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In vivo competitive autoradiographic study of [3H]corticosterone and [3H]aldosterone binding sites within mouse brain hippocampus
.I. smoid Biochem. Vol. 28, No. I, pp. 29-34, 1987
Printed in Great Britain. All rights reserved
Copyright 0
0022-4731/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
IN v1VO COMPETITIVE AUTORADIOGRAPHIC STUDY OF [‘HICORTICOSTERONE AND [‘HIALDOSTERONE BINDING SITES WITHIN MOUSE BRAIN HIPPOCAMPUS MICHI?LE COUTARD, DOMINIQUE DUVAL INSERM
and MARY J. OSBORNE-PELLEGRIN
U7, HBpital Necker, 161 rue de S&es,
75015 Paris, France
(Received 30 October 1986)
Summary-The binding sites for [3H]corticosterone (‘HB) and [3H]aldosterone (‘HA) within the hippocampal area of the mouse brain have been studied by autoradiography in competition experiments. Excess
unlabelled aldosterone (A) or corticosterone (B) both abolished the nuclear accumulation of radioactivity within neurons observed after injection of either IHA or ‘HB. Experiments where a subcutaneous injection of a “pure glucocorticoid’ RU26988 was given before injection of ‘HA alone showed a marked accumulation of radioactivity within neuronal nuclei of the hippocampus suggesting the presence of ‘HA binding sites distinct from classical type II glucocorticoid receptors. In addition, when RU26988 was given before the injection of 3HA associated with a 30- or IOO-fold excess of either A or B, the cell nuclear accumulation of radioactivity was no longer observed. These results showed that in our in vivo experimental conditions, B displayed the same ability as A to occupy 3HA binding sites, supporting the view that in mouse hippocampal neuronal nuclei, the aldosterone-binding and corticosterone-preferring sites represent the same molecular entity.
INTRODUCTION
authors [ 19,221 reported that in hippocampal cytosol Results from previous in vivo autoradiographic B displayed a higher affinity than A for both ‘HB and studies in mouse and rat brains with labelled cortico- 3HA binding sites which were distinct from classical sterone (B) [l-1] and dexamethasone (DEX) [5-71 type II glucocorticoid receptors, others [23] showed showed that these glucocorticoids are preferentially that A and B have a similar affinity for these sites. In retained in cell nuclei from different brain areas. A addition, the latter group demonstrated that these slower rate of penetration into the brain of DEX [6] 3HA and 3HB binding sites display an identical may at least in part explain these observed differ- steroid specificity suggesting that they represent the ences. However, various experiments have suggested same molecular entity. the presence of two sets of glucocorticoid receptors In order to examine the hippocampal binding sites [g-12]. for 3HB and 3HA at a cell nuclear level and in an in Several autoradiographic studies have pointed out vivo situation, we have performed competitive autothe similarity of the distribution of the radioactivity radiographic experiments with these labelled steroids. within rat brain of the tritiated mineralocorticoid We have studied the distribution of the radioactivity aldosterone (A) [13-151 with that observed for la- in mouse hippocampal autoradiograms after injecbelled corticosterone, although within some areas of tion of 3HA and ‘HB alone or in presence of an the hippocampus the intensity of the labelling differed excess of unlabelled A or B. The synthetic steroid [15]. If, as suggested by most of the above-reported RU26988 (RU) has been previously shown to have an data, the hippocampus contains (at least) two distinct affinity similar to DEX for classical ‘HDEX glucoclasses of nuclear glucocorticoid receptors, one neur- corticoid receptors (171. In further experiments deonal B preferring and one glial DEX preferring, the signed to study the 3HA binding sites distinct from question arises as to whether the binding sites for A classical type II glucocorticoid receptors, we have and B in the hippocampal neuronal nuclei are the used RU as previously described by others [17]. same molecular entity or distinct receptors. BiochemAlthough RU has been reported to display in vivo ical studies on whole rat brain [16] and on hipposome glucocorticoid effects on enzymatic activity in campal cytosols [17-201 established the presence of an rat liver and brain [24] and on blood pressure [25], we aldosterone binding site coexisting with a gluco- have performed some biochemical studies to detercorticoid receptor. However, it has been reported that mine whether under our experimental conditions RU rat brain aldosterone receptors display a high affinity was able to cross the blood-brain barrier and occupy for corticosterone [21]. Results of further studies on hippocampal glucocorticoid receptors. Part of this the relative affinity of A and B for 3HA and 3HB study has already been published in abstract form binding sites are somewhat divergent. Whereas some 1261. 29
30
MIC&LE
COUTARD
EXPERIMENTAL
