- Email: [email protected]
Pituitary-adrenal response to stress in rats with hypothalamic islands
BRAIN RESEARCH
395
P I T U I T A R Y - A D R E N A L RESPONSE TO STRESS IN RATS W I T H H Y P O T H A L A M I C ISLANDS
JON DUNN* AND VAUGHN CR1TCHLOW Department of Anatomy, Baylor College of Medicine, Houston, Texas 77025 (U.S.A.)
(Accepted May 20th, 1969)
INTRODUCTION It is generally recognized that extrahypothalamic structures are capable of influencing pituitary-adrenal function under both resting or non-stress conditions and during the response to stress 4,1°. However, the extent to which hypothalamic regulation of adrenocorticotropic hormone (ACTH) secretion is dependent upon neural connections with extrahypothalamic structures is not clear. Using the 'deafferentation' technique of Halfisz and Pupp 7, recent studies have indicated that hypothalamic control of pituitary-adrenal function is to some extent independent of neural connections with other parts of the central nervous system. Although circadian periodicity in A C T H release appears to be disrupted in rats with isolated ('deafferented') hypothalami 8,13, pituitary-adrenal function under resting or non-stress conditions is appreciable. Furthermore, these preparations demonstrate significant corticosteroid responses to several types of stress 5,s,13 and show feedback suppression of non-stress corticosterone levels 13 with relatively low doses of dexamethasone. The results obtained using the 'deafferentation' technique to isolate the hypothalamus from contiguous parts of the central nervous system were in part corroborated by our recent study which pursued the question of hypothalamic autonomy using forebrain removal as a means of isolating the hypothalamus 3. Brain removal was used to assure completeness of isolation and to preclude the possibility of surrounding brain tissue exerting an influence on the isolated hypothalamus. Twenty-four hours after surgery, rats with hypothalamic or pituitary islands had physiologic levels of plasma corticosterone in the absence of overt or acutely applied stress, and these levels were suppressed with dexamethasone. The present study describes the effects of stress on pituitary-adrenal function 24 h after partial or complete forebrain removal.
* Present address: Department of Anatomy, Louisiana State University Medical Center, New Orleans, La. 70112, U.S.A. Brain Reseoreh, 16 (1969) 395-403
396
J. I)UNN AN[) V. ¢'RIT(~HLOW
METHODS
All animals used in these studies were adult (200-280 g) female SpragueDawley rats (Cheek-Jones, Houston) acclimated to controlled lighting (fluorescenl illumination from 04:00 to 18:00) and temperature (26 _+ I°C) conditions for at least 3 weeks. Purina Lab Chow and tap water were available ad libitum. Fig. I illustrates the island preparations used in this study. The mesencephalo-diencephalic .junction was severed and graded amounts of forebrain were removed leaving either a hypothalamic island (HI), a basal hypothalamic island (BHI) or a pituitary island (PI). Sham-operated and intact animals served as controls. The method of Matsuda et al. 11 was used for brain removal and subsequent hemostasis. Preparation of shamoperated rats as well as postoperative care of experimentals has been detailed elsewhere and will not be elaborated upon here 3. All rats stopped eating and drinking following brain removal; therefore, food and water were withheld from control animals. However, all rats received i.p. 5-10 ml of 5 ~ dextrose in physiologic saline. In all ex-
:
I
II
Fig. 1. Diagrams of experimental preparations used in the present study. Brain Research, 16 (1969) 395-403
397
FOREBRAIN REMOVAL AND RESPONSE TO STRESS
periments, treatment and collection procedures were done outside the animal quarters 24 h after forebrain removal (09:00). Plasma concentrations of corticosterone, measured fluorometrically 6, were used as an index of pituitary-adrenal function. Correction for residual fluorescence was not made. In this laboratory fluorescence in plasma from adrenalectomized female rats is equivalent to approximately 6/~g corticosterone/100 ml plasma.
Effect of forebrain removal on pituitary-adrenal response to ether stress To determine the pituitary-adrenal response to ether stress, 'non-stress' and stress levels of plasma corticosterone were obtained 24 h after partial or complete forebrain removal. The method used for obtaining 'non-stress' and stress blood samples was similar to that described by Zimmermann and Critchlow 15. In the present study, plasma samples obtained within 3 min of initial handling and in absence of overt stress for 16-18 h will be referred to as 'non-stress' even though plasma levels of corticosterone may reflect responses to the stress associated with brain removal and/or food and water deprivation. In this procedure, rats were removed from the animal room, placed in an ether environment, the external jugular vein was exposed and 1.0-1.5 ml of blood was collected in a heparinized syringe within 3 min of initial handling. Exposure to ether was terminated at 3 rain and rats were placed in individual holding cages. For collection of stress samples, the animals were decapitated 12 rain later ( 15 min after initiation of stress) and trunk blood collected in heparinized beakers. Blood samples were immediately centrifuged and plasma collected for determination of 'non-stress' and stress plasma corticosterone concentrations. Differences between 'non-stress' and stress levels of corticosterone in plasma were calculated for individual rats and used as a measure of stress-induced increments in pituitary-adrenal function.
