Photic induction and circadian expression of Fos-like protein. Immunohistochemical study in the retina and suprachiasmatic nuclei of hamster

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Photic induction and circadian expression of Fos-like protein. Immunohistochemical study in the retina and suprachiasmatic nuclei of hamster I. Chambille, S. Doyle and J. Servi~re Laboratoire de Physiologie Sensorielle, 1NRA, Jouy-en-Josas (France) (Accepted 15 December 1992)

Key words: Circadian rhythm; Immediate early gene; Ganglion cell layer; Suprachiasmatic nucleus; Light induction; Endogenous expression; Phase shift

Fos-immunohistochemistry was performed in the retina and at four rostro-caudal levels of the suprachiasmatic nuclei (SCN) in hamsters. Animals were sacrificed at four circadian times (CT) relative to activity onset (CTI2): CT07, 11, 14, 19 either in permanent darkness (DD) or 1 h after light stimulation. Quantification of immunoreactive nuclei showed (i) endogenous CT related changes exclusively within the rostral SCN with maximum immunoreactivity at CT07, (ii) CT related responses to light in retinal displaced amacrines, ganglion cells and caudal SCN (maximum at CT19), (iii) expression differences in four subsets of SCN cells according to CT. The rostral subset could be implicated in the endogenous clock mechanism since it exhibited a fluctuation of Fos immunoreactivity in DD and expression of Fos protein at CTs 06 and 18 when light provokes transients in the free-running period. In the caudal SCN, a ventro-laterally localized set responded to light at CTs 13 and 18, a dorsal crescent of cells responded only at CT18 and a group located laterally between these two responded at CT18. These cellular subsets may have different functions in the light-entrainment mechanism since light stimuli at CTI3 induced phase-delays and, at CT18, phase-advances in the onset of the free-running locomotor activity rhythm.

INTRODUCTION In the natural environment, mammalian circadian rhythms (CR) are most effectively entrained by the day-night cycle (LD) 4°. This is accomplished by a daily light-induced phase shift of one or more underlying endogenous or intrinsic oscillators 6'35, the spontaneous period of which is thus lead to match the period of the external synchronizer. In a constant environment such as permanent darkness (DD), i.e. when lacking any external synchronizer, circadian rhythms 'free-run' with a period close to 24 h. Among these free-running rhythms, the locomotor activity rhythm can be shifted by light pulses in a phase dependent manner 6. During the active part of the cycle (subjective night) of nocturnal rodents, a light pulse delivered in early subjective night permanently delays the onset of activity of the next cycle but, when given in the middle late subjective night, permanently advances the onset of activity. When delivered during the inactive part of the cycle (subjec-

tive day), light stimulation is without effect on the phase of the rhythm. These phase-shifting effects are formalized in 'phase response curves' 11. The suprachiasmatic nucleus (SCN) acts as the central pacemaker in many kinds of C R 27'4°. Complete destruction abolishes activity rhythms in rodents 4~', while some retinal rhythms are preserved 4s. In mammals, the retina is the only transducer for the LD synchronizer. Photic information from the retina reaches the SCN via two pathways, the direct retinohypothalamic tract 12'2'~ and the indirect geniculohypothalamic tract 5 originating in the intergeniculate leaflet of the lateral geniculate body. Although the electrophysiological responses of SCN cells to light are well documented 23, little is known about how a light pulse would initiate the cellular events that would lead to a phase shift in the circadian rhythm of locomotor activity. Recent studies have shown that expression of several immediate early genes can be induced in the SCN at the circadian times (CT) when light has

Correspondence: J. Servi6re, Laboratoire de Physiologie Sensorielle, INRA. 78352 Jouy-en-Josas Cedex, France. Fax: (33) 34.65.25.05.

139 phase-shifting effects. Among these genes are cfos 1'2'3'8'9"15'16'17"36'37"41'42'44,c-jun 42, jun B 16'42, erg-1 ~° a n d NGFI-A41'42. In the retina, a few studies have established that light induces c-fos in amacrine cells of the inner nuclear layer and in displaced amacrine and ganglion cells of the ganglion cell layer 8'43. In spite of the growing amount of evidence indicating that these immediate early genes could be part of the resetting mechanism of the circadian clock, no attempt has been made to study the retinal response although it is a mandatory step in the process of entrainment by light. The present study was undertaken to establish (i) whether light induction of c-fos in the retina fluctuates according to circadian time and if so, (ii) how this fluctuation is related to c-los expression in the SCN as a function of CT. Since the endogenous circadian activity of the SCN is well established, we also looked for (iii) a fluctuation of endogenous expression in four rostro-caudal, levels of the SCN. The quantification of the number of Fos immunoreactive (Fos-ir) nuclei was performed simultaneously at retinal and suprachiasmatic levels in DD and in DD at times when light induces phase advances, phase delays or does not change the free running locomotor activity rhythm. MATERIAL

AND METHODS

Animals Approximately one hundred 3-month-old male golden hamsters

(Mesocricetus auratus) from the laboratory colony, weighing 100-125 g were used in this study. They were born under a long day photoperiod regimen (LD 16:8), transferred at the time of weaning to LD 12:12 and at 6 weeks of age put into individual cages fitted with running-wheels for the recording of locomotor activity. After 3 weeks of recording under LD 12:12, all animals were put into permanent darkness (DD) for at least two weeks. Only animals showing clear activity onsets and robust activity were used in this experiment. Some of the animals also received two identical light stimulations a week apart in order to provide a correlation between behavioral and cellular responses.