Animals and Reagents Mature female C57BL/6 mice were adrenalectomized 3 days before use and given a 0.9% NaCl solution to drink. [ 1,2-3 H] corticosterone (‘HB) (43 Ci/mmol) and [ 1,2-‘H] aldosterone (‘HA) (41 Ci/mmol) were purchased from Amersham-Searle (U.K.) and all non-radioactive compounds from Sigma, St Louis, except for RU26988 (11 fi,17fidihydroxy - 17a -propynylandrost - 1,4,6 - trien - 3 -one) which was generously supplied by Roussel-Uclaf (France). The day of the experiment mice were anaesthetized with Nembutal (40 mg/kg, i.p.) and were injected via the jugular vein with 100 ~1 of radioactive solution. Autoradiographic Studies Experiments with 3HB In this series of experiments, 21 mice were used. Five mice were injected with 20 PCi (about 5 x 10-‘Omol) of ‘HB alone, or with a lOO-fold excess (about 100 pg/lOO g b. wt) of unlabelled B or A. Three mice received ‘HB with a 30-fold excess (about 3Opg/lOOg b.wt) of A or B. Experiments with 3HA A total of 32 mice were used (4 for each experimental condition). Injection of ‘HA alone or with an excess of unIabelled A or B. Mice received 2OpCi (about 5 x lo-” mol) of ‘HA alone, or with a loo-fold excess unlabelled A or B. Subcutaneous injection of RU26988 1 h before the injection of3 HA alone or with an excess of unlabelled A or B. 5 x lo-‘mol (about 1 mg/lOO g b.wt) of RU26988 were injected subcutaneously 1 h before the i.v. injection of 5 x 10-‘Omol of ‘HA alone, or with a 30- or lOO-fold excess of A or B. Thirty minutes after steroid administration mice were killed by decapitation and the brain was quickly removed from the skull and frozen in liquid nitrogencooled isopentane. The thaw-mounted autoradiographic method used has been previously described [7]. Briefly, brain sections of about 10pm thickness were made by means of a cryostat (American Optical, type Cryocut) maintained at a temperature of - 18°C. The sections were transferred in the dark on to slides precoated with Kodak AR 10 stripping film emulsion. After about 4 months of exposure at a temperature of about - 20°C autoradiographs were allowed to reach ambient temperature and thereafter were plunged into methanol to fix the brain sections, rinsed with distilled water and then developed for 5 min with Kodak D19B and fixed in 30% (wt/vol) sodium hyposulphite. After being rinsed in distilled water, sections were stained with methyl green pyronin (Gurr’s reagent), rinsed, left to dry and mounted with Eukitt medium.
ef al
Biochemical Analysis The animals received a S.C. injection of either 5 x lo-‘mols of unlabelled RU26988 or the same volume of the diluent alone (control). One hour after injection 9-10 animals in each group were killed by decapitation. The brains were removed quickly and the hippocampal regions dissected and homogenized using a polytron in ice-cold medium (10 mM Tris pH 7.4 containing 1.5 mM EDTA, 0.5 mM DTT and 20mM sodium molybdate). The homogenates were then centrifuged for 60 min at 105,OOOg. Aliquots of the cytosol(250 pLO.4 mg protein) were incubated in the presence of increasing concentrations of tritiated corticosterone (final concentation 10 -’ to 5 x 1O-8 M). Non-specific binding was determined in a parallel set of samples incubated with a 500-fold excess of non-radioactive corticosterone. After a 4 h incubation at 4°C each sample was treated by 1 ml of Dextran-coated charcoal (0.5% Norit A, 0.05 Dextran T,,) for 15 min at 4°C. After 2 min centrifugation at 10,000 g, the protein bound radioactivity was determined by scintillation spectrometry and expressed as fmol of specific binding/mg protein. The number of binding sites and the affinity of the receptor for the radioactive ligand were determined according to Scatchard. Protein concentrations were measured according to Lowry using bovine serum albumin as standard. RESULTS
Autoradiographic Experiments Experiments with ’ HB In mouse brain, ‘HB and/or its metabolites accumulated within cell nuclei of the hippocampus (Figs la, b) as previously shown [4]. Autoradiograms from mice which received a concomitant injection of ‘HB and a 30-fold excess of unlabelled B showed no radioactivity concentrated within nuclei of hippocampal neurons (Fig. lc). The same results were obtained after the injection of ‘HB in the presence of a 30-fold excess of unlabelled A (Fig. Id). Experiments with 3HA Injection of ‘HA alone or with an excess of unlabelled A or B. Neurons from hippocampal areas in mouse brain accumulate ‘HA and/or its metabolites (Figs 2a, b). However, the intensity of the labelling appears to be lower than that observed after injection of ‘HB. This cell nuclear concentration of the radioactivity was no longer observed when )HA was injected with a loo-fold excess of either A (Fig. 2c) or B (Fig. 2d). S.C. injection of RU26988 1 h before the injection of ‘HA alone or with an excess of unlabelled A or B. After a S.C. injection of a lOOO-fold excess of RU, ‘HA and/or its metabolites concentrated within nuclei of the hippocampal neurons (Figs 3a, b). The intensity of the labelling appeared more marked than
[‘HIsteroid
autoradiography
in mouse
brain
Fig. 1. Autoradiograms from mouse brain hippocampus (cornu ammonis) after injection of [‘Hlcorticosterone (3HB). (a) Field CA, and (b) field CA, after injection of 3HB alone. (c) Field CA, after injection of ‘HB with a 30-fold excess of corticosterone. (d) Field CA, after injection of rHB with a 30-fold excess of aldosterone.
Fig. 2. Autoradiograms from mouse brain hippocampus after injection of [3H]aldosterone (‘HA). (a) Field CA, and (b) indusium griseum after injection of ‘HA alone. (c) Field CA, after injection of ‘HA with a IOO-fold excess of aldosterone. (d) Field CA, after injection of 3HA with a IOO-fold excess of corticosterone. Note the absence of nuclear accumulation of silver grains in c and d.
