuberalis of the hypothalamus and, in rats, the area postrema 37. Melatonin has an inhibitory action on SCN neuronal discharge ~6'3~ and a biphasic effect on SCN metabolic activity, with decreases observed in the late subjective day and increases in the late subjective night s. Melatonin has also been shown to reset the phase of the circadian pacemaker in the SCN in vitro, causing phase advances of the rhythm of firing rate of SCN neurons when applied during the late subjective day ~7. Since both light and melatonin phase-shift the circadian pacemaker in rodents and Fos has been shown to be induced in the SCN by photic stimulation, we have investigated whether melatonin administration affects expression of Fos in the rat SCN and in other brain regions in which melatonin receptors have been described. MATERIALS

AND METHODS

ImmunohLs'tochemical studies Male Wistar rats (250-350 g) were maintained under a light-dark, 12 h:12 h (LD 12:12) photoperiod. On the day prior to treatment, animals were released into constant darkness (D D) at circadian time 12 (CT 12). At nine circadian times separated by 4 h intervals commencing 2 h after release into DD (CT 14, 18, 22, 2, 6, 10, 14, 18, or 22), different groups of animals (n = 6 per group) were administered subcutaneous injections of melatonin (100 /~g/kg) or vehicle (1% ethanol in physiological saline) under dim red light (15 W bulb covered with a Kodak 1A filter; < 2 Ix) and t h e n : r e t u r n e d to darkness. Additional animals were injected with 1 /zg/kg or 10 p.g/kg melatonin at CT 22 (10 h after release into DD). The doses used were based on those reported to phase advance circadian rhythms 3 and to inhibit SCN metabolism in rats 7. After a 1 or 2 h incubation period, animals were deeply anesthetized under dim red light ( < 2 Ix) with a solution of 7.5% ketamine/0.8% xylazine/0.15% acepromazine and perfused transcardially in the light with 100 ml saline containing 20U heparin followed by 500 ml 4% paraformaldehyde in phosphate-buffered saline (pH 7.4; PBS). The brain and, in some cases, retinae were removed, post-fixed in the same fIxative for 4 h at 4°C, placed into PBS and cut into ~.00-p,m-thick coronal sections on a vibratome within 24 h. Because of previous descriptions of melatonin receptor distribution within the rat brain, particular attention was paid during sectioning to the hypothalamus adjacent to the SCN, to the median e m i n e n c e / p a r s tuberalis, and to the brainstem adjacent to the area postrema. Fos immunohistochemistry. Sections were treated with 8% normal horse serum (NHS) containing 0.2% Triton X-100 and 0.1% bovine serum albumin in PBS (NHS solution) for 2 h and then incubated in a monoclonal Fos antiserum (LA041; Microbiological Associates, Bethesda, MD) diluted 1 : 10,000 in NHS solution for 48-72 h at 4°C. The primary antiserum was a monoclonal antibody to residues 4-17 of the Fos protein. Unlike other antibodies used in studies of the SCN to date, the N peptide has little homology with known Fos-related antigens and recognizes a single band on Western blots 1°. The

time course of appearance and disappearance oi tl~c protein ~'ecog nized by LA041 closely parallels c:/'os mRNA whereas the protein recognized by M-peptide-directed antibodies remain elevated fc,r several days2'~; consequently, LA041 is presumed to be specific fo~ Fos z~. Fos immunoreactivity (Fos-ir) was visualized using the avidinbiotin-peroxidase method (Elite Vectastain kit, Vector Labs, San Carlos, CA) with diaminobenzidine (Sigma) as the chromagcn. Control sections in the presence of the synthetic N peptide showed no nuclear staining, as previously documented 29. Data analysis. On the basis of in situ hybridization studies ~42~''', basal levels of Fos (as opposed to Fos-related antigens) in the SCN were expected to be low. Consequently, an analysis based on cell counts would be skewed because of the expected large number of zero values. Therefore, it was determined that sections would be scored by a rater blind to the experimental condition simply as either Fos-positive or Fos-negative. The immunohistochemical data are therefore reported as the proportion of animals within each group exhibiting Fos-ir in the SCN and statistical significance assessed using the test for significance of difference between two proportions (z statistic). Indeed, the majority of the rats examined in the current study (61/108) showed no Fos-ir in the SCN. However, Fos-ir was observed in both vehicle and melatonin-injected animals in the periventricular thalamus and piriform cortex at all circadian times; these patterns of immunoreactivity served as an internal control for each assay.

