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Uptake of corticosterone by rat brain and its concentration by certain limbic structures
BRAIN RESEARCH
227
U P T A K E OF C O R T I C O S T E R O N E BY RAT BRAIN A N D ITS C O N C E N T R A T I O N BY C E R T A I N LIMBIC S T R U C T U R E S
BRUCE S. McEWEN, JAY M. WEISS AND LESLIE S. SCHWARTZ
Rockefeller University, New York, N.Y. 10021 (U.S.A.) (Accepted April 17th, 1969)
INTRODUCTION
Certain steroid hormones act directly on the brain to influence neural and behavioral events 1,2,29 31,34. In the case of estradiol, neurons of brain regions such as the preoptic area and hypothalamus on which the hormone acts to affect sexual behavior and gonadotrophic hormone release 19 tend to accumulate and retain labeled estradiolg,13,2~,27, 32. Because we are interested in the action of corticosterone on brain biochemistry, neural processes, and behavior, we have attempted to answer the following questions concerning the availability of this hormone to the brain: (1) Does labeled corticosterone, the principal adrenal steroid in the rat 5, enter the brain from the blood in sufficient amounts to enable us to believe that circulating corticosterone released during stress can directly affect nerve cells? (2) Is there any evidence of a selective retention of corticosterone by regions of the limbic system which may be related to the control of A C T H secretion or to the behavioral response to stress? Since normal rats secrete large amounts of corticosterone as a result of the stress of injection and handling, we decided to begin our study of radioactive hormone uptake by removing the source of endogenous hormone, the adrenal glands. In this way possible binding sites in the brain would remain unsaturated and available to accumulate labeled hormone. In this paper we wish to present evidence that there are two simultaneous uptake processes for corticosterone in the brain : a non-specific entry of the hormone into all parts of the brain which shows increasing uptake with increasing blood level of the hormone; and a limited-capacity retention of the hormone in certain parts of the limbic system, namely, the hippocampus and septum. A preliminary report of this work has already appeared in print 23. EXPERIMENTAL PROCEDURE
Male albino rats (Sprague-Dawley strain) of 250-400 g body weight were used for the study. Adrenalectomies were carried out in the laboratory, and uptake experiments were performed usually 1 week after the operation and never more than 3 Brain Research, 16 (1969) 227-241
228
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weeks later. Adrenalectomized animals were maintained on 0.8(!, sodium chloridc solution, and standard laboratory chow ad/ibitum. Early in the study we found great variability in corticosterone uptake which we traced to the fact that some animals were incompletely adrenalectomized. This phenomenon has been shown to be due either to the rupture of the adrenal during removal (leaving a small amount of steroid-producing tissue in the body cavity) or to the presence of accessory nodules of adrenal tissue '~8. We have therefore screened all operated rats for completeness of adrenalectomy by sampling blood by heart puncture 20 rain after a standard ether stress and assaying the plasma for corticosterone by the wellknown acid fluorimetric technique ll,2G. Under the conditions of the ether stress and heart puncture, normal rats show blood levels of 32.2 -~ 14.4/~g/100 ml (compared with resting levels of 7-12 #g/100 ml), while completely adrenalectomized rats have plasma blood levels of 3.7 ~ 0.9 ,ug/100 ml. This figure is presumably due to other substances than corticosterone, and it is also obtained for hypophysectomized rats in which the adrenal has atrophied ~~. A rat was rejected as incompletely adrenalectomized if it gave a plasma level in excess of 6 #g/100 ml during screening. [1,2-aH]Corticosterone was obtained from New England Nuclear Corporation,
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Fig. 1. A, Longitudinal section of rat brain, showing some of the brain areas sampled in fresh dissection procedure, B, Rat brain with cortex of right hemisphere removed showing=hippocampus and septum. Roman numerals refer to I, dorsal; II, medial; and III, ventral portions of hippocampus removed for experiment described in Table IIL Brain Research, 16 (1969) 227-241
229
CORTICOSTERONEUPTAKEBY RAT BRAIN
and its purity was checked by thin-layer chromatography (see below). Fifty #C of the isotopic compound, containing 0.7 #g of steroid and dissolved in 50 #1 of benzeneethanol (1 : 1, v/v), were injected intraperitoneally by means of a Hamilton microsyringe. Injected rats were sacrificed by decapitation at the indicated time intervals after isotope administration and a sample of trunk blood was collected. The brain was removed from the skull and dissected into major anatomical subdivisions, according to an atlas of the rat brain 15 after consultation with a neuroanatomist, Dr. George Wolf of Mount Sinai School of Medicine. The division of the brain is illustrated in the diagrams in Fig. l. The structures routinely taken were as follows: pituitary, septum (medial plus lateral), hippocampus, hypothalamus, thalamus, midbrain behind thalamus and including superior colliculi, brain stem (including posterior midbrain, pons, and medulla oblongata), cerebellum, amygdala with overlying cortex, and samples of pyriform and cingulate cortex (anterior portion), and the rest of the cortex. In view of the similarity in concentration of radioactive hormone in many of these brain regions, data from some regions will be omitted in the presentation of the results. Brain samples and approximately 200 ,ul aliquots of blood obtained at decapitation were placed in weighed (to 0.1 rag) glass vials of the type used for scintillation counting. The vial plus sample was weighed again on a balance so as to obtain the wet sample weight to the nearest 0.2 rag. Depending on the weight of the tissue, 3, 6, or 9 ml of dichloromethane (Matheson, Coleman and Bell Spectrograde) was added, and the vial was capped tightly and shaken gently on a metabolic shaker for 16-18 h (usually overnight). One ml aliquots were then transferred to other scintillation vials together with 15 ml of a toluene-based scintillation fluid (5 g/l 2,5-diphenyloxazole (PPO) and 0.25 g/l dimethyl-l,4-bis-(5-phenyloxazolyl-2)-benzene (dimethyl-POPOP) in toluene) and TABLE I P E R C E N T A G E OF R A D I O A C T I V I T Y
EXTRACTED IN A REPRESENTATIVE EXPERIMENT
Samples were re-extracted with dichloromethane in order to estimate the radioactivity remaining after the first extraction. Sample
Wet weight (mg)
Volume DCM (ml)
DPM extracted
% Radioactivity extracted
Blood Blood Pituitary Septum Hippocampus
232.61 236.12 9.39 7.07 160.93
9 9 3 3 6
6680 6739 282 77 1427
95.2 94.2 99.0 100.0 90.1
Hypothalamus Pyriform cortex Midbrain Thalamus Cortex
132.24 31.53 63.34 38.69 92.43
6 6 6 6 6
1209 234 680 387 891
93.5 85.7 94.2 92.1 94.0
Brain stem Cerebellum Cingulate cortex Amygdala
209.05 275.29 25.30 150.61
9 9 3 6
2595 2619 251 1277
93.8 91.6 87.4 88.4
Brain Research, 16 (1969) 227-241
230
m s . MCliWiTNcl ai.