31
MICH&LECOUTARDet al.
32
Fig. 3. Autoradiograms from hippocampus after a subcutaneous injection of RU26988 prior to the injection of 3HA. (a) Field CA, and (b) indusium griseum after injection of ‘HA alone. (c) Field CA, after injection of ‘HA with a 30-fold excess of aldosterone, (d) Field CA, after injection of ‘HA with a 30.fold excess of corticosterone.
without prior injection of the pure glucocorticoid (compare Figs 2a, b with Figs 3a, b). The subiculum, the indusium griseum and the area CA, of the cornu ammonis (CA) showed the highest labelling. Autoradiograms obtained from RU-treated mice injected with ‘HA in presence of a lOO- or even a 30-fold excess of A (Fig. 3c) or B (Fig. 3d) did not show preferential accumulation of radioactivity within nuclei of hippocampal neurons. Biochemical
Analysis
The mean values (+ SD) of the number of binding sites and affinities obtained in three different experiments for control and RU26988 treated animals are shown in Table 1. The in vivo treatment by the glucocorticoid agonist led to a significant decrease in the apparent number of corticosterone binding sites
Table I. Affinity (KJ and number of sites (Rn) for corticosterone in cytosols from mice receiving SC injection of RU 26988 or vehicle (control) I h before killing Rn fmol/mg protem Kd nM
Control
RU 26988
178 + I?
48 * 19*
6.3 f 2.1
4.0 f 2.2t
Values are mean i SD, n = 3. *P < 0.001 using the Student I-test tN.S.
measured in vitro without any significant alteration the affinity of these sites for the tracer.
of
DISCUSSION
Brain autoradiograms from mice which have received 3HA alone showed that, as in the rat [13, IS], the radioactivity accumulated within cell nuclei of structures from the hippocampal complex. The comparative autoradiographic study of Birmingham et al. [15] in the rat CNS showed that although a similar pattern of distribution of the radioactivity was observed after injection of ‘HA or ‘HB, the highest concentration for ‘HB occurred in the areas CA, and CA, of the hippocampus whereas the highest concentration for 3HA was in the indusium griseum (supracallosal hippocampus). However, data with ‘HB was obtained in the adrenalectomized and castrated rat whereas that with ‘HA was obtained in the adrenalectomized rat. In our competition experiments with ‘HB as radioactive tracer, a lOO- or even a 30-fold excess of unlabelled A or B are equally effective in abolishing the accumulation of radioactivity within neuronal nuclei of the hippocampus. These results agree with the in vivo biochemical study of Veldhuis et al. [19] showing that B and A have the same ability to block 3HB uptake in cell nuclei of the hippocampus from
[3H]steroid
autoradiography
adrenalectomized rats. In addition, our data are in agreement with those obtained using in vitro autoradiography of rat brain which demonstrated that “mineral0-like” receptors for B were mainly located in the septal-hippocampal complex [ 1I]. The effect on serotonin turnover in the raphe-hippocampal complex induced by B may be prevented by a pretreatment with aldosterone in the adrenalectomized rat [27]. Furthermore, an excess of another mineralocorticoid, deoxycorticosterone acetate, abolished the nuclear accumulation of radioactivity observed in autoradiograms after injection of ‘HB [lS]. In order to determine the nature of the hippocampal sites binding 3HA, but apparently distinct from classical type II glucocorticoid receptors, we have used RU26988. This compound has been shown to be a pure glucocorticoid both in vitro [19] and in viuo [18]. When RU was injected prior to ‘HA, neuronal nuclei of the hippocampus markedly accumulated the radioactivity suggesting the presence of 3HA binding sites different from classical glucocorticoid receptors within nuclei of hippocampal neurons. This result is in agreement with previous in vitro biochemical data on hippocampal cytosols [ 17, 19,20,22]. In our autoradiograms, the intensity of the radioactivity within neuronal nuclei of hippocampus was higher than after the injection of ‘HA alone for reasons as yet unexplained. As for the competition experiments with injection of ‘HA as labelled tracer, a IOO-fold excess of unlabelled A or B similarly abolished the cell nuclear concentration of radioactivity within the hippocampal areas studied. However, Birmingham et nl. [15] reported that the pretreatment of adrenalectomized rats with an excess of unlabelled B prior to -‘HA injection prevented the nuclear localization of radioactivity in most brain areas studied except in the indusium griseum and in CA, and CA, hippocampal areas where a slight nuclear concentration of radioactivity remained. The discrepancy between these data and our own may be due to species differences or to the very small number of animals [l-2] used in Birmingham’s study. In the competition experiments when RU was used to occupy classical glucocorticoid receptors, a lOO- or even a 30-fold excess of unlabelled A or B suppressed the cell nuclear accumulation of 3HA within hippocampal neurons. These results showed that, in nuclei of hippocampal neurons, )HA binding sites different from type II glucocorticoid receptors displayed a high affinity for A and also for B. This suggestion is further substantiated by our ex uivo binding experiments. In contrast to what is usually observed when the binding of an exogenous tracer is competed for by an endogenous hormone, i.e. an apparent decrease in the affinity of the ligand for the receptors without any significant alteration of the number of binding sites, we showed that the in vivo treatment by RU26988 induced a significant reduction in the number of