In situ hybridization histochemical studies To determine whether melatonin would induce transcription of

c-]bs m R N A as well as Fos protein in the SCN at CT 22 and to further verify the suggestion of a circadian rhythm of Fos expression obtained from the immunohistochemical results, c-los m R N A was specifically examined in the SCN at CT 22 after rats had been released into DD for 58 h. A total of 24 Wistar rats were examined in four groups (n = 6 per group): (1) melatonin-treated (100 ~g/kg); (2) vehicle (1% ethanol in physiological saline); (3) 45 rain photic stimulation using a GE cool white fluorescent bulb (approx. 1700 Ix); and (4) dark control. Melatonin and saline injections were administered subcutaneously under dim red light (15 W bulb covered with Kodak 1A filter; < 2 Ix) before animals were returned to darkness. Animals were allowed to survive 45 rain after initiation of treatment before being deeply anesthetized under red light with a solution of 7.5% ketamine/0:8% xylazine/0.15% acepromazine and then decapitated within 3 min. Brains were removed under red light, frozen in 2-methylbutane cooled to - 4 0 ° C on dry ice and stored at -70°C. Synthesis and labelling of RNA probes. [35S]-labeled sense and antisense c-los probes were transcribed from a full-length rat cDNA 9 cloned in both orientations in pSP65. Both plasmids were linearized with Barn H1 to produce templates of approximately 2.1 Kb. Linear DNA templates (1 ug) were transcribed in 20 ul containing 400 uCi [35S]-UTP (1250 Ci/mmol, Amersham), 2 mM ATP, CTP and GTP (Promega), 2 units RNase block (Stratagene), 10 mM dithiothreitol (DTT), 1 unit of SP6 RNA polymerase, and 1 × R N A polymerase buffer (BRL). Following RNA synthesis (1 h, 37°C), the DNA template was degraded by the addition of DNase (10 units, BRL; 20 min 37°C), after which the R N A was extracted with phenol/chloroform and ethanol precipitated. In situ hybridization histochemistry. In situ hybridization histochemistry was performed by using a slight modification of protocols which have been utilized successfully with other probes ~'32'3s. Frozen tissue was warmed to - 1 8 ° C in a cryostat and 12 /zm sections were cut through the hypothalamus containing the SCN from its rostro-caudal pole and thaw mounted onto twice-coated gelatin chrome-alum slides. The slides were then placed upon a heat plate at 65°C until dry and then stored desiccated at -80°C. Before hybridization, sections were warmed to room temperature and allowed to dry. Sections were then fixed in 4% formaldehyde in PBS at 4°C for 10 min. Slides were rinsed twice (1 rain each) in PBS, incubated in 0,25% acetic anhydride in 0.1 M triethanolamine/0.9c7 NaCI (pH 8) for 10 minutes at room temperature, and then transferred successively through 70% (1 min), 80% (1 min), 95% (2 min), and 100% ethanol (1 min), 100% chloroform (5 min), 100% and 95% ethanol and air dried.