Photic stimulations It has been demonstrated, in the golden hamster, that 1 s light pulses can reset the circadian clock 7. In another mammal, similar shifting effects were obtained using 500 ms flashes 13. In addition, repetitive light-stimulation at a rate ranging between 5 - 1 0 / s are reported to increase the dicharge activity of suprachiasmatic cells 3°. We then decided to use a light stimulation made of 30 flashes (200 ms each) delivered at 6 per min at four different circadian times (CT06, 10, 13, 18); CT12 corresponding to locomotor activity onset. Light flashes were provided by a generator unit driving a xenon flash-tube delivering a pulse of white light with a radiant energy of 0.36 J / m 2 (United Detector Technology 81 Optometer equiped with a silicon photodiode probe) measured at the level of the animal in its cage. One hour after our repetitive light-flashes sequence, the animals were deeply anaesthetized with an overdose of pentobarbital in dim red illumination. A minimum of 4 animals per CT was processed for immunohistochemistry under a DD + Flash condition ( D D + FI). Immunohistochemistry of non-light-stimulated control animals was performed on 2 animals sacrificed at CT06 and 2 at CT07. Thus, 4

control animals were used for each ' C T - C T + 1' time span: CT06-07, 10-11, 13-14 and 18-19.

Immunohistochemistry procedures and anti-Fos antibodies Hamsters were perfused with 250 ml of warm (37°C) saline solution containing 1% sodium nitrite, followed by 300 ml of 4% paraformaldehyde in 0.1 M (pH 7.4) phosphate buffer solution. Brains were removed and post-fixed 4 h in the same fixative at 4°C before being cryoprotected in 10, 20 and 30% successive sucrose phosphate buffer solutions. Serial coronal sections (60/xm) were cut at - 2 0 ° C and immunostained for Fos protein with the avidin-biotin procedure. Free floating sections were collected in 0.1 M, pH 7.4 phosphate buffer solution (PB), incubated for 30 min at room temperature in a blocking solution of 3% normal goat serum in PB with 0.3% Triton X-100 (NGSTPB) and rinsed three times in 1% NGSTPB. They were then incubated at 4°C for 24 h in the primary antibody serum diluted in 1% NGSTPB. Although we worked with the polyclonal antibody provided by Oncogene Science (NY, USA), two other polyclonal antibodies were used for comparison. The Oncogene Science antibody (Abe), raised in rabbit against a synthetic peptide sequence corresponding to amino acids 4-17 of the N-terminal sequence of the human Fos protein was used at a dilution of 1:50,000. lmmunoblot analysis (Oncogene technical data sheet for batch no. 40890207, 1990) indicates that it recognizes a 62 kD Fos protein but does not react with the 39 kD Jun. This sequence, conserved in both the mouse and human Fos molecules was reported to have no homologies with the Fos-related proteins (Fra) 49. The Cambridge Research Biochemicals antibody (Ab 2) (Cambridge, UK), raised in sheep against a synthetic peptide sequence corresponding to amino acids 2-16 of the human Fos protein, was used at a dilution of 1 : 2,000 in normal rabbit serum triton phosphate buffer (NRSTPB). Immunoblot analysis (CRB technical data sheet) showed that it recognizes Fos (62 kD) and several Fos related antigens (48-49 and 70 kD). The last antibody (Ab 3) was a rabbit antiserum directed against the whole protein product of an in vitro translated c-los gene kindly provided by Dr. D. Menetrey 24. This antiserum was used at a dilution of 1:5,000 in NGSTPB. After primary antibody incubations, three rinses in NGSTPB or NRSTPB over 24 h were performed before 24 h incubations in biotinylated secondary antibodies, either an antirabbit antisera for primary Ab I and Ab 3 (Elite-ABC kit; Vector Labs) or an antisheep antiserum for Ab 2 (ABC kit PK4006; Vector Labs.). Sections were washed repeatedly with phosphate saline buffer (PBS) and then reacted for 4 min with the chromogen solution consisting of 0.02% DAB, 0.3% nickel ammonium sulfate in PBS and 0.035% hydrogen peroxide. The nickel-enhanced diaminobenzidine reaction produced a blue-black reaction product. After mounting on glass slides, the sections were counterstained with neutral red and dehydrated before being coverslipped. For control purposes, the Oncogene science c-fos antiserum was preabsorbed by preincubating the antiserum for 24 h with its corresponding peptide (c-fos peptide-2, Oncogene science) diluted at different concentrations in PBS. Preabsorption with the peptide abolished the nuclear staining between 2 × 10 -5 and 4 × 10 6 p.g/ml of diluted antibody. Further specificity of the staining was controlled by omitting the primary antibody, incubations with this control produced no immunoreactivity. After perfusion of the animals, eye-balls were taken, cornea and lenses removed and immersed 4 h in cold phosphate buffer solution containing 4% paraformaldehyde for post-fixation. Retinae were then dissected in phosphate buffer and cleaned from the vitreous. The immunohistochemical reactions were identical to the ones used on brain sections and were performed simultaneously. In this case only Ab I was used at a dilution of 1:20,000. For comparison purposes, the retinae and the brain sections of control and light-stimulated animals taken at the four circadian times were processed simultaneously in the same reaction baths.

Immunohistochemical reading procedures Comparison of topographical and temporal distributions of Fos-ir nuclei was performed using the three different antibodies. Immuno-

140 Fos-ir nuclei was made by moving a 0.2 × 0.2 nun*: square graticulc sample surface over the whole retina at 500 # m intervals. Retinal surfacc was measured using the image analysis system but the number of Fos-ir nuclei was counted by eye. The total number of immunoreactive nuclei was obtained by summing all the samples taken over the total area of the retina. The total number of cells within the ganglion cell layer was determined with a protocol used in a previous study (Chambille and Servi~re, submitted). The number of Fos-ir nuclei in the SCN was quantify in the following way. The number of SCN immunoreactive nuclei was counted for each of the 4 rostro-caudal levels prcviously described. These values were expressed as a density of Fos-ir nuclei over the SCN area at each level. In addition, we also used the density calculated by dividing the total number of Fos-ir cells in the whole SCN extent over the total area analysed. The non parametrical M a n n - W h i t n e y U-test for small samples was used.