in mouse
brain
33
binding sites for corticosterone without any decrease in their affinity. This indicates first that RU26988 crosses the brain-blood barrier and occupies some sites in the hippocampal region. Given the known properties of RU26988, we interpret our results as follows: about 2/3 of the corticosterone binding sites in hippocampus represent true glucocorticoid receptors which can be occupied by RU26988; those which still bind corticosterone despite the presence of RU26988 correspond with the sites which bind aldosterone. The amounts of unlabelled steroids used in our autoradiographic studies were still too large to discriminate an eventual difference in the potency of A and B in binding to these nuclear receptors. However, we may assume that B has a high affinity for these 3HA binding sites, since in the adrenalectomized mouse B is able to occupy them even when a large proportion of B is bound to CBG in plasma. In conclusion, these competition autoradiographic studies showed that, in the nuclei of hippocampal neurons, binding sites are present which differ from the classical type II glucocorticoid receptors in that they display a high affinity for both A and B. These data support the view of Krozowski and Funder [23] that the in vitro aldosterone-binding and corticosterone preferring sites in the hippocampal nuclei may be the same molecular entity. Acknowledgements-We thank Professor J. W. Funder for his helpful advice during this study and Dr D. Philibert of Roussel-Uclaf for the generous gift of RU26988. REFERENCES
1 Gerlach J. L. and McEwen B. S.: Rat brain binds adrenal steroid hormones: radioautography of hippo175 (1972) campus with corticosterone. Science 1133-l 136. 2. Rhees R. W., Grosser B. I. and Stevens W.: Effect of steroid competition and time on the uptake on 3H-corticosterone in the rat brain; an autoradiographic study. Bruin Res. 83 (1975) 293-300. M.: Radioautographic study of the rat 3. Warembourg brain after injection of (l,23H)-corticosterone. Brain Rex 89 (1975) 61-70. M. and Osborne-Pellegrin M. J.: Auto4. Coutard radiographic studies of a glucocorticoid agonist and antagonist: localization of ’ H-corticosterone and 3H-cortexolone in mouse brain. Cell liss. Rex 197 (1979) 531-538. M.: Radioautographic study of the rat 5. Warembourg brain after injection of ‘H-dexamethasone. Cell tiss. Res. 161 (1975) 183-191. W. E. and Sar M.: Auto6. Rees H. D., Stumpf radiographic studies with )H-dexamethasone in the rat brain and pituitary. In Anatomical Neuroendocrinology (Edited by W. E. Stumpf and C. D. Grant). S. Karger, Base1 (1975) p. 262-269. M. J. and Funder J. W.: 7 Coutard M., Osborne-Pellegrin Tissue distribution and specific binding of tritiated dexamethasone in vivo: autoradiographic and cell fractionation studies in the mouse. Endocrinology 103 (1978) 114&1152. 8. De Kloet E. R., Wallach G. and McEwen B. S.: Differences in corticosterone and dexamethasone binding to rat brain and pituitary. Endocrinology 96 (1975) 598409.
MICI&LECOUTARDet al.
34
9. De Kloet E. R. and McEwen B. S.: Differences between cytosol receptor complexes with corticosterone and dexamethasone in hippocampal tissue from rat brain. Biochim. biophys. Acta 421 (1976) 124-132.
10. Nestler E. J., Rainbow T. C., McEwen B. S. and Greengard P.: Corticosterone increases the amount of protein 1, a neuron-specific protein, in rat hippocampus. Science 212 (1981) 1162-l 164. 11. Reul J. M. and De Kloet E. K.: Anatomical resolution of two types of corticosterone receptor sites in the rat brain with in vitro autoradiography and computerized image analysis. J. steroid Biochem. 24 (1986) 269-272. 12. Sheppard K. E. and Funder J. W.: Cortisol 178 acid, transcortin, and the heterogeneity of rat brain glucocorticoid receptors. J. steroid Biochem. 25 (1986) 285-288. 13. Ermish A. and Ruhl H. J.: Autoradiographic demonstration of aldosterone-concentrating neuron population in rat brain. Brain Res. 147 (1978) 154-158. 14. Birmingham M. K., Stumpf W. E. and Sar M.: Nuclear localization of aldosterone in rat brain cells assessed by autoradiography. Experientia 35 (1979) 124&1241. 15. Birmingham M. K., Sar M. and Stumpf W. E.: Localization of aldosterone and corticosterone in the central nervous system, assessed by quantitative autoradioaranhv. Neurochem. Res. 9 (1984) 333-350. III N. S. and Fanestil D. D.: Corticoid 16. .&d&on receptors in rat brain: evidence for an aldosterone receptor. Endocrinology 98 (1976) 676684. 17. Moguilewsky M. and Raynaud J. P.: Evidence for a specific mindralocorticoid receptor in rat pituitary and brain. J. steroid Biochem. 12 (1980) 309-314. 18. De Nicola A. F., Tornello S., Weiseinberg, L., Fridman 0. and Birmingham M. K.: Uptake and binding of JH-aldosterone by the anterior pituitary and brain regions in adrenalectomized rats. Horm. Metab. Res. 13 (1981) 103-106.
