Immunohistochemical studies of noradrenergic-induced expression of c-fos in the rat CNS

Brain Research, 592 (1992) 57-62

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© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

BRES 18118

Immunohistochemical studies of noradrenergic-induced expression of c-fos in the rat CNS G u o y i n g Bing, Eric A. Stone, Yi Z h a n g and David Filer Department of Psychiatry, New York UniversitySchool of Medicine, New York, NY 10016 (USA) (Accepted 5 May 1992)

Key words: c-los; Noradrenergic system; Immunohistochemistry; Stress;/3-Adrenoceptor

Previous studies have shown that stimulation of adrenergic receptors in the rat brain causes increased levels of mRNA of the immediate early gene, c.fos. The present studies were undertaken to determine if this stimulation also induces increased levels of c-los immunoreactivity in the central nervous system (CNS). Rats were treated with the alpha-2 adrenoceptor blockers, yohimbine or atipamezole, or with restraint stress to activate central noradrenergie activity and were perfused 2 h later for immnnohistochemical analysis of the cerebral cortex. Yohimbine, atipamezole and restraint stress each was found to cause increases in e-los-like immunoreactivity (c.fos-li). Western blot analysis revealed increased c-los protein in the cortex after yohimbine treatment. The c-fos-li response to yohimbine was blocked by prior administration of the beta receptor antagonist, dl-propranolol, and to a lesser degree by the alpha-I antagonist, prazosin. It is concluded that adrenergic receptor stimulation in the cortex causes increased production of c-los or los related antigens and that this (these) immediate early gene product(s) may play a role in noradrenergic function in the CNS.

INTR'3DUCTION

The c.fos gene codes for a protein (c-fos) that, together with c.jun, serves to regulate the transcription of genes having the AP-I consensus element. This gene is a member of a group of immediate early genes (lEGs), which have, among other characteristics, the ability to be activated independently of protein synthesis by various neurotransmitters, hormones and growth factors 8'27. IEGs may serve a variety of functions in mediating genomic responses to extracellular stimu-

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Recently, investigators have shown that the transcription of a number of lEGs including c-fos, nur77, zif-268, tis-7 and t/s-21 in the rat cerebral cortex is under the control of beta and alpha-] adrenergic receptors 5'~3. The mRNA of these genes can be markedly stimulated by drugs or stressors which release brain norepinephrine (NE) onto these receptors 14. The present study was undertaken to determine if there is a corresponding increase in the translation of this message into protein. To this end we have conducted

immunohistochemical studies of the product of one of these genes, c-Jbs, in the brains of rats after activation of the noradrenergic system. The latter was accomplished by the use of blockers of inhibitory presynaptic alpha-2 receptors (yohimbine or atipamezole) or by exposure to a mild stre:;s (restraint). MATERIALS AND METHODS

Behat'ioral Male Sprague-Dawley rats (180-200 g, Taconic Farms) were used. The animals were brought to the laboratory two days prior to the experiment and housed two per cage. They were gently handled for 1 rain on the day prior to the experiment. We have found in previous work that these housing and handling procedures yield reliably low baseline levels of c.fos mRNA in the rat cerebral cortex (Stone, Bing and Filer, unpublished observations). The rats were injected with yohimbine (5 mg/kg), atipamezole (5 mg/kg), propranolol (10 mg/kg) and/or prazosin (5 mg/kg). All injections were i.p. and were given rapidly by two people so as to minimize the stress to the rat. For stress experiments the animals were restrained in cylindrical cages (6x 15 cm) for 1 h.

lmmunohistochemistry All rats were anesthetized with pentobarbital-(70 mg/kg) 2 h after injection or stress and were perfused transcardially with 50 ml

Correspondence: E.A. Stone, Psychiatry - TH HN 510, New York University Medical Center, 550 First Ave., New York, NY 10016, USA. Fax: (i) (212) 263-5740.