Effect of forebrain removal on pituitary-adrenal response to immobilization stress The effect of immobilization stress on pituitary-adrenal function was assessed in a procedure similar to that described above. 'Non-stress' plasma samples were obtained by rapidly immobilizing rats in the supine position, exposing the jugular vein following infiltration of the overlying skin with procaine and removing 1.0- I. 5 ml of blood within 3 min of initial handling. Following 3-min immobilization, each rat was placed in a holding cage. Twelve minutes later (15 min after initial handling), rats were decapitated and trunk blood collected in heparinized beakers. 'Non-stress' and stress blood samples were processed as described above for corticosterone determinations. Although the above procedures are designated as ether and immobilization stresses, they included such additional stress stimuli as handling, skin incision, superficial neck dissection and acute loss of 1.0-1.5 ml of blood. To determine whether these additional stress stimuli were essential to the response to immobilization, a protocol was used which avoided the double bleeding procedure. Five BHI rats were decapitated immediately on removal from cage and 'non-stress' blood samples were
Brain Research, 16 (1969) 395-403
398
J. D U N N A N D \ .
( RII'(TFILOt,\,
collected. An additional 5 BHI animals were subjected to 3-rain immobilization and decapitated at 15 min for collection of stress blood samples. Adrenal response to A C T H
To test the responsiveness of adrenals of rats a large dose of A C T H (l 8 mU Acthar Get/100 g body tered i.v. to 4 BHI and to 4 intact rats anesthetized capitated 15 min following injection and trunk blood plasma corticosterone levels.
following forebrain removal, weight, Armour) was adminiswith ether. Animals were decollected for determination of
Rat heads were placed in 1 0 ~ formalin following decapitation. After fixation they were decalcified in 1 0 ~ formic acid and processed for histological examination of in situ brain and pituitary. Treatments were assigned and performed according to a randomized block design. Because of missing values due to a mortality rate of approximately 15 ~o, the data were analyzed according to a completely randomized design. Statistical probabilities were determined by analysis of variance and the multiple range test o f Duncan ] performed by the Common Research Computer Facility* with the Program of Sakiz 14. RESULTS Twenty-four hours after forebrain removal, approximately 85 ° o of the operated animals were alive and responded to handling or immobilization with vigorous escape efforts and squeeling. Most rats responded to loud noises with startle reflexes and some exhibited grooming behavior. In the ether and immobilization experiments that utilized the double bleeding procedure, the highest 'non-stress' corticosterone levels were observed in H! prep"Non-stress"
. 80-
I
I--'-I
G. -~ 6 o 0 9
i
40
Stress
-
-
u 20-
0 I2
I~.I..!. il
Intact
Sham
Hypothal. Island
hl¢l Hypothal. Island
Pituitary Island
Fig. 2. Effectof ether stress on plasma levels of corticosterone in femalerats. Concentratio_lsin control animals and 24 h after forebram removal. In this and subsequent illustrations number of animals per treatment group is indicated at the base of the columns: vertical lines indicate standard error. * Supported by U.S. Public Health Service Grant FR-00254-01. Brain Research, 16 (1969) 395-403
399
FOREBRAIN REMOVAL AND RESPONSE TO STRESS
arations (Figs. 2, 3). These levels were not statistically higher than those in intact controls, but they were elevated (P < 0.05) in comparison to those of BHl and PI rats. Effect of forebrain removal on pituitary-adrenal response to ether stress Fig. 2 illustrates the effect of ether stress on plasma levels of corticosterone. Whereas HI and PI rats failed to show significant responses to this stress, intact, sham-operated and BHI rats had stress levels of corticosterone which were higher (P < 0.05) than 'non-stress' levels. However, the stress-induced increments in BHI preparations were smaller (P < 0.05) than those in control groups. Effect of./orebrain removal on pituitary-adrenal response to immobilization stress The effect of immobilization stress on plasma levels of corticosterone is summarized in Figs. 3 and 4. Fig. 3 illustrates the effect of the composite stress which included immobilization, handling, skin incision and hemorrhage on plasma cortico"Non-stress" I
=•80
I Stress
_= O.
r
6o
o
I 7,JJJ
L-",-',-" v/// v/// v///
g,'/L
o
~= 20
111.
N
~51 e
9
Intact
Sham
Hypothalamic Basal Pituitary island Hypothalamie Island Island
Fig. 3. Effect of immobilization stress on plasma levels o f c o r t i c o s t e r o n e in female rats. C o n c e n t r a t i o n s in control animals a n d 24 h after forebrain removal.
== K
60
o _o 4 0
ii)
2O
o
o
o
O
"Non-stress"
Stress
Fig. 4. Effect o f immobilization, in the absence of skin incision a n d h e m o r r h a g e , on plasma levels of corticosterone in female rats with basal h y p o t h a l a m i c islands.
Brain Research, 16 (1969) 395-403
400
.f. DUNN AND V. fRI1-CHLOW
sterone levels. Whereas neither H I nor PI preparations responded to the stress with increased plasma corticosterone concentrations, intact, sham-operated and BHi rats showed significant responses (P <~ 0.05). As with ether stress, the response in B H I rats was smaller (P < 0.01) than in controls. The effect of immobilization in the absence of skin incision and hemorrhage is shown in Fig. 4; plasma concentrations of corticosterone were higher (P < 0.05) in BHI rats subjected to stress than in those killed under 'non-stress' conditions. Adrenal response to A C T H As illustrated in Fig. 5. both BH! and intact rats showed marked increments in plasma corticosterone concentrations 15 min following i.v. A C T H . Although B H I rats did not show as great a response as intact rats. the mean steroid level (78.9 ~3.6/ag/100 ml) was higher than those observed following ether or immobilization stress. Histology Brains of all experimental animals were histologically evaluated for completeness of island preparation and intactness of median eminence-stalk-pituitary complex. Although there was some variation, islands of most HI preparations included rostrally the medial preoptic area, caudally the premammillary nuclei and dorsolaterally the arcuate and ventromedial nuclei. BHI preparations consisted primarily of the basal tuberal region and contained arcuate and medial part of the ventromedial nuclei. All PI rats had complete forebrain removal, as illustrated in Fig. 1, and extensive pituitary infarction. N o infarcts were observed in pituitaries of H I and B H I preparations.