19. Veldhuis H. D., Van Koppen C., Van Ittersum M. and De Kloet E. R.: Specificity of the adrenal steroid receptor system in rat hippocampus. Endocrinology 110 (1982) 2044-2051. 20. Coirini H., Marusic E., De Nicola A. F., Rainbow T. C. and McEwen B. S.: Identification of mineralcorticoid binding sites in rat brain by competition studies and density gradient centrifugation. Neuroendocrinology 37 (1983) 354-360. 21. Beaumont K. and Fanestil D. D.: Characterization of rat brain aldosterone receptors reveals high affinity for corticosterone. Endocrinology 113 (1983) 2043-2051. 22. De Kloet E. R., Veldhuis H. D., Wagenaars J. L. and Bergink E. W.: Relative binding affinity of steroids for the corticosterone receptor system in rat hippocampus. J. steroid Biochem. 21 (1984) 173-178. 23. Krozowski Z. S. and Funder J. W.: Renal mineralocorticoid receptors and hippocampal corticosteronebinding species have identical intrinsic steroid specificity. Proc. natn. Acad. Sci. U.S.A. 80 (1983) 605tiO60.
24. Cousin M. A., Lando D. and Moguilewsky M.: Ornithine decarboxylase induction by glucocorticoids in brain and liver of adrenalectomized rats. J. Neurochem. 38 (1982) 12961304.
25. Griinfeld J. P., Eloy L., Moura A. M., Ganeval D., Ramos-Frendo B. and Worcel M.: Effects of antiglucocorticoids on glucocorticoid hypertension in the rat. Hypertension 7 (1985) 292-299. 26. Coutard M. and Osborne-Pellegrin M. J.: “In vivo” autoradiographic study of 3H-corticosterone and 3H-aldosterone binding sites within mouse central nervous system. J. steroid Biochem. Suppl. 19 (1983) 167s abstr. 488. 27. De Kloet E. R., Versteeg D. H. G. and Kovacs G. L.: Aldosterone blocks the response to corticosterone in the raphe-hippocampal serotonin system. Brain Res. 264 (1983) 323-327.
Printed in Great Britain. All rights reserved
Copyright 0
0022-4731/87 $3.00 + 0.00 1987 Pergamon Journals Ltd
IN v1VO COMPETITIVE AUTORADIOGRAPHIC STUDY OF [‘HICORTICOSTERONE AND [‘HIALDOSTERONE BINDING SITES WITHIN MOUSE BRAIN HIPPOCAMPUS MICHI?LE COUTARD, DOMINIQUE DUVAL INSERM
and MARY J. OSBORNE-PELLEGRIN
U7, HBpital Necker, 161 rue de S&es,
75015 Paris, France
(Received 30 October 1986)
Summary-The binding sites for [3H]corticosterone (‘HB) and [3H]aldosterone (‘HA) within the hippocampal area of the mouse brain have been studied by autoradiography in competition experiments. Excess
unlabelled aldosterone (A) or corticosterone (B) both abolished the nuclear accumulation of radioactivity within neurons observed after injection of either IHA or ‘HB. Experiments where a subcutaneous injection of a “pure glucocorticoid’ RU26988 was given before injection of ‘HA alone showed a marked accumulation of radioactivity within neuronal nuclei of the hippocampus suggesting the presence of ‘HA binding sites distinct from classical type II glucocorticoid receptors. In addition, when RU26988 was given before the injection of 3HA associated with a 30- or IOO-fold excess of either A or B, the cell nuclear accumulation of radioactivity was no longer observed. These results showed that in our in vivo experimental conditions, B displayed the same ability as A to occupy 3HA binding sites, supporting the view that in mouse hippocampal neuronal nuclei, the aldosterone-binding and corticosterone-preferring sites represent the same molecular entity.