58 of ~line followed by 2(10 ml of 4% paraformaldehyde. The 2 h interval was chosen because previous investigations have revealed a strong induction of c-los.like iraraunoreactivity (c-.fos-li) at this time after a variety of c-los raRNA inducing agents ~'21''Z''~. Brains were removed and 30 ~tra coronal frozen sections cut on a sliding knife raicrotome. The sections were collected in a cryoprotective solution, iraraunohistocheraical staining for c-los was carried out via the avidin-biotin procedure, in brief, the sections were first incubated for 30 rain in 10% normal serum plus 1% BSA in PBS to block non-specific binding and then were incubated overnight at 4~C with rabbit polyclonal c-los antiserum (Oncogene Science, 1:2,000 dilution). For control purposes the sections were either incubated with antiserum that had been exposed to saturating levels of the c-los peptide (preabsorbtion control) or incubated in the absence of the primary antibody. The sections were then incubated with a secondary antiserum (Vector) for 60 rain and subsequently reacted with avidin-biotin complex (ABC) (Vector), The peroxidase reaction was developed in a chromagen solution containing 100 raM nickel sulfate, 125 mM sodium acetate, l0 raM imidazole, 0,03% diarainobenzidine (DAB) and 0.01% hydrogen peroxide at pH 6.5. The sections were then mounted and photoraicrographed.

Western blot analysis This analysis was performed using the procedure of Towbin et al? a with several modifications. Briefly, cerebral cortex was dissected on ice immediately after decapitation, The tissue was dispersed with a polytron and dissolved in suspension buffer (0,1 M NaCI, 0.01 M Tris-HCI (pH 7.6), 0,l}01 M EDTA, I p.g/ral aprotinin and 100 #g/ral phenylmethylsulfonyl fluoride), Protein concentration was measured with the Coumassie Blue reagent. The protein samples were loaded onto a 7.5% SDS-acrylaraide gel and run with a Bio-Rad mini electrophoresis systera for 45 rain at 2(10 V, The proteins were then transferred frora the gel to a nitrocellulose filter using the Bio.Rad mini Trans-Blot apparatus at 100 V for I h, The nitrocellulose membrane, after air dwing, was incubated with apprnprialely diluted primary lmtiscrum overnight. The membranes were then Iransferred to a solution of the secondary antibody which had been conjugated with ;,Ikaline phosphatase (Vector), Color was devel()ped by subsequent re,orion with ttlkaline phos. phaluse substrate solution.

Counting of c.fos stained cells All individual nucle,r staining cells of each of 6 sections per

rat were counted manually on a grid objective t,nder the microscope. The area included all tissue in the superior right quad. rant at tile levelof the anterior commissure,Coordinates for the cell counting were 0-2 ram anterior to Bregma, !-4 ram late[~l to midline and 2,5 ram from the plal surface, All of the counting was performed by a person who was unaware of the animals' treatments, The total numberof los positivecells in each experiment was derived by the Abercrombiemethodt, The results were analyzed by ANOVA followedby the Newraan-Keulscomparison method. cortex per

RESULTS

Western blot analysis A Western blot for c.fos antiserum using cortex from a control rat or a rat treated 2 h earlier with yohimbine is shown in Fig. 1. As can be seen, a single band of antigen was detected at the molecular weight of c-fos (62 kDa) for both the noninjected and yohimbine injected rats. The yohimbine treated rat showed a darker band than the control. No staining was found at the weights of los related antigens (35 and 46 kDa). Similar results were obtained in an additional 2 control and 2 yohimbine treated rats.