49 106 CPM probe in 50 /~1 buffer containing 0.01% DTI', 50% formamide, 4 × SSC (standard sodium citrate; l x SSCffi0.15 M NaCI, 0.15 M sodium citrate, pH. 7.2), 1 × Denhardt's, 2 5 0 / x g / m l yeast tRNA, 500 t t g / m l single-stranded DNA, and 10% dextran sulfate were applied to each section. Sections were covered with a glass coverslip and incubated overnight at 37"C in a humid chamber. The coversfips were removed and the sections rinsed twice in 1 × SSC with 10 mM DTT at room temperature. Slides hybridized with the c-fos riboprobe were then incubated in RNase A 2 5 / t g / m l at 37"C for 30 rain. All sections were then washed in four 15-rain changes of 2 × SSC/50% formamide at 40"C, then for two 30-min washes at room temperature in 1 x SSC, rinsed in water, dehydrated in 70% and then 95% ethanol and air dried. Autoradiographic localization of bound probe was performed by exposure of the sections to XAR.5 film for 4-8 days. For higher resolution, the sections were dipped into Kodak NTB3 nuclear emulsion diluted 1:1 with water. Exposures ranged from 2 to 4 weeks after which the slides were developed by using Kodak D-19 developer followed by Kodak fixer and counterstained with 0.1% thionin. Data analysis. Specific hybridization was quantified using a computer-assisted image analysis system (MCID; Imaging Research, Inc., St. Catherines, Ont.). For each animal, the autoradiograms from two coronal brain sections were chosen on the basis of thionin stains to contain the mid-region of the SCN. From each autoradiogram, bilateral optical density (O.D.) measurements were made of the ventrolateral (VL-SCN), dorsomedial SCN (DM-SCN), anterior hypothalamus (AH) and optic chiasm (OC). To control for slight variation in section thickness, time of exposure, and specific activity

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of the probes, relative O.D. (ROD) values were calculated within each animal for the VL-SCN, DM-SCN, and OC using the AH as denominator. The mean ROD value for each of the three dependent variables was then calculated within each experimental group. Comparisons were made between light vs. dark and melatonin-treated vs. saline groups on the three dependent variables using Hotelling's t 2 test followed by the appropriate univariate F-tests. RESULTS

Immunohistochemical studies Initial efforts focussed on CT 10 and CT 22, times at which melatonin inhibits or facilitates, respectively, SCN metabolism. Administration of 100/~g/kg melatonin subcutaneously to rats at CT 22 resulted in robust expression of Fos immunoreaetivity (Fos-ir) in ceils located within the ventral SCN (Fig. 1A) of all animals examined (n -- 6). Injection of 10/~g/kg melatonin at the same time of day also resulted in Fos expression but in many fewer cells concentrated in the most ventral portion of the SCN (Fig. 1B). In contrast, 100/~g/kg melatonin administered at CT 10 was ineffective in inducing Fos-ir (Fig. 1C) in any animal (n =

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100 pm Fig. 1. Sections through the left suprachiasmatic nuclei (SCN) of rat brains stained for Fos immunoreactivity with a monoclonal antibody. A: rat injected with 100/~g/kg melatonin at CT 22. Arrows indicate Fos-immunoreactive (Fos-IR) nuclei in ventral SCN near border with dorsomedial SCN. B: rat injected with 10/~g/kg melatonin at CT 22. Arrows indicate two Fos-IR nuclei in ventral SCN. C: rat injected with 100/~g/kg melatonin at CT 10. D: rat injected with vehicle solution at CT 22. dSCN, dorsomedial SCN; OC, optic chiasm; 3V, 3rd ventricle.

50 I

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cadian times; these patterns of immunoreactivity served as an internal control for each assay. When Fos expression was examined at other circadian times, a greater proportion of vehicle-treated animals exhibited Fos-ir early in the first subjective night after release into DD (CT 14 and CT 18: Table 1) than at CT 22. On the following subjective day (CT 2, 6, or 10), Fos expression was not observed in the SCN of either vehicle- or melatonin-treated animals (Table I). During the second subjective night, a high proportion of vehicle-treated animals ( 5 / 6 ) showed endogenous Fos expression at CT 18; melatonin treatment (100 /xg/kg) was effective in inducing Fos in the SCN (relative to vehicle-treated controls) at other times examined during the second subjective night (Table 1: CT 14 - P < 0.001; CT 22 - P < 0.0001).