INTRODUCTION
authors [ 19,221 reported that in hippocampal cytosol Results from previous in vivo autoradiographic B displayed a higher affinity than A for both ‘HB and studies in mouse and rat brains with labelled cortico- 3HA binding sites which were distinct from classical sterone (B) [l-1] and dexamethasone (DEX) [5-71 type II glucocorticoid receptors, others [23] showed showed that these glucocorticoids are preferentially that A and B have a similar affinity for these sites. In retained in cell nuclei from different brain areas. A addition, the latter group demonstrated that these slower rate of penetration into the brain of DEX [6] 3HA and 3HB binding sites display an identical may at least in part explain these observed differ- steroid specificity suggesting that they represent the ences. However, various experiments have suggested same molecular entity. the presence of two sets of glucocorticoid receptors In order to examine the hippocampal binding sites [g-12]. for 3HB and 3HA at a cell nuclear level and in an in Several autoradiographic studies have pointed out vivo situation, we have performed competitive autothe similarity of the distribution of the radioactivity radiographic experiments with these labelled steroids. within rat brain of the tritiated mineralocorticoid We have studied the distribution of the radioactivity aldosterone (A) [13-151 with that observed for la- in mouse hippocampal autoradiograms after injecbelled corticosterone, although within some areas of tion of 3HA and ‘HB alone or in presence of an the hippocampus the intensity of the labelling differed excess of unlabelled A or B. The synthetic steroid [15]. If, as suggested by most of the above-reported RU26988 (RU) has been previously shown to have an data, the hippocampus contains (at least) two distinct affinity similar to DEX for classical ‘HDEX glucoclasses of nuclear glucocorticoid receptors, one neur- corticoid receptors (171. In further experiments deonal B preferring and one glial DEX preferring, the signed to study the 3HA binding sites distinct from question arises as to whether the binding sites for A classical type II glucocorticoid receptors, we have and B in the hippocampal neuronal nuclei are the used RU as previously described by others [17]. same molecular entity or distinct receptors. BiochemAlthough RU has been reported to display in vivo ical studies on whole rat brain [16] and on hipposome glucocorticoid effects on enzymatic activity in campal cytosols [17-201 established the presence of an rat liver and brain [24] and on blood pressure [25], we aldosterone binding site coexisting with a gluco- have performed some biochemical studies to detercorticoid receptor. However, it has been reported that mine whether under our experimental conditions RU rat brain aldosterone receptors display a high affinity was able to cross the blood-brain barrier and occupy for corticosterone [21]. Results of further studies on hippocampal glucocorticoid receptors. Part of this the relative affinity of A and B for 3HA and 3HB study has already been published in abstract form binding sites are somewhat divergent. Whereas some 1261. 29
30
MIC&LE
COUTARD
EXPERIMENTAL
Animals and Reagents Mature female C57BL/6 mice were adrenalectomized 3 days before use and given a 0.9% NaCl solution to drink. [ 1,2-3 H] corticosterone (‘HB) (43 Ci/mmol) and [ 1,2-‘H] aldosterone (‘HA) (41 Ci/mmol) were purchased from Amersham-Searle (U.K.) and all non-radioactive compounds from Sigma, St Louis, except for RU26988 (11 fi,17fidihydroxy - 17a -propynylandrost - 1,4,6 - trien - 3 -one) which was generously supplied by Roussel-Uclaf (France). The day of the experiment mice were anaesthetized with Nembutal (40 mg/kg, i.p.) and were injected via the jugular vein with 100 ~1 of radioactive solution. Autoradiographic Studies Experiments with 3HB In this series of experiments, 21 mice were used. Five mice were injected with 20 PCi (about 5 x 10-‘Omol) of ‘HB alone, or with a lOO-fold excess (about 100 pg/lOO g b. wt) of unlabelled B or A. Three mice received ‘HB with a 30-fold excess (about 3Opg/lOOg b.wt) of A or B. Experiments with 3HA A total of 32 mice were used (4 for each experimental condition). Injection of ‘HA alone or with an excess of unIabelled A or B. Mice received 2OpCi (about 5 x lo-” mol) of ‘HA alone, or with a loo-fold excess unlabelled A or B. Subcutaneous injection of RU26988 1 h before the injection of3 HA alone or with an excess of unlabelled A or B. 5 x lo-‘mol (about 1 mg/lOO g b.wt) of RU26988 were injected subcutaneously 1 h before the i.v. injection of 5 x 10-‘Omol of ‘HA alone, or with a 30- or lOO-fold excess of A or B. Thirty minutes after steroid administration mice were killed by decapitation and the brain was quickly removed from the skull and frozen in liquid nitrogencooled isopentane. The thaw-mounted autoradiographic method used has been previously described [7]. Briefly, brain sections of about 10pm thickness were made by means of a cryostat (American Optical, type Cryocut) maintained at a temperature of - 18°C. The sections were transferred in the dark on to slides precoated with Kodak AR 10 stripping film emulsion. After about 4 months of exposure at a temperature of about - 20°C autoradiographs were allowed to reach ambient temperature and thereafter were plunged into methanol to fix the brain sections, rinsed with distilled water and then developed for 5 min with Kodak D19B and fixed in 30% (wt/vol) sodium hyposulphite. After being rinsed in distilled water, sections were stained with methyl green pyronin (Gurr’s reagent), rinsed, left to dry and mounted with Eukitt medium.