I00
J ~-
-g
BO
0
9
~_ 4O .Q "2 o 0
~ 2o INTACT
BHI Before ACTH
After ACTH
Before AGTH
After ACTH
Fig. 5; Effect o f i.v. A C T H (18 m U A c t h a r Gel/lO0 g body weight, A r m o u r ) on plasma leVels o f corticosterone in female rats 24 h after forebrain removal.
Brain Research, 16 (1969) 395-403
FOREBRAIN REMOVAL AND RESPONSE TO STRESS
40l
DISCUSSION
The results of the present study are consistent with our previous observations that forebrain removal is compatible, 24 h after surgery, with physiologic 'nonstress' plasma corticosterone levels 2,3. Although 'non-stress' corticosterone levels did not differ significantly between HI preparations and intact rats as in previous experiments, concentrations in H I preparations were higher (P < 0.05) than those of BHI rats in both experimental situations. This finding agrees with our previous observation that 'non-stress' plasma corticosterone levels following forebrain removal may be related to the size and/or contents of residual hypothalamic tissue 3. In the present study, BHI but not HI or PI preparations showed significant increments in plasma corticosterone concentrations following 3-min ether or immobilization stress. This seemingly paradoxic finding, that basal hypothalamic island (BHI) rats but not rats with larger hypothalamic islands (HI) responded to stress, may in part be related to the differential effect of hypothalamic island size on 'nonstress' levels of plasma corticosterone. Thus, 24 h after forebrain removal, the hypothalamo-pituitary unit may respond to stress only if the initial 'non-stress' level of activity is not elevated. The basis for the inability of rats with hypothalamic islands to show corticosterone responses to stress equivalent to those in control groups is unknown. The decreased responses observed may reflect a partial dependence upon neural projections to the hypothalamus and compromised secretion of ACTH resulting from destruction of such connections. On the other hand, the reduced responses obtained may be due to alterations in peripheral processes that affect the delivery of ACTH to the adrenal, the responsiveness of adrenocortical tissue to ACTH, and/or the half-life of corticosterone in circulating plasma. The responses of BHI rats to the large dose of A C T H used in these studies do not shed much light on this problem; the data obtained suggest only that adrenals in rats with forebrain ablation are capable of secreting more corticosterone in response to large amounts of exogenous ACTH than in response to stress stimuli. More experiments will be needed to determine the basis for the deficient responses in these preparations. The ability of rats with hypothalamic islands to respond to stress is consistent with the findings of Matsuda et al. 12. Twenty-four hours after forebrain removal, male rats with hypothalamic or median eminence, but not pituitary islands, responded significantly to ether stress with or without additional traumatic stress. If Nembutal anesthesia were used, traumatic stress did not induce a rise in adrenocortical secretion unless a large peninsula of neural tissue connecting dorsal mesencephalon with hypothalamus was left intact. Matsuda et al. concluded that ether directly stimulates the median eminence whereas ACTH secretion induced by traumatic stress involves ascending neural pathways. These findings were recently corroborated by the study of Greer and Rockie 5 which demonstrated that ether induced ACTH release in hypothalamic deafferented rats was blocked with pentobarbital anesthesia. Our data support the suggestion that ether may act directly on hypothalamus and/or median eminence to stimulate pituitary-adrenal function. In addition, the increments in corticosterone levels observed in basal hypothalamic island preparations in reBrain Research, 16 (1969) 395-403
402
J. D U N N
AND
x
~ R[I(:HLO~,
sponse to immobilization suggest that this stress may also utilize, in part, no,~neural pathways to activate the hypothalamo-pituitary-adrenal system. On the basi~ of the experiment which avoided prior bleeding, it appears that neither hemorrhage nor the other stimuli associated with obtaining 'non-stress' plasma samples are essential to the corticosteroid responses to immobilization in rats with most of lbrebrain removed. Although the mechanism by which stress, especially immobilization. caused an elevation in plasma concentrations of corticosterone in BH 1 rats is unknown. the possibility of a stimulus mediated by the vascular system must be considered. The fact that pituitary island rats did not respond to ether or immobilization does not necessarily suggest that these stresses influence pituitary-adrenal function by effecting C R F release from the hypothalamus and/or median eminence: lhe lack of response to stress in the PI group may reflect a lack of functional anterior pJtuttary tissue since all pituitary island rats had large pituitary infarcts. However. preliminary studies indicate that lysine-vasopressin, in a dose of 20 mU/100 g body weight, will induce a significant increase in plasma corticosterone concentration in pituitary island rats. Recent studies indicate that isolation of the hypothalamus in the rat. using the 'deafferentation' technique of Halfisz and Pupp 7, is compatible with retention of" considerable pituitary-adrenal function. In brief, rats subjected to 'deafferentatton show appreciable resting or 'non-stress' levels of corticosterone in plasma 5.8.