Brain Research, 105 (1976) 121-128
121
© Elsevier ScientificPublishingCompany,Amsterdam- Printed in The Netherlands
GLUCOCORTICOID BINDING TO RECEPTOR-LIKE PROTEINS IN RAT BRAIN AND PITUITARY: ONTOGENETIC AND EXPERIMENTALLY INDUCED CHANGES
H A N S - R U D O L F OLPE AND B R U C E S. M c E W E N
The Rockefeller University, New York, N. Y. 10021 (U.S.A.) (Accepted August 19th, 1975)
SUMMARY
Cytosol binding of [aH]corticosterone and [3H]dexamethasone was measured in various brain areas and the pituitary of perfused rats 12 h and 72 h, respectively, after adrenalectomy (ADX). A considerable regional hetelogeneity was found 12 h post ADX representing differences of the normal cytosol binding capacities between various areas. When the second phase of the adrenalectomy-induced increase in binding capacity was allowed to develop (72 h post ADX), the cytosol binding of all regions increased to various extents. The highest percentage increases were found in those areas with the highest glucocorticoid binding capacity, namely the hippocampus and the septum. The ontogeny of the cytosol glucocorticoid binding macromolecules was investigated in the hippocampus, hypothalamus and pituitary using [3H]corticosterone and [~H]dexamethasone. The concentration of corticosterone binding sites is lowest in all three areas around day one and then increases by a factor of 2-3 reaching adult levels around day 32. For [~H]dexamethasone a similar pattern was observed in the hippocampus and hypothalamus. In the pituitary, however, the concentration of binding sites was slightly higher at day 1 than at any later developmental stage. Intenuption of the fimbria in 3-day-old rats did not affect the development of the binding sites in the hippocampus. In an attempt to interfere with the normal glucocorticoid binding of the hippocampus as well as with the postadrenalectomy increase of the cytosol binding sites, bilateral transection of the fimbria was performed either 3 days or 80 days before ADX. In neither case did fimbria transection prevent the increase of the binding sites. The intrinsic (12 h post ADX) cytosol binding capacity of the hippocampus was also not affected by this lesion.
122 INTRODUCTION
Corticosterone, the principal glucocorticoid of the rat, is taken up from the blood and bound in a distinctive regional pattern to both cytosol and nuclear proteins of various brain regions of adrenalectomized rats 2,10,13. The highest concentration of bound hormone is found in the hippocampus 10. Previous work from this laboratory has shown that the binding capacity of the hippocampus for corticosterone increases in a biphasic pattern following adrenalectomy11. A rapid initial increase occurs during the first 2 h. The binding capacity measured at that time and up to 12 h post adrenalectomy (ADX) reflects the normal binding of the structure in the absence of endogenous hormone. A second rise was observed to begin more than 12 h post ADX, reaching a plateau 3-5 days after the operation. We investigated in the present study whether the second postadrenalectomy increase of glucocorticoid binding sites is a general property shared by other brain areas, which also bind glucocorticoids. To study the mechanism of the postadrenalectomy increase, we performed experiments in which the hippocampus was deprived of its rostral efferents and afferents. The hippocampus appeared to be an ideal structure in which to attack this problem since it is anatomically well defined, with distinct connections to other brain areas through the fimbria and alvear pathways. We also tried to change the concentration of binding sites in the hippocampus by transecting the fimbria in young rats in which binding levels were not yet fully developed. To select a proper developmental stage we first studied the ontogenetic pattern of glucocorticoid binding sites in the brain and pituitary. METHODS
Male Sprague-Dawley rats (CD strain, Charles River Breeding Labs., Wilmington, Mass.) of 300-400 g body weight were used. The animals were housed in group cages in a room lighted between 5 a.m. and 7 p.m. and with Purina Rat Chow and water available ad libitum. Bilateral adrenalectomy was performed either 12 h or 3 days before sacrificing the animals. All rats used in the ontogeny study were adrenalectomized 14-15 h before the experiment. Adult rats were maintained post ADX on 0.9 70 saline in drinking water in groups of 4-5 rats/cage. The pups were isolated from their mother after the operation and kept at a temperature of 37 °C. [l,2-aH]Corticosterone ([all]B; 40 Ci/mmole; New England Nuclear Corp., Boston, Mass.) and [1,2-3H]dexamethasone ([aH]Dex; 29 Ci/mmole; AmershamSearle, Chicago, Ill.) were checked for purity by thin layer chromatography. The in vitro determination of the binding of both [aH]B and [aH]Dex was carried out according to the procedure described elsewhere 5. Only the main methodological steps and alterations of the method will be mentioned. The rats were killed between 8 and 10 a.m. After perfusion of the rats with 6 70 dextran in 0.86 70 saline, the brains were quickly removed and dissected into the following areas as described elsewhere, namely: hippocampus, septum, amygdala, hypothalamus and cortex9,1~. The pituitary was also included as a separate sample. The labeled hormones [aH]B or [aH]Dex