ef al
Biochemical Analysis The animals received a S.C. injection of either 5 x lo-‘mols of unlabelled RU26988 or the same volume of the diluent alone (control). One hour after injection 9-10 animals in each group were killed by decapitation. The brains were removed quickly and the hippocampal regions dissected and homogenized using a polytron in ice-cold medium (10 mM Tris pH 7.4 containing 1.5 mM EDTA, 0.5 mM DTT and 20mM sodium molybdate). The homogenates were then centrifuged for 60 min at 105,OOOg. Aliquots of the cytosol(250 pLO.4 mg protein) were incubated in the presence of increasing concentrations of tritiated corticosterone (final concentation 10 -’ to 5 x 1O-8 M). Non-specific binding was determined in a parallel set of samples incubated with a 500-fold excess of non-radioactive corticosterone. After a 4 h incubation at 4°C each sample was treated by 1 ml of Dextran-coated charcoal (0.5% Norit A, 0.05 Dextran T,,) for 15 min at 4°C. After 2 min centrifugation at 10,000 g, the protein bound radioactivity was determined by scintillation spectrometry and expressed as fmol of specific binding/mg protein. The number of binding sites and the affinity of the receptor for the radioactive ligand were determined according to Scatchard. Protein concentrations were measured according to Lowry using bovine serum albumin as standard. RESULTS
Autoradiographic Experiments Experiments with ’ HB In mouse brain, ‘HB and/or its metabolites accumulated within cell nuclei of the hippocampus (Figs la, b) as previously shown [4]. Autoradiograms from mice which received a concomitant injection of ‘HB and a 30-fold excess of unlabelled B showed no radioactivity concentrated within nuclei of hippocampal neurons (Fig. lc). The same results were obtained after the injection of ‘HB in the presence of a 30-fold excess of unlabelled A (Fig. Id). Experiments with 3HA Injection of ‘HA alone or with an excess of unlabelled A or B. Neurons from hippocampal areas in mouse brain accumulate ‘HA and/or its metabolites (Figs 2a, b). However, the intensity of the labelling appears to be lower than that observed after injection of ‘HB. This cell nuclear concentration of the radioactivity was no longer observed when )HA was injected with a loo-fold excess of either A (Fig. 2c) or B (Fig. 2d). S.C. injection of RU26988 1 h before the injection of ‘HA alone or with an excess of unlabelled A or B. After a S.C. injection of a lOOO-fold excess of RU, ‘HA and/or its metabolites concentrated within nuclei of the hippocampal neurons (Figs 3a, b). The intensity of the labelling appeared more marked than
[‘HIsteroid
autoradiography
in mouse
brain
Fig. 1. Autoradiograms from mouse brain hippocampus (cornu ammonis) after injection of [‘Hlcorticosterone (3HB). (a) Field CA, and (b) field CA, after injection of 3HB alone. (c) Field CA, after injection of ‘HB with a 30-fold excess of corticosterone. (d) Field CA, after injection of rHB with a 30-fold excess of aldosterone.
Fig. 2. Autoradiograms from mouse brain hippocampus after injection of [3H]aldosterone (‘HA). (a) Field CA, and (b) indusium griseum after injection of ‘HA alone. (c) Field CA, after injection of ‘HA with a IOO-fold excess of aldosterone. (d) Field CA, after injection of 3HA with a IOO-fold excess of corticosterone. Note the absence of nuclear accumulation of silver grains in c and d.
31
MICH&LECOUTARDet al.
32
Fig. 3. Autoradiograms from hippocampus after a subcutaneous injection of RU26988 prior to the injection of 3HA. (a) Field CA, and (b) indusium griseum after injection of ‘HA alone. (c) Field CA, after injection of ‘HA with a 30-fold excess of aldosterone, (d) Field CA, after injection of ‘HA with a 30.fold excess of corticosterone.
without prior injection of the pure glucocorticoid (compare Figs 2a, b with Figs 3a, b). The subiculum, the indusium griseum and the area CA, of the cornu ammonis (CA) showed the highest labelling. Autoradiograms obtained from RU-treated mice injected with ‘HA in presence of a lOO- or even a 30-fold excess of A (Fig. 3c) or B (Fig. 3d) did not show preferential accumulation of radioactivity within nuclei of hippocampal neurons. Biochemical
Analysis
The mean values (+ SD) of the number of binding sites and affinities obtained in three different experiments for control and RU26988 treated animals are shown in Table 1. The in vivo treatment by the glucocorticoid agonist led to a significant decrease in the apparent number of corticosterone binding sites
Table I. Affinity (KJ and number of sites (Rn) for corticosterone in cytosols from mice receiving SC injection of RU 26988 or vehicle (control) I h before killing Rn fmol/mg protem Kd nM
Control
RU 26988
178 + I?
48 * 19*
6.3 f 2.1
4.0 f 2.2t
Values are mean i SD, n = 3. *P < 0.001 using the Student I-test tN.S.
measured in vitro without any significant alteration the affinity of these sites for the tracer.
of
DISCUSSION
Brain autoradiograms from mice which have received 3HA alone showed that, as in the rat [13, IS], the radioactivity accumulated within cell nuclei of structures from the hippocampal complex. The comparative autoradiographic study of Birmingham et al. [15] in the rat CNS showed that although a similar pattern of distribution of the radioactivity was observed after injection of ‘HA or ‘HB, the highest concentration for ‘HB occurred in the areas CA, and CA, of the hippocampus whereas the highest concentration for 3HA was in the indusium griseum (supracallosal hippocampus). However, data with ‘HB was obtained in the adrenalectomized and castrated rat whereas that with ‘HA was obtained in the adrenalectomized rat. In our competition experiments with ‘HB as radioactive tracer, a lOO- or even a 30-fold excess of unlabelled A or B are equally effective in abolishing the accumulation of radioactivity within neuronal nuclei of the hippocampus. These results agree with the in vivo biochemical study of Veldhuis et al. [19] showing that B and A have the same ability to block 3HB uptake in cell nuclei of the hippocampus from
[3H]steroid
autoradiography
