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Diurnal changes in endogenous corticosterone content of some brain regions of rats
Brabl Research, 124 (19771 571-575 ;(') Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
571
Diurnal changes in endogenous corticosterone content of some brain regions of rats
I. LENGVA.Ri and ZS. L1POSITS
Department ~f Anatomy, University Medical School, P&s (Hungary) (Accepted December 17th, 1976)
The presence of corticosterone-binding molecules in the rat brain has been reported in earlier publications4,"L Stevens et al. 1'~ have demonstrated that the binding of [3H]corticosterone is inversely related to the circulating amounts of endogenous corticosterone, indicating that the amount of endogenous corticosterone bound in the brain appears to be related to circulating blood levels. A similar conclusion was drawn from the experiments of McEwen et al. 1°, who found that previously administered non-labelled corticosterone reduced the capacity of brain areas to bind [3H]corticosterone. These experiments, however, did not provide information on the amount of endogenous corticosterone. Butte et al. 1 reported a method for determination of endogenous brain corticosterone. They found that the corticosterone content of whole brain and plasma corticosterone levels changed in parallel in mice during acute stress, and recently the same group reported that the corticosterone content of the whole rat brain shows a daily trough and crest with timing similar to that observed for the plasma steroid 2. The diurnal variation in plasma corticosterone levels has been well documented. In rats, the lowest levels of plasma corticosterone are seen in the morning, and highest values prior to or immediately after the onset of darkness. The possibility arises that the interaction of corticosterone with specific brain receptors forms a link in this feedback control system ; therefore, experiments were performed in which diurnal changes in endogenous amounts of corticosterone of various brain areas were investigated in normal rats and in rats in which the anterior connections of the medial basal hypothalamus were interrupted. Frontal deafferentation of the medial basal hypothalamus disrupts diurnal variations in plasma corticosterone levelsV,lL Adult male rats of our inbred Wistar strain were used. The medial basal hypothalamus was frontally deafferented in a group of animals by the method of Halfisz and Pupp 6, and the animals were sampled 18-24 days after surgery. Both intact and operated rats were maintained under standard environmental conditions (25 ~ 2 °C, 60 jo~,relative humidity, lights on 4 a.m. to 6 p.m.). Standard rat pellets and tap water were available ad libitum. Samplingprocedure. Under light ether anesthesia 7 control and 7 operated animals were exsanguinated through the abdominal aorta and decapitated at 7:00, [1:00,
572 15:00 and 19:00 h. The brain and anterior pituitary were quickly removed and placed on dry ice. Elapsed time between initial handling of an animal and removal or" the brain was always less than 2 rain. The following parts were excised from the brains oi" intact animals: whole hypothalamus, preoptic region, basis et tegmentum mesencephali. amygdala with the overlying cortex, dorsal hippocampus, basis pontis and a piece of parietal cortex. The hypothalamus and preoptic region were investigated in operated animals. Individual brain samples were weighed to tile nearest 0.1 mg and !cspective samples pooled and frozen. Similar sampling procedures were repeated 10 t i rues over a period of three months (March-May). Brain corticosterone content was determined by the method of Butic et al. ~, Briefly, the pooled brain samples were homogenized in double-distilled watt,~ and the steroids were extracted by dichloromethane followed by evaporation of an 'aliquot of the extract. After resuspension in water the samples were washed with hep~ane, reextracted in chloroform and an aliquot of the extract was evaporated. The residue was dissolved in absolute ethanol, diluted with water and chromatographed iCelite 535). The hormone content of the "corticosterone fraction" was assayed fluorimetrically. The overall recovery of [aH]corticosterone added to brain homogenate was 65 75'!,,; similar figures for Jail]progesterone and [aH]estradiol in the "corticostet'onc fraction ~ were less than 1 ,~, and less than 5'!,,, respectively. The method of Guillemin et al.., was used for plasma corticosterone content determination in general, but m some cases the method of Butte and Noble "~was employed for the same purpose. In our c ~,pe rience, using internal standard for the whole procedure, the two methods give similar results. especially on male rats. The assays were performed on the days following the sampling days and, as no consistent variation was detected using the same stock of chemicals during the entire experimental period, the results were combined. The daily rhythm of endogenous corticosterone content of various brain areas and that of the anterior pituitary of intact male rats is illustrated in F'ig. i. t~gethm with plasma corticoster one rhythm. The latter shows a pattern which isconslstent w~thin our st rain and is in accordance with data in the literature. The endogenous corHcosterone content of the preoptic region, hippocampus, mesencephalon, amygdala and anterior pituitary exhibits a diurnal pattern similar to that of the plasma corticosterone level. Endogenous corticosterone contents of the pons and cortex are rather low. and do not show daily changes. Interestingly, the hypothalamic corticosterone content shows a rhythmicity which is inversely correlated with the rhythm of plasma corticosterone content. Fig. 2 summarizes the results obtained from operated animals together with data of respective intact controls killed at the same time. The daily rhythm of endogenous corticosterone content of the preoptic region of control animals again is parallel with, and that of the hypothalamus is inversely related to plasma corticostero~ae rhythm. After frontal deafferentation of the medial basal hypothatamus, the plasma corticosterone content does not show rhythmicity comparable to that of controls, and no rhythm was found in the endogenous corticosterone content of the preoptic region and hypothalamus, it can be noted, however, that in these operated animals the endogenous corticosterone content of both the hypothalamus and preoptic region showed an inverse relationship to plasma corticosterone levels.