123 were added at a final concentration of 1.8 × 10-s M to the homogenized tissues and these were kept in an ice bath for 4 h in order to allow equilibration with the binding proteins. During the incubation the homogenates were centrifuged at 4 °C for 1 h at 105,000 × gay. At the end of 4 h a 0.1-ml aliquot of the supernatant (cytosol) was passed over an LH 20 (Sephadex) column as described by deKloet et al. 5 and the eluate containing the protein-bound hormone was collected in a scintillation vial. Aliquots of 0.1 ml of the eluate were used for protein determinations according to the method of Lowry et al. s. Protein values in eluates of LH 20 columns are higher by two times than in eluates of G25 (Sephadex) columns, due to the presence of lower molecular weight peptides in the former. These binding values are approximately half of those obtained from the G25 method 5,11. The radioactivity of the remainder was counted in a 10~ Biosolve (BBS 3, Beckman Instruments) toluene scintillation mixture at 40 ~o efficiency. A bilateral transection of the fimbria was performed in the iats by lowering a knife vertically into the brain according to the method of Lengvari and Hal~sz 7 using a David Kopf stereotaxic apparatus. The coordinates and the operation procedure has been described elsewhere 14. Sham operated rats were opened at the skull and the superior sagittal sinus ruptured by a knife cut. In baby rats the fimbria transection was performed in a free hand cut by lowering a surgical knife 3 mm into the skull and cutting 2 mm laterally at a coronal plane situated half a millimeter rostral to the bregma. Only those rats were used for measuring cytosol binding which upon inspection after sacrifice showed a complete transection of their fimbria. RESULTS
The long-term increase of glucocorticoid binding sites in brain following adrenalectomy was originally observed in the hippocampus 11, but not studied in other brain areas. To further investigate the mechanism of this phenomenon, the obvious next step was to repeat the same kind of experiments including additional brain areas which from our previous work are known to display considerable glucocorticoid binding. In Table I the binding capacity of various structures at two different time intervals following adrenalectomy is shown for both [3H]corticosterone ([3H]B) and [3H]dexamethasone ([3H]Dex). The amount of bound hormone per area (12 h post ADX) shows considerable regional heterogeneity. For [SH]B a high binding was found in both the hippocampus and pituitary, whereas the hypothalamus bound significantly less hormone. For [aH]Dex highest levels of bound hormone were detected in the septum and hippocampus. The hypothalamus, amygdala and cortex displayed lower binding. The lowest level of bound [3H]Dex was observed in the pituitary. These binding values, measured 12 h post ADX, probably indicate the normal binding of each structure in the absence of endogenous hormone 11. The binding capacity determined 72 h post ADX is higher in all structures than the values found 12 h post ADX (Table I), indicating that the postadrenalectomy increase occurs in all structures. However, the percentage of the postadrenalectomy increase appears to be larger
124 TABLE I BINDING OF [3H]CORTICOSTERONE AND [3H]DEXAMETHASONE in vitro TO CYTOSOL BINDING SITES 12 n AND 72 H RESPECTIVELY AFTER ADRENALECTOMY
(Expt. No.)
A D X 12 h
A D X 72 h
Increase
(1) (2) (1) (2) (1) (2)
144 ~ 25 151 ± 24 105 ± 11 108 ± 18 139 A__25 244 :~: 40
319 ± 9 292 -~_30 137 ± 21 161 +_ 39 269 -2:23 434 ± 82
121%* 93 ~* 30%** 39~** 93 ~* 78 ~*
(3) (4) (3) (4) (3) (4) (5) (5) (5)
173 ~: 27 173 ~ 29 125 ± 13 120 ± 8 63 -J: 9 66 ~ 6 151 -2:26 113 _-jz15 121 ± 11
284 ± 17 303 :~ 12 181 _L 13 172 ~ 17 77 5_ 12 81 ± 11 271 ± 31 177 ± 19 200 ± 29
For i 'aH]eorticosterone
Hippocampus Hypothalamus Pituitary For [ 3H]dexamethasone
Hippocampus Hypothalamus Pituitary Septum§ Amygdala§ Cortex§
75 %* 65%** 45%** 43~** 22~*** 20~*** 80~* 56~* 65~*
§ Results are expressed in femtomoles of bound radioactivity/mg protein. Each value is the average of data from 4 animals -k S.E.M., except for the values marked with a §. These results represent the average of data from 12 animals each. Students t-test: * P < 0.01; ** P < 0.05; *** P < 0.2.