histochemical procedures were performed on consecutive sections from the same brain, incubating alternate sections with either Ab I and Ab e, or Ab 1 and Ab 3. The nuclei stained with the c-los Oncogene antiserum were counted and the total number of immunostained nuclei within the limits of the two SCN at four antero-posterior coronal planes were calculated. The determination of each coronal plane was based on the m e a s u r e m e n t of the width of the optic chiasm thus allowing a comparison of position between animals. The following criteria were used to define four different rostrocaudal levels: level 1: chiasm length 2.1-2.3 mm, narrow distance of 60 p,m between IIIrd ventricle lumen and dorsal border of chiasm, average SCN height 190 p,m, nuclei separated at their medial borders. level 2: chiasm length 2.5-2.7 mm, distance between Illrd ventricle lumen and chiasm 90-101) /,m, average height 234 ttm, nuclei not yet fused medially. level 3: chiasm length 2.9-3.1 ram, distance between llIrd ventricle lumen and chiasm: 160 p,m, nuclei with characteristic oval tk~rm at their ventral border, average height 2 9 4 / , m , ventromedial aspects fused. level 4: chiasm length 3.2-3.4 mm, increased distance between Illrd ventricle and chiasm 235 /xm, ventromedial aspects still fused, contours more difficult to delineate.

Tracing proeedures This parallel study was performed to compare the distribution of Fos-ir nuclei to the terminal field of direct retinal projections. The procedure was similar to the one already used by one of us 2¢). Briefly, animals under anaesthesia (50 m g / k g i.p.; Imalg~ne 500, Merieux, France) received a 6/zl injection of cholera toxin-horseradish peroxidase conjugates (ChT-HRP) in the posterior chamber of one eye. Two days later, animals were deeply anaesthetized and perfused for histochemistry. H R P products were visualized by the tetramethyl henzidine procedure (TMB) and observed under dark-field illumination.

Counting procedures The counting of Fos-ir nuclei and m e a s u r e m e n t s of a given area of SCN were performed using an image analysis system (Histo 2000 from Biocom, France) linked to a microscope equiped with a X-Y stage recording device. Quantitative topographical data were also combined with immunoreactive 'intensity-level' data measured by observers according to three categories: (cat. 1) uniformly darkstained nucleus, (cat. 2) picnotic dark-grey nucleus well above the basal backround staining of surrounding tissue, (cat. 3) weak grey nuclei faintly visible above background level. Observers blind to the experiment were told to take into account and plot only those nuclei corresponding to cat. 1 and cat. 2. The number of Fos-ir nuclei in the retinal ganglion cell layer was quantified in the following way. An estimation of the total n u m b e r of

RESULTS

Locomotor activity rhythms The onset of wheel-running locomotor activity has been used as a marker of the course of the circadian clock. In a 24 h cycle the time position of activity onset, referred to as CT12, can be shifted by light pulses. The effect of our 5 min sequence of flashes on the locomo-

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D Fig. 1. Effects of light stimulation on the free-running locomotor activity rhythm of hamsters in D D recorded over 24 days. Each horizontal line represents a 24 h recording period begining at the left. A stimulation of 6 flashes per min for 5 rain was given at four CTs (stars): A, CT06; B, CT10; C, CT13: D, CT18. CT12 corresponds to activity onset. Arrows in C and D indicate the direction of the phase shift observed on the next cycle: ~ phase delay and ~- phase advance, respectively.

141 tor activity rhythm changed according to the CT at which it was delivered. The representative examples of phase shifting presented Fig. 1 were obtained from randomly selected free-running animals kept for 2 weeks in DD and given flashes either during their subjective day or their subjective night. The effects of light-flashes presented during subjective day were usually minor, in no case a shift in activity onset was observed. When presented at mid-subjective day (CT06), light induced a slight modification in the freerunning period (2 animals out of 10) and the modification never exceeded 10 min (3 min in the case of locomotor activity illustrated in Fig. 1A). In 3 cases out of 10, the slope of a line connecting subsequent onsets changed for 3 cycles (transients) and returned to the previous one. In 5 animals out of 10, no change in activity onset nor permanent modification of the freerunning period were observed. When flashes were delivered at the end of subjective day (CT10) light remained without any measurable effect (Fig. 1B). Light stimulation during the subjective night was always followed by a shift in activity onset of the next cycle, viz. a delay was observed when light was given at the begining of subjective night (CT13) (Fig. 1C), while flashes presented at CT18 resulted in a clear phase advance. As a rule, final stabilization to the former free-running period was reached through successive minor advances of activity onset (transients) (Fig. 1D).

Induction of Fos immunoreactivity by light in the retina No immunoreactivity could be detected in the retinae of animals (4 per CT) kept and sacrificed in DD whatever the CT. After light stimulation, Ab 1 Fos-immunoreactive nuclei were observed within the ganglion cell layer over the entire retina (4 animals per CT). Fos-ir nuclei were always more numerous during subjective night (Fig. 2). To further define this difference, Fos-ir nuclei were counted in one animal per CT. The quantification demonstrated a fluctuation according to CT with a maximum at CT18. At CT06, the density was 8 9 / m m z, thus corresponding to one Fos-ir cell out of 40 amacrine and ganglion neurons of the ganglion cell layer (Fig. 2A). The density of immunoreactive nuclei was almost multiplied by three at CT18 (331/mm 2, Fig. 2D) with intermediate values at CT10 ( 2 0 0 / m m 2, Fig. 2B) and CT13 ( 2 3 0 / m m z, Fig. 2C). At the time of maximal response to light (CI'18), one out of 13 cells was Fos-immunoreactive within the ganglion cell layer. At this CT the distribution of Fos-ir nuclei diameters ranged from 3 to 8 /zm indicating that more than one type of cell could be involved in induction of the protein Fos.

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Fig. 2. Fos immunohistochemistryin the retinal ganglion cell layer after light stimulation. Examples from retinae of four animals kept under DD for two weeks and flash stimulated at CT06 (A), CT10 (B), CT13 (C) or CT18 (D). Bar = 100/~m for all photomicrographs.

