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The effect of dexamethasone blockade of ACTH release on avoidance learning
Physiology and Behavior. Vol. 5, pp. 361-363. Pergamon Press, 1970. Printed in Great Britain
The Effect of Dexamethasone Blockade of ACTH Release on Avoidance Learning' P H Y L L I S K A S P E R - P A N D I I, R U T H
H A N S I N G s A N D D A V I D R. U S H E R
University of Wisconsin-Madison, Department of National Health and Welfare, Ottawa, Canada (Received 16 O c t o b e r 1969) P., R. HANSINGAND D. R. USHER. Effect of dexamethasone blockade of ACTH release on avoidance learning. PHYSIOL.BEHAV. 5(3) 361-363, 1970.--Forty-eight male albino rats received 25 avoidance training trials in a KASPER-PANDI,
shuttle-box, and a second block of 25 trials after a 0-hr, l-hr or 4-hr inters~sion interval. Half of the rats were injected with 400 ixg of dexarnethasone 21-phosphate 2 hr before the first 25 trials. Animals tested after the 0-hr intersession interval avoided most often, and tended to make more spontaneous intertrial crossings than the l-hr group. Dexarnethasone 21-phosphate blocked adrenocortieotropin (ACTH) release in response to the stress of training as reflected in plasma corticosterone levels, but it did not affect avoidance behavior. U-shaped relationship
Active avoidance
Dexamethasone 21-phosphate
Stress
ACTH
Corticosterone
Apparatus
T m ~ is a U-shaped relationship between performance of an incompletely learned shuttle-box response and the interval between the initial block of training trials and a subsequent block of trials [7]. A group of rats tested at a 1-hr interval made fewer avoidance responses than groups tested at 1-min, 24-hr and 19-day intervals. The U-shaped relationship appears to be a general phenomenon, since it occurs in passive [12] as well as active avoidance [2--4, 7, 8, 10]. Little effort was made to account for the U-shaped effect, until a recent demonstration [2] of the relationship between the downward limb of the U-shaped response curve and levels of circulating corticosterone measured at appropriate intervals after one block of trials. Low levels of corticosterone occurred at 1-hr and 4-hr intervals as did the poorest performance. In the following experiment, the release of A C T H (adrenocorticotropin) in response to shuttle-box training was blocked with dexamethasone 21-phosphate [13, 16]. It was predicted that the downward limb of the effect would not occur in the absence of normal pituitary-adrenal function.
Two automated shuttle-boxes housed in sound-attenuating enclosures were used [15]. A high voltage a.c. power supply was connected to the grid floor through a Lehigh Valley shock scrambler. Intertrial intervals and CS and US presentations were controlled automatically. The latencies of escape and conditioned avoidance, and the number of intertrail crossings were recorded automatically.
Procedures All rats were tested and killed between 11 a.m. and 5 p.m. to minimize the effects of diurnal fluctuations in adrenal function on the measures of plasma corticosterone. The test times for the different experimental groups were balanced within this interval. Forty-eight rats were divided into two major experimental groups, Control and Dexamethasone. These groups were each further subdivided into three groups; 0-hr, 1-hr and 4-hr. The latter group designations refer to the intervals between an initial block of 25 training trials and a subsequent block of 25 trials. The Dexamethasone animals received a subcutaneous injection in the abdominal region of 400 gg of dexamethasone 21-phosphate (Merck Sharp and Dohme, DEC A D R O N Phosphate, 4 mg/cm 3) 2 hr prior to the start of training. Control animals received a similarly placed saline injection. Animals remained in their home cages until the start of training.
METHOD
Subjects The animals were 57 male albino rats weighing 230-280 g when obtained from the Holtzman Co. of Madison, Wisconsin. They were housed individually under conditions of constant illumination for three weeks prior to training to decrease diurnal fluctuations in plasma cortieosterone level.
