Daily cycle of fos expression within hypothalamic POMC neurons of the male rat

Brain Research 771 Ž1997. 45–54

Research report

Daily cycle of fos expression within hypothalamic POMC neurons of the male rat A.K. Jamali ) , G. Tramu Laboratoire de Neurocytochimie Fonctionnelle, UniÕersite´ de Bordeaux I, CNRS URA 339, AÕenue des Facultes, ´ 33405 Talence, France Accepted 10 June 1997

Abstract The activity of hypothalamic pro-opiomelanocortin ŽPOMC. neurons is known to display a circadian cycle. We hypothesized that the existence of a c-Fos responsive element ŽAP-1 site. within the POMC gene sequence might reflect the ability of POMC neurons to express c-fos proto-oncogene during circadian increase of their neuronal activity. To this aim, adult male rats previously kept under a controlled 12 h lightr12 h dark schedule were sacrificed every 4 h throughout the 24 h cycle and their brains processed for Fos andror POMC immunocytochemistry. Here we show that, specifically during the dark period of the cycle, the mediobasal hypothalamic area spontaneously exhibits a strong Fos immunoreactivity, whereas very low Fos labelling was detected during the light period. As postulated, the simultaneous visualisation of both Fos and POMC antigens allowed us to show that this nocturnal induction of Fos occurs almost exclusively at the nuclear level of POMC-producing neurons. These results not only highlight the mechanisms underlying the physiological functioning of the hypothalamic POMC system, but also demonstrate the feasibility of using c-fos expression as a useful tool to assess the pharmacological effect of drugs on the activity of POMC neurons as is the case for many other neuronal systems. Such drugs might be relevant in the treatment of psychosis since an alteration of POMC-related peptide transmission has been reported in the brains of both schizophrenic and depressive patients. q 1997 Elsevier Science B.V. Keywords: b-Endorphin; Mediobasal hypothalamus; Biological rhythm; Pro-opiomelanocortin neuron; Schizophrenia; Depression

1. Introduction Within the mammalian brain, hypothalamic proopiomelanocortin ŽPOMC. neurons constitute an important peptidergic neuromodulatory system acting upon both neuroendocrine and behavioural expression. The hypothalamic POMC system has interested investigators in almost all fields of neuroscience. Thus, this system has been implicated in analgesia, thermoregulation, cardiovascular, respiratory and neuroendocrine regulation as well as in consummatory, grooming, locomotor, sexual and defensiveraggressive behaviors. POMC neurons have also been implicated both in mediating endogenous reward and in modulating learningrmemory processes. Furthermore, brain POMC-related peptides Žsuch as b-endorphin, b-END. have also been involved in human reproductive function and psychiatric disorder states such as schizophrenia and depression Žfor review see w1,21,24,37,55–58x.. Yet, despite the existence of a vast literature, the mechanisms

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0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 7 6 7 - 1

underlying the physiological functioning of this system remain unclear. Interestingly, the basal activity of hypothalamic POMC neurons is known to display a circadian cycle ŽCC. in the sense that the expression of POMC gene products ŽmRNA and its related peptide. was found to fluctuate spontaneously throughout the CC. In fact, such fluctuation was reported for the hypothalamic b-END content in both male w23x and female w11,12x rats as well as in male Syrian hamsters w42x. Moreover, a diurnal rhythm in the level of POMC mRNA expression within the MBH was reported for the male rat w53x. All together, these results strongly suggest that hypothalamic POMC neurons are likely to be subjected to changes in the level of physically active stimulatory andror inhibitory afferents during the CC. On the other hand, using the corticotrope-derived AtT20 cell line, Boutillier et al. w2,3x have shown that the c-fos proto-oncogene product, Fos protein, stimulates both basal and corticotrophin-releasing hormone ŽCRH.-induced POMC transcription through a c-Fos-responsive element Ža non-canonical but functional AP-1 site. localized in the first exon of the POMC gene sequence w2,3x.

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A.K. Jamali, G. Tramur Brain Research 771 (1997) 45–54

On the basis of all these results, we postulated that Fos immunohistochemistry ŽIHC., which has been successfully u s e d a s a n e u ro n a l a c tiv ity m a rk e r w4,8,9,19,22,28,33,36,45,47 –50x coupled to POMC-related peptide immuno-detection w6,10,17,18,30–32,41x, could provide a powerful tool to reveal the endogenous activation of hypothalamic POMC neurons throughout the CC.