142

Endogenous Fos immunoreactiHty within SCN In thc SCN of animals sacrificed in DD (4 per CT), the position and the number of immunoreactive nuclei depended on the CT. Such a result is evident on pseudo 3D-reconstitutions of the endogenous Fos immunoreactivity (left rows in Fig. 3). Fos-ir ceils were mainly observed during subjective day in rostral levels (Fig. 4) while almost no endogenous Fos-immunoreactivity was detected in caudal levels (Fig. 5). This endogenous fluctuation was confirmed by analysis of the densities of immunoreactive cells calculated for the entire rostro-caudal extent of the SCN. The non parametric M a n n - W h i t n e y test indicated that time-based counting of Fos-ir cclls were significantly different ( P = 0.057) between subjective day (n = 4) and subjective night (n = 4). Moreover, this fluctuation was mainly restricted to the rostral SCN during subjective day ( C T 0 7 + C T l l ) since the two rostral levels (1 and 2) exhibited a population of Fos-ir cells significantly greater ( P = 0.014) than the two caudal levels (3 and 4) (Figs. 4 and 5). Furthermore, plotting the density of Fos-ir cells along the rostro-caudal extent in two ani-

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mals at each CT shows a trend towards highest densities in level 2 at CT07 and C T l l (Fig. 6).

Induction of Fos immunoreactiuity by light in the SCN In the whole SCN, light had a maximal effect during subjective night. Fos-ir patterns observed at CT14 and CT19 differed qualitatively according to the position of immunoreactive nuclei and were quantitatively significant in number ( P = 0.04). The highest immunoreactivity was observed at CTI9 in the caudal SCN (Fig. 5H). When light was given at CT06, the number of Fos-ir nuclei was increased in the rostral part (Figs. 3A and 7A,B), the response being restricted to this subdivision (Fig. 6). The comparison (Mann-Whitney test) of densities counted in levels 1 and 2 at CT06 in D D (n = 2) vs. DD + FI animals (n = 2) demonstrated a significant effect of light-flashes ( P = 0.014). In rostral SCN, the light had almost no effect at C T l l , a weak one at CT14 and a strong one at CT19 (Figs. 3 and 6). In levels 3 and 4, light stimulation did not induce any Fos-immunoreactivity during subjective day (Fig. 5B,D) but induced a very strong immunoreactivity when given

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Fig. 3. Phase dependent spatial distribution of Fos-ir nuclei within the SCN. Unstimulated animals (DD) compared to light-stimulated animals (DD + FI). CTs indicate the time of sacrifice: light-stimulus was delivered 1 h before, l~ -~ 14, successive rostro-caudal coronal levels.

143 during subjective night (Figs. 3, 5F,H and 6). While the density of Fos-ir nuclei was the highest in level 4 (Fig. 6), their distributions differed between CT14 and CT19 (Fig. 3). AT CT14, Fos-ir nuclei were exclusively packed in the ventrolateral SCN (level 4) just above the optic chiasm (Fig. 3). AT CT19, this ventrolateral group was also seen, and in addition two other subsets of Fos-ir nuclei could be distinguished; the first one, observed in level 3, formed a crescent lining the dorsolateral boundaries of SCN, the second subset, observed only in level 4, was located along the lateral margins of the SCN (Fig. 3). At the same CT19, the two rostral levels also exhibited a strong Fos-immunoreactivity after light stimulation (Figs. 6 and 7C,D). As observed on the 2 animals presented in Fig. 6, the densities of immunoreactive nuclei were not significantly different from those calculated in DD + F1 at CT07. A second parameter relating to the strengh of induction was the density of immunoreactive staining products. According to this criterion, in all the immunostained preparations, the number of picnotic dark-grey nuclei (defined as cat. 2, cf methods section) was always greater than the number of uniformly dark-stained nuclei (cat. 1) except at level 4 with animals stimulated at CT18. At this time, dense darkstained and dark-grey nuclei were in equal proportion, thus indicating a stronger induction. We were also interested in comparing the relative distributions of Fos-ir cells to the terminal field of retinal projections to the SCN. Retinal fibers, as revealed by anterograde transport of ChT-HRP, did enter the two rostral levels (1 and 2) of the nucleus (Fig. 8, panel 1,2). In the medial (panel 3) and caudal (panel 4) levels, terminals had a characteristic distribution, being very dense all over the entire nucleus, extending into the lateral and dorsal parts and beyond SCN borders (Fig. 8, panel 3,4). This pattern of distribution was in good register with the Fos-ir nuclei following light stimulation (Fig. 3).

Fig. 4. Endogenous Fos immunoreactivity in the rostral SCN (level 2). Level defined by width of chiasm and distance between IIIrd ventricule lumen and chiasm (see Materials and Methods). Animals kept under DD for 2 weeks and sacrificed at four CTs: A, 07; B, 11; C, 14; D, 19. Bar = 100/xm.

Comparison of SCN immunoreactive stainings as observed with different anti-Fos polyclonal antibodies In order to assess whether the fluctuations in Ab t Fos immunoreactivity would also be found whatever the anti-Fos antibody, two other antibodies were used. The results obtained with Ab 1 have been taken as reference. Ab 2 and Ab 3 Fos immunoreactivities were compared to Ab I Fos immunoreactivity observed under the same conditions (DD and DD + F1) and at the same CT on immediately adjacent sections from the same brain. Comparisons were made at times when Ab~ Fos immunoreactivity was maximum, that is, level

144

Fig. 5. CT dependent Fos immunoreactivity obtained in caudal level 4 of SCN. Light stimulated animals (right column) are compared to controls (left column). Animals sacrificed at four CTs: 07 (A,B), 11 (C,D), 14 (E,F) and 19 (G,H). Light-stimulus, as described in the legend to Fig. 1, was given 1 h before sacrifice. Bar = 100 ~m.