for structures having highest binding as m e a s u r e d 12 h p o s t A D X . F o r [3H]B, the highest increase occurs in the h i p p o c a m p u s a n d for [3H]Dex, in the h i p p o c a m p u s a n d septum. (The s e p t u m was n o t studied for [3H]B binding.) A D X does n o t p r o d u c e the same increase for the two h o r m o n e s in a given structure. This difference o f the binding c a p a c i t y between [3H]Dex a n d [3H]B 72 h p o s t A D X is m o s t a p p a r e n t in the h i p p o c a m p u s and pituitary. In the h i p p o c a m p u s the binding is some 35 ~ lower and in the p i t u i t a r y a b o u t 60 ~ lower for [3H]Dex t h a n for [3H]B. H a v i n g f o u n d that the p o s t a d r e n a l e c t o m y increase o f glucocorticoid binding sites is m o s t p r o n o u n c e d in the h i p p o c a m p u s and septum, we designed the fimbria lesion experiments which we expected w o u l d tell us if this increase is triggered by a neural i n p u t entering the h i p p o c a m p u s t h r o u g h the fimbria. F i r s t we investigated the effect of the fimbria transection on the n o r m a l cytosol binding o f [3H]B in the h i p p o c a m p u s , h y p o t h a l a m u s a n d pituitary. W e f o u n d that this o p e r a t i o n p e r f o r m e d either 6 or 80 days before the sacrifice o f the rat did n o t affect the level of b o u n d [3H]B (Table I I A a n d B). In these experiments, the time between A D X a n d the b i n d i n g assay was 13 h. To investigate if the fimbria transection affects the p o s t a d r e n a l e c t o m y increase, the same kind o f e x p e r i m e n t was repeated in this case with an interval o f 3 days between A D X a n d the binding d e t e r m i n a t i o n . The levels o f b o u n d [3H]Dex
125 TABLE II BINDING OF [3H]CORTICOSTERONE A N D [3H]DEXAMETHASONE in vitro TO CYTOSOL BINDING SITES I N RATS W I T H TRANSECTED FIMBRIA AND I N SHAM OPERATED ANIMALS
Results are expressed in femtomoles of bound radioactivity per mg protein. Each value is the mean of data from 4 rats 4- S.E.M. Time interval between fimbria transection and sacrifice of the rats: A, 6 days; B, 80 days; C, 40 days (age of the rats at the time of fimbria transection: 3 days old); D, 6 days. Sham Operated
Fimbria Operated Increase
Time after A D X
1704-24 1104-14 180 4- 21
1844-18 115 4- 11 169 :~ 13
8% 4% --6%
13h
151 4-30 107 ± 18 1734-26
165 4-27 119 4- 14 1794-20
9~ 11% --3%
13h
1964- 15
191 4-17
--2%
13 h
193 4- 16 198 4- 43 107 4- 22 157 -4- 18 63± 5 139 4- 9
240 -4- 22 238 -4- 37 139 4- 32 164 4- 25 67 4- 2 148 4- 15
24%* 22%** 30~** 5%** 6~** 6%**
72 h
For corticosterone
(A) Adult rats Hippocampus Hypothalamus Pituitary (B) Adult rats Hippocampus Hypothalamus Pituitary (C) Young rats Hippocampus For dexamethasone
(D) Adult rats Hippocampus Hypothalamus Pituitary
* P < 0.05; ** P < 0.2 (Student's t-test).
were not found to be significantly changed (Table liD). Only a tendency for an increase related to fimbria transection could be seen in the hippocampus. We then tried to alter the amount of bound hormone in adult rats by transecting the fimbria at an early developmental stage at which the cytosol receptors were not yet fully developed. To select a proper postnatal age, we first studied the developmental pattern of glucocorticoid binding proteins. The binding capacity of the hippocampus, hypothalamus and pituitary for both [ZH]B and [aH]Dex during ontogeny is shown in Fig. I. For [aH]B a considerable postnatal increase of the concentration of binding sites was observed in all 3 areas reaching adult levels around day 32. In the hippocampus a 3-fold, and in the hypothalamus and pituitary a 2-fold, increase of the concentration of binding sites could be observed. The two latter areas display an almost linear increase with time of [ZH]B binding capacity, in contrast to the pituitary where there appears to be a stepwise development. The developmental pattern found with [3H]Dex was different from the one determined with [aH]B. In the hippocampus and hypothalamus the number of binding sites increases considerably during the first 15 days. At day 15 levels of bound hor-
126 200 Hipp°campusIHipPT~II "~
#,
. . . . . . . . .
100 ~
f
t -I
$
200 Hypothalamus Io0
"-I
0
n- 2OO m 100
~II
8
. . . . . . . 15
24
32 Doys
--1" Adult
Fig. 1. Developmental time course of the binding of [aH]dexamethasone (dashed line) and [3H]corticosterone (solid line) to cytosol macromolecules of rats adrenalectomized 14-15 h before sacririce and perfused through the heart at sacrifice. Binding, expressed as fmoles aH radioactivity bound/ mg of cytosol protein, is presented as a function of postnatal age (in days) of the rats at sacrifice.