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97K 66KP45K Fig. 1. lmmunoblot (Western) analysis rabbit polyclonal c-los antibody (Oncogene Science) in cortex of control and yohimbine treated rat. Lanes 1 and 2 from representative control rat (undisturbed), lanes 3 and 4 from representative yohimbine treated (5 rag/kg, 2 h) rat.

c-los immunoreactivity in brain Immunohistochemicai analysis revealed the presence of c-fos-li in the cortex of both control and yohimbine treated rats (Fig. 2). The staining was in all cases nuclear as has been previously reported for this protein 9. Preabsorbtion of the antiserum with the c-los peptide to which the antiserum had been raised resulted in a total abolition of staining (Fig. 2D) as did omission of the primary antiserum (Fig. 2B). Yohimbine produced a marked increase in c-fos reactivity in the cortex. Cell counts of positively stained nuclei were as follows (mean and S,E.M., n = 5 - 6 rats): noninjected control, 6.9 + 4.6; yohimbine, 1157 + 199, P < 0.001 vs. control. A similar increase was seen in the locus cocruleus (Fig. 3): control, 0.8 +0,5; yohimbine, 93.6 ± 20.7, P < 0.001 vs control. Yohimbine also markedly increased c.fos.li in a variety of other subcortical structures including the paraventricu. lar nucleus, Islands of Calleja and supraoptic nucleus but no attempt to quantify these subcortical increases were made in the present experiment. To estimate the degree to which the stress of injection itself contributed to this effect, two rats were injected with saline and processed as above for c-los. Compared to the yohimbine treated rats, these animals showed very little cortical and LC staining (cell counts: frontal cortex, 10.5 ± 1.5; locus coeruleus, 2.0 :I: 1.0). Two other agents for releasing brain NE, atipamezo!e and restraint stress (I h) produced similar increases in cortical c-fos-li at 2 h, Cell counts were as follows: atipamezole, 726 + 208, n = 7, P < 0.01 vs. control; restraint stress, 427.5 + 77.4, n = 5, P < 0.001 vs. control. To determine whether the response to yohimbine or restraint stress was mediated by adrenergic receptors animals were pretreated with either propranolol a n d / o r prazosin. The blocking agents were given as separate injections I0 min prior to yohimbine with the yohimbine alone group receiving an injection of saline at this time. Representative animals are shown in Fig.

59 4. It can be seen that propranolol greatly reduced the c-fos-li whereas prazosin had a smaller inhibitory effect. Cell counts were as follows (all n's = 6) yohimbine alone, 1157 + 199, yohimbine + propranolol, 15.2 + 5.1 ( P < 0.001 vs. yohimbine); yohimbine + prazosin, 767.2 + 276.2 (NS vs. yohimbine); yohimbine + propranolol + prazosin (27.5 + 13.4 ( P < 0.001 vs. yohimbine). In the restraint stressed rats, only propranolol was tested.

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The antagonist produced a similar inhibition in the frontal cortex which was not as great as that seen with yohimbine (restraint alone, 427.5 + 77.4; restraint + propranolol, 67.3 + 17.1, P < 0.01, t-test). DISCUSSION Previous studies have shown that stimulation of central adrenergic receptors by injection of alpha-2 recep-

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Fig. 2. Fos immunoreactivity in various cortical regions of the rat brain after yohimbine injection (5 mg/kg, 2 h) or control (no injection). A: piriform cortex in yohimbine rat, arrowheads indicate lateral olfactory tract. B: section corresponding to A incubated without primaryantibody. C: frontal cortex of yohimbine treated rat. D: preabsorbtion control corresponding to section C (antiserum preincubated with fos pcptide). E: entorhinai cortex and amygdaloid nucleus (arrowhead) in yohimbine treated rat. F: section corresponding to E in noLl-injected control rat. Bar = 500/zm (in all figures).

60

sponse and prazosin partially blocked it is similar to the effects of these blocking agents on the cyclic AMP response to NE in this brain region 7'~. This suggests that, like the case for cyclic AMP, the c-los response to yohimbine is mediated predominantly by beta receptors and is modulated by alpha-1 receptor stimulation. The similarities of the two responses suggests that in the cortex cyclic AMP may be involved in the genera-

Fig. 3. los reactivity in locus coeruleus of yohimbine-treated rat.