145 2 under DD (endogenous expression) and level 4 under DD + FI (induced expression). Under DD, Ab2 Fos immunoreactivity was lacking. The absence of Fos-ir nuclei was evident at CT07 (Fig. 9B) when Ab 1 gave positive results (Fig. 9A). Thus, no fluctuation in endogenous expression could be detected. Furthermore, Ab 2 immunoreactivity was always weaker than with Ab~ after light induction, either during subjective day at CT07 (Fig. 9F) or subjective night at CT14 and CT19. The results obtained with Ab 3 were qualitatively similar to those obtained with Ab] showing an endogenous immunoreactivity with a maximum at CT07 in level 2 (Fig. 9D), an increase by light of immunoreactivity at this level at CT07 (Fig. 9H) and a massive induction, in caudal levels, during the subjective night. However, in all cases, the Fos-ir nuclei appeared less numerous with Ab 3 than with Ab,.

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Rostro-caudal level Fig. 6. Rostro-caudal distribution (4 levels) of the density of Fos-ir suprachiasmatic cell nuclei (nuclei/mm 2) as a function of CT. Nuclei were counted at four CTs in two independent series of animals (right and left columns). For each CT, one animal stimulated 1 h before sacrifice (DD+FI, hatched boxes) and one control animal (DD, filled blocks) is shown.

DISCUSSION Recent s t u d i e s 3'8'15-17'36'37'4]'42 demonstrate an increase in Fos-like immunoreactivity and c-los mRNA in the SCN of rat and hamster following exposure to light pulses. These studies stress the fact that c-fos was induced only when light was given at those CTs that could phase shift the free-running locomotor activity rhythm. This experimental fact was regarded as evidence that c-fos participates in the cellular cascade of events involved in the entrainment of the circadian clock by light. The present work supports this conclusion and in addition shows that there is (i) a circadian rhythm in the retinal response to light at the level of the ganglion cell layer, (ii) an endogenous rhythm of Fos immunoreactivity in the anterior part of the SCN with a maximum at mid-subjective day, (iii) a spatiotemporal circadian fluctuation in the response to light with greatest amplitude during subjective night in the caudal SCN. The main body of data has been obtained with an antibody raised against a synthetic peptide corresponding to amino acids 4-17 in the N-terminal sequence of the Fos protein (Abt). Although no quantification of the number of Fos-ir nuclei was attempted with the two other antibodies, some conclusions can be drawn. The Ab 3 antiserum, directed against the whole protein product of an in vitro translated c-fos gene, provided the same trends as Ab,, that is (i) the endogenous Fos immunoreactivity was maximal at mid-subjective day (CT07) in rostral SCN, (ii) the response to light had a greater amplitude during subjective night in the caudal SCN than during subjective day. However Ab 1 Fos-ir was always higher than Ab 3 Fos-ir for both endogenous and light induced expression. The other antibody (Ab 2) has been used in previous works TM. Although raised against a peptide sequence (2-16) close to the sequence of Ab,, the number of immunoreactive cells was smaller with light induction and endogenous changes were absent. Could these differences in immunoreactivity be significant or are they simply due to the fact that Ab, has more non specific binding activity than the two other antibodies? A complementary study with in situ hybridization is necessary to resolve this question.

Behavioral effects of light flashes The effect of our light stimulation on the locomotor activity rhythm changed according to CT in a manner similar to that classically described 11. Light stimuli used in the literature to obtained phase response curves are continuous with durations ranging from 1 s to several hours ]'. For a given wavelenghth of light (maxi-

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Fig. 7. Fos immunoreactivityobtained within the two rostral levels of SCN 1 h after stimulation by light at CT06 (A,B) and CT18 (C,D). A,C, level 1; B,D, level 2. Bar = 100 ;~m. A,B: CT07; C,D: CT19.

mum sensitivity 503 nm), the amplitude of the phase shift is related only to the number of photons whatever the duration of the pulse, up to 45 min 42. In our case, the intense and discontinuous stimulus over 5 rain has been seen to induce large phase shifts at CT18, up to 3 h. In the present experiments, no phase shift was observed during subjective day but transients and changes in the free-running period were obtained in 50% of the animals. Small phase s h i f t s 11"37'45 o r transitory changes in the free-running period 34 have been reported in the golden hamster after light stimulation during subjective day.

Circadian fluctuation of Fos-immunoreactive response to light in the retina As reported in the rat s and the rabbit retinae 43, the present data shows that the synthesis of Fos in the retinal ganglion cell layer is only present after photic stimulation• Contrary to the SCN, no endogenous fluctuation of Fos immunoreactivity was found in retinal cells. Nevertheless, the response to light showed a circadian dependence since the number of Fos-ir cells in this layer fluctuated according the CT. Various

circadian related processes have already been described in the retina such as disc-shedding of rod outer segments, absolute threshold for scotopic vision, amplitude of the response to light (b-wave of the electroretinogramm), melatonin synthesis and opsin mRNA synthesis 3s. Some of these rhythms persist in constant darkness a n d / o r in SCN-lesioned animals thus indicating the possible existence of a retinal ciracadian pacemaking mechanism 4s. We have shown that a circadian component in induction by light of the Fos protein in the retina can now be added to these various circadian phenomena. In the present results, the number of Fos-ir retinal cells was maximal when flashes were presented in the middle of the night (CT18). The large distribution of the nuclei diameters of Fos-ir cells (3-8 /xm) might indicate that this retinal cell population is composed of true ganglion and displaced amacrine cells as suggested for the rabbit 43 and the rat s retinae. It is tempting to speculate a direct correlation between the Fos-ir cells of the retinal ganglion layer and those found in the SCN since both populations showed the same temporal distribution in responses to flashes. In the hamster, true ganglion cells represent 40% of the total ganglion cell layer population (unpublished

147 observations). If we consider the number of Fos-ir cells at CT18 with respect to this proportion, then at most, 8% are true ganglion cells. This value is clearly far too high to lend plausibility to the hypothesis that the entire population of retinal ganglion Fos-ir cells projects to the SCN since in rodents it has been estimated to be as small as 0.1-0.4% 4'21'3l. However, many anatomical studies have demonstrated that in addition to the SCN ~2, ganglion cells also project to the anterior 31'32 and lateral hypothalamic areas 14'39, the ventral part of paraventricular nuclei 12 and the supraoptic nuclei 18'25 where we often observed Fos-ir nuclei. A few Fos-ir cells have also been observed in the leaflet of the lateral geniculate nuclei, a structure implicated in the regulation of entrainment of the circadian system by light 33 while, in spite of a massive retinal projection, almost no Fos-ir cells were observed in the ventral and dorsal parts of the lateral geniculate nuclei known to process visual information. Additional studies are needed to examine the hypothesis that the Fos-ir ganglion cells (i) contain the population which projects to the SCN and thus (ii) could be causally linked to the induction of this immmediate early gene in the SCN and related hypothalamic structures.