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In situ hybridization histochemical studies To determine whether melatonin induced transcription of c-los m R N A as well as Fos protein in the SCN at CT 22, we examined c-fos m R N A expression at CT 22 by in situ hybridization histochemistry on the third subjective night after release into D D Cthe third subjective night was chosen to be comparable with the procedures employed by Rusak et al.2S). Since previous investigators had demonstrated that c-los m R N A could be induced in the SCN by photic stimulation 14'2s. we included photic stimulated and dark control groups as well as the melatonin and vehicle-injected groups. Inspection of the autoradiograms revealed that all animals within the photic stimulation (Fig. 3A vs. 3C) and melatonin treatment (Fig. 3E vs. 3G) groups exhibited c-los m R N A expression specifically in the VL-SCN. However, considerable variability existed in the amount of c-los m R N A detected among subjects within the dark (Fig. 3B,D) and vehicle-treated groups (Fig. 3F.H).

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Fig. 2. Rostral/caudal distribution of nuclei of Fos-immunostaining cells within the suprachiasmatic nucleus of a rat treated with 100 ~g/kg melatonin at CT 22. SCN, suprachiasmatic nucleus; OC, optic chiasm; III, 3rd ventricle.

6), as were vehicle injections administered at CT 10 (n = 6) and in 5 of 6 vehicle-treated animals at CT 22 (Fig. ID; P < 0.0001, Table I). Fig. 2 schematically illustrates the rostral-caudal distribution of Fos-ir cells within the SCN of a representative animal. Although the SCN, median e m i n e n c e / p a r s tuberalis, intergeniculate leaflet, and area postrema were examined in all animals, and retinae in some cases, melatonin induced Fos-ir exclusively in the SCN. Fos-ir was also observed in both vehicle and melatonin-injected animals in the periventricular thalamus and piriform cortex at all cir-

TABLE I Proportion of animals exhibiting Fos immunoreactivity in the suprachiasmatic nucleus of melatonin- and vehicle-treated groups across the circadian day

N.S., not significant. Condition

Circadian time

Hours after dark onset

Vehicle

Melatonin (100 Iz g / kg)

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Subjective night 1

CT 14 CT 18 CT 22

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-0.92

N.S.

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N.S. 0.0001

CT 02 CT 06 CT 10

14 18 22

0/6 0/6 0/6

0/6 0/6 0/6

0

N.S.

0 0

N.S. N.S.

CT 14 CT 18 CT 22

26 30 34

0/6 5/6 1/6

3/6 5/6 6/6

Subjective day

1

Subjective night 2

- 3.46 0 - 7.65

0.001 N.S. 0.0001

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Fig. 3. Representative in situ hybridization autoradiograms of rat brain sections showing regional localization of c-fos mRNA under the following experimental conditions. Arrows indicate location of SCN in all panels. A: rat subjected to photic stimulation at CT 22 and hybridized with a c-fos antisense cRNA probe. B: rat sacrificed in darkness at CT 22 and hybridized with a c-fos antisense cRNA probe showing relatively high level of c-fos mRNA expression in the SCN. C: adjacent section from rat illustrated in A but hybridized with sense strand riboprobe. D: rat treated identically to rat in B but showing relatively low level of c-fos mRNA expression in the SCN. E: rat administered 100 ~ g / k g melatonin s.c. under dim red light at CT 22 and hybridized with a c-fos antisense cRNA probe. F: rat administered vehicle injection under dim red light at CT 22 and hybridized with a c-los antisense cRNA probe showing relatively high level of c-fos mRNA expression in the SCN. (3: adjacent section from rat illustrated in E but hybridized with sense strand riboprobe. H: rat treated identically to rat in F but showing relatively low level of c-fos mRNA expression in the SCN.

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REGION Fig. 4. Relative optical density (ROD) measurements for the four groups of animals (n = 6) treated at CT 22 in the in situ hybridization study. ROD values are calculated relative to the OD of the AH. A: ROD values obtained from sections hybridized with c-los antisense cRNA probe. B: ROD values obtained from sections hybridized with a control c-los sense strand riboprobe. VL-SCN, ventrolateral SCN; DM-SCN, dorsomedial SCN; OC, optic chiasm.