counted at 26~o efficiency in a Packard Tricarb, Model 3375, scintillation counter, Since this procedure has not previously been described in the literature, it was necessary to show that it results in complete and reproducible extraction ol' the radioactive steroid. A representative experiment is presented in Table 1, which illustrates the typical tissue weights, extraction volumes and the extent of extraction. It can be seen that the procedure consistently removes 90 o{ or more of the dichloromethane-soluble steroid, independently of the sample weight or volume of extraction fluid Thin-layer chromatography was used to identify labeled corticosterone in the dichloromethane extracts of brain and blood. This step was particularly important in view of the demonstration that the brain is capable of converting corticosterone to 1 ldehydrocortisone t0. Before chromatography, the dichloromethane extracts were washed one time each with one-third volume of I N N a O H , water, and 40!.i0 aqueous methanol. The first 2 washes removed 15'?/£ and 29 °4; of the radioactivity from brain and blood extracts, respectively. The methanol wash removed almost no radioactive material but did remove a substance which seemed to interfere with the thin-layer chromatography. The washed dichloromethane extracts were then evaporated to dryness and redissolved in 0.1-0.3 ml of absolute ethanol for the chromatography. Chromatography was carried out on 250 l* layers of Silica Gel HFes4 (Merck AG, Darmstadt, obtained from Brinkman Instruments, Westbury, N.Y. 11590) using chloroform-ethanol (9 : I, v/v) for development 18. Non-radioactive corticosterone and 1l-dehydrocorticosterone (Steraloids, Inc., Pawling, N.Y.) were added to
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Fig. 2. Time course of disappearance of radioactivity injected as [1,2-3H] corticosterone from blood and 6 brain regions in adrenalectomized rats. Medulla plus ports = brain stem. Brain Research, 16 (1969) 227-241
231
CORTICOSTERONE UPTAKE BY RAT BRAIN
each sample before chromatography and each spot was visualized under ultraviolet light. The spots were scraped from the plate, and steroid was eluted with 0.5 ml of absolute ethanol and counted in 15 ml of the toluene-based scintillator described above. The rest of the plate was also scraped and radioactive material eluted and counted. Compared to an equal aliquot of the sample, which was also spotted on a plate (but not chromatographed), eluted, and counted, recovery of radioactive material from the chromatograph was virtually complete in every case. RESULTS
Uptake by and disappearance of radioactivity from various brain regions Adrenalectomized rats were given 50 #C (0.7 /~g) of [l,2-3H]corticosterone intraperitoneally and were sacrificed 30 rain, or 2, 4, or 6 h later. The average concentrations of dichloromethane-extractable radioactivity (expressed as DPM/mg wet weight of tissue or blood) are plotted in Fig. 2A on a logarithmic scale as a function of time after the injection. All 4 brain regions shown in Fig. 2A have a similar concentration of radioactive material which is half the concentration in the blood up to the 4th h. The fall in concentration in these 4 areas (and in other regions of the brain which were omitted from this figure) parallels very closely the disappearance of radioactivity from the blood, indicating that a free and rapid exchange exists between the radioactive hormone in the brain and that in the blood. Because the disappearance of radioactive material from the cortex parallels that from the blood and shows no indication of retention of radioactivity over the entire time course, the uptake in this region will be referred to as 'non-specific' and will be used in the next section as a reference for the calculation of retention by other brain regions. An important feature of this 'non-specific' uptake of hormone is that the concentration in the brain increases with an increase in the concentration ofcorticosterone circulating in the blood. In order to demonstrate this phenomenon, we injected adrenalectomized rats with 3 different doses of unlabeled hormone mixed with the same amount of labeled hormone (50/tC). Two hours later the usual brain regions were dissected and extracted for estimation of radioactive hormone uptake. The concentrations of radioactive hormone, at each dose, in 2 representative brain regions, the cortex and hypothalamus, are presented in Table II. When these concentrations are divided by the specific activity of corticosterone (radioactive plus non-radioactive) at each dose, the quotient represents the concentration of corticosterone in that brain region expressed as picograms of steroid per gram of tissue. It can be seen from the right-hand column of Table II that this concentration increases in both cortex and hypothalamus according to the dose injected. This indicates that increased circulating corticosterone produced by stress leads to corresponding increases in the brain level of the hormone. It is apparent from Fig. 2B that the hippocampus and septum concentrate labeled corticosterone compared to the blood (dotted line) and to the other brain regions shown in Fig. 2A. Moreover, the rate of disappearance of labeled cortico-
Brain Research, 16 (1969) 227-241
232
TABLE
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11
I N C R E A S E D N O N ° S P E C I F I C E N T R Y ()[- C I ) R Y I C O S T E R O N E I N T O B R A I N W I T H I N C R E A S E D B L O O D C O N C E N T R A T I O N OF T H E H O R M O N E
Dose (t~g)
Specific activity o f corticosterone (DPM/pg) *
Radioactive concentration ( DPM/mg wet wt. tissue ~ S E M ) ;
Concentration o f steroid (pg/g wet wt. tissue {- S E M ) ; n : 4
n :4
Cortex 0.7 5.0 50.0
157.0 19.3 2.17
49 ±: 18 34 =: 14 28 +- 8
315 ~ I1(/ 175l 4 : 6 9 9 13008 :t: 3610
Hypothalamus 0.7 5.0 50.0
157.0 19.3 2.17
26 {: 16 33 ± 13 32 ± 9
293 ± 102 1743 -2_ 689 14855 ~ 4045
* pg = picogram = 10-12 g. sterone from these 2 structures is lower than that from the blood and from the other brain regions between 30 rain and 2 h after hormone administration. Thereafter, the rate of disappearance does not appear to differ from that in the blood and may actually be somewhat faster than that from the other brain regions. This time course suggests that there is in both hippocampus and septum of adrenalectomized rats an interval of retention of the hormone up to 2 h, followed by a period of accelerated release. The existence of rapidly turning-over binding sites for the hormone and the intracetlular site of binding will be considered in the Discussion. C o n c e n t r a t i o n o f r a d i o a c t i v i t y in b r a i n r e g i o n s r e l a t i v e to b l o o d a n d c o r t e x
We have used the concentration of radioactive corticosterone in the blood and cortex as references for comparison of the time course of disappearance of radioactivity from other brain regions. For each animal in the experiment, the concentration of radioactivity in each brain region was divided first by the concentration in the blood and then by the concentration in the cortex. These relative concentrations were then averaged for each structure at each time and are presented in Fig. 3A and 3B, as a function of the time between injection and sacrifice. These figures demonstrate striking differences in retention of radioactive material between the septum and hippocampus on the one hand and the cortex, hypothalamus, amygdala, and medulla plus pons on the other. These figures confirm the demonstration in Fig. IB of a time-dependent retention and release of radioactive material in the septum and hippocampus, although, according to this means of expressing the data, the retention phase persists to the 4th h. It should also be pointed out that, in spite of the similarity among the other brain structures, there is a slight tendency for the medulla plus pons to show a time course similar to the septum and hippocampus (Fig. 3B). This and the tendency for the relative concentration of the hypothalamus Brain Research, 16 (1969) 227-241
CORTICOSTERONEUPTAKE BY RAT BRAIN
233
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Fig. 3. Time course of concentration of radioactivity injected as [1,2-3H]corticosterone in 6 brain regions expressed as a ratio to the concentration in A: blood, and B: cerebral cortex.
and amygdala, compared to cortex, to increase with time may be of some interest for a future investigation.
Distribution of uptake along the hippocampus The hippocampus from adrenalectomized rats which had been injected 2 h before sacrifice with 50 ffC [l,2-3H]corticosterone was divided into 3 approximately equal-length segments, as shown in Fig. IC. As shown in Table |II, the highest concentration was observed in the most dorsal and anterior segment, nearest to the septum and fornix. Uptake in the next segment of the hippocampus is also very high compared to cortex, and it is slightly higher than in the third (and most ventral) segment of the hippocampus but lower than that in the first segment. This dorsal-ventral 'gradient' of radioactivity may reflect the decreasing density of certain neuron cell bodies along the Brain Research, 16 (1969) 227-241
234
R.s. MCEWEN et al.
TABLE Ill D I S T R I B U T I O N OF R A D I O A C T I V E CORTICOSTERONE ALONG H I P P O C A M P U S
Sample
Blood Pituitary Septum Hippocampus I Dorsal II Medial II1 Ventral Hypothalamus Cortex
~oo
Concentration (DPM/mg) ~: S E M
Relative concentration Blood =]=S E M
Cortex ~ S E M
101 ± 38 129 i 54 95 F 32
1.0 1.19 ~+ 0.06 0.95 ± 0.14
2.35 iv 0.23 1.83 ~1 0.23
211 163 163 46 49
2.08 ± 1.67 ± 1.51 ± 0.50 ~ 0.51 ~
4.01 3.19 2.80 0.96 1.0
-5 ~ ~ ± ±
69 52 58 14 16
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x Hypothalamus
z~ S e p t u m u Hippocampus
• Cortex • B r a i n stem
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0.38 0.34 0.32 0.03 0.02
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T i m e , hr Fig. 4. A, Time course of disappearance of radioactivity injected as [1,2-8H]corticosterone from blood and 5 brain regions in normal rats. B, Same data expressed as a ratio to concentration of radioactivity in blood. C, Same data expressed as a ratio to concentration of radioactivity in cerebral cortex.
l e n g t h o f t h e h i p p o c a m p u s . A u t o r a d i o g r a p h i c studies o f t h e c e l l u l a r l o c a l i z a t i o n o f c o r t i c o s t e r o n e u p t a k e in t h e h i p p o c a m p u s a n d s e p t u m a r e in p r o g r e s s . Brain Research, 16 (1969) 227-241
CORTICOSTERONE UPTAKE BY RAT BRAIN
235 Two hour corlicosterone uploke
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Fig. 5. Selective saturation of retention process for [1,2-3H]corticosterone by unlabeled steroids in adrenalectornized rats and by endogenous steroid in normal animals. Data are expressed as concentration in a given brain region relative to concentration in cortex of that animal. Brackets indicate standard error of the mean for 4-5 rats. ADX adrenalectomized rats; N.D. ~- not done.