2. Materials and methods 2.1. Animals and procedure Adult male Wistar rats Ž175–200 g. were purchased from Iffa-Credo ŽFrance.. Prior to sacrifice, animals were first acclimatised to laboratory conditions for at least 3 weeks. They were housed in individual boxes, under controlled conditions of lighting: 12 h of lightr12 h of total darkness Ž12 : 12 LD cycle. with light onset Ždawn. at CT 6 and light offset Ždusk. at CT 18. Light intensity was 100–200 lux at each cage level. Room temperature was fixed at 22 " 0.58C. Food and water were provided ad libitum twice a week. All rats were sacrificed when weighing 250–280 g Ž8–9 weeks of age.. Access to the animal house was prohibited for at least 48 h before the day of sacrifice in order to avoid stress-induced Fos-IR. Rats were sacrificed every 4 h, along the CC at CTs: 2, 6, 10, 14, 18 and 22. At each time point the number of animals sacrificed was n s 4–6. 2.2. Perfusion and preparation of tissue At the designated CT, each rat was injected with an overdose of Na-pentobarbital Ž100 mgrkg bodyweight, i.p.., rapidly transferred to an adjacent room and then immediately prepared for IHC. Rats were perfused transcardially first with 150 ml of heparinized normal saline Ž0.9% NaCl., second with 300 ml of paraformaldehyde Ž4%. in 0.1 M phosphate buffer with picric acid Ž0.014%.. Brain was removed from the cranium, post-fixed in the same fixative for 8 h at 48C, soaked overnight in 20% buffered sucrose, frozen at y608C and then cut on a freezing microtome ŽMicrom. into 40 mm thick coronal sections throughout the entire rostrocaudal extent of the MBH and also of some other brain structures Žsee below.. Adjacent sections were divided in two series. The first series was processed for Fos-IHC. Sections of the second series were immunostained for b-END Žor ACTH. or for both Fos and b-END Žor both Fos and ACTH. antigens.

2.3. Immunohistochemistry Fos-IHC was performed using the commercially available polyclonal rabbit antibody Ab2 raised to the Ž4–17. synthetic peptide from the N-terminal end of Fos ŽOncogene Science.. This peptide has little homology with known Fos-related antigen ŽFRAs. and is presumed to be specific for the authentic Fos. Two antisera raised in the rabbit, respectively, against Ž1–27. b-END and ACTH peptides w7,54x, were used as markers for POMC-synthesizing neurons. Since they give rise to similar stainings only results obtained with the anti-b-END are shown here. Sections were first rinsed 3 times in PBS for 30 min per rinse to remove residual fixativersucrose. The same buffer was used in the following steps: PBS with 0.4% Triton. Sections were incubated with primary antibodies: Ži. the Ab2 was used at 1 : 2000 dilution for 24 h at room temperature ŽRT.; Žii. the b-END antiserum was used at 1 : 40 000 dilution for 48 h at 48C. After incubation with primary antibodies, sections were washed and then incubated with biotinylated goat anti-rabbit IgG antibody ŽJackson. at 1 : 2500 dilution for 2 h at RT. Tissues were rinsed and incubated with streptavidin–peroxidase complex ŽJackson. at 1 : 2000 dilution for 45 min at RT. Nickel-intensified diaminobenzidine ŽDAB, Sigma. was used as a chromogen according to Shu et al. w51x. Sections from different groups were mixed around and processed together for Fos-IHC with the same batches of reagents, in order to avoid generating artifactual differences. Within the single-stained sections, detection of Fos protein or b-END antigen appeared as black reaction products restricted, respectively, to the nucleus or to the cytoplasmrprocesses. Sections treated with DAB-Ni only were mounted on subbed slides, dehydrated, coverslipped and examined with a Leitz Aristoplan microscope. Controls for the specificity of Fos-IR consisted of deletion of the primary antibody or incubation of tissues in Ab2 that had been preabsorbed with synthetic peptide Ž4–17. Fos at a final concentration of 10y6 M. These procedures blocked all staining and particularly the Fos-IR seen within the MBH. The same result was obtained when omitting the biotinylated goat anti-rabbit IgG or the streptavidin–peroxidase complex. In the same manner the specificity of b-END staining was controlled. In the dual-staining technique b-END-IHC was initiated after the Fos-IHC, just requiring a change of the chromogen for the second step: 3-amino-9-ethylcarbazole ŽAEC, Sigma. was used for b-END visualisation. FosrbEND-positive neurons were easily distinguishable for their

