Effects of Early Experience on the Metabolism and Production of Corticosterone in Rats
Rats were handled for 3 min daily for 3 weeks before or after weaning or were left totally undisturbed. At 80 days of age, animals from each group were selected for corticosterone half-life determination. Adrenal tissue from the remaininganimals was incubated in vim in the presence or absence of ACTH. Handled and unmanipulated animals did not differ in half-life or in adrenocortical output. These data do not support the hypothesis that the reduced adrenocortical rcactivity that resultt from manipulating animals is due to either the metabolism of corticosterone or the rcsponse of the adrenal cortex to ACTH.
The experience of handling or shocking infant rats has been reported by many to result in reduced adrenocortical function at adulthood (Ader, 1975; Ader & Grota, 1973; Denenberg & Zarrow, 1971). The basis for this inference is that following stimulation, animals nlanipulated during infancy have lower plasma corticosterone levels or a temporal pattern of plasma corticosterone which returns t o base line sooner than animals undisturbed during infancy. The reduced adrenocortical reactivity among manipulated animals has been observed at weaning (Ader, Friedman, Grota, & Schaefer, 1968; Grota, 1975) and at adulthood (Ader & Grota, 1969; Hess, Denenberg, Zarrow, & Pfeifer, 1969; Levine, Haltmeyer, Karas, & Denenberg, 1967) in spite of differences in the conditions of the experiments. Three possibilities might account for such differences in the response curves of control and manipulated animals: (1) the differences might reflect increases in the rate of metabolism of corticosterone by the liver as a result of early experience; (2) the differences could arise from a decreased o u t p u t of corticosterone in response to adrenocorticotrophic hormone (ACTH) by animals manipulated in infancy; and (3) the ACTH response of manipulated animals may be less than that of controls. In the present paper, the metabolism of corticosterone as measured by half-life in blood and the capacity of adrenocortical tissue to respond t o ACTH it? vifro as a function of early experience are examined.
Received for publication 29 November 1974 Revised for publication 24 March 1975 Developmental Psychobiology, 9 (3) :2 1 1-215 ( 19 76 ) @ 1976 by John Wiley & Sons, Inc.
21 1
313
GKOTA
Mc tll od Ch;trIcs River ( C D 1 ) rats (Rattlrs ~ 7 0 r ~ g i c u obtained s) comnlercially werc rnaint:iiiiecl ;tiid bred in o u r laboratory as needed. At birth. litters were niaclc up to X-I0 aniiiials by poolins across littci-s born in a 24-hr period and assigiied to Iiancllcd or uiitii~inip\ilated groups. I-landling consisted of holding the pup in the experiincriter's Iiatid f o r 3 inin daily (Ader c t 31.. 1968; Ader & Crota. 196')). Some of the pups werc Iiandlcd t'rotii 1-3I days of age; others were handled from 2 1-43 days of age. !\fter wcaning at 21 clays of age, the aninials were housed individually until 80 days of a y wlieti they were tested for adrenal function. Food and water were ad l i h f i i i i i a n d the 1;iboratory was kept a t 32°C on ;I 13-lir light: 12-hr dark schedule.
I la II-I i fc Ilc t crin i 112t ion Half-life determinations were begun 3.5 hr prior to darkness. I F x h aninid was a n e s t l i c t i d with pentobarbital ( 3 5 mg/kg o f body weight) a r ~ l15 miti later ;I short piece uf the tail was cut. The tail was lightly clamped t o retard blood flow. After 30 i n i r i o f ;iricstliesia. 35 pCi of f-I-corticosteroiic ( ' I l i ; 50 Ci/rnrnole) was injected iiitrapcritoneally. Three minutes after injection 100 PI of blood was collected in Iieinatoci-it tubes froti1 the tail. At 10-min intervals after the injection o f labeled c o i ticustcrotie. siniilat- 1 OO-pI samples of blood were collected froin the tail (veil)) foi- ;I tot:tl of 8 saniples. Thc blood was centrifuged and 50 pl ot' plusrn:t frozcti until assayed. T I ) c x l i p l a s m sample was added a known amount of IJC-corticosterctuc ( 1 4 C ) I'or rccovcry clctermination and .5 in1 of water. The saiiiples were extracted twice with 1 1111 o f ctliyl acetate. ctironiatographed on thin layer (Eastrnan 6060) in chloroforiii: niettiailol (95:s) and the are:i corresponding to anthen t i c corticosteronc eluted wit11 ctlianol. dried. and counted by liquid scintillation spectrometry. Tlie a m o u n t of -'l{ arid I4C was estimated arithmetically by a channels ratio method. The : m o u n t of' ' 1 I was adjusted according to ~ I i crecovery of I4c and the half-life was estiinateci SrapIiically f r o i n the logarithmic transformation of t h e exponential (Y = he - k ' ) rcluting counts iri the sample (Y) and time ( t) .