~:~ and these levels are completely suppressed with a relatively low dose of dexamethasonO3; such rats also demonstrate corticosteroid responses to several types of stressS,S, 13. To date, the findings in rats with hypothalamic islands produced b? forebrain removal are remarkably similar in that resting or 'non-stress' eort~costerone levels are maintained within or near the physiologic range 2,3,9,a'~, and such levels can be depressed with dexamethasone '-',~,9. Furthermore, as demonstrated by Matsuda e t al. ~z and by the present study, such preparations are capable of demonstrating corticosterone responses to stress stimuli. Thus, results obtained with hypothalamlc 'deafferentation' and forebrain removal collectively suggest that hypothalamlc regulation of ACTH secretion is surprisingly independent of neural connections with other central nervous system structures. This is not to say, however, that limbic. midbrain, and other neural structures do not influence pituitary-adrenal function in the intact animal. SUMMARY
To pursue the question of hypothalamic autonomy, 'non-stress' and stress levels of corticosterone (Cpd B) were determined fluorometricalty in plasma obtained before and after stress from adult female rats with hypothalamic (HI), basal hypothalamic (BHI) and pituitary (P1) islands. Sham-operated and intact rats served as controls. Twenty-four hours after partial brain removal, blood samples were obtained rapidly ( < 3 min) for determination of 'non-stress' levels of Cpd B. Stress plasma samples were obtained 15 min following onset of 3-rain ether or immobilization stress. Whereas BH1, sham-operated and intact rats had comparable 'nonBrain Research,
t6 (1969) 395-403
FOREBRAIN REMOVAL AND RESPONSE TO STRESS
403
stress' Cpd B levels and showed significant stress responses, HI rats had higher 'non-stress' C p d B levels and did not show stress responses. PI rats did not respond to stress but had 'non-stress' Cpd B levels c o m p a r a b l e to those o f controls. These results suggest that the isolated h y p o t h a l a m o - p i t u i t a r y unit responds to stress if 'non-stress' Cpd B levels are not elevated. The inability o f PI rats to r esp o n d to stress implies that the h y p o t h a l a m u s a n d / o r median eminence are essential to the stress response. ACKNOWLEDGEMENTS
The technical assistance o f D e n a Buck, Jessie K r o n i n g and Lucille Perkins is gratefully acknowledged. This investigation was supported by U.S. Public Health Service G r a n t AM-3385. J. D. was a U.S. Public Health Service Postdoctoral Fellow l - F 2 - A M - 2 5 , 828-01. V.C. was supported by U.S. Public Health Service Research Career P r o g r a m A w a r d 3 K 3 - G M - 1 5 , 364-09. REFERENCES 1 DUNCAN, D. B., Multiple range and multiple F tests Biometrics, 11 (1955) 142. 2 DUNN, J., AND CR1TCHLOW, V., Plasma corticosterone suppression in rats with pituitary islands, Life Sci., 8 (1969) 9-16. 3 DUNN, J., AND CR|TCHLOW, V., Feedback suppression of 'non-stress' pituitary-adrenal function in rats with forebrain removed, Neuroendocrinology, 4 (1969) 296 308. 4 GANONG, W. F., The central nervous system and the synthesis and release of adrenocorticotropic hormone. In A. V. NALBANDOV(Ed.), Advances in Neuroendocrinology, Univ. of Illinois Press, Urbana, 111., 1963, pp. 92-149. 5 GREER, M. A., AND ROCKIE,C., Inhibition by pentobarbital of ether induced ACTH secretion in the ret, Endocrinology, 83 (1968) 1247-1252. 6 GUILLEMIN, R., CLAYTON, G. W., L1PSCOMB, H. S., AND SMITH, J. D., Fluorometric measurement of rat plasma and adrenal corticosterone concentration, J. Lab. clin. Med., 53 (1958) 830-832. 7 HAL~SZ, 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. 8 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. 9 KENDALL, J. W., MATSUDA, K., DUYCK, C.~ AND GREER, M. A., Studies of the location of the receptor site for negative feedback control of ACTH release, Endocrinology, 74 (1964) 279-283. 10 MANGILI, G., MOTTA, M., AND MARTINI, L., Control of adrenocorticotropic hormone secretion. In L. MARTINIAND W. F. GANONG(Eds.), Neuroendocrinology, Vol. 1, Academic Press, New York, 1966, pp. 297-370. 11 MATSUDA, K., KENDALL, J. W., DUYCK, C., AND GREER, M. A., Neural control of ACTH secretion: Effect of acute decerebration in the rat, Endocrinology, 72 (1963) 845-852. 12 MATSUDA, K., KENDALL, J. W., DUYCK, C., AND GREER, M. A., Pathways by which traumatic stress and ether induce increased ACTH release in the rat, Endocrinology, 75 (1964) 981-985. 13 PALKA, V., COYER, D. D., AND CRITCHLOW, V., Hypothalamic deafferentation and adrenal function, Fed. Proc., 27 (1968) 217. 14 SAKIZ, E., Program Exbiol on file at the Common Research Computer Facility, Texas Medical Center, Houston, Texas. 15 ZIMMERMANN, E., AND CRITCHLOW, V,, Effects of diurnal variation in plasma corticosterone levels on adrenocortical responses to stress, Proc. Soc. exp. Biol. Med. (N. Y.), 125 (1967) 658-663.