A N O V A performed on the optical density measurements (Fig. 4A) documented a significant difference between the light and dark groups in both the VL-SCN ( P < 0.0001; F = 21.53, df = 1,20) and DM-SCN ( P < 0.005; F = 10.61, df = 1,20). However, densitometric measurements suggested only a trend toward increased c-fos m R N A in the VL-SCN of melatonin-treated animals vs. vehicle-injected controls ( P < 0.1; F = 3.06, df = 1,20) despite the fact that c-fos m R N A was observed in the VL-SCN of all animals in the melatonintreated group. Although the variability in c-los m R N A expression within the VL-SCN was similar in the vehicle-treated and dark control groups (Fig. 4A), photic stimulation was more effective in inducing c-fos m R N A than melatonin treatment. The level of c-fos m R N A detected in the optic chiasm (OC) was invariant across experimental groups. Fig. 4B indicates that hybridization of the sense strand riboprobe did not significantly differ among experimental groups. Although melatonin treatment at this dose was a weaker stimulus than photic stimulation at the intensity used in this study, c-fos m R N A was evident in the same region of the SCN in both groups (Fig. 5A,B).

Previous reports 4'11'23"28'33 have indicated that photic stimulation induces expression of Fos-LI or c-los m R N A in the rat SCN. The present study utilized monoclonal antibody LA041 which is thought to be specific for Fos 29 because (l) it was raised against an N-terminal peptide of Fos which has little homology with Fos-related antigens; (2) it produces a single band on Western blots1°; and (3) the time course of the protein recognized by this antibody is similar to that of c-fos m R N A and contrasts with the prolonged time course of proteins recognized by M-peptide-directed antibodies 29. Our results indicate that exogenous administration of metatonin, a neurohormone that normally acts on the SCN and known to play an important role in the circadian system, can affect immediate early gene expression in the SCN. The concentration of melatonin which induces Fos expression at CT 22 is comparable to that which is effective for entrainment of activity rhythms in rats by daily injections of melatonin. The EDs0 for entrainment is 5 / z g / k g 6 ; significant expression of Fos was observed in the SCN of rats treated at both 1 0 / z g / k g (Fig. 1B) and 1 /zg/kg. The effect of melatonin on Fos induction is circadian phase-specific: melatonin administration during the subjective day is ineffective, and the maximal effect is observed late in the subjective night at CT 22. This effect was also SCN-specific; Fos expression was not observed in other brain regions in which high-affinity melatonin receptors have been described in the rat (area postrema, median e m i n e n c e / p a r s tuberalis). This observation suggests that melatonin receptors in the SCN may be distinct from those in other brain regions with respect to coupling to the 'third messenger' c-los. Despite the fact that the maximal effect of melatonin treatment was at CT 22, a large proportion of vehicle-treated rats exhibited Fos-ir early in the first subjective night (Table I). We attribute Fos expression in the SCN of these animals to an after-effect of the prior light cycle. Although some investigators have reported only transient expression of Fos after photic stimulation 28, we have observed significant levels of Fos in mid-day if the lights are kept on. Consequently, light appears to have a tonic effect on Fos induction in the SCN, not simply a phasic effect. Therefore, Fos observed at CT 14 and CT 18 of the first subjective night (Table I) is likely to reflect, at least in part, the

53

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100 pm Fig. 5. Emulsion-dipped in situ hybridization autoradiograms showing distribution of c-los mRNA in the ventral SCN. A: dark-field autoradiogram of rat exposed to light for 30 min at CT 22. B: dark field autoradiogram of rat treated with 100/~g/kg melatonin at CT 22. Black arrows in A and B indicate the dorsolateral border of the SCN. C: light-field autoradiogram of an enlarged area from A showing distribution of autoradiographic grains in proximity to SCN cells. White arrows in A and C indicate corresponding cells that contain c-los mRNA. D: light-field autoradiogram of an enlarged area from B showing distribution of autoradiographic grains in proximity to SCN cells. White arrows in B and D indicate corresponding cells that contain c-los mRNA. OC, optic ehiasm; 3V, third ventricle.