Endogenous fluctuation of Fos immunoreactiuity within SCN The present data show an endogenous circadian fluctuation of Fos-ir in the SCN with a maximum during mid-subjective day. The topography of this expression is such that it only involves the anterior SCN. Comparison with results from other reports is often quite difficult since the photographs presented as taken from control animals (DD) always concerned the posterior a n d / o r caudal 2 / 3 of the S C N 1'2'36'37. In our case this corresponds to the part where almost no Fos-ir fluctuation occurred (levels 3 and 4). Nevertheless, Ebling et al. 1° who considered three rostro-caudal levels in the SCN, report that in DD, a few Fos-ir ceils were always observed in the dorsolateral part of hamster SCN at each CT examined. Under another constant condition, LL, Earnest et al. 9 report a circadian fluctuation with a maximum at CT18 in the ventrolateral part of the rat SCN. When observations were made under LD conditions 8,15,41, Fos-ir was always greater in the ventrolateral SCN during the light phase than during the dark phase.

Induction by light of Fos immunoreactivity within SCN Fig. 8. Direct retinal projections within the SCN and adjacent hypothalamic areas 48 h after intra-ocular injection of ChT-HRP. H R P products were observed under dark-field illumination in the SCN at four rostro-caudal coronal levels (1, 11; 2, 12; 3, 13; 4, 14). Bar = 100/zm.

The present data provide additional evidence that

c-los is involved in the cellular mechanism of entrainment of the circadian clock by light. Light stimulation during subjective night always phase shifted free-run-

148

Fig. 9. Fos-ir nuclei observed within SCN at rostro-caudal level 2 with three different polyclonal antibodies (Ab). Hamsters were sacrificed during subjective day (CT07) either in the dark, DD ( A - D ) , or 1 h after light-stimulation, D D + F L ( E - H ) . Ab I (Oncogene Science)= A, C, E, G; Ab 2 (Cambridge Research Biochemicals) = B, F; Ab 3 ('whole-protein antibody')= D, H. Bar = 100 p,m.

ning locomotor activity and parallely induce a large Fos-immunoreaetivity in the SCN. Moreover, our data confirm that the localization and number of suprachiasmatic Fos-ir cells differed according to CT. These spatial and temporal results led to the recognition of four cellular sub-types fluctuating endogenously a n d / o r responding differentially to light according to the circadian time. A 1st group of Fos-ir cells was defined as the population endogenously expressing c-los at CT07 in the rostral SCN at levels 1 and 2. This group seems to be the same as the one responding to light at CT06 and C r l 8 in the rostral SCN. The three other groups were defined by the distribution of Fos-ir cells after light stimulation. The 2nd and 3rd groups, observed only at CT19, correspond to the dorsal crescent (level 3) and to the dorso-lateral group (level 4). The 4th group would be constituted by the tightly packed group of cells observed at CT14 and CT19 in the ventro-lateral SCN (level 4). These immunoreactive cell groups are located within the anatomical boundaries of the SCN. After anterograde migration of C h T - H R P injected into the eye, the profile of labelled terminals in the

SCN was in good register with the Fos-ir cell distribution in each rostro-caudal coronal level. Ebling et al. 1° using the same technique in the hamster did not observe R H T terminals or Fos-ir cells in their rostral SCN level corresponding to our levels 1 and 2. Precise descriptions of rostral Fos-ir cells are not reported in other related works 2m. In the same species, it was recently demonstrated 37 that the pattern of Fos-immunoreactivity changes according to whether light is phase-advancing or phase-delaying. The effect of light was described at three CTs and four rostro-caudal levels. Light presented at CT18 was correlated with the presence of a dorsal cluster of Fos-ir nuclei located in a position corresponding to our dorsal crescent (2nd group). However, the comparison with patterns observed in our two rostral levels is difficult since the most anterior level described is located 400 ~ m from the rostral border of the SCN, a level corresponding to our level 3. This might account for the absence of light effect at CT06 in anterior SCN. It is worth to note that although we used a light stimulation different from the one used by Rea 37 (15 min of lower intensity white

149 light), the Fos-immunoreactive patterns are similar in subjective night in the caudal levels. Other works using pharmacological induction of Fos-ir, provide information supporting the present proposition that several sub-groups of cells are located within the SCN. These lines of evidence indicate that glutamatergic neurotransmission has a role in mediating the effects of light on SCN function 19'23'26. The subdivision into four cellular sub-groups is reinforced by the following observations: (i) blockade of light-induced c-fos by two N-methyl-o-aspartate (NMDA) receptor antagonists (non competitive MK8011'1° and competitive CPP 2) in the ventro-lateral SCN equivalent to our 4 th group and in another part regarded as rostral by Abe et aI.1'2 but which in fact corresponds to our 2 nd group (dorsal crescent), (ii) impossibility of blocking light-induced c-los with these two antagonists in the dorso-lateral SCN 1'2'1° corresponding to our 3 rd group (dorso-lateral extension), and finally, (iii) the rostral or i st population could be regarded as homologous to the Fos-ir cell population observed in the rostral SCN when the glutamate agonist NMDA was locally administered ~°. With respect to possible functional roles for the four sub-groups of SCN cells identified by Fos immunohistochemistry, the following propositions could be considered. The anterior group of cells, exhibiting an endogenous fluctuation in Fos immunoreactivity may be involved in the intrinsic clock mechanism itself, whereas the three other posterior populations, would be implicated in the entrainment of the clock by light. The differential activation by light of these three latter populations is phase dependent in the same way as light induces phase shifts of the locomotor activity rhythm; a similar parallelism was recently reported for two of these subsets 37. We noticed that the same anterior group of cells exhibited Fos immunoreactivity after light stimulation at both at CT06 and CT18. When flashes were given at CT06, the locomotor activity rhythm quite often exhibited transient modifications in the free-running period over several consecutive days. At this CT, Fos-ir cells are present in the rostral SCN. When flashes were given at CT18, in addition to the phase advance shifting effect, small transient decreases of the period of the free-running locomotor activity rhythm were always recorded over several consecutive days. Such a phenomenon has been reported and analysed by Pittendrigh and Daan 35. It is to be stressed that, at CT18, light induced Fos immunoreactivity was observed in cells located in both the rostral and caudal SCN. If the anterior group of cells, exhibiting an endogenous fluctuation in Fos immunoreactivity, is also implicated in c-fos induction when light