mone are equivalent to those in the adult. The pituitary, in contrast, showed a higher [aH]Dex binding during the very first postnatal days than in the adult. DISCUSSION
The regional study of glucocorticoid binding to receptor-like proteins presented in this paper is consistent with earlier findings from this laboratory12, showing the highest binding to occur in the hippocampus and septum. Regional studies of glucocorticoid cytosol binding have been presented also by Grosser et aL 3 using [all]B, and by Lassman and Mulrow 6 for [3H]deoxycorticosterone. In agreement with our data both authors demonstrated the highest cytosol binding in the hippocampus. However, the pattern of binding to cytosol from other brain areas differs considerably among the three glucocorticoids. With [aH]deoxycorticosterone the next highest binding capacity following the hippocampus was found in the cortex and amygdala and less binding was found in the hypothalamus. With [3H]Dex we observed a higher binding in the hypothalamus, but less in the amygdala relative to the hippocampus. Comparing our findings with the [3H]B binding pattern observed by Grosser et al. 3, we found a similar high binding of [SH]Dex in the septum relative to the hippocampus but less was bound to the hypothalamus. These discrepencies might reflect real differences in the avidity of various corticoids for a single type of binding protein or they could be interpreted as an indication of the presence of various kinds of receptor molecules s. The second postadrenalectomy increase of the glucocorticoid binding capacity originally detected in the hippocampus 11 was found to be a property of all areas
127 investigated in this study. However, the largest increase of the [aH]Dex binding sites (72 h post ADX) is shown to occur in the hippocampus and septum, followed by the hypothalamus, amygdala and cortex. The pituitary, having the lowest binding capacity for [3H]Dex, also showed the smallest postadrenalectomy increase of its binding activity. With [SH]B this area bound almost as much hormone as the hippocampus and displayed a lalge increase of its binding sites post ADX. The high [3H]B binding and the large second post-adrenalectomy increase of its binding can be explained by the presence of a transcortin-like protein known to be present in the pituitary, which binds corticosterone and not dexamethasone (deKloet, personal communication). It is known that transcortin levels in blood rise after ADX 15,1~,18, but the origin of pituitary transcortin-like material is presently not known. It does not appear to be an artifact of blood contamination of pituitary tissue (deKloet, personal communication), deKloet et al. 5 presented evidence for the existence of two different populations of glucocorticoid binding proteins. Their conclusion is based on the observation that steroid binding activities toward [3H]B and [3H]Dex are inactivated at different rates in the absence of steroid, and that [3H]B and [3H]Dex show distinct and different regional distributions for in vivo nuclear binding. This study adds further support to this idea. First, the in vitro cytosol binding of the hippocampus, hypothalamus and pituitary are not identical for the two hormones, even though the dissociation constants are the same 5. Second, the size of the second postadrenalectomy increase differs for the two hormones. This is best illustrated by the pituitary, where the binding capacity differs by a factor of 2-3 and where the postadrenalectomy increase is about 4 times larger with [3H]Dex than with [aH]B. From our previous work we knew already that the second postadrenalectomy increase is independent of the thyroid gland or any hypophyseal hormone secretion11. It was conceivable therefore that the increase is induced through the trophic influence of some afferent nerve fibers. Transection of the fimbria 6 or 80 days before measuring the hormone binding capacity did not affect the amount of normally present binding sites in any of the investigated areas. Also, the postadrenalectomy increase was not affected by this operation, suggesting that this process is not triggered by rostral afferents of the hippocampus. It is still possible, however, that afferents of the alvear pathway might be involved in the regulation of this phenomenon. Despite these negative findings, it was conceivable that these rostral afferents are perhaps more important for the establishment of the binding sites at an early developmental stage. Fimbria transection was performed in 3-day-old rats for the following two reasons. First, the cytosol binding capacity is low at that developmental stage. Second, the hippocampus has not yet received the cholinergic afferents at this stage 17. Transection of the fimbria at day 3 did not affect the adult level of binding sites (12 h post ADX) in these rats, suggesting that the ontogeny of these molecules is probably an autonomous property of the hippocampus or is regulated by the alvear pathway. The amount of [3H]B and [aH]Dex binding sites increases considerably during ontogeny in all investigated areas. A similar developmental pattern of [3H]Dex binding sites has been described also for the rat liver by Henning et al. a. In the
128 intestine and lungs, however, the same authors f o u n d a higher b i n d i n g capacity in y o u n g t h a n in adult rats, which is a similar finding to the p a t t e r n described in this work for the [aH]Dex b i n d i n g proteins in the pituitary. ACKNOWLEDGEMENTS This work was supported by Research G r a n t NS 07080 from the U n i t e d States Public Health Service (to Bruce McEwen) a n d by fellowships (to H a n s - R u d o l f Olpe) f r o m the U S P H S F o r e i g n Postdoctoral Program, The H o l d e r b a n k C e m e n t Factory ( H o l d e r b a n k , Switzerland), a n d from the J a n g g e n - P o e h n Stiftung (St. Gallen, Switzerland).