Saturation of the uptake process In contrast to the situation for adrenalectomized rats, corticosterone is not accumulated selectively by the hippocampus of normal rats. This finding is illustrated in Fig. 4, which presents for normal rats the concentration o f h o r m o n e in the brain (A) and the relative concentration c o m p a r e d to blood (B) and cortex (C), as a function of time after the administration o f radioactive corticosterone. It can be seen that the concentration o f h o r m o n e and time course o f disappearance o f h o r m o n e from the hippocampus is very similar to that from the cortex of the normal rat. A likely explanation for this observation is that endogenous levels o f corticosterone in the blood o f the normal rat completely saturate the uptake sites in the hippocampus. In order to test this explanation o f a limited-capacity retention process in the hippocampus, adrenalectomized rats were injected with a 3-mg dose o f unlabeled corticosterone 30 min before administration of a 0.7 /~g (50/zC) dose of [l,2-3H]corticosterone. The rats were sacrificed 2 h later. It can be seen from Fig. 5, which presents the concentration of radioactivity in 10 brain regions relative to cortex, that this dose o f unlabeled corticosterone reduced the concentration of radioactivity in the hippocampus o f an adrenalectomized rat to that o f the cortex. Two other steroid hormones compete with radioactive corticosterone for uptake sites in the hippocampus of adrenalectomized rats. A 3-mg dose o f hydrocortisone partially reduced corticosterone uptake in hippocampus,
Brain Research, 16 (1969) 227-241
B . s . MC~WEN et al.
236
while a 3-rag dose of dexamethasone completely saturated the uptake process (Fig. 5). As noted at the beginning of this section, the hippocampus of normal rats shows no tendency to accumulate labeled corticosterone, and this finding is also presented in Fig. 5. The septum is the only other structure in the brain that is affected by the administration of unlabeled corticosterone. The septum nevertheless tends to accumulate radioactive corticosterone in normal (Fig. 4) as well as adrenalectomized rats (Fig. I). The uptake process in the septum therefore appears to have a larger capacity for the circulating levels of corticosterone found in normal rats than does the hippocampus. However, Fig. 5 shows that 3-rag doses of corticosterone, dexamethasone, and hydrocortisone reduce the uptake by the septum of [1,2-3H]corticosterone.
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Time (hr) after ADX Fig. 6. Uptake of radioactivity injected as [l,2-3H]corticosterone in 4 brain regions as a function of time after adrenalectomy. A 2-h interval elapsed between radioactive hormone injection and sacrifice. A, Concentration of radioactivity vs. time. B, Concentration of radioactivity relative to cortex vs. time. At each time the values represent averages from 3 rats. Brain R e s e a r c h , 16 (1969) 227-241
237
CORTICOSTERONE UPTAKE BY RAT BRAIN TABLE IV COMPOSITIDN OF DCM-EXTRACTABLE RADIOACTIVEMATERIAL EXTRACTED FROM HIPPOCAMPUS Cort.
corticosterone; 11-DHC = 1 l-dehydrocorticosterone.
Time interval (injection to sacrifice)
Percent o f DCM-extractable R.A. with RF o f Blood
Hippocampus
Rest o f br~zin
(h) 2
4 6
Cort.
1 I-DHC
Corl,
11-DHC
Corr.
11-DHC
45 62 38 58 69 15
26 7 5 9 7 4.5
75 62 76 89 82 41
11 2 9 7 8 6.5
76 65 66 72 61 36.4
13 11 10 9 5 7
Time after adrenalectomy Since the hippocampus of normal rats does not accumulate radioactive corticosterone, it was interesting to see the time course whereby the hippocampal uptake of labeled hormone appears after adrenalectomy. Normal rats were adrenalectomized at 9 a.m., and radioactive corticosterone was given for a 2-h uptake period at 2, 4, 6, and 20 h after removal of the adrenals. Analysis of blood samples taken from rats at various times after adrenalectomy indicate that there is a very rapid decline in circulating corticosterone, reaching levels comparable to those in long-term adrenalectomized rats within I h of adrenalectomy and remaining at that level thereafter. Because corticosterone leaves the hippocampus and other parts of the brain quite rapidly (Fig. 2), we expected to see the appearance of binding sites for corticosterone in the hippocampus as early as 2 h after adrenalectomy. It can be seen from Fig. 6A that the concentration of radioactive corticosterone in the hippocampus, which is the same as that in the cortex in normal rats, is greater than that in the cortex 2 h after adrenalectomy and continues to increase with time thereafter. When the data are expressed as the concentration relative to cortex (Fig. 6B), it can be seen that the hippocampal concentration of hormone is twice that in the cortex at 2 h after adrenalectomy and 2.5 times as great at 6 h after adrenalectomy. It is somewhat surprising that at 20 h after adrenalectomy the relative concentration had increased to 3.5 and then tended to decrease to 3.0 in long-term adrenalectomies. Possible explanations for this phenomenon will be considered in the Discussion. It should also be noted that at all times after adrenalectomy the relative concentration of radioactivity in the septum remains constant and the same as that in rats with adrenals intact. The fact that in Fig. 6A the concentration of radioactive hormone in all brain regions can be seen to increase with time after adrenalectomy may be due to a gradual decline in the effectiveness of the system for removing corticosterone from the blood, since blood levels of radioactive hormone (not shown in Fig. 6) also increased with time after adrenalectomy. Brain Research, 16 (1969) 227 241
238
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Identification o ( radioaetive material in the brain as cortieosterone
In order to estimate the proportion of dichloromethane-extractable radioactivity from the brain and blood which remains as corticosterone, the extracts from the uptake experiments were concentrated and subjected to thin-layer chromatography as is described in the experimental procedure. Chromatography was carried out in the presence of 2 carrier steroids, corticosterone and 1 l-dehydrocorticosterone. The latter was chosen because it is known to be a metabolite ofcorticosterone which is found in nervous tissue TM. Table IV shows that at 2 and 4 h, respectively, after [l~2-aH]corti costerone administration, 70 and 80 °,~; respectively, of the radioactivity was recovered as a steroid with the mobility of corticosterone. A very small proportion of the total radioactivity was recovered in the I I-dehydrocorticosterone spot. The fact that the percentage of corticosterone in hippocampus is somewhat higher than that in the rest of the brain is consistent with the preferential retention of corticosterone by the hippocampus. DISCUSSION In this paper we have shown that radioactivity injected as [1,2-3H]corticosterone is retained and concentrated by a limited-capacity uptake process in the hippocampus and septum of adrenalectomized rats. Ovariectomized female rats tend to concentrate and retain estradiol in other brain regions, namely, the preoptic area, hypothalamus, and pituitary 9,13,22,27,a2, indicating that the limbic system of the rat brain is differentiated with respect to hormone sensitivity. The septum, however, tends to accumulate and retain both corticosterone (see above) and estradiol 9,1a,z2,'zv,3'z as well as testosterone, hydrocortisone, and aldosterone2L Weiss et al. 3a and Levine 17 have suggested that corticosterone has inhibitory effects on behavior which tend to counteract the excitatory or disinhibiting effects of ACTH3,16,17, ~. Such hormone effects fit well with the present findings that corticosterone is retained by the hippocampus and septum, since several investigators have suggested that both hippocampus and septum have inhibitory influences on behavior14,21,aS. The hippocampus and septum may also exert an inhibitory influence on A C T H release from the pituitary 6,2°. We do not yet know whether corticosterone actually facilitates the activity of the septum and hippocampus, but electrophysiotogical and hormone implantation studies are in progress. Radioactive corticosterone uptake and retention by the septum occurs in normal rats and appears to be saturated only at high and unphysiological concentrations of the unlabeled hormone, whereas the hippocampal uptake mechanism is saturated by the circulating levels of corticosterone found in normal rats subjected to the stress of radioactive hormone injection. In normal rats, the concentration of hormone found in the hippocampus at all times after injection is the same as that in the cortex. If radioactive corticosterone is injected 2 h after adrenalectomy, a time interval at which endogenous hormone should have been extensively released from the hippocampus, we observed a 2-fold greater concentration of labeled corticosterone in the hippocampus Brain Research, 16 (1969) 227-241
CORTICOSTERONE UPTAKE BY RAT BRAIN
239