induces transient shifts in the free-running period, this subset of cells might be an obligatory component of the endogenous mechanism of the clock. Acknowledgements. We are grateful to Drs. R. Kado and B. Dumortier for their intellectual help and comments in manuscript preparation. We are grateful to G. Gendrot and S. Venla for technical assistance.

REFERENCES 1 Abe, H., Rusak, B. and Robertson, H.A., Photic induction of Fos protein in the suprachiasmatic nucleus is inhibited by the NMDA receptor antagonist MK-801, Neurosci. Lett., 127 (1991) 9-12. 2 Abe, H., Rusak, B. and Robertson, H.A., NMDA and non-NMDA receptor antagonists inhibit photic induction of Fos protein in the hamster suprachiasmatic nucleus, Brain Res. Bull., 28 (1992) 831-835. 3 Aronin, N., Sagar, S.M., Sharp, F.R. and Schwartz, W.J., Light regulates expression of a fos-related protein in rat suprachiasmatic nuclei, Proc. Natl. Acad. Sci. USA, 87 (1990) 5959-5962. 4 Balkema, G. and Dr~iger, U.C., Origins of uncrossed retinofugal projections in normal and hypopigmented mice, Visual Neurosci., 4 (1990) 595-604. 5 Card, J.P. and Moore, R.Y., Organization of lateral geniculatehyptothalamic connections in the rat, J. Comp. Neurol., 284 (1989) 135-147. 6 De Coursey, P.J., Function of a light response rhythm in hamsters, J. Cell. Comp. Physiol., 63 (1964) 189-196. 7 Earnest, D.J. and Turek, F.W., Effect of one-second light pulses on testicular function and locomotor activity in the golden hamster, Biol. Reprod., 28 (1983) 557-565. 8 Earnest, D.J., Iadarola, M., Yeh, H.H. and Olschowka, J.A., Photic regulation of c-los expression in neural components governing the entrainment of circadian rhythms, Exp. Neurol., 109 (1990) 353-361. 9 Earnest, D.J., Ouyang, S. and Olschowka, J.A., Rhythmic expression of fos-related proteins within the rat suprachiasmatic nucleus during condtant retinal illumination, Neurosci. Lett., 140 (1992) 19-24. 10 Ebling, F.J.P., Maywood, E.S., Staley, K., Humby, T., Hancock, D.C., Waters, C.M., Evan, G.I. and Hastings, M.H., The role of N-methyl-D-aspartate-type glutamatergic neurotransmission in the photic induction of immediate-early gene expression in the suprachiasmatic nuclei of the Syrian Hamster, J. Neuroendocrinol., 3(6) (1991) 641-652. 11 Johnson, C.H., An Atlas of Phase Response Curves for Circadian and Circatidal Rhythms, Vol. H, Vanderbilt University, 1990. 12 Johnson, R.F., Morin, L.P. and Moore, R.Y., Retinohypothalamic projections in the hamster and rat demonstrated using cholera toxin, Brain Res., 462 (1988) 301-312. 13 Joshi, D. and Chandrashekaran, M.K., Bright light flashes of 0.5 milliseconds reset the circadian clock of a microchiropteran bat, J. Exp. Zool., 230 (1984) 325-328. 14 Kita, H. and Oomura, Y., An anterograde HRP study of retinal projections to the hypothalamus in the rat, Brain Res. Bull., 8 (1982) 249-253. 15 Kononen, J., Koistinaho, J. and Alho, H., Circadian rhythm in c-Fos-like immunoreactivity in the rat brain, Neurosci. Lett., 120 (1990) 105-108. 16 Kornhauser, J.M., Nelson, D.E., Mayo, K.E. and Takahashi, J.S., Photic and circadian regulation of c-Fos gene expression in the hamster suprachiasmatic nucleus, Neuron, 5 (1990) 127-134. 17 Kornhauser, J.M., Nelson, D.E., Mayo, K.E. and Takahashi, J.S., Regulation of jun-b messenger RNA and AP-1 activity by light and a circadian clock, Science, 255 (1992) 1581-1584. 18 Levine, M.L., Weiss, M.L., Rosenwasser, A.M. and Miselis, R.R., Retinohypothalamic tract in the female albino rat: a study using horseradish peroxidase conjugated to cholera toxin, J. Comp. Neurol., 306 (1991) 344-360.