Fig. 1. Coronal sections through the rostral MBH stained for Fos ŽA–C. and b-END ŽD. antigens of adult male rats sacrificed at CT 18 ŽA., CT 22 ŽB. and CT 6 ŽC and D.. It is evident that Fos-IR is almost totally absent within the MBH at lights off ŽCT 18; A., strongly induced 4 h after dark onset ŽCT 22; B. and intermediate at the lights-on time point on the following day ŽCT 6; C.. The arrowhead in A points to some strong Fos-immunopositive nuclei located dorsally to the MBH, thus providing evidence that the absence of such labelled nuclei within the MBH at CT 18 is not the result of a decreased sensitivity of the staining procedure. The distribution pattern of MBH Fos-positive nuclei in C is very similar to that established for b-END-containing neurons ŽD.. Asterisks third ventricle; ME s median eminence; MBHs mediobasal hypothalamus. Magnification: =100.

A.K. Jamali, G. Tramur Brain Research 771 (1997) 45–54

47

A.K. Jamali, G. Tramur Brain Research 771 (1997) 45–54

48

black nuclei and red cytoplasms. Specificity of the double staining was also evaluated. Specifically, omission of the b-END antiserum or its preabsorption with the synthetic Ž1–27. b-END peptide did not result in any alteration of the first image obtained with the Fos antibody. Furthermore, no cross-reactivity due to the use of two rabbit antisera was noted since the two antigens visualized were localised in different compartments of the cell.

2.4. QuantitatiÕe and statistical analysis In the single-stained preparations, counts of Fos- or b-END-immunoreactive neurons were performed on consecutive sections. In the double-stained preparations, Fospositive neurons were quantified on the same sections both before and after b-END immunostaining. In all cases, counts were obtained from camera lucida drawings of the MBH of 4–6 rats per group Žmagnification used for the single-stained sections: 100 = ; for the double-stained sections: 250 = .. For each rat, the MBH area was divided into 3 arbitrary rostrocaudal subdivisions of equal length Žinteraural coordinates according to the atlas of Paxinos and Watson w40x: rostral MBH: 7.2–6.4 mm; middle MBH: 6.6–5.8 mm and caudal MBH: 5.8–5.0 mm.. Results are given as the mean number" S.D. of Fos-, b-END- or both Fosrb-END-positive neurons per section per subdivision. At least three sections per subdivision were bilaterally quantified. Statistical analysis was performed using oneway ANOVA Žanalysis of variance. followed by the Scheffe F-test.

3. Results 3.1. Fos expression within the MBH Using a polyclonal rabbit Fos antibody, we found that nuclear Fos immunoreactivity ŽFos-IR. was spontaneously expressed within a specific hypothalamic area including the caudal part of the retrochiasmatic nucleus ŽRCN., the arcuate nucleus ŽAN. and the peri-arcuate area ŽPAA.. We refer to these brain structures, collectively, as the mediobasal hypothalamus ŽMBH, i.e. the area lying between the caudal part of the RCN and the premammillary recess.. Fos-IR was particularly induced in all animals sacrificed during the dark period of the cycle ŽFig. 1 and Table 1.. In fact, this spontaneous Fos-IR was intense in all the rats killed at the CTs 22, 2 and 6. The maximum labelling of Fos protein was seen in the rostral third of the MBH. Across the light period of the cycle ŽCTs 10, 14 and 18., the MBH area was very lightly stained for Fos ŽFig. 1 and Table 1.. 3.2. Comparison of the distribution of Fos and POMC within the MBH b-END antiserum, used as a marker for the POMC population, resulted in the staining of numerous neurons distributed throughout the entire rostrocaudal extent of the MBH area and also of dense intra-arcuate and arcuatofugal innervations. b-END-immunoreactive neurons were largely localized in an area including the caudal part of the RCN, the AN and its immediate basolateral vicinity. This pattern

Table 1 Expression of Fos and b-END immunoreactivities in the male rat MBH throughout the CC Circadian time of sacrifice