over that in the cortex. This concentration factor increases to 2.5 at 6 h after adrenalectomy when all endogenous steroid should have disappeared. The tendency of labeled corticosterone to accumulate in the hippocampus increases still further with time up to 20 h after adrenalectomy, and remains at or slightly below this level in long-term adrenalectomies. This increase in binding capacity beyond 6 h after adrenalectomy might be explained by some kind of metabolic transformation in the hippocampus, although until we are able to measure endogenous steroid in the hippocampus it is impossible to rule out a very slow loss of bound hormone from this structure. The idea of a metabolic process responsible for the binding of hormone by the hippocampus is attractive from another standpoint, since it was noted above that, in the time course of corticosterone disappearance from the hippocampus of long-term adrenalectomies, there is a phase of retention, up to 2 h after administration, followed by a period of release of radioactive hormone. A possible explanation for this phenomenon might be the existence of binding factors having a rapid turnover in vivo, and the release of bound hormone would therefore be due to the destruction of the binding material. Such an explanation further suggests that the hormone might stimulate the synthesis of its own binding factor, an idea which has been proposed by another laboratory for estradiol retention in the hypothalamus 2~. These possibilities are currently under investigation. In order to discuss the possible types of action of steroid hormones, such as corticosterone, on the brain and on particular regions of the brain, it must be emphasized that we are dealing with 2 simultaneous processes : (1) The entry of hormone into all regions of the brain from the blood in significant quantities. (2) The retention of hormone by some cells which may themselves be concentrated in certain brain regions. We were able to show that increasing the amount of corticosterone injected into the peritoneal cavity resulted in a nearly proportional increase in the amount of hormone entering the cerebral cortex and hypothalamus over a dose range from 0.7 to 50 #g per 250 g rat. Furthermore, concentrations of radioactive hormone per unit wet weight in structures such as the cortex and hypothalamus are approximately half of those in the blood, and the kinetics of disappearance of hormone from the cortex and hypothalamus indicate free and rapid exchange. It is thus evident that increases in the circulating corticosterone in rats subjected to stressful situations are reflected in increases in the brain level of the hormone. Such hormones may act at the level of the cell membrane, as electrophysiological studies with corticosteroids, testosterone, estradiol, and progesteronO, 29-al would tend to suggest. The rat hypothalamus appears to be acted upon by corticosteroids both functionally and electrically 29 31, and it is important to stress that we have not seen any tendency for corticosterone to accumulate in this region. Therefore, binding of hormone may not be required for this type of action on the rat brain. In this connection, however, Eik-Nes and Brizzee 8 have observed that labeled cortisol concentrates in certain regions of the dog hypothalamus. Their results suggest that cortisol, unlike corticosterone, may act on the hypothalamus by way of a mechanism which requires binding of the hormone. Superimposed on top of the general uptake of corticosterone by the rat brain is the tendency for cells concentrated in the hippocampus and septum to concentrate and Brain Research, 16 (1969) 227 241
240
f~, s. v~Cr~WF~Ne t a / .
retain the hormone by a limited-capacity mechanism which we do not completely understand at this time. A clue to the intracellular site of action of bound hormone is the recent observation in this laboratory "4 that highly purified cell nuclei isolated from the hippocampus retain up to 5 0 ~ of the radioactive material found in theentire structure. Similar observations have been made for estradiol bound by the preoptic area and hypothalamus 24 and are supported by recent autoradiographic evidenceZL Since it is well-established that nuclear binding of steroid hormones in tissues such as the uterus and prostate gland leads to an increase in ribonucleic acid and protein synthesis in the tissue 2,4,12, it is likely that nuclear binding of hormones in brain cell nuclei leads to similar alterations in macromolecular synthesis in those brain cells. SUMMARY [l,2-3H]Corticosterone is taken up by all parts of the brain of normal and adrenalectomized rats. As the blood concentration of hormone increases, a situation which occurs following stress, the concentration of hormone in the brain also increases. Superimposed on top of the brain-wide uptake of corticosterone is the tendency of 2 limbic structures, the hippocampus and septum, to concentrate and retain labeled corticosterone. The hippocampal uptake sites are saturated by levels of corticosterone found in normal rats, while the septal sites are not. Hippocampal uptake is therefore observed only in adrenalectomized rats and can be seen as early as 2 h after adrenalectomy. The hippocampal uptake sites in adrenalectomized rats are saturated by exogenous unlabeled corticosterone, hydrocortisone, and dexamethasone. The relevance of hormone retention to the function of the hippocampus and septum in behavior and control of ACTH release is discussed. The possible cellular sites of action of corticosterone are also considered. ACKNOWLEDGEMENTS This research was supported by U.S. Public Health Service Grants NB 07080 to Dr. McEwen and M H 13189 to Dr. N. E. Miller. Dexamethasone was the generous gift of Merck, Sharp and Dohme, Rahway, N.J.
REFERENCES 1 BARRACLOUGH, C., AND CROSS, B., Unit activity in the hypothalamus of the cyclic female rat:
Effects of genital stimuli and progesterone, J. Endocr., 26 (1963) 339-359. 2 BARTON, R. W., AND LIAO, S., A similarity in the effect of estrogen and androgen on the synthesis
of RNA in the cell nuclei of gonadohormone-sensitivetissues, Endocrinology, 81 (1967) 409-412. 3 Bonus, B., AND DEWIED, O., Inhibitory and facilitatory effect of two related peptides on extinction of avoidance behavior, Science, 153 (1966) 318-320. 4 BRUCHEVSKY,N., AND WILSON, J. O., The intranuclear binding of testosterone and 5a-androstane17fl-ol-3-one by rat prostate, J. biol. Chem., 243 (1968) 5953-5960. 5 BUSH,I. E., Species differences in adrenocortical secretion, J. Endocr., 9 (t953) 95-100. Brain Research, 16 (1969) 227-241
CORTICOSI'ERONE UPTAKE BY RAT BRAIN
241
6 DALLMAN, M. F., AND YATES, F. E., Anatomical and functional mapping of central input and feedback pathways of the adrenocortical system, Mere. Soc. Endocr., 17 (1968) 39-77. 7 DEWtED, D., Inhibitory effects of ACTH and related peptides on extinction of conditioned avoidance behavior in rats, Proc. Soc. exp. Biol. (N. Y.), 122 (1966) 28-32. 8 EIK-NEs, K. B., AND BR1ZZEE, K. R., Concentration of tritium in brain tissue of dogs given [1,2-~H2] cortisol intravenously, Biochim. biophys. Acta (Amst.), 97 (1965) 320-333. 9 EISENFELD, A. J., AND AXELROD, J., Selectivity of estrogen distribution in tissues, J. Pharmacol. exp. Ther., 150 (1965) 469-475. 10 GROSSER, B. I., 11 (~-Hydroxysteroid metabolism by mouse brain and glioma 261, J. Neurochem., 13 (1966) 475-478. 11 GUILLEMIN,R., CLAYTON, G. W., SMITH, J. D., AND LIPSCOMB,H. S., Measurement of free corticosteroid in rat plasma: Physiological validation of a method, Endocrinology, 63 (1958) 349-358. 12 HAMILTON, T. H., Control by estrogen of genetic transcription and translation, Science, 161 (1968) 649-661. 13 KATO, J., AND VILLEE, C. A., Preferential uptake of estradiol by the anterior hypothalamus in the rat, Endocrinology, 80 (1967) 567-575. 14 KIMBLE, D. P., Hippocampus and internal inhibition, Psychol. Bull., 70 (1968) 285 295. 15 KON1G, J. F. R., AND KLIPPEL, R. A., The Rat Brain, Williams and Wilkins, Baltimore, 1963, 162 pp. 16 KORANYI, L., ENDROCZI, E., AND TARNOK, F., Sexual behavior in the course of avoidance conditioning in male rabbits, Neuroendocrinology, 1 (1965/66) 144 157. 17 LEVINE, S., Hormones and behavior. In Nebraska Symposium on Motivation, 1968, in press. 18 LISBOA,B. P., Separation and characteristics of AI-3 ketosteroids of the pregnane-series by means of thin layer chromatography, Acta endocr. (Kbh.), 43 (1963) 47-66. 19 LISK, R. D., Sexual behavior: hormonal control. In L. MARTINI AND W. GANONG (Eds.), Neuroendocrinology, Vol. 2, Academic Press, New York, 1967, pp. 197-240. 20 MANGIL1,G., MOTTA, M., AND MARTINI, L., Control of adrenocorticotrophic hormone secretion. In L. MARTINI AND W. GANONG (Eds.), Neuroendocrinology, Vol. 1, Academic Press, New York, [ 966, pp. 297-370. 21 McCLEARY, R. A., Response specificity in the behavioral effects of limbic system lesions in the cat, J. comp. physiol. P~ychol., 54 (1961) 605-613. 22 MCEwEN, B. S., PFAEE, D. W., AND ZIGMOND, R., Unpublished experinaents. 23 MCEWEN, B. S., WFI~S, J. M., AND SCHWARTZ, L. S., Selective retention of corticosterone by limbic structures in rat brain, Nature (Lond.), 220 (1968) 911-912. 24 McEwEN, B. S., AND ZIGMOND, R., Unpublished observations. 25 McGUIRE, J. L., AND LISK, R. D., Estrogen receptors in the intact rat, Proc. nat. Acad. Sci. (Wash.), 61 (1968) 497-503. 26 PETERSON, R. E., The identification of corticosterone in human plasma and its assay by isotope dilution, J. biol. Chem., 225 (1957) 25-37. 27 PFAFF, D. W., Uptake of H3-estradiol by the female rat brain: An autoradiographic study, Endocrinology, 82 (1968) 1149 1155. 28 RICHTER, C. P., Sodium chloride and dextrose appetite of untreated adrenalectomized rats, Endocrinology, 29 (1941) 115 125. 29 SAWYER, C. H., Effects of hormonal steroids on certain mechanisms in the adult brain. In L. MARTINI, E. FRASCH1NI AND M. MOTTA (Eds.), Hormonal Steroids, Excerpta reed. (Amst.), Congr. Ser. No. 132, 1968, pp. 123 135. 30 SAWYER, C. H., KAWAKAMI, M., MEYERSON, B., WHITMOYER, O. 1., AND LILLEY, J. J., Effects of ACTH, dexamethasone, and asphyxia on electrical activity of the rat hypothalamus, Brah7 Researeh, 10 (1968) 213 216. 31 STEINER, F. A., RUE, K., AND AKERT, F., Steroid-sensitive neurones in rat brain: Anatomical localization and responses of neurohumors to ACTH, Brain Research, 12 (1969) 74-85. 32 STUMPF, W. E., Estradiol-concentrating neurons: Topography in the hypothalamus by drymount autoradiography, Science, 162 (1968) 1001-1003. 33 WEISS, J. M., MCEWEN, B. S., SILVA, M. T., AND KALKUT, M., Pituitary-adrenal influences on fear responding, Science, 163 (1969) 197-199. 34 WOODBURY, D. M., AND VERNADAKIS, A., Effects of steroids in the cerebral nervous system. In R. 1. DOREMAN (Ed.), Methods in Hormone Research, Vol. 5, Academic Press, New York, 1966, pp. 1-57. 35 ZUCKER, I., AND MCCLEARY, R., Perseveration in septal cats, Psychonom. Sei., 1 (1964) 387-388.