tor blockers increases the level of c-fos mRNA in the rat cortex 5''3. The present studies show that this change is accompanied by increased levels of c-fos-li. Treatment with three agents that stimulated brain norepinephrine (NE) release, yohimbine, atipamezole and restraint stress all led to marked increases in c-los immunoreactivity in the cortex. The protein response was localized to the cell nucleus as has been reported previously for other c-fos inducing agents '~.:~,2.~. An increase in binding to c-fos protein after yohimbine was also detected in Western blots from the cortices of these animals. Blockers of noradrenergic receptors, propranolol and prazosin produced reductions in cortical c-fos-li. In agreement with previous results on mRNA responses, the protein response to yohimbinc was primarily mediated by beta adrenoceptors .~,L~.Propranolol also significantly attenuated the c-Jbs.li response of the cortex to restraint stress. The increase in c.fos.li after yohimbin¢ could reflect c-fos protein or los related antigens. Both types of proteins have been observed to be elevated at 2 h after a number of c-fos-indueing pharmacological agents "~''~~''¢' 2~. The Western blot analysis showed an increase in binding to c.fos protein which is suggestive that the latter protein is involved. However, it is not known whether the data from the Western analysis can be extrapolated to the different conditions of the immunohistochemical assay. For thi~ reason the more conservative conclusion, that noradrenergic beta transmission in the cortex produces an increase in c-fos-li is being made in this study. The present Western analysis did not detect fos related antigens in the cortices of these animals, The latter proteins have been reported to be present in the cortices of untreated rats ~.2~, The reason for this discrepaacy is not known at present, The pattern of adrenergic blockade found above in whiO propranolol totally blocked the yohimbine re-

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g Fig, 4, Effects of propranolol (10 mg/kg)and prazosin (5 mg/kg)on the c-fos-li response to yohimbine (5 mg/kg) in various regions of CN$, Rats were injected with blocking agents or saline 10 min before receiving yohimbine, Saline plus yohimbine (A), prazosin + yohimbine (B), propranolol + yohimbine (C),

61 tion of the c-fos response as has been reported previously for other tissues 4'24. Previous studies by Gubits et al. 13 and by our group 5 have shown that the simple stress of handling and i.p. saline injection is sufficient to significantly elevate c-los mRNA in the rat cortex although to a lesser degree than yohimbine injection. In the present study saline injection stress had a relatively trivial effect on c-fos-li compared to that of yohimbine. This suggests that for injection stress there may be some dissociation between increases in mRNA and protein production. Yohimbine can affect other monoaminergic systems in the brain including the serotonergic and dopaminergic 2°. This raises the possibility that the latter systems contributed to the above effect. This is unlikely for several reasons. First, the beta adrenoceptor blocker, propranolol, produced a virtually complete inhibition of the cortical response to yohimbine. Second, a more specific alpha-2 adrenergic blocker, atipamezole, which has no direct actions on serotonergic or dopaminergic receptors "~4 produced results similar to those of yohimbine in this brain area. Third, we have recently shown that direct infusion of norepinephrine into the cerebral cortex via a microdialysis probe induces a similar local increase in c-los immunoreactivity "~2. And fourth, in agreement with the findings by others with agents that activate LC units 6'~t't4 there was an elevated c.fos.li in the LC after yohimbine in the present experiment, and we have recently found that lesion of this nucleus markedly attenuates the c.fos immunoreactive response to yohimbine .~'. In summary, therefore, the pre~ent results show that the increase in c.fos mRNA caused by noradrenergic stimulation in the cerebral cortex is accompanied by a similar rise in c-fos-li. The c-los gene has been suggested to be involved in a variety of neuronal functions including the activation of enzymes for neurotransmitter synthesis t2'3° and processes involved in trophic mechanisms and learning 2'~s'35. The present results support the hypothesis that c-fos or related proteins may be involved in the actions of the noradrenergic system on these processes. Acknowledgements. Supported in part by Grants AFOSR 89-0208, MH45265 and MH08(,i8.