Brain Research, 16 (1969) 395-403
395
P I T U I T A R Y - A D R E N A L RESPONSE TO STRESS IN RATS W I T H H Y P O T H A L A M I C ISLANDS
JON DUNN* AND VAUGHN CR1TCHLOW Department of Anatomy, Baylor College of Medicine, Houston, Texas 77025 (U.S.A.)
(Accepted May 20th, 1969)
INTRODUCTION It is generally recognized that extrahypothalamic structures are capable of influencing pituitary-adrenal function under both resting or non-stress conditions and during the response to stress 4,1°. However, the extent to which hypothalamic regulation of adrenocorticotropic hormone (ACTH) secretion is dependent upon neural connections with extrahypothalamic structures is not clear. Using the 'deafferentation' technique of Halfisz and Pupp 7, recent studies have indicated that hypothalamic control of pituitary-adrenal function is to some extent independent of neural connections with other parts of the central nervous system. Although circadian periodicity in A C T H release appears to be disrupted in rats with isolated ('deafferented') hypothalami 8,13, pituitary-adrenal function under resting or non-stress conditions is appreciable. Furthermore, these preparations demonstrate significant corticosteroid responses to several types of stress 5,s,13 and show feedback suppression of non-stress corticosterone levels 13 with relatively low doses of dexamethasone. The results obtained using the 'deafferentation' technique to isolate the hypothalamus from contiguous parts of the central nervous system were in part corroborated by our recent study which pursued the question of hypothalamic autonomy using forebrain removal as a means of isolating the hypothalamus 3. Brain removal was used to assure completeness of isolation and to preclude the possibility of surrounding brain tissue exerting an influence on the isolated hypothalamus. Twenty-four hours after surgery, rats with hypothalamic or pituitary islands had physiologic levels of plasma corticosterone in the absence of overt or acutely applied stress, and these levels were suppressed with dexamethasone. The present study describes the effects of stress on pituitary-adrenal function 24 h after partial or complete forebrain removal.
* Present address: Department of Anatomy, Louisiana State University Medical Center, New Orleans, La. 70112, U.S.A. Brain Reseoreh, 16 (1969) 395-403
396
J. I)UNN AN[) V. ¢'RIT(~HLOW
METHODS
All animals used in these studies were adult (200-280 g) female SpragueDawley rats (Cheek-Jones, Houston) acclimated to controlled lighting (fluorescenl illumination from 04:00 to 18:00) and temperature (26 _+ I°C) conditions for at least 3 weeks. Purina Lab Chow and tap water were available ad libitum. Fig. I illustrates the island preparations used in this study. The mesencephalo-diencephalic .junction was severed and graded amounts of forebrain were removed leaving either a hypothalamic island (HI), a basal hypothalamic island (BHI) or a pituitary island (PI). Sham-operated and intact animals served as controls. The method of Matsuda et al. 11 was used for brain removal and subsequent hemostasis. Preparation of shamoperated rats as well as postoperative care of experimentals has been detailed elsewhere and will not be elaborated upon here 3. All rats stopped eating and drinking following brain removal; therefore, food and water were withheld from control animals. However, all rats received i.p. 5-10 ml of 5 ~ dextrose in physiologic saline. In all ex-
:
I
II
Fig. 1. Diagrams of experimental preparations used in the present study. Brain Research, 16 (1969) 395-403
397
FOREBRAIN REMOVAL AND RESPONSE TO STRESS
periments, treatment and collection procedures were done outside the animal quarters 24 h after forebrain removal (09:00). Plasma concentrations of corticosterone, measured fluorometrically 6, were used as an index of pituitary-adrenal function. Correction for residual fluorescence was not made. In this laboratory fluorescence in plasma from adrenalectomized female rats is equivalent to approximately 6/~g corticosterone/100 ml plasma.
Effect of forebrain removal on pituitary-adrenal response to ether stress To determine the pituitary-adrenal response to ether stress, 'non-stress' and stress levels of plasma corticosterone were obtained 24 h after partial or complete forebrain removal. The method used for obtaining 'non-stress' and stress blood samples was similar to that described by Zimmermann and Critchlow 15. In the present study, plasma samples obtained within 3 min of initial handling and in absence of overt stress for 16-18 h will be referred to as 'non-stress' even though plasma levels of corticosterone may reflect responses to the stress associated with brain removal and/or food and water deprivation. In this procedure, rats were removed from the animal room, placed in an ether environment, the external jugular vein was exposed and 1.0-1.5 ml of blood was collected in a heparinized syringe within 3 min of initial handling. Exposure to ether was terminated at 3 rain and rats were placed in individual holding cages. For collection of stress samples, the animals were decapitated 12 rain later ( 15 min after initiation of stress) and trunk blood collected in heparinized beakers. Blood samples were immediately centrifuged and plasma collected for determination of 'non-stress' and stress plasma corticosterone concentrations. Differences between 'non-stress' and stress levels of corticosterone in plasma were calculated for individual rats and used as a measure of stress-induced increments in pituitary-adrenal function.
