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Effect of steroid competition and time on the uptake of [3H]corticosterone in the rat brain; An autoradiographic study
Brain Research, 83 (1975) 293-300
293
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
E F F E C T OF STEROID C O M P E T I T I O N A N D T I M E O N T H E U P T A K E OF [aH]CORTICOSTERONE IN T H E RAT BRAIN; A N A U T O R A D I O G R A P H I C STUDY*
R. W.
RHEES**, B. I. GROSSER ANDW. STEVENS
Departments of Anatomy and Psychiatry, University of Utah, College of Medicine, Salt Lake City, Utah 84112 (U.S.A.)
(Accepted September 16th, 1974)
SUMMARY The localization of [SH]corticosterone in the brain and pituitary of the adrenalectomized rat was studied by autoradiography. Corticosterone was concentrated preferentially by neurons in the hippocampus, septum, amygdala, and neocortex, with the hippocampal and septal neurons having the greatest concentration. [aH]Corticosterone localization was significantly greater in neurons from adrenalectomized animals. Administration of unlabeled corticosterone (3 mg) 30 min before injection with [aH]corticosterone significantly decreased cellular localization of the labeled corticosterone, whereas [3H]corticosterone localization was not affected by progesterone pretreatment. Animals were sacrificed 15, 30 and 120 min after the injection of [aH]corticosterone to study the distribution of the hormone over the cytoplasm and nuclei. Silver grains were localized primarily over the cytoplasm of the concentrating neurons 15 min after injection; however, by 30 min after injection they were evenly distributed over both nuclei and cytoplasm.
INTRODUCTION Recent biochemical studies have shown that [3H]corticosterone is bound to a protein(s) which has been isolated from cytosol~-7,14 and nuclei s-l° of rat brain. This * Presented in part at the IV International Congress of Endocrinology, June, 1972, Washington, D. C., U.S.A.
** Present address: Department of Zoology, Brigham Young University, Provo, Utah 84601, U.S.A.
294
R.W. RHEES~'l al.
protein(s) has many of the characteristics of a steroid receptor molecule i.e., selectivity, stereospecificity, high affinity and limited capacity. In addition, significantly greater quantities of this protein(s) are found in limbic structures than in other brain regions .9, 14. These biochemical data, however, cannot provide information about the cellular localization of corticosterone in intact tissues. Therefore, in this study, we present autoradiographic data which confirm and extend earlier histochemical observationsa, 15,16,18,19 and supplement recent biochemical evidence. MATERIALS AND METHODS
Male 320-375 g Sprague-Dawley rats, were adrenalectomized by the dorsal approach, 6-8 days prior to use, and maintained on standard rat chow and normal saline, ad libitum. Completeness of adrenalectomy was checked by the water imbibition technique. The effect of endogenous and exogenous corticosterone on the saturation of binding sites was studied in adrenalectomized, non-adrenalectomized (intact), and adrenalectomized rats pretreated with 3 mg of unlabeled corticosterone, i.p., 30 rain before the injection of [3H]corticosterone. In addition, rats pretreated with 3 mg of unlabeled progesterone, i.p., 30 min before the injection of [3H]corticosterone were used to study further the specificity of the [aH]corticosterone binding sites. Each animal received a single injection of 300/~Ci of [3H]corticosterone (40 Ci/mmole, New England Nuclear Corporation), i.p., dissolved in a 25 ~ ethanol-saline solution. At the end of 15, 30, and 120 min, 3 rats from each experimental group were sacrificed by decapitation. A modification of the autoradiographic technique originally developed by Stumpf and Roth 17 which minimizes the translocation of the injected labeled hormone during tissue preparation was used. The brains and pituitaries were quickly removed, carefully wrapped in foil, and frozen by submersion in liquid nitrogen. Tissues were stored at --40 °C until they were sectioned. Frozen sections were cut at 6-8 #m in a cryostat maintained at --20 to --30 °C. Under a red safety light the frozen-hydrated sections were moved with a small camel hair brush TABLE I AVERAGE NUMBER OF SILVER G R A I N S LOCALIZED OVER N E U R O N S IN DIFFERENT REGIONS OF THE BRAIN
FROM ADRENALECTOMIZEO RATS SACRIFICED 30 m i n AFTER THE INJECTION OF [SH]coRTICOSTERONE M e a n ± s t a n d a r d deviation for 100 n e u r o n s per brain region.