573 /ug/lOOg 25P L A S MA PITUITARY ill Z O
20
uJ Io') O (J
15PREOPTIC
REGION
I.-
O O
MESENCEPHALON
10.
"HIPPOCAMPUS AMYGDALA
HYPOTHALAMUS
CORTEX PONS 7 ' O0
11t00
15' O0
19 , 130
HOURS OF
DAY
Fig. 1. Diurnal changes in brain corticosterone content of different brain regions and in plasma corticosterone level of intact adult male rats. Each point represents the mean of 10 pooled samples. Horizontal bars indicate standard error of mean. The present experiment does not prove the original assumption that the amount of endogenous steroid bound in the brain is always parallel with the circulating blood level of hormone. This is true for the preoptic region, hippocampus, amygdala and mesencephalon of intact animals, but a reverse correlation exists between the circulating blood corticosterone level and the hypothalamic endogenous hormone content. Moreover, the hormone content of some brain areas does not follow the changes in blood hormone content (e.g. pons and cortex in the present experiment). Kakihana et al. ~ have seen non-parallel changes in blood corticosterone levels and whole brain corticosterone content : while plasma corticosterone levels did not increase significantly 15 min after histamine stress during the stress-responsive period of neonatal mice, brain corticosterone levels did increase slightly, but significantly, during the same period. On the basis of the relationship between the plasma corticosterone level and hormone content of brain regions, the various brain areas can be divided into three groups. (1) The hormone content of some regions (preoptic region, hippocampus, mesencephalon and amygdala) follow the changes in blood corticosterone level. All of these limbic structures participate in the regulation of A C T H secretion. (2) The hypothalamic tissue, which is responsible for the production of corticotrophic hormone releasing hormone, contains the largest amount of corticosterone when the blood level is the lowest, and vice versa. (3) Finally, some brain structures which, according to our present knowledge
574 ~g~ lOOg
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FD H Y P O T H A L A M U $
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7 00
11 O0
15 00
19 00
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Fig. 2. Dirunal changes in hypothalamic and preoptic corticosterone content and plasma corticosterof]e levels of control rats and of rats with frontally deafferented hypothalamus (FD)I For further explanations see Fig. 1.
do not act directly on A C T H secretion (pons and cortex), exhibit a ~ery tow corticosterone content that does not change with the blood level. It should be pointed out. however, that these extremely low levels of hormone content of these structures are at the borderline of sensitivity to the assay method, and therefore some fluctuations could be overlooked, it is suggested from this classification of brain regions that there is a peculiar relationship between the corticosterone binding characteristics of these brain structures and their participation in the regulation of A C T H secretion. Further experiments are needed to prove this hypothesis. Frontal deafferentation of the medial basal hypothalamus disrupts diurnal changes in plasma corticosterone levels, which is well documented in the literature 7,1~. it is not surprising that after this intervention the investigated brain structures do not show diurnal rhythm in endogenous corticosterone content; however, it ~as unexpected that after this operation the hormone content of the preoptic region was inversely correlated to the blood hormone level, which is characteristic only of the hypothalamus of intact animals. No explanation is readily apparent to account for this latter observation. The authors wish to thank Ms. M/trta Szelier for excellent technicai assistance and Ms. MS.rta Solt6sz for photographic work.