Effect of forebrain removal on pituitary-adrenal response to immobilization stress The effect of immobilization stress on pituitary-adrenal function was assessed in a procedure similar to that described above. 'Non-stress' plasma samples were obtained by rapidly immobilizing rats in the supine position, exposing the jugular vein following infiltration of the overlying skin with procaine and removing 1.0- I. 5 ml of blood within 3 min of initial handling. Following 3-min immobilization, each rat was placed in a holding cage. Twelve minutes later (15 min after initial handling), rats were decapitated and trunk blood collected in heparinized beakers. 'Non-stress' and stress blood samples were processed as described above for corticosterone determinations. Although the above procedures are designated as ether and immobilization stresses, they included such additional stress stimuli as handling, skin incision, superficial neck dissection and acute loss of 1.0-1.5 ml of blood. To determine whether these additional stress stimuli were essential to the response to immobilization, a protocol was used which avoided the double bleeding procedure. Five BHI rats were decapitated immediately on removal from cage and 'non-stress' blood samples were
Brain Research, 16 (1969) 395-403
398
J. D U N N A N D \ .
( RII'(TFILOt,\,
collected. An additional 5 BHI animals were subjected to 3-rain immobilization and decapitated at 15 min for collection of stress blood samples. Adrenal response to A C T H
To test the responsiveness of adrenals of rats a large dose of A C T H (l 8 mU Acthar Get/100 g body tered i.v. to 4 BHI and to 4 intact rats anesthetized capitated 15 min following injection and trunk blood plasma corticosterone levels.
following forebrain removal, weight, Armour) was adminiswith ether. Animals were decollected for determination of
Rat heads were placed in 1 0 ~ formalin following decapitation. After fixation they were decalcified in 1 0 ~ formic acid and processed for histological examination of in situ brain and pituitary. Treatments were assigned and performed according to a randomized block design. Because of missing values due to a mortality rate of approximately 15 ~o, the data were analyzed according to a completely randomized design. Statistical probabilities were determined by analysis of variance and the multiple range test o f Duncan ] performed by the Common Research Computer Facility* with the Program of Sakiz 14. RESULTS Twenty-four hours after forebrain removal, approximately 85 ° o of the operated animals were alive and responded to handling or immobilization with vigorous escape efforts and squeeling. Most rats responded to loud noises with startle reflexes and some exhibited grooming behavior. In the ether and immobilization experiments that utilized the double bleeding procedure, the highest 'non-stress' corticosterone levels were observed in H! prep"Non-stress"
. 80-
I
I--'-I
G. -~ 6 o 0 9
i
40
Stress
-
-
u 20-
0 I2
I~.I..!. il
Intact
Sham
Hypothal. Island
hl¢l Hypothal. Island
Pituitary Island
Fig. 2. Effectof ether stress on plasma levels of corticosterone in femalerats. Concentratio_lsin control animals and 24 h after forebram removal. In this and subsequent illustrations number of animals per treatment group is indicated at the base of the columns: vertical lines indicate standard error. * Supported by U.S. Public Health Service Grant FR-00254-01. Brain Research, 16 (1969) 395-403
399
FOREBRAIN REMOVAL AND RESPONSE TO STRESS
arations (Figs. 2, 3). These levels were not statistically higher than those in intact controls, but they were elevated (P < 0.05) in comparison to those of BHl and PI rats. Effect of forebrain removal on pituitary-adrenal response to ether stress Fig. 2 illustrates the effect of ether stress on plasma levels of corticosterone. Whereas HI and PI rats failed to show significant responses to this stress, intact, sham-operated and BHI rats had stress levels of corticosterone which were higher (P < 0.05) than 'non-stress' levels. However, the stress-induced increments in BHI preparations were smaller (P < 0.05) than those in control groups. Effect of./orebrain removal on pituitary-adrenal response to immobilization stress The effect of immobilization stress on plasma levels of corticosterone is summarized in Figs. 3 and 4. Fig. 3 illustrates the effect of the composite stress which included immobilization, handling, skin incision and hemorrhage on plasma cortico"Non-stress" I
=•80
I Stress
_= O.
r
6o
o
I 7,JJJ
L-",-',-" v/// v/// v///
g,'/L
o
~= 20
111.
N
~51 e
9
Intact
Sham
Hypothalamic Basal Pituitary island Hypothalamie Island Island
Fig. 3. Effect of immobilization stress on plasma levels o f c o r t i c o s t e r o n e in female rats. C o n c e n t r a t i o n s in control animals a n d 24 h after forebrain removal.
== K
60
o _o 4 0
ii)
2O
o
o
o
O
"Non-stress"
Stress
Fig. 4. Effect o f immobilization, in the absence of skin incision a n d h e m o r r h a g e , on plasma levels of corticosterone in female rats with basal h y p o t h a l a m i c islands.
Brain Research, 16 (1969) 395-403
400
.f. DUNN AND V. fRI1-CHLOW
sterone levels. Whereas neither H I nor PI preparations responded to the stress with increased plasma corticosterone concentrations, intact, sham-operated and BHi rats showed significant responses (P <~ 0.05). As with ether stress, the response in B H I rats was smaller (P < 0.01) than in controls. The effect of immobilization in the absence of skin incision and hemorrhage is shown in Fig. 4; plasma concentrations of corticosterone were higher (P < 0.05) in BHI rats subjected to stress than in those killed under 'non-stress' conditions. Adrenal response to A C T H As illustrated in Fig. 5. both BH! and intact rats showed marked increments in plasma corticosterone concentrations 15 min following i.v. A C T H . Although B H I rats did not show as great a response as intact rats. the mean steroid level (78.9 ~3.6/ag/100 ml) was higher than those observed following ether or immobilization stress. Histology Brains of all experimental animals were histologically evaluated for completeness of island preparation and intactness of median eminence-stalk-pituitary complex. Although there was some variation, islands of most HI preparations included rostrally the medial preoptic area, caudally the premammillary nuclei and dorsolaterally the arcuate and ventromedial nuclei. BHI preparations consisted primarily of the basal tuberal region and contained arcuate and medial part of the ventromedial nuclei. All PI rats had complete forebrain removal, as illustrated in Fig. 1, and extensive pituitary infarction. N o infarcts were observed in pituitaries of H I and B H I preparations.