Region
Number o f silver grains
Hippocampus Septum Amygdala Neocortex B a c k g r o u n d (over adjacent intercellular space o f equivalent size)
41.7 27.4 18.8 9.2 4.2
3_ 18.6 :~ 10.9 ~ 9.2 L: 3.1 ~: 1.9
CORTICOSTERONE LOCALIZATION IN THE BRAIN
295
Fig. 1. A low-power autoradiogram of [3H]corticosterone in the hippocampal region of the rat brain. The neurons of the pyramidal layer of the cornu ammonis (CA), the gyrus dentatus (GD), and the subiculum (S), contained larger numbers of silver grains than neurons from other brain regions. Adrenalectomized [ZHlcorticosterone injected rat. × 25.
from the cold cryostat knife and melted onto glass slides, which had been previously coated with Kodak NTB-3 emulsion and desiccated before use. The slides were packed in light-proof boxes containing small bags of Drierite, exposed for 6-12 months at 4 °C, developed in Kodak Dektol developer, and stained with either cresyl violet, Darron red and light green, or hematoxylin. The autoradiograms were systematically studied to determine the localization of silver grains in the various tissues from each of the treatment groups. For quantitative purposes, silver grains were counted over 100 neurons from each treatment group (10 randomly selected neurons from each of 10 randomly selected slides per group). RESULTS
In adrenalectomized rats injected with [3H]corticosterone, silver grains were concentrated over neurons in the hippocampus, septum, amygdala, and neocortex (Table I). There were relatively few silver grains localized over neurons in other brain regions. In the hippocampus, the most heavily labeled structure, silver grains were localized over neurons of the pyramidal layer of the cornu ammonis, the dentate gyrus, and the subiculum (Fig. 1). Most of the neurons in these structures were labeled with silver grains, some being more heavily labeled than others (Fig. 2). The heavily labeled neurons contained 10 times as many silver grains as found over the adjacent intercellular space of equivalent size (background, Table I). In intact animals, a small number of hippocampal neurons were labeled with a few silver grains and it was difficult to differentiate between the localized and unlocalized grains (Fig. 3, Table II). In adrenalectomized rats pretreated with nonradioactive corticosterone, none of the neurons concentrated the radioactive corticosterone since grain counts over the neurons were not significantly different from
296
~{. w . RHEtiS ~'l a/.
CORTICOSTERONELOCALIZATIONIN THE BRAIN
297
b a c k g r o u n d counts (Fig. 4, Table II). The i n c o r p o r a t i o n o f [aH]corticosterone by neurons f r o m adrenalectomized animals pretreated with progesterone was n o t significantly different f r o m that observed in the untreated adrenalectomized rat injected with [aH]corticosterone (Fig. 5, Table II). In adrenalectomized rats sacrificed 15 min after the injection o f [aH]corticosterone, the silver grains were distributed primarily over the cytoplasm o f h i p p o c a m p a l neurons, while only a few silver grains were localized over the nuclei (Fig. 6, Table III). In a u t o r a d i o g r a m s made f r o m the brains o f adrenalectomized animals sacrificed 30 rain and 120 rain after injection o f [SH]corticosterone, silver grains were also f o u n d over the cytoplasm, but an equivalent n u m b e r o f silver grains were f o u n d over the nuclei o f m o s t neurons examined (Fig. 7, Table III). Large numbers o f silver grains were distributed over most o f the cells o f the anterior pituitary. The tissue sections were n o t well e n o u g h preserved to determine whether [SH]corticosterone was preferentially concentrated by any specific anterior pituitary cell. The cells o f the pars intermedia and posterior pituitary were only lightly labeled. There was a significant reduction in the n u m b e r o f silver grains over the cells in the anterior pituitary o f intact and adrenalectomized animals pretreated with corticosterone when c o m p a r e d to adrenalectomized and adrenalectomized progesterone treated animals. Grain counts o f pituitary cells f r o m adrenalectomized animals sacrificed after the 3 different time periods showed no significant changes in the distribution o f the labeled steroid over cellular nuclei and cytoplasm. DISCUSSION The data presented in this study complement and extend existing biochemical 510,14 and autoradiographica,15,16,1s, 19 data which demonstrate that corticosterone is concentrated in and b o u n d by specific cells in the brain o f the rat. The localization o f corticosterone by a u t o r a d i o g r a p h y not only serves as a control for the biochemical
Fig. 2. Neurons of the pyramidal layer of the cornu ammonis from an adrenalectomized rat sacrificed 15 min after the injection of laH]corticosterone. Most of the neurons are labeled with silver grains (arrows), while a few of the neurons are unlabeled (U). The heavily labeled neurons contain 10 times as many silver grains as found over the adjacent intercellular space. × 525. Fig. 3. Pyramidal neurons of the cornu ammonis from an intact animal sacrificed 15 rain after the injection of laH]corticosterone. Only a small number of the neurons are lightly labeled. × 650. Fig. 4. Pyramidal neurons from an adrenalectomized animal pretreated with unlabeled eorticosterone 0.5 h before the injection of laH]corticosterone. None of the neurons concentrated the radioactive corticosterone. × 650. Fig. 5. Pyramidal neurons from an adrenalectomized animal pretreated with unlabeled progesterone 0.5 h before the injection of [aH]corticosterone. Most of the neurons concentrated the radioactive corticosterone. × 650. Fig. 6. A neuron of the pyramidal layer from an adrenalectomized animal sacrificed 15 min after the injection of [aH]corticosterone. Silver grains are distributed primarily over the cytoplasm with only a few localized over the nucleus (arrows). x 920. Fig. 7. A neuron of the pyramidal layer from an adrenalectomized animal sacrificed 30 min after the injection of [aH]corticosterone. Silver grains are localized over the nucleus (arrows) and the cytoplasm. × 920.