575 1 Butte, J. C., Kakihana, R. and Noble, E. P., Rat and mouse brain corticosterone, Endocrinology, 90 (1972) 1091 1100. 2 Butte, J. C., Kakihana, R. and Noble, E. P., Circadian rhythm of corticosterone levels in rat brain, J. Endocr., 68 (1976) 235 239. 3 Butte, J. C. and Noble, E. P., Simultaneous determination of plasma or whole blood cortisol and corticosterone, Acta endocr. (Kbh.), 61 (1969) 678 686. 4 Grosset, B. l., Stevens, W., Bruenger, F. W. and Reed, D. J., Corticosterone binding by rat brain cytosol, J. Nemvchem., 18 (1971) 1725-1732. 5 Guillemin, R., Clayton, G. W., Smith, J. D. and Lipscomb, H., Measurement of free corticosteroids in rat plasma. Physiological validation of a method, Endocrinology, 63 (1958) 349-358. 6 Hal~tsz, B. and Pupp, L., Hormone secretion of the anterior pituitary gland after physical interruption of all nervous pathways to the hypophysiotrophic area, Endocrinology, 77 (1965) 553 562. 7 Hal~.sz, B., Slusher, M. A. and Gorski, R. A., Adrenocorticotrophic hormone secretion in rats after partial or total deafferentation of the medial basal hypothalamus, Neuroendocrinology, 2 (1967) 43-55. 8 Kakihana, R., Blum, S. and Kessler, S., Developmental study of pituitary-adrenocortical response in mice: plasma and brain corticosterone determination after histamine stress, J. Endocr., 60 (1974) 353-358. 9 McEwen, B. S., Magnus, C. and Wallach, G., Soluble corticosterone-binding macromolecu[es extracted from rat brain, Endocrinology, 90 (1972) 217 226. 10 McEwen, B.S., Weiss, J. M. and Schwartz, L. S., Retention ofcorticosterone by cell nuclei from brain regions of adrenalectomized rats, Brain Research, 17 (1970) 471-482. I I Palka, Y., Coyer, D. and Critchlow, V., Effects of isolation of medial basal hypothalamus on pituitairy-adrenal and pituitary-ovarian functions, Neuroendocrinology, 5 (1969) 339 349. 12 Stevens, W., Reed, D. J., Erickson, S. and Grosset, B. I., The binding of corticosterone to brain proteins: diurnal variation, Endocrinology, 93 (1973) 1152 1156.
571
Diurnal changes in endogenous corticosterone content of some brain regions of rats
I. LENGVA.Ri and ZS. L1POSITS
Department ~f Anatomy, University Medical School, P&s (Hungary) (Accepted December 17th, 1976)
The presence of corticosterone-binding molecules in the rat brain has been reported in earlier publications4,"L Stevens et al. 1'~ have demonstrated that the binding of [3H]corticosterone is inversely related to the circulating amounts of endogenous corticosterone, indicating that the amount of endogenous corticosterone bound in the brain appears to be related to circulating blood levels. A similar conclusion was drawn from the experiments of McEwen et al. 1°, who found that previously administered non-labelled corticosterone reduced the capacity of brain areas to bind [3H]corticosterone. These experiments, however, did not provide information on the amount of endogenous corticosterone. Butte et al. 1 reported a method for determination of endogenous brain corticosterone. They found that the corticosterone content of whole brain and plasma corticosterone levels changed in parallel in mice during acute stress, and recently the same group reported that the corticosterone content of the whole rat brain shows a daily trough and crest with timing similar to that observed for the plasma steroid 2. The diurnal variation in plasma corticosterone levels has been well documented. In rats, the lowest levels of plasma corticosterone are seen in the morning, and highest values prior to or immediately after the onset of darkness. The possibility arises that the interaction of corticosterone with specific brain receptors forms a link in this feedback control system ; therefore, experiments were performed in which diurnal changes in endogenous amounts of corticosterone of various brain areas were investigated in normal rats and in rats in which the anterior connections of the medial basal hypothalamus were interrupted. Frontal deafferentation of the medial basal hypothalamus disrupts diurnal variations in plasma corticosterone levelsV,lL Adult male rats of our inbred Wistar strain were used. The medial basal hypothalamus was frontally deafferented in a group of animals by the method of Halfisz and Pupp 6, and the animals were sampled 18-24 days after surgery. Both intact and operated rats were maintained under standard environmental conditions (25 ~ 2 °C, 60 jo~,relative humidity, lights on 4 a.m. to 6 p.m.). Standard rat pellets and tap water were available ad libitum. Samplingprocedure. Under light ether anesthesia 7 control and 7 operated animals were exsanguinated through the abdominal aorta and decapitated at 7:00, [1:00,