10 14 18 22 2 6

b-END

Fos Rostral

Middle

Caudal

Rostral

Middle

Caudal

11.2 " 3.5 14.3 " 6.1 20.3 " 6.8 115.6 " 14.5 Ž F s 23.71. a 114 " 14.5 Ž F s 22.89. a 80 " 15 Ž F s 9.28. a

12 " 3.5 6"1 12.6 " 4.5 82 " 10.7 Ž F s 34.04. a 67 " 7.5 Ž F s 20.51. a 44 " 8.1 Ž F s 6.82. a

9.5 " 3.6 6.3 " 2 11 " 1.7 53 " 8.1 Ž F s 15.01. a 43 " 9.1 Ž F s 8.71. a 42.3 " 7 Ž F s 8.35. a

165.3 " 17.2 145 " 17.3 185 " 18 111.6 " 12.5 Ž F s 4.79. a 108.6 " 16.6 Ž F s 5.1. a 188.3 " 25.6 Ž F s 0.01. NS

131.6 " 29.2 135.6 " 15 164.3 " 14.6 88.3 " 13 Ž F s 5.22. a 113.6 " 10.5 Ž F s 2.32. NS 183.3 " 20.2 Ž F s 0.32. NS

120.3 " 20.7 121.6 " 23.7 140.3 " 14.2 83.3 " 9.6 Ž F s 3.01. NS 103.3 " 13.6 Ž F s 1.27. NS 146 " 21.5 Ž F s 0.03. NS

Quantitative analysis of Fos and b-END IRs in the MBH of male rats throughout a 12 : 12 LD cycle Žlight-on was at CT 6.. Counts were obtained from camera lucida drawings of the MBH of 4–6 rats per group. For each rat the MBH area was divided into 3 rostrocaudal subdivisions of equal length. Results are given as the mean number Ž"S.D.. of Fos- or b-END-positive neuronsrsection. Three sectionsrsubdivision were quantified for the estimation of the level of Fos expression and their respective adjacent sections were used for the estimation of the number of b-END-positive perikarya. The results show that Fos-IR is only occasionally detected within the MBH during the light phase of the cycle ŽCTs: 10, 14 and 18., but is dramatically increased during the dark phase of the cycle ŽCTs: 22, 2 and 6.. In contrast to the circadian evolution of Fos-IR within the MBH, the number of b-END-positive perikarya is maximal throughout the light phase of the cycle ŽCTs: 10, 14 and 18. but is, at least in the rostral MBH, dramatically decreased in the early and the middle stages of the dark phase of the cycle ŽCTs: 22 and 2.. At the end of the dark phase ŽCT 6., the level of b-END expression returns to the diurnal level. F is given for the Scheffe F-test. a Indicates a significant difference from the CT 18 group, P - 0.05. NS s non-significant.

A.K. Jamali, G. Tramur Brain Research 771 (1997) 45–54

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Fig. 2. Coronal sections through the rostral MBH stained for Fos ŽA. and for both Fos and b-END antigens ŽB–F. from rats sacrificed at CT 22. ŽA–D. represent photomicrographs of the same section successively stained for Fos ŽA. and then for b-END ŽB, which is also magnified in C and D.. The major part of the Fos-positive nuclei shown in ŽA. also display b-END-immunoreactive cytoplasms in B while only a very few are negative. Conversely, a large number of b-END-positive neurons also show Fos-positive nuclei. At a more rostral level, F represents a high-power magnification of doubly stained neurons framed in E. Arrowheads, open arrows and full arrows illustrate, respectively, Fosqrb-ENDq, Fosyrb-ENDq and Fosqrb-ENDy stained neurons. Asterisks third ventricle; ME s median eminence. Magnification: =95 ŽA.; =100 ŽB.; =170 ŽC and D.; =150 ŽE. and =120 ŽF..