298
R.W.
R H E E S ~;l ¢3/.
TABLE II AVERAGE NUMBER OF SILVER GRAINS PER NEURON IN THE HIPPOCAMPUS OF RATS SACRIFICED 3 0
rain
AFTER THE INJECTION OF [3H]CORTICOSTERONE
Mean ± standard deviation for 100 neurons per treatment group. Number of silver grains
Adrenalectomized Non-adrenalectomized (intact) Adrenalectomized and pretreated with non-labeled corticosterone Adrenalectomized and pretreated with non-labeled progesterone
41.7 -~ 18.6 7.4 ~ 3.8 4.3 -:- 1.6 37.6 ~ 12.7
data but also makes it possible to identify and quantitate the cells which concentrate the labeled steroid. In adrenalectomized rats, [aH]corticosterone was localized preferentially in neurons of the hippocampus, septum, amygdala and neocortex. The hippocampal and septal neurons were the most heavily labeled, followed by the neurons of the amygdala and neocortex. These autoradiographic data are in agreement with the biochemical data reported from this laboratory 14 which demonstrate that brain cytosols taken from the hippocampus and septum contained the highest concentration of corticosterone bound per mg of protein and with the data presented by McEwen and co-workers 1° that cell nuclei from the hippocampus and septum bind more corticosterone than nuclei from other brain regions. However, the autoradiograms show that within the heavily labeled brain regions there is a differential quantitative incorporation of the hormone from one cell to another. This difference in cellular uptake suggests that not all of the corticosterone receptor neurons are in the same physiological state or that some of the neurons in the heavily labeled areas may not be specific receptor cells for
TABLE III COMPARISON OF NUCLEAR AND CYTOPLASMIC DISTRIBUTION OF SILVER GRAINS OVER HIPPOCAMPAL NEURONS OF ADRENALECTOMIZED ANIMALS SACRIFICED AT 15, 3 0 , AND 120 min AFTER THE INJECTION
OF [SH]coRTICOSTERONE Mean dz standard deviation for 100 hippocampal neurons. Time (min)
Region
Number of silver grains
15
Nucleus Cytoplasm Nucleus Cytoplasm Nucleus Cytoplasm
6.3 ± 2.3 31.6 ± 13.6 19.2 ± 11.7 20.7 ~- 10.4 15.9 ± 9.8 18.1 :~: 11.3
30 120
CORTICOSTERONELOCALIZATION1N THE BRAIN
299
corticosterone. Unlabeled neurons in the heavily labeled brain regions may also be receptors for steroid hormones other than corticosterone as has been suggested for receptor neurons in the hippocampus and septum of birds 12. [aH]Corticosterone concentration was significantly greater in neurons from the heavily labeled regions of adrenalectomized rats than from intact animals. Pretreatment with non-radioactive corticosterone significantly reduced cellular uptake in adrenalectomized animals, whereas localization was not affected by progesterone pretreatment. Therefore, the binding of labeled corticosterone is significantly influenced by the presence of endogenous and/or exogenous corticosterone. The fact that progesterone pretreatment did not inhibit cellular uptake provides further evidence for the specificity of the receptor protein. This observation is consistent with the biochemical data 5 which demonstrate that 11-dehydrocorticosterone, cortisol and cortisone significantly decrease corticosterone uptake, whereas, progesterone, 17fl-estradiol, and testosterone do not interfere with the binding of [aH]corticosterone by brain cytosols. Since corticosterone receptor molecules have been isolated from brain nuclei and cytosol, the temporal cellular uptake was investigated to test the hypothesis that the mechanism for corticosterone binding in the brain is similar to the two-step mechanism suggested for steroid binding in the uterus xA, prostate z, and oviducOL Silver grains were found to be concentrated primarily over the cytoplasm of the receptor neurons at 15 rain after injection and then by 30 min the silver grains were evenly distributed over both nuclei and cytoplasm. The nuclear and cytoplasmic concentrations of the radioactive corticosterone are approximately equal in the animals sacrificed at 30 and 120 min after injection (Table III), although earlier investigatorsa, ~5,~6have reported that in animals sacrificed 30 min or more after injection, the radiochemical is concentrated predominately over nuclei of receptor neurons. Recent biochemical studies of the interaction between nuclear and cytosol binding of corticosterone further demonstrate that the in vitro nuclear uptake of [SH]corticosterone by brain nuclei isolated and purified from adrenalectomized rats is dependent on an initial association of the corticosterone with brain cytosol proteins 18. Therefore, these autoradiographic data, together with biochemical data, indicate that such a twostep mechanism may exist in the brain.