150 19 Liou, S.Y., Shibata, S., lwasaki, K. and Ueki, S., Optic nerve stimulation-induced increase of release of ~H-glatamate and 3Haspartate but nol 3I-t-gaba from the suprachiasmatic nucleus in slices of rat hypothalamus, Brain Res. Bull., 16 (1986)527-531. 20 Martinet, L, Servi~re. J. and Peytevin. J. Direct retinal projeclions of the 'non-image forming' system to the hypothalamus and basal telencephalon of mink (Mustela rison) brain. Exp. Brain Res., 89 (1992) 373 382. 21 Mason, C.A. and Lincoln, D.W., Vizualization of the retino-hypotalamic projection in the rat by cobalt precipitation. Cell Tissue Res., 168 (1976) 117-131. 22 Meijer, J.H., Van der Zee, E.A. and Dietz, M., Glutamate phase shifts circadian activity rhythms in hamsters, Neurosci. Lett., 86 (19881 177-183. 23 Metier, J.H. and Rietveld, W.J., Neurophysiology of the suprachiasmatic circadian pacemaker in rodents, Physiol. Reu., 69 (19891 671 7(t7. 24 Menetrey, D., Gannon, A., Levine, J.D. and Basbaum, A.I., The expression of c-los protein in presumed nociceptive interneurons and projection neurons of the rat spinal cord: anatomical mapping of the central effects of noxious somatic, articular and visceral stimulation, J. Comp. Neurol,, 285 (1989) 177-195. 25 Mikkelsen, J.D. and ServiEre, J., Demonstration of a direct projection from the retina to the hypothalamic supraoptic nucleus of the hamster, Neurosci. Lett., 139 (1992) 149-152. 26 Moffet, J.R., Williamson, L., Palkovits, M. and Namboodiri, M.A.A., N-Acetylaspartylglutamate: a transmitter candidate for the retinohypothalamic tract, Proc. Natl. Acad. Sci. USA, 87 (1990) 8065-8069. 27 Moore, R.Y., Organization and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus, Fed. Proc., 42 (1983) 2783-2789. 28 Moore, R.Y. and Eichler, V.B., Loss of a circadian adrenal corticosterone rhythm following suprachiasmatic nucleus lesions in the rat, Brain Res., 42 (1972) 201-2116. 29 Moore, R.Y. and Lenn, N.J., A retinohypothalamic projection in the rat, J. Comp. NeuroL, 146 (1972) 1-14. 30 Nishino, H., Koizumi. K. and Brooks, C. McC., The role of suprachiasmatic nuclei of the hypothalamus in the production of circadian rhythm, Brain Res., 112 (1976) 45-59. 3I Pickard, G.E., The afferent connections of the suprachiasmatic nucleus of the golden hamster with emphasis on the retinohypothalamic projection, J. Comp. Neurol., 211 (1982) 65-83. 32 Pickard, G.E. and Silverman, A.J., Direct retinal projections to the hypothalamus, piriform cortex, and accessory optic nuclei in the golden hamster as demonstrated by a sensitive anterograde horseradish peroxidase technique, J. Comp. Neurol., 196 (19811 155 172. 33 Pickard, G.E., Ralph, M.R. and Menakcr, M., The intergeniculate leaflet partially mediates effects of light on circadian rhythms, .1. Biol. Rhythms, 2 (1987) 35-56.

34 Pittendrigh, C.S., Bruce, V. and Kaus. P., ()n Ihe significance t~l transients in daily rhythms. Proc. Nat. Acad. Sci. USA. 44 (19581 965 973. 35 Pittendrigh, C.S. and Dann, S., A functional analysis of circadian pacemakers in nocturnal rodents. IV. Entrainment: pacemaker as clock., ./. Comp. Physiol., 1(16 (19761 291-331 36 Rea, M.A., kight increases Fos-related protein immunoreactivity in the rat suprachiasmatic nuclei. Brain Res. Bull., 23 (19891 577-581. 37 Rea, M.A., Different population of cells in the suprachiasmatic nuclei express c-fbs in association with light-induced phase delays and advances of the free-running activity rhythm in hamsters, Brain Res., 579 (1992) 1117- 112. 38 Remd, C.E., Wirz-Justice, A. and Terman, M., The visual inpul stage of the mammalian circadian pacemaking system. I. ls there a clock in the mammalian eye'? Ji Biol. Rhythms, 6 (19911 5-29. 39 Riley, J.N., Card, J.P. and Moore, R.Y., A retinal projection to the lateral hypothalamus in the rat, Cell Tiss. Res.. 217 (1981) 257 269. 4(1 Rusak, B. and Zucker, 1., Neural regulation of circadian rhythms, Physiol, Rec., 3 (1979) 449-525. 41 Rusak, B., Robertson, H.A., Wisden, W. and Hunt, S.P., Light pulses that shift rhythms induce gene expression in the suprachiasmatic n ucteus, Science, 248 (1990) 1237-1240. 42 Rusak, B., Mc Naughton, L., Robertson, H.A. and Hunt, S.P., Circadian variation in photic regulation of immediate-early gene mRNAs in rat suprachiasmatic nucleus ceils, Mol. Brain Res., 14 (19921 124-130. 43 Sagar, S.M. and Sharp, F.R., Light induces a Fos-like nuclear antigcn in retinal neurons, Mol. Brain Res., 7 (1990) 17-21. 44 Smeyne, R.J., Schilling, K.. Robertson, L., Luk, D., Oberdick, J., Curran. T. and Morgan, J.l., Fos-LacZ transgenic mice: mapping sites of gene induction in the central nervous system, Neuron, 8 (19921 13-23. 45 Smith, R.D., Turek, F.W. and Takahashi, J.S., Two families of phase-response curves characterize the resetting of the hamster circadian clock, Am. J. Physiol., 262 (19921 R1149-R1153. 46 Stephan, F.K. and Zucker, I., Circadian rhythms in drinking behavior and locomotor activity are eliminated by suprachiasmatte lesions, Proc. Natl. Acad. Sci. USA, 54 (1972) 1521-1527. 47 Takahashi, J.S., DeCoursey, P.J., Bauman, L. and Menaker, M., Spectral sensitivity of a novel photoreceptive system mediating entrainment of mammalian circadian rhythms, Nature, 308 (1984) 186-188. 48 Terman, M. and Terman, J., A circadian pacemaker R)r visual sensitivity'.) Ann. NYAcad. Sci., 453 (1985) 147- 161. 49 Zerial, M., Toschi, L., Ryseck, R.P., Schuermann, M., Muller, R. and Bravo, R., The product of a novel growth factor activated gone, fi~s B, interacts with jun proteins enhancing their DNA binding activity, EMBO J.. 8 (1989) 805-813.