XThis research was supported by National Science Foundation Postdoctoral Fellowship 46025 held by Dr. Kasper-Pandi in 1966 and by N.I.M.H. grant MH-10801 held by Dr. Schwartzbaum. The authors wish to thank Alec Bayne of the Canadian Department of National Health and Welfare for the corticosterone assays, and Robert Silber and Sanford Stcelman of the Merck Institute for detailed information concerning the use of dexamethasone 21-phosphate. ~Present address: Rudolf Magnus Institute for Pharmacology, Vondellaan 6, Utrecht, The Netherlands. Send reprint requests to Dr. David Usher, Food and Drug Directorate, Department of National Health and Welfare, Ottawa, Canada. aPresent address: Naval Personnel Research Activity, San Diego, California 92152. 361
362
KASPER-PANDI, HANSING AND USHER
Animals were allowed 2 min to explore the shuttle-box. The CS was a slight increase in illumination in the safe compartment. Animals were allowed 10 sec to move to the safe compartment before the 1.0 mA shock was applied until a response occurred. Shock levels commonly used in this type of research are 1.1 mA [7, 8], 1.7 mA [4] and 0.3-0.25 mA [2, 3, 10]. Both CS and US were response-terminated. The intertrial interval was constant at 30 sec. Rats in the 0-hr group received 50 uninterrupted trials. The l-hr and 4-hr groups were returned to their home cages after 25 trials for the intervals between blocks of trials. They then received a second block of training trials. Immediately after training, each animal was etherized to the point where its breathing had stopped but its heart was still beating. The heart was exposed and 2 ml of blood was removed from the right ventricle into a heparinized syringe. The blood was immediately centrifuged for 7 min, and the plasma was quickly removed and frozen. Plasma levels of corticosterone were determined [6] as indications of ACTH release. The plasmas of one 0-hr Control and one 4-hr Dexamethasone animal were lost. Although dexamethasone 21phosphate has glucocorticoid activity, its presence does not interferewiththefluorometricassayofcorticosterone[13]. Nine untrained and uninjected rats were killed to provide base line measures of plasma corticosterone. RESULTS
The plasma corticosterone and avoidance data are summarized in Table 1. The corticosterone levels of the Control group are significantly higher than those of the Dexamethasone group (F = 35.88, d f = 1/40, p < 0.001). The interval and interaction effects did not approach significance. The corticosterone levels of the Control group were high after the second block of trials at all intervals, suggesting that adrenal responsiveness at the 1-hr and 4-hr intervals was not grossly inferior to responsiveness at the 0-hr interval. In comparison, rats given only one block of trials and killed after 1-hr or 4-hr intervals would be expected to have plasma corticosterone levels equal to or slightly lower than those of unstressed rats [2, 10]. The mean levels ofcorticosteronefound in the pooled data of the Dexamethasone groups did not differ from the mean of 28 t~g/100 ml found in the unstressed uninjected control animals, demonstrating that treatment effectively suppressed ACTH release. The development of some nonspecific fluorescence during plasma storage (6 months) may have elevated the values slightly [1]. However, the 28 vtg/100 ml found in the unstressed controls is not strikingly different from the 21 and 18 tzg/100ml reported for unstressed Long-Evans rats housed in constant light [2, 10]. Dexamethasone 21-phosphate had no effect upon the number of avoidance responses made by the 0-hr, 1-hr and 4-hr
groups (Table 1), or upon the latencies of escape or avoidance. No effect was observed during either the first or second block of trials. The avoidance data from the Control and Dexamethasone groups were pooled and analyzed for the U-shaped effect. The number of responses made by the pooled 0-hr group exceeded that of the 1-hr group, p < 0.02 (Mann-Whitney U, two-tailed), but there was no difference between the l-hr and 4-hr groups. The downward limb of the U-shaped curve was clearly obtained. The 0-hr group made slightly more spontaneous intertrial crossings than the l-hr group, p :~ 0.05 (Mann-Whitney U, one-tailed). TABLE 1 SUMMARY OF MEAN PLASMA CORTICOSTERONE VALUES AFTER Two BLOCKS OF 25 SHUTrLE-BOX TRAINING TRIALS AND MEAN NUMBER OF CONDITIONEDAVOIDANCERESPONSES MADE DURING THE SECOND BLOCK OF 25 TRIALS
Control Dexamethasone
Mean plasma corticosterone level in t~g/100ml 0-hr l-hr 4-hr 66.0 64.4 60.2 19.8 22.5 12.4
Control Dexamethasone
Mean number of avoidance responses 0-hr 1-hr 4-hr 7.5 1.9 2.5 8.0 0.5 1.6
DISCUSSION
No support was found for the hypothesis that the downward limb of the U-shaped curve is dependent upon circulating levels of ACTH and/or corticosterone. The tremendous difference in ACTH release between the Dexarnethasone and Control groups was not accompanied by a behavioral difference. Furthermore, since dexamethasone 21-phosphate has potent glucocorticoid activity and since the control animals had high levels of plasma corticosterone after the second block of trials, both groups could be said to have high levels of steroids at all three intervals. Yet the performance of the 0-hr groups was superior to that of the 1-hr group. Our findings are surprising in view of the substantial literature demonstrating effects of pituitary-adrenal hormones on fear-motivated behavior [5, 9-11, 17, 18]. One possible explanation lies in recent data suggesting that the role of pituitary-adrenal hormones is strongest when interacting with weak fear drive [18]. If motivation is intense enough, hormonal effects on behavior may be obscured.