I00
J ~-
-g
BO
0
9
~_ 4O .Q "2 o 0
~ 2o INTACT
BHI Before ACTH
After ACTH
Before AGTH
After ACTH
Fig. 5; Effect o f i.v. A C T H (18 m U A c t h a r Gel/lO0 g body weight, A r m o u r ) on plasma leVels o f corticosterone in female rats 24 h after forebrain removal.
Brain Research, 16 (1969) 395-403
FOREBRAIN REMOVAL AND RESPONSE TO STRESS
40l
DISCUSSION
The results of the present study are consistent with our previous observations that forebrain removal is compatible, 24 h after surgery, with physiologic 'nonstress' plasma corticosterone levels 2,3. Although 'non-stress' corticosterone levels did not differ significantly between HI preparations and intact rats as in previous experiments, concentrations in H I preparations were higher (P < 0.05) than those of BHI rats in both experimental situations. This finding agrees with our previous observation that 'non-stress' plasma corticosterone levels following forebrain removal may be related to the size and/or contents of residual hypothalamic tissue 3. In the present study, BHI but not HI or PI preparations showed significant increments in plasma corticosterone concentrations following 3-min ether or immobilization stress. This seemingly paradoxic finding, that basal hypothalamic island (BHI) rats but not rats with larger hypothalamic islands (HI) responded to stress, may in part be related to the differential effect of hypothalamic island size on 'nonstress' levels of plasma corticosterone. Thus, 24 h after forebrain removal, the hypothalamo-pituitary unit may respond to stress only if the initial 'non-stress' level of activity is not elevated. The basis for the inability of rats with hypothalamic islands to show corticosterone responses to stress equivalent to those in control groups is unknown. The decreased responses observed may reflect a partial dependence upon neural projections to the hypothalamus and compromised secretion of ACTH resulting from destruction of such connections. On the other hand, the reduced responses obtained may be due to alterations in peripheral processes that affect the delivery of ACTH to the adrenal, the responsiveness of adrenocortical tissue to ACTH, and/or the half-life of corticosterone in circulating plasma. The responses of BHI rats to the large dose of A C T H used in these studies do not shed much light on this problem; the data obtained suggest only that adrenals in rats with forebrain ablation are capable of secreting more corticosterone in response to large amounts of exogenous ACTH than in response to stress stimuli. More experiments will be needed to determine the basis for the deficient responses in these preparations. The ability of rats with hypothalamic islands to respond to stress is consistent with the findings of Matsuda et al. 12. Twenty-four hours after forebrain removal, male rats with hypothalamic or median eminence, but not pituitary islands, responded significantly to ether stress with or without additional traumatic stress. If Nembutal anesthesia were used, traumatic stress did not induce a rise in adrenocortical secretion unless a large peninsula of neural tissue connecting dorsal mesencephalon with hypothalamus was left intact. Matsuda et al. concluded that ether directly stimulates the median eminence whereas ACTH secretion induced by traumatic stress involves ascending neural pathways. These findings were recently corroborated by the study of Greer and Rockie 5 which demonstrated that ether induced ACTH release in hypothalamic deafferented rats was blocked with pentobarbital anesthesia. Our data support the suggestion that ether may act directly on hypothalamus and/or median eminence to stimulate pituitary-adrenal function. In addition, the increments in corticosterone levels observed in basal hypothalamic island preparations in reBrain Research, 16 (1969) 395-403
402
J. D U N N
AND
x
~ R[I(:HLO~,
sponse to immobilization suggest that this stress may also utilize, in part, no,~neural pathways to activate the hypothalamo-pituitary-adrenal system. On the basi~ of the experiment which avoided prior bleeding, it appears that neither hemorrhage nor the other stimuli associated with obtaining 'non-stress' plasma samples are essential to the corticosteroid responses to immobilization in rats with most of lbrebrain removed. Although the mechanism by which stress, especially immobilization. caused an elevation in plasma concentrations of corticosterone in BH 1 rats is unknown. the possibility of a stimulus mediated by the vascular system must be considered. The fact that pituitary island rats did not respond to ether or immobilization does not necessarily suggest that these stresses influence pituitary-adrenal function by effecting C R F release from the hypothalamus and/or median eminence: lhe lack of response to stress in the PI group may reflect a lack of functional anterior pJtuttary tissue since all pituitary island rats had large pituitary infarcts. However. preliminary studies indicate that lysine-vasopressin, in a dose of 20 mU/100 g body weight, will induce a significant increase in plasma corticosterone concentration in pituitary island rats. Recent studies indicate that isolation of the hypothalamus in the rat. using the 'deafferentation' technique of Halfisz and Pupp 7, is compatible with retention of" considerable pituitary-adrenal function. In brief, rats subjected to 'deafferentatton show appreciable resting or 'non-stress' levels of corticosterone in plasma 5.8.