half-life of Fos p r o t e i n i n d u c e d by the tonic effect of light while the rats were m a i n t a i n e d in L D 12 : 12. P e r h a p s the most i n t r i g u i n g aspect of o u r d a t a are

the levels of Fos observed within the SCN of vehiclet r e a t e d rats at C T 18 of the second subjective night ( T a b l e I) a n d at C T 22 o n the third subjective night

54 (Fig. 3B, 3F and Fig. 4A). In these cases, too much time had elapsed to attribute the Fos observed to be an after-effect of photic stimulation, yet Fos is observed in the SCN in a large proportion of the control animals. We suggest that the levels observed in the control animals may reflect endogenous Fos expression in the SCN. Since c-los m R N A and Fos protein expression were observed only in a subset of the vehicle-treated rats examined at these times during the subjective night, we further suggest that the levels reported above reflect true variability among rats and are indicative of Fos expression in the SCN that may be near the detection limits of the procedures described here. Konenen et al. 13 have previously reported a circadian rhythm of Fos-LI in the rat brain. However, from the procedures described, these investigators did not release animals into constant conditions and thus are more likely to have documented a diurnal, rather than a circadian, change in Fos-LI. In that study, the greatest number of Fos-positive cells in the SCN were found at 12.00 h and minimum number at 24.00 h, results that would be consistent with an increase of Fos-Ll due to photic stimulation. A recent preliminary report suggests a circadian rhythm of c-los m R N A expression in several brain areas that may be related to behavioral activity .w. We have considered the possibility that our results were due to inadvertent photic stimulation of our rats at a time of maximum photic sensitivity. This possibility was suggested because the time at which we observe Fos expression due to melatonin treatment (CT 22) and the time at which we see endogenous Fos-ir in some animals (CT 18 and CT 22) are close to the times at which photic stimulation is most effective in inducing c-fos m R N A in the rat 28 and hamster SCN 14. We have rejected this possibility for several reasons. First, all melatonin-injected and vehicle-treated animals were treated identically in terms of exposure to the dim red light during the injection procedure, yet Fos-ir was observed in only a subset of vehicle-treated animals at any circadian time (Table I). Secondly, half of the 24 animals in the in situ hybridization histochemical study at CT 22 were sacrificed on each of two different days. Within the dark control group, some, but not all, animals sacrificed on each day showed endogenous c-fos m R N A expression despite the fact that all animals were treated identically. Thirdly, we conducted another small scale (n = 4) in situ hybridization histochemical study at CT 22 as a further check on our procedures in which half of the rats were handled under conditions identical to the injection and then returned to darkness for 45 min ('handled' group) and the other half were simply kept in the dark. In this

study, c-fos m R N A was observed in the SCN of one ol the two animals kept in the dark and in neither of the two handled animals. On the basis of these lines ol evidence, we conclude that our observations of c-Jos m R N A and Fos protein expression in the rat SCN during the subjective night reported above reflect true variability among rats and indicative of endogenous Fos expression in the SCN that may be near the detection limits of the procedures described here. Given that Fos can be induced in the SCN by exogenous administration of melatonin at CT 22, we further suggest that the circadian rhythm of endogenous Fos expression in the SCN may be regulated by release of pineal melatonin. In rats kept under an LD 12:12 photoperiod, pineal melatonin levels increase 2 - 4 h after onset of darkness (CT 14-16), peak approximately 6 h after lights out (CT 18), and have already declined by CT 2221. Release of mclatonin by the pineal could activate Fos expression in SCN cells containing high-affinity melatonin receptors by a mechanism similar to that described in other neuronal systems 2°. This pattern of melatonin secretion may also have contributed to the high level of Fos expression observed in the SCN of the vehicle-treated animals at CT 18 on the first and second subjective nights (Table I); the absence of a detectable level of Fos expression in the SCN of most control rats at CT 22 may reflect that, by this time, endogenous melatonin levels have already decreased subsequent to melatonin's nocturnal peak. An experimental test of this hypothesis would be to evaluate Fos expression in the SCN of pinealectomized rats. Previous reports have called attention to the fact that the times at which Fos-LI or c-)'os mRNA are induced by light corresponds to the 'active' portion of the circadian pacemaker's phase-response curve. These observations have led to the suggestion that Fos induction may be a necessary component of a phase-shift of the circadian pacemaker. Melatonin administration at 50 tzg/kg at CT 9 to CT 11 has been shown to induce phase advances in rats 3, yet we have not seen Fos induction by melatonin administration of either 10 or 100 tzg/kg at CT 10 (Table 1). These observations suggest that Fos induction is not a necessary component of all phase-shift responses in rats, at least Fos induction at levels detectable by immunohistochemistry. The conclusions of this investigation rely, in part, on the specificity of the probe used for in situ hybridization histochemistry. Several factors argue for the specificity of the c-los riboprobe used in this study. First, discrete localization of the c-fos probe occured in several specific brain areas (i.e., VL-SCN, cerebral