Brain Research, 16 (1969) 227 241
227
U P T A K E OF C O R T I C O S T E R O N E BY RAT BRAIN A N D ITS C O N C E N T R A T I O N BY C E R T A I N LIMBIC S T R U C T U R E S
BRUCE S. McEWEN, JAY M. WEISS AND LESLIE S. SCHWARTZ
Rockefeller University, New York, N.Y. 10021 (U.S.A.) (Accepted April 17th, 1969)
INTRODUCTION
Certain steroid hormones act directly on the brain to influence neural and behavioral events 1,2,29 31,34. In the case of estradiol, neurons of brain regions such as the preoptic area and hypothalamus on which the hormone acts to affect sexual behavior and gonadotrophic hormone release 19 tend to accumulate and retain labeled estradiolg,13,2~,27, 32. Because we are interested in the action of corticosterone on brain biochemistry, neural processes, and behavior, we have attempted to answer the following questions concerning the availability of this hormone to the brain: (1) Does labeled corticosterone, the principal adrenal steroid in the rat 5, enter the brain from the blood in sufficient amounts to enable us to believe that circulating corticosterone released during stress can directly affect nerve cells? (2) Is there any evidence of a selective retention of corticosterone by regions of the limbic system which may be related to the control of A C T H secretion or to the behavioral response to stress? Since normal rats secrete large amounts of corticosterone as a result of the stress of injection and handling, we decided to begin our study of radioactive hormone uptake by removing the source of endogenous hormone, the adrenal glands. In this way possible binding sites in the brain would remain unsaturated and available to accumulate labeled hormone. In this paper we wish to present evidence that there are two simultaneous uptake processes for corticosterone in the brain : a non-specific entry of the hormone into all parts of the brain which shows increasing uptake with increasing blood level of the hormone; and a limited-capacity retention of the hormone in certain parts of the limbic system, namely, the hippocampus and septum. A preliminary report of this work has already appeared in print 23. EXPERIMENTAL PROCEDURE
Male albino rats (Sprague-Dawley strain) of 250-400 g body weight were used for the study. Adrenalectomies were carried out in the laboratory, and uptake experiments were performed usually 1 week after the operation and never more than 3 Brain Research, 16 (1969) 227-241
228
..
s.
~lcl.wt:N
el
al.
weeks later. Adrenalectomized animals were maintained on 0.8(!, sodium chloridc solution, and standard laboratory chow ad/ibitum. Early in the study we found great variability in corticosterone uptake which we traced to the fact that some animals were incompletely adrenalectomized. This phenomenon has been shown to be due either to the rupture of the adrenal during removal (leaving a small amount of steroid-producing tissue in the body cavity) or to the presence of accessory nodules of adrenal tissue '~8. We have therefore screened all operated rats for completeness of adrenalectomy by sampling blood by heart puncture 20 rain after a standard ether stress and assaying the plasma for corticosterone by the wellknown acid fluorimetric technique ll,2G. Under the conditions of the ether stress and heart puncture, normal rats show blood levels of 32.2 -~ 14.4/~g/100 ml (compared with resting levels of 7-12 #g/100 ml), while completely adrenalectomized rats have plasma blood levels of 3.7 ~ 0.9 ,ug/100 ml. This figure is presumably due to other substances than corticosterone, and it is also obtained for hypophysectomized rats in which the adrenal has atrophied ~~. A rat was rejected as incompletely adrenalectomized if it gave a plasma level in excess of 6 #g/100 ml during screening. [1,2-aH]Corticosterone was obtained from New England Nuclear Corporation,
eptum
I I--
~
::] I~':;
:'...,~........
:~ypothalamus~:ii~-i2~!:i~iiii~;~
A
~
"
.
" .
.
:: . . . . ::~:,!;:~@il Brain stem
.
.
Fig. 1. A, Longitudinal section of rat brain, showing some of the brain areas sampled in fresh dissection procedure, B, Rat brain with cortex of right hemisphere removed showing=hippocampus and septum. Roman numerals refer to I, dorsal; II, medial; and III, ventral portions of hippocampus removed for experiment described in Table IIL Brain Research, 16 (1969) 227-241
229
CORTICOSTERONEUPTAKEBY RAT BRAIN
and its purity was checked by thin-layer chromatography (see below). Fifty #C of the isotopic compound, containing 0.7 #g of steroid and dissolved in 50 #1 of benzeneethanol (1 : 1, v/v), were injected intraperitoneally by means of a Hamilton microsyringe. Injected rats were sacrificed by decapitation at the indicated time intervals after isotope administration and a sample of trunk blood was collected. The brain was removed from the skull and dissected into major anatomical subdivisions, according to an atlas of the rat brain 15 after consultation with a neuroanatomist, Dr. George Wolf of Mount Sinai School of Medicine. The division of the brain is illustrated in the diagrams in Fig. l. The structures routinely taken were as follows: pituitary, septum (medial plus lateral), hippocampus, hypothalamus, thalamus, midbrain behind thalamus and including superior colliculi, brain stem (including posterior midbrain, pons, and medulla oblongata), cerebellum, amygdala with overlying cortex, and samples of pyriform and cingulate cortex (anterior portion), and the rest of the cortex. In view of the similarity in concentration of radioactive hormone in many of these brain regions, data from some regions will be omitted in the presentation of the results. Brain samples and approximately 200 ,ul aliquots of blood obtained at decapitation were placed in weighed (to 0.1 rag) glass vials of the type used for scintillation counting. The vial plus sample was weighed again on a balance so as to obtain the wet sample weight to the nearest 0.2 rag. Depending on the weight of the tissue, 3, 6, or 9 ml of dichloromethane (Matheson, Coleman and Bell Spectrograde) was added, and the vial was capped tightly and shaken gently on a metabolic shaker for 16-18 h (usually overnight). One ml aliquots were then transferred to other scintillation vials together with 15 ml of a toluene-based scintillation fluid (5 g/l 2,5-diphenyloxazole (PPO) and 0.25 g/l dimethyl-l,4-bis-(5-phenyloxazolyl-2)-benzene (dimethyl-POPOP) in toluene) and TABLE I P E R C E N T A G E OF R A D I O A C T I V I T Y
EXTRACTED IN A REPRESENTATIVE EXPERIMENT
Samples were re-extracted with dichloromethane in order to estimate the radioactivity remaining after the first extraction. Sample
Wet weight (mg)
Volume DCM (ml)
DPM extracted
% Radioactivity extracted
Blood Blood Pituitary Septum Hippocampus
232.61 236.12 9.39 7.07 160.93
9 9 3 3 6
6680 6739 282 77 1427
95.2 94.2 99.0 100.0 90.1
Hypothalamus Pyriform cortex Midbrain Thalamus Cortex
132.24 31.53 63.34 38.69 92.43
6 6 6 6 6
1209 234 680 387 891
93.5 85.7 94.2 92.1 94.0
Brain stem Cerebellum Cingulate cortex Amygdala
209.05 275.29 25.30 150.61
9 9 3 6
2595 2619 251 1277
93.8 91.6 87.4 88.4
Brain Research, 16 (1969) 227-241
230
m s . MCliWiTNcl ai.