REFERENCES 1 CLARK,J. H., ANI~GORSKI,J., Estrogen receptors, an evaluation of cytoplasmic-nuclear interaction in a cell-free system and a method for assay, Biochim. biophys. Acta (Amst.), 192 (1959) 508-515. 2 FANG, S., ANDERSON, K. H., AND LIOS, S., Receptor proteins for androgens, J. biol. Chem., 244 (1969) 6584--6595. 3 GERLACH,J. L., AND McEWEN, B. S., Rat brain binds adrenal steroid hormone: radioautography of hippocampus with corticosterone, Science, 175 (1972) 1133-1136. 4 GIANNOPOULOS,G., AND GORSKI,J., Estrogen receptors. Quantitative studies of transfer of estradiol from cytoplasmic to nuclear binding sites, d. biol. Chem., 246 (1971) 2524--2529. 5 GROSSER,B. I., STEVENS,W., BRUENGER,F. W., AND REED, D. J., Corticosterone binding in rat brain cytosol, J. Neurochem., (1971) 1725-1732.
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6 GROSSER, ]3. I., STEVENS, W., AND REED, D. J., Properties of corticosterone-binding macromolecules from rat brain cytosol, Brain Research, 57 (1973) 387-395. 7 MCEWEN, B. S., MAGNUS,C., AND WALLACH,G., Soluble corticosterone-binding macromolecules extracted from rat brain, Endgcrinabgy, 90 0972) 217-226. 8 McEWEN, B. S., AND PLAPLINGER, L., Association of 3H-corticosterone-l,2 with macromolecules extracted from cell nuclei, Nature (Lond.), 226 (1970) 263-264. 9 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. 10 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) 471482. 11 O'MALLEY, B. W., TOFT, D. O., AND SHERMAN, M. R., Progesterone-binding components of chick oviduct, II. Nuclear components, J. biol. Chem., 246 (1971) 1117-1122. 12 RHEES,R. W., ABEL, J. H., AND HAACK, D. W., Uptake of tritiated steroids in the brain of the duck (Anas platyrhynchos). An autoradiographic study, Gen. comp. Endocrinol., 18 (1972) 292-300. 13 RHEES,R. W., STEVENS,W., AND GROSSER,B. 1., Relationship between nuclear and cytosol binding of aH-corticosterone in the rat brain, Sec. ann. Meet. Sac. Neurosci., 1972, p. 244. 14 STEVENS,W., GROSSER,B. I., AND REED, D. J., Corticosterone-binding molecules in rat brain cytosols: regional distribution, Brain Research, 35 (1971) 602-607. 15 STUMPF,W. E., Autoradiographic techniques and the localization of estrogen, androgen and glucocorticoid in pituitary and brain, Amer. Zool., 11 (1971) 725-739. 16 STUMPF, W. E., Estrogen, androgen and adrenal hormone attracting neurons in the periventricular brain, Fed. Proc., 30 (I 97t) 309 (abstract). 17 STUMPF, W. E., AND ROTH, L. J., High resolution autoradiography with dry mounted, freezedried frozen sections. Comparative study of six methods using two diffusible compounds 3H-estradiol and 3H-mesobilirubinogen, J. Histochem. Cytochem., 14 (1966) 274-287. t8 STUMI'F, W. E., AND SAR, M., Topography of extrahypothalamic glucocorticosteroid "feedback' sites in the rat brain. In IVth Int. Cong. Endocrinol., Excerpta Medica ICS, 256, 1972, pp. 120-121 (abstract). 19 STUMPF, W. E., AND SAR, M., Hormonal inputs to releasing factor cells, feedback sites. In E. ZIMMERMAN,W. H. GISPEN, B. H. MARKSAND D. DE WIED (Eds.), Drug Effects on Neuroendocrhle Regulation, Progr. Brain Res., Vol. 39, Elsevier, Amsterdam, 1973, pp. 53-71.