REFERENCES
I. Bowman, R. E. and R. E. De Luna. Protein-bindingassays for adrenocorticoids. Behav. res. Meth. Instrum. 1: 135-138, 1969. 2. Brush, F. R. and S. Levine. Adrenocortieal activity and avoidance learning as a function of time after fear conditioning. Physiol. Behav. 1: 309-311, 1966. 3. Brush, F. R., J. S. Meyer and M. E. Palmer. Joint effects of intertrial and intersession interval upon avoidance learning. PsychoL Rep. 14: 31-37, 1964.
4. Denny, M. R. and R. E. Ditchman. The locus of maximal "Kamin effect" in rats. J. comp. physiol. Psychol: 55: 10691070, 1962. 5. De Wied, D. Opposite effects of ACTH and glucocorticosteroids on extinction of conditioned avoidance behavior. Prec. Second Internat. Cong. Hormonal Steroids, Milan, 1966. Amsterdam: Excerpta Medica, 1967, 945-951. 6. Givner, M. L. and G. J. Rocbefort. An improved assay of corticosterone in rat serum and adrenal tissue. Steroids 6: 485--489, 1965.
DEXAMETHASONE AND AVOIDANCE 7. Kamin, L. J. The retention of an incompletely learned avoidance response. J. comp. physiol. Psychol. 50: 457-460, 1957. 8. Kamin, L. J. Retention of an incompletely learned avoidance response: Some further analyses. J. comp. physiol. Psychol. 56: 713-718, 1963. 9. KorAnyi, L., E. Endr6czi, K. Lissfik and l~. Szepes. The effect of ACTH on behavioral processes motivated by fear in mice. Physiol. Behav. 2: 439--445, 1967. 10. Levine, S. and F. R. Brush. Adrenocortical activity and avoidance learning as a function of time after avoidance training. Physiol. Behav. 2: 385-388, 1967. 11. Levine, S. and L. E. Jones. Adrenocorticotropic hormone (ACTH) and passive avoidance learning. J. comp. physiol Psychol. 59: 357-360, 1965. 12. Pinel, J. P. J. and R. M. Cooper. Demonstration of the Kamin effect after one-trial avoidance learning. Psychonom. Sci. 4: 17-18, 1966.
363 13. Purvcs, H. D. and N. E. Sirett. Assay of corticotrophin in dexamethasone-treated rats. Endocrinology 77: 366-374, 1965. 14. Rocbefort, G. J., J. Roscnberger and M. Saffran. Depletion of pituitary corticotrophin by various stresses and by neurohypophysial preparations. J. Physiol. 146: 105-116, 1959. 15. Schwartzbaum, J. S., R. H. Green, W. W. Beatty and J. B. Thompson. Acquisition of avoidance behavior following septal lesions in the rat. J. comp. physiol. Psychol. 63: 95-104, 1967. 16. Stark, E., A. Gy6vai, Zs. Acs, K. Sz. Szalay and B. Varga. The site of the blocking action of dexamethasonc on ACTH secretion: In vivo and in vitro studies. Neuroendocrinology 3: 275-284, 1968. 17. Weiss, J. M., B. S. McEwcn, M. T. A. Silva and M. F. Kalkut. Pituitary-adrenal influences on fear responding. Science 163: 197-199, 1969. 18. Wcrtheim, G. A., R. L. Conner and S. Lcvine. Avoidance conditioning and adrenocortical function in the rat. Physiol. Behav. 4: 41-44, 1969.