50

Table 2 Co-expression of Fos and b-END immunoreactivities in the male rat MBH throughout the CC Circadian time of sacrifice

10"2.5

Middle

Caudal

Fosrb-END

Rostral

Middle

Caudal

Rostral

Middle

Caudal

9.2"3.4

6.7"2.9

141.3"17

111.3"18.6

116.3"24.7

5"1

4.6"2

141"19

123.3"12.5

111.3"21.5

9.2"1.7 92%; 6% 8"3.6 83%; 5% 18"3.5 94%; 10% 101"12.1 Ž F s 42.38. a 96%; 98% 80.3"9 Ž F s 23.84. a 95%; 84% 59.6"7.3 Ž F s10.59. a 96%; 34%

8.2"2.9 93%; 8% 5"2 92%; 3% 9.6"5 95%; 6% 69.3"8.1 Ž F s 38.13. a 93%; 79% 56"4 Ž F s 22.99. a 96%; 48% 37"7.5 Ž F s8.00. a 95%; 21%

6.7"2.8 90%; 6% 4.6"2.5 100%; 4% 8.6"3 95%; 6% 45.3"5 Ž F s19.17. a 95%; 57% 32.6"8 Ž F s8.21. a 84%; 23% 28"4.3 Ž F s 5.33. a 90%; 21%

14

9.6"3.7

18

16.5"6.2

10.7"3.5

10.5"3.8

178.3"17.5

156.6"14.3

135.6"18.2

22

105.6"12.5

66.6"5.5

47.6"6

103.6"10.5

78.3"8.7

78.3"7.6

115.3"10.5

92.3"11.5

2

84"9.6

50.6"7.7

38.6"5.5

6

61.6"7.2

38.6"6.5

31"5.2

95"9.8

172.3"20.8

173.6"17.6

131"22.6

Quantitative analysis of Fos-, b-END- and both Fosrb-END-positive neurons in the MBH of male rats throughout a 12 : 12 LD cycle. Counts Žmean number"S.D.. were obtained from camera lucida drawings of the MBH. The same 3 sectionsrsubdivision previously used for the quantification of the MBH Fos-positive nuclei were used for a subsequent b-END staining. Then, Fos-positive, b-END-positive perikarya and Fosrb-END-doubly stained neurons were quantified. The percentage of Fos-positive neurons which also express b-END Žfirst percentage. and the percentage of b-END perikarya that express Fos Žsecond percentage. are given in the last 3 columns. The results show that the nocturnal induction of Fos expression almost exclusively occurs in b-END neurons. It may also be noted that a large population of b-END IR neurons are also positive for Fos. However, not all b-END neurons express Fos. F is given for the Scheffe F-test. a Indicates a significant difference from the CT 18 group, P - 0.05.

A.K. Jamali, G. Tramur Brain Research 771 (1997) 45–54

Rostral 10

b-END

Fos

A.K. Jamali, G. Tramur Brain Research 771 (1997) 45–54

of distribution of b-END-IR closely agrees with that previously reported for POMC-containing neurons w20,24x. Comparative study of the distribution patterns of Fosimmuno-positive ŽIP. nuclei and POMC-IP perikarya revealed their obvious neuroanatomical overlap throughout the MBH. Thus, both IRs were seen in the caudal part of the RCN and across the entire rostrocaudal extent of the AN and its vicinity ŽFig. 1, Tables 1 and 2.. Our results also revealed that the number of b-END-immunoreactive neurons observed during the light period is dramatically decreased 4 h after the onset of the dark period ŽTable 1.. Although this decrease was seen in all of the rostrocaudal subdivisions of the MBH, it was more pronounced in its rostral third, the area that also shows the highest number of Fos-labelled nuclei. Furthermore, the results also showed that the MBH pool of b-END-IP perikarya seen across the light period is reconstituted in the late stage of the dark period, when Fos-IR has significantly decreased within the corresponding areas. 3.3. Fos and POMC colocalization within MBH neurons Dual-staining techniques against both Fos and b-END antigens were performed in all rats sacrificed throughout the 24 h cycle. This double staining resulted in a decrease of the number of both Fos- and b-END-positive neurons as compared to the corresponding singly stained sections. However, this decrease remained moderate, probably because quantifications on singly and doubly stained sections were made under, respectively, 100 = and 250 = magnification. Then, some faintly Fos-labelled nuclei which were not counted under low magnification, became visible under high magnification. In any way, this double staining showed that, in the MBH, the majority of unambiguously identified Fos-positive neurons were also clearly showing b-END-positive cytoplasm ŽFig. 2.. Thus, at all the CTs studied, nearly 82–100% of the MBH Fos-immunoreactive neurons were clearly identified as b-END-producing neurons ŽTable 2.. However, 0–18% of Fos-positive neurons did not exhibit b-END-IP perikarya. Conversely, not all of the b-END-positive neurons contained Fos-IR. Maximum number of neurons colocalizing both Fos and b-END antigens was seen in the rostral MBH as compared to its middle and caudal parts.