REFERENCES 1 Abercrombie, M., Estimation of nuclear population from microtome sections, Anat. Rec., 94 (1946) 239-247. 2 Anokhin, K.V. and Rose, S.P.R., Learning-induced increase of immediate early gene messenger RNA in the chick forebrain, Eur. J. Neurosci., 3 (1991) 162-167.

3 Arenander, A.T., De Vellis, J. and Herschman, H.R., Induction of c-los and TIS genes in cultured rat astrocytes by neurotransmitters, J. NeuroscL Res., 24 (1989) 107-114. 4 Berkowitz, L.A., Riabowol, K.T. and Gilman, M.Z., Multiple sequence elements of a single functional class are required for cyclic AMP responsiveness of the mouse c-los promoter, Mol. Cell. Biol., 9 (1989) 4272-4281. 5 Bing, G., Filer, D., Miller, J.C. and Stone, E.A., Noradrenergic activation of immediate early genes in rat cerebral cortex, /Viol. Brain Res., 11 (1991) 43-46. 6 Ceccatelli, S., Villar, M.J., Goldstein, M. and Hokfelt, T., Expression of c-Fos immunoreactivity in transmitter-characterized neurons after stress, Prec. Natl. Acad. Sci. USA, 86 (1989) 9569-9573. 7 Daly, J.W., Padgett, W., Nimitkitpaison, Y., Creveling, C.R., Cantacuzene, D. and Kirk, K.L., Fluoronurepinephrines: specific agonists for the activation of alpha and beta adrenergic-sensitive cyclic AMP-generating systems in brain slices, J. Pi~armacol. Exp. Ther., 212 (1980) 382-389. 8 Dragunow, M., C-'urrie, R.W., Faull, R.L.M., Robertson, H.A. and Jansen, K., Immediate-early genes, kindling and long-term potentiation, Neurosci. Biobehav. Rev., 13 (1989) 301-313. 9 Dragunow, M. and Faull, R., The use of c-los as a metabolic marker in neuronal pathway tracing, J. Neurosci. Methods, 29 (1989) 261-265. 10 Dragunow, M., Robertson, G.S., Faull, R.L.M., Robertson, H.A. and Jansen, I¢', D2 dopamine receptor antagonists induce los and related proteins in rat striatal neurons, Neuroscience, 3"/ (1990) 287-294. 11 Fritschy, J.-M., Frondoza, C.G. and Grzanna, R., Differential effects of reserpine on brainstem catecholaminergic neurons revealed by los protein immunohistochemistry, Brabt Res., 562 (1991) 48-56. 12 Gizang-Ginsberg, E. and Ziff, E.B., Nerve growth factor regulates tyrosine hydroxylase gene transcript ion through a nucleoprotein complex that contains c-los, Genes De~,.,4 (1990) 477-491. 13 Gubits, R.M., Smith, T.M., Fairhurst, J.L. and Yu, H., Adrenergic receptors mediate changes in c.fos mRNA levels in brain, Mol. Brain Res., 6 (1989) 39-45. 14 Hayward, M.D., Duman, R.S. and Nestler, E.J., Induction of the c-J~s prolo-oncogene during opiate withdrawal in the locus couruleus :rod other regions of rat brain, Brain Res., 525 (1990) 256-266. 15 ttengerer, B., I.indholm, D., Heumann, R., Ruther, U. and Wagner, E.F., I.esion-induced increase in nerve growth fitctor mRNA is medial:ed by e.fos, Proc. Natl, Acad. Sei. USA, 87 (19911) 3899-3903. 16 Leblanc, G.G. und Ciaranello, R.D., AIpha-noradrenergic poten. tiation of neut,,transmitter.stimulated cAMP production in rat striatal slices, Brain Res., 293 (1984) 57-65. 17 Lira, R.W., Varnum, B.C. and Herschman, H.R., Cloning of tetracedanoyl ]zhorbol ester-induced 'primary response' se. quences and their expression in density-arrested Swiss 3T3 cells and a TPA non.proliferative variant, Oncogene, 1 (1987) 263-270. 18 Moccheti, I., De Bernardi, M.A., Szekely, A.M., AIho, H., Brooker, G. and Costa, E., Regulation of nerve growth factor biosynthesis b}' beta-adrenergic receptor activation in astrocytoma cells: a p~4ential role of c.fos protein, Prec. Natl. Acad. Sei. USA, 86 (1989~ 3891-3895. 19 Mugnaini, E., Berrebi, A.S., Morgan, J.I. and Curran, T., Fos-like immunoreactivity induced by seizure in mice is specifically associated with euehromatin in neurons, Eur, J. Neurosci., 1 (1989) 46-52. 20 Papeschi, R. and Theiss, R., The effect of yohimbine on the turnover of brain catecholamines and serotonin, Eur. J. Pharmacol., 33 (1975) 1-12. 21 Robertson, H.A., Peterson, M,R., Murphy, K. and Robertson, G.S., Dl-dopamine receptor agonists selectively activate striatal c-los independent of rotational behaviour, Bra#r Res., 503 (1989) 346-349. 22 Sagar, S.M. and Sharp, F.R., Light induces a Fos-like nuclear antigen in retinal neurons, Mol. Brain Res., 7 (1990) 17-21. 23 Sagar, S.M., Sharp, F.R. and Curran, T., Expression of c-los