adrenalectomized rats. In addition, our data are in agreement with those obtained using in vitro autoradiography of rat brain which demonstrated that “mineral0-like” receptors for B were mainly located in the septal-hippocampal complex [ 1I]. The effect on serotonin turnover in the raphe-hippocampal complex induced by B may be prevented by a pretreatment with aldosterone in the adrenalectomized rat [27]. Furthermore, an excess of another mineralocorticoid, deoxycorticosterone acetate, abolished the nuclear accumulation of radioactivity observed in autoradiograms after injection of ‘HB [lS]. In order to determine the nature of the hippocampal sites binding 3HA, but apparently distinct from classical type II glucocorticoid receptors, we have used RU26988. This compound has been shown to be a pure glucocorticoid both in vitro [19] and in viuo [18]. When RU was injected prior to ‘HA, neuronal nuclei of the hippocampus markedly accumulated the radioactivity suggesting the presence of 3HA binding sites different from classical glucocorticoid receptors within nuclei of hippocampal neurons. This result is in agreement with previous in vitro biochemical data on hippocampal cytosols [ 17, 19,20,22]. In our autoradiograms, the intensity of the radioactivity within neuronal nuclei of hippocampus was higher than after the injection of ‘HA alone for reasons as yet unexplained. As for the competition experiments with injection of ‘HA as labelled tracer, a IOO-fold excess of unlabelled A or B similarly abolished the cell nuclear concentration of radioactivity within the hippocampal areas studied. However, Birmingham et nl. [15] reported that the pretreatment of adrenalectomized rats with an excess of unlabelled B prior to -‘HA injection prevented the nuclear localization of radioactivity in most brain areas studied except in the indusium griseum and in CA, and CA, hippocampal areas where a slight nuclear concentration of radioactivity remained. The discrepancy between these data and our own may be due to species differences or to the very small number of animals [l-2] used in Birmingham’s study. In the competition experiments when RU was used to occupy classical glucocorticoid receptors, a lOO- or even a 30-fold excess of unlabelled A or B suppressed the cell nuclear accumulation of 3HA within hippocampal neurons. These results showed that, in nuclei of hippocampal neurons, )HA binding sites different from type II glucocorticoid receptors displayed a high affinity for A and also for B. This suggestion is further substantiated by our ex uivo binding experiments. In contrast to what is usually observed when the binding of an exogenous tracer is competed for by an endogenous hormone, i.e. an apparent decrease in the affinity of the ligand for the receptors without any significant alteration of the number of binding sites, we showed that the in vivo treatment by RU26988 induced a significant reduction in the number of
in mouse
brain
33
binding sites for corticosterone without any decrease in their affinity. This indicates first that RU26988 crosses the brain-blood barrier and occupies some sites in the hippocampal region. Given the known properties of RU26988, we interpret our results as follows: about 2/3 of the corticosterone binding sites in hippocampus represent true glucocorticoid receptors which can be occupied by RU26988; those which still bind corticosterone despite the presence of RU26988 correspond with the sites which bind aldosterone. The amounts of unlabelled steroids used in our autoradiographic studies were still too large to discriminate an eventual difference in the potency of A and B in binding to these nuclear receptors. However, we may assume that B has a high affinity for these 3HA binding sites, since in the adrenalectomized mouse B is able to occupy them even when a large proportion of B is bound to CBG in plasma. In conclusion, these competition autoradiographic studies showed that, in the nuclei of hippocampal neurons, binding sites are present which differ from the classical type II glucocorticoid receptors in that they display a high affinity for both A and B. These data support the view of Krozowski and Funder [23] that the in vitro aldosterone-binding and corticosterone preferring sites in the hippocampal nuclei may be the same molecular entity. Acknowledgements-We thank Professor J. W. Funder for his helpful advice during this study and Dr D. Philibert of Roussel-Uclaf for the generous gift of RU26988. REFERENCES
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19. Veldhuis H. D., Van Koppen C., Van Ittersum M. and De Kloet E. R.: Specificity of the adrenal steroid receptor system in rat hippocampus. Endocrinology 110 (1982) 2044-2051. 20. Coirini H., Marusic E., De Nicola A. F., Rainbow T. C. and McEwen B. S.: Identification of mineralcorticoid binding sites in rat brain by competition studies and density gradient centrifugation. Neuroendocrinology 37 (1983) 354-360. 21. Beaumont K. and Fanestil D. D.: Characterization of rat brain aldosterone receptors reveals high affinity for corticosterone. Endocrinology 113 (1983) 2043-2051. 22. De Kloet E. R., Veldhuis H. D., Wagenaars J. L. and Bergink E. W.: Relative binding affinity of steroids for the corticosterone receptor system in rat hippocampus. J. steroid Biochem. 21 (1984) 173-178. 23. Krozowski Z. S. and Funder J. W.: Renal mineralocorticoid receptors and hippocampal corticosteronebinding species have identical intrinsic steroid specificity. Proc. natn. Acad. Sci. U.S.A. 80 (1983) 605tiO60.
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25. Griinfeld J. P., Eloy L., Moura A. M., Ganeval D., Ramos-Frendo B. and Worcel M.: Effects of antiglucocorticoids on glucocorticoid hypertension in the rat. Hypertension 7 (1985) 292-299. 26. Coutard M. and Osborne-Pellegrin M. J.: “In vivo” autoradiographic study of 3H-corticosterone and 3H-aldosterone binding sites within mouse central nervous system. J. steroid Biochem. Suppl. 19 (1983) 167s abstr. 488. 27. De Kloet E. R., Versteeg D. H. G. and Kovacs G. L.: Aldosterone blocks the response to corticosterone in the raphe-hippocampal serotonin system. Brain Res. 264 (1983) 323-327.