counted at 26~o efficiency in a Packard Tricarb, Model 3375, scintillation counter, Since this procedure has not previously been described in the literature, it was necessary to show that it results in complete and reproducible extraction ol' the radioactive steroid. A representative experiment is presented in Table 1, which illustrates the typical tissue weights, extraction volumes and the extent of extraction. It can be seen that the procedure consistently removes 90 o{ or more of the dichloromethane-soluble steroid, independently of the sample weight or volume of extraction fluid Thin-layer chromatography was used to identify labeled corticosterone in the dichloromethane extracts of brain and blood. This step was particularly important in view of the demonstration that the brain is capable of converting corticosterone to 1 ldehydrocortisone t0. Before chromatography, the dichloromethane extracts were washed one time each with one-third volume of I N N a O H , water, and 40!.i0 aqueous methanol. The first 2 washes removed 15'?/£ and 29 °4; of the radioactivity from brain and blood extracts, respectively. The methanol wash removed almost no radioactive material but did remove a substance which seemed to interfere with the thin-layer chromatography. The washed dichloromethane extracts were then evaporated to dryness and redissolved in 0.1-0.3 ml of absolute ethanol for the chromatography. Chromatography was carried out on 250 l* layers of Silica Gel HFes4 (Merck AG, Darmstadt, obtained from Brinkman Instruments, Westbury, N.Y. 11590) using chloroform-ethanol (9 : I, v/v) for development 18. Non-radioactive corticosterone and 1l-dehydrocorticosterone (Steraloids, Inc., Pawling, N.Y.) were added to
I000
A
• Cortex
F
x Hypothalamus
I
o Blood
o
• Amygdolo + Medulla and pans
J~ '
"~
I00
t-
B
o Blood z~ Septurn o Hippocompus
\
k%
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g I0 "E
i
~P
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c 0 L)
I
I
I
I
I
I
I
I
0.5
2
4
6
0.5
2
4
6
T i m e , hr
Fig. 2. Time course of disappearance of radioactivity injected as [1,2-3H] corticosterone from blood and 6 brain regions in adrenalectomized rats. Medulla plus ports = brain stem. Brain Research, 16 (1969) 227-241
231
CORTICOSTERONE UPTAKE BY RAT BRAIN
each sample before chromatography and each spot was visualized under ultraviolet light. The spots were scraped from the plate, and steroid was eluted with 0.5 ml of absolute ethanol and counted in 15 ml of the toluene-based scintillator described above. The rest of the plate was also scraped and radioactive material eluted and counted. Compared to an equal aliquot of the sample, which was also spotted on a plate (but not chromatographed), eluted, and counted, recovery of radioactive material from the chromatograph was virtually complete in every case. RESULTS
Uptake by and disappearance of radioactivity from various brain regions Adrenalectomized rats were given 50 #C (0.7 /~g) of [l,2-3H]corticosterone intraperitoneally and were sacrificed 30 rain, or 2, 4, or 6 h later. The average concentrations of dichloromethane-extractable radioactivity (expressed as DPM/mg wet weight of tissue or blood) are plotted in Fig. 2A on a logarithmic scale as a function of time after the injection. All 4 brain regions shown in Fig. 2A have a similar concentration of radioactive material which is half the concentration in the blood up to the 4th h. The fall in concentration in these 4 areas (and in other regions of the brain which were omitted from this figure) parallels very closely the disappearance of radioactivity from the blood, indicating that a free and rapid exchange exists between the radioactive hormone in the brain and that in the blood. Because the disappearance of radioactive material from the cortex parallels that from the blood and shows no indication of retention of radioactivity over the entire time course, the uptake in this region will be referred to as 'non-specific' and will be used in the next section as a reference for the calculation of retention by other brain regions. An important feature of this 'non-specific' uptake of hormone is that the concentration in the brain increases with an increase in the concentration ofcorticosterone circulating in the blood. In order to demonstrate this phenomenon, we injected adrenalectomized rats with 3 different doses of unlabeled hormone mixed with the same amount of labeled hormone (50/tC). Two hours later the usual brain regions were dissected and extracted for estimation of radioactive hormone uptake. The concentrations of radioactive hormone, at each dose, in 2 representative brain regions, the cortex and hypothalamus, are presented in Table II. When these concentrations are divided by the specific activity of corticosterone (radioactive plus non-radioactive) at each dose, the quotient represents the concentration of corticosterone in that brain region expressed as picograms of steroid per gram of tissue. It can be seen from the right-hand column of Table II that this concentration increases in both cortex and hypothalamus according to the dose injected. This indicates that increased circulating corticosterone produced by stress leads to corresponding increases in the brain level of the hormone. It is apparent from Fig. 2B that the hippocampus and septum concentrate labeled corticosterone compared to the blood (dotted line) and to the other brain regions shown in Fig. 2A. Moreover, the rate of disappearance of labeled cortico-
Brain Research, 16 (1969) 227-241
232
TABLE
~, s. Mc~Wt~N el a/.
11
I N C R E A S E D N O N ° S P E C I F I C E N T R Y ()[- C I ) R Y I C O S T E R O N E I N T O B R A I N W I T H I N C R E A S E D B L O O D C O N C E N T R A T I O N OF T H E H O R M O N E
Dose (t~g)
Specific activity o f corticosterone (DPM/pg) *
Radioactive concentration ( DPM/mg wet wt. tissue ~ S E M ) ;
Concentration o f steroid (pg/g wet wt. tissue {- S E M ) ; n : 4
n :4
Cortex 0.7 5.0 50.0
157.0 19.3 2.17
49 ±: 18 34 =: 14 28 +- 8
315 ~ I1(/ 175l 4 : 6 9 9 13008 :t: 3610
Hypothalamus 0.7 5.0 50.0
157.0 19.3 2.17
26 {: 16 33 ± 13 32 ± 9
293 ± 102 1743 -2_ 689 14855 ~ 4045
* pg = picogram = 10-12 g. sterone from these 2 structures is lower than that from the blood and from the other brain regions between 30 rain and 2 h after hormone administration. Thereafter, the rate of disappearance does not appear to differ from that in the blood and may actually be somewhat faster than that from the other brain regions. This time course suggests that there is in both hippocampus and septum of adrenalectomized rats an interval of retention of the hormone up to 2 h, followed by a period of accelerated release. The existence of rapidly turning-over binding sites for the hormone and the intracetlular site of binding will be considered in the Discussion. C o n c e n t r a t i o n o f r a d i o a c t i v i t y in b r a i n r e g i o n s r e l a t i v e to b l o o d a n d c o r t e x
We have used the concentration of radioactive corticosterone in the blood and cortex as references for comparison of the time course of disappearance of radioactivity from other brain regions. For each animal in the experiment, the concentration of radioactivity in each brain region was divided first by the concentration in the blood and then by the concentration in the cortex. These relative concentrations were then averaged for each structure at each time and are presented in Fig. 3A and 3B, as a function of the time between injection and sacrifice. These figures demonstrate striking differences in retention of radioactive material between the septum and hippocampus on the one hand and the cortex, hypothalamus, amygdala, and medulla plus pons on the other. These figures confirm the demonstration in Fig. IB of a time-dependent retention and release of radioactive material in the septum and hippocampus, although, according to this means of expressing the data, the retention phase persists to the 4th h. It should also be pointed out that, in spite of the similarity among the other brain structures, there is a slight tendency for the medulla plus pons to show a time course similar to the septum and hippocampus (Fig. 3B). This and the tendency for the relative concentration of the hypothalamus Brain Research, 16 (1969) 227-241
CORTICOSTERONEUPTAKE BY RAT BRAIN
233
• Cortex 7O O O
× Hypothalamus
O
A
g z
D
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Amygdala Brain stern
o Hippocampus z~ S e p t u m
2.0
g g c~
1,0
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o
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LI
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I
5.0 a
o
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g .z o
3.0
c c
o 2.0
o _
_
I
1
I
i
0.5
2
4
6
Time,
hr
Fig. 3. Time course of concentration of radioactivity injected as [1,2-3H]corticosterone in 6 brain regions expressed as a ratio to the concentration in A: blood, and B: cerebral cortex.
and amygdala, compared to cortex, to increase with time may be of some interest for a future investigation.