REFERENCES 1 FONNUM,F., Topographical and subceUular localization of choline acetyltransferase in rat hippocampal region, J. Neurochem., 17 (1970) 1029-1037. 2 GROSSER,B. I., STEVENS,W., BRUENGER,F. W., AND REED, D. J., Corticosterone binding by rat brain cytosol, J. Neuroehem., 18 (1971) 1725-1732. 3 GROSSER,B. I., STEVENS,W., AND REED, D. J., Properties of corticosterone-binding macromolecules from rat brain cytosol, Brain Research, 57 (1973) 387-395. 4 HENNING, S. J., BALLARD, P. L., AND KRETCHMER, N., A study of the cytoplasmic receptors for glucocorticoids in intestine of pre- and postwearding rat, J. bioL Chem., 250 (1975) 2073-2079. 5 DEKLOET,E. R., WALLACH,G., AND MCEWEN, B. S., Differences in corticosterone and dexamethasone binding to rat brain and pituitary, Endocrinology, 96 (1975) 598-609. 6 LASSMAN,M. N., AND MULROW,P. J., Deficiency of deoxycorticosterone-binding protein in the hypothalamus of rats resistent to deoxycorticosterone-induced hypertension, Endocrinology, 94 (1974) 1541-1546. 7 LENGVARI, I., AND HALASZ, B., Evidence for a diurnal fluctuation in plasma corticosterone levels after fornix transection in the rat, Neuroendocrinology, 11 (1973) 113-136. 8 LOWRY, O. H., ROSEBROUOH,N. J., FARR. A. L., AND RANDALL,R. J.. Protein measurement with the Folin phenol reagent J. biol. Chem., 193 (1951) 265-275. 9 MCEWEN,B. S., ANDPFAFE,D. W., Factors influencing sex hormone uptake by rat brain regions. I. Effects of neonatal treatment, hypophysectomy, and competing steroid on estradiol uptake, Brain Research, 21 (1970) 1-16. 10 McEWEN,B. S., ANDWALLACH,G., Corticosterone binding to hippocampus: Nuclear and cytosol binding in vitro, Brain Research, 57 (1973) 373-386. 11 McEWEN, B. S., WALLACH, G., AND MAGNUS, C., Corticosterone binding to hippocampus: Immediate and delayed influences of the absence of adrenal secretion, Brain Research, 70 (1974) 321 334. 12 McEWEN, B. S., WEISS,J. M., AND SCHWARTZ, L. S., Uptake of corticosterone by rat brain and its concentration by certain limbic structures, Brain Research, 16 (1969) 227-241. 13 MCEWEN,B. S., WEISS,J. M., AND SCHWARTZ,L. S., Retention of corticosterone by cell nuclei from brain regions of adrenalectomized rats, Brain Research, 17 (1970) 471-482. 14 OLPE, H. R., ANDMCEWEN,B. S., Axonal transport in the efferent pathways of the hippocampus: labeling of projection areas after [3H]valine injections, Brain Research, (1976) in press. 15 PERRIN, F. M., AND FOREST, M. G., Time course of the effect of adrenalectomy on transcortin binding characteristics: appraisal of different methods of calculation, Endocrinology, 96 (1975) 869-878. 16 SEAL, U. S., AND DOE, R. P., Vertebrate distribution of corticosteroid-binding globulin and some endocrine effects on concentration, Steroids, 5 (1965) 827-841. 17 SORIMACHI, M., AND KATAOKA, K., High affinity choline uptake: an early index of cholinergic innervation in rat brain, Brain Research, 94 (1975) 325-336. 18 WESTPHAL,U., WILLIAMS,W. C., JR., ASHLEY, B. D., UND DE VENUTO, F;'., Proteinbindung der Corticosteroide im Serum adrenalektomierter und hypophysektomierter Ratten, Hoppe-Seylers z. physiol. Chem., 332 (1963) 54-69.
121
© Elsevier ScientificPublishingCompany,Amsterdam- Printed in The Netherlands
GLUCOCORTICOID BINDING TO RECEPTOR-LIKE PROTEINS IN RAT BRAIN AND PITUITARY: ONTOGENETIC AND EXPERIMENTALLY INDUCED CHANGES
H A N S - R U D O L F OLPE AND B R U C E S. M c E W E N
The Rockefeller University, New York, N. Y. 10021 (U.S.A.) (Accepted August 19th, 1975)
SUMMARY
Cytosol binding of [aH]corticosterone and [3H]dexamethasone was measured in various brain areas and the pituitary of perfused rats 12 h and 72 h, respectively, after adrenalectomy (ADX). A considerable regional hetelogeneity was found 12 h post ADX representing differences of the normal cytosol binding capacities between various areas. When the second phase of the adrenalectomy-induced increase in binding capacity was allowed to develop (72 h post ADX), the cytosol binding of all regions increased to various extents. The highest percentage increases were found in those areas with the highest glucocorticoid binding capacity, namely the hippocampus and the septum. The ontogeny of the cytosol glucocorticoid binding macromolecules was investigated in the hippocampus, hypothalamus and pituitary using [3H]corticosterone and [~H]dexamethasone. The concentration of corticosterone binding sites is lowest in all three areas around day one and then increases by a factor of 2-3 reaching adult levels around day 32. For [~H]dexamethasone a similar pattern was observed in the hippocampus and hypothalamus. In the pituitary, however, the concentration of binding sites was slightly higher at day 1 than at any later developmental stage. Intenuption of the fimbria in 3-day-old rats did not affect the development of the binding sites in the hippocampus. In an attempt to interfere with the normal glucocorticoid binding of the hippocampus as well as with the postadrenalectomy increase of the cytosol binding sites, bilateral transection of the fimbria was performed either 3 days or 80 days before ADX. In neither case did fimbria transection prevent the increase of the binding sites. The intrinsic (12 h post ADX) cytosol binding capacity of the hippocampus was also not affected by this lesion.