62

24

26

27 28

29

protein in brain: metabolic mapping at the cellular level, Scieuce, 240 (1988) 1328-1331. Sassone-Corsi, P., Visvader, J.. Ferland, L., Mellon, P,L. and Verma, I.M., Induction of proto-oncogene los transcription through the adenylate cyclase pathway: characterization of a cAMP-responsive element, Genes Def., 2 (1988) 1529-1538. Sharp, F.R., Sagar, S.M., Hicks, K., Lowenstein, D. and Hisanaga, K., c.fos mRNA, Fos, and Fos-related antigen induction by hypertonic saline and stress, J. Neurosci., 11 (1991) 2321-2331. Sharp, J.W., Sagar, S.M., Hisanaga, K., Jasper, P. and Sharp, F.R., The NMDA receptor mediates cortical induction of los and los-related antigens following cortical injury, Exp. NeuroL, 109 (1990) 323-332. Sheng, M. and Greenberg, M.E., The regulation and function of c-los and other immediate early genes in the nervous system. Neuron, 4 (1990)477-485. Sonnenberg, J.L., MacGregor-Leon, P.F., Curran, T. and Morgan, J.l. Dynamic alterations occur in the levels and composition of transcription factor AP-I complexes after seizure, Neuron, 3 (1989) 359-365. Sonnenberg, J.L., Mitchelmore, C., Macgregor-Leon, P.R., Hempstead..I., Morgan, J.l. and Curran, T., Glutamate receptor

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agonists increase the expression of los, ffa, and AP-I DNA binding activity in the mammalian brain, J. Neurosci. Re~:, 24 (1989) 72-80. Sonnenberg, J.L., Rauscher 111, F.J., Morgan, J.l. and Curran, T., Regulation of proenkephalin by Fos and Jun, Science, 246 (1989) 1622-1625. Stone, E.A., Zhang, Y., Filer, D. and Bing, G., Effect of locus coeruleus lesion on c-los expression in the cerebral cortex caused by yohimbine injection or stress, submitted. Stone, E.A., Zhang, Y., John, S.M. and Bing, G., c-fos response to administration of catecbolamines into brain by microdialysis, Neurosci. Lett., 133 (1991) 33-35. Towbin, H., Strehelin, T. and Gordon, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA, 76 (1979) 4350-4354. Virtanen, R., Savola, J.-M. and Saano, V., Highly selective and specific antagonism of central and peripheral alpha 2-adrenoceptors by atipamezole, Arch. Int. Pharmacodyn., 297 (1990) 190-204. Wen, L., Huang, J. and Blackshear, P. Rat ornithine decarboxylase gene. J. Biol. Chem., 264 (1989) 9016-9021.