~:~ and these levels are completely suppressed with a relatively low dose of dexamethasonO3; such rats also demonstrate corticosteroid responses to several types of stressS,S, 13. To date, the findings in rats with hypothalamic islands produced b? forebrain removal are remarkably similar in that resting or 'non-stress' eort~costerone levels are maintained within or near the physiologic range 2,3,9,a'~, and such levels can be depressed with dexamethasone '-',~,9. Furthermore, as demonstrated by Matsuda e t al. ~z and by the present study, such preparations are capable of demonstrating corticosterone responses to stress stimuli. Thus, results obtained with hypothalamlc 'deafferentation' and forebrain removal collectively suggest that hypothalamlc regulation of ACTH secretion is surprisingly independent of neural connections with other central nervous system structures. This is not to say, however, that limbic. midbrain, and other neural structures do not influence pituitary-adrenal function in the intact animal. SUMMARY
To pursue the question of hypothalamic autonomy, 'non-stress' and stress levels of corticosterone (Cpd B) were determined fluorometricalty in plasma obtained before and after stress from adult female rats with hypothalamic (HI), basal hypothalamic (BHI) and pituitary (P1) islands. Sham-operated and intact rats served as controls. Twenty-four hours after partial brain removal, blood samples were obtained rapidly ( < 3 min) for determination of 'non-stress' levels of Cpd B. Stress plasma samples were obtained 15 min following onset of 3-rain ether or immobilization stress. Whereas BH1, sham-operated and intact rats had comparable 'nonBrain Research,
t6 (1969) 395-403
FOREBRAIN REMOVAL AND RESPONSE TO STRESS
403
stress' Cpd B levels and showed significant stress responses, HI rats had higher 'non-stress' C p d B levels and did not show stress responses. PI rats did not respond to stress but had 'non-stress' Cpd B levels c o m p a r a b l e to those o f controls. These results suggest that the isolated h y p o t h a l a m o - p i t u i t a r y unit responds to stress if 'non-stress' Cpd B levels are not elevated. The inability o f PI rats to r esp o n d to stress implies that the h y p o t h a l a m u s a n d / o r median eminence are essential to the stress response. ACKNOWLEDGEMENTS
The technical assistance o f D e n a Buck, Jessie K r o n i n g and Lucille Perkins is gratefully acknowledged. This investigation was supported by U.S. Public Health Service G r a n t AM-3385. J. D. was a U.S. Public Health Service Postdoctoral Fellow l - F 2 - A M - 2 5 , 828-01. V.C. was supported by U.S. Public Health Service Research Career P r o g r a m A w a r d 3 K 3 - G M - 1 5 , 364-09. REFERENCES 1 DUNCAN, D. B., Multiple range and multiple F tests Biometrics, 11 (1955) 142. 2 DUNN, J., AND CR1TCHLOW, V., Plasma corticosterone suppression in rats with pituitary islands, Life Sci., 8 (1969) 9-16. 3 DUNN, J., AND CR|TCHLOW, V., Feedback suppression of 'non-stress' pituitary-adrenal function in rats with forebrain removed, Neuroendocrinology, 4 (1969) 296 308. 4 GANONG, W. F., The central nervous system and the synthesis and release of adrenocorticotropic hormone. In A. V. NALBANDOV(Ed.), Advances in Neuroendocrinology, Univ. of Illinois Press, Urbana, 111., 1963, pp. 92-149. 5 GREER, M. A., AND ROCKIE,C., Inhibition by pentobarbital of ether induced ACTH secretion in the ret, Endocrinology, 83 (1968) 1247-1252. 6 GUILLEMIN, R., CLAYTON, G. W., L1PSCOMB, H. S., AND SMITH, J. D., Fluorometric measurement of rat plasma and adrenal corticosterone concentration, J. Lab. clin. Med., 53 (1958) 830-832. 7 HAL~SZ, 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. 8 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. 9 KENDALL, J. W., MATSUDA, K., DUYCK, C.~ AND GREER, M. A., Studies of the location of the receptor site for negative feedback control of ACTH release, Endocrinology, 74 (1964) 279-283. 10 MANGILI, G., MOTTA, M., AND MARTINI, L., Control of adrenocorticotropic hormone secretion. In L. MARTINIAND W. F. GANONG(Eds.), Neuroendocrinology, Vol. 1, Academic Press, New York, 1966, pp. 297-370. 11 MATSUDA, K., KENDALL, J. W., DUYCK, C., AND GREER, M. A., Neural control of ACTH secretion: Effect of acute decerebration in the rat, Endocrinology, 72 (1963) 845-852. 12 MATSUDA, K., KENDALL, J. W., DUYCK, C., AND GREER, M. A., Pathways by which traumatic stress and ether induce increased ACTH release in the rat, Endocrinology, 75 (1964) 981-985. 13 PALKA, V., COYER, D. D., AND CRITCHLOW, V., Hypothalamic deafferentation and adrenal function, Fed. Proc., 27 (1968) 217. 14 SAKIZ, E., Program Exbiol on file at the Common Research Computer Facility, Texas Medical Center, Houston, Texas. 15 ZIMMERMANN, E., AND CRITCHLOW, V,, Effects of diurnal variation in plasma corticosterone levels on adrenocortical responses to stress, Proc. Soc. exp. Biol. Med. (N. Y.), 125 (1967) 658-663.
Brain Research, 16 (1969) 395-403