Distribution of uptake along the hippocampus The hippocampus from adrenalectomized rats which had been injected 2 h before sacrifice with 50 ffC [l,2-3H]corticosterone was divided into 3 approximately equal-length segments, as shown in Fig. IC. As shown in Table |II, the highest concentration was observed in the most dorsal and anterior segment, nearest to the septum and fornix. Uptake in the next segment of the hippocampus is also very high compared to cortex, and it is slightly higher than in the third (and most ventral) segment of the hippocampus but lower than that in the first segment. This dorsal-ventral 'gradient' of radioactivity may reflect the decreasing density of certain neuron cell bodies along the Brain Research, 16 (1969) 227-241
234
R.s. MCEWEN et al.
TABLE Ill D I S T R I B U T I O N OF R A D I O A C T I V E CORTICOSTERONE ALONG H I P P O C A M P U S
Sample
Blood Pituitary Septum Hippocampus I Dorsal II Medial II1 Ventral Hypothalamus Cortex
~oo
Concentration (DPM/mg) ~: S E M
Relative concentration Blood =]=S E M
Cortex ~ S E M
101 ± 38 129 i 54 95 F 32
1.0 1.19 ~+ 0.06 0.95 ± 0.14
2.35 iv 0.23 1.83 ~1 0.23
211 163 163 46 49
2.08 ± 1.67 ± 1.51 ± 0.50 ~ 0.51 ~
4.01 3.19 2.80 0.96 1.0
-5 ~ ~ ± ±
69 52 58 14 16
o Blood
x Hypothalamus
z~ S e p t u m u Hippocampus
• Cortex • B r a i n stem
A ,
0.38 0.34 0.32 0.03 0.02
[ B ~
~ 0.63 ± 0.55 ~: 0.47 ~ 0.03
C
3.0
3.0
°
g
g
gzo
,o
B ~.o
I
I
O.5
2
B~
-
|
,.o
I
I
I
I
0.5
2
0.5
2
T i m e , hr Fig. 4. A, Time course of disappearance of radioactivity injected as [1,2-8H]corticosterone from blood and 5 brain regions in normal rats. B, Same data expressed as a ratio to concentration of radioactivity in blood. C, Same data expressed as a ratio to concentration of radioactivity in cerebral cortex.
l e n g t h o f t h e h i p p o c a m p u s . A u t o r a d i o g r a p h i c studies o f t h e c e l l u l a r l o c a l i z a t i o n o f c o r t i c o s t e r o n e u p t a k e in t h e h i p p o c a m p u s a n d s e p t u m a r e in p r o g r e s s . Brain Research, 16 (1969) 227-241
CORTICOSTERONE UPTAKE BY RAT BRAIN
235 Two hour corlicosterone uploke
!
w x
z°
•
Normol
[]
ADX
[] []
ADX + hydrocortisone ADX + dexomethosone
i
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rt0 i Pituitory
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Midbroin
Tholomus
HippocompusHypotholomus Amygdolo
3.0 o r
2.0 O O
1.0
Medullo ond pons
Cerebellum Pyriform codex
Fig. 5. Selective saturation of retention process for [1,2-3H]corticosterone by unlabeled steroids in adrenalectornized rats and by endogenous steroid in normal animals. Data are expressed as concentration in a given brain region relative to concentration in cortex of that animal. Brackets indicate standard error of the mean for 4-5 rats. ADX adrenalectomized rats; N.D. ~- not done.
Saturation of the uptake process In contrast to the situation for adrenalectomized rats, corticosterone is not accumulated selectively by the hippocampus of normal rats. This finding is illustrated in Fig. 4, which presents for normal rats the concentration o f h o r m o n e in the brain (A) and the relative concentration c o m p a r e d to blood (B) and cortex (C), as a function of time after the administration o f radioactive corticosterone. It can be seen that the concentration o f h o r m o n e and time course o f disappearance o f h o r m o n e from the hippocampus is very similar to that from the cortex of the normal rat. A likely explanation for this observation is that endogenous levels o f corticosterone in the blood o f the normal rat completely saturate the uptake sites in the hippocampus. In order to test this explanation o f a limited-capacity retention process in the hippocampus, adrenalectomized rats were injected with a 3-mg dose o f unlabeled corticosterone 30 min before administration of a 0.7 /~g (50/zC) dose of [l,2-3H]corticosterone. The rats were sacrificed 2 h later. It can be seen from Fig. 5, which presents the concentration of radioactivity in 10 brain regions relative to cortex, that this dose o f unlabeled corticosterone reduced the concentration of radioactivity in the hippocampus o f an adrenalectomized rat to that o f the cortex. Two other steroid hormones compete with radioactive corticosterone for uptake sites in the hippocampus of adrenalectomized rats. A 3-mg dose o f hydrocortisone partially reduced corticosterone uptake in hippocampus,
Brain Research, 16 (1969) 227-241
B . s . MC~WEN et al.
236
while a 3-rag dose of dexamethasone completely saturated the uptake process (Fig. 5). As noted at the beginning of this section, the hippocampus of normal rats shows no tendency to accumulate labeled corticosterone, and this finding is also presented in Fig. 5. The septum is the only other structure in the brain that is affected by the administration of unlabeled corticosterone. The septum nevertheless tends to accumulate radioactive corticosterone in normal (Fig. 4) as well as adrenalectomized rats (Fig. I). The uptake process in the septum therefore appears to have a larger capacity for the circulating levels of corticosterone found in normal rats than does the hippocampus. However, Fig. 5 shows that 3-rag doses of corticosterone, dexamethasone, and hydrocortisone reduce the uptake by the septum of [1,2-3H]corticosterone.
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Time (hr) after ADX Fig. 6. Uptake of radioactivity injected as [l,2-3H]corticosterone in 4 brain regions as a function of time after adrenalectomy. A 2-h interval elapsed between radioactive hormone injection and sacrifice. A, Concentration of radioactivity vs. time. B, Concentration of radioactivity relative to cortex vs. time. At each time the values represent averages from 3 rats. Brain R e s e a r c h , 16 (1969) 227-241
237
CORTICOSTERONE UPTAKE BY RAT BRAIN TABLE IV COMPOSITIDN OF DCM-EXTRACTABLE RADIOACTIVEMATERIAL EXTRACTED FROM HIPPOCAMPUS Cort.
corticosterone; 11-DHC = 1 l-dehydrocorticosterone.
Time interval (injection to sacrifice)
Percent o f DCM-extractable R.A. with RF o f Blood
Hippocampus
Rest o f br~zin
(h) 2
4 6
Cort.
1 I-DHC
Corl,
11-DHC
Corr.