4. Discussion 4.1. Our Fos-IHC reflects the known circadian fluctuation of Fos protein synthesis throughout the rat brain In all the rats examined, irrespective of the stage of the CC, a small number of Fos-positive nuclei were seen in the midline thalamus, the zona incerta, the piriform and sensorimotor cortices as well as in the amygdalar complex. This observation indicates that the sensitivity of the staining

51

procedure remained comparable across the entire experiment. However, Fos-IR within these areas was noticeably stronger during the earliest activity period ŽCTs 22 and 2. than that of the other times studied Žnot illustrated.. This is in good agreement with previous studies that investigated the circadian oscillation of c-fos mRNA expression in the rat brain under basal conditions, throughout a 12 : 12 LD cycle w14,15x. Thus, using Northern blot analysis, these studies showed that, in the major brain structures Žincluding the diencephalon which includes the MBH area., the highest circadian level of c-fos mRNA expression was reached during the earliest activity period. This concordance in the results suggests that our Fos-IHC reflects a physiological circadian oscillation of Fos protein synthesis within the rat brain. 4.2. c-Fos, Fos-related antigens (FRAs) or both? When examining brain Fos-IR, special attention was given to the caudate putamen ŽCP., dentate gyrus ŽDG., supraoptic nucleus ŽSON. and the suprachiasmatic nucleus ŽSCN.. All these areas are known to display large fluctuations Žexcept for the SON which maintains a high but constant level. in the circadian level of FRAs expression as was previously revealed by the use of a polyclonal rabbit antibody raised to the synthetic M-peptide, a highly conserved region in Fos and FRAs w25x. However, our Fos-IHC showed only occasional labelling within these structures across the CC Žnot illustrated.. This indicates that, in the present study, there is little likelihood of FRAs detection in the MBH. Moreover, preabsorption of the polyclonal antibody used in our study with the Ž4–17. Fos synthetic peptide Ža peptide sequence not shared with FRAs. blocked all staining. Taken together these observations suggest that our methods very likely detect the authentic Fos Žthe transcription factor c-Fos, i.e. the product of the c-fos proto-oncogene.. 4.3. Fos expression shows a robust daily rhythm within MBH POMC neurons This study clearly demonstrates that the c-fos protooncogene product, Fos phosphoprotein, is rhythmically and spontaneously produced within the MBH of the young adult male rat. Indeed, throughout the light period, Fos-IR was almost totally absent in the MBH. This lack of detectable Fos expression provides evidence that our methods were not detecting either the basal Fos protein Žconstitutive neuronal Fos. or some other constitutive antigen which could be permanently expressed within this brain area. This observation also shows that, under our experimental conditions, handling, induction of anesthesia and preparation of animals for perfusion cannot be considered as responsible for the induction of Fos-IR in the MBH. This is rendered even more likely since these procedures were performed too rapidly Ž10 min. to enable the induc-

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A.K. Jamali, G. Tramur Brain Research 771 (1997) 45–54