122 INTRODUCTION
Corticosterone, the principal glucocorticoid of the rat, is taken up from the blood and bound in a distinctive regional pattern to both cytosol and nuclear proteins of various brain regions of adrenalectomized rats 2,10,13. The highest concentration of bound hormone is found in the hippocampus 10. Previous work from this laboratory has shown that the binding capacity of the hippocampus for corticosterone increases in a biphasic pattern following adrenalectomy11. A rapid initial increase occurs during the first 2 h. The binding capacity measured at that time and up to 12 h post adrenalectomy (ADX) reflects the normal binding of the structure in the absence of endogenous hormone. A second rise was observed to begin more than 12 h post ADX, reaching a plateau 3-5 days after the operation. We investigated in the present study whether the second postadrenalectomy increase of glucocorticoid binding sites is a general property shared by other brain areas, which also bind glucocorticoids. To study the mechanism of the postadrenalectomy increase, we performed experiments in which the hippocampus was deprived of its rostral efferents and afferents. The hippocampus appeared to be an ideal structure in which to attack this problem since it is anatomically well defined, with distinct connections to other brain areas through the fimbria and alvear pathways. We also tried to change the concentration of binding sites in the hippocampus by transecting the fimbria in young rats in which binding levels were not yet fully developed. To select a proper developmental stage we first studied the ontogenetic pattern of glucocorticoid binding sites in the brain and pituitary. METHODS
Male Sprague-Dawley rats (CD strain, Charles River Breeding Labs., Wilmington, Mass.) of 300-400 g body weight were used. The animals were housed in group cages in a room lighted between 5 a.m. and 7 p.m. and with Purina Rat Chow and water available ad libitum. Bilateral adrenalectomy was performed either 12 h or 3 days before sacrificing the animals. All rats used in the ontogeny study were adrenalectomized 14-15 h before the experiment. Adult rats were maintained post ADX on 0.9 70 saline in drinking water in groups of 4-5 rats/cage. The pups were isolated from their mother after the operation and kept at a temperature of 37 °C. [l,2-aH]Corticosterone ([all]B; 40 Ci/mmole; New England Nuclear Corp., Boston, Mass.) and [1,2-3H]dexamethasone ([aH]Dex; 29 Ci/mmole; AmershamSearle, Chicago, Ill.) were checked for purity by thin layer chromatography. The in vitro determination of the binding of both [aH]B and [aH]Dex was carried out according to the procedure described elsewhere 5. Only the main methodological steps and alterations of the method will be mentioned. The rats were killed between 8 and 10 a.m. After perfusion of the rats with 6 70 dextran in 0.86 70 saline, the brains were quickly removed and dissected into the following areas as described elsewhere, namely: hippocampus, septum, amygdala, hypothalamus and cortex9,1~. The pituitary was also included as a separate sample. The labeled hormones [aH]B or [aH]Dex
123 were added at a final concentration of 1.8 × 10-s M to the homogenized tissues and these were kept in an ice bath for 4 h in order to allow equilibration with the binding proteins. During the incubation the homogenates were centrifuged at 4 °C for 1 h at 105,000 × gay. At the end of 4 h a 0.1-ml aliquot of the supernatant (cytosol) was passed over an LH 20 (Sephadex) column as described by deKloet et al. 5 and the eluate containing the protein-bound hormone was collected in a scintillation vial. Aliquots of 0.1 ml of the eluate were used for protein determinations according to the method of Lowry et al. s. Protein values in eluates of LH 20 columns are higher by two times than in eluates of G25 (Sephadex) columns, due to the presence of lower molecular weight peptides in the former. These binding values are approximately half of those obtained from the G25 method 5,11. The radioactivity of the remainder was counted in a 10~ Biosolve (BBS 3, Beckman Instruments) toluene scintillation mixture at 40 ~o efficiency. A bilateral transection of the fimbria was performed in the iats by lowering a knife vertically into the brain according to the method of Lengvari and Hal~sz 7 using a David Kopf stereotaxic apparatus. The coordinates and the operation procedure has been described elsewhere 14. Sham operated rats were opened at the skull and the superior sagittal sinus ruptured by a knife cut. In baby rats the fimbria transection was performed in a free hand cut by lowering a surgical knife 3 mm into the skull and cutting 2 mm laterally at a coronal plane situated half a millimeter rostral to the bregma. Only those rats were used for measuring cytosol binding which upon inspection after sacrifice showed a complete transection of their fimbria. RESULTS
The long-term increase of glucocorticoid binding sites in brain following adrenalectomy was originally observed in the hippocampus 11, but not studied in other brain areas. To further investigate the mechanism of this phenomenon, the obvious next step was to repeat the same kind of experiments including additional brain areas which from our previous work are known to display considerable glucocorticoid binding. In Table I the binding capacity of various structures at two different time intervals following adrenalectomy is shown for both [3H]corticosterone ([3H]B) and [3H]dexamethasone ([3H]Dex). The amount of bound hormone per area (12 h post ADX) shows considerable regional heterogeneity. For [SH]B a high binding was found in both the hippocampus and pituitary, whereas the hypothalamus bound significantly less hormone. For [aH]Dex highest levels of bound hormone were detected in the septum and hippocampus. The hypothalamus, amygdala and cortex displayed lower binding. The lowest level of bound [3H]Dex was observed in the pituitary. These binding values, measured 12 h post ADX, probably indicate the normal binding of each structure in the absence of endogenous hormone 11. The binding capacity determined 72 h post ADX is higher in all structures than the values found 12 h post ADX (Table I), indicating that the postadrenalectomy increase occurs in all structures. However, the percentage of the postadrenalectomy increase appears to be larger
124 TABLE I BINDING OF [3H]CORTICOSTERONE AND [3H]DEXAMETHASONE in vitro TO CYTOSOL BINDING SITES 12 n AND 72 H RESPECTIVELY AFTER ADRENALECTOMY
(Expt. No.)