11-DHC
45 62 38 58 69 15
26 7 5 9 7 4.5
75 62 76 89 82 41
11 2 9 7 8 6.5
76 65 66 72 61 36.4
13 11 10 9 5 7
Time after adrenalectomy Since the hippocampus of normal rats does not accumulate radioactive corticosterone, it was interesting to see the time course whereby the hippocampal uptake of labeled hormone appears after adrenalectomy. Normal rats were adrenalectomized at 9 a.m., and radioactive corticosterone was given for a 2-h uptake period at 2, 4, 6, and 20 h after removal of the adrenals. Analysis of blood samples taken from rats at various times after adrenalectomy indicate that there is a very rapid decline in circulating corticosterone, reaching levels comparable to those in long-term adrenalectomized rats within I h of adrenalectomy and remaining at that level thereafter. Because corticosterone leaves the hippocampus and other parts of the brain quite rapidly (Fig. 2), we expected to see the appearance of binding sites for corticosterone in the hippocampus as early as 2 h after adrenalectomy. It can be seen from Fig. 6A that the concentration of radioactive corticosterone in the hippocampus, which is the same as that in the cortex in normal rats, is greater than that in the cortex 2 h after adrenalectomy and continues to increase with time thereafter. When the data are expressed as the concentration relative to cortex (Fig. 6B), it can be seen that the hippocampal concentration of hormone is twice that in the cortex at 2 h after adrenalectomy and 2.5 times as great at 6 h after adrenalectomy. It is somewhat surprising that at 20 h after adrenalectomy the relative concentration had increased to 3.5 and then tended to decrease to 3.0 in long-term adrenalectomies. Possible explanations for this phenomenon will be considered in the Discussion. It should also be noted that at all times after adrenalectomy the relative concentration of radioactivity in the septum remains constant and the same as that in rats with adrenals intact. The fact that in Fig. 6A the concentration of radioactive hormone in all brain regions can be seen to increase with time after adrenalectomy may be due to a gradual decline in the effectiveness of the system for removing corticosterone from the blood, since blood levels of radioactive hormone (not shown in Fig. 6) also increased with time after adrenalectomy. Brain Research, 16 (1969) 227 241
238
r~. s. x~c~wf~Nel a/,
Identification o ( radioaetive material in the brain as cortieosterone
In order to estimate the proportion of dichloromethane-extractable radioactivity from the brain and blood which remains as corticosterone, the extracts from the uptake experiments were concentrated and subjected to thin-layer chromatography as is described in the experimental procedure. Chromatography was carried out in the presence of 2 carrier steroids, corticosterone and 1 l-dehydrocorticosterone. The latter was chosen because it is known to be a metabolite ofcorticosterone which is found in nervous tissue TM. Table IV shows that at 2 and 4 h, respectively, after [l~2-aH]corti costerone administration, 70 and 80 °,~; respectively, of the radioactivity was recovered as a steroid with the mobility of corticosterone. A very small proportion of the total radioactivity was recovered in the I I-dehydrocorticosterone spot. The fact that the percentage of corticosterone in hippocampus is somewhat higher than that in the rest of the brain is consistent with the preferential retention of corticosterone by the hippocampus. DISCUSSION In this paper we have shown that radioactivity injected as [1,2-3H]corticosterone is retained and concentrated by a limited-capacity uptake process in the hippocampus and septum of adrenalectomized rats. Ovariectomized female rats tend to concentrate and retain estradiol in other brain regions, namely, the preoptic area, hypothalamus, and pituitary 9,13,22,27,a2, indicating that the limbic system of the rat brain is differentiated with respect to hormone sensitivity. The septum, however, tends to accumulate and retain both corticosterone (see above) and estradiol 9,1a,z2,'zv,3'z as well as testosterone, hydrocortisone, and aldosterone2L Weiss et al. 3a and Levine 17 have suggested that corticosterone has inhibitory effects on behavior which tend to counteract the excitatory or disinhibiting effects of ACTH3,16,17, ~. Such hormone effects fit well with the present findings that corticosterone is retained by the hippocampus and septum, since several investigators have suggested that both hippocampus and septum have inhibitory influences on behavior14,21,aS. The hippocampus and septum may also exert an inhibitory influence on A C T H release from the pituitary 6,2°. We do not yet know whether corticosterone actually facilitates the activity of the septum and hippocampus, but electrophysiotogical and hormone implantation studies are in progress. Radioactive corticosterone uptake and retention by the septum occurs in normal rats and appears to be saturated only at high and unphysiological concentrations of the unlabeled hormone, whereas the hippocampal uptake mechanism is saturated by the circulating levels of corticosterone found in normal rats subjected to the stress of radioactive hormone injection. In normal rats, the concentration of hormone found in the hippocampus at all times after injection is the same as that in the cortex. If radioactive corticosterone is injected 2 h after adrenalectomy, a time interval at which endogenous hormone should have been extensively released from the hippocampus, we observed a 2-fold greater concentration of labeled corticosterone in the hippocampus Brain Research, 16 (1969) 227-241
CORTICOSTERONE UPTAKE BY RAT BRAIN
239
over that in the cortex. This concentration factor increases to 2.5 at 6 h after adrenalectomy when all endogenous steroid should have disappeared. The tendency of labeled corticosterone to accumulate in the hippocampus increases still further with time up to 20 h after adrenalectomy, and remains at or slightly below this level in long-term adrenalectomies. This increase in binding capacity beyond 6 h after adrenalectomy might be explained by some kind of metabolic transformation in the hippocampus, although until we are able to measure endogenous steroid in the hippocampus it is impossible to rule out a very slow loss of bound hormone from this structure. The idea of a metabolic process responsible for the binding of hormone by the hippocampus is attractive from another standpoint, since it was noted above that, in the time course of corticosterone disappearance from the hippocampus of long-term adrenalectomies, there is a phase of retention, up to 2 h after administration, followed by a period of release of radioactive hormone. A possible explanation for this phenomenon might be the existence of binding factors having a rapid turnover in vivo, and the release of bound hormone would therefore be due to the destruction of the binding material. Such an explanation further suggests that the hormone might stimulate the synthesis of its own binding factor, an idea which has been proposed by another laboratory for estradiol retention in the hypothalamus 2~. These possibilities are currently under investigation. In order to discuss the possible types of action of steroid hormones, such as corticosterone, on the brain and on particular regions of the brain, it must be emphasized that we are dealing with 2 simultaneous processes : (1) The entry of hormone into all regions of the brain from the blood in significant quantities. (2) The retention of hormone by some cells which may themselves be concentrated in certain brain regions. We were able to show that increasing the amount of corticosterone injected into the peritoneal cavity resulted in a nearly proportional increase in the amount of hormone entering the cerebral cortex and hypothalamus over a dose range from 0.7 to 50 #g per 250 g rat. Furthermore, concentrations of radioactive hormone per unit wet weight in structures such as the cortex and hypothalamus are approximately half of those in the blood, and the kinetics of disappearance of hormone from the cortex and hypothalamus indicate free and rapid exchange. It is thus evident that increases in the circulating corticosterone in rats subjected to stressful situations are reflected in increases in the brain level of the hormone. Such hormones may act at the level of the cell membrane, as electrophysiological studies with corticosteroids, testosterone, estradiol, and progesteronO, 29-al would tend to suggest. The rat hypothalamus appears to be acted upon by corticosteroids both functionally and electrically 29 31, and it is important to stress that we have not seen any tendency for corticosterone to accumulate in this region. Therefore, binding of hormone may not be required for this type of action on the rat brain. In this connection, however, Eik-Nes and Brizzee 8 have observed that labeled cortisol concentrates in certain regions of the dog hypothalamus. Their results suggest that cortisol, unlike corticosterone, may act on the hypothalamus by way of a mechanism which requires binding of the hormone. Superimposed on top of the general uptake of corticosterone by the rat brain is the tendency for cells concentrated in the hippocampus and septum to concentrate and Brain Research, 16 (1969) 227 241
240
f~, s. v~Cr~WF~Ne t a / .
retain the hormone by a limited-capacity mechanism which we do not completely understand at this time. A clue to the intracellular site of action of bound hormone is the recent observation in this laboratory "4 that highly purified cell nuclei isolated from the hippocampus retain up to 5 0 ~ of the radioactive material found in theentire structure. Similar observations have been made for estradiol bound by the preoptic area and hypothalamus 24 and are supported by recent autoradiographic evidenceZL Since it is well-established that nuclear binding of steroid hormones in tissues such as the uterus and prostate gland leads to an increase in ribonucleic acid and protein synthesis in the tissue 2,4,12, it is likely that nuclear binding of hormones in brain cell nuclei leads to similar alterations in macromolecular synthesis in those brain cells. SUMMARY [l,2-3H]Corticosterone is taken up by all parts of the brain of normal and adrenalectomized rats. As the blood concentration of hormone increases, a situation which occurs following stress, the concentration of hormone in the brain also increases. Superimposed on top of the brain-wide uptake of corticosterone is the tendency of 2 limbic structures, the hippocampus and septum, to concentrate and retain labeled corticosterone. The hippocampal uptake sites are saturated by levels of corticosterone found in normal rats, while the septal sites are not. Hippocampal uptake is therefore observed only in adrenalectomized rats and can be seen as early as 2 h after adrenalectomy. The hippocampal uptake sites in adrenalectomized rats are saturated by exogenous unlabeled corticosterone, hydrocortisone, and dexamethasone. The relevance of hormone retention to the function of the hippocampus and septum in behavior and control of ACTH release is discussed. The possible cellular sites of action of corticosterone are also considered. ACKNOWLEDGEMENTS This research was supported by U.S. Public Health Service Grants NB 07080 to Dr. McEwen and M H 13189 to Dr. N. E. Miller. Dexamethasone was the generous gift of Merck, Sharp and Dohme, Rahway, N.J.
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