tion of Fos protein synthesis, which is known to require a minimum time of 45 min w9,36,48x. In the same context, all the rats were left undisturbed and access to their room was prohibited for at least 48 h before sacrifice. This delay is thought to be sufficient to prevent stress-induced Fos-IR w48x. On the other hand, during the dark period of the cycle, transcription factor Fos is strongly expressed within the MBH area. In fact, Fos expression is dramatically induced 4 h after the light offset, still detectable 8 h after the light offset but noticeably decreased at the time of the light onset on the following day. As expected, nearly 90% of the Fos-expressing neurons were also showing b-END-IR. This high percentage of colocalization clearly indicates that, in the MBH area, the circadian nuclear binding of Fos occurs specifically within POMC neurons. The results also showed that the nocturnal induction of Fos expression occurs, almost exclusively, within hypothalamic POMC neurons which were not characterised by a total cytoplasmic depletion of the b-END peptide. Such depletion is suggested by the nocturnal decrease of the number of b-END-immunoreactive neurons. However, as demonstrated by the double-staining technique, a large number of Fos-IP neurons was not concerned with this hypothetical total b-END depletion. One plausible explanation is that, at the studied times, the b-END-positive Fos-expressing neurons were in fact recently activated POMC neurons Ž1–2 h previously.. Thus, the apparent-total cytoplasmic b-END depletion could have occurred either only in POMC neurons which had already expressed Fos Žup to 3–4 h, since Fos expression is known to be transient. or in a subpopulation that never expresses Fos. 4.4. Functional considerations The c-fos proto-oncogene is known to operate Žvia its protein product, Fos. as an important element in the stimulus–transcription coupling cascade w13,16,36,39,43,52,59x. In neurons, its induction might require extracellular receptor activation and induction of second messengers w5,34,35,38,44x. Similarly, the spontaneous nocturnal increase of Fos expression within hypothalamic POMC neurons, reported here, might reflect the occurrence of cyclic changes in neurophysiological events at the membrane level andror modifications of gene expression at the nuclear level. In fact, a previous study that investigated, by means of in situ hybridization, the spontaneous oscillation of the hypothalamic POMC mRNA expression throughout the 12 : 12 LD cycle, reported a dramatic increase in POMC mRNA levels 6 h after the light offset w53x. Besides this, other authors have demonstrated the existence of an AP1 site on the POMC gene promoter in the corticotrope AtT20 cell line w2,3x. However, many questions have to be answered to determine the transcriptional modulating effect of Fos on POMC gene expression within the hypothalamic POMC neurons Žis the AP-1 site functional in these

neurons?, which memberŽs. of the Jun family w26,27,46x does Fos colocalize and dimerize with?.. Our data also suggest the existence of a possibly cyclic modification in the release of POMC-related peptides at the target structureŽs. of hypothalamic POMC neurons throughout the CC. Indeed, the nocturnal decrease in the number of b-END-positive perikarya, reported in this paper, presumably might reflect axonal exportation andror increased-activity release of b-END peptide within specific hypothalamic POMC subpopulationŽs.. This is rendered even more likely since previous studies have reported a positive correlation between Fos induction and neuronal firing under specific experimental conditions w29x. To our knowledge this is the first demonstration that under physiological conditions, transcription factor Fos is expressed spontaneously and cyclically within a large population of hypothalamic POMC neurons, an observation which is compatible with the suggestion that these neurons play a critical role in the mediation of neuroendocrine andror behavioral rhythmŽs.. Moreover, our results enable the use of c-fos expression as a tool to study the pharmacological effects of drugs on the neuronal activity of POMC neurons and to investigate the potential therapeutic value of this strategy in the treatment of a number of psychiatric disorders. Acknowledgements We would like to thank Dr. T. Durkin for his helpful comments on the manuscript and for correcting the English. We are also grateful to Mr. M. Chaigniau for his excellent photographic assistance. A part of this study was presented as an abstract at the 2nd Meeting of the European Society for Neuroscience, 24–28 September 1996, Strasbourg, France. References w1x H. Akil, S.J. Watson, b-Endorphin and biosynthetically related peptides in the central nervous system, in: L. Iversen, S.D. Iversen, S.H. Snyder ŽEds.., Handbook of Psychopharmacology, Vol. 16, Plenum, New York, 1983, pp. 209–253. w2x A.L. Boutillier, D. Monnier, D. Lorang, J.R. Lundblad, J.L. Roberts, J.P. Loeffler, Corticotrophin-releasing hormone stimulates proopiomelanocortin transcription by c-Fos-dependent and -independent pathways: characterization of an AP1 site in exon 1, Mol. Endocrinol. 9 Ž1995. 745–755. w3x A.L. Boutillier, P. Sassone-Corsi, J.P. Loeffler, The proto-oncogene c-fos is induced by corticotropin-releasing factor and stimulates pro-opiomelanocortin gene transcription in pituitary cells, Mol. Endocrinol. 5 Ž1991. 1301–1310. w4x R.K.W. Chan, P.E. Sawchenko, Hemodynamic regulation of tyrosine hydroxylase messenger RNA in medullary catecholamine neurons: a c-fos-guided hybridization histochemical study, Neuroscience 66 Ž1995. 377–390. w5x A.J. Cole, D.W. Saffen, J.M. Baraban, P.F. Worley, Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation, Nature 340 Ž1989. 474–476.

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