572 15:00 and 19:00 h. The brain and anterior pituitary were quickly removed and placed on dry ice. Elapsed time between initial handling of an animal and removal or" the brain was always less than 2 rain. The following parts were excised from the brains oi" intact animals: whole hypothalamus, preoptic region, basis et tegmentum mesencephali. amygdala with the overlying cortex, dorsal hippocampus, basis pontis and a piece of parietal cortex. The hypothalamus and preoptic region were investigated in operated animals. Individual brain samples were weighed to tile nearest 0.1 mg and !cspective samples pooled and frozen. Similar sampling procedures were repeated 10 t i rues over a period of three months (March-May). Brain corticosterone content was determined by the method of Butic et al. ~, Briefly, the pooled brain samples were homogenized in double-distilled watt,~ and the steroids were extracted by dichloromethane followed by evaporation of an 'aliquot of the extract. After resuspension in water the samples were washed with hep~ane, reextracted in chloroform and an aliquot of the extract was evaporated. The residue was dissolved in absolute ethanol, diluted with water and chromatographed iCelite 535). The hormone content of the "corticosterone fraction" was assayed fluorimetrically. The overall recovery of [aH]corticosterone added to brain homogenate was 65 75'!,,; similar figures for Jail]progesterone and [aH]estradiol in the "corticostet'onc fraction ~ were less than 1 ,~, and less than 5'!,,, respectively. The method of Guillemin et al.., was used for plasma corticosterone content determination in general, but m some cases the method of Butte and Noble "~was employed for the same purpose. In our c ~,pe rience, using internal standard for the whole procedure, the two methods give similar results. especially on male rats. The assays were performed on the days following the sampling days and, as no consistent variation was detected using the same stock of chemicals during the entire experimental period, the results were combined. The daily rhythm of endogenous corticosterone content of various brain areas and that of the anterior pituitary of intact male rats is illustrated in F'ig. i. t~gethm with plasma corticoster one rhythm. The latter shows a pattern which isconslstent w~thin our st rain and is in accordance with data in the literature. The endogenous corHcosterone content of the preoptic region, hippocampus, mesencephalon, amygdala and anterior pituitary exhibits a diurnal pattern similar to that of the plasma corticosterone level. Endogenous corticosterone contents of the pons and cortex are rather low. and do not show daily changes. Interestingly, the hypothalamic corticosterone content shows a rhythmicity which is inversely correlated with the rhythm of plasma corticosterone content. Fig. 2 summarizes the results obtained from operated animals together with data of respective intact controls killed at the same time. The daily rhythm of endogenous corticosterone content of the preoptic region of control animals again is parallel with, and that of the hypothalamus is inversely related to plasma corticostero~ae rhythm. After frontal deafferentation of the medial basal hypothatamus, the plasma corticosterone content does not show rhythmicity comparable to that of controls, and no rhythm was found in the endogenous corticosterone content of the preoptic region and hypothalamus, it can be noted, however, that in these operated animals the endogenous corticosterone content of both the hypothalamus and preoptic region showed an inverse relationship to plasma corticosterone levels.
573 /ug/lOOg 25P L A S MA PITUITARY ill Z O
20
uJ Io') O (J
15PREOPTIC
REGION
I.-
O O
MESENCEPHALON
10.
"HIPPOCAMPUS AMYGDALA
HYPOTHALAMUS
CORTEX PONS 7 ' O0
11t00
15' O0
19 , 130
HOURS OF
DAY
Fig. 1. Diurnal changes in brain corticosterone content of different brain regions and in plasma corticosterone level of intact adult male rats. Each point represents the mean of 10 pooled samples. Horizontal bars indicate standard error of mean. The present experiment does not prove the original assumption that the amount of endogenous steroid bound in the brain is always parallel with the circulating blood level of hormone. This is true for the preoptic region, hippocampus, amygdala and mesencephalon of intact animals, but a reverse correlation exists between the circulating blood corticosterone level and the hypothalamic endogenous hormone content. Moreover, the hormone content of some brain areas does not follow the changes in blood hormone content (e.g. pons and cortex in the present experiment). Kakihana et al. ~ have seen non-parallel changes in blood corticosterone levels and whole brain corticosterone content : while plasma corticosterone levels did not increase significantly 15 min after histamine stress during the stress-responsive period of neonatal mice, brain corticosterone levels did increase slightly, but significantly, during the same period. On the basis of the relationship between the plasma corticosterone level and hormone content of brain regions, the various brain areas can be divided into three groups. (1) The hormone content of some regions (preoptic region, hippocampus, mesencephalon and amygdala) follow the changes in blood corticosterone level. All of these limbic structures participate in the regulation of A C T H secretion. (2) The hypothalamic tissue, which is responsible for the production of corticotrophic hormone releasing hormone, contains the largest amount of corticosterone when the blood level is the lowest, and vice versa. (3) Finally, some brain structures which, according to our present knowledge
574 ~g~ lOOg
CONTROL
PLASMA
FD PLASM~
pp=.tlq
t'~,~ oo,~/~¸ CONTROL PREQPTtC REGION
FD PREOPT~C
~EGION
FD H Y P O T H A L A M U $
CONTROL HYPOTHALAMUS
r
7 00
11 O0
15 00
19 00
HOURS OF DAY
Fig. 2. Dirunal changes in hypothalamic and preoptic corticosterone content and plasma corticosterof]e levels of control rats and of rats with frontally deafferented hypothalamus (FD)I For further explanations see Fig. 1.