293
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
E F F E C T OF STEROID C O M P E T I T I O N A N D T I M E O N T H E U P T A K E OF [aH]CORTICOSTERONE IN T H E RAT BRAIN; A N A U T O R A D I O G R A P H I C STUDY*
R. W.
RHEES**, B. I. GROSSER ANDW. STEVENS
Departments of Anatomy and Psychiatry, University of Utah, College of Medicine, Salt Lake City, Utah 84112 (U.S.A.)
(Accepted September 16th, 1974)
SUMMARY The localization of [SH]corticosterone in the brain and pituitary of the adrenalectomized rat was studied by autoradiography. Corticosterone was concentrated preferentially by neurons in the hippocampus, septum, amygdala, and neocortex, with the hippocampal and septal neurons having the greatest concentration. [aH]Corticosterone localization was significantly greater in neurons from adrenalectomized animals. Administration of unlabeled corticosterone (3 mg) 30 min before injection with [aH]corticosterone significantly decreased cellular localization of the labeled corticosterone, whereas [3H]corticosterone localization was not affected by progesterone pretreatment. Animals were sacrificed 15, 30 and 120 min after the injection of [aH]corticosterone to study the distribution of the hormone over the cytoplasm and nuclei. Silver grains were localized primarily over the cytoplasm of the concentrating neurons 15 min after injection; however, by 30 min after injection they were evenly distributed over both nuclei and cytoplasm.
INTRODUCTION Recent biochemical studies have shown that [3H]corticosterone is bound to a protein(s) which has been isolated from cytosol~-7,14 and nuclei s-l° of rat brain. This * Presented in part at the IV International Congress of Endocrinology, June, 1972, Washington, D. C., U.S.A.
** Present address: Department of Zoology, Brigham Young University, Provo, Utah 84601, U.S.A.
294
R.W. RHEES~'l al.
protein(s) has many of the characteristics of a steroid receptor molecule i.e., selectivity, stereospecificity, high affinity and limited capacity. In addition, significantly greater quantities of this protein(s) are found in limbic structures than in other brain regions .9, 14. These biochemical data, however, cannot provide information about the cellular localization of corticosterone in intact tissues. Therefore, in this study, we present autoradiographic data which confirm and extend earlier histochemical observationsa, 15,16,18,19 and supplement recent biochemical evidence. MATERIALS AND METHODS
Male 320-375 g Sprague-Dawley rats, were adrenalectomized by the dorsal approach, 6-8 days prior to use, and maintained on standard rat chow and normal saline, ad libitum. Completeness of adrenalectomy was checked by the water imbibition technique. The effect of endogenous and exogenous corticosterone on the saturation of binding sites was studied in adrenalectomized, non-adrenalectomized (intact), and adrenalectomized rats pretreated with 3 mg of unlabeled corticosterone, i.p., 30 rain before the injection of [3H]corticosterone. In addition, rats pretreated with 3 mg of unlabeled progesterone, i.p., 30 min before the injection of [3H]corticosterone were used to study further the specificity of the [aH]corticosterone binding sites. Each animal received a single injection of 300/~Ci of [3H]corticosterone (40 Ci/mmole, New England Nuclear Corporation), i.p., dissolved in a 25 ~ ethanol-saline solution. At the end of 15, 30, and 120 min, 3 rats from each experimental group were sacrificed by decapitation. A modification of the autoradiographic technique originally developed by Stumpf and Roth 17 which minimizes the translocation of the injected labeled hormone during tissue preparation was used. The brains and pituitaries were quickly removed, carefully wrapped in foil, and frozen by submersion in liquid nitrogen. Tissues were stored at --40 °C until they were sectioned. Frozen sections were cut at 6-8 #m in a cryostat maintained at --20 to --30 °C. Under a red safety light the frozen-hydrated sections were moved with a small camel hair brush TABLE I AVERAGE NUMBER OF SILVER G R A I N S LOCALIZED OVER N E U R O N S IN DIFFERENT REGIONS OF THE BRAIN
FROM ADRENALECTOMIZEO RATS SACRIFICED 30 m i n AFTER THE INJECTION OF [SH]coRTICOSTERONE M e a n ± s t a n d a r d deviation for 100 n e u r o n s per brain region.