The Effect of Dexamethasone Blockade of ACTH Release on Avoidance Learning' P H Y L L I S K A S P E R - P A N D I I, R U T H
H A N S I N G s A N D D A V I D R. U S H E R
University of Wisconsin-Madison, Department of National Health and Welfare, Ottawa, Canada (Received 16 O c t o b e r 1969) P., R. HANSINGAND D. R. USHER. Effect of dexamethasone blockade of ACTH release on avoidance learning. PHYSIOL.BEHAV. 5(3) 361-363, 1970.--Forty-eight male albino rats received 25 avoidance training trials in a KASPER-PANDI,
shuttle-box, and a second block of 25 trials after a 0-hr, l-hr or 4-hr inters~sion interval. Half of the rats were injected with 400 ixg of dexarnethasone 21-phosphate 2 hr before the first 25 trials. Animals tested after the 0-hr intersession interval avoided most often, and tended to make more spontaneous intertrial crossings than the l-hr group. Dexarnethasone 21-phosphate blocked adrenocortieotropin (ACTH) release in response to the stress of training as reflected in plasma corticosterone levels, but it did not affect avoidance behavior. U-shaped relationship
Active avoidance
Dexamethasone 21-phosphate
Stress
ACTH
Corticosterone
Apparatus
T m ~ is a U-shaped relationship between performance of an incompletely learned shuttle-box response and the interval between the initial block of training trials and a subsequent block of trials [7]. A group of rats tested at a 1-hr interval made fewer avoidance responses than groups tested at 1-min, 24-hr and 19-day intervals. The U-shaped relationship appears to be a general phenomenon, since it occurs in passive [12] as well as active avoidance [2--4, 7, 8, 10]. Little effort was made to account for the U-shaped effect, until a recent demonstration [2] of the relationship between the downward limb of the U-shaped response curve and levels of circulating corticosterone measured at appropriate intervals after one block of trials. Low levels of corticosterone occurred at 1-hr and 4-hr intervals as did the poorest performance. In the following experiment, the release of A C T H (adrenocorticotropin) in response to shuttle-box training was blocked with dexamethasone 21-phosphate [13, 16]. It was predicted that the downward limb of the effect would not occur in the absence of normal pituitary-adrenal function.
Two automated shuttle-boxes housed in sound-attenuating enclosures were used [15]. A high voltage a.c. power supply was connected to the grid floor through a Lehigh Valley shock scrambler. Intertrial intervals and CS and US presentations were controlled automatically. The latencies of escape and conditioned avoidance, and the number of intertrail crossings were recorded automatically.
Procedures All rats were tested and killed between 11 a.m. and 5 p.m. to minimize the effects of diurnal fluctuations in adrenal function on the measures of plasma corticosterone. The test times for the different experimental groups were balanced within this interval. Forty-eight rats were divided into two major experimental groups, Control and Dexamethasone. These groups were each further subdivided into three groups; 0-hr, 1-hr and 4-hr. The latter group designations refer to the intervals between an initial block of 25 training trials and a subsequent block of 25 trials. The Dexamethasone animals received a subcutaneous injection in the abdominal region of 400 gg of dexamethasone 21-phosphate (Merck Sharp and Dohme, DEC A D R O N Phosphate, 4 mg/cm 3) 2 hr prior to the start of training. Control animals received a similarly placed saline injection. Animals remained in their home cages until the start of training.
METHOD
Subjects The animals were 57 male albino rats weighing 230-280 g when obtained from the Holtzman Co. of Madison, Wisconsin. They were housed individually under conditions of constant illumination for three weeks prior to training to decrease diurnal fluctuations in plasma cortieosterone level.
