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Ontogeny of Type I and Type II corticosteroid receptors in the rat hippocampus
Developmental Brain Research, 42 (1988) 113-118
113
Elsevier BRD 50776
Ontogeny of Type I and Type II corticosteroid receptors in the rat hippocampus Patricia Rosenfeld 2, Winardi Sutanto 1, Seymour Levine 2 and E. Ronald De Kloet 1 1RudolfMagnus Institutefor Pharmacology, MedicalFaculty, Universityof Utrecht, Utrecht(The Netherlands) and 2Department of Psychiatry, Stanford University, Stanford, CA 94305 (U.S.A.) (Accepted 8 March 1988)
Key words: Corticosteroid receptor; Hippocampus, Ontogeny; Rat
The ontogeny of the corticoid receptors in the rat hippocampuswas examined by in vitro [3H]corticosterone(CORT) binding to soluble molecules in the cytosol, using the selective Type II glucocorticoidagonist, RU 28362, to discriminate between Type I and Type II receptor sites. Type I receptors were undetectable until 8 days after birth. From this age on, the receptor showed adult characteristics for both the binding capacity (Bmax)and affinity (Kd). The Type II receptor concentration increased gradually over the observed period; however, at 3 weeks of age concentrations were still only about 65% those found in adults. The binding affinity of Type II to CORT was high during the first week of life but decreased thereafter towards adult value. These data thus suggest clear distinctions in the developmental patterns of Type I and Type II receptors for corticosteroidsin the rat.
INTRODUCTION Glucocorticoids (GCs) exert, in the adult animal, a wide variety of regulatory and permissive actions on a number of systems, aimed basically at maintaining homeostasis 13. Most of these effects are readily reversible. During development, however, many of these systems are not yet operational. Moreover, GCs have been shown to produce, in the immature animal, permanent morphological, physiological and behavioral effects, i.e. they influence the structural organization of the CNS 2"6'9. A common factor of all (or at least most) GC actions, is that they are mediated via receptors. Recent research has confirmed the existence of two types of receptors for corticosteroids in the rat brain: Type I and Type II receptors. They can be distinguished on the basis of their primary structure, affinity, localization and function 1'5"7"16.The characterization of these receptor sites, however, has been carried out in the adult organism. The ontogeny of the corticosteroid
receptors in the brain has been examined in the past using either cytosol binding techniques 3'11'14 or in vivo autoradiography 12,24. However, none of these studies discriminated between the two types of receptors. In this study, we examined the ontogeny of both the Type I and Type II receptors in the hippocampus of the infant rat. We chose to look at the hippocampus because it is known to contain, at least in the adult, high concentrations of both receptor types 16"23. The receptor binding constants were measured using an in vitro cytosol binding assay. Our findings demonstrate clearly the distinctions in the developmental patterns of both receptor systems for corticosteroids in the rat. MATERIALS AND METHODS
Animals The day after giving birth (= day 1) individually housed, multiparous Wistar rats were given 10 male one-day-old pups. From this moment on the animals
Correspondence: E.R. de Kloet, Rudolf Magnus Institute for Pharmacology,Medical Faculty, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands. 0165-3806/88/$03.50© 1988Elsevier Science Publishers B.V. (Biomedical Division)
114 were not handled in any way (except for the brief period in which they were removed for adrenalectomy ADX), nor were the cages cleaned, until time of testing. Food and water (0.9% saline after ADX) were available ad libitum. Animals were maintained under standard lighting (lights on between 06.00 and 20.00 h) and temperature (23 °C) conditions. All procedures were carried out during the morning. Animals were sacrificed on 2, 4, 8, 12, 16 and 20 days of age. All animals for a given time point proceeded from the same litter. Adult animals (150-200 g body weight) were used as reference. A D X was performed on all the pups and their dam. The operation was carried out via the dorsal approach under ether (8-, 12-, 16- and 20-day-olds and adults) or hypothermia (2- and 4-day-olds) anaesthesia. After allowing sufficient time for recovery, pups and dam were reunited in the home cage and left undisturbed until immediately prior to sacrifice. A D X was performed 24 h previously in order to clear the receptor of endogenous ligand; this time has been shown to be sufficient for this to occur 14.
Tissue collection Animals were anaesthetized (hypothermia in 2and 4-day-olds; Nembutal in 12-, 16- and 20-day-olds and in adults) and perfused through the heart with ice-cold saline to remove traces of corticosteroid binding globulin (CBG, transcortin). After perfusion. the hippocampus was rapidly dissected from the brain and frozen on dry-ice. Tissues were stored at -80 °C until further use.