Region
Number o f silver grains
Hippocampus Septum Amygdala Neocortex B a c k g r o u n d (over adjacent intercellular space o f equivalent size)
41.7 27.4 18.8 9.2 4.2
3_ 18.6 :~ 10.9 ~ 9.2 L: 3.1 ~: 1.9
CORTICOSTERONE LOCALIZATION IN THE BRAIN
295
Fig. 1. A low-power autoradiogram of [3H]corticosterone in the hippocampal region of the rat brain. The neurons of the pyramidal layer of the cornu ammonis (CA), the gyrus dentatus (GD), and the subiculum (S), contained larger numbers of silver grains than neurons from other brain regions. Adrenalectomized [ZHlcorticosterone injected rat. × 25.
from the cold cryostat knife and melted onto glass slides, which had been previously coated with Kodak NTB-3 emulsion and desiccated before use. The slides were packed in light-proof boxes containing small bags of Drierite, exposed for 6-12 months at 4 °C, developed in Kodak Dektol developer, and stained with either cresyl violet, Darron red and light green, or hematoxylin. The autoradiograms were systematically studied to determine the localization of silver grains in the various tissues from each of the treatment groups. For quantitative purposes, silver grains were counted over 100 neurons from each treatment group (10 randomly selected neurons from each of 10 randomly selected slides per group). RESULTS
In adrenalectomized rats injected with [3H]corticosterone, silver grains were concentrated over neurons in the hippocampus, septum, amygdala, and neocortex (Table I). There were relatively few silver grains localized over neurons in other brain regions. In the hippocampus, the most heavily labeled structure, silver grains were localized over neurons of the pyramidal layer of the cornu ammonis, the dentate gyrus, and the subiculum (Fig. 1). Most of the neurons in these structures were labeled with silver grains, some being more heavily labeled than others (Fig. 2). The heavily labeled neurons contained 10 times as many silver grains as found over the adjacent intercellular space of equivalent size (background, Table I). In intact animals, a small number of hippocampal neurons were labeled with a few silver grains and it was difficult to differentiate between the localized and unlocalized grains (Fig. 3, Table II). In adrenalectomized rats pretreated with nonradioactive corticosterone, none of the neurons concentrated the radioactive corticosterone since grain counts over the neurons were not significantly different from
296
~{. w . RHEtiS ~'l a/.
CORTICOSTERONELOCALIZATIONIN THE BRAIN
297
b a c k g r o u n d counts (Fig. 4, Table II). The i n c o r p o r a t i o n o f [aH]corticosterone by neurons f r o m adrenalectomized animals pretreated with progesterone was n o t significantly different f r o m that observed in the untreated adrenalectomized rat injected with [aH]corticosterone (Fig. 5, Table II). In adrenalectomized rats sacrificed 15 min after the injection o f [aH]corticosterone, the silver grains were distributed primarily over the cytoplasm o f h i p p o c a m p a l neurons, while only a few silver grains were localized over the nuclei (Fig. 6, Table III). In a u t o r a d i o g r a m s made f r o m the brains o f adrenalectomized animals sacrificed 30 rain and 120 rain after injection o f [SH]corticosterone, silver grains were also f o u n d over the cytoplasm, but an equivalent n u m b e r o f silver grains were f o u n d over the nuclei o f m o s t neurons examined (Fig. 7, Table III). Large numbers o f silver grains were distributed over most o f the cells o f the anterior pituitary. The tissue sections were n o t well e n o u g h preserved to determine whether [SH]corticosterone was preferentially concentrated by any specific anterior pituitary cell. The cells o f the pars intermedia and posterior pituitary were only lightly labeled. There was a significant reduction in the n u m b e r o f silver grains over the cells in the anterior pituitary o f intact and adrenalectomized animals pretreated with corticosterone when c o m p a r e d to adrenalectomized and adrenalectomized progesterone treated animals. Grain counts o f pituitary cells f r o m adrenalectomized animals sacrificed after the 3 different time periods showed no significant changes in the distribution o f the labeled steroid over cellular nuclei and cytoplasm. DISCUSSION The data presented in this study complement and extend existing biochemical 510,14 and autoradiographica,15,16,1s, 19 data which demonstrate that corticosterone is concentrated in and b o u n d by specific cells in the brain o f the rat. The localization o f corticosterone by a u t o r a d i o g r a p h y not only serves as a control for the biochemical
Fig. 2. Neurons of the pyramidal layer of the cornu ammonis from an adrenalectomized rat sacrificed 15 min after the injection of laH]corticosterone. Most of the neurons are labeled with silver grains (arrows), while a few of the neurons are unlabeled (U). The heavily labeled neurons contain 10 times as many silver grains as found over the adjacent intercellular space. × 525. Fig. 3. Pyramidal neurons of the cornu ammonis from an intact animal sacrificed 15 rain after the injection of laH]corticosterone. Only a small number of the neurons are lightly labeled. × 650. Fig. 4. Pyramidal neurons from an adrenalectomized animal pretreated with unlabeled eorticosterone 0.5 h before the injection of laH]corticosterone. None of the neurons concentrated the radioactive corticosterone. × 650. Fig. 5. Pyramidal neurons from an adrenalectomized animal pretreated with unlabeled progesterone 0.5 h before the injection of [aH]corticosterone. Most of the neurons concentrated the radioactive corticosterone. × 650. Fig. 6. A neuron of the pyramidal layer from an adrenalectomized animal sacrificed 15 min after the injection of [aH]corticosterone. Silver grains are distributed primarily over the cytoplasm with only a few localized over the nucleus (arrows). x 920. Fig. 7. A neuron of the pyramidal layer from an adrenalectomized animal sacrificed 30 min after the injection of [aH]corticosterone. Silver grains are localized over the nucleus (arrows) and the cytoplasm. × 920.