do not act directly on A C T H secretion (pons and cortex), exhibit a ~ery tow corticosterone content that does not change with the blood level. It should be pointed out. however, that these extremely low levels of hormone content of these structures are at the borderline of sensitivity to the assay method, and therefore some fluctuations could be overlooked, it is suggested from this classification of brain regions that there is a peculiar relationship between the corticosterone binding characteristics of these brain structures and their participation in the regulation of A C T H secretion. Further experiments are needed to prove this hypothesis. Frontal deafferentation of the medial basal hypothalamus disrupts diurnal changes in plasma corticosterone levels, which is well documented in the literature 7,1~. it is not surprising that after this intervention the investigated brain structures do not show diurnal rhythm in endogenous corticosterone content; however, it ~as unexpected that after this operation the hormone content of the preoptic region was inversely correlated to the blood hormone level, which is characteristic only of the hypothalamus of intact animals. No explanation is readily apparent to account for this latter observation. The authors wish to thank Ms. M/trta Szelier for excellent technicai assistance and Ms. MS.rta Solt6sz for photographic work.
575 1 Butte, J. C., Kakihana, R. and Noble, E. P., Rat and mouse brain corticosterone, Endocrinology, 90 (1972) 1091 1100. 2 Butte, J. C., Kakihana, R. and Noble, E. P., Circadian rhythm of corticosterone levels in rat brain, J. Endocr., 68 (1976) 235 239. 3 Butte, J. C. and Noble, E. P., Simultaneous determination of plasma or whole blood cortisol and corticosterone, Acta endocr. (Kbh.), 61 (1969) 678 686. 4 Grosset, B. l., Stevens, W., Bruenger, F. W. and Reed, D. J., Corticosterone binding by rat brain cytosol, J. Nemvchem., 18 (1971) 1725-1732. 5 Guillemin, R., Clayton, G. W., Smith, J. D. and Lipscomb, H., Measurement of free corticosteroids in rat plasma. Physiological validation of a method, Endocrinology, 63 (1958) 349-358. 6 Hal~tsz, B. and Pupp, L., Hormone secretion of the anterior pituitary gland after physical interruption of all nervous pathways to the hypophysiotrophic area, Endocrinology, 77 (1965) 553 562. 7 Hal~.sz, B., Slusher, M. A. and Gorski, R. A., Adrenocorticotrophic hormone secretion in rats after partial or total deafferentation of the medial basal hypothalamus, Neuroendocrinology, 2 (1967) 43-55. 8 Kakihana, R., Blum, S. and Kessler, S., Developmental study of pituitary-adrenocortical response in mice: plasma and brain corticosterone determination after histamine stress, J. Endocr., 60 (1974) 353-358. 9 McEwen, B. S., Magnus, C. and Wallach, G., Soluble corticosterone-binding macromolecu[es extracted from rat brain, Endocrinology, 90 (1972) 217 226. 10 McEwen, B.S., Weiss, J. M. and Schwartz, L. S., Retention ofcorticosterone by cell nuclei from brain regions of adrenalectomized rats, Brain Research, 17 (1970) 471-482. I I Palka, Y., Coyer, D. and Critchlow, V., Effects of isolation of medial basal hypothalamus on pituitairy-adrenal and pituitary-ovarian functions, Neuroendocrinology, 5 (1969) 339 349. 12 Stevens, W., Reed, D. J., Erickson, S. and Grosset, B. I., The binding of corticosterone to brain proteins: diurnal variation, Endocrinology, 93 (1973) 1152 1156.