In vitro binding assays of cytosolic receptors The number of Type I and Type II receptors in cytosol obtained from neonatal and adult tissues was determined using in vitro [3H]corticosteroid binding assays. Hippocampal tissue was homogenized in Tris-HCl buffer (pH 7.4) containing 5 mM Tris, 1 mM E D T A , 1 mM fl-mercaptoethanol, 10 mM sodiummolybdate and 5% glycerol. Generally, for 1 ml of the buffer, 2 hippocampi from adult, 20- and 16-dayold animals, or 4 from 12-, 8-, 4- and 2-day-old animals were used. The homogenate was centrifuged at 100,000 g (35,000 rpm) at 2 °C and 1 h to obtain cytosol. [3H]CORT ([1,2,6,7-3H]corticosterone; spec. act. 82.5 Ci/mmol, New England Nuclear, Dreieich,
F.R.G.) was added to aliquots of cytosol at a concentration range of 0.5-15.0 nM. Parallel incubations included a 100-fold excess of unlabeled RU 28362 (RousseI-Uclaf Pharmaceutical Co., Romainville, France), which was used to exclusively occupy the Type II receptor 15"17'23. The remaining [~H]CORT binding after inclusion of RU 28362 represented binding to the Type I receptor and to remnants of CBG due to blood contaminations. Non-specific binding was determined in the presence of a 500-fold excess of unlabeled CORT. Binding to CBG was measured by subtracting non-specific binding from the binding of [3H]CORT obtained in the presence of a 500-fold excess of dexamethasone (DEX). Unlabeled C O R T and DEX were generous gifts from Organon International, Oss, The Netherlands. The non-specific binding was determined by co-incubating parallel samples with a 500-fold molar excess of RU 28362. All assays were carried out on the same day in order to minimize inter-assay variability. To determine the relative binding affinities (RBA) of CORT, DEX, RU 28362 and aldosterone (ALDO) to hippocampal cytosol, aliquots of the cytosol obtained from 2-day-old and adult rats were incubated with 4.0 nM [3H]RU 28362 (for Type II sites) or 1.5 nM [3H]CORT in the presence of a 100fold excess of RU 28362 (which would occupy the Type I1 sites). The binding thus measured was that to the Type I receptor. In each case, the concentration range of the unlabelled steroids was 0.1-1000.0 nM. Non-specific binding was determined as described previously. In each of these experiments, an incubation of 3 h at 0 °C was sufficient to reach binding equilibrium. Separation of the bound from unbound steroid was performed using Sephadex LH-20 gel fiitration 4'16"23. The cytosolic protein content was determined by the method of Lowry et al. using bovine serum albumin as standard 1°.
Calculation and statistical analyses of data The values of apparent (molar) binding affinity [(Kd) expressed as nM] and the apparent (maximum) binding capacity (Bmax) expressed as fmol/mg protein were evaluated using Scatchard analysis of the binding data 22. The number of Type I sites was estimated from Bmax of [3H]CORT in the presence of a 100-fold excess of RU 28362 and corrected for the presence of
115 CBG traces. Type II sites are those [3H]C-ORT binding sites that are displaceable by R U 28362 and corrected for traces of CBG. Hence, the binding constants of the Type II sites can be calculated by subtracting the number of [3H]CORT binding sites in the presence of R U 28362 from those in the absence of RU 28362. The values of RBA (1(750) were determined as described in the legend.
day-old hippocampal cytosol is shown in Fig. lB. The linear Scatchard plot for the Type II receptor gives a K d value of 3.2 nM and a Bmax of 92.7 fmol/mg protein, while for the Type I receptor, the K d is 0.9 nM and the Bmax is 32.6 fmol/mg protein. Table I shows the binding constants for Type I and Type II receptors at different ages in rat hippocampus. These values are corrected for traces of CBG (5-10 fmol/mg protein) which remained in the tissue after perfusion. Type I receptors are absent in the cytosol of 2- and 4-day-old hippocampi. From 8 days of age on, the receptor is present and shows adult values for both the binding affinity and capacity. The Type II receptor concentration is low in 2-day-old animals (approximately 1/10 adult levels). Concentrations increase gradually up to 12 days of age, but then in the 16-dayold animals the values return to those observed in the 8-day-old rats. At 20 days of age, levels are substantially higher again, although still considerably below those found in the adult (Table I). The binding affinity was high at the younger ages (2- and 4-day-olds) but by 8 days of age was already within the range of
RESULTS
Heterogeneity of corticosteroid binding to cytosol of neonatal and adult rats The binding data of [3H]CORT to rat hippocampal cytosol obtained from 4-day-old pups and plotted according to the method of Scatchard is shown in Fig. 1A. The linear plot for the Type II receptor gives K d values of 1.7 nM and a Brnax of 48.3 fmol/mg protein. There was total displacement of [3H]CORT binding by inclusion of a 100-fold excess R U 28362. This indicates that all binding sites correspond to the Type II receptor, as the Type I receptor concentration was below detection levels. [3H]CORT binding to the 16-
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Fig. 1. Scatchard plots of [3H]CORT binding to rat hippocampal cytosolic receptors in (A) 4-day-old and (B) 16-day-old animals, in the absence (~1"--~) and presence (O---C)) of a 100-fold excess RU 28362. (Inset: isotherms of the binding data from which the Scatchard plots were derived.) In the presence of a 100-fold excess RU 28362, the binding is to Type I receptor. Dashed areas: [3H]CORT binding to Type II receptor. See Materials and Methods for calculation of values of Kd (in nM) and Bmax (in fmol/mg protein). These values are also shown in Table I. In all binding assays, rco~ ranged from 0.92 to 0.98. Insets: isotherms of the binding data from which the Scatchard plots were derived.