298
R.W.
R H E E S ~;l ¢3/.
TABLE II AVERAGE NUMBER OF SILVER GRAINS PER NEURON IN THE HIPPOCAMPUS OF RATS SACRIFICED 3 0
rain
AFTER THE INJECTION OF [3H]CORTICOSTERONE
Mean ± standard deviation for 100 neurons per treatment group. Number of silver grains
Adrenalectomized Non-adrenalectomized (intact) Adrenalectomized and pretreated with non-labeled corticosterone Adrenalectomized and pretreated with non-labeled progesterone
41.7 -~ 18.6 7.4 ~ 3.8 4.3 -:- 1.6 37.6 ~ 12.7
data but also makes it possible to identify and quantitate the cells which concentrate the labeled steroid. In adrenalectomized rats, [aH]corticosterone was localized preferentially in neurons of the hippocampus, septum, amygdala and neocortex. The hippocampal and septal neurons were the most heavily labeled, followed by the neurons of the amygdala and neocortex. These autoradiographic data are in agreement with the biochemical data reported from this laboratory 14 which demonstrate that brain cytosols taken from the hippocampus and septum contained the highest concentration of corticosterone bound per mg of protein and with the data presented by McEwen and co-workers 1° that cell nuclei from the hippocampus and septum bind more corticosterone than nuclei from other brain regions. However, the autoradiograms show that within the heavily labeled brain regions there is a differential quantitative incorporation of the hormone from one cell to another. This difference in cellular uptake suggests that not all of the corticosterone receptor neurons are in the same physiological state or that some of the neurons in the heavily labeled areas may not be specific receptor cells for
TABLE III COMPARISON OF NUCLEAR AND CYTOPLASMIC DISTRIBUTION OF SILVER GRAINS OVER HIPPOCAMPAL NEURONS OF ADRENALECTOMIZED ANIMALS SACRIFICED AT 15, 3 0 , AND 120 min AFTER THE INJECTION
OF [SH]coRTICOSTERONE Mean dz standard deviation for 100 hippocampal neurons. Time (min)
Region
Number of silver grains
15
Nucleus Cytoplasm Nucleus Cytoplasm Nucleus Cytoplasm
6.3 ± 2.3 31.6 ± 13.6 19.2 ± 11.7 20.7 ~- 10.4 15.9 ± 9.8 18.1 :~: 11.3
30 120
CORTICOSTERONELOCALIZATION1N THE BRAIN
299
corticosterone. Unlabeled neurons in the heavily labeled brain regions may also be receptors for steroid hormones other than corticosterone as has been suggested for receptor neurons in the hippocampus and septum of birds 12. [aH]Corticosterone concentration was significantly greater in neurons from the heavily labeled regions of adrenalectomized rats than from intact animals. Pretreatment with non-radioactive corticosterone significantly reduced cellular uptake in adrenalectomized animals, whereas localization was not affected by progesterone pretreatment. Therefore, the binding of labeled corticosterone is significantly influenced by the presence of endogenous and/or exogenous corticosterone. The fact that progesterone pretreatment did not inhibit cellular uptake provides further evidence for the specificity of the receptor protein. This observation is consistent with the biochemical data 5 which demonstrate that 11-dehydrocorticosterone, cortisol and cortisone significantly decrease corticosterone uptake, whereas, progesterone, 17fl-estradiol, and testosterone do not interfere with the binding of [aH]corticosterone by brain cytosols. Since corticosterone receptor molecules have been isolated from brain nuclei and cytosol, the temporal cellular uptake was investigated to test the hypothesis that the mechanism for corticosterone binding in the brain is similar to the two-step mechanism suggested for steroid binding in the uterus xA, prostate z, and oviducOL Silver grains were found to be concentrated primarily over the cytoplasm of the receptor neurons at 15 rain after injection and then by 30 min the silver grains were evenly distributed over both nuclei and cytoplasm. The nuclear and cytoplasmic concentrations of the radioactive corticosterone are approximately equal in the animals sacrificed at 30 and 120 min after injection (Table III), although earlier investigatorsa, ~5,~6have reported that in animals sacrificed 30 min or more after injection, the radiochemical is concentrated predominately over nuclei of receptor neurons. Recent biochemical studies of the interaction between nuclear and cytosol binding of corticosterone further demonstrate that the in vitro nuclear uptake of [SH]corticosterone by brain nuclei isolated and purified from adrenalectomized rats is dependent on an initial association of the corticosterone with brain cytosol proteins 18. Therefore, these autoradiographic data, together with biochemical data, indicate that such a twostep mechanism may exist in the brain.