Ad rc nal 011t p LI t Corticostcrone output by adrenocortical tissue in v i m in the presence or Cibsence ul. K T I I was determined as suggested by Birmingham and Kurlents (19%). KaIs werc killed b y decapitation. and the adrenals removed and cleared of fat. Eacli :idrenal was c i u a ~ t c i - e darid half of each adrenal weighed and placed i n I of 3 Ilasks. t;iw 1111 oi' K t - c l w R i n p tical-boriitte buffer containing 300 iiig%, gliicose itnd saturated wrtli 05!S O 2 weie added tu each flask. The flasks werc incubated f o r 60 riiin a t 37°C 111 9S'J 0 2 . I I i c but'i'cr was replaced and 1 International Unit of ACTH/100 ins adrenal tissue ail~lctl to 1 I l x k I'roni each aninial. The f l x k s were incubated agairi a n d a t 30 2nd 60 ti1111 o f incubation, .OS nil of the liquid medium was removed. Corticosterone in the iiictliurii was dctcriiiinctl lluorornetrically as described by Frieclman. Adcr. G I o t a . a n d l,~llsol-l( l ~ ) 6 7 ) ,
EARLY EXPERIENCE AND ADRENAL FUNCTION
213
Results and Discussion The half-life of corticosterone for both sexes as a function of early experience is presented in Table 1. As expected, females had lower half-lives than males: 23.2 2 1.3 min (mean 2 S.E.) for females and 30.7 2 1.56 min for males. These values are somewhat longer than the 10-13 rnin for females and 14-30 rnin for males reported in the literature (Glenister & Yates, 1961; Kitay, 1961; Saroff & Wexler, 1969; Schapiro. Percin, & Kotichas, 1971; Ulrich & Long, 1956). Wide differences in methods may account for the disparity of absolute values of the half-lives but all authors found the sex difference observed in this experiment. However, manipulated and unmanipulated animals did not differ. These data d o not support a hypothesis that differences in the metabolism of corticosterone account for the decreased plasma corticosterone levels that results from daily handling or shocking. Analysis of corticoid output as measured by corticosterone levels in the incubation mediurn revealed an interaction between the presence or absence of ACTH and time of sampling (F = 23.3, d f = l / l 1 7 , p < .01). (See Table 2.) The interaction occurred because the increment in corticosterone level a t 60 min relative to 30 min was greater in the presence of ACTH than in its absence. As n o differences existed between nianipulated and control animals, these data d o not support a hypothesis that differences in adrenocortical function that result from manipulation are due to differences in tlic capacity of adrenocortical tissue to respond to ACTH. The findings that the rate of metabolism of corticosterone and the response to ACT11 by cortical tissue do not differ among manipulated and unmanipulated animals suggest that the decrease in adrenocortical reactivity that results from daily manipulation is the result of differences in the amount of circulating ACTH. This same suggestion has been made by Pfeifer and Davis (1974) who observed reduced tyrosine liydroxylase activity in the adrenals of animals handled in infancy compared to unmanipulatcd animals. These data are also consistent with the observation of Zarrow, Campbell, and Denenberg (1972) that the corticotropliin releasing activity of hypothalamic tissue from manipulated animals is less than that in uninanipulated animals. Thus. the reduced adrenocortical reactivity that results from manipulating infant rats apparently reflects changes in the neural control of ACTH secretion.