55 cortex, pyriform cortex). Second, Kornhauser et al. 14, using a slightly shorter (1.8 kb) c-los cRNA probe transcribed from the same eDNA clone, and Rusak et al. 28, using a c-los oligonucleotide probe, described similar patterns of c-los mRNA in the SCN after photic stimulation. The distribution of c-fos mRNAs observed with in situ hybridization histochemistry is also in good agreement with the localization of the Fos protein as reported above and in previous immunocytochemical reports focusing on the SCN 4'11'23'28. Furthermore, a sense strand RNA probe used in these studies served as a measure of nonspecific labeling; no signal above background was detected with this control probe (Fig. 4B). Since c-los is induced rapidly and transiently by a variety of extraeellular signals and intracellular second messengers, Fos expression has been proposed to couple short-term membrane events to long-term changes in expression of 'target' genes that have AP-1 binding sites in their regulatory regions 2°'s°. The target genes of interest in the circadian system are those whose protein products are involved in phase-shifts and entrainment of the circadian pacemaker in the SCN. Although the identity of such target genes is unknown at the present time, some clues may be suggested from the location of Fos expression. Within the SCN itself, Fos expression at CT 22 was localized to the ventral and/or ventrolateral subregion (Figs. 1B and 5), the region in which retino-hypothalamic input terminates 12'~9 and vasoactive intestinal polypeptide-containing cell bodies occur 5. Given the non-uniform distribution of neuropeptides and transmitters in the SCN, efforts directed toward identification of the particular cell type(s) that express Fos after melatonin treatment or photic stimulation may indicate target genes regulated by Fos. At this point, however, it is not even clear whether glial or neuronal cells express Fos consequent to melatonin treatment. Our results indicate that melatonin, a pineal hormone known to be part of the circadian system, influences the transcriptional machinery of cells within the SCN in a circadian phase-dependent manner. The significance of the c-los mRNA and Fos protein expression documented here would be strengthened by direct evidence of melatonin induction of AP-1 binding activity in SCN cells from gel-shift assays. However, a preliminary report of changes in protein synthesis in the SCN induced by melatonin administration supports the argument for physiological function of the results documented h e r e 26. Given the recent report of a phase response curve for melatonin on SCN activity in vitro 17, changes in gene transcription induced by melatonin administration and mediated by c-los or other tran-

scriptional activators may provide further insights into the molecular machinery of the biological clock. Acknowledgements. We thank Dr. Tom Curran for supplying the c-fos eDNA clone, Louise Bitting and Dr. Joseph Miller for invaluable discussions and Drs. Jeffrey Elliott, Bruce O'Hara and Susan Welch for comments on the manuscript. This work supported by a grant from the Upjohn Company.

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Note added in proof In rats kept under an LD 12:12 photoperiod, c-los mRNA (Carter, D.A., Biochem. Biophys. Res. Commun., 166 (1990) 589-594)andFos-tike immunoreactivity (Koistinaho, J. and Yang, G., Histochemistry, 95 (1990) 73-76) are transiently expressed in the pineal gland in the middle of the dark phase.