116 TABLE 1
TABLE II
('orticosteroid binding constants to neonatal and adult rat hippocampi
Relative binding affinities (IC5o) for Type 1 and Type 11 receptors in 2-day-old and adult rat hippocampi
Binding constants of [3H]CORT binding to receptors in 2, 4, 8, 12, 16, 20 day and adult rat hippocampal cytosol. The apparent binding affinity (K~, expressed as nM) and capacity (BmaX, as fmol/mg protein) were calculated from the Scatchard analyses of the binding data of [3H]CORT, each in the presence or absence of a 100-fold excess RU 28362 (see Materials and Methods). In all binding assays, rcorrranged from 0.92 to 0.98.
To determine binding to Type 1 site samples were incubated with 1.5 nM [3H]CORT in the presence of a t00-fold excess RU 28362. To determine binding to Type II site, parallel samples were run with 4.0 nM [3H]RU 28362. In each case, competing steroids (concentration range 1.0-1000.0 nM) were simultaneously included in parallel samples. 1C5o is calculated as the concentration of the unlabelled steroid required to decrease the binding capacity of the labelled steroid by 50%.
Age (days)
Type I receptor Type 11receptor . . . . . . . . . . . . . . . . . . . . K,t (nM) B ..... Kd (nM) Bmax (frnol/mg) (fmol/mg)
Receptor
Competitors
Neonate ICy)(nM)
A dutt 1C~o (nM)
2 4 8 12 16 20 Adult
0.9 1.3 0.9 0.9 1.0
Type I
RU 28362 CORT ALDO DEX
-
425.0 1.4 6,0 10.0
Type I I
RU 28362 CORT ALDO DEX
3.7 4.5 70.0 10.0
32.1 31.7 32.6 51.9 42.1
1.0 1.7 2.3 3.2 3.2 6.4 5.0
28.7 48.3 80.9 136.6 92.7 148.3 3(Xl.5
3.0 10.0 62.0 6.8
values observed in adults (Table I). Relative binding affinities (ICso) o f C O R T , R U 28362, A L D O and D E X f o r Type I and Type H receptors in neonatal and adult hippocampi Table II shows the IC5o of C O R T , R U 28362, A L D O and D E X for T y p e I and T y p e II receptors. ICso is d e t e r m i n e d as the effective concentration at which 50% binding of the labeled steroid is displaced by the competitor, as p o i n t e d out in the legend to Table If. Results shown in Table II clearly indicate that in the adult h i p p o c a m p u s , R U 28362, the p o t e n t glucocorticoid agonist, binds to T y p e II sites with a much higher affinity than to T y p e I sites (IC5o 3.0 nM vs 425.0 nM) while C O R T and A L D O show the opposite. A L D O has also a very low affinity for the Type II site in the 2-day-old rat (IC50 3 0 - 7 0 nM). The R B A of Type II for C O R T was again higher in the neonate than in the adult. Note that the T y p e I receptor is.not present in the 2-day-old animal, and that binding to the Type II receptor was m e a s u r e d using [3H]RU 28362. DISCUSSION The results of our study point to clear differences in the d e v e l o p m e n t a l patterns of Type I and Type II corticoid receptors in the hippocampus. Thus, T y p e I
receptors are still undetectabte in the 4-day-old rat. In the next few days, however, concentrations rise dramatically so that by 8 days of age the T y p e I receptor system is already similar to that found in the adult. As from this age on, no changes are found in Type I binding constants. In contrast, Type II receptors are present already at birth, but subsequent development is a slow and uneven process. Affinity is high in the neonate, but by the end of the first week of life it has decreased to adult values, whereas the receptor concentration (Bmax) is still increasing at 20 days of age. These data not only extend b u t also differ in some i m p o r t a n t aspects from previous reports on the ontogeny of G C receptors in the brain 3'n'12'14. As mentioned previously, these studies did not discriminate between r e c e p t o r types. H o w e v e r , taking into account the present data, it can be assumed that they p r o b a b l y m e a s u r e d T y p e II receptors during the first week of life (when the Type I r e c e p t o r is absent) and the total T y p e I + II r e c e p t o r pool thereafter. A c o m p a r a b l e study to ours has been carried out in the pituitary t9. Type I r e c e p t o r ontogeny in the brain resembles that described for the pituitary in that the receptor is not present until a p p r o x i m a t e l y the end of the first week. H o w e v e r , in the pituitary, r e c e p t o r concentrations rise gradually and do not achieve adult levels until the 3rd or 4th week of life. This con-