A D X 12 h
A D X 72 h
Increase
(1) (2) (1) (2) (1) (2)
144 ~ 25 151 ± 24 105 ± 11 108 ± 18 139 A__25 244 :~: 40
319 ± 9 292 -~_30 137 ± 21 161 +_ 39 269 -2:23 434 ± 82
121%* 93 ~* 30%** 39~** 93 ~* 78 ~*
(3) (4) (3) (4) (3) (4) (5) (5) (5)
173 ~: 27 173 ~ 29 125 ± 13 120 ± 8 63 -J: 9 66 ~ 6 151 -2:26 113 _-jz15 121 ± 11
284 ± 17 303 :~ 12 181 _L 13 172 ~ 17 77 5_ 12 81 ± 11 271 ± 31 177 ± 19 200 ± 29
For i 'aH]eorticosterone
Hippocampus Hypothalamus Pituitary For [ 3H]dexamethasone
Hippocampus Hypothalamus Pituitary Septum§ Amygdala§ Cortex§
75 %* 65%** 45%** 43~** 22~*** 20~*** 80~* 56~* 65~*
§ Results are expressed in femtomoles of bound radioactivity/mg protein. Each value is the average of data from 4 animals -k S.E.M., except for the values marked with a §. These results represent the average of data from 12 animals each. Students t-test: * P < 0.01; ** P < 0.05; *** P < 0.2.
for structures having highest binding as m e a s u r e d 12 h p o s t A D X . F o r [3H]B, the highest increase occurs in the h i p p o c a m p u s a n d for [3H]Dex, in the h i p p o c a m p u s a n d septum. (The s e p t u m was n o t studied for [3H]B binding.) A D X does n o t p r o d u c e the same increase for the two h o r m o n e s in a given structure. This difference o f the binding c a p a c i t y between [3H]Dex a n d [3H]B 72 h p o s t A D X is m o s t a p p a r e n t in the h i p p o c a m p u s and pituitary. In the h i p p o c a m p u s the binding is some 35 ~ lower and in the p i t u i t a r y a b o u t 60 ~ lower for [3H]Dex t h a n for [3H]B. H a v i n g f o u n d that the p o s t a d r e n a l e c t o m y increase o f glucocorticoid binding sites is m o s t p r o n o u n c e d in the h i p p o c a m p u s and septum, we designed the fimbria lesion experiments which we expected w o u l d tell us if this increase is triggered by a neural i n p u t entering the h i p p o c a m p u s t h r o u g h the fimbria. F i r s t we investigated the effect of the fimbria transection on the n o r m a l cytosol binding o f [3H]B in the h i p p o c a m p u s , h y p o t h a l a m u s a n d pituitary. W e f o u n d that this o p e r a t i o n p e r f o r m e d either 6 or 80 days before the sacrifice o f the rat did n o t affect the level of b o u n d [3H]B (Table I I A a n d B). In these experiments, the time between A D X a n d the b i n d i n g assay was 13 h. To investigate if the fimbria transection affects the p o s t a d r e n a l e c t o m y increase, the same kind o f e x p e r i m e n t was repeated in this case with an interval o f 3 days between A D X a n d the binding d e t e r m i n a t i o n . The levels o f b o u n d [3H]Dex
125 TABLE II BINDING OF [3H]CORTICOSTERONE A N D [3H]DEXAMETHASONE in vitro TO CYTOSOL BINDING SITES I N RATS W I T H TRANSECTED FIMBRIA AND I N SHAM OPERATED ANIMALS
Results are expressed in femtomoles of bound radioactivity per mg protein. Each value is the mean of data from 4 rats 4- S.E.M. Time interval between fimbria transection and sacrifice of the rats: A, 6 days; B, 80 days; C, 40 days (age of the rats at the time of fimbria transection: 3 days old); D, 6 days. Sham Operated
Fimbria Operated Increase
Time after A D X
1704-24 1104-14 180 4- 21
1844-18 115 4- 11 169 :~ 13
8% 4% --6%
13h
151 4-30 107 ± 18 1734-26
165 4-27 119 4- 14 1794-20
9~ 11% --3%
13h
1964- 15
191 4-17
--2%
13 h
193 4- 16 198 4- 43 107 4- 22 157 -4- 18 63± 5 139 4- 9
240 -4- 22 238 -4- 37 139 4- 32 164 4- 25 67 4- 2 148 4- 15
24%* 22%** 30~** 5%** 6~** 6%**
72 h
For corticosterone
(A) Adult rats Hippocampus Hypothalamus Pituitary (B) Adult rats Hippocampus Hypothalamus Pituitary (C) Young rats Hippocampus For dexamethasone
(D) Adult rats Hippocampus Hypothalamus Pituitary
* P < 0.05; ** P < 0.2 (Student's t-test).
were not found to be significantly changed (Table liD). Only a tendency for an increase related to fimbria transection could be seen in the hippocampus. We then tried to alter the amount of bound hormone in adult rats by transecting the fimbria at an early developmental stage at which the cytosol receptors were not yet fully developed. To select a proper postnatal age, we first studied the developmental pattern of glucocorticoid binding proteins. The binding capacity of the hippocampus, hypothalamus and pituitary for both [ZH]B and [aH]Dex during ontogeny is shown in Fig. I. For [aH]B a considerable postnatal increase of the concentration of binding sites was observed in all 3 areas reaching adult levels around day 32. In the hippocampus a 3-fold, and in the hypothalamus and pituitary a 2-fold, increase of the concentration of binding sites could be observed. The two latter areas display an almost linear increase with time of [ZH]B binding capacity, in contrast to the pituitary where there appears to be a stepwise development. The developmental pattern found with [3H]Dex was different from the one determined with [aH]B. In the hippocampus and hypothalamus the number of binding sites increases considerably during the first 15 days. At day 15 levels of bound hor-
126 200 Hipp°campusIHipPT~II "~
#,
. . . . . . . . .