XThis research was supported by National Science Foundation Postdoctoral Fellowship 46025 held by Dr. Kasper-Pandi in 1966 and by N.I.M.H. grant MH-10801 held by Dr. Schwartzbaum. The authors wish to thank Alec Bayne of the Canadian Department of National Health and Welfare for the corticosterone assays, and Robert Silber and Sanford Stcelman of the Merck Institute for detailed information concerning the use of dexamethasone 21-phosphate. ~Present address: Rudolf Magnus Institute for Pharmacology, Vondellaan 6, Utrecht, The Netherlands. Send reprint requests to Dr. David Usher, Food and Drug Directorate, Department of National Health and Welfare, Ottawa, Canada. aPresent address: Naval Personnel Research Activity, San Diego, California 92152. 361
362
KASPER-PANDI, HANSING AND USHER
Animals were allowed 2 min to explore the shuttle-box. The CS was a slight increase in illumination in the safe compartment. Animals were allowed 10 sec to move to the safe compartment before the 1.0 mA shock was applied until a response occurred. Shock levels commonly used in this type of research are 1.1 mA [7, 8], 1.7 mA [4] and 0.3-0.25 mA [2, 3, 10]. Both CS and US were response-terminated. The intertrial interval was constant at 30 sec. Rats in the 0-hr group received 50 uninterrupted trials. The l-hr and 4-hr groups were returned to their home cages after 25 trials for the intervals between blocks of trials. They then received a second block of training trials. Immediately after training, each animal was etherized to the point where its breathing had stopped but its heart was still beating. The heart was exposed and 2 ml of blood was removed from the right ventricle into a heparinized syringe. The blood was immediately centrifuged for 7 min, and the plasma was quickly removed and frozen. Plasma levels of corticosterone were determined [6] as indications of ACTH release. The plasmas of one 0-hr Control and one 4-hr Dexamethasone animal were lost. Although dexamethasone 21phosphate has glucocorticoid activity, its presence does not interferewiththefluorometricassayofcorticosterone[13]. Nine untrained and uninjected rats were killed to provide base line measures of plasma corticosterone. RESULTS
The plasma corticosterone and avoidance data are summarized in Table 1. The corticosterone levels of the Control group are significantly higher than those of the Dexamethasone group (F = 35.88, d f = 1/40, p < 0.001). The interval and interaction effects did not approach significance. The corticosterone levels of the Control group were high after the second block of trials at all intervals, suggesting that adrenal responsiveness at the 1-hr and 4-hr intervals was not grossly inferior to responsiveness at the 0-hr interval. In comparison, rats given only one block of trials and killed after 1-hr or 4-hr intervals would be expected to have plasma corticosterone levels equal to or slightly lower than those of unstressed rats [2, 10]. The mean levels ofcorticosteronefound in the pooled data of the Dexamethasone groups did not differ from the mean of 28 t~g/100 ml found in the unstressed uninjected control animals, demonstrating that treatment effectively suppressed ACTH release. The development of some nonspecific fluorescence during plasma storage (6 months) may have elevated the values slightly [1]. However, the 28 vtg/100 ml found in the unstressed controls is not strikingly different from the 21 and 18 tzg/100ml reported for unstressed Long-Evans rats housed in constant light [2, 10]. Dexamethasone 21-phosphate had no effect upon the number of avoidance responses made by the 0-hr, 1-hr and 4-hr
groups (Table 1), or upon the latencies of escape or avoidance. No effect was observed during either the first or second block of trials. The avoidance data from the Control and Dexamethasone groups were pooled and analyzed for the U-shaped effect. The number of responses made by the pooled 0-hr group exceeded that of the 1-hr group, p < 0.02 (Mann-Whitney U, two-tailed), but there was no difference between the l-hr and 4-hr groups. The downward limb of the U-shaped curve was clearly obtained. The 0-hr group made slightly more spontaneous intertrial crossings than the l-hr group, p :~ 0.05 (Mann-Whitney U, one-tailed). TABLE 1 SUMMARY OF MEAN PLASMA CORTICOSTERONE VALUES AFTER Two BLOCKS OF 25 SHUTrLE-BOX TRAINING TRIALS AND MEAN NUMBER OF CONDITIONEDAVOIDANCERESPONSES MADE DURING THE SECOND BLOCK OF 25 TRIALS
Control Dexamethasone
Mean plasma corticosterone level in t~g/100ml 0-hr l-hr 4-hr 66.0 64.4 60.2 19.8 22.5 12.4
Control Dexamethasone
Mean number of avoidance responses 0-hr 1-hr 4-hr 7.5 1.9 2.5 8.0 0.5 1.6
DISCUSSION
No support was found for the hypothesis that the downward limb of the U-shaped curve is dependent upon circulating levels of ACTH and/or corticosterone. The tremendous difference in ACTH release between the Dexarnethasone and Control groups was not accompanied by a behavioral difference. Furthermore, since dexamethasone 21-phosphate has potent glucocorticoid activity and since the control animals had high levels of plasma corticosterone after the second block of trials, both groups could be said to have high levels of steroids at all three intervals. Yet the performance of the 0-hr groups was superior to that of the 1-hr group. Our findings are surprising in view of the substantial literature demonstrating effects of pituitary-adrenal hormones on fear-motivated behavior [5, 9-11, 17, 18]. One possible explanation lies in recent data suggesting that the role of pituitary-adrenal hormones is strongest when interacting with weak fear drive [18]. If motivation is intense enough, hormonal effects on behavior may be obscured.