REFERENCES 1 CLARK,J. H., ANI~GORSKI,J., Estrogen receptors, an evaluation of cytoplasmic-nuclear interaction in a cell-free system and a method for assay, Biochim. biophys. Acta (Amst.), 192 (1959) 508-515. 2 FANG, S., ANDERSON, K. H., AND LIOS, S., Receptor proteins for androgens, J. biol. Chem., 244 (1969) 6584--6595. 3 GERLACH,J. L., AND McEWEN, B. S., Rat brain binds adrenal steroid hormone: radioautography of hippocampus with corticosterone, Science, 175 (1972) 1133-1136. 4 GIANNOPOULOS,G., AND GORSKI,J., Estrogen receptors. Quantitative studies of transfer of estradiol from cytoplasmic to nuclear binding sites, d. biol. Chem., 246 (1971) 2524--2529. 5 GROSSER,B. I., STEVENS,W., BRUENGER,F. W., AND REED, D. J., Corticosterone binding in rat brain cytosol, J. Neurochem., (1971) 1725-1732.
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R . W . RHEES cl a/.
6 GROSSER, ]3. I., STEVENS, W., AND REED, D. J., Properties of corticosterone-binding macromolecules from rat brain cytosol, Brain Research, 57 (1973) 387-395. 7 MCEWEN, B. S., MAGNUS,C., AND WALLACH,G., Soluble corticosterone-binding macromolecules extracted from rat brain, Endgcrinabgy, 90 0972) 217-226. 8 McEWEN, B. S., AND PLAPLINGER, L., Association of 3H-corticosterone-l,2 with macromolecules extracted from cell nuclei, Nature (Lond.), 226 (1970) 263-264. 9 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. 10 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) 471482. 11 O'MALLEY, B. W., TOFT, D. O., AND SHERMAN, M. R., Progesterone-binding components of chick oviduct, II. Nuclear components, J. biol. Chem., 246 (1971) 1117-1122. 12 RHEES,R. W., ABEL, J. H., AND HAACK, D. W., Uptake of tritiated steroids in the brain of the duck (Anas platyrhynchos). An autoradiographic study, Gen. comp. Endocrinol., 18 (1972) 292-300. 13 RHEES,R. W., STEVENS,W., AND GROSSER,B. 1., Relationship between nuclear and cytosol binding of aH-corticosterone in the rat brain, Sec. ann. Meet. Sac. Neurosci., 1972, p. 244. 14 STEVENS,W., GROSSER,B. I., AND REED, D. J., Corticosterone-binding molecules in rat brain cytosols: regional distribution, Brain Research, 35 (1971) 602-607. 15 STUMPF,W. E., Autoradiographic techniques and the localization of estrogen, androgen and glucocorticoid in pituitary and brain, Amer. Zool., 11 (1971) 725-739. 16 STUMPF, W. E., Estrogen, androgen and adrenal hormone attracting neurons in the periventricular brain, Fed. Proc., 30 (I 97t) 309 (abstract). 17 STUMPF, W. E., AND ROTH, L. J., High resolution autoradiography with dry mounted, freezedried frozen sections. Comparative study of six methods using two diffusible compounds 3H-estradiol and 3H-mesobilirubinogen, J. Histochem. Cytochem., 14 (1966) 274-287. t8 STUMI'F, W. E., AND SAR, M., Topography of extrahypothalamic glucocorticosteroid "feedback' sites in the rat brain. In IVth Int. Cong. Endocrinol., Excerpta Medica ICS, 256, 1972, pp. 120-121 (abstract). 19 STUMPF, W. E., AND SAR, M., Hormonal inputs to releasing factor cells, feedback sites. In E. ZIMMERMAN,W. H. GISPEN, B. H. MARKSAND D. DE WIED (Eds.), Drug Effects on Neuroendocrhle Regulation, Progr. Brain Res., Vol. 39, Elsevier, Amsterdam, 1973, pp. 53-71.