TABLE I . Half-life (niin)of H-Corticostcrotiein Adult Ruts. (Aleuii t S.E., 11 = number in puren theses. ) Group
Male
Unmanipulated
31.9 t 2.5 ( I 2) 28.8 ? 2.4 (11) 31.3 t 3.4 (1 1)
Handled Early Handled Late
Female
24.4
t
1.8
(11)
22.2 2 2.3 (10) 2 3 . 0 t 2.8 (1 1)
TAB1.L’2. Adrenocortical output iri vrtro as a Functioii 01 ACTH arid Previous L:xperiencr with Handlirlg Corticostcroizc Concerztrutron (pg) in the M.lcdiuni/lOOnig Adretial Tissue is Presented (Mean S.L.)
*
Time of‘ Sampling
C; ro u p 30 min
50 rnin
Umiianipul;itcd liandled Ilarly Handled Late
1.62 * .07 1.65 i .08 1.83 k . I 2
2.88 + . I 2 2.82 + .17 3.55 ? .23
ACT1 I IJninanipulatcd Handlid I k l y I-1and 1ed La tc
3.72 3.82 3.93
.I 8 .19 .19
9.47 i S O 9.50 i .41 7.88 4 .66
N o ACT11 ~
~
~
I
t +
i
- _ _ - ” . - - ~
Notes This research was supported b y Grant riumbcr 08362 f r o m t h e National Institute of (’hild t Ic;il t Ii J nd 1 lu i n a n I k v c Iopiiivn t . TIic autlior would like to tliank Mrs. ‘I’. I’octcr and Mrs. U. Risen for technical m i s t a n c e . K q u c s t reprint\ from I)r I.cc (;rota. I k p a r t m c n t of Psychiatry, Univercity 01‘ Roi,liestcr. School ol Mcdiciiic and I k n t i s l r y , liochestcr, New York 14642, U . S . A .
R e fcre 11ce s A t l ~.~ K. i ( I 975). I.:arly expcricricc and horrnones: Emotional hchavior and adrenocurtical fuiii.tion. I n 13. I:. I:leftlicriou a n d K. Spr;itt (Eds.), Iloriizonul Corrrlutcs of B(Jl/(zvior. New Y o r k :
I’Icnun1. I’p. 7-33. Adcr. I~M?cliaiiisi?is. Ncw Y o r k : Ac;itIeniic Prcs\. I’p. 39-64. Iricdin,in. S. t 3 . . Adcr, K.,
Rats were handled for 3 min daily for 3 weeks before or after weaning or were left totally undisturbed. At 80 days of age, animals from each group were selected for corticosterone half-life determination. Adrenal tissue from the remaininganimals was incubated in vim in the presence or absence of ACTH. Handled and unmanipulated animals did not differ in half-life or in adrenocortical output. These data do not support the hypothesis that the reduced adrenocortical rcactivity that resultt from manipulating animals is due to either the metabolism of corticosterone or the rcsponse of the adrenal cortex to ACTH.
The experience of handling or shocking infant rats has been reported by many to result in reduced adrenocortical function at adulthood (Ader, 1975; Ader & Grota, 1973; Denenberg & Zarrow, 1971). The basis for this inference is that following stimulation, animals nlanipulated during infancy have lower plasma corticosterone levels or a temporal pattern of plasma corticosterone which returns t o base line sooner than animals undisturbed during infancy. The reduced adrenocortical reactivity among manipulated animals has been observed at weaning (Ader, Friedman, Grota, & Schaefer, 1968; Grota, 1975) and at adulthood (Ader & Grota, 1969; Hess, Denenberg, Zarrow, & Pfeifer, 1969; Levine, Haltmeyer, Karas, & Denenberg, 1967) in spite of differences in the conditions of the experiments. Three possibilities might account for such differences in the response curves of control and manipulated animals: (1) the differences might reflect increases in the rate of metabolism of corticosterone by the liver as a result of early experience; (2) the differences could arise from a decreased o u t p u t of corticosterone in response to adrenocorticotrophic hormone (ACTH) by animals manipulated in infancy; and (3) the ACTH response of manipulated animals may be less than that of controls. In the present paper, the metabolism of corticosterone as measured by half-life in blood and the capacity of adrenocortical tissue to respond t o ACTH it? vifro as a function of early experience are examined.
Received for publication 29 November 1974 Revised for publication 24 March 1975 Developmental Psychobiology, 9 (3) :2 1 1-215 ( 19 76 ) @ 1976 by John Wiley & Sons, Inc.