REFERENCES
I. Bowman, R. E. and R. E. De Luna. Protein-bindingassays for adrenocorticoids. Behav. res. Meth. Instrum. 1: 135-138, 1969. 2. Brush, F. R. and S. Levine. Adrenocortieal activity and avoidance learning as a function of time after fear conditioning. Physiol. Behav. 1: 309-311, 1966. 3. Brush, F. R., J. S. Meyer and M. E. Palmer. Joint effects of intertrial and intersession interval upon avoidance learning. PsychoL Rep. 14: 31-37, 1964.
4. Denny, M. R. and R. E. Ditchman. The locus of maximal "Kamin effect" in rats. J. comp. physiol. Psychol: 55: 10691070, 1962. 5. De Wied, D. Opposite effects of ACTH and glucocorticosteroids on extinction of conditioned avoidance behavior. Prec. Second Internat. Cong. Hormonal Steroids, Milan, 1966. Amsterdam: Excerpta Medica, 1967, 945-951. 6. Givner, M. L. and G. J. Rocbefort. An improved assay of corticosterone in rat serum and adrenal tissue. Steroids 6: 485--489, 1965.
DEXAMETHASONE AND AVOIDANCE 7. Kamin, L. J. The retention of an incompletely learned avoidance response. J. comp. physiol. Psychol. 50: 457-460, 1957. 8. Kamin, L. J. Retention of an incompletely learned avoidance response: Some further analyses. J. comp. physiol. Psychol. 56: 713-718, 1963. 9. KorAnyi, L., E. Endr6czi, K. Lissfik and l~. Szepes. The effect of ACTH on behavioral processes motivated by fear in mice. Physiol. Behav. 2: 439--445, 1967. 10. Levine, S. and F. R. Brush. Adrenocortical activity and avoidance learning as a function of time after avoidance training. Physiol. Behav. 2: 385-388, 1967. 11. Levine, S. and L. E. Jones. Adrenocorticotropic hormone (ACTH) and passive avoidance learning. J. comp. physiol Psychol. 59: 357-360, 1965. 12. Pinel, J. P. J. and R. M. Cooper. Demonstration of the Kamin effect after one-trial avoidance learning. Psychonom. Sci. 4: 17-18, 1966.
363 13. Purvcs, H. D. and N. E. Sirett. Assay of corticotrophin in dexamethasone-treated rats. Endocrinology 77: 366-374, 1965. 14. Rocbefort, G. J., J. Roscnberger and M. Saffran. Depletion of pituitary corticotrophin by various stresses and by neurohypophysial preparations. J. Physiol. 146: 105-116, 1959. 15. Schwartzbaum, J. S., R. H. Green, W. W. Beatty and J. B. Thompson. Acquisition of avoidance behavior following septal lesions in the rat. J. comp. physiol. Psychol. 63: 95-104, 1967. 16. Stark, E., A. Gy6vai, Zs. Acs, K. Sz. Szalay and B. Varga. The site of the blocking action of dexamethasonc on ACTH secretion: In vivo and in vitro studies. Neuroendocrinology 3: 275-284, 1968. 17. Weiss, J. M., B. S. McEwcn, M. T. A. Silva and M. F. Kalkut. Pituitary-adrenal influences on fear responding. Science 163: 197-199, 1969. 18. Wcrtheim, G. A., R. L. Conner and S. Lcvine. Avoidance conditioning and adrenocortical function in the rat. Physiol. Behav. 4: 41-44, 1969.