100 ~
f
t -I
$
200 Hypothalamus Io0
"-I
0
n- 2OO m 100
~II
8
. . . . . . . 15
24
32 Doys
--1" Adult
Fig. 1. Developmental time course of the binding of [aH]dexamethasone (dashed line) and [3H]corticosterone (solid line) to cytosol macromolecules of rats adrenalectomized 14-15 h before sacririce and perfused through the heart at sacrifice. Binding, expressed as fmoles aH radioactivity bound/ mg of cytosol protein, is presented as a function of postnatal age (in days) of the rats at sacrifice.
mone are equivalent to those in the adult. The pituitary, in contrast, showed a higher [aH]Dex binding during the very first postnatal days than in the adult. DISCUSSION
The regional study of glucocorticoid binding to receptor-like proteins presented in this paper is consistent with earlier findings from this laboratory12, showing the highest binding to occur in the hippocampus and septum. Regional studies of glucocorticoid cytosol binding have been presented also by Grosser et aL 3 using [all]B, and by Lassman and Mulrow 6 for [3H]deoxycorticosterone. In agreement with our data both authors demonstrated the highest cytosol binding in the hippocampus. However, the pattern of binding to cytosol from other brain areas differs considerably among the three glucocorticoids. With [aH]deoxycorticosterone the next highest binding capacity following the hippocampus was found in the cortex and amygdala and less binding was found in the hypothalamus. With [3H]Dex we observed a higher binding in the hypothalamus, but less in the amygdala relative to the hippocampus. Comparing our findings with the [3H]B binding pattern observed by Grosser et al. 3, we found a similar high binding of [SH]Dex in the septum relative to the hippocampus but less was bound to the hypothalamus. These discrepencies might reflect real differences in the avidity of various corticoids for a single type of binding protein or they could be interpreted as an indication of the presence of various kinds of receptor molecules s. The second postadrenalectomy increase of the glucocorticoid binding capacity originally detected in the hippocampus 11 was found to be a property of all areas
127 investigated in this study. However, the largest increase of the [aH]Dex binding sites (72 h post ADX) is shown to occur in the hippocampus and septum, followed by the hypothalamus, amygdala and cortex. The pituitary, having the lowest binding capacity for [3H]Dex, also showed the smallest postadrenalectomy increase of its binding activity. With [SH]B this area bound almost as much hormone as the hippocampus and displayed a lalge increase of its binding sites post ADX. The high [3H]B binding and the large second post-adrenalectomy increase of its binding can be explained by the presence of a transcortin-like protein known to be present in the pituitary, which binds corticosterone and not dexamethasone (deKloet, personal communication). It is known that transcortin levels in blood rise after ADX 15,1~,18, but the origin of pituitary transcortin-like material is presently not known. It does not appear to be an artifact of blood contamination of pituitary tissue (deKloet, personal communication), deKloet et al. 5 presented evidence for the existence of two different populations of glucocorticoid binding proteins. Their conclusion is based on the observation that steroid binding activities toward [3H]B and [3H]Dex are inactivated at different rates in the absence of steroid, and that [3H]B and [3H]Dex show distinct and different regional distributions for in vivo nuclear binding. This study adds further support to this idea. First, the in vitro cytosol binding of the hippocampus, hypothalamus and pituitary are not identical for the two hormones, even though the dissociation constants are the same 5. Second, the size of the second postadrenalectomy increase differs for the two hormones. This is best illustrated by the pituitary, where the binding capacity differs by a factor of 2-3 and where the postadrenalectomy increase is about 4 times larger with [3H]Dex than with [aH]B. From our previous work we knew already that the second postadrenalectomy increase is independent of the thyroid gland or any hypophyseal hormone secretion11. It was conceivable therefore that the increase is induced through the trophic influence of some afferent nerve fibers. Transection of the fimbria 6 or 80 days before measuring the hormone binding capacity did not affect the amount of normally present binding sites in any of the investigated areas. Also, the postadrenalectomy increase was not affected by this operation, suggesting that this process is not triggered by rostral afferents of the hippocampus. It is still possible, however, that afferents of the alvear pathway might be involved in the regulation of this phenomenon. Despite these negative findings, it was conceivable that these rostral afferents are perhaps more important for the establishment of the binding sites at an early developmental stage. Fimbria transection was performed in 3-day-old rats for the following two reasons. First, the cytosol binding capacity is low at that developmental stage. Second, the hippocampus has not yet received the cholinergic afferents at this stage 17. Transection of the fimbria at day 3 did not affect the adult level of binding sites (12 h post ADX) in these rats, suggesting that the ontogeny of these molecules is probably an autonomous property of the hippocampus or is regulated by the alvear pathway. The amount of [3H]B and [aH]Dex binding sites increases considerably during ontogeny in all investigated areas. A similar developmental pattern of [3H]Dex binding sites has been described also for the rat liver by Henning et al. a. In the
128 intestine and lungs, however, the same authors f o u n d a higher b i n d i n g capacity in y o u n g t h a n in adult rats, which is a similar finding to the p a t t e r n described in this work for the [aH]Dex b i n d i n g proteins in the pituitary. ACKNOWLEDGEMENTS This work was supported by Research G r a n t NS 07080 from the U n i t e d States Public Health Service (to Bruce McEwen) a n d by fellowships (to H a n s - R u d o l f Olpe) f r o m the U S P H S F o r e i g n Postdoctoral Program, The H o l d e r b a n k C e m e n t Factory ( H o l d e r b a n k , Switzerland), a n d from the J a n g g e n - P o e h n Stiftung (St. Gallen, Switzerland).
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