21 1
313
GKOTA
Mc tll od Ch;trIcs River ( C D 1 ) rats (Rattlrs ~ 7 0 r ~ g i c u obtained s) comnlercially werc rnaint:iiiiecl ;tiid bred in o u r laboratory as needed. At birth. litters were niaclc up to X-I0 aniiiials by poolins across littci-s born in a 24-hr period and assigiied to Iiancllcd or uiitii~inip\ilated groups. I-landling consisted of holding the pup in the experiincriter's Iiatid f o r 3 inin daily (Ader c t 31.. 1968; Ader & Crota. 196')). Some of the pups werc Iiandlcd t'rotii 1-3I days of age; others were handled from 2 1-43 days of age. !\fter wcaning at 21 clays of age, the aninials were housed individually until 80 days of a y wlieti they were tested for adrenal function. Food and water were ad l i h f i i i i i a n d the 1;iboratory was kept a t 32°C on ;I 13-lir light: 12-hr dark schedule.
I la II-I i fc Ilc t crin i 112t ion Half-life determinations were begun 3.5 hr prior to darkness. I F x h aninid was a n e s t l i c t i d with pentobarbital ( 3 5 mg/kg o f body weight) a r ~ l15 miti later ;I short piece uf the tail was cut. The tail was lightly clamped t o retard blood flow. After 30 i n i r i o f ;iricstliesia. 35 pCi of f-I-corticosteroiic ( ' I l i ; 50 Ci/rnrnole) was injected iiitrapcritoneally. Three minutes after injection 100 PI of blood was collected in Iieinatoci-it tubes froti1 the tail. At 10-min intervals after the injection o f labeled c o i ticustcrotie. siniilat- 1 OO-pI samples of blood were collected froin the tail (veil)) foi- ;I tot:tl of 8 saniples. Thc blood was centrifuged and 50 pl ot' plusrn:t frozcti until assayed. T I ) c x l i p l a s m sample was added a known amount of IJC-corticosterctuc ( 1 4 C ) I'or rccovcry clctermination and .5 in1 of water. The saiiiples were extracted twice with 1 1111 o f ctliyl acetate. ctironiatographed on thin layer (Eastrnan 6060) in chloroforiii: niettiailol (95:s) and the are:i corresponding to anthen t i c corticosteronc eluted wit11 ctlianol. dried. and counted by liquid scintillation spectrometry. Tlie a m o u n t of -'l{ arid I4C was estimated arithmetically by a channels ratio method. The : m o u n t of' ' 1 I was adjusted according to ~ I i crecovery of I4c and the half-life was estiinateci SrapIiically f r o i n the logarithmic transformation of t h e exponential (Y = he - k ' ) rcluting counts iri the sample (Y) and time ( t) .
Ad rc nal 011t p LI t Corticostcrone output by adrenocortical tissue in v i m in the presence or Cibsence ul. K T I I was determined as suggested by Birmingham and Kurlents (19%). KaIs werc killed b y decapitation. and the adrenals removed and cleared of fat. Eacli :idrenal was c i u a ~ t c i - e darid half of each adrenal weighed and placed i n I of 3 Ilasks. t;iw 1111 oi' K t - c l w R i n p tical-boriitte buffer containing 300 iiig%, gliicose itnd saturated wrtli 05!S O 2 weie added tu each flask. The flasks werc incubated f o r 60 riiin a t 37°C 111 9S'J 0 2 . I I i c but'i'cr was replaced and 1 International Unit of ACTH/100 ins adrenal tissue ail~lctl to 1 I l x k I'roni each aninial. The f l x k s were incubated agairi a n d a t 30 2nd 60 ti1111 o f incubation, .OS nil of the liquid medium was removed. Corticosterone in the iiictliurii was dctcriiiinctl lluorornetrically as described by Frieclman. Adcr. G I o t a . a n d l,~llsol-l( l ~ ) 6 7 ) ,
EARLY EXPERIENCE AND ADRENAL FUNCTION
213
Results and Discussion The half-life of corticosterone for both sexes as a function of early experience is presented in Table 1. As expected, females had lower half-lives than males: 23.2 2 1.3 min (mean 2 S.E.) for females and 30.7 2 1.56 min for males. These values are somewhat longer than the 10-13 rnin for females and 14-30 rnin for males reported in the literature (Glenister & Yates, 1961; Kitay, 1961; Saroff & Wexler, 1969; Schapiro. Percin, & Kotichas, 1971; Ulrich & Long, 1956). Wide differences in methods may account for the disparity of absolute values of the half-lives but all authors found the sex difference observed in this experiment. However, manipulated and unmanipulated animals did not differ. These data d o not support a hypothesis that differences in the metabolism of corticosterone account for the decreased plasma corticosterone levels that results from daily handling or shocking. Analysis of corticoid output as measured by corticosterone levels in the incubation mediurn revealed an interaction between the presence or absence of ACTH and time of sampling (F = 23.3, d f = l / l 1 7 , p < .01). (See Table 2.) The interaction occurred because the increment in corticosterone level a t 60 min relative to 30 min was greater in the presence of ACTH than in its absence. As n o differences existed between nianipulated and control animals, these data d o not support a hypothesis that differences in adrenocortical function that result from manipulation are due to differences in tlic capacity of adrenocortical tissue to respond to ACTH. The findings that the rate of metabolism of corticosterone and the response to ACT11 by cortical tissue do not differ among manipulated and unmanipulated animals suggest that the decrease in adrenocortical reactivity that results from daily manipulation is the result of differences in the amount of circulating ACTH. This same suggestion has been made by Pfeifer and Davis (1974) who observed reduced tyrosine liydroxylase activity in the adrenals of animals handled in infancy compared to unmanipulatcd animals. These data are also consistent with the observation of Zarrow, Campbell, and Denenberg (1972) that the corticotropliin releasing activity of hypothalamic tissue from manipulated animals is less than that in uninanipulated animals. Thus. the reduced adrenocortical reactivity that results from manipulating infant rats apparently reflects changes in the neural control of ACTH secretion.
TABLE I . Half-life (niin)of H-Corticostcrotiein Adult Ruts. (Aleuii t S.E., 11 = number in puren theses. ) Group
Male
Unmanipulated
31.9 t 2.5 ( I 2) 28.8 ? 2.4 (11) 31.3 t 3.4 (1 1)
Handled Early Handled Late
Female
24.4
t
1.8
(11)
22.2 2 2.3 (10) 2 3 . 0 t 2.8 (1 1)
TAB1.L’2. Adrenocortical output iri vrtro as a Functioii 01 ACTH arid Previous L:xperiencr with Handlirlg Corticostcroizc Concerztrutron (pg) in the M.lcdiuni/lOOnig Adretial Tissue is Presented (Mean S.L.)
*
Time of‘ Sampling
C; ro u p 30 min
50 rnin
Umiianipul;itcd liandled Ilarly Handled Late
1.62 * .07 1.65 i .08 1.83 k . I 2
2.88 + . I 2 2.82 + .17 3.55 ? .23
ACT1 I IJninanipulatcd Handlid I k l y I-1and 1ed La tc
3.72 3.82 3.93
.I 8 .19 .19
9.47 i S O 9.50 i .41 7.88 4 .66
N o ACT11 ~
~
~
I
t +
i
- _ _ - ” . - - ~
Notes This research was supported b y Grant riumbcr 08362 f r o m t h e National Institute of (’hild t Ic;il t Ii J nd 1 lu i n a n I k v c Iopiiivn t . TIic autlior would like to tliank Mrs. ‘I’. I’octcr and Mrs. U. Risen for technical m i s t a n c e . K q u c s t reprint\ from I)r I.cc (;rota. I k p a r t m c n t of Psychiatry, Univercity 01‘ Roi,liestcr. School ol Mcdiciiic and I k n t i s l r y , liochestcr, New York 14642, U . S . A .
R e fcre 11ce s A t l ~.~ K. i ( I 975). I.:arly expcricricc and horrnones: Emotional hchavior and adrenocurtical fuiii.tion. I n 13. I:. I:leftlicriou a n d K. Spr;itt (Eds.), Iloriizonul Corrrlutcs of B(Jl/(zvior. New Y o r k :
I’Icnun1. I’p. 7-33. Adcr. I~M?cliaiiisi?is. Ncw Y o r k : Ac;itIeniic Prcs\. I’p. 39-64. Iricdin,in. S. t 3 . . Adcr, K.,