117 trasts sharply with what occurs in the hippocampus, where adult concentrations are reached in a matter of days. Type II receptors in the pituitary are present at birth in adult concentration and affinity, and change very little with age. Again, this contrasts with that found in the hippocampus, where perinatal levels are only about 1/10 of adult concentrations and there are marked changes with age both in the affinity and capacity of the Type II receptor system. With regard to the Type II capacity in the neonates, the value increases gradually up to the age of 20 days with the exception of that at age 12, the value of which is greater than that of day 8 and day 16. This phenomenon is as yet unexplained, but it is interesting to note that in our immunocytochemical studies TM, the observed immunoreactive staining at day 12 is less than that seen at day 8. When evaluating biochemical data, it is necessary to take certain factors into account. First, animals must be ADX in order to eliminate endogenous corticosteroids. Beyond the non-specific effects (of unknown consequences) of the operation itself, ADX has been shown in the adult animal to have a differential effect on the number of receptor sites available for in vitro radioligand binding in cytosol. Thus, Type I availability shows a marked increase within 4-7 h, presumably due to the depletion of endogenous hormone, and no further increase thereafter; while Type II availability, on the other hand, remains relatively constant in the first 24 h post-surgery, but then increases gradually for several days 17. Although autoregulation does not seem to occur perinatally 2~, it cannot be ruled out with certainty until it is studied in the individual receptor types. Second, this being an in vitro assay, caution should be exercised when interpreting in vivo phenomena. The presence of a cytosolic receptor capable of binding CORT in vitro does not necessarily imply that this same receptor is capable of binding to DNA in vivo. Immunocytochemical data in fact suggest that the 'activated' state of the receptor, capable of binding to cell nuclear DNA 25, also shows an ontogenetic change TM. Third, although the cytosol binding assay does permit the estimation of the binding constants of the receptor types present in the hippocampus, the immunocytochemical data show that the Type II receptor immunoreactivity changes dramatically both in intensity
and localization as a function of age within small areas of the hippocampus itself. However, in spite of the limitations of the technique, the cytosol binding assay provides important information on the apparent maximum capacity and affinity of the receptor system, not available otherwise. The functional implications of these findings have yet to be explored. It is interesting to note though that the Type I receptor does not appear until the end of the first postnatal week. This is true both for the pituitary and the hippocampus. This fact could be of value in discriminating the receptor types underlying the different effects of CORT. Thus, the effects of CORT administered during the first few days of life must be assumed to be mediated by the Type II receptor. Similarly, one would expect hippocampal Type I receptor-mediated functions to show a sudden 'turning on' during development concordant with the rapid rise in receptor concentrations. It is of interest in this respect that before the appearance of the Type I receptor, the affinity of the Type II receptor is in the range of the Type I receptor affinity. The Type II receptor affinity approaches adult value only after day 8. This observation raises the possibility that the Type II receptor displays Type I characteristics in the binding of CORT during the first post-natal week. Hippocampal glucocorticoid receptors have been implicated in the turning off of the stress-induced CORT response s'2°'2J. This negative feedback has been shown to be deficient in the immature animals only acquiring adult characteristics around the end of the first month of life. This correlates with the gradual increase in Type II receptor concentrations, suggesting that this receptor may in fact mediate this function. Clearly these data, in conjunction with other data obtained from immunocytochemical studies indicate a pattern of development of corticoid receptors in the brain that is far more complex than the existing descriptions of the ontogeny of these receptors. It has been well documented that the corticoid receptors show dramatic alterations during a critical period of developmental changes in the functional regulation of the HPA system. The relationship between the functional aspects of the system and the observed changes in corticoid receptors in the brain remains to be established.
118 REFERENCES 1 Arriza, J.L., Weinberger, C., Cerelli, G., Glaser, T.M., Handelin, B.L., Housman, D.E. and Evans, R.M., Cloning of human mineralocorticoid receptor complementary D N A : structural and functional kinship with the glucocorticold receptor, Science, 237 (1987) 268-275. 2 Bohn, M.C., Glucocorticoid induced teratologies of the nervous system. In J. Yanai (Ed.), Neurobehavioral Teratologies of the Nervous System, Elsevier, Amsterdam, 1984, pp. 365-387. 3 Clayton, C.J., Grosser, B.I. and Stevens, W., The ontogeny of corticosterone and dexamethasone receptors in the rat brain, Brain Res., 134 (1977) 445-453. 4 De Kloet, E.R., Wallach, G. and McEwen, B.S., Differences in corticosterone and dexamethasone binding to rat brain and pituitary, Endocrinology, 96 (1975) 598-609. 5 De Kloet, E.R. and Reul, J.M.H.M., Feedback action and tonic influence of corticosteroids on brain function: a concept arising from heterogeneity of brain receptor systems, Psychoneuroendocrinology, 12 (1987) 83-105. 6 Doupe, A.J. and Patterson, P.H., Glucocorticoids and the developing nervous system. In D. Ganten and D. Pfaff' (Eds.), Current Topics in Neuroendocrinology, Vol. 2, Springer, Berlin, 1982, pp. 23-43. 7 Funder, J.W. and Sheppard, K., Adrenocortical steroids and the brain, Ann. Rev. Physiol., 49 (1987) 397-411. 8 Henning, S.J., Plasma concentrations of total and free corticosterone during development in the rat, Am. J. Physiol., 235 (1978) E451-456. 9 Levine, S. and Mullins, R.F., Jr., Hormonal influences on brain organization in infant rats, Science, t52 (1966) 1585-1592. 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 11 Meaney, M.J., Sapolsky, R.M. and McEwen, B.S., The development of the glucocorticoid receptor system in the rat limbic brain. I. Ontogeny and autoregulation, Dev. Brain Res., 18 (1985) 159-164. 12 Meaney, M.J., Sapolsky, R.M. and McEwen, B.S., The development of the glucocorticoid receptor system in the rat limbic brain. II. An autoradiographic study, Dev. Brain Res., 18 (1985) 165-168. 13 Munck, A., Guyre, P.M. and Holbrook, N.J., Physiological functions of glucocorticoids in stress and their relation to
pharmacological actions, Endocr. Rev., 5 (19~4) 25 44~ 14 Olpe, H.-R. and McEwen, B.S., Glucocorticoid binding to receptor-like proteins in rat brain and pituitary: ontogenctic and experimentally induced changes, Brain Re.~., 105 (1976) 121-128. 15 Philibert, D. and Moguilewsky, M., RU 28362, a useful too[ for the characterization of glucocorticoid and mineralocorticoid receptors, Proceedings of 65th Annual Meeting of the Endocrine Society, U.S.A., (1983) pp. 335. 16 Reul, J.M.H.M. and De Kloet, E.R., Two receptor systems for corticosteroids in the brain: microdistribution and differential occupation, Endocrinology, ]17 (1985) 2505-2511. 17 Reul, J.M.H.M., van den Bosch, F.R. and De Kloet, E.R., Relative occupation of type-I and type-If corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications, J: Endocrinol., 108 (1987) 459-467. 18 Rosenfeld, P., Van Eekelen, J.A.M., Westphal, H.M., Levine, S. and De Kloet, E.R., Ontogeny of the Type 2 glucocorticoid receptor in discrete rat brain regions: an immunocytochemical study, Dev. Brain Res., 42 (1988) 119-127. 19 Sakly, M. and Koch, B., Ontogenesis of the glucocorticoid receptor in the anterior pituitary gland: transient dissociation among cytoplasmic receptor density, nuclear uptake and regulation of corticotropic activity, Endocrinology, 108 (1981) 591-596. 20 Sapolsky, R.M. and Meaney, M.J., Maturation of the adrenal stress response: neuroendocrine control mechanisms and the stress hyporesponsive period, Brain Res'. Re~,., 11 (1986) 65-76. 21 Sapolsky, R.M., Krey, L.C. and McEwen, B.S., The neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis, Endocr. Rev., 7 (1986) 284-301. 22 Scatchard, G., The attractions of proteins for small molecules and ions, Ann. N. Y. A cad. Sci., 51 (1949)660-672. 23 Sutanto, W. and De Kloet, E.R., Species-specificity of corticosteroid receptor in hamster and rat brains, Endocrinology, 121 (1987) 1405-1411. 24 Turner, B.B., Ontogeny of glucocorticoid binding in the rodent brain, Am. Zool., 18 (1978) 461-475. 25 Van Eekelen, J.A.M., Kiss, J.Z., Westphal, H.M. and De KIoet, E.R., Immunocytochemical study of the intracellular localization of the Type 2 glucocorticoid receptor in the rat brain, Brain Res., 436 (1987) 120-128.
113
Elsevier BRD 50776
Ontogeny of Type I and Type II corticosteroid receptors in the rat hippocampus Patricia Rosenfeld 2, Winardi Sutanto 1, Seymour Levine 2 and E. Ronald De Kloet 1 1RudolfMagnus Institutefor Pharmacology, MedicalFaculty, Universityof Utrecht, Utrecht(The Netherlands) and 2Department of Psychiatry, Stanford University, Stanford, CA 94305 (U.S.A.) (Accepted 8 March 1988)
Key words: Corticosteroid receptor; Hippocampus, Ontogeny; Rat
The ontogeny of the corticoid receptors in the rat hippocampuswas examined by in vitro [3H]corticosterone(CORT) binding to soluble molecules in the cytosol, using the selective Type II glucocorticoidagonist, RU 28362, to discriminate between Type I and Type II receptor sites. Type I receptors were undetectable until 8 days after birth. From this age on, the receptor showed adult characteristics for both the binding capacity (Bmax)and affinity (Kd). The Type II receptor concentration increased gradually over the observed period; however, at 3 weeks of age concentrations were still only about 65% those found in adults. The binding affinity of Type II to CORT was high during the first week of life but decreased thereafter towards adult value. These data thus suggest clear distinctions in the developmental patterns of Type I and Type II receptors for corticosteroidsin the rat.
INTRODUCTION Glucocorticoids (GCs) exert, in the adult animal, a wide variety of regulatory and permissive actions on a number of systems, aimed basically at maintaining homeostasis 13. Most of these effects are readily reversible. During development, however, many of these systems are not yet operational. Moreover, GCs have been shown to produce, in the immature animal, permanent morphological, physiological and behavioral effects, i.e. they influence the structural organization of the CNS 2"6'9. A common factor of all (or at least most) GC actions, is that they are mediated via receptors. Recent research has confirmed the existence of two types of receptors for corticosteroids in the rat brain: Type I and Type II receptors. They can be distinguished on the basis of their primary structure, affinity, localization and function 1'5"7"16.The characterization of these receptor sites, however, has been carried out in the adult organism. The ontogeny of the corticosteroid
receptors in the brain has been examined in the past using either cytosol binding techniques 3'11'14 or in vivo autoradiography 12,24. However, none of these studies discriminated between the two types of receptors. In this study, we examined the ontogeny of both the Type I and Type II receptors in the hippocampus of the infant rat. We chose to look at the hippocampus because it is known to contain, at least in the adult, high concentrations of both receptor types 16"23. The receptor binding constants were measured using an in vitro cytosol binding assay. Our findings demonstrate clearly the distinctions in the developmental patterns of both receptor systems for corticosteroids in the rat. MATERIALS AND METHODS
Animals The day after giving birth (= day 1) individually housed, multiparous Wistar rats were given 10 male one-day-old pups. From this moment on the animals
Correspondence: E.R. de Kloet, Rudolf Magnus Institute for Pharmacology,Medical Faculty, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands. 0165-3806/88/$03.50© 1988Elsevier Science Publishers B.V. (Biomedical Division)
114 were not handled in any way (except for the brief period in which they were removed for adrenalectomy ADX), nor were the cages cleaned, until time of testing. Food and water (0.9% saline after ADX) were available ad libitum. Animals were maintained under standard lighting (lights on between 06.00 and 20.00 h) and temperature (23 °C) conditions. All procedures were carried out during the morning. Animals were sacrificed on 2, 4, 8, 12, 16 and 20 days of age. All animals for a given time point proceeded from the same litter. Adult animals (150-200 g body weight) were used as reference. A D X was performed on all the pups and their dam. The operation was carried out via the dorsal approach under ether (8-, 12-, 16- and 20-day-olds and adults) or hypothermia (2- and 4-day-olds) anaesthesia. After allowing sufficient time for recovery, pups and dam were reunited in the home cage and left undisturbed until immediately prior to sacrifice. A D X was performed 24 h previously in order to clear the receptor of endogenous ligand; this time has been shown to be sufficient for this to occur 14.
Tissue collection Animals were anaesthetized (hypothermia in 2and 4-day-olds; Nembutal in 12-, 16- and 20-day-olds and in adults) and perfused through the heart with ice-cold saline to remove traces of corticosteroid binding globulin (CBG, transcortin). After perfusion. the hippocampus was rapidly dissected from the brain and frozen on dry-ice. Tissues were stored at -80 °C until further use.
In vitro binding assays of cytosolic receptors The number of Type I and Type II receptors in cytosol obtained from neonatal and adult tissues was determined using in vitro [3H]corticosteroid binding assays. Hippocampal tissue was homogenized in Tris-HCl buffer (pH 7.4) containing 5 mM Tris, 1 mM E D T A , 1 mM fl-mercaptoethanol, 10 mM sodiummolybdate and 5% glycerol. Generally, for 1 ml of the buffer, 2 hippocampi from adult, 20- and 16-dayold animals, or 4 from 12-, 8-, 4- and 2-day-old animals were used. The homogenate was centrifuged at 100,000 g (35,000 rpm) at 2 °C and 1 h to obtain cytosol. [3H]CORT ([1,2,6,7-3H]corticosterone; spec. act. 82.5 Ci/mmol, New England Nuclear, Dreieich,
F.R.G.) was added to aliquots of cytosol at a concentration range of 0.5-15.0 nM. Parallel incubations included a 100-fold excess of unlabeled RU 28362 (RousseI-Uclaf Pharmaceutical Co., Romainville, France), which was used to exclusively occupy the Type II receptor 15"17'23. The remaining [~H]CORT binding after inclusion of RU 28362 represented binding to the Type I receptor and to remnants of CBG due to blood contaminations. Non-specific binding was determined in the presence of a 500-fold excess of unlabeled CORT. Binding to CBG was measured by subtracting non-specific binding from the binding of [3H]CORT obtained in the presence of a 500-fold excess of dexamethasone (DEX). Unlabeled C O R T and DEX were generous gifts from Organon International, Oss, The Netherlands. The non-specific binding was determined by co-incubating parallel samples with a 500-fold molar excess of RU 28362. All assays were carried out on the same day in order to minimize inter-assay variability. To determine the relative binding affinities (RBA) of CORT, DEX, RU 28362 and aldosterone (ALDO) to hippocampal cytosol, aliquots of the cytosol obtained from 2-day-old and adult rats were incubated with 4.0 nM [3H]RU 28362 (for Type II sites) or 1.5 nM [3H]CORT in the presence of a 100fold excess of RU 28362 (which would occupy the Type I1 sites). The binding thus measured was that to the Type I receptor. In each case, the concentration range of the unlabelled steroids was 0.1-1000.0 nM. Non-specific binding was determined as described previously. In each of these experiments, an incubation of 3 h at 0 °C was sufficient to reach binding equilibrium. Separation of the bound from unbound steroid was performed using Sephadex LH-20 gel fiitration 4'16"23. The cytosolic protein content was determined by the method of Lowry et al. using bovine serum albumin as standard 1°.
Calculation and statistical analyses of data The values of apparent (molar) binding affinity [(Kd) expressed as nM] and the apparent (maximum) binding capacity (Bmax) expressed as fmol/mg protein were evaluated using Scatchard analysis of the binding data 22. The number of Type I sites was estimated from Bmax of [3H]CORT in the presence of a 100-fold excess of RU 28362 and corrected for the presence of
115 CBG traces. Type II sites are those [3H]C-ORT binding sites that are displaceable by R U 28362 and corrected for traces of CBG. Hence, the binding constants of the Type II sites can be calculated by subtracting the number of [3H]CORT binding sites in the presence of R U 28362 from those in the absence of RU 28362. The values of RBA (1(750) were determined as described in the legend.
day-old hippocampal cytosol is shown in Fig. lB. The linear Scatchard plot for the Type II receptor gives a K d value of 3.2 nM and a Bmax of 92.7 fmol/mg protein, while for the Type I receptor, the K d is 0.9 nM and the Bmax is 32.6 fmol/mg protein. Table I shows the binding constants for Type I and Type II receptors at different ages in rat hippocampus. These values are corrected for traces of CBG (5-10 fmol/mg protein) which remained in the tissue after perfusion. Type I receptors are absent in the cytosol of 2- and 4-day-old hippocampi. From 8 days of age on, the receptor is present and shows adult values for both the binding affinity and capacity. The Type II receptor concentration is low in 2-day-old animals (approximately 1/10 adult levels). Concentrations increase gradually up to 12 days of age, but then in the 16-dayold animals the values return to those observed in the 8-day-old rats. At 20 days of age, levels are substantially higher again, although still considerably below those found in the adult (Table I). The binding affinity was high at the younger ages (2- and 4-day-olds) but by 8 days of age was already within the range of
RESULTS
Heterogeneity of corticosteroid binding to cytosol of neonatal and adult rats The binding data of [3H]CORT to rat hippocampal cytosol obtained from 4-day-old pups and plotted according to the method of Scatchard is shown in Fig. 1A. The linear plot for the Type II receptor gives K d values of 1.7 nM and a Brnax of 48.3 fmol/mg protein. There was total displacement of [3H]CORT binding by inclusion of a 100-fold excess R U 28362. This indicates that all binding sites correspond to the Type II receptor, as the Type I receptor concentration was below detection levels. [3H]CORT binding to the 16-
A
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120
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80
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SOUND(SM)OOST(m,wm~
Fig. 1. Scatchard plots of [3H]CORT binding to rat hippocampal cytosolic receptors in (A) 4-day-old and (B) 16-day-old animals, in the absence (~1"--~) and presence (O---C)) of a 100-fold excess RU 28362. (Inset: isotherms of the binding data from which the Scatchard plots were derived.) In the presence of a 100-fold excess RU 28362, the binding is to Type I receptor. Dashed areas: [3H]CORT binding to Type II receptor. See Materials and Methods for calculation of values of Kd (in nM) and Bmax (in fmol/mg protein). These values are also shown in Table I. In all binding assays, rco~ ranged from 0.92 to 0.98. Insets: isotherms of the binding data from which the Scatchard plots were derived.
116 TABLE 1
TABLE II
('orticosteroid binding constants to neonatal and adult rat hippocampi
Relative binding affinities (IC5o) for Type 1 and Type 11 receptors in 2-day-old and adult rat hippocampi
Binding constants of [3H]CORT binding to receptors in 2, 4, 8, 12, 16, 20 day and adult rat hippocampal cytosol. The apparent binding affinity (K~, expressed as nM) and capacity (BmaX, as fmol/mg protein) were calculated from the Scatchard analyses of the binding data of [3H]CORT, each in the presence or absence of a 100-fold excess RU 28362 (see Materials and Methods). In all binding assays, rcorrranged from 0.92 to 0.98.
To determine binding to Type 1 site samples were incubated with 1.5 nM [3H]CORT in the presence of a t00-fold excess RU 28362. To determine binding to Type II site, parallel samples were run with 4.0 nM [3H]RU 28362. In each case, competing steroids (concentration range 1.0-1000.0 nM) were simultaneously included in parallel samples. 1C5o is calculated as the concentration of the unlabelled steroid required to decrease the binding capacity of the labelled steroid by 50%.
Age (days)
Type I receptor Type 11receptor . . . . . . . . . . . . . . . . . . . . K,t (nM) B ..... Kd (nM) Bmax (frnol/mg) (fmol/mg)
Receptor
Competitors
Neonate ICy)(nM)
A dutt 1C~o (nM)
2 4 8 12 16 20 Adult
0.9 1.3 0.9 0.9 1.0
Type I
RU 28362 CORT ALDO DEX
-
425.0 1.4 6,0 10.0
Type I I
RU 28362 CORT ALDO DEX
3.7 4.5 70.0 10.0
32.1 31.7 32.6 51.9 42.1
1.0 1.7 2.3 3.2 3.2 6.4 5.0
28.7 48.3 80.9 136.6 92.7 148.3 3(Xl.5
3.0 10.0 62.0 6.8
values observed in adults (Table I). Relative binding affinities (ICso) o f C O R T , R U 28362, A L D O and D E X f o r Type I and Type H receptors in neonatal and adult hippocampi Table II shows the IC5o of C O R T , R U 28362, A L D O and D E X for T y p e I and T y p e II receptors. ICso is d e t e r m i n e d as the effective concentration at which 50% binding of the labeled steroid is displaced by the competitor, as p o i n t e d out in the legend to Table If. Results shown in Table II clearly indicate that in the adult h i p p o c a m p u s , R U 28362, the p o t e n t glucocorticoid agonist, binds to T y p e II sites with a much higher affinity than to T y p e I sites (IC5o 3.0 nM vs 425.0 nM) while C O R T and A L D O show the opposite. A L D O has also a very low affinity for the Type II site in the 2-day-old rat (IC50 3 0 - 7 0 nM). The R B A of Type II for C O R T was again higher in the neonate than in the adult. Note that the T y p e I receptor is.not present in the 2-day-old animal, and that binding to the Type II receptor was m e a s u r e d using [3H]RU 28362. DISCUSSION The results of our study point to clear differences in the d e v e l o p m e n t a l patterns of Type I and Type II corticoid receptors in the hippocampus. Thus, T y p e I
receptors are still undetectabte in the 4-day-old rat. In the next few days, however, concentrations rise dramatically so that by 8 days of age the T y p e I receptor system is already similar to that found in the adult. As from this age on, no changes are found in Type I binding constants. In contrast, Type II receptors are present already at birth, but subsequent development is a slow and uneven process. Affinity is high in the neonate, but by the end of the first week of life it has decreased to adult values, whereas the receptor concentration (Bmax) is still increasing at 20 days of age. These data not only extend b u t also differ in some i m p o r t a n t aspects from previous reports on the ontogeny of G C receptors in the brain 3'n'12'14. As mentioned previously, these studies did not discriminate between r e c e p t o r types. H o w e v e r , taking into account the present data, it can be assumed that they p r o b a b l y m e a s u r e d T y p e II receptors during the first week of life (when the Type I r e c e p t o r is absent) and the total T y p e I + II r e c e p t o r pool thereafter. A c o m p a r a b l e study to ours has been carried out in the pituitary t9. Type I r e c e p t o r ontogeny in the brain resembles that described for the pituitary in that the receptor is not present until a p p r o x i m a t e l y the end of the first week. H o w e v e r , in the pituitary, r e c e p t o r concentrations rise gradually and do not achieve adult levels until the 3rd or 4th week of life. This con-
117 trasts sharply with what occurs in the hippocampus, where adult concentrations are reached in a matter of days. Type II receptors in the pituitary are present at birth in adult concentration and affinity, and change very little with age. Again, this contrasts with that found in the hippocampus, where perinatal levels are only about 1/10 of adult concentrations and there are marked changes with age both in the affinity and capacity of the Type II receptor system. With regard to the Type II capacity in the neonates, the value increases gradually up to the age of 20 days with the exception of that at age 12, the value of which is greater than that of day 8 and day 16. This phenomenon is as yet unexplained, but it is interesting to note that in our immunocytochemical studies TM, the observed immunoreactive staining at day 12 is less than that seen at day 8. When evaluating biochemical data, it is necessary to take certain factors into account. First, animals must be ADX in order to eliminate endogenous corticosteroids. Beyond the non-specific effects (of unknown consequences) of the operation itself, ADX has been shown in the adult animal to have a differential effect on the number of receptor sites available for in vitro radioligand binding in cytosol. Thus, Type I availability shows a marked increase within 4-7 h, presumably due to the depletion of endogenous hormone, and no further increase thereafter; while Type II availability, on the other hand, remains relatively constant in the first 24 h post-surgery, but then increases gradually for several days 17. Although autoregulation does not seem to occur perinatally 2~, it cannot be ruled out with certainty until it is studied in the individual receptor types. Second, this being an in vitro assay, caution should be exercised when interpreting in vivo phenomena. The presence of a cytosolic receptor capable of binding CORT in vitro does not necessarily imply that this same receptor is capable of binding to DNA in vivo. Immunocytochemical data in fact suggest that the 'activated' state of the receptor, capable of binding to cell nuclear DNA 25, also shows an ontogenetic change TM. Third, although the cytosol binding assay does permit the estimation of the binding constants of the receptor types present in the hippocampus, the immunocytochemical data show that the Type II receptor immunoreactivity changes dramatically both in intensity
and localization as a function of age within small areas of the hippocampus itself. However, in spite of the limitations of the technique, the cytosol binding assay provides important information on the apparent maximum capacity and affinity of the receptor system, not available otherwise. The functional implications of these findings have yet to be explored. It is interesting to note though that the Type I receptor does not appear until the end of the first postnatal week. This is true both for the pituitary and the hippocampus. This fact could be of value in discriminating the receptor types underlying the different effects of CORT. Thus, the effects of CORT administered during the first few days of life must be assumed to be mediated by the Type II receptor. Similarly, one would expect hippocampal Type I receptor-mediated functions to show a sudden 'turning on' during development concordant with the rapid rise in receptor concentrations. It is of interest in this respect that before the appearance of the Type I receptor, the affinity of the Type II receptor is in the range of the Type I receptor affinity. The Type II receptor affinity approaches adult value only after day 8. This observation raises the possibility that the Type II receptor displays Type I characteristics in the binding of CORT during the first post-natal week. Hippocampal glucocorticoid receptors have been implicated in the turning off of the stress-induced CORT response s'2°'2J. This negative feedback has been shown to be deficient in the immature animals only acquiring adult characteristics around the end of the first month of life. This correlates with the gradual increase in Type II receptor concentrations, suggesting that this receptor may in fact mediate this function. Clearly these data, in conjunction with other data obtained from immunocytochemical studies indicate a pattern of development of corticoid receptors in the brain that is far more complex than the existing descriptions of the ontogeny of these receptors. It has been well documented that the corticoid receptors show dramatic alterations during a critical period of developmental changes in the functional regulation of the HPA system. The relationship between the functional aspects of the system and the observed changes in corticoid receptors